**Contents**


Reprinted from: *Medicina* **2022**, *58*, 739, doi:10.3390/medicina58060739 . . . . . . . . . . . . . . . **99**


## **About the Editors**

#### **Costantino Balestra**

Prof. Dr. Costantino Balestra first studied the neurophysiology of fatigue, then started studies on environmental and aging physiology issues. He teaches physiology, biostatistics, and research methodology, as well as other subjects. He is the Director of the Integrative Physiology Laboratory and a full-time professor at the Haute Ecole Bruxelles-Brabant (Brussels). He is Vice President (VP) of DAN Europe for research and education, Past President of the European Underwater and Baromedical Society (EUBS), VP of The Belgian Society of Hyperbaric and Subaquatic Medicine (SBMHS-BVOOG), and VP of the Underwater and Hyperbaric Medical Society (UHMS) (USA). His personal research interests include integrative physiology as well as challenging and extreme environments. ORCID: 0000-0001-6771-839X.

#### **Jacek Kot**

Dr. Jacek Kot is a specialist in anesthesiology and intensive care; currently he is a full-time Professor in Diving and Hyperbaric Medicine at Medical University of Gdansk, Poland, also serving as the Chief of Research and Development of the National Centre for Hyperbaric Medicine in Gdynia, Poland. The Centre is equipped with several multi-place hyperbaric chambers, a dry saturation simulator with a 'wet pot', as well as a six-bed intensive care unit within the University Hospital. Jacek Kot is the current President of the European Committee for Hyperbaric Medicine (ECHM), and from 2015 to 2018, he was President of the European Underwater and Baromedical Society (EUBS). He has been involved in international cooperation between European diving and hyperbaric centers (COST-B14, OXYNET, PHYPODE, DAN), as well as in the UHMS International Web-based Education Initiative. His professional interests mainly include HBOT in deep dives and saturation decompressions, as well as critically ill patients, especially with severe soft tissue infections. ORCID: 0000-0001-5604-8407.

## **Preface to "Hyperbaric Medicine"**

Oxygen (both its presence and absence) acts as a potent signaling mechanism in many, if not most, cellular processes.

Oxygen has been used therapeutically mainly to alleviate or correct hypoxia, and has been administered in supra-atmospheric doses in the form of hyperbaric oxygen (a method derived from diving medicine practices, but has been used in non-diving applications since the 1960s).

Hyperbaric oxygen is, by definition, administered in an intermittent way, and in recent years, even the effects of intermittent oxygen administration at non-hyperbaric doses have been investigated and showed interesting results. Since the very beginning, because hyperbaric oxygen therapy relies on pure physics and just applying increased environmental pressure will increase partial pressure of the breathed gases (namely, oxygen), the therapeutic mechanisms involved were believed to be fully understood.

Nowadays, all reported data demonstrate how hyperoxic and hypoxic states can potentially be manipulated if oxygen is considered as a multifaceted molecule more than just a gas.

The certainties and the dogma of when hyperbaric medicine was in its adolescence are slowly being replaced.

Hyperbaric medicine is slowly moving out of its infancy. However, as in real life, with the progression away from infancy, the certainties disappear. It is now our task, as researchers, to reflect upon these uncertainties and distil out of them a coherent, balanced advice towards readers. Let us not jump to conclusions too fast, as our new "certainties"may very well prove to be "not the whole story"again. This reprint is dedicated to increase knowledge in this very interesting field, and present hyperbaric medicine in not so usual applications.

> **Costantino Balestra and Jacek Kot** *Editors*

## *Editorial* **Oxygen: A Stimulus, Not "Only" a Drug**

**Costantino Balestra <sup>1</sup> and Jacek Kot 2,\***


**Abstract:** Depending on the oxygen partial pressure in a tissue, the therapeutic effect of oxygenation can vary from simple substance substitution up to hyperbaric oxygenation when breathing hyperbaric oxygen at 2.5–3.0 ATA. Surprisingly, new data showed that it is not only the oxygen supply that matters as even a minimal increase in the partial pressure of oxygen is efficient in triggering cellular reactions by eliciting the production of hypoxia-inducible factors and heat-shock proteins. Moreover, it was shown that extreme environments could also interact with the genome; in fact, epigenetics appears to play a major role in extreme environments and exercise, especially when changes in oxygen partial pressure are involved. Hyperbaric oxygen therapy is, essentially, "intermittent oxygen" exposure. We must investigate hyperbaric oxygen with a new paradigm of treating oxygen as a potent stimulus of the molecular network of reactions.

**Keywords:** oxygen; hyperbaric oxygen; epigenetics; normobaric oxygen paradox; hyperoxic-hypoxic paradox

#### **1. Background**

The usual path in medical sciences is typically the same: starting with understanding mechanisms; then conducting cellular tests using tissues, small animals, larger animals, then small human studies; and finally extensive clinical studies. Nevertheless, hyperbaric medicine has developed in an unusual way.

In fact, in the 1950s, a Dutch cardiac surgeon, Prof. Ite Boerema, began to use a hyperbaric chamber that was considered efficient in curing divers affected with the socalled "caissons' disease" to help his newborn patients called "blue babies". The procedure to mend cardiac septal defects needed a cardiac arrest, and the time for such a cardiac arrest was only minutes to secure a cardiovascular restart [1]. At that moment, an unacceptable number of patients were not surviving the surgery, and this surgeon wanted to find a method that would allow him to achieve better outcomes. His experiments were published in a famous paper entitled "Life without blood", and he proved that it was possible to survive critical exsanguination while remaining in a hyperbaric chamber breathing high oxygen pressures (3.0 ATA).

Increasing circulating oxygen levels in the body by increasing the barometric pressure in the operating room permits survival even after a more prolonged cardiac arrest. Of course, some years later, extracorporeal circulation became available, and the "hyperbaric operating room" was no longer needed. Hyperbaric oxygen is no longer used for maintaining general oxygenation levels, and its use has been somewhat limited for the treatment of specific diseases that are not necessarily combined with hypoxemia. Since that very moment, many hyperbaric centers have been developed, and a long list of indications and procedures have been produced.

This medical field has been so "dispersive" that reactions arose, and hyperbaric medicine was even "coined" as a "therapy in search of diseases" [2].

Significant progress has been made since the 1960s, and investigations into the mechanisms that are underlying oxygen variations were awarded a Nobel prize in 2019 [3]. This

**Citation:** Balestra, C.; Kot, J. Oxygen: A Stimulus, Not "Only" a Drug. *Medicina* **2021**, *57*, 1161. https:// doi.org/10.3390/medicina57111161

Received: 9 October 2021 Accepted: 22 October 2021 Published: 25 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

is an essential milestone in the field as, today, we have sufficient understanding to tackle two significant points that still need to be investigated in hyperbaric medicine: how much and how often. Hyperbaric medicine should be called "oxygen medicine"; an extensive range of oxygen levels may be used; and, according to these levels, repetitions of sessions must be adapted.

#### **2. The Challenge of Oxygen Partial Pressure**

Depending on the oxygen partial pressure of tissues, the therapeutic effect of oxygenation can vary from simple substance substitution, when normobaric hyperoxia is used to restore tissue oxygen levels to normal values from (120 torr in arterial blood to approx. 30–40 torr in soft tissues), up to hyperbaric oxygenation when breathing of hyperbaric oxygen at 2.5–3.0 ATA gives tissue hyperoxia well above 1000 torr.

Interestingly, the dose-effect relation is not linear but is instead "U-shaped". It is already the clinical standard that, at normobaric conditions, the arterial partial pressure must be kept in the relatively narrow range of 10–20 kPa (75–150 torr), as increased mortality was observed in critically ill patients exposed either to hypoxic or hyperoxic levels [4].

On the other hand, it is well known that exposing humans to hyperoxia induces oxygen toxicity (Paul Bert's effect on the brain and the Lorrain–Smith effect on the lungs).

#### **3. The Challenge of Oxygen Toxicity**

Today, the hyperbaric exposure dose (pressure and time) and repetitions are mainly limited by pulmonary and neurological toxicity. To remain on the safe side and not harm the patient, the OTUs (Oxygen Toxicity Units) or UTPDs (Units of Pulmonary Toxicity Dose) are calculated, even if those units are challenged in new approaches [5–8]. Permanent exposure to hyperbaric oxygen is not a clinical option. Fortunately, it seems that intermittent switching between low and high oxygen pressure is sufficiently potent to induce significant therapeutic molecular actions (Figure 1).

**Figure 1.** Oxygen levels and their therapeutic use (PO<sup>2</sup> , partial pressure of oxygen).

#### **4. Oxygen as a Trigger**

Surprisingly, new data show that even a minimal increase in the partial pressure of oxygen efficiently triggers cellular reactions [9,10]. As we already expressed in previous works, "some decades ago, on the physiological side, the two parameters that characterize extreme environments were identified as eliciting the production of two particular elements: hypoxia-inducible factors and heat-shock proteins. The two are ubiquitous and essential for cellular life" [11,12].

These "Hypoxia-Inducible Factors can trigger several hundred genes", but it has been shown that hyperoxia, more specifically, coming back to normoxia after hyperoxic exposure (relative hypoxia), can trigger this essential factor responsible for vascular, cellular, and metabolic homeostasis and apoptosis [10,13,14]. Its beneficial actions in the fight against cancer cells have recently been advocated [12]. The second is a family of proteins acting as chaperones for other proteins and resetting impaired proteic structures triggered by many environmental stressors [15].

#### **5. Epigenetics and the Challenge of Cellular Responses**

Epigenetics seems to play a significant role in exercise and extreme environments [11]. Recently, it was shown that external stressors could indeed interact with the genome, especially when changes in the oxygen partial pressure are involved [16].

This is may not be all that surprising as we know that physical exercise can produce extensive cellular reactions, including epigenetic reactions. Physical exercise is actually an intermittent oxidative stress variation. The oxygen level is very low in the mitochondria (Figure 2) [17]. We can, therefore, understand how potent a minute variation of oxygen tension may be at that level. We may need to consider oxygen variations in the mitochondrion as the most potent homeorhetic trigger in nature.

**Figure 2.** Oxygen cascade from ambient air down to mitochondria (figure taken from [17]).

Considering this no wonder that even variations of 10% of the inspired oxygen fraction may be effective in humans and cultured cells [9,10,18]. It is now known that genes are not always activated. They are not mandatorily expressed. They can be "turned on" or "off" by external interferences that do not change the DNA sequence. There are two major mechanisms for this: DNA methylation and histone modifications. Acute environmental changes can induce epigenetic modifications; cells constantly receive all kinds of signals informing them about their surrounding environment and adjust their activity to the situation.

Recent data showed an epigenetic change through methylation in alpinists exposed to hypoxia, demonstrating rapid changes that were even recently not considered possible in the human epigenome following acute oxygen variation [16].

Several other articles have discussed the fact that pulsed hyperoxia induces hypoxiainducible factor 1α (HIF-1α) activation and the expression of genes involved in the response to low oxygen describing a "normobaric oxygen paradox" (NOP), i.e., that relative changes of oxygen availability, rather than steady-state hypoxic (or hyperoxic) conditions, coordinate HIF-1α transcriptional effects [14,19]. This phenomenon has different names, either "Hyperoxic–Hypoxic Paradox" [17] or "Normobaric Oxygen Paradox" [20] depending on the range of variation of PO<sup>2</sup> imposed. Nevertheless, a general term could be a "relative hypoxia" without reaching tissular hypoxic levels [20].

These studies investigated the activation of oxygen-sensitive transcription factors in peripheral blood mononuclear cells (PBMC) obtained from a human after breathing, increasing PO2, generating mild, high, and very high hyperoxia (30%, 100%, and 140% O2, respectively). The responses were followed for 24 h post exposure. It is possible that, for higher levels of hyperoxia (high and very-high), a longer time is needed to see reactive adaptations, such as the expression of nuclear factor erythroid 2-related factor 2 (NRF2) and HIF-1α. Further investigations are required with prolonged periods.

All treatments were associated with significant activation of NRF2 and HIF-1α. Conversely, the nuclear factor kappa B (NF-kB) transcription factor was significantly activated only by higher oxygen concentrations. The intracellular levels of total glutathione paralleled the nuclear transfer of NRF2 and remained elevated up to the end of the experimental observation time along with the plasmatic level of matrix metalloproteinase 9 (MMP-9). We confirmed that mild hyperoxia is sensed as hypoxic stress in vivo within the first 24 h, activating HIF-1α and NRF2, but not NF-kB.

Conversely, high hyperoxia was associated with a progressive loss of the NOP response and increased oxidative stress signals leading to NRF2 and NF-kB activation, accompanied by the synthesis of GSH. After very high hyperoxia, HIF-1α activation was absent in the first 24 h, and the oxidative stress response accompanied by NF-kB activation was prevalent. The glutathione (GSH) levels paralleled the nuclear transfer of NRF2 and remained elevated during the observation time together with the MMP-9 plasma levels. Further confirmation was published on the activation of microRNA during different oxygen exposures [21].

Interestingly, several articles have been interested in increasing hemoglobin and oxygen exposure [9,14,22–24]. To achieve such an increase in a fast way, small deltas were elicited; for a more extended response, higher oxygen variations and more distant (less frequent) variations were needed [21]. If the oxygen variations were relatively small, a repeated and frequent exposure had a fast answer (a few days) [22].

#### **6. New Levels of Oxygen**

Recent data confirmed that, in vivo, the return to normoxia after mild hyperoxia is sensed as hypoxic stress characterized by HIF-1α activation [10]. On the contrary, high hyperoxia and very high hyperoxia induce a shift toward an oxidative stress response, characterized by NRF2 and NF-κB activation in the first 24 h post-exposure.

Even though it is possible that higher levels of hyperoxia (high hyperoxia from 50% to 100%, very high hyperoxia at hyperbaric levels) may induce late responses to recover homeostasis over a longer window of time, previous studies in cultured cells [14] and in vivo [12,25] suggested that critical adaptive responses occur within shorter times. However, further investigations are needed to investigate if pulsed hyperoxia induces specific compensatory reactive adaptations at more extended periods. Future studies should focus on the two components of this paradigm: the oxygen exposure (time and PO2) and time between sessions (intermittent exposures) [12,23].

Overall, already published data [10] suggested the occurrence of a "hormetic" adaption to high oxygen triggered by the activation of signaling cascades leading to the expression of antioxidant systems; this is in agreement with data obtained in rodents undergoing either hyperbaric or normobaric oxygen that initially induced significant oxidative stress that was eventually resolved after continued exposure [26].

To optimize applicable clinical protocols from this paradigm, future studies are expected to focus also on the "down-stream" effects of HIF-1α transcriptional activation. Depending on the therapeutic target, using mild hyperoxia (from 30% to 40–50%) may be more desirable [12,27], or, on the other hand, eliciting oxidative stress utilizing HH/VHH administration may be considered a more appropriate desirable effect.

#### **7. Intermittent Oxygen, the Clue?**

Hyperbaric oxygen therapy is, by essence, "intermittent oxygen" exposure. It is clear from previous data that we must investigate this direction with a new paradigm of treating oxygen rather as a potent stimulus of the molecular network of reactions. The vital task is answering the questions of "how much", "how long", and "how often" this stimulus should be given. The non-linearity of the dose-response curve complicates the picture. Still, to the end, we should be able to answer what oxygen dose should be given to reach a specific clinical or molecular effect.

We hope that the authors of the papers in this special issue of *Medicina* and active readers using those ideas in their research will help to shape the future of "oxygen medicine".

**Author Contributions:** Conceptualization, C.B. and J.K.; writing—review and editing, C.B. and J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This submission received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **A General Overview on the Hyperbaric Oxygen Therapy: Applications, Mechanisms and Translational Opportunities**

**Miguel A. Ortega 1,2,3,\* , Oscar Fraile-Martinez 1,2,\* , Cielo García-Montero 1,2 , Enrique Callejón-Peláez <sup>4</sup> , Miguel A. Sáez 1,2,5 , Miguel A. Álvarez-Mon 1,2 , Natalio García-Honduvilla 1,2 , Jorge Monserrat 1,2 , Melchor Álvarez-Mon 1,2,6, Julia Bujan 1,2 and María Luisa Canals <sup>7</sup>**


**Abstract:** Hyperbaric oxygen therapy (HBOT) consists of using of pure oxygen at increased pressure (in general, 2–3 atmospheres) leading to augmented oxygen levels in the blood (Hyperoxemia) and tissue (Hyperoxia). The increased pressure and oxygen bioavailability might be related to a plethora of applications, particularly in hypoxic regions, also exerting antimicrobial, immunomodulatory and angiogenic properties, among others. In this review, we will discuss in detail the physiological relevance of oxygen and the therapeutical basis of HBOT, collecting current indications and underlying mechanisms. Furthermore, potential areas of research will also be examined, including inflammatory and systemic maladies, COVID-19 and cancer. Finally, the adverse effects and contraindications associated with this therapy and future directions of research will be considered. Overall, we encourage further research in this field to extend the possible uses of this procedure. The inclusion of HBOT in future clinical research could be an additional support in the clinical management of multiple pathologies.

**Keywords:** hyperbaric oxygen therapy (HBOT); Hyperoxia; wound healing; antimicrobial properties; Coronavirus Disease-19 (COVID-19)

#### **1. Introduction**

Hyperbaric oxygen therapy (HBOT) is a therapeutical approach based on exposure to pure concentrations of oxygen (O2) in an augmented atmospheric pressure. According to the Undersea and Hyperbaric Medical Society (UHMS), this pressure may equal or exceed 1.4 atmospheres (atm) [1]. However, all current UHMS-approved indications require that patients breathe near 100% oxygen while enclosed in a chamber pressurized to a minimum of 2 ATA [2].

The first documented use of hyperbaric medical therapy was in 1662 by Henshaw, a British physician who placed patients in a container with pressurized air. Interestingly, it was conducted before the formulation of the Boyle-Mariotte Law, which described the

**Citation:** Ortega, M.A.;

Fraile-Martinez, O.; García-Montero, C.; Callejón-Peláez, E.; Sáez, M.A.; Álvarez-Mon, M.A.; García-Honduvilla, N.; Monserrat, J.; Álvarez-Mon, M.; Bujan, J.; et al. A General Overview on the Hyperbaric Oxygen Therapy: Applications, Mechanisms and Translational Opportunities. *Medicina* **2021**, *57*, 864. https://doi.org/10.3390/ medicina57090864

7

Academic Editors: Costantino Balestra and Jacek Kot

Received: 26 July 2021 Accepted: 20 August 2021 Published: 24 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

relationship between the pressure and volume of a gas, and prior to the discovery of O<sup>2</sup> by John Priestly over 100 years later [3]. Afterwards, the pathway of HBOT in medical care was retarded by the observation of possible O2-derived adverse effects at 100% concentrations by Lavoisier and Seguin in 1789. Years later, in 1872 Paul Bert, considered the "father of the hyperbaric physiology", described the physiological basis of pressurized air in the human body, also defining the neurotoxic effects of O<sup>2</sup> in the human body, consequently named the Paul Bert effect [4], followed by the description of the pulmonary toxicity of O<sup>2</sup> by Lorrain Smith [5]. Simultaneously, a growing interest in the use of HBOT in the treatment of different affections was reported, including treatment for divers who suffered decompression sickness during World War II [6]. Since then, a plethora of studies were prompted, with hundreds of facilities based on HBOT being established at the beginning of the 21st century [7].

Currently, there are 14 approved indications for HBOT, including a wide variety of complications like air embolism, severe anemia, certain infectious diseases or idiopathic sensorial hearing loss. In addition, in the last European Consensus Conference on Hyperbaric Medicine highlighted the use of HBOT as a primary treatment method for certain conditions according to their moderate to high degree of evidence (e.g., after carbon monoxide (CO) poisoning), or as a potential adjuvant to consider in other conditions with a moderate amount of scientific evidence (e.g., Diabetic foot) [8]. In this work we will review in detail the basis of O<sup>2</sup> as a therapeutical agent and the principles of hyperbaric medicine regarding most relevant applications concerning HBOT, and potential implications for different approaches including COVID-19.

#### **2. Physiological Role of Oxygen in the Organism**

O<sup>2</sup> is a frequently disregarded nutrient because of its particular access inside the human body, through the lungs instead of the gastrointestinal tract, typical of all other nutrients [9]. O<sup>2</sup> is key for human cells to perform so-called aerobic respiration, which takes places in the mitochondria. Here, O<sup>2</sup> acts as an electron acceptor finally leading to ATP synthesis in a process known as oxidative phosphorylation. From an evolutionary perspective, the uptake of O<sup>2</sup> was the origin of eukaryotic cells, emerging as a result of an endosymbiotic relationship between prokaryotic cells (archaea and eubacteria) which were capable of using this nutrient [10]. This fact represented an adaptative advantage with regard to those cells unable to utilize it, complex organisms were coevolving with O2, thus becoming an essential nutrient for our cells [11].

In a simple manner, O<sup>2</sup> is introduced in our body by two distinguished process: ventilation, in which gases are transported from the environment to the bronchial tree and diffusion, where an equilibrium in the distribution of O<sup>2</sup> between alveoli space and blood is reached. Given that the partial pressure of O<sup>2</sup> (PO2) here is low, and rich in carbon dioxide (CO2), gas exchange occurs [12]. Simultaneously, the difference in the pressure and volume in the chest wall and lungs are essential to permit the oxygen flow, as atmospheric pressure does not vary at all [13]. Once in the bloodstream, O<sup>2</sup> is mostly bound to haemoglobin (Hb) in the erythrocytes, and to a little extent in a dissolved form, being systemically distributed. Then, oxygen exchange is produced between the microcirculatory vessels—Not only capillaries, but also arterioles and venules-and the rest of the tissues, due to the different partial pressure of O<sup>2</sup> and the Hb oxygen saturation (SO2), which is also dependant on other variables like temperature, PCO<sup>2</sup> and pH, among others [14]. If, however there is a lack of oxygen in the tissue it may appear a condition designed as hypoxia. This may be due to low O<sup>2</sup> content in the blood (Hypoxemia), which may be a consequence of either a disruption in the blood flow to the lungs (Perfusion), airflow to the alveoli (Ventilation) or problems in the gas diffusion in the haemato-alveolar barrier. Furthermore, low blood supply (ischaemia) or difficulties in the O<sup>2</sup> delivery, may also be responsible for tissue hypoxia [15]. Consequently, within cells there are specific sensors named as Hypoxia-inducible factors (HIF) that under hypoxic conditions will bind to the hypoxia response element (HRE), thereby regulating a wide variety of cellular pro-

cesses [16]. Occasionally, hypoxia might provide favourable implications for health, for instance during early developmental stages [17] or in the case of intermittent exposures [18]. Nonetheless, hypoxia mostly induce a pathological stress for cells that is closely related with the appearance and progress of a broad spectrum of diseases [19]. As a result, oxygen has been proposed as a potential therapeutic agent for patients undergoing different acute or chronic conditions [20,21]. As targeting cellular hypoxia is a promising, but still an emerging approach [22], clinical management of hypoxia is directed to modulate global hypoxemia and oxygen delivery within the tissues [23]. In this context, HBOT arises as an extraordinary support in the handling of hypoxia and other hypoxia-related phenomena by increasing blood and tissue levels of oxygen [24]. Hereunder, we will describe the principles and mechanisms of action of HBOT, regarding its therapeutical basis and specific considerations of this therapy.

#### **3. Principles of Hyperbaric Oxygen Therapy. Therapeutical Basis**

As above mentioned, HBOT consist of the supply of pure oxygen under augmented pressure. This procedure is conducted in a monoplace or multiplace chamber if there are only one or various patients undergoing this procedure, respectively. In the first case, the chambers are usually compressed with O<sup>2</sup> whereas in the second, people breath oxygen individually through a face mask, hood, or an endotracheal tube [25]. In the case of critically ill patients, it seems that multiplace chambers allow a better monitoring of the vital functions in comparison to monoplace chambers, although the use of the latter are also safe and well tolerated by patients [26,27]. Depending on the protocol, the estimated duration of session varies from 1.5 to 2 h and may be performed from one to three times daily, being given among 20 to 60 therapeutical doses depending on the condition [28]. Frequently, this method utilizes between 2 to 3 atms of pressure. Nevertheless, it has also been obtained promising results in some studies from <2 atms (1.5 atms) for certain conditions [29,30], although according to all UHMS currently approved indications it is required a chamber pressurized to a minimum of 2 ATA [2]. Despite some protocols accept the use of 6 atms (i.e., treatment of gas embolism), little benefits are usually reported from >3 atms as it may be associated with a plethora of adverse effects [31]. Moreover, it is not possible to breath pure O<sup>2</sup> at higher pressures than 2.8 atm, and in those cases it is accompanied with other gases like helium, nitrogen or ozone. The alternative, normobaric oxygen therapy (NBOT), utilizes oxygen at 1 atm of pressure. In comparison with HBOT, NBOT is cheaper and easier to apply, and it could be found in almost all hospitals, as it does not require hyperbaric chambers [32]. However, some studies have reported a reduced efficacy of NBOT in comparison with HBOT [33,34], therefore showing the relevance of HBOT for certain conditions. Conversely, the use of NBOT could be critical for patients suffering from some maladies in absence of HBOT facilities.

The therapeutical basis of hyperbaric oxygenation are consequence of three main factors: (1) By breathing 100% O2, a positive gradient is created, hence favouring diffusion for hyperoxigenated lungs to hypoxic tissues; (2) due to the high pressure, O<sup>2</sup> concentration in the blood raises according to Henry's Law (the amount of dissolved gas within a liquid is directly proportional to its partial pressure) and (3) it decreases the size of gas bubbles in the blood following Boyle-Mariotte Law and Henry's Law [6]. In other words, the creation of a hyperbaric environment with pure oxygen permits a significant increment of the oxygen supply to blood (Hyperoxemia) and to the tissues (Hyperoxia) even without the contribution from Hb [35]. Thus, HBOT provides multiple effects in the organism, and it could be used to correct tissue hypoxia, chronic hypoxemia and to aid in the clinical management of different pathological processes including wound healing, necrosis, or reperfusion injuries [36].

Contrary to hypoxia, the human body has not developed any specific adaptation to hyperoxia. Interestingly, the exposure to intermittent hyperoxia, share many of the mediators and cellular mechanisms which are induced by hypoxia. This is called the hyperoxic-hypoxic paradox [37]. Importantly, it does not have to be considered a negative property. As occurring with intermittent hypoxia, the submitting of short-term hyperoxia may provide favourable outcomes in the cell. The explanation resides in a crucial concept in biology, the hormesis, which correlates the type of response obtained with the dose received [38]. From a molecular perspective, high PO<sup>2</sup> in the tissues may have important implications in the cellular signalling, particularly through increasing the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These changes induce multiple effects in the organism, including the synthesis of different growth factors, improving neovascularization or showing immunomodulatory properties, among others, therefore exerting its clinical efficacy [39,40]. Moreover, HBOT upregulates HIF, by ROS/RNS and Extracellular Regulated Kinases (ERK1/ERK2) pathway [37,41]. In the same manner, an excessive production of ROS and RNS due to hyperoxia may lead to the appearance of oxidative stress, DNA damage, metabolic disturbances, endothelial dysfunction, acute pulmonary injury and neurotoxicity [42]. As hyperbaric O<sup>2</sup> may provide both beneficial and adverse effects, it is essential to balance the different factors to clinically recommend or reject HBOT [43]. Due to the physics of HBOT, it is not easy to design adequate studies and clinical trials to fully endorse its use. Despite this, there are some predictive models that may be an additional tool to evaluate what patients may benefit the most from receiving this therapy, considering distinct therapeutical approaches if necessary [44].

In Figure 1 conditions and characteristics of hyperbaric chambers are illustrated, besides the main effect of pressurized O<sup>2</sup> administration. Below, main applications and translational applications of HBOT will be subsequently discussed, in order to review the actual importance of this procedure in current clinical practice and potential uses.

**Figure 1.** Illustration of a monoplace hyperbaric chamber and the effect of hyperbaric O<sup>2</sup> . Pressurized O<sup>2</sup> (2–3 atm) at 100% concentration is administered normally during 1.5–2 h per session and repeated three times a day. Depending on the clinical condition sessions vary in number, from 20 to 60. The inhalated air comes from an external elevated PO<sup>2</sup> , hence positive gradient allows higher O<sup>2</sup> entry, which per diffusion will be higher also in alveoli, bloodstream and therefore there will be greater arrival to tissues. This effect of "hyperoxemia" and "hyperoxia" is independent from haemoglobin (Hb), then will lessen hypoxia in tissues. This will result in a major supply of reactive oxygen species (ROS) and reactive nitrite species (RNS), with a consequent higher expression of growth factors and promotion of neovascularization and enhanced immunomodulatory properties.

#### **4. Approved Indications for HBOT**

Due to the multiple characteristics of HBOT, the possible applications of this procedure are numerous. For instance, HBOT may be used as an urgent treatment for acute pathologies but also as an additional support for chronic diseases [41]. Currently, there are 14 approved indications for HBOT are represented in Table 1. Most of these uses, can be grouped according to three main effects (a) in the wound healing acceleration and angiogenesis enhancement (b) exerting antimicrobial effects, and (c) as a medical emergency.

#### **Table 1.** Approved indications for HBOT.


#### *4.1. HBOT and Wound Healing: The Angiogenesis Enhancement*

In clinical practice, it has been observed how HBOT can speed wound healing. As wounds need oxygen to regenerate tissues properly, an exposure of 100% oxygen accelerates this process. The application in this field is quite extensive, comprising microbial-infected wounds (e.g., Clostridial myonecrosis and Fournier's gangrene), traumatic wounds, thermal burns, skin grafts, radiation-induced wounds, diabetic and vascular insufficiency ulcers [45].

In the field of diabetes, there is a critical complication called "diabetic foot ulcers", an open wound at the bottom of the foot that affects 15% of patients. HBOT has been specially regarded for this injury, being implicated many inflammatory and tissue repairing parameters. For instance, there was some evidence that HBOT may improve the healing rate of wounds, by increasing nitric oxide (NO) levels and the number of endothelial progenitor cells, in the non-healing vasculitis, calcific uremic arteriolopathy (CUA), livedoid vasculopathy (LV), pyoderma gangrenosum (PG) ulcers [46]. Some trials show a prominent angiogenesis while reducing inflammation: angiogenic markers like epithelial growth factor (EGF) and VEGF become enhanced, and positively associates to Nrf2 transcription factor increase [47]. Furthermore, anaerobic infections have a lower occurrence and amputation rates immensely decrease [48,49]. Different systematic reviews support the adjuvant use of systemic but not topical HBOT in the wound healing of diabetic foot ulcers [50,51]. However, studies results are quite heterogeneous, and it is still necessary to define which group of patients may benefit most from this intervention [52]. For instance, patients with diabetic foot ulcers and peripheral arterial occlusive disease may not improve wound healing [53]. Another recent study demonstrated that the use of HBOT may be associated with improved six-year survival in patients with diabetic foots [54]. Further studies and greater samples are required to identify the most suitable candidates for HBOT.

Additionally, HBOT may be an excellent adjuvant in surgery injuries resolutions, and it is key as it may provide better outcomes if it is earlier administered. When wounds do not follow conventional treatments for healing, an extra aid can be found in HBOT. Animal models have described the importance of this procedure in the wound healing by the acceleration of epithelialization and neovascularization [55,56]. Reported effects on these events resides in the up-regulation of host factors like tumour necrosis factor-α (TNF-α), matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) [57]. In a rabbit model of irradiated tissue, NBOT O<sup>2</sup> was compared to hyperbaric demonstrating once again that O<sup>2</sup> is required at higher pressures to provoke an angiogenic effect [56]. More studies in vivo have alleged tension exerted by hyperbaric O<sup>2</sup> modulates proliferation rate of stem cells in small intestinal crypts and raises angiogenesis in chorioallantonic membrane in *Gallus gallus* embryos [58]. In a clinical trial of patients with chronic non-healing wounds (more than 20 months without healing), HBOT was standardized for 20 sessions (five sessions/week). The results were increased levels of vascular endothelial growth factor (VEGF) and interleukine-6 (IL-6), and lower levels of endothelin-1. These facts entail an activation of host wound resolution factors, angiogenesis and vascular tone [59]. Vasculogenesis gains efficiency thanks to HBOT upregulation of nitric oxide (NO) and associates to a decrease in lesions area [60].

Multiple lines of research have also been opened to evaluate the enhanced angiogenesis and healing of tissues following HBOT. For instance, a phase 2A clinical trial demonstrated the possible benefits from HBOT in combination with steroids for patients with ulcerative colitis in terms of achieving higher rates of clinical remission, and a reduced probability of progression to second-line therapy during the hospitalization [61]. However, there are few studies in this field, and soon an updated meta-analysis and systematic review of the available evidence will be published [62]. Similar conclusions might be extrapolated to radiation-induced hemorrhagic cystitis and proctitis [63]. Osteoradionecrosis is also a frequent and worrisome condition in oncological patients after receiving radiotherapy. Frequently, this condition affects to the jaw and consists of the development aseptic, avascular necrosis which can lead to infection, tooth loss, and even pathological fracture of the jaw. Moreover, it often results in an ulceration and necrosis of the mucosa with exposed bone. HBOT plays a critical role in the treatment of this condition, improving the tissue response to surgical wounding, and even as prophylactic approach in patients with previous head and neck irradiation undergoing dental extractions or complete exodontia [64]. The enhancing angiogenesis and wound healing make HBOT an adequate adjuvant treatment in a wide variety of conditions, although future studies should be directed to evaluate the most effective dose and to identify the most suitable candidates for submitting this procedure.

#### *4.2. HBOT and Infections: The Antimicrobial Activity*

The use of HBOT as an antimicrobial adjuvant is particularly useful in healing context now that microbial infections are the most important cause of non-healing wounds: meta-analysis affirm that prevalence of bacterial biofilms in chronic wounds is 78.2% [65]. HBOT is considered a non-conventional strategy for non-healing wounds consisting in a modification of biophysical parameters in the wound microenvironment, breaking the bacterial biofilms [66]. HBOT upregulates HIF that induces the expression of Nitric Oxide Synthases (NOS) and virus killing peptides (defensins and cathelicidins such as cathelinrelated antimicrobial peptide) with consequent neutrophil and monocyte phagocytosis of the microbes [67–69]. Increased cathelicidins in mice lungs provide a better response to the flu virus [70]. Cathelicidin-deficient mice show higher susceptibility to viral damage [71].

The most important applications of the antimicrobial activity of HBOT are under necrotizing soft tissue infections (NSTIs), including necrotizing fasciitis, Fournier's gangrene and gas gangrene. There is a calamitous soft tissue infection implying a wide variety of gram-positive, gram-negative, aerobic and anaerobic bacteria. It happens under conditions of trauma or minor lesions that become more complicated, normally, due to systemic problems like diabetes or vascular disfunctions [45,72]. An early and combined HBOT therapy plus current practices may be crucial as a lifesaving and cost-efficacy therapy, particularly in the most critical patients [73]. Clinical practice agrees on the necessity of HBOT in the event of an anaerobic infection, as anaerobic bacteria are killed by a much

higher amount of pressurized O<sup>2</sup> [74,75]. For instance, the use of HBOT in the anaerobic *Clostridium perfringens* bacteria is specially recommended [76]. This bacterium produces more than 20 recognized toxins. However, two toxins, alpha and theta are the main mediators of the infection caused by this agent. *Clostridium perfringens* growth is restricted at O<sup>2</sup> tensions up to 70 mm Hg, and alpha-toxin production is halted at tensions of 250 mm Hg, also achieving bacteriostasis and other antimicrobial effects. Thus, recommended treatment is O<sup>2</sup> at 3 ATA for 90 min three times in the first 24 h and twice a day for the next 2 to 5 days, always in combination with proper antibiotic use [77]. The anti-inflammatory potential of HBOT also aids to lessen tissue damage and infection expansion [72], also explained by a decrease in neutrophil activation, eviting rolling and accumulation of white blood cells (WBCs), hence limiting the production of ROS by neutrophils and avoiding reperfusion injury [45]. Moreover, this is observed in In vitro studies, having been demonstrated the biofilm shrinkage ability with the significant decreases in cellular load of anaerobic bacteria and fungi after HBOT [75]. A sepsis mouse model showed a significant increase in survival rate, >50%, with early HBOT compared to a control group that did not receive the treatment and was associated with lower expression of TNF-α, IL-6 and IL-10 [78]. Translation to clinical experience reports that the improvements in oxygenation follow the neovascularization, which avoid undesired events like amputation [28]. This is the case, for example, of Fournier's gangrene, where bacteremia and sepsis are top factors of fatality, which can be avoided by adjuvant HBOT, providing much higher survival rates in clinical trials [79]. Sometimes unwanted events are underestimated until it is late and polymicrobial infection has bursted into surgical bone and joint lesions [80]. For that reason, molecular assessments of bacterial identification like mass spectrometry, are every time more accomplished to consider if HBOT is worthy for patients' better recovering.

On the other hand, the use of HBOT might provide a central therapeutical option in the intracranial abscess (ICA). ICA presentation includes cerebral abscess, subdural empyema, and epidural empyema, and it is caused by an encapsulated infection in which the proper inflammatory response may damage the surrounding brain parenchyma [81]. The etiological agent might be bacteria, fungi, or a parasite, and it might appear as a consequence of a dissemination of previous infections like sinusitis, otitis, mastoiditis, dental infection; hematogenous seeding or cranial trauma [82]. Due to the high morbidity and mortality, along with the urgency of a non-invasive and effective method, HBOT has been proposed as a well-accepted adjunctive therapy for ICA, being regarded as a safe and tolerated method [83]. The main mechanisms by which HBOT represent an additional tool in the management of ICA resides on the impairment of the acidotic and hypoxic environment in ICAs due to the proper infection and the use of antibiotics [84]. Similarly, osteomyelitis is a chronic infection in the bone marrow frequently caused by bacteria or mycobacteria. It is a difficult condition to treat, as many antimicrobials do not penetrate in the bone properly. When this condition does not respond to the treatment or reemerge after receiving the therapy it is designed refractory osteomyelitis [85]. HBOT is a potential indication of refractory osteomyelitis as it provides synergist antibiotic activity, while enhancing angiogenesis, leukocyte oxidative killing and osteogenesis process [86]. A recent systematic review [87] reported that adjuvant HBOT provided almost a 75% of therapeutic success in patients with chronic refractory osteomyelitis, hence showing the importance of this treatment in bacterial infections. Malignant otitis externa, another infection, a necrotizing infection of the soft tissue of the external auditory canal which may rapidly cause skull base osteomyelitis may also benefit from the use of HBOT, although further studies are needed to conclude its effects [88].

Finally, some authors have also proposed a potential clinical use of HBOT as a medical emergency treatment of mucormycosis, a fungal infection [89]. Despite there still being few studies supporting its use, a compelling evidence show its potential use in a similar manner than necrotizing fasciitis, although further research is needed in this area.

#### *4.3. HBOT in Medical Emergencies*

Apart from the previously discussed applications, there has been further conditions in which HBOT may be considered. Some of them are designed as medical emergencies, in which the use of HBOT is an urgent indication for these patients. These are the cases of some infections above mentioned, decompression sickness, air or gas embolism, acute arterial insufficiencies such as central retinal arterial occlusion (CRAO), crush injury, compartment syndrome and acute traumatic ischemia, along with CO/Cyanide poisoning [89]. In this context, the central role of HBOT is derived from the rapid and effective response of the tissues under certain conditions that may be severe and even life-threatening [90].

A. Decompression sickness is a condition occurring due to the formation of bubbles caused by a reduction in ambient pressure that introduced dissolved gases within the body accidents. In turn, these bubbles drive to mechanical disruption of tissues, blood flow occlusion, endothelial dysfunction, platelet activation and capillary leakage. [91]. However, the term decompression sickness has been abandoned by the ECHM to be replaced by "Decompression illness" (DCI) [92], so in this article we will refer this malady as DCI. Clinical manifestations are at least one of more of the following: generalized fatigue or rash, joint pain, hypesthesia and in serious cases motor weakness, ataxia, pulmonary edema, shock and death [71]. DCI can occur in aviators, divers, astronauts, compressed air workers and, in some cases, it may appear due to iatrogenic causes [93]. HBOT/recompression therapy tables (US Navy Treatment Table 6 or helium/oxygen (Heliox Comex Cx30 or equivalent) are recommended for the initial treatment of DCI (Type 1 recommendation, Level C evidence). US Navy Treatment Table 5 can be used as the first recompression schedule for selected mild cases [94]. Therapies at higher pressure could be administered in exceptional cases, but it entails higher difficulties and risks. To maximize its efficacy, different adjunctive therapies are used in combination with HBOT including fluid administration, non-steroidal anti-inflammatory drugs and prophylactic agents to prevent venous thromboembolism events, particularly in paralyzed patients [93,95]. Overall, because of the high pressure, HBOT provide the opposite effects of the pathological mechanisms of DCIs, therefore exerting its therapeutical efficacy.

B. Air embolism. Apart from DCI, bubble formation of large arterial air embolism during operations are unusual occurrences but also ruinous and life-threatening. For bubble gas formation in veins from lung biopsy, arterial catheterization, cardiopulmonary bypass, HBOT is strictly necessary as there are no better alternatives in time. It provides tissue oxygenation by promoting gas reabsorption, and hence reduces ischemic injuries [96]. In this context, retrograde cerebral air embolism is a worrisome condition that may appear in major procedures (neurosurgery and cardiovascular operations, endoscopy), or during minor interventions (peripheral or central venous access), being particularly lethal when presented with encephalopathy [97]. The therapeutic basis of air embolism is similar to DCS, with HBOT as first-line therapy [98]. Some reports have emphasized the importance of an early HBOT, in the first 6 h since diagnosis, for this complication to obtain better outcomes, less sequelae or death rate [99]. However, there is some evidence of late benefits from its use, up to 60 h after the onset [100]. Even when there is no gas seen in on image test, patients may benefit from the use of HBOT [101]. On the other hand, recent data indicates that less cases appears to be treated by HBOT probably by the lack of belief of some physicians in HBOT, particularly in UK [102]. However, available evidence supports the use of this therapy to prevent and improve the outcome of such a dangerous condition.

C. CRAO is an ophthalmological complication caused by a permanent occlusion of the central retinal artery, mostly due to a embolus at its narrowest part that is typically associated with a sudden, massive loss of vision in the affected eye [103]. Prognosis for visual recovery is poor, as the retinal tissue is not tolerant to hypoxia, and it presents the highest oxygen consumption rate in the body at 13 mL/100 g per min [104]. As a result, HBOT is a robust indication for patients with CRAO and many studies have reported encouraging results from its use, minimum at eight sessions, with some advantages presented in comparison to other lines of treatment such as being a non-invasive method with low

adverse effects [104–107]. However, and despite these benefits, HBOT is rarely offered for patients with CRAO [108], probably due to the lack of facilities in the hospital services.

D. Another approved indication for HBOT is crush injury and acute ischemia occurred as result of a trauma. Presentations of these damage vary from mild contusions to limb threatening damage, involving multiple tissues, from skin to muscles and bones. A severe consequence of trauma is the skeletal muscle-compartment syndrome (SMCS), a condition affecting both muscle and nerves [1]. Subsequently to trauma, the affected tissue will suffer from hypoxia, edema and ischemia. Here, the efficacy of angiogenesis has been also proved to be boosted by HBOT in animal models for ischemic limbs when combined with bone marrow derived mononuclear cells transplantation [109]. Some translational studies of multicenter randomized trials did not show a significant complete progress of healing [110], but in contrast, other trials showed the advantage of HBOT as adjunct for ischemic limbs when reconstructive surgery was not possible [111]. Evaluating skin peripheral circulation as well, the outcomes showed significant improvements in revascularization [112], therefore demonstrating the important role of HBOT in this condition.

E. CO poisoning is a problem that happens when household devices which use gas or coal produce CO due to an uncomplete combustion. Inhalation of this gas can be lethal and cause long-term problems particularly cognitive and brain deficits, presented up to a 40% of the patients and approximately one in three people develop cardiac dysfunction, like arrhythmia, left ventricular systolic dysfunction, and myocardial infarction [113]. To address these problems, HBOT has been applied [114] being associated to neurological sequelae reduction [115] and when applied in the first 24 h can reduce the risk of cognitive sequelae months later more efficiently [116]. In general, NBOT is immediately used after CO poisoning until HBOT is available [117]. Evidence indicates that HBOT should be considered for all cases of serious acute CO poisoning, loss of consciousness, ischemic cardiac changes, neurological deficits, significant metabolic acidosis, or COHb greater than 25% [113]. Another kind of poisoning in which HBOT has its application is cyanide toxicity. This issue appears with uncomplete combustion, this time, of materials like plastics, vinyl, acrylics, nylon, etc. HBOT is the primary treatment, but it exceeds when is combined with the antidote hydroxycobalamin, ameliorating mitochondrial oxidative phosphorylation function [118,119]. Potential uses of HBOT in a wide range of urgent conditions at least might be considered as an important tool in medical emergencies.

F. Severe anemias and idiopathic sudden sensorineural hearing loss. Despite not being considered a medical emergency, the use of HBOT is also indicated for these conditions [89]. In the first case, as Hb levels critically drops, O<sup>2</sup> delivery to the tissues may be impaired. In this line, the use of 100%, hyperbaric O<sup>2</sup> might solve this issue, simultaneously exerting a wide range of favorable effects in the hematological profile [120]. This could be especially important in patients who cannot be transfused for religion, immunologic reasons, or blood availability problems. Idiopathic sudden sensorineural hearing loss or acute acoustic trauma (AAT) are also important conditions in which HBOT could be a valuable tool. In fact, a recent systematic review and meta-analysis conducted by Rhee et al. [121] showed that the addition of HBOT to standard medical therapy is a valuable treatment option particularly for patients with severe to profound hearing loss and in those patients which received, at least 1200 min of HBOT. Apart from the regulation of ROS and inflammatory response, previous research has demonstrated the protective role of HBOT in the hair cell stereocilia, probably through hormetic mechanisms [122].

G. Finally HBOT can significantly improve symptoms and quality of life of patients affected by femoral head necrosis (ECHM recommendation type II level of evidence B) [123] as well as the previously mentioned NSTI, gas gangrene and urgent HBO alpha toxin neutralized

#### **5. Translational and Potential Applications of HBOT**

Besides approved indications, further lines of research have demonstrated the potential applications and translation of HBOT in the field of inflammatory and systemic conditions, cancer, COVID-19 and other conditions are summarized.

#### *5.1. HBOT and Inflammation: Immunomodulatory Properties*

HBOT might also be applicated in the regulation of inflammatory responses and its derived complications. Among the most important immunomodulatory effects, HBOT drives an alteration in CD4+:CD8+ ratio, a reduced proliferation of lymphocytes, and an activation of neutrophils with migration to hyperoxic regions [124]. Thus, HBOT might be used in a wide variety of conditions presenting an altered immune system as part of its pathogenesis. In this sense, it has been proposed the role of HBOT in the management of autoimmune diseases (ADs). A study conducted by Xu et al. [125] observed the overall effect of HBOT in general immune populations and particular Th1 and B lymphocytes subsets, proving its promising role in certain ADs. Furthermore, long-term exposure to HBOT was proven to supress the development of autoimmune symptoms, including proteinuria, facial erythema and lymphadenopathy [126]. In the same manner, the use of HBOT in early and middle stage of disease mice also show a significant increase in survival with a decrease in inflammatory cells, anti-dsDNA antibody titers, and amelioration of immune-complex deposition in comparison to later stage of disease [127] The use of HBOT has also proven its efficacy on rheumatoid arthritis, particularly due to the polarization of Th17 cells to T reg, with a significative reduction of cell hypoxia [128].

Similarly, these results could be extrapolated to other inflammatory conditions. For instance, HBOT provides an anti-inflammatory response in DSS-induced colitis. Through direct effects on HIF, HBOT induces antioxidant expression and the downregulation of proinflammatory cytokines like IL-6, therefore reducing colonic inflammation [129]. In vitro studies with lymphocytes from type 1 diabetes mellitus have proved effects of HBOT on inducible NOS expression, observing lower activity with a consequent decreased levels of NFkB [130]. Additionally, HBOT comprises another potential approach regarding musculoskeletal dysfunctions. Fibromyalgia represents an incapacitant disorder characterized by a widespread muscle and joint pain, frequently accompanied by systemic symptoms including cognitive dysfunction, mood disorders, fatigue, and insomnia [131]. HBOT exerts direct effects on brain activity, chronic pain and immune dysregulation, therefore improving quality of life of affected patients [132]. Interestingly, Woo et al. [133] also observed that HBOT could be considered an interesting alternative to attenuate exercise-induced inflammation and muscle damage.

Overall, previous research has indicated the favourable effects of HBOT in the immune system and also on the whole body.

#### *5.2. Role of HBOT in the COVID-19 Pandemic*

COVID-19 pandemic has challenged healthcare systems worldwide, overloading them with a huge burden in economy and our normalcy [134,135]. The urge of conducting massive vaccination programs besides finding better therapies for clinical management, have been the focus these months. In this context, HBOT has been proposed as an adjuvant for clinical practice in severe patients, and also for recovery after SARS-CoV-2 infection. Results from clinical trials have already demonstrated the potential uses of this treatment to redirect O<sup>2</sup> diffusion avoided by hypoxemia, and its ability to eliminate inflammatory cytokines.

Nevertheless, not only hyperbaric O<sup>2</sup> may be worthy for severe patients, but also for treating the named "silent" hypoxemia in those patients that do not have a bad clinical course yet [136]. This silent hypoxemia is not characterized by typical respiratory distress in critically ill patients, but it may be dangerous if it is not sooner detected as a prompt deterioration can occur without noticing [137]. In fact, previous studies have demonstrated the association between hypoxemia with fatal outcomes in patients with COVID-19 [138].

In the same manner, physicians observed that patients exhibit hypoxemia without dyspnea, being crucial to find care solutions to anticipate a problem with more patients at important risk [139]. Some cases of people with mild or even without symptoms, that contracted multi-organ failure and then died, have emphasized the importance of self-monitoring of pulse oximetry, which typically presents reduced readings in these patients [140]. Collected data from patients that did not present problems of breathing at admission, agreed with the suggestion of utilizing pulse oximetry to predict the outcome of hypoxemia/hypocapnia syndrome that defines asymptomatic hypoxia [141]. Steps forward in the understanding of our complex respiratory system have also launched reviews about the higher oxygenation rate in prone position, concerning variables like gravity, lung structure and the higher expression of nitric oxide (NO) in dorsal lung vessels than in ventral ones [142]. It has been demonstrated that HBOT increases the production of NO and ROS/RNS, inhibiting SARS-CoV-2 replication in previous In vitro models [41].

Moreover, all these facts have shed a light on finding better treatments to prevent fast hypoxia, fatality or even the need for mechanical ventilation [143,144] being HBOT a suggested adjuvant for its promising outcomes from previous animal models and clinical cases of sepsis and inflammatory diseases [145]. Preliminary comparisons of HBOT applications in COVID-19 to other maladies, like livedoid vasculopathy, have exposed the possible mechanisms that may occur: anti-inflammatory actions (decreased ICAM-1, proinflammatory cytokines and neutrophil rolling), anticoagulant actions (boosted fibrinolysis and increased plasminogen activator) and tissue healing actions (increased fibroblasts and stem cells) [146].

First studies in a severe patient affirmed that, compared to normobaric oxygen supply, the better empiric outcome agreed with the theoretic expectance of the potential uses of HBOT in COVID-19 [147]. Although it is still being evaluated scientifically, positive results are arising for COVID-19 treatment, finding an attenuation of the innate immune system, and increasing hypoxia tolerance [148]. In every report, this therapy has been rated as a potential support in the relieving of cytokine storm [149]. Now that mechanical ventilation may be long lasting and, preferably, avoided, in a controlled trial, safety and efficacy of HBOT for COVID-19 patients was successfully evaluated [150]. Another preliminary study showed rapid alleviation of hypoxemia from the beginning of the treatment in patients with COVID-19 pneumonia [151].

Anatomically, pathologic examinations of lung with early-phase COVID-19 have shown edema, proteinaceous exudate, inflammatory cellular infiltration, and interstitial thickening that entails a disproportional gas exchange. This is due to CO<sup>2</sup> diffuses through tissues much faster than O2, about 20 times, what leads to hypocapnia [152]. Alveolar structure is altered in the COVID-19 patient, there is also hyaline membrane formation, there is thickness in alveolar membrane and the space for the diffusion of oxygen generates a lot of exudate and inflammation. Hence, diffusion from the alveolus through the haematoalveolar membrane does not occur correctly, the concentration of oxygen in the blood and in the tissues begins to fall and the exchange of the dioxide also becomes difficult. Due to possible viral interactions with Hb [153] and a hypoxemia-induced shift in the oxyhemoglobin dissociation curve to the left, there is O<sup>2</sup> saturation but low arterial blood pressure [154].

Clinical evidence from few studies about COVID-19 patients undergoing HBOT, notes that this therapy may make possible to contribute to reverse hypoxemia and ameliorating the pulmonary capillary circulation diffusion despite the thickness in alveolar membrane in disease. According to Henry's Law, HBOT allows to increase pressure of O<sup>2</sup> in the alveoli above ambient pressure. In this way, there will be a large increase of O<sup>2</sup> diffusion into the pulmonary capillary circulation, more than 10 times, for its arrival in the plasma and reach the tissues independently of Hb. There will be a gain of O<sup>2</sup> supply to the tissues mediated by the increase in pressure. Experimentally, hematological, biochemical and inflammatory parameters were significantly improved after HBOT. In first trials the observation of lymphocyte count was increased, whereas lactate and fibrinogen were decreased [147,151]. However, during this procedure patients may suffer from desaturation reflexes. Despite the etiology of this reflex is unclear, it might be probably caused by a vasoconstriction affecting the pulmonary arteries, due to the oxidative stress as well as direct damage in type II pneumocytes and thrombus associated with COVID-19 [124].

Notwithstanding the ongoing clinical trials and the efforts of standardize better protocols for safety, COVID-19 is not yet an accepted indication for HBOT, but this may be recommended for post-viral sequelae [155]. In order to guarantee its beneficial effects, there is still a need of more controlled trials to measure different inflammatory and hematological parameters that demonstrate that exudate and inflammation are reduced besides the improvements in alveolar circulation diffusion. This would confirm the potential of this adjuvant, also for considering the financial investment in hyperbaric chambers in hospitals.

#### *5.3. HBOT and Cancer*

Cancer is a complex entity which encompasses a broad spectrum of unique pathologies that share the following hallmarks: Immune system evasion, tumor-promoting inflammation, genome instability, enabling replicative immortality, activating invasion and metastasis sustaining proliferative signaling, evading growth suppressors, resisting cell death, inducing angiogenesis, and metabolic reprogramming [156]. Tumor-hypoxia plays a central role in many of these carcinogenic features, promoting an aggressive phenotype besides limit the effectiveness of radiotherapy, chemotherapy, and immunotherapy thereby worsening prognosis in the oncological patients [157]. Thus, targeting tumoral hypoxia and its downstream effectors have been proposed as a potential therapeutical approach in cancer management [158–160]. In this line, accumulating evidence supports the role of HBOT in the inhibition of tumor growth and therapy success, by three main mechanisms: (1) By limiting cancer-associated hypoxia, (2) through the generation of ROS and RNS and (3) restoring immune function [161]. Actual investigations show the promising role of HBOT in a wide variety of malignancies, including breast cancer, prostate cancer, head and neck cancer, colorectal cancer, leukemia, brain tumors, cervical cancer and bladder cancer [162]. Main applications derived from HBOT in oncology may be (a) As part of the treatment (b) as a radiotherapy adjuvant and (c) as a chemotherapy adjuvant [163].

The use of HBOT as part of the cancer therapy is not currently an approved indication, although some promising results have arisen recently. In this context, Thews & Vaupel [164] compared the efficacy of NBOT (1 atm) versus HBOT (2 atm) oxygenation reporting broader reductions of hypoxia under hyperbaric conditions. However, even at high pressure oxygenation, tumor hypoxia was not completely removed, hence showing that HBOT alone efficacy is limited. Importantly, as previously described HBOT was associated with increased angiogenesis, these effects are not significative in tumour cells, so its use could be important in the cancer management [165]. Conversely, a study conducted by Pande et al. [166] revealed that notwithstanding HBOT-treated mice initially induced a decrease in tumor progression, a tumorigenic effect was observed post-therapy, probably due to impaired DNA repair, mutagenicity and chromosomic aneuploidies together with an altered blood supply and nutrients. On the other hand, some authors suggest that the lack of therapeutical efficacy of HBOT might be due to the difficulty on creating a hyperoxic environment in the tumor and that, by combining HBOT with other methods it could act a as a potential cure in certain types of cancer. In this line, Lu et al. [167] proposed a combined use in prostate cancer patients of HBOT with ultrasound guided transrectal prostate puncture, in order to create a hyperoxic environment within the tumor, which may lead to DNA damage and a detention in the G2/M cycle, hence establishing the basis for future research. Similarly, tumor hypoxia is associated with the metabolic reprogramming of tumour cells, also known as the aerobic glycolysis or "Warburg effect". This consists of a glycolytic switch of cancer cells, which refrain from performing oxidative phosphorylation [168]. In this sense, Poff et al. [169] described the combined effects of HBOT in combination with ketogenic diet in a murine model, preventing tumoral metastasis while expanding overall survival. Furthermore, HBOT alone or combined with

18

low glucose and ketone supplementation also exert multiple benefits against late-stage metastatic cancers, by increasing the production of ROS and oxidative stress [170]. Despite the encouraging results, further research is required to establish the efficacy of HBOT in the different types of cancer, also searching for the most adequate use of this therapy in a global context.

Radiotherapy (RT) is a central component in cancer management, with approximately 50% of patients receiving this therapy contributing up to a 40% of curative success for cancer [171]. Through ionizing radiation, it creates a ROS and RNS overproduction, leading to double strand breaks, chromosomal aberrations and rearrangements with subsequent cell death or dysfunction, thus exerting its anti-tumoral effects. The effect of HBOT on human glioblastoma (GBM) was investigated, in laboratory, on patient-derived cells and on microglia cell biology (CHME-5). The results obtained from the combination of HBO and RT clearly showed a radiosensitising effect of HBO on GBM cells grown [172]. Hypofractionated stereotactic radiotherapy (HSRT) after HBO (HBO-RT) appears to be effective for the treatment of recurrent high-grade glioma (rHGG), as pointed out on a cohort of 9 adult rHGG patients. It could represent an alternative, with low toxicity, to systemic therapies for patients who cannot or refuse to undergo such treatments [173]. However, although non-tumour cells are less sensitive, radiation could also affect them, altering multiple cellular signaling pathways or inducing apoptosis, hence explaining its multiple adverse effects [174] One of the most severe consequences resulted from irradiation is the appearance of post-radiation injuries, a process starting during radiotherapy that involves the dysregulation of multiple bioactive compounds, particularly fibrogenic cytokines like TGFβ [175]. Similarly, almost all tissues with delayed irradiation injury present a histological feature named as obliterative endarteritis, finally leading to a tissue damage characterized by hypoxia, hypovascularity and hypocellularity [176]. In this line, HBOT has consistently demonstrated its therapeutical effectivity against radiation-induced injury also approved by the UHMS [177] and the ECHM [8]. Last 2016 Cochrane review [178] evidenced that the use of HBOT in head, neck, anus and rectum injured tissues were associated with improved outcomes and, at some extent with osteoradionecrosis following tooth extraction in an irradiated field. According to ECHM recommendation the use of HBOT is recommended in the treatment of radiation proctitis (Type 1 recommendation, Level A evidence), mandibular osteoradionecrosis and haemorrhagic radiation cystitis (Type 1 recommendation, Level B evidence) and suggested in the treatment of osteoradionecrosis of other bone than the mandible, for preventing loss of osseointegrated implants in irradiated bone and in the treatment of soft-tissue radionecrosis (other than cystitis and proctitis), in particular in the head and neck area (Type 2 recommendation, Level C evidence). Furthermore, it would be reasonable to use HBOT for treating or preventing radio-induced lesions of the larynx, in the treatment of radio-induced lesions of the central nervous system (Type 3 recommendation, Level C evidence) [8]

Finally, the combined use of HBOT plus chemotherapy have reported certain benefits. In this line, a recent study conducted by Brewer et al. [179] demonstrated the effectiveness of using HBOT to prevent chemotherapy-induced neuropathy In vivo. This fact appears to be due to the various implications of HBOT in the neuronal activity and signaling [180–182] Kawasoe et al. also observed [183] that an integrative strategy of carboplatin plus HBOT significantly reduced mortality in C3H mice with inoculated osteosarcoma cells Similar results were obtained with HBOT and chemotherapy in lung cancer cultures and animal models [184]. In particular, the combination of paclitaxel and carboplatin plus HBOT and hyperthermia show promising results for treating patients with non-small cell lung cancer and multiple metastasis [185]. Despite these results, the use of HBOT and chemotherapy may also represent a contraindication for the patients. For instance, the combination of HBOT with doxorubicin, bleomycin, or cisplatin may exert synergic cardiotoxicity, pulmonary toxicity or impaired wound healing, respectively [186]. This is an important issue to address in the oncologic patient. In these cases, it is important to separate chemotherapy from the use of necessary HBOT, to avoid undesired effects. In addition, further strategies

could be considered targeting tumour hypoxia and functioning as therapeutic adjuvants like physical activity [187]. Overall, the benefits of HBOT in cancer management is a potential field to keep on exploring.

#### *5.4. Other Applications*

In the same manner, other novel lines of research are exploring potential uses of HBOT in a plethora of conditions. For instance, some studies related to microvascular or macrovascular insufficiencies causing erectile dysfunction (ED) have hypothesized the effects of HBOT in patients with this problem. Empirical data suggests that it can induce penile angiogenesis and improve erectile function in men suffering from ED. This is due to vasodilatation relies on proper blood vessels in corpora cavernosa. Then, being a major concentration of oxygen in tissues, there is an increased angiogenesis by VEGF and endothelial cells differentiation [188]. This application has not provided significant data on rehabilitation after prostatectomy [189] but it has obtained good symptoms resolution for other clinical manifestations like ED in diabetes mellitus [190] or in recovery after urethral reconstruction [191]

Equally, the use of HBOT for ischemic stroke and brain injury is an interesting point of study. For instance, different studies have demonstrated the importance of this procedure as a prophylactic approach for sequestration of inflammation inherent in stroke and traumatic brain injury, preventing neuronal death [192]. Other uses such as brain preconditioning before stem cells transplantation have also been explored [193]. However, the efficacy and safety of HBOT in these conditions remains to be fully elucidated, although some basic and clinical research have shown encouraging results [194].

Finally, the use of HBOT could be potentially extended to novel fields like aging. Hachmo et al. [195] reported the effect of hyperbaric oxygen in the prevention of telomere shortening and immunosenescence by the clearance of senescent immune cells. In this line, other studies have reported the same results in the aging skin, through the acceleration of epidermal basal cells proliferation [196], in the endothelial cells, where it induces antioxidants expression [197] and also in the brain, where HBOT appears to improve the cerebral blood flow [198], restoring cognitive parameters, hippocampal functions and even improved insulin resistance in both normal-weigh and obese aging rats [199].

As summarized in Figure 2, the main consequences of HBOT and its related hyperoxemia and hyperoxia in the human body could be related with the angiogenesis enhancement, antimicrobial properties and immunomodulatory effects. Approved indications for this therapy could also be grouped according to its emergency.

**Figure 2.** Summary of top properties of HBOT and its clinical applications. Firstly, it can provide an angiogenesis enhancement, observed by the prime production of NO which subsequently brings an upregulation of Nrf2 and growth factors like epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and endothelin-1. TNF-α, matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) will be boosted too. Secondly, the antimicrobial activity is visible due to bacterial killing by O<sup>2</sup> , removing biofilm and lessening white blood cells (WBCs) rolling and neutrophils recruitment, hence promoting a downregulation of proinflammatory cytokines (TNF-α, IL-6 and IL-10). The immunomodulation properties are observed by a downregulation of transcriptional factor NFkB, involving a proinflammatory response switch off (IL-6) and a polarization from Th17 lymphocytes to Treg. Summarized applications include: indications for which HBOT is approved (mostly wound healing and infections), primary emergencies (like CO/CN poisoning or air embolism), and translational research (comprising COVID-19, cancer, inflammatory conditions or aging among others).

#### **6. Adverse Effects and Contraindications**

Notwithstanding the multiple benefits and applications of HBOT, there are important adverse effects that may appear during this procedure. As a result of the hyperoxia and the hyperbaric environment, there are some issues when using this therapy. The two most common complications during HBOT are claustrophobia and barotrauma. Both occur during monoplace or multiplace chamber compression [200]. In the case of barotrauma, it could be defined as an injury caused by an inability to equalize pressure from an air-containing space and the surrounding environment. Ear barotrauma is the most frequent condition affecting the middle ear, although sinus/paranasal, dental or pulmonary barotrauma could also be reported [201]. Despite the incidence of this complication being extremely rare [202], its seriousness should be taken into account, considering clinical history of patients at risk of suffering from these complications while implementing different strategies to prevent this complication, such as anti-epileptic therapy, prolonged air brakes or controlling treatment pressure [203]. The last event is associated with the appearance of the Paul Bert effect because of the formation of seizures that may bring transient but negative consequences for cognitive functioning and behavioural patterns [204]. These effects are primarily due to the toxic properties of oxygen at high concentrations. However, to date, no threshold has been described to precisely assess the pathological levels of oxygen, which could be an important issue for critical patients [205]. Pulmonary toxicity is not associated with the use of repeated hyperbaric oxygen following current protocols [206]. Ocular manifestations

from HBOT may also be described, particularly hyperbaric myopia, transitory in most cases. Other ophthalmological complications less frequent observed are cataracts, keratoconus or retinopathy of prematurity, in the case of pregnant women exposed to HBOT [207,208]. All these adverse effects may be ameliorated prominently by an adequate screening, through the use of certain devices and the adjustment of the treatment protocols [200,201]

On the other hand, there are certain conditions in which HBOT might be absolutely contraindicated or relatively contraindicated. The first case is exclusively represented by untreated pneumothorax, as it could be a life-threatening procedure [209]. The rest of contraindications are relative, its indication will depend on the real necessity of this therapy. Aside from the chemotherapheutic agents previously described other treatments like sulfamylon (Mafenide), could also share the same action than cisplatin impeding wound healing effects derived from HBOT, and it should also be interrupted before this therapy [45]. If patient has a pacemaker or any type of implantable devices, it is necessary to verify its safety with increased pressure or with pure concentrations of oxygen. Hereditary spherocytosis may also be a contraindication, as hyperbaric oxygen could cause severe haemolysis [43]. Pregnancy is another potential contraindication for this therapy in exception of CO poisoning [210]. Although rare in non-diabetic individuals, patients may also suffer from hypoglycaemia during this procedure, and it is important to evaluate their blood glucose levels before HBOT, as it could aggravate their hypoglycaemic profile [211]. Similarly, patients with underlying respiratory pathologies like chronic obstructive pulmonary disease (COPD), asthma and even upper respiratory infections might be also possible contraindications from receiving HBOT, as it could increase the risk of hypercapnia, pulmonary barotrauma and sinus or middle ear barotrauma, respectively [209]. An additional effect derived from HBOT is the increment of blood pressure [212]. Hyperbaric oxygen may also induce pulmonary oedema and cardiovascular difficulties in patients with heart failure or in patients with reduced cardiac ejection fractions [213]. Finally, the history of epilepsy, hypoglycaemia, hyperthyroidism, current fever, and certain drugs such as penicillin and disulfiram are also thought to lower the seizure threshold during this therapy [214]. Diabetic patients may be warned from regulating its doses of HBOT in order to prevent the hypoglycaemic effect of this therapy.

To summarize, despite the multiple applications of HBOT it is equally important to consider its potential adverse effects and underlying conditions in which this therapy is not going to exert its efficacy, also representing a potential risk for these patients.

#### **7. Conclusions and Future Directions**

HBOT is an effective method to increase blood and tissue oxygen levels, independently from Hb transportation. Its therapeutical basis could be understood from three different perspectives: Physical (Hyperbaric 100% oxygen), physiological (Hyperoxia and hyperoxemia) and cellular/molecular effects. All these effects provide HBOT its efficacy in the management of hypoxia derived conditions and hypoxemia, respectively, also exerting direct effects in infectious agents and immune cells, modulating a wide variety of cellular signaling pathways, cytokine production and tissue processes such as angiogenesis. Herein, the use of HBOT might be extended to a broad spectrum of pathologies, from infections and inflammatory/systemic maladies to wound healing and vascular complications, also reporting its efficacy in the management of medical emergencies like air embolism or gas poisoning. Although respiratory infections and diseases have been mentioned as contraindications for HBOT, the case of SARS-CoV-2 is an exception. Nowadays, the potential use of HBOT in the COVID-19 has been specially regarded, exposing results in numerous controlled clinical trials. Moreover, the use of this procedure in different types of malignancies represents an important support in the delayed radiation injury. In the same manner, the use of HBOT as a therapeutical agent have shown promising results in trials as an adjunctive substance with other approved treatments like chemotherapy and even, recent research have also reported significative improvements in nanomedicine approaches when combined with HBOT [215].

Despite its benefits, there are still certain challenges which need to be overcome to improve the current and potential applications of HBOT. In this line, a worrisome issue would be to develop sophisticated strategies to address tissue hypoxia, as for certain conditions like tumoral cells, the HBOT induced hyperoxia does not completely eliminate tumour hypoxia. An adequate combination of HBOT with another procedure might be interesting to targeting this problem [167]. On the other hand, it is equally important to determine and quantify potential adverse effects derived from HBOT, as well as potential contraindications from receiving this therapy. Future research should be destinated on developing accurate systems to determine potential benefits and risks for patients before submitting HBOT. In this line, the development of predictive models as previously mentioned or novel strategies could be interesting approaches in these fields.

Currently, there are only 14 approved indications for this therapeutical approach. We encourage further studies to extend the possible uses of this procedure, always considering individual benefits and risks from receiving this therapy. The inclusion of HBOT in future clinical research could be an additional support in the clinical management of multiple pathologies.

**Author Contributions:** Conceptualization, M.A.O., O.F.-M., C.G.-M., M.Á.-M., J.B., M.L.C.; Methodology, M.A.O., O.F.-M., C.G.-M.; Formal Analysis, M.A.O., O.F.-M., C.G.-M.; Investigation, M.A.O., O.F.-M., C.G.-M., E.C.-P., M.A.S., M.A.Á.-M., N.G.-H., J.M., M.Á.-M., J.B., M.L.C.; Data Curation, M.A.O., O.F.-M., C.G.-M.; Writing-Original Draft Preparation, M.A.O., O.F.-M., C.G.-M., E.C.-P., M.A.S., M.A.Á.-M., N.G.-H., J.M., M.Á.-M., J.B., M.L.C.; Writing-Review & Editing, M.A.O., O.F.-M., C.G.-M., E.C.-P., M.A.S., M.A.Á.-M., N.G.-H., J.M., M.Á.-M., J.B., M.L.C.; Supervision, M.Á.-M., J.B., M.L.C.; Project Administration, M.Á.-M., J.B.; Funding Acquisition, M.Á.-M., J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the Comunidad de Madrid (B2017/BMD-3804 MITIC-CM), Univer-sidad de Alcalá (32/2013, 22/2014, 26/2015) and Halekulani S.L.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data used to support the findings of the present study are available from the corresponding author upon request.

**Acknowledgments:** Oscar Fraile-Martinez had a predoctoral fellowship from the University of Alcalá during the course of this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Analysis of the Increase of Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression and the Effect of Exposure in a Hyperbaric Chamber on VCAM-1 in Human Blood Serum: A Cross-Sectional Study**

**Katarzyna Van Damme-Ostapowicz 1,\* , Mateusz Cybulski <sup>2</sup> , Mariusz Kozakiewicz <sup>3</sup> , Elzbieta Krajewska-Kułak ˙ 2 , Piotr Siermontowski <sup>4</sup> , Marek Sobolewski <sup>5</sup> and Dorota Kaczerska <sup>6</sup>**


**Abstract:** *Background and Objectives:* Vascular cell adhesion molecule-1 (VCAM-1) was identified as a cell adhesion molecule that helps to regulate inflammation-associated vascular adhesion and the transendothelial migration of leukocytes, such as macrophages and T cells. VCAM-1 is expressed by the vascular system and can be induced by reactive oxygen species, interleukin 1 beta (IL-1β) or tumor necrosis factor alpha (TNFα), which are produced by many cell types. The newest data suggest that VCAM-1 is associated with the progression of numerous immunological disorders, such as rheumatoid arthritis, asthma, transplant rejection and cancer. The aim of this study was to analyze the increase in VCAM-1 expression and the impact of exposure in a hyperbaric chamber to VCAM-1 levels in human blood serum. *Materials and Methods*: The study included 92 volunteers. Blood for the tests was taken in the morning, from the basilic vein of fasting individuals, in accordance with the applicable procedure for blood collection for morphological tests. In both groups of volunteers, blood was collected before and after exposure, in heparinized tubes to obtain plasma and hemolysate, and in clot tubes to obtain serum. The level of VCAM-1 was determined using the immunoenzymatic ELISA method. *Results*: The study showed that the difference between the distribution of VCAM-1 before and after exposure corresponding to diving at a depth of 30 m was at the limit of statistical significance in the divers group and that, in most people, VCAM-1 was higher after exposure. Diving to a greater depth had a much more pronounced impact on changes in VCAM-1 values, as the changes observed in the VCAM-1 level as a result of diving to a depth of 60 m were statistically highly significant (*p* = 0.0002). The study showed an increase in VCAM-1 in relation to the baseline value, which reached as much as 80%, i.e., VCAM-1 after diving was almost twice as high in some people. There were statistically significant differences between the results obtained after exposure to diving conditions at a depth of 60 m and the values measured for the non-divers group. The leukocyte level increased statistically after exposure to 60 m. In contrast, hemoglobin levels decreased in most divers after exposure to diving at a depth of 30 m (*p* = 0.0098). *Conclusions*: Exposure in the hyperbaric chamber had an effect on serum VCAM-1 in the divers group and non-divers group. There is a correlation between the tested morphological parameters and the VCAM-1 level before and after exposure in the divers group and the non-divers group. Exposure may result in activation of the endothelium.

**Citation:** Van Damme-Ostapowicz, K.; Cybulski, M.; Kozakiewicz, M.; Krajewska-Kułak, E.; Siermontowski, P.; Sobolewski, M.; Kaczerska, D. Analysis of the Increase of Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression and the Effect of Exposure in a Hyperbaric Chamber on VCAM-1 in Human Blood Serum: A Cross-Sectional Study. *Medicina* **2022**, *58*, 95. https://doi.org/10.3390/ medicina58010095 4.0/). *medicina*

Academic Editors: Costantino Balestra and Jacek Kot

Received: 23 November 2021 Accepted: 4 January 2022 Published: 8 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/

**Keywords:** atmosphere exposure chambers; diving physiology; decompression

#### **1. Introduction**

Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell adhesion factor. It is expressed on endothelium activated by cytokines, but may also occur in a soluble form in serum [1]. VCAM-1 was identified as a cell adhesion molecule that helps regulate inflammation-associated vascular adhesion and the transendothelial migration of leukocytes, such as macrophages and T cells. VCAM-1 is expressed by the vascular system and can be induced by reactive oxygen species, interleukin 1 beta (IL-1β) or tumor necrosis factor alpha (TNFα), which are produced by many cell types. The newest data suggests that VCAM-1 is associated with the progression of numerous immunological disorders, such as rheumatoid arthritis, asthma, transplant rejection and cancer [1]. Homeostasis is a necessary condition for health and the proper functioning of the body, and hence, diseases result from disturbances in mechanisms that maintain homeostasis [2]. Commercial saturation divers work in high-pressure environments, in which their bodies must acclimatize to a variety of physiological stress factors [3].

Research shows that intercellular adhesion molecule-1 (ICAM-1) and VCAM-1 adhesive molecules are potential markers of changes in the endothelium [4]. These molecules are of great interest both in order to understand the mechanisms of their action and their usefulness in the diagnosis and treatment of diseases [4,5]. Brubakk et al. [6], in their studies on vesicle formation and endothelial function in human and animal models, showed a decrease in the arterial endothelial function after a single dive using air.

The researchers [7–9] indicated that the endothelium was sensitive to oxidative stress and the shear rate, leading to vascular remodeling and a release of micro molecules. According to Freyssinet [9], endothelial microparticles are constantly shed into the circulation of healthy individuals and have been shown to be elevated in many diseases, most notably those characterized by endothelial dysfunction. This was supported by Horstman et al. [10].

It would be interesting to deepen the knowledge of the role of the VCAM-1 biomarker in the human body during decompression. The aim of this study was to analyze the increase in VCAM-1 expression and the impact of exposure in a hyperbaric chamber to the VCAM-1 level in human blood serum.

The research problem was to answer the following questions:


The following hypotheses were formulated:


#### **2. Materials and Methods**

#### *2.1. Design of the Study*

The cross-sectional study involved four exposures. Short-term simulated hyperbaric air exposures corresponding to diving at a depth of 30 and 60 m were carried out. The exposure corresponding to a 60 m dive was chosen because it was the maximum allowable depth for a dive using air as a breathing mixture, and 30 m as half the maximum depth. Air was used for breathing in the hyperbaric chamber during dives. This was an experimental **Exposition Depth Bottom** 

**Time** 

chamber complex DGKN 120 belonging to the Department of Underwater Works Technology of the Naval Academy in Gdynia. It consists of 3 chambers located at the same level: dry, wet and transient. In a dry chamber, where the study was carried out on short-term exposures, there may be 7 people, with longer ones—4, and with saturated ones—2. The maximum working pressure is 120 m of water column, i.e., 12 at or 13 ata. Additional inhalers (beeps) allow you to breathe, e.g., oxygen in a different atmosphere in the chamber. Pressure unit converters were the following: 760 mmHg = 760 tracks ~ 1 atm. = 1.033227 at. = 1.01325 N/m<sup>2</sup> = 1.01325 Pa = 14.69 psi. ber. Pressure unit converters were the following: 760 mmHg = 760 tracks ~ 1 atm. = 1.033227 at. = 1.01325 N/m2 = 1.01325 Pa = 14.69 psi. Exposures were based on the Naval Table for the decompression and recompression of divers (Table 1). Exposures were carried out by compressing the subjects in a hyperbaric chamber to a pressure of 400 kPa, corresponding to a dive at a depth of 30 m, and to a pressure of 700 kPa, corresponding to a dive at a depth of 60 m. This pressure was maintained for 30 min. The entire time of exposure to 4 atm was 1 h, and to 7 atm—2 h. The

The cross-sectional study involved four exposures. Short-term simulated hyperbaric

air exposures corresponding to diving at a depth of 30 and 60 m were carried out. The exposure corresponding to a 60 m dive was chosen because it was the maximum allowable depth for a dive using air as a breathing mixture, and 30 m as half the maximum depth. Air was used for breathing in the hyperbaric chamber during dives. This was an experimental chamber complex DGKN 120 belonging to the Department of Underwater Works Technology of the Naval Academy in Gdynia. It consists of 3 chambers located at the same level: dry, wet and transient. In a dry chamber, where the study was carried out on shortterm exposures, there may be 7 people, with longer ones—4, and with saturated ones—2. The maximum working pressure is 120 m of water column, i.e., 12 at or 13 ata. Additional inhalers (beeps) allow you to breathe, e.g., oxygen in a different atmosphere in the cham-

*Medicina* **2022**, *58*, x FOR PEER REVIEW 3 of 14

Exposures were based on the Naval Table for the decompression and recompression of divers (Table 1). Exposures were carried out by compressing the subjects in a hyperbaric chamber to a pressure of 400 kPa, corresponding to a dive at a depth of 30 m, and to a pressure of 700 kPa, corresponding to a dive at a depth of 60 m. This pressure was maintained for 30 min. The entire time of exposure to 4 atm was 1 h, and to 7 atm—2 h. The plateau of both exposures was 30 min. The pressure exposure profiles are shown on Figures 1 and 2. Exposures were performed at the Department of Underwater Works Technology of the Naval Academy in cooperation with the Department of Maritime and Hyperbaric Medicine of the Military Medical Institute in Gdynia. Exposures were carried out by a qualified physician and a technical employee of the Department of Underwater Works Technology of the Naval Academy in Gdynia. plateau of both exposures was 30 min. The pressure exposure profiles are shown on Figures 1 and 2. Exposures were performed at the Department of Underwater Works Technology of the Naval Academy in cooperation with the Department of Maritime and Hyperbaric Medicine of the Military Medical Institute in Gdynia. Exposures were carried out by a qualified physician and a technical employee of the Department of Underwater Works Technology of the Naval Academy in Gdynia. **Table 1.** 3 MW decompression tables. **Time Decompression Stops (mH2O) Total Ascent Time 42 39 36 33 30 27 24 21 18 15 12 9 6 3** 

**Table 1.** 3 MW decompression tables. **to First Stop Time on the Stops (min) Air Oxygen** 

**2. Materials and Methods** 

*2.1. Design of the Study* 


**Figure 1.** Pressure exposure profile 30 m/30 min. **Figure 1.** Pressure exposure profile 30 m/30 min.

**Figure 2.** Pressure exposure profile 60 m/30 min. **Figure 2.** Pressure exposure profile 60 m/30 min.

#### *2.2. Characteristics of Subject Population*

*2.2. Characteristics of Subject Population*  Volunteers also completed a questionnaire, providing information concerning their age, sex, place of residence, education, seniority, type of work, type of physical exertion, smoking status, coffee consumption and a self-assessment of their physical condition. A total of 45 professional divers volunteered to participate in the study. They were subjected to hyperbaric exposure in a pressure chamber. A total of 47 volunteers—non-divers who had never been subjected to hyperbaric exposure—were also included. The non-divers group stayed in the same chamber for the same time period and breathed in the same pattern with the same (identical) breathing mixture to best reflect the effect of the same pressure during exposure and decompression. The non-divers group, who were not ex-Volunteers also completed a questionnaire, providing information concerning their age, sex, place of residence, education, seniority, type of work, type of physical exertion, smoking status, coffee consumption and a self-assessment of their physical condition. A total of 45 professional divers volunteered to participate in the study. They were subjected to hyperbaric exposure in a pressure chamber. A total of 47 volunteers—non-divers who had never been subjected to hyperbaric exposure—were also included. The non-divers group stayed in the same chamber for the same time period and breathed in the same pattern with the same (identical) breathing mixture to best reflect the effect of the same pressure during exposure and decompression. The non-divers group, who were not exposed, sat in the in the same chamber, with the same temperature and lighting conditions and breathed the same breathing mixture.

posed, sat in the in the same chamber, with the same temperature and lighting conditions and breathed the same breathing mixture. Criteria for inclusion in the divers group were professionally active diver, mentally and physically healthy and aged from 24 to 55 years. Exclusion criteria were respiratory tract infection, age under 24 and over 55 years, using intoxicating drugs, using any other medications and resignation from participation in the study. Ultimately, 18 people participated in the study. Inclusion criteria for the non-divers group were non-divers, who had Criteria for inclusion in the divers group were professionally active diver, mentally and physically healthy and aged from 24 to 55 years. Exclusion criteria were respiratory tract infection, age under 24 and over 55 years, using intoxicating drugs, using any other medications and resignation from participation in the study. Ultimately, 18 people participated in the study. Inclusion criteria for the non-divers group were non-divers, who had never been subjected to hyperbaric exposure before, mentally and physically healthy and aged 24 to 55 years. Exclusion criteria were respiratory tract infection, aged under 24 and over 55, using intoxicating drugs, using any other medications and resignation from participation in the study Ultimately, 14 people participated in the study.

never been subjected to hyperbaric exposure before, mentally and physically healthy and aged 24 to 55 years. Exclusion criteria were respiratory tract infection, aged under 24 and All the people who participated in the study breathed air and were not subjected to physical exertion.

over 55, using intoxicating drugs, using any other medications and resignation from participation in the study Ultimately, 14 people participated in the study. All the people who participated in the study breathed air and were not subjected to physical exertion. Basic information about the divers group (N = 18) and the non-divers group (N = 14) Basic information about the divers group (N = 18) and the non-divers group (N = 14) is presented in Table 2. There were statistically significant differences between the sex (*p* = 0.0391), type of work (*p* = 0.0436) and physical effort (*p* = 0.0043) between the groups. In the divers group, manual workers predominated. The descriptive statistics presented below show that both groups were completely comparable in terms of age and occupational seniority.

is presented in Table 2. There were statistically significant differences between the sex (*p* = 0.0391), type of work (*p* = 0.0436) and physical effort (*p* = 0.0043) between the groups. In the divers group, manual workers predominated. The descriptive statistics presented below show that both groups were completely comparable in terms of age and occupational

**Group** 

**N % N %** 

**Sex** 0.0391 \* female 0 0.0% 3 21.4%

**Table 2.** Demographic and occupational status of subjects.

seniority.

**Demographic and Occupational Status** 


**Table 2.** Demographic and occupational status of subjects.

Abbreviations: \*—*p* < 0.05; \*\*—*p* < 0.01.

#### *2.3. Methods*

Blood for the tests was taken in the morning, from the basilic vein of fasting individuals, in accordance with the applicable procedure for blood collection for morphological tests. Tests were performed by a certified medical analytical laboratory. VCAM-1 measurements were performed with serum and BCC with plasma.

In both groups, blood was collected at the same time, before and after exposure, to heparin anticoagulant tubes to obtain plasma and hemolysate and to clot tubes to obtain serum. The level of VCAM-1 was determined using the immunoenzymatic ELISA method, with the DIACLONE kit.

#### *2.4. Procedural and Ethical Considerations*

The study was performed from September 2018 to June 2019 and the study obtained ethical approval from the Bioethics Committee of the Medical University in Bialystok, Poland (R-I-002/237/2015). Members of the research team provided oral and written information about the study. Subjects gave their informed consent for participation in the study. Each participant received written and oral information about the possibility of withdrawing from the study at any time and without any consequences. The research conformed with the Good Clinical Practice guidelines, and the procedures were in accordance with the principles of the 1975 Declaration of Helsinki, as revised in 2000, and with the ethical standards of the institutional committee on human experimentation.

#### *2.5. Statistical Analysis*

To present listings as elements of the description of both groups, summary tables contain numbers and percentages, and for age and occupational seniority—means (M) ± standard deviations (SD); the *p*-value was calculated using the chi-square test of independence (for comparison of percentage structure) or the Mann–Whitney U test (for comparison of numerical values—age and occupational seniority of the subjects).

Selected numerical characteristics of the examined parameters were determined: arithmetic mean (M), median (Me), the highest (maximum) and the lowest (minimum) value and standard deviation (SD).

A test of the statistical significance of the relationship under study was performed. For all statistical analyses, the significance level was set at *p* < 0.05.

Additionally, information on the 95% confidence interval for the average VCAM-1 level measured in the four tested situations is presented. The normality of VCAM-1 level distribution in various tested situations was assessed using the Shapiro–Wilk test. The statistical significance of differences in the distribution of VCAM-1 before and after exposure was analyzed. As the measurements were made on the same group of divers, the Wilcoxon test was used for the analysis. Results were graphically illustrated using scatter plots. The Mann–Whitney U test was used to compare the distribution of VCAM-1 among non-divers (in various tested situations). The difference between the results obtained in both groups was assessed using the Mann–Whitney U test. The Wilcoxon test was also used to examine the significance of changes between tests performed before and after exposure.

Spearman's rank correlation coefficient (*r*S) was used to assess the strength of relationships between the BCC and VCAM-1.

#### **3. Results**

There were no significant differences between the divers and non-divers groups in terms of coffee consumption, smoking status or self-assessment of physical condition.

The *p*-value calculated for "coffee consumption" was *p* = 0.3365, for "smoking status" —*p* = 0.7876 and for "self-assessment of physical condition"—*p* = 0.0842 (Table 3).


**Table 3.** Subject lifestyle.

The table below (Table 4) provides information on the 95% confidence intervals for the average level of VCAM-1 in the four tested situations.


**Table 4.** 95% confidence intervals for the average level of VCAM-1 parameter measured in four tested situations in the divers group.

Abbreviations: CI—confidence interval; VCAM-1—vascular cell adhesion molecule-1. *Medicina* **2022**, *58*, x FOR PEER REVIEW 7 of 14

> The difference between the VCAM-1 distribution before and after the exposure with a diving depth of 30 m was on the limit of statistical significance (*p* = 0.0582). In most people, VCAM-1 after exposure was higher, on average, by about 1.5 ng/mL (but a decrease in the VCAM-1 level was also noted in some people). Diving to a greater depth had a much more pronounced impact on the changes in the VCAM-1 value. In all the subjects, VCAM-1 increased after exposure by at least 0.8, and at most by 24.2 ng/mL. On average, the change was about 5.9 ng/mL, although the average was somewhat overestimated by the quite outlying peak value of VCAM-1 growth of 24.2. Therefore, a median of 4.2 ng/mL may be a better measure of the average level of VCAM-1 changes after a dive (Table 5). The difference between the VCAM-1 distribution before and after the exposure with a diving depth of 30 m was on the limit of statistical significance (*p* = 0.0582). In most people, VCAM-1 after exposure was higher, on average, by about 1.5 ng/mL (but a decrease in the VCAM-1 level was also noted in some people). Diving to a greater depth had a much more pronounced impact on the changes in the VCAM-1 value. In all the subjects, VCAM-1 increased after exposure by at least 0.8, and at most by 24.2 ng/mL. On average, the change was about 5.9 ng/mL, although the average was somewhat overestimated by the quite outlying peak value of VCAM-1 growth of 24.2. Therefore, a median of 4.2 ng/mL

> **Table 5.** Distribution of VCAM-1 before and after the exposure corresponding to a 30 m and 60 m dive in the divers group. may be a better measure of the average level of VCAM-1 changes after a dive (Table 5). The changes in VCAM-1 as a result of diving to a depth of 60 m were highly statistically significant (the *p*-value determined using the Wilcoxon test was 0.0002).


Abbreviations: M—mean; Me—median; SD—standard deviation; Min.—minimum; Max.—maximum; \*\*\*—*p* < 0.001. [ng/mL] 60 After dive 18.7 17.5 11.8 6.7 55.3

The changes in VCAM-1 as a result of diving to a depth of 60 m were highly statistically significant (the *p*-value determined using the Wilcoxon test was 0.0002). Abbreviations**:** M—mean; Me—median; SD—standard deviation; Min.—minimum; Max.—maximum; \*\*\*—*p* < 0.001.

Change (*p* = 0.0002 \*\*\*) 5.9 4.2 5.3 0.8 24.2

As seen in the chart below, the relative increase in VCAM-1 in some people was as high as 60% of the initial value (Figure 3). As seen in the chart below, the relative increase in VCAM-1 in some people was as high as 60% of the initial value (Figure 3).

diving).

**Figure 3.** Distribution of VCAM-1 before and after exposure to a 30 m dive in the study group. **Figure 3.** Distribution of VCAM-1 before and after exposure to a 30 m dive in the study group.

As presented in the graph below (Figure 4), the relative increase in VCAM-1 over the

As presented in the graph below (Figure 4), the relative increase in VCAM-1 over the baseline was as high as 80% (i.e., in some subjects, VCAM-1 was almost twice as high after diving). *Medicina* **2022**, *58*, x FOR PEER REVIEW 8 of 14

**Figure 4.** VCAM-1 values with diving to 60 m in the study group. Abbreviations**:** \*\*\*—*p* < 0.001. **Figure 4.** VCAM-1 values with diving to 60 m in the study group. Abbreviations: \*\*\*—*p* < 0.001.

The distribution of results obtained before and after exposure to the conditions corresponding to diving at a depth of 30 m did not differ in a statistically significant way from the distribution of results in the non-divers group. Statistically significant differences existed between the results obtained after expo-The distribution of results obtained before and after exposure to the conditions corresponding to diving at a depth of 30 m did not differ in a statistically significant way from the distribution of results in the non-divers group.

sure to diving conditions at a depth of 60 m and the values measured for the non-divers group (*p* = 0.0494; Table 6). Statistically significant differences existed between the results obtained after exposure to diving conditions at a depth of 60 m and the values measured for the non-divers group (*p* = 0.0494; Table 6).

**Table 6.** Values of descriptive statistics characterizing the distribution of VCAM-1 in the compared groups. **Group Table 6.** Values of descriptive statistics characterizing the distribution of VCAM-1 in the compared groups.


The graph (Figure 5) shows the values of position statistics of the VCAM-1 distribu-Abbreviations: \*—*p* < 0.05.

tion in the compared groups and test series. The graph (Figure 5) shows the values of position statistics of the VCAM-1 distribution in the compared groups and test series.

The leukocyte count increased in a statistically significant manner after exposure to a 60 m dive. However, exposure to the conditions corresponding to diving at a depth of 30 m did not affect the unequivocally directed change in the leukocyte count. Longer exposure results in greater tissue saturation with gases (among others with nitrogen) during decompression, which lasts significantly longer; in this case, many more microbubbles are formed, which activate the immune system. The hemoglobin level decreased in most divers after exposure to a 30 m dive (*p* = 0.0098). Detailed data presenting the distribution of leukocytes and hemoglobin counts in individual tests, showing the significance of changes between the tests completed before and after exposure, are presented in Table 7.

Leukocytes [G/L]

Hemoglobin [g/dL]

**Figure 5.** Values of position statistics of VCAM-1 distribution in the compared groups and test **Figure 5.** Values of position statistics of VCAM-1 distribution in the compared groups and test series.

series. The leukocyte count increased in a statistically significant manner after exposure to a 60 m dive. However, exposure to the conditions corresponding to diving at a depth of **Table 7.** Descriptive statistics characterizing the distribution of leukocytes and hemoglobin counts in individual tests, showing the significance of changes between tests completed before and after exposure.


Before dive 30 m 6.20 6.19 1.05 4.23 9.03 After dive 30 m 6.43 6.22 1.23 4.42 8.87 Abbreviations: G/L—billion per liter; M—mean; Me—median; SD—standard deviation; Min.—minimum; Max.—maximum; \*\*—*p* < 0.01; \*\*\*—*p* < 0.001.

Change (*p* = 0.2485) 0.23 0.18 0.74 −0.76 1.56

Before dive 60 m 5.95 6.01 0.96 3.70 7.56 After dive 60 m 7.10 7.26 1.28 4.55 9.33 Change (*p* = 0.0004 \*\*\*) 1.17 0.94 0.85 0.12 3.20 Before dive 30 m 15.2 15.3 0.8 14.0 16.8 After dive 30 m 15.0 15.0 0.9 13.4 16.8 Change (*p* = 0.0098 \*\*) −0.2 −0.2 0.3 −1.0 0.4 The results of BCC after the exposure of the subjects in the divers group to conditions corresponding to a 30 m dive were not correlated with the VCAM-1 values. All the correlation coefficients were statistically insignificant (*p* > 0.05). Those few correlations that were close to the level of statistical significance, and also in a single case that was statistically significant, occurred between BCC and VCAM-1 after exposure to conditions corresponding to a 60 m dive.

These relationships were as follows:


corresponding to a 30 m dive were not correlated with the VCAM-1 values. All the

Changes in the BCC and VCAM-1 level after exposure to conditions corresponding to a 30 m dive were not statistically significantly correlated with each other.

More statistically significant relationships existed between the changes in BCC and the changes in VCAM-1 after exposure to the conditions corresponding to a 60 m dive. There were the following relationships:


#### **4. Discussion**

The aim of this study was to analyze the increase in VCAM-1 expression and the impact of exposure in a hyperbaric chamber on VCAM-1 in human blood serum. We believe that the results that have been obtained will allow for a better understanding of the biological changes that take place in our body during pressure changes at various depths, a deeper knowledge about the role of the VCAM-1 biomarker in our body and the possible impact of exposure in a hyperbaric chamber on human blood serum.

Madden and Laden [11], in their study, suggested that endothelial microparticles (MP) can be used as a decompression sickness (DCI) stress marker by assessing the antigenic markers of circulating MP that not only allow a specific origin, but also reflect endothelial integrity. According to these researchers, after endothelial disruption, the expression of adhesion molecules was expressed in accordance with the adopted configuration, and one such molecule, the VCAM-1 molecule, is an attractive marker due to the fact that it was only expressed on the activated endothelium, which was achieved after vascular trauma and is therefore a prognostic marker of a pro-inflammatory endothelium.

In this study, the statistical significance of differences in the distribution of VCAM-1 before and after exposure was analyzed. The difference between the VCAM-1 distribution before and after the exposure corresponding to a 30 m dive was on the limit of statistical significance (*p* = 0.0582). For most people, VCAM-1 was higher after exposure; on average, it was around 1.5 ng/mL, but there were also subjects who experienced a decrease in VCAM-1. The relative increase in VCAM-1 in some subjects was as high as 60% of the baseline value. Vince et al. [12] reported a significant increase in VCAM-1 positive microparticles (VCAM + MP), observed 1 h after diving using air compared to the non-divers group (*p* = 0.013), which was not observed after oxygen diving (*p* = 0.095).

The discussed study showed that diving at a greater depth had a much more pronounced effect on changes in VCAM-1 values. In all the subjects, VCAM-1 increased after exposure—at least by 0.8, and at most by 24.2 ng/mL. The average change was about 5.9 ng/mL. The changes in VCAM-1, as a result of diving to a depth of 60 m, were highly statistically significant. A study by Bao et al. [13] found that diving caused significantly reduced VCAM-1 levels.

Our research showed that the relative increase in VCAM-1 compared to the baseline value was as high as 80%, i.e., VCAM-1 after diving almost doubled in some subjects. A study performed by Zhang et al. [14] showed that VCAM-1 levels increased post-decompression in DCI rats.

In the presented study, a comparison of VCAM-1 distribution among divers before and after exposure to a 30 m and 60 m dive and the non-divers group was performed.

The distribution of the results obtained before and after exposure to the conditions corresponding to a 30 m dive did not statistically significantly differ from the distribution of results in the non-divers group. The research showed that there were statistically significant differences between the results obtained after exposure to conditions of a 60 m dive and the values measured for the non-divers group (*p* = 0.0494). VCAM-1 is expressed exclusively on the activated endothelium following vascular insult, and therefore, is a marker of a proinflammatory endothelium, as a study by Bao et al. [13] showed.

Research by Vince et al. [12] in which the VCAM + MP was quantified before diving (09:00 a.m. and 1:00 p/m.) and after diving (+1, +3 and +15 h), showed that both for diving with air and oxygen, and compared to control samples collected from the same subjects, VCAM + MP showed a similar trend in all the experiments. However, both dives resulted in a change in endothelial status as measured by VCAM + MP. A significant increase in VCAM + MP was observed 1 h after diving using air compared to the controls (*p* = 0.013), which was not observed after oxygen diving (*p* = 0.095). The researchers [12] observed an increase in the circulating VCAM + MP population after simulated diving with both compressed air and oxygen, compared to their non-dive controls taken at the same time of day. Due to its expression only on the activated endothelium, VCAM + MP can be used as a sensitive marker of endothelial function/dysfunction. Researchers hypothesized that the increase in circulating VCAM + MP could reflect changes in the state of the endothelium and could be potentially used as a biomarker of sensitivity to, for example, decompression sickness, when vascular mechanisms are involved [12].

This research showed that the results of BCC after the exposure of the subjects in the divers and non-divers group to conditions corresponding to a 30 m dive were not correlated with values of VCAM-1. However, the leukocyte count increased in a statistically significant manner after exposure to a 60 m dive, and exposure to conditions corresponding to a 30 m dive did not affect the unequivocally targeted change in the leukocyte count, which could be explained by an insufficient number of subjects in the divers group. A study performed by Glavas et al. [15] showed that the microbubbles produced during the decompression process induce endothelial damage and affect leukocyte mobilization.

In our study, the hemoglobin level decreased in most divers after exposure to diving conditions at a depth of 30 m and this effect should be considered as not accidental (*p* = 0.0098), and after a stronger exposure—to conditions corresponding to a depth of 60 m—such effects were not observed. The scale of the decrease in hemoglobin levels was small—only 0.2 g/dL on average; therefore, it was not a change affecting the health of the divers. Several studies have reported an altered hematological status and hemoglobin reduction after saturation diving [16–19].

A decrease in the number of neutrophils in the blood in our study is unlikely to indicate inflammation. Most likely, these results decrease from the fact that microbubbles are treated as hostile pathogens by neutrophils; as a result of the so-called oxygen burst (respiratory burst), microbubbles are eliminated, and thus the neutrophil cells are destroyed. This would confirm the increased generation of reactive oxygen species and the intensification of oxidative stress (we also observed an increase in oxidative stress in our volunteers).

More statistically significant relationships existed between the changes in the BCC parameters and the changes in VCAM-1 after exposure to conditions corresponding to a 60 m dive in the non-divers group. The relationships that occurred were as follows: higher increments of VCAM-1 were associated with a greater increase (in some cases a smaller decrease) in MCH and MCHC—these were relationships of average strength and statistically significant; quite a strong correlation was found between changes in neutrophils count and changes in VCAM-1—the greater the increase in the number of neutrophils is, the smaller the increase in VCAM-1 is.

Our study showed that the number of blood platelets decreased in a statistically significant (*p* = 0.0382) manner after exposure to the conditions corresponding to a dive to a depth of 60 m, and the level of neutrophils increased in a statistically significant manner after exposure to conditions corresponding to a dive to a depth of 60 m. Olsza ´nski et al. [20] demonstrated in his study that the diving technology employed did not generate substantial changes in the examined parameters of blood in divers, and the increase in neutrophils, blood platelets and the fibrinogen concentration in the blood plasma immediately after diving is of a temporary character, being a typical reaction observed during diving.

In turn, the increase in the number of lymphocytes after exposure to conditions corresponding to a 60 m dive correlated with the increase in VCAM-1 (R = 0.54; *p* = 0.0304); similar (i.e., positive) correlations apply to VCAM-1 and the lymphocyte ratio. This research also showed that those few correlations that were close to the level of statistical significance, and in one case statistically significant, occurred between BCC and VCAM-1 after exposure to conditions corresponding to a 60 m dive in the divers group. The relationships were as follows: there was a relationship between the neutrophils count and VCAM-1—the higher the number of neutrophils was, the lower the value of VCAM-1 was (this correlation was statistically significant—*p* = 0.0456; its strength was average—R = −0.51); a similar relationship was found between the number of leukocytes and VCAM-1 (it was slightly weaker and only close to the level of statistical significance); VCAM-1 was higher in the subjects with a higher lymphocyte ratio (R = 0.44)—this correlation was close to the level of statistical significance (*p* = 0.0848). In a study performed by Bao et al. [13], deep heliox diving caused a significant decrease in red blood cells (RBC) but had no significant effect on hemoglobin (HGB) levels. These changes can be explained by the oxidative damage-induced fragility of the RBC membrane. A study by Perovic et al. [21] showed that neutrophils increased, and monocytes decreased immediately after 30 m-depth compresseddiving using air. Since the number of intermediate cells in humans is small, the reduction in the percentage of intermediate cells may be a transient response to external stimuli. Obad at al. [22] have shown in their study that this may be caused by trans-endothelial migration due to altered vascular/endothelial function after diving. Sureda et al. [23] has shown in his study that scuba diving at 50 m deep for a total time of 35 min was enough to induce a post-diving neutrophil mobilization in normobaria, suggesting the initiation of an immune-like response, similar to that which occurs after an infection or an acute bout of exercise. Exactly these results could be expected, because VCAM-1 is a key cell adhesion molecule involved in inflammation that is closely implicated in various immunological disorders [24,25]. The VCAM-1 protein mediates the adhesion of neutrophils, monocytes, eosinophils and basophils to the vascular endothelium [14,26]. It is also active in the signal transduction of leukocytes and endothelial cells [24,27–29]. A study by Glavas et al. [15] showed that the absolute number of monocytes was slightly, but not significantly, increased after a dive, and this study suggests that biochemical changes induced by scuba diving primarily activate existing monocytes rather than increase the number of monocytes at a time of acute arterial endothelial dysfunction.

#### *Strengths and Limitations of the Study*

A strength of this study was the well-matched divers and non-divers groups.

The limitations of the present study should be noted. Due to the small sample size, the present study has limited power. We believe that further research with a larger population is required and warranted. In searching for the relationship between the BCC results of blood counts and the values of VCAM-1 in the test group (conditions corresponding to a 30 m dive), the lack of a relationship with white blood cells can also be explained by the group of respondents being too small.

#### **5. Conclusions**


3. We believe that exposure in a hyperbaric chamber may result in the activation of the endothelium.

**Author Contributions:** Conceptualization, K.V.D.-O., M.C., M.K., E.K.-K., P.S. and D.K.; data curation, K.V.D.-O., M.C., M.K., P.S. and D.K.; formal analysis, K.V.D.-O., M.C., M.K., P.S. and M.S.; funding acquisition, K.V.D.-O.; investigation, K.V.D.-O., M.C., M.K., P.S. and M.S.; methodology, K.V.D.-O., M.K., P.S. and D.K.; project administration, K.V.D.-O.; supervision, M.C., M.K., E.K.-K., P.S. and D.K.; writing—original draft, K.V.D.-O., M.K., P.S., M.S. and D.K.; writing—review and editing, K.V.D.-O., M.C., M.K.,E.K.-K., P.S. and M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded with grant no. N/ST/ZB/15/001/3310 from the Ministry of Science and Higher Education in Poland. The funders had no role in the study design, data collection and analysis, preparation of the manuscript or decision about its publication.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Medical University of Białystok, Poland (no. R-I-002/237/2015; approval date: 25 June 2015).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data are available upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Clinical Assessment of the Hyperbaric Oxygen Therapy Efficacy in Mild to Moderate Periodontal Affections: A Simple Randomised Trial**

**Alexandru Burcea <sup>1</sup> , Laurenta Lelia Mihai 1,\*, Anamaria Bechir 1,\* , Mircea Suciu <sup>2</sup> and Edwin Sever Bechir <sup>2</sup>**


**Abstract:** *Background and Objectives:* Gum disease represents the condition due to the dental plaque and dental calculus deposition on the surfaces of the teeth, followed by ulterior destruction of the periodontal tissues through the host reaction to the pathogenic microorganisms. The aim of study was to present aspects regarding the efficacy of hyperbaric oxygen therapy (HBOT) as an adjuvant therapy for the treatment of periodontal disease, started from the already certified benefits of HBOT in the general medicine specialties. *Materials and Methods:* The participant patients in this study (71) required and benefited from specific periodontal disease treatments. All patients included in the trial benefited from the conventional therapy of full-mouth scaling and root planing (SRP) within 24 h. HBOT was performed on the patients of the first group (31), in 20 sessions, of one hour. The patients of the control group (40) did not benefit from HBO therapy. *Results:* At the end of study, the included patients in HBOT group presented significantly better values of oral health index (OHI-S), sulcus bleeding index (SBI), dental mobility (DM), and periodontal pocket depth (PD) than the patients of the control group. *Conclusions:* HBOT had beneficial effects on the oral and general health of all patients, because in addition to the positive results in periodontal therapy, some individual symptoms of the patients diminished or disappeared upon completion of this adjuvant therapy.

**Keywords:** periodontal disease; oral health index; dental mobility; periodontal pockets depth; hyperbaric oxygen therapy

#### **1. Introduction**

Dental biofilm is the main etiologic factor for caries, periodontal and peri-implant infections. Gum disease represents a disease with dental plaque and calculus formation on the surfaces of the teeth, followed by ulterior destruction of the periodontal tissues due to the host reaction to pathogenic microorganisms [1,2].

The spreading of periodontal disease is between 20 and 50% in the world, and represents one of important causes of indentations, which jeopardize the functions of oro-facial system, including mastication, aesthetics, self-reliability, and life quality [3]. This prevalence of periodontal disease is presumed to be rising in future years because of increased aging in population and of maintenance of natural teeth of dental arches in the elderly [4,5]. The development of periodontal disease in the context of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions presents four stages, defined based on severity (primarily periodontal breakdown with reference to root length and periodontitis-associated tooth loss), complexity of management (pocket depth, infrabony defects, furcation involvement, tooth hypermobility, masticatory dysfunction) and additionally described as extent (localized or generalized). The grade of periodontitis is estimated with direct or indirect evidence of progression rate in three categories:

**Citation:** Burcea, A.; Mihai, L.L.; Bechir, A.; Suciu, M.; Bechir, E.S. Clinical Assessment of the Hyperbaric Oxygen Therapy Efficacy in Mild to Moderate Periodontal Affections: A Simple Randomised Trial. *Medicina* **2022**, *58*, 234. https:// doi.org/10.3390/medicina58020234

Academic Editor: Gaetano Isola

Received: 1 January 2022 Accepted: 1 February 2022 Published: 4 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). *medicina* slow, moderate and rapid progression (Grade A–C). Risk factor analysis is used as grade modifier [6,7].

Cellular oxygenation is carried out by transporting the oxygen (that is fixed to haemoglobin), from inspired air to the tissues and cells, through the circulatory system. Disruption or interruption of oxygen transport induces hypoxia through changes in haemoglobin, capillary network, or blood flow [8–10]. Oxygen has a very small molecule, which allows its increased diffusibility in tissues, in comparison to any other substance [11]. Increasing the oxygen pressure in the environment over a certain point, increases the amount of dissolved oxygen in the plasma (quantitative increase) and the penetration rate of oxygen into the tissues (qualitative increase) [12].

The Undersea and Hyperbaric Medical Society (UHMS) defines hyperbaric oxygen therapy (HBOT) as a treatment in which the patient intermittently inspires 100% oxygen, in a pressurized treatment chamber at a higher pressure than at the sea level (1 atm absolute, ATA) [11,13,14]. In HBO therapy, the patient inspires pure or enriched oxygen, which causes a reduction in the amount of nitrogen in the blood [15]. The mechanisms of therapeutic action of HBOT are based on raising the partial pressure of inspired oxygen and increasing hydrostatic pressure, by compressing the gas in all spaces in the body, according to Boyle's law [16,17]. Increasing the oxygen's partial pressure raises its diffusibility in tissues. Even if there is a similar amount of oxygen in the plasma and in the transported oxygen by haemoglobin, by increasing the oxygen's partial pressure, its effectiveness is enhanced at a cellular level [18]. Increasing the pressure of the oxygen in the environment over a certain point induces the rising of the amount of oxygen dissolved in the plasma (increase in volume), and the penetration of the oxygen in the tissues (increase in quality) [19,20]. Therefore, HBO treatment leads to a considerable development in bone formation, so that the lamellar bone grows [21].

In the study effectuated by Giacon et al. [22], the authors consider that through HBO therapy pre-treatment, the protection proteins of oxidative stress are activated and tissues are prepared for surgery entailing transient ischemia. They presented a case report of an immediate dental implant, which depicted the utilisation of HBO therapy and of advanced platelet-rich fibrin (A-PRF) for pre-treating the implant site in a case of severe periodontitis with tooth attachment loss. Three sessions of HBO therapy effectuated for preconditioning by increasing the positive results, presumably through sterilization of surgical site and development of antioxidant protection. The authors underlined that future studies should specifically address this topic.

Altug et al. [23], studied the consequences of HBO therapy on implant osseointegration in experimental diabetes rabbits. The authors concluded that the disclosures in histomorphometry hint that HBO therapy has a certain impact on the osseointegration of dental implants, in the early healing time in diabetic rabbits. The authors underlined that dental implant stability is not influenced by HBO therapy.

The administration of oxygen in HBOT is realized in hyperbaric chambers, which can be multiplace (type A), and monoplace (type B, that provides treatment for a single patient) [24]. Both chambers are used to treat various diseases, from simple injuries to serious illness [25].

The comparative study started from the already certified benefits of HBOT in the general medicine specialties. The aim of this comparative study was to find out if the adjuvant HBO therapy presents beneficial effects in the treatment of adult patients affected by periodontal disease, after the conventional therapy of performing professional dental hygienization measures, and of full-mouth scaling and root planing (SRP) with EMS Piezon device and EMS Air-Flow Master. The expected result of the trial should potentially contribute to an advanced treatment strategy for periodontal disease, with an ideal clinical outcome.

#### **2. Materials and Methods**

The study was realized in conformity to the ethical principles of the Declaration of Helsinki and of the good clinical practice. The protocol was approved by the Ethics Committee of Dental Medicine Faculty, Titu Maiorescu University of Bucharest (No. 2 of 5 May 2017). All patients were informed about the research requirements, attended only by those that entered voluntarily in the research program. The study phases were explained to each recruited patient, including the need for monitoring. Included patients signed the written informed consent prior to the beginning of this study. The working hypothesis started from the premise that the benefits of HBOT are already certified in general medicine specialties, but can it be used as an adjuvant therapy in Romanian patients with periodontal disease?

The study was conducted in the Clinics of Dental Medicine Faculties, between of May 2017 and May 2021, but the COVID-19 pandemic epidemiological context determined a 12-month intermission. The authors followed calibration trainings to ensure: the precision and correctness of patients' anamneses, of clinical examination and diagnosis; the proper use of EMS Piezon and EMS Air-Flow Master Device; the use of the same standardized clinical measurements. The calibration trainings were realized in order to ensure the validity and reliability of clinical study and of obtained results. Adjuvant HBO therapy was performed in Hypermed Care SRL Clinic, by using the Revitalair®430 monoplace equipment, produced by Biobarica (Medley, FL, USA).

Clinical examinations and interviews were accomplished to evaluate eligibility. Patients were randomly screened, and then asked to participate in the study. Study initially enrolled 89 patients, but 12 subjects withdrawn voluntarily during the study and 6 subjects were excluded for lack of cooperation. The remaining patients in the study (71), had the age range of 38–59 years (means 48.5 years, ±10.5 years). Table 1 presents the sample patients and Figure 1 presents the flow diagram of the study.


**Table 1.** Sample patients.

Oral examinations and X-rays were effectuated at the time of patients selection, to differentiate the periodontal affection from other diseases (e.g., apical periodontitis, tooth fracture, etc.). The oral examinations consisted of the assessment of periodontal status by determination of the oral hygiene conditions and of Simplified Oral Health Index (OHI-S), Sulcus Bleeding Index (SBI), examination of clinical attachment level (CAL), checking for dental mobility degree (MD), of the pocket depth (PD), and of furcation involvement. All periodontal examinations were effectuated by using mirrors, tweezers, probes, and a calibrated periodontal UNC-PCP15 Color-Coded Probe (Hu-Friedy Europe, The Netherlands) probe.

Inclusion criteria for this study consisted of non-smoking patients aged 38–59 years old, having at least 6 natural teeth on a half dental arch (excluding third molars), having periodontal symptomatology at the presentation in the dental office (like gum redness or/and bleeding, gum swelling, persistent metallic taste, halitosis, painful chewing, sensitive teeth, minimum 4 teeth with first or second degree of dental mobility, periodontal pockets), or with a confirmed diagnosis of mild to moderate periodontal disease.

**Figure 1.** Flow Diagram of the study. **Figure 1.** Flow Diagram of the study.

Oral examinations and X-rays were effectuated at the time of patients selection, to differentiate the periodontal affection from other diseases (e.g., apical periodontitis, tooth fracture, etc.). The oral examinations consisted of the assessment of periodontal status by All selected patients for admission into the first group of patients with HBO therapy completed and signed a fact sheet with information regarding their general and specific health conditions (which contained affections which determined the exclusion criteria of study). Inclusion criteria of patients are depicted in Table 2.

determination of the oral hygiene conditions and of Simplified Oral Health Index (OHI-**Table 2.** Inclusion criteria of patients in study.


periodontal symptomatology at the presentation in the dental office (like gum redness or/and bleeding, gum swelling, persistent metallic taste, halitosis, painful chewing, sensitive teeth, minimum 4 teeth with first or second degree of dental mobility, periodontal pockets), or with a confirmed diagnosis of mild to moderate periodontal disease. All selected patients for admission into the first group of patients with HBO therapy completed and signed a fact sheet with information regarding their general and specific Exclusion criteria for this study were represented by smoker patients, maximum scores of all studied periodontal indices, periodontal treatment and antibiotic therapy in the last six months, aggressive periodontitis, endodontic affections, orthodontic patients, patients with infections, systemic disorders, upper respiratory and pulmonary disorders (e.g., untreated pneumothorax, pneumonia, asthma, chronic obstructive pulmonary disease, etc.), cataract, Eustachian tube dysfunction, hereditary spherocytosis, fever, claustrophobia, convulsions, cardiac simulators or other implanted or external devices that control body

old, having at least 6 natural teeth on a half dental arch (excluding third molars), having

health conditions (which contained affections which determined the exclusion criteria of

functions, patients with unstable cardiovascular disease, pregnant woman, hospitalized patients, those with cancer, with uncontrolled diabetes mellitus, with parafunction of chewing habit, with severe malocclusion, those with missing data, patients with mental disability, uncooperative patients, and patients who refused to be included in the study.

According to the safety requirements of patients, those with general medical conditions presented in exclusion criteria of patients cannot be accepted for HBO therapy, thus patients with these affections were not be admitted in study group 1 (with HBOT). Exclusion criteria are presented in Table 3.

**Table 3.** Exclusion criteria of patients in study.


Supra- and subgingival debridement, scaling, and root planing with EMS Piezon device and EMS Air-Flow Master device, followed by manual root planing were effectuated in every selected patient in 24 h. Guided Biofilm Therapy with EMS device is a treatment protocol built in conformity of each patient diagnosis and risk evaluation for achieving optimum outcomes. The treatment is realized in a minimally invasive way, with the highest comfort, safety and efficiency for the patients. Oral hygiene instructions were presented to every participant patient, and dental plaque disclosing gel (GC Tri Plaque ID Gel) was used before any assessment sessions. Patients included in the study used the same toothpaste (Colgate Total Gum Protection Toothpaste), same dental brush (Colgate Gum Health Extra Soft Toothbrush for Sensitive Gums with Deep Cleaning Floss-Tip Bristles), and same interdental pick (GUM-6326RA Soft-Picks Original Dental Picks, small). Participants brushed their teeth twice a day, morning and evening, at least two minutes each time (in conformity with the American Dental Association (ADA) suggestions), with Stillman method of tooth brushing. An amount of 1 cm tooth paste was used. The inter-dental pick was indicated to be utilized only in the evening, before teeth brushing. Two tubes of plaque revealing gel were handed to the patients participating in the study, for checking their dental hygiene 3 times weekly. The gel was applied after tooth brushing, and if the sanitization was not correct, the patients had to perform their tooth brushing again. At the end of the study, the used quantity of the plaque disclosing gel from the tubes was verified, in order to verify the compliance of participants to the study protocol.

The selected patients (71), were divided into two groups, the first group of patients (group I) who agreed upon and benefited of HBOT adjuvant therapy (31 patients, 15 women and 16 men), and the second group of patients (group II / control group), who did not undergo HBO therapy (40 patients, 19 women and 21 males). Both patient groups were selected according to the same inclusion/exclusion criteria and benefited from the same dental treatment protocol for periodontal disease, excepting HBO adjuvant therapy (which was realized only in 1st group of patients). The type of study design was simple randomized trial, by centralized randomization into groups, than participant patients were divided into the two groups by centralized randomization.

The applied clinical protocol to all patients consisted of: consultation and complementary radiographic examinations; diagnosis of general health and of oral/periodontal

tissues; first clinical evaluation and registration of dental and periodontal status by determination of OHI-S index, of the sulcus bleeding index, the pathological tooth mobility and the depth of periodontal pockets; establishing of the treatment plan; filing the general and specific information sheet (by the patient for admission in the HBOT group), and signature on the informed consent; awareness of patients on the state of periodontal illness at presentation; training and insisting upon artificial oral hygiene procedures by using the Stillman technique of tooth brushing; taking of the same brand and type of toothbrushes, toothpastes, interdental brushes, and dental plaque disclosing gel; conducting of the corrective treatment (performing professional oral hygiene, and of specific therapy for dental and periodontal diseases with EMS devices); second clinical evaluation of the patients at 1 month after periodontal treatment; applying HBO therapy in the first group of patients; third clinical evaluation at 2 months after the completion of HBO therapy and recording of the results; comparison of results. Figure 2 depict the flow chart of the clinical study. *Medicina* **2022**, *58*, 234 7 of 18

**Figure 2.** Flow chart of the clinical study. **Figure 2.** Flow chart of the clinical study.

After the effectuation of specific periodontal treatment in all patients, each patient belonging to the first group (with HBO adjuvant therapy) was set to perform 20 sessions of hyperbaric oxygen therapy for one hour, with the applied pressure of 1.4 ATM. The frequency of the therapy sessions was three sessions per week. HBO therapy was performed at the Biobarica Hypermed Care Clinic in Bucharest, in a monoplace hyperbaric chamber (Figure 3). After the effectuation of specific periodontal treatment in all patients, each patient belonging to the first group (with HBO adjuvant therapy) was set to perform 20 sessions of hyperbaric oxygen therapy for one hour, with the applied pressure of 1.4 ATM. The frequency of the therapy sessions was three sessions per week. HBO therapy was performed at the Biobarica Hypermed Care Clinic in Bucharest, in a monoplace hyperbaric chamber (Figure 3).

**Figure 3.** The monoplace hyperbaric chamber used in Biobarica Hypermed Care Clinic.

**Figure 2.** Flow chart of the clinical study.

chamber (Figure 3).

**Figure 3.** The monoplace hyperbaric chamber used in Biobarica Hypermed Care Clinic. **Figure 3.** The monoplace hyperbaric chamber used in Biobarica Hypermed Care Clinic.

Stillman method of tooth brushing consists of thoroughly brushing around and under the gum line, and it helps to clean the debris deposited between the teeth, because the toothbrush bristles reach under the gums [25].

After the effectuation of specific periodontal treatment in all patients, each patient belonging to the first group (with HBO adjuvant therapy) was set to perform 20 sessions of hyperbaric oxygen therapy for one hour, with the applied pressure of 1.4 ATM. The frequency of the therapy sessions was three sessions per week. HBO therapy was performed at the Biobarica Hypermed Care Clinic in Bucharest, in a monoplace hyperbaric

Oral hygiene status can be determined by using the oral health index (OHI), calculated by the oral debris score and the dental calculus score found on the buccal and lingual surfaces of each of the three segments of the dental arches. The calculation of the numerical values of simplified oral health index (OHI-S) is in conformity to the existing dental plaque and calculus deposits. The simplified oral hygiene index (OHI-S) allows the separate evaluation of the soft and hard deposits, present on the buccal/labial or oral dental crown surfaces of 6 teeth of both dental arches, one tooth for each sextant: maxillary dental arch, teeth 1.6, 1.1, 2.6 on buccal/labial surfaces, respectively mandibular dental arch teeth 3.6, 3.1, 4.6 on lingual surfaces (enumerated teeth are noted after FDI notation system). The selected surfaces used for scoring were the buccal for maxillary molar and the lingual for mandibular molars, respectively, the labial for the maxillary right central incisors and for the mandibular left central incisors. In the absence of first molar, the second or third molar were examined, and in the case of the incisors, the neighbouring incisor.

The quantification of dental deposits can be performed visually or by staining solution. The surfaces of dental crowns are examined with the probe, extending the examination to the level of contact points of the proximal coronary surfaces, including the subgingival area. Debris index-simplified (DI-S) calculation method: 0 = absence of dental plaque; 1 = microbial plaque present up to 1/3 of the tooth surface; 2 = microbial plaque present between 1/3 and 2/3 of the tooth surface; 3 = microbial plaque present over 2/3 of the tooth surface. Calculus index-simplified (CI-S) calculation method: 0 = absence of dental calculus; 1 = calculus present up to 1/3 of the tooth surface; 2 = calculus present between 1/3 and 2/3 of the tooth surface; 3 = calculus present over 2/3 of the tooth surface. The averages for the plaque and tartar indices will be calculated and then added together; their sum will represent the OHI-S index [26,27]. OHI-S score is summed and then divided to the number of examined dental crown surfaces, for the mean oral hygiene score [28,29]. An arithmetic mean of the individual scores for debris and calculus index was performed, and subsequently the highest determined score was taken into consideration. The values of OHI-S index, necessary for interpretation, are: excellent 0; good 0.1–1.2; satisfactory 1.3–3.0; and unsatisfactory 3.1–6 [30]. Index interpretation of OHI-S scores used in this study was: excellent = 0; good = 1; satisfactory = 2; and unsatisfactory = 3. The scores were calculated according to the results of the determinations performed on each patient. An arithmetic mean of the individual scores was performed on each tooth, and subsequently the highest determined final score was taken into consideration.

Sulcus Bleeding Index (SBI, M˝uhlemann and Son) on gentle probing of the sulcus represents one of the initial signs of periodontal disease. In SBI, four gingival units are scored systematically for each tooth: the labial and lingual marginal gingival (M units) and the mesial and distal gingival papilla (P units). After probing, the examiner should wait for 30 s for scoring. Scores for these units are summed and then distributed to 4. Adding the obtained scores of the studied teeth and dividing them by the number of studied teeth establishes the sulcus bleeding index (SBI). Criteria of SBI scoring are: 0—healthy aspect of papilla and of marginal gingiva, without bleeding on probing; 1—healthy gingival aspect, but bleeding on probing; 2—bleeding on probing, modified colour, without edema; 3—bleeding on probing, modified colour, slight edema; 4—bleeding on probing, modified colour, evident edema; 5—spontaneous bleeding, modified colour, pronounced edema. SBI scoring is effectuated on the eight upper and lower anterior teeth, and four gingival areas are included for each tooth: mesial-labial, mesial-lingual, oro-mesial, oro-distal [31]. Index interpretation of BSI scores in this study was: excellent = 0; very good = 1; good = 2; satisfactory = 3; and unsatisfactory = 4.

The clinical sign of dental/tooth mobility depicts the periodontal destruction degree determined by local affections of gums and surrounding structures of the teeth. Tooth mobility presents 4 degrees: grade 0 represents the physiological mobility; in grade 1, the teeth present more than 1 mm mobility in a buccal-oral direction: in grade 2, the teeth present more than 1 mm mobility in buccal-oral and mesial-distal direction; and in grade 3, the teeth present mobility in three directions, buccal-oral, mesial-distal and incisal/occlusal-apical direction. Tooth mobility can represent a possible aggravation factor of the establishment of periodontal disease [32].

Periodontal pockets represent a pathological feature characterized by the displacement of the gingival attachment, respectively, the deepening of the gingival sulcus apically, due to the expansion of dental plaque and dental calculus towards the dental root. It can be classified as supra-alveolar (when the bottom of the pocket is situated at the crown of the alveolar bone), and intra-alveolar (when the bottom of the periodontal pocket is situated apical to the alveolar bone). Periodontal pockets can imply one or more tooth surfaces, and they can present various depths on different surfaces of the tooth. Periodontal pocket depth (PD) is a primary sign of periodontitis. The size and severity can be divided into normal (1 to 3 mm), early/mild periodontitis (4 to 5 mm), moderate periodontitis (5 to 7 mm), and severe periodontitis (7 to 12 mm). Periodontal examinations are effectuated with a periodontal probe [33]. In this study, an arithmetic mean of the individual scores for pockets was performed on each tooth, and subsequently the highest determined score was taken into consideration. Rationale of classifying periodontal pockets is to realize a correct evaluation, and then a correct prognosis of periodontal disease after the stage and grade of the disease, including the contributory factors, and after that, to effectuate the adequate treatment management of disease [6].

All the statistical analysis were performed in SPSS 24 Software. The considered level of significance is 0.05, otherwise mentioned. Data was analysed through means of the Chi-Square test for group differences.

#### **3. Results**

Two months after the end of HBO therapy in the first group of patients, we summarized and compared the recorded data of all patients. The obtained results in both studied groups, according to the three clinical examinations, are presented in Table 4.


**Table 4.** Obtained results in the studied groups, according to the three clinical examinations.

By comparing the listed values in Table 4, we found the following: index Score 1 11 (=35.48%) 12 (=30.00%) Score 2 5 (=16.12%) 11 (=27.50%)

*Medicina* **2022**, *58*, 234 10 of 18

bleeding index (SBI)

Dental mobility degree (DM)

Pockets depth (PD)

Oral health

finalization of periodontal

treatments Sulcus


Score 0 3 (=9.67%) 5 (=12.50%) Score 1 5 (=16.12%) 7 (=17.50%) Score 2 7 (=22.58%) 9 (=22.50%) Score 3 14 (=45.16%) 16 (=40.00%) Score 4 2 (=6.45%) 3 (=7.50%)

Normal 3 (=9.67%) 5 (=12.50%)

Score 0 12 (=38.40%) 9 (=22.50%)

Physiological mobility 4 (=12.90%) 7 (=17.50%) 1st degree mobility 16 (=51.61%) 19 (=47.50%) 2nd degree mobility 11 (=35.48%) 14 (=35.00%)

Early/mild periodontitis 17 (=54.83%) 19 (=47.50%) Moderate periodontitis 11 (=35.48%) 16 (=40.00%)

	- Dental mobility (DM): We found that the DM has diminished in both groups; at the presentation all of patients had I-st and II-nd degree dental mobility; in the other two clinical examinations, we found that DM was reduced till physiological mobility, especially in patients who received HBO adjuvant therapy. Figure 6 depicts the patient distribution based on DM index at first (a), second (b), and third (c) examination. control), because of SRP treatment, but probably also as a result of patient awareness in the need for correct oral hygiene through brushing exercises (initially done at the clinic, until all patients have acquired the correct technique), respectively, by revealing the bacterial plaque (performed at each presentation of the patient in the prac-
	- Pockets depth (PD): the measurements were effectuated with the periodontal probe and registered in the patients' record; we found that the PD values were reduced in both groups of patients in the second and third clinical examination, but the reduction of PD was more significant in patients belonging to the HBOT group. In Figure 7, the patient distribution based on PD index at first (a), second (b), and third (c) examination is presented. tice); at the second and third determination, we found that the OHI-S decreased in the patients of both groups, to 0, 1, and 2; and in the third assessment, the OHI-S values were lower in the first group (which benefited from HBOT) compared to the control group. In Figure 4, the patient distribution based on OHI-S index, are presented at the first (a), second (b), and third (c) examination.

**Figure 4.** Patient distribution based on OHI index at (**a**) first examination, (**b**) second examination, and (**c**) third examination. **Figure 4.** Patient distribution based on OHI index at (**a**) first examination, (**b**) second examination, and (**c**) third examination.

*Medicina* **2022**, *58*, 234 11 of 18

**Figure 5.** Patient distribution based on SB index at (**a**) first examination, (**b**) second examination, and (**c**) third examination. **Figure 5.** Patient distribution based on SB index at (**a**) first examination, (**b**) second examination, and (**c**) third examination. pecially in patients who received HBO adjuvant therapy. Figure 6 depicts the patient distribution based on DM index at first (a), second (b), and third (c) examination.

clinical examinations, we found that DM was reduced till physiological mobility, es-

‐ Sulcus bleeding index (SBI): at their presentation, the patients of both groups had only values of 1, 2, 3, and 4 in SBI score, without value 0; in second clinical examination we found that SBI scores were significantly reduced in both study groups (HBOT and control), because of the same reasons (SRP treatment, correct teeth brushing); at the second and third determination, we found that the SBI scores decreased in the patients of both groups; and in third assessment, the SBI values were lower in the first group (HBOT) compared to the control group. In Figure 5, the patient distribution based on SBI index at first (a), second (b), and third (c) examination are depicted.

‐ Sulcus bleeding index (SBI): at their presentation, the patients of both groups had only values of 1, 2, 3, and 4 in SBI score, without value 0; in second clinical examination we found that SBI scores were significantly reduced in both study groups (HBOT and control), because of the same reasons (SRP treatment, correct teeth brushing); at the second and third determination, we found that the SBI scores decreased in the patients of both groups; and in third assessment, the SBI values were lower in the first group (HBOT) compared to the control group. In Figure 5, the patient distribution based on SBI index at first (a), second (b), and third (c) examination are depicted.

**Figure 6.** Patient distribution based on DM index at (**a**) first examination, (**b**) second examination, and (**c**) third examination. **Figure 6.** Patient distribution based on DM index at (**a**) first examination, (**b**) second examination, and (**c**) third examination. *Medicina* **2022**, *58*, 234 12 of 18

examination is presented. **Figure 7.** Patient distribution based on PD at (**a**) first examination, (**b**) second examination, and (**c**) third examination. **Figure 7.** Patient distribution based on PD at (**a**) first examination, (**b**) second examination, and (**c**) third examination.

7, the patient distribution based on PD index at first (a), second (b), and third (c)

In conformity with the inclusion criteria, only patients with mild and moderate periodontal disease were admitted in study. Only two patients out of a total of 71 had a slight involvement of the furcation, therefore assessment of the involvement of furcation was not introduced in the study. In conformity with the inclusion criteria, only patients with mild and moderate periodontal disease were admitted in study. Only two patients out of a total of 71 had a slight involvement of the furcation, therefore assessment of the involvement of furcation was not introduced in the study.

Table 5 present Chi-Square test *p*-values for test differences between control and test groups for all variables, at all three levels of investigation, with *p*-values. There are no significant differences, at any level, between the frequencies of the control and test groups, for any of the observed variables, no matter the treatment type. Table 5 present Chi-Square test *p*-values for test differences between control and test groups for all variables, at all three levels of investigation, with *p*-values. There are no significant differences, at any level, between the frequencies of the control and test groups, for any of the observed variables, no matter the treatment type.

**Table 5.** Chi-Square test *p*-values for test differences between groups, for all variables, at all "three

OHI-S1 0.75 SBI1 0.99 DM1 0.65 PD1 0.91 OHI-S2 0.99 SBI2 0.91 DM2 0.82 PD2 0.81 OHI-S3 0.27 SBI3 0.21 DM3 0.66 PD3 0.28

In Table 6, Chi-Square test p-values are presented for test differences between groups for all variables, for test and control groups. The significance level is 0.1. Exceptions appear for the SBI index in the control group, which is significant at a 0.1 significance level, and all differences are significant at a 0.05 level. It can be noticed that there are significant

**Table 6.** Chi-Square test *p*-values for test differences between both groups (SRP+HBOT and SRP)

OHI-S 0 0.016 SBI 0 0.078 \* DM 0.018 0.013 PD 0 0.026

At the finalisation of study, it was noted that, although patients did not initially mention their mild vertigo, mild tinnitus, and fatigability, at the end of HBO therapy, 21 patients (= 67.74%) stated, without being asked, that they no longer had mild vertigo, mild

**Test Control** 

differences in the effect of the three assessments, both in test or control groups.

**Variable** *p***-Value** 

tinnitus symptoms and that they no longer present signs of chronic fatigue.

levels of investigation".

for all variables.

\* Significance level 0.1.


**Table 5.** Chi-Square test *p*-values for test differences between groups, for all variables, at all "three levels of investigation".

In Table 6, Chi-Square test p-values are presented for test differences between groups for all variables, for test and control groups. The significance level is 0.1. Exceptions appear for the SBI index in the control group, which is significant at a 0.1 significance level, and all differences are significant at a 0.05 level. It can be noticed that there are significant differences in the effect of the three assessments, both in test or control groups.

**Table 6.** Chi-Square test *p*-values for test differences between both groups (SRP+HBOT and SRP) for all variables.


\* Significance level 0.1.

At the finalisation of study, it was noted that, although patients did not initially mention their mild vertigo, mild tinnitus, and fatigability, at the end of HBO therapy, 21 patients (= 67.74%) stated, without being asked, that they no longer had mild vertigo, mild tinnitus symptoms and that they no longer present signs of chronic fatigue.

#### **4. Discussion**

The current standards of periodontal disease for the assessment of periodontitis is based primarily upon attachment and bone loss, and classifies the disease into four stages based on severity (I, II, III or IV) and three grades based on disease susceptibility (A, B or C). Therefore, is possible to create a staging and grading system for periodontitis [34].

Complete radiographic examination represents a part of the initial periodontal assessment for establishing the degree of horizontal and vertical alveolar bone loss. According to the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions, a new periodontitis classification categorizes the disease based on a multi-dimensional staging and grading system [6,7]. Staging is determined by the severity of the disease at initial presentation and the complexity of disease management [35]. Wandawa et al. [36] applied 16 HBOT meetings after SRP, and their results were not significant from a statistical point of view. Soranta et al. [37] studied the action of HBOT on MMP-8 level in the saliva of chronic periodontitis patients. They applied 8 HBOT sessions, and observed that the results were meaningfully better than in monotherapy with SRP. The study of Robo et al. [38] and Chen et al. [39] proved that HBOT meaningfully decreases the anaerobic flora in subgingival sulcus. In their research, Balestra et al. [40] consider that studies in reference with HBOT should be extended, because this therapy can produce a strong stimulus at the level of the molecular reactions, but the requirements are in reference with "how much", "how long", and "how often" should this adjuvant therapy be used.

HBOT has a triggering role in bone remodelling, and the research effectuated by Lu et al. [41] demonstrated that the major impact of oxygen pressure is at the incipient phase of differentiation of osteoblasts. Salmón-González et al. [42] consider that there is a correlation between increasing oxygen pressure and increasing osteoblastic and osteoclastic activity. Studies of Huang et al. [43] mention that HBOT stimulates the fibroblast activity, the angiogenesis, and proceeds on leukocyte function for promoting lesion healing. In the

article published by El-Baz et al. [44], it is highlighted that HBOT is a treatment that has become quite popular in the community of autistic patients and that this type of therapy has many benefits. In the research conducted by Bennett et al. [45], it is emphasized that there was some evidence of HBOT efficacy in the treatment of acute migraine, although study participants belonged to an unselected population. Devaraj et al. [46] believe that although HBOT has broad indications in various medical cases, the effective use of this type of dental treatment requires evidence, so research should also be undertaken in the field of dentistry to develop adjunctive therapy options with hyperbaric oxygen.

During the Tenth European Consensus Conference on Hyperbaric Medicine, recommendations for accepted and non-accepted clinical indications and practice of HBOT have been established [47]. In certain situations, it is necessary to assess the cost–benefit ratio, especially when patients pay full treatment or in case of presumably long treatments.

HBOT can be used as a monotherapy, or as a multimodal therapeutic variant. After a study published in 2017, Chhabra et al. [48] concluded, that ensuring the local hyperbaric oxygen atmosphere, the administration of growth factors, skin-substitutes, electric stimulation and local drainage, may constitute the conditions for local wound healing, a fact which may be applicable in the treatment of periodontal diseases. Marcinkowska et al. [49] consider that through properly addressing and evaluating methodological issues referring to HBOT, this therapy may have potential for the treatment of neuropsychological deficits in a wide range of neurological states, with importance in the treatment of trigeminal nerve affections.

HBOT as a preventive therapy may diminish the peril of dental implant failures in the maxillofacial area, including the irradiated patients [50,51]. The research of Hollander et al. [52], show that HBOT could be beneficial in nonirradiated patients with intraoral compromised wound healing. The healing of wounds after application of HBOT is underlined by Re et al. [21], Hollander et al. [52], and Shih et al. [53], especially in periodontal disease and oral submucous fibrosis. HBOT as adjuvant therapy in dentistry, associated with the other specific dental treatments, has benefits and facilitates the healing process, notwithstanding the potential complications that may appear [21,54].

According to the safety requirements of patients, HBO therapy should not be accepted to those with general medical conditions presented in exclusion criteria of patients. Adverse effects and complications that may arise during HBO therapy can be absolute, relative or potential [55,56]. The most common complications during HBO therapy are represented by middle and inner ear barotrauma, pulmonary barotrauma, sinus/paranasal and dental barotrauma, claustrophobia, and ophthalmological manifestations (as progressive myopia, new cataracts) [56–58]. Potential contraindications of HBOT are represented by the presence of pacemakers or any implantable devices, hereditary spherocytosis, pregnancy, hypoglycaemia, chronic obstructive pulmonary disease, allergic rhinitis, asthma, upper respiratory infections, and acute pulmonary edema. In defiance to its numerous uses, potential adverse effects of HBOT, which represent a possible hazard for patients, should be taken into account. All these impose that this knowledge referring to the complications and adverse effects of HBO therapy should be presented in the informed consent [59,60].

Medical conditions along with the comprehension of necessity in preservation of dental and gingival health represent the potential predictable of general and oral health status [61]. Diseases of the periodontal tissues also affect the tooth mobility degree [2,62]. The mobility of the teeth represents a utilized symptom in the evaluation of the health status of periodontal tissues, respectively, in obtaining success or failure of the periodontal treatment [35,63]. The extension of periodontal disease cannot be correctly estimated without the evaluation of the mobility degree [64]. The used clinical method for determining the tooth mobility is based on individual's perception of tooth movement by application of a force on the tooth crown [65].

The acquaintances regarding the periodontal disease are significant for the prevention, and also for the preservation of the periodontal tissues health, in order to prevent severe

subsequent disease. Incorrect oral hygiene induces the triggering of oral cavity tissue diseases, in particular of periodontal affections [5,66].

Future research directions will be correlated with the inclusion of a larger number of patients in clinical trial, longer duration of follow-up, thoroughgoing study of HBOT effects in severe periodontitis, respectively, by the enlargement of the researched items (such as patients which presents gingival recession, TMJ disorders).

The absence of assessments referring to severe periodontitis represents a limitation of this study. Another limitation of this clinical trial is represented by the relatively reduced number of patients in groups. The cost of HBO therapy is rather high, and represents another limitative reason. Additionally, because the price of HBO therapy in oral diseases is not discounted by the Romanian health care insurance system, the implementation of HBO therapy in real life presents difficulties.

#### **5. Conclusions**

Within the limits of the study, we concluded that the HBOT group of patients presented better values of OHI-S, SBI, DM and PD two months after the completion of HBO adjuvant therapy than patients in the second control group, but Chi-Square test *p*-values for test differences between groups, for all variables, at all the three levels of investigation, with *p*-values shown that there are no significant differences, at any level, between the frequencies of the control and test groups, for any of the observed variables, no matter the treatment type.

The majority of the patients of the first group declared that HBOT had beneficial effects on their general health status in symptoms prior to this adjuvant therapy, as tinnitus, vertigo, chronic fatigue, and migraines, but it is necessary to emphasise that all these are subjective statements.

Clinical trials with a greater the number of patients and longer follow-up time are required.

**Author Contributions:** Conceptualization, A.B. (Anamaria Bechir) and E.S.B.; methodology, L.L.M. and A.B. (Anamaria Bechir); software, M.S. and E.S.B.; validation, A.B. (Alexandru Burcea) and A.B. (Anamaria Bechir); investigation, A.B., A.B. (Alexandru Burcea), L.L.M. and E.S.B.; resources, M.S. and E.S.B.; original draft preparation, A.B. (Alexandru Burcea) and E.S.B.; review and editing, A.B. (Anamaria Bechir) and L.L.M.; supervision, A.B. (Alexandru Burcea) and A.B. (Anamaria Bechir). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Dental Medicine Faculty, Titu Maiorescu University of Bucharest (No. 2/5 May 2017).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The material, technical and administrative support for HBO therapy was covered by Biobarica Hypermed Care Clinic.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Hyperbaric Oxygen Therapy with Iloprost Improves Digit Salvage in Severe Frostbite Compared to Iloprost Alone**

**Marie-Anne Magnan 1,\* , Angèle Gayet-Ageron <sup>2</sup> , Pierre Louge <sup>1</sup> , Frederic Champly <sup>3</sup> , Thierry Joffre <sup>4</sup> , Christian Lovis <sup>5</sup> and Rodrigue Pignel <sup>1</sup>**


**Abstract:** *Background and Objectives*: Frostbite is a freezing injury that can lead to amputation. Current treatments include tissue rewarming followed by thrombolytic or vasodilators. Hyperbaric oxygen (HBO) therapy might decrease the rate of amputation by increasing cellular oxygen availability to the damaged tissues. The SOS-Frostbite study was implemented in a cross-border program among the hyperbaric centers of Geneva, Lyon, and the Mont-Blanc hospitals. The objective was to assess the efficacy of HBO + iloprost among patients with severe frostbite. *Materials and Methods:* We conducted a multicenter prospective single-arm study from 2013 to 2019. All patients received early HBO in addition to standard care with iloprost. Outcomes were compared to a historical cohort in which all patients received iloprost alone between 2000 and 2012. Inclusion criteria were stage 3 or 4 frostbite and initiation of medical care <72 h from frostbite injury. Outcomes were the number of preserved segments and the rate of amputated segments. *Results*: Thirty patients from the historical cohort were eligible and satisfied the inclusion criteria, and 28 patients were prospectively included. The number of preserved segments per patient was significantly higher in the prospective cohort (mean 13 ± SD, 10) compared to the historical group (6 ± 5, *p* = 0.006); the odds ratio was significantly higher by 45-fold (95%CI: 6-335, *p* < 0.001) in the prospective cohort compared to the historical cohort after adjustment for age and delay between signs of freezing and treatment start. *Conclusions*: This study demonstrates that the combination of HBO and iloprost was associated with higher benefit in patients with severe frostbite. The number of preserved segments was two-fold higher in the prospective cohort compared to the historical group (mean of 13 preserved segments vs. 6), and the reduction of amputation was greater in patients treated by HBO + iloprost compared with the iloprost only.

**Keywords:** frostbite; classification; hyperbaric oxygen therapy; cold disease; prognosis; amputation; medical outcome

## **1. Introduction**

Frostbite is an injury caused by freezing of the skin and underlying tissues. Severe frostbite is a relatively uncommon event that can lead to early arthritis, tissue loss, or amputation. Frostbite comprises on average 2% of mountain emergencies in the western Alps [1]. Frostbite takes place in three phases: pre-freeze/freezing, thawing/rewarming, and mummification.

**Citation:** Magnan, M.-A.; Gayet-Ageron, A.; Louge, P.; Champly, F.; Joffre, T.; Lovis, C.; Pignel, R. Hyperbaric Oxygen Therapy with Iloprost Improves Digit Salvage in Severe Frostbite Compared to Iloprost Alone. *Medicina* **2021**, *57*, 1284. https://doi.org/10.3390/ medicina57111284

Academic Editors: Costantino Balestra and Jacek Kot

Received: 21 October 2021 Accepted: 8 November 2021 Published: 22 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Pre/freeze is an acute ischemia with peripheral vasoconstriction. During freezing, cell death is triggered by intracellular dehydration and direct damage to cell membrane by ice microcrystals. Thawing is best accomplished by the immersion of frozen limbs in warm water. After blood flow is restored, cyanotic lesions can occur. During rewarming, there is a vascular stasis with a prothrombotic environment (hypoxia and acidosis), interstitial edema, and ischemia–reperfusion injuries. It leads to the destruction of microcirculation and cell death [2,3]. Frostbite outcome is related to the initial cyanotic lesion. The Cauchy classification defines four grades that predict the amputation risk after rapid thawing in warm water when there is no targeted frostbite care [3]. It is based on the extent of the initial cyanotic lesion. Frostbite is classified as grade 1 if cyanosis disappears, grade 2 if only distal phalanges are cyanotic (amputation risk below 1%), grade 3 if cyanosis involves the intermediate or proximal phalanges (amputation risk: 30–83% greater in the hands than feet), and grade 4 if cyanosis involves the metacarpals or metatarsals (amputation risk: 99%) [3] (Figure 1).

**Figure 1.** The frostbite classification by E. Cauchy (*drawings@copyright ifremmont*).

The goal of treatment is to limit tissue damage from hypoxia and acidosis, mitigate the subsequent prothrombotic cascade, reduce edema and the inflammatory response, and minimize the impact of the ischemic–reperfusion syndrome. Prior studies have demonstrated the efficacy of thrombolytics such as recombinant tissue plasminogen activator (rt-PA) [4]

and vasodilator such as iloprost [5–7] in improving outcome [8]; medical care must be initiated within 24 h for rt-PA [9,10] and 48 h for iloprost [11]. Currently, the Wilderness Medical Society guidelines do not recommend HBO treatment for frostbite [8]. However, HBO may improve frostbite outcome by increasing the cellular oxygen availability to the damaged tissues. This may help to mitigate the negative impact of the inflammatory cascade and the ischaemia–reperfusion syndrome [12] . Few case reports suggest that HBO might improve frostbite injury outcome [13–23]. There are no randomized controlled trials (RCT) with HBO conducted so far. It is arduous to carry out a double-blinded RCT for HBO because frostbite is uncommon, and blinding subjects to HBO or not HBO could be difficult.

We implemented a cross-border European program (INTERREG-IV FRANCE-SUISSE) to foster and coordinate the care management of patients who suffer frostbite in the French and Swiss Alps.

#### **2. Materials and Methods**

#### *2.1. Study Oversight*

The SOS-Frostbite research program was a multicenter prospective, non-randomized study from 2013 to 2019. The study was conducted by the hyperbaric centers of Geneva and Lyon, and the Mont-Blanc hospitals in Chamonix and Sallanches. The statistical analysis was performed independently by the unit of methodological support from the CTU of Geneva University Hospital. The study aim was to assess whether the early addition of HBO to standard care with iloprost (prospective group) was associated with better frostbite outcomes compared to standard care alone (retrospective group).

#### *2.2. Setting and Participants*

Patients were eligible for the SOS-Frostbite protocol after screening determined no contraindication to aspirin, iloprost, or HBO. The inclusion criteria for both groups were grade 3 or 4 frostbite according to the Cauchy classification [3] and start of medical care within 72 h from frostbite injury, which was defined previously in the historical cohort as the onset of frostbite. Physicians involved in the study systematically searched for the onset of loss of sensitivity in the fingers or toes through the medical history to determine this time period.

To identify the historical cohort, we retrospectively collected data of all frostbite medical files treated at the Mont-Blanc hospital from 2000 to 2012. Before 2000, as the Cauchy classification had not yet been established, no patients could be included. All eligible patients who met the inclusion criteria from the retrospective analysis were included in the historical cohort. They were all grade 3 or 4 frostbitten patients who received a standardized protocol including iloprost, which was initiated no longer than 72 h from frostbite injury.

The standardized frostbite treatment: frostbitten extremities were rewarmed by immersion in warm water (38 ◦C) for 60 min, and patients were given aspirin 250 mg orally. During the hour following the rewarming, the frostbite classification was determined. Grade 3 or 4 frostbite patients received the first iloprost infusion immediately (by infusion pump, 8–10 mcg/h for 6 h, 48–60 mcg/day). Patients were hospitalized for 7 days to continue daily iloprost (by infusion pump, 8 to 10 mcg/h for 6 h, 48–60 mcg/day), aspirin (250 mg/day; orally), antibiotics (amoxicillin/clavulanate: 1 g/125 mg 3 times daily, orally for 7 days), and daily wound care with topical hyaluronic acid.

To identify the SOS-Frostbite group, data were prospectively collected from patients satisfying inclusion criteria who received the same standardized frostbite treatment protocol plus early HBO from 2013 to 2019.

The SOS Frostbite protocol: The SOS-Frostbite protocol was initiated upon hospital arrival. Patients were treated with the same standardized protocol as the historical cohort with the addition of HBO. The first HBO (150 min at 2.5ATA) session was done as soon as possible after the first iloprost infusion (from 1 to 6 h after the end of the iloprost infusion,

as some patients were transferred from other hospitals to the Geneva or Lyon hyperbaric chamber for HBO). Then, patients were hospitalized for 7 days and received the same treatment protocol as in the historical cohort plus HBO sessions (150 min, 2.5 ATA, 1 daily) (Appendix A, Figure A1). After hospital discharge, the patient completed daily HBO sessions for 7 additional days (14 HBO session in total). Hyperbaric chambers involved in the study used multiplace chambers and patients breathed oxygen via a mask or a hood.

#### *2.3. The Follow-Up*

A Technecium 99 (Tc99) bone scan was performed at day 3 and day 7 (control group and prospective cohort). Results were considered pathological when the bone scan demonstrated absent or markedly decreased uptake of the Tc99 tracer in the bone tissue (severe bone ischemia). An additional Tc 99 bone scan was conducted at the end of the HBO sessions if radiological improvement (recovery of bone activity) was identified on the day 7 Tc99 bone scan compared to the day 3 Tc99 bone scan. All patients had a clinical examination at 6 months, 1 year. Patients enrolled in the first 4 years of the study also had a follow-up at 2 years and 3 years to evaluate early and delayed sequelae such as arthritis.

#### *2.4. Outcomes*

The study's primary outcome was the number of preserved segments at 12 months, which was defined as the difference between the number of segments with frostbite after rewarming and lost segments. Each phalanx and each metacarpal or metatarsal is defined as a segment; 4 segments comprise a ray (3 segments for the thumb or the hallux), and 3 out 4 segments make a digit (2 out 3 segments make the digit for the thumb or the hallux). To align with the eligibility criteria regarding frostbite severity (grade 3 or 4), we only considered rays with at least 2 segments damaged. The secondary outcomes were the number of amputated segments at 12 months and the ratio of the number of amputated segments at 12 months divided by the number of segments with initial frostbite injury.

#### *2.5. Data Collection*

All data from the prospective and the historical cohorts were collected on site using a standardized case report form. All observations were coded to preserve patient anonymity and data confidentiality.

#### *2.6. Statistical Analysis*

There was no preliminary estimation of study sample size; we used all available data on 31 December 2019 and obtained a fixed sample size of 58 patients. In the control group, we described 6 (mean ± SD, 5.3) preserved segments at 12 months post-treatment. We had 80% power to detect a two-fold increase in the number of preserved segments (+6) in the standard care plus HBO group, considering a larger variability of the difference of number of preserved segments (±10).

Continuous variables were reported as mean ± SD, median, and interquartile range. Categorical variables are reported as frequencies and percentages. We compared two cohorts of patients: those included between 2000 and 2012 (historical cohort) and those included after 2013 (prospective cohort). We compared continuous variables between the two cohorts of patients with the use of nonparametric Mann–Whitney test, as we anticipated that continuous variables are non-normally distributed and do not respect the assumptions for using Student's *t*-test; we compared categorical variables between the two cohorts with the use of chi-square or Fisher's exact tests, depending on assumptions, and *p*-values of less than 0.05 were considered to indicate statistical significance. Since the main outcome (number of preserved segments) was an ordinal variable (0, 1, 2, 3, and 4 preserved segments) and because one patient could have several data points for the main outcome (repeated measurements), we performed mixed ordinal logistic regressions with the patient identifier as a random factor. We compared the main outcome between the two cohorts of patients (HBO plus standard care vs. standard care alone). We adjusted the

analysis for patient age, delay between signs of freezing and medical treatment received (<6 h, 6–12 h, 12–24 h, 24–48 h, and 48–72 h). For secondary outcome, we also performed mixed ordinal logistic regressions models as the number of amputations was also ordinal (3–4, 2, 1, 0 amputation), and we also adjusted the analysis for patient age and the delay between signs of freezing and medical treatment received. All analyses were performed with the use of STATA 16 IC (StatCorp, College Station, TX, USA).

#### **3. Results**

*3.1. Description*

3.1.1. Patients

The prospective cohort: Thirty-nine patients with grade 3 or 4 frostbite were treated from 2013 to 2019 with the SOS-Frostbite protocol; 11 patients were excluded because medical care delay was over 72 h from frostbite injury or the treatment protocol was interrupted or changed. For statistical analysis, 28 patients were prospectively included in the SOS-Frostbite group. None of the patients from the prospective cohort suffered from HBO side effects.

The retrospective cohort (control group): After reviewing all frostbite medical files in the Mont-Blanc hospitals (168 medical files), 30 patients met the inclusion criteria (standardized frostbite treatment with iloprost, grade 3 or 4 frostbite and medical care initiated within 72 h from frostbite injury) (Figure 2).

**Figure 2.** Study flow chart.

The SOS-Frostbite group and the historical control group both consisted of a similar number of patients with identical inclusion criteria.

The comparison of patient characteristics is presented in Table 1. Percentages of patients with delays of 12 to 24 h or 24 to 48 h were more frequent in the prospective cohort compared to the historical cohort. Patients were significantly older in the prospective than in the historical cohort. A higher proportion of patients with three or four segments with frostbite were observed in the prospective cohort compared to the control group (*p* < 0.001).


**Table 1.** Description of patients included in the study (*n* = study), the number of preserved digits, and the number of amputated segments.

\* Mann–Whitney nonparametric test; \*\* Fisher's exact test; \*\*\* Chi-square test.

#### 3.1.2. Outcomes

A significantly higher mean number of preserved segments per patient was observed in the prospective SOS-Frostbite group (13 SD ± 10) compared to the historical control group (6 SD ± 5) (*p* = 0.006). In the prospective cohort, 57% of patients had three to four preserved segments (respectively 43% for three segments and 14% for four segments) compared to 13% in the control group (respectively 13% for three segments and 0% for four segments). (*p* < 0.001, Table 2). At baseline, a higher but not statistically significant number of frostbitten segments was observed in the prospective than in the control group. However, a significantly higher number of frostbitten amputated segments was observed in the control than in the prospective group (*p* = 0.014, Table 2).

The odds ratio of the number of preserved segments was significantly higher by 20-fold (95%CI: 4-101, *p* < 0.001) in the prospective group who received standard care plus HBO compared to the control group (Table 3, model 1). This association remained after adjustment for patient age and delay between signs of freezing and medical treatment start (Table 3, model 2).

The association between the treatment received (cohort group) and a lower number of amputated segments was assessed. The odds of fewer amputated segments were significantly higher in the prospective group with standard care plus HBO compared to the control group with standard care alone (odds ratio 0.015; 95% CI: 0.0009; 0.25, *p* = 0.003). This association was reinforced after adjustment for patient age and delay between signs of freezing and onset of medical treatment, but due to very small numbers, the imprecision of the estimates was very large (odds ratio 0.0004; 95% CI: 0.00003; 0.06, *p* = 0.002).


**Table 2.** Comparison of outcomes between retrospective and prospective cohort studies.

\* Mann–Whitney nonparametric test. \*\* Mixed ordinal logistic regression model with number of preserved digits coded as 0, 1, 2, 3, and 4 (five categories) as the dependent variable and group as the independent variable. \*\*\* Mixed logistic regression model with beam with frostbite (yes/no) as the dependent variable and group as the independent variable among observations with at least one segment with frostbite. \*\*\*\* Fisher's exact test.


**Table 3.** Association between treatment group and study outcome, univariate and multivariate analyses.

If we consider the ratio of segment amputation to all injured segments, a higher proportion of patients with one-third, half, two-thirds, or the total of segments amputated in the control group were observed compared to the standard care plus HBO group after 1-year follow-up (Table 2).

#### **4. Discussion**

This observational study is the first published prospective study reporting data on severe frostbite treated by early HBO.

In this study, HBO is a positive adjunct to treatment with iloprost. When started within 48 h from injury, iloprost can increase the segment salvage rate up to 78% in severe frostbite [24]. Iloprost has the highest recommendation level in frostbite treatment [8] and should be considered on grade 3 or 4 frostbites when rt-PA is contraindicated or is used in the field. Frostbite treatment with iloprost is strongly recommended, as it decreases the risk of amputation; HBO further improves segment salvage even if initiated after 48 h from frostbite injury.

This study did not compare the combined effect of thrombolytics and HBO. Thrombolytics are another recommended treatment that can lower the amputation rate from 41% to 10% when done within 24 h from frostbite injury [4]; a risk–benefit analysis should always be performed regarding bleeding risk and all contraindication to the treatment.

HBO is a non-invasive treatment; side effects are self-limiting and can mostly be avoided with appropriate screening [25]. In appropriate indications, the benefits of HBO frequently outweigh the risks. The US Food and Drug administration approved HBO for the treatment of acute ischemia, whereas iloprost has not yet been approved for such treatment. It can be performed on some people with contraindication to rt-PA due to the bleeding risk or in children. When available, HBO may be considered as an alternative treatment when there are contraindications to iloprost or thrombolytics. In our study, we showed that HBO plus standard care including iloprost significantly reduced the amputation risk even over 48h from frostbite injury.

The physiological mechanism of HBO action is well known [12–23], but there are no previous randomized controlled trials conducted to evaluate the added value of HBO on frostbite injury outcomes. Regarding frostbite physiopathology, there are good reasons as to why HBO could improve frostbite injury outcomes. HBO has a direct action on tissue ischemia, increasing dissolved oxygen and improving oxygen transportation in the blood. The HBO decreases blood viscosity and minimizes the inflammatory cascade. There is a hyperoxic vasoconstriction in the micro vascularization of healthy tissues, inducing a redistribution of blood to hypoxic territories. Those effects of HBO on vasoconstriction decrease edema and the incidence of compartment syndrome. There is a reduction of the deleterious influences of ischemia–reperfusion [12,26,27] besides diminishing damages due to the thaw–rewarming phase; HBO has an anti-infective activity due to its bactericidal effect on anaerobic germs and bacteriostatic action on aerobic germs so it can prevent infection during the mummification phase [12,28]. Finally, when repeated every day, HBO sessions induce vascular endothelium growth factor activation, fibroblast and collagen production, and thus the progression toward the resolution of tissue damage. HBO promotes the formation of the healing sulcus between necrotic and healthy tissues [12,28]. These clinical effects were described in recent retrospective studies [13].

Regarding the longer delay for medical care in the prospective cohort, the second aim of this INTERREG project was to set up a network for severe frostbite management. A SOS-Frostbite call center has been created. Some patients have been repatriated from far away to benefit from this research protocol, which could explain the longer delay for medical care from frostbite injury in the SOS-Frostbite group. Despite the longer delay for medical care in the SOS-Frostbite group, segment salvage was still significantly improved.

#### **5. Conclusions**

The SOS-Frostbite program is the first controlled prospective study that evaluates the effect of early HBO additive to iloprost on severe frostbite. Results show more favorable outcome in terms of the functionality and quality of life in patients treated by HBO: HBO added to the standard care with iloprost might improve frostbite injury outcomes by doubling the chance to preserve the number of injured segments from amputation.

Moreover, the benefits of HBO frequently outweigh the risks as contraindications and side effects are limited, in comparison to standard treatments such as rt-PA and iloprost. Transferring the patient suffering from severe frostbite to a hyperbaric center could be considered even if it implies delayed HBO, as it still improves frostbite outcomes after 48 h. Our findings should be tested in a randomized controlled trial before concluding that HBO should be added to standard care of severe frostbite in patients receiving iloprost.

#### **6. Patents**

The decision to design a prospective single arm study instead of two-arm randomized study was made because severe frostbite is an infrequent event [1,2]. We collected data on a small sample of 28 patients prospectively and compared the prospective cohort with data from a retrospective cohort from a previous double blinded RCT [5]. In both series, patients were mostly healthy, had little comorbidity, and had good access to medical care. Frostbite also occurs secondary to occupational exposure and in the homeless and migrant populations. The prognosis and outcome of frostbite for members of socially disadvantaged groups is likely much more severe. The fact our patients were healthy was an advantage, as frostbite was the only injury studied, inducing less bias from other pathologies. The Lyon hyperbaric site was more focused on the treatment of occupational accidents and injuries sustained by homeless patients. These patients were often hospitalized on medical services to treat comorbid conditions with an unfortunate delay in frostbite treatment. These patients were excluded if frostbite treatment was not initiated with 72 h.

Our study was not a randomized controlled trial. We tried to minimize selection and information biases using strict eligibility criteria. The allocation of the treatment group was not at random in our study, but we prespecified a list of criteria to select patients with very similar characteristics in this observational study in order to allow an unbiased comparison of treatment effects between the two treatment groups.

The two groups have a comparable number of patients, but those from the prospective group were older, had more severe frostbite, and the medical care delay was longer in comparison with the control group.

Another hypothesis is that HBO might prevent other side effects such as early arthritis by augmenting the healing process. It is still too early to present data, and it will not be possible to compare data with the historical cohort as there was no long-term follow up over 12 months.

**Author Contributions:** Conceptualization M.-A.M., R.P. and F.C.; methodology M.-A.M., R.P., P.L. and F.C.; software: C.L.; validation, M.-A.M., A.G.-A., P.L., F.C., T.J., C.L. and R.P.; formal analysis, A.G.-A.; investigation M.-A.M., P.L., F.C. and T.J.; data curation R.P. and M.-A.M.; writing—original draft preparation M.-A.M. and A.G.-A.; writing—review and editing, M.-A.M., A.G.-A., P.L. and R.P.; visualization M.-A.M., R.P. and P.L.; supervision M.-A.M. and R.P.; project administration: M.-A.M.; funding acquisition M.-A.M., R.P. and F.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** The SOS-Frostbite program had financial support by the Interreg IV France-Switzerland and the Swiss Confederation. The Interreg committee and the Swiss Confederation had no influence on the design and the conduct of the trial and were not involved in data collection or analysis in writing of or submitting the manuscript. There was no commercial support for this study.

**Institutional Review Board Statement:** This trial was conducted according to the guidelines of the Declaration of Helsinki and was approved by the institutional review board at the University of Geneva and the French Committee on the Protection of the Persons in Biomedical Research (CCPPRB) approved the study protocol (protocol code 14-053 on 14 October 2015).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study and written informed consent form has been obtained from the patient(s) to publish this paper. Each patient received oral and written information about the treatment and signed a written consent form.

**Data Availability Statement:** All data from the prospective and the historical cohorts were collected on site using a standardized case report form in the international frostbite registry. All observations were coded to preserve patient anonymity and data confidentiality.

**Acknowledgments:** We honor the memory of Emmanuel Cauchy, who unfortunately died in an avalanche in 2018. He was a medical doctor specialized in mountain medicine and a mountain guide. He initiated the SOS-Frostbite program in 2013. We thank the INTERREG France-Suisse IV program and the Swiss Confederation for its financial support.

**Conflicts of Interest:** The authors declare no conflict of interest. The Interreg committee and the Swiss Confederation had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

## **Appendix A**

**Figure A1.** The SOS-Frostbite protocol during hospitalization at university hospitals of Geneva.

## **References**


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