**A Systematic Review to Assess the Impact of Hyperbaric Oxygen Therapy on Glycaemia in People with Diabetes Mellitus**

**Sudhanshu Baitule 1,\* ,†, Aaran H. Patel 1,2,\* ,† , Narasimha Murthy 1,2, Sailesh Sankar 1,2, Ioannis Kyrou 1,2,3,4 , Asad Ali 1,2, Harpal S. Randeva 1,2 and Tim Robbins 1,2,3,5,\***


**Abstract:** *Background and Objectives*: Hyperbaric oxygen is a recognised treatment for a range of medical conditions, including treatment of diabetic foot disease. A number of studies have reported an impact of hyperbaric oxygen treatment on glycaemic control in patients undergoing treatment for diabetic foot disease. There has been no systematic review considering the impact of hyperbaric oxygen on glycaemia in people with diabetes. *Materials and Methods*: A prospectively PROSPEROregistered (PROSPERO registration: CRD42021255528) systematic review of eligible studies published in English in the PUBMED, MEDLINE, and EMBASE databases, based on the following search terms: hyperbaric oxygen therapy, HBO2, hyperbaric oxygenation, glycaemic control, diabetes, diabetes Mellitus, diabetic, HbA1c. Data extraction to pre-determined piloted data collection form, with individual assessment of bias. *Results*: In total, 10 eligible publications were identified after screening. Of these, six articles reported a statistically significant reduction in blood glucose from hyperbaric oxygen treatment, while two articles reported a statistically significant increase in peripheral insulin sensitivity. Two articles also identified a statistically significant reduction in HbA1c following hyperbaric oxygen treatment. *Conclusions*: There is emerging evidence suggesting a reduction in glycaemia following hyperbaric oxygen treatment in patients with diabetes mellitus, but the existing studies are in relatively small cohorts and potentially underpowered. Additional large prospective clinical trials are required to understand the precise impact of hyperbaric oxygen treatment on glycaemia for people with diabetes mellitus.

**Keywords:** diabetes; hyperbaric oxygen therapy; glycaemia

#### **1. Introduction**

The use of hyperbaric oxygen in treating decompression sickness in deep-sea divers and people with carbon monoxide poisoning is well-established [1]. Hyperbaric oxygen therapy (HBOT) is also an approved medical treatment for various conditions including necrotizing soft tissue infection, diabetic wounds, osteomyelitis, compartment syndrome, crush and reperfusion injuries, and acute sensorineural hearing loss [1]. HBOT has been postulated to have a positive impact on diabetic foot ulcers, suggesting its incorporation as an adjunct treatment with further scope for research in this area [2,3].

**Citation:** Baitule, S.; Patel, A.H.; Murthy, N.; Sankar, S.; Kyrou, I.; Ali, A.; Randeva, H.S.; Robbins, T. A Systematic Review to Assess the Impact of Hyperbaric Oxygen Therapy on Glycaemia in People with Diabetes Mellitus. *Medicina* **2021**, *57*, 1134. https://doi.org/10.3390/ medicina57101134

Academic Editors: Costantino Balestra, Jacek Kot and Enrico Camporesi

Received: 6 September 2021 Accepted: 16 October 2021 Published: 19 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/).

HBOT involves oxygen delivery at a concentration of 100% with a pressure of 2 to 3 atmosphere absolute (ATA) in a hyperbaric chamber. The mechanism of HBOT is to increase tissue oxygen levels resulting in accelerated wound healing, decreased oedema, and killing of anaerobic bacteria [4,5]. postulated to have a positive impact on diabetic foot ulcers, suggesting its incorporation as an adjunct treatment with further scope for research in this area [2,3]. HBOT involves oxygen delivery at a concentration of 100% with a pressure of 2 to 3 atmosphere absolute (ATA) in a hyperbaric chamber. The mechanism of HBOT is to in-

*Medicina* **2021**, *57*, x FOR PEER REVIEW 2 of 13

in the potential impact of HBOT on glycaemia in diabetes.

In diabetes, meticulous glycaemic control has been shown to reduce the risk of microvascular, macrovascular, and neurological complications [6,7]. There is emerging evidence demonstrating blood glucose level changes in people with diabetes undergoing hyperbaric oxygen treatment [8–12]. However, these studies have involved diverse methodologies, whilst, to date, there has been no systematic review of the impact of HBOT on glycaemia in people with diabetes. crease tissue oxygen levels resulting in accelerated wound healing, decreased oedema, and killing of anaerobic bacteria [4,5]. In diabetes, meticulous glycaemic control has been shown to reduce the risk of microvascular, macrovascular, and neurological complications [6,7]. There is emerging evidence demonstrating blood glucose level changes in people with diabetes undergoing hyperbaric oxygen treatment [8–12]. However, these studies have involved diverse methodologies, whilst, to date, there has been no systematic review of the impact of HBOT on

Here, we present the first systematic review considering the effect of HBOT on the glycaemia in people with diabetes. We also explore the proposed mechanisms involved in the potential impact of HBOT on glycaemia in diabetes. glycaemia in people with diabetes. Here, we present the first systematic review considering the effect of HBOT on the glycaemia in people with diabetes. We also explore the proposed mechanisms involved

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

The systematic review was performed according to the PRISMA protocol as shown in Figure 1 [13]. The review was prospectively registered on the NIHR PROSPERO Database (PROSPERO registration ID: CRD42021255528). **2. Materials and Methods**  The systematic review was performed according to the PRISMA protocol as shown in Figure 1 [13]. The review was prospectively registered on the NIHR PROSPERO Database (PROSPERO registration ID: CRD42021255528).

**Figure 1.** Diagram demonstrating the search strategy used according to the PRISMA protocol [13]. **Figure 1.** Diagram demonstrating the search strategy used according to the PRISMA protocol [13].

#### *2.1. Study Selection 2.1. Study Selection*

The literature search was conducted in the PUBMED, MEDLINE, and EMBASE databases. The search terms used to identify the relevant medical literature were Hyperbaric oxygen therapy/HBO2/hyperbaric oxygenation; Glycaemic control; Diabetes/diabetic/diabetes mellitus; HbA1c. The search strategies used are detailed in the Appendix A (Tables A1–A3). Only studies involving humans which were published in English language journals were considered eligible, with no restriction to the publication date. Furthermore, filters were applied to set participant's age as 18 years and above, as this research looked at only the adult population with any type of diabetes (excluding diabetes insipidus) who The literature search was conducted in the PUBMED, MEDLINE, and EMBASE databases. The search terms used to identify the relevant medical literature were Hyperbaric oxygen therapy/HBO2/hyperbaric oxygenation; Glycaemic control; Diabetes/diabetic/diabetes mellitus; HbA1c. The search strategies used are detailed in the Appendix A(Tables A1–A3). Only studies involving humans whichwere published in English language journals were considered eligible, with no restriction to the publication date. Furthermore, filters were applied to set participant's age as 18 years and above, as this research looked at only the adult population with any type of diabetes (excluding diabetes insipidus) who had undergone HBOT. Any study that focused only on animals, children, or hyperbaric combination therapies was excluded. Any studies focusing on wound care and insulin sensitivity were also excluded. Studies focused on insulin sensitivity but mentioning glycaemia as an outcome in their abstracts were included.

#### *2.2. Data Extraction*

Data were extracted to a pre-defined, data-extraction proforma which was based on the following variables: year of publication, type of study, location of research and publication, sample size including the baseline characteristics of the population, any biases, single centre or multicentre study, length of follow up comprising of a number of session of interventions, statistical methods used for analysis showing the statistically significant outcome. Miscellaneous variables relevant to this systematic review were also extracted. Data extraction was performed independently by two authors (S.B. and A.P.), with any discrepancies resolved by a third author (T.R.).

#### *2.3. Quality Assessment*

All studies included in the review were assessed for study quality. Due to the small number of studies and diverse methodologies a single formal tool was not used. Instead, a narrative review was conducted for bias considering sample size, study methodology, any evidence of randomisation, and blinding. All studies were assessed independently for bias by two authors (S.B. and A.P.). Papers were not excluded based on producing a negative outcome or being of low-quality.

#### *2.4. Data Synthesis*

The diversity among the identified eligible studies, in terms of their study design, sample size and population, did not allow a meta-analysis to be conducted. A qualitative analysis and narrative summary of the studies reporting any change or any factors that pre-dispose to changes in HbA1c were performed. Where possible, these changes have then been grouped under broader categories in a tabulated form.

#### **3. Results**

The performed systematic search yielded 428 records. Of these, 208 were duplicates and, thus, were removed prior to screening of titles and abstracts. Of the 220 records screened, 11 articles were selected for a detailed review. One of these was excluded after detailed review as it was a letter to editor and not an original research article [10]. In total, 10 studies were eligible for inclusion in this systematic review, which were all available as full text articles. The designs and locations of these studies are summarised in Tables 1 and 2. The characteristics and findings of the individual eligible studies are summarised in Table 3.

**Table 1.** Summary of study designs included in this systematic review.


**Table 2.** Summary of locations (countries) of the studies included in this systematic review.






Of the included studies, seven were prospective studies in cohorts of patients with diabetes, two presented retrospective analyses of prospectively collected data, and one study was a randomised, prospective, placebo-controlled trial in patients with type 2 diabetes mellitus. Most of these studies included participants with diabetes mellitus who were receiving HBOT for various indications, including non-healing wounds, diabetic foot ulcers, radio-induced cystitis and neurological deficits such as sudden deafness.

The majority of the included studies demonstrated a reduction in blood glucose levels following a single session of HBOT in patients with type 2 diabetes mellitus. This effect was consistent across different session lengths and treatment conditions used in the different studies. A prospective cross-over study by Ekanayake & Doolette demonstrated that blood glucose levels in five patients with diabetes mellitus reduced following exposure to both hyperbaric and normobaric conditions, but this decrease only reached significance following exposure to HBOT for at least 45 min [12]. However, this study did not find a significant reduction in blood glucose levels in control subjects without diabetes mellitus in either condition. A significant reduction in blood glucose following a HBOT session in patients with diabetes mellitus was also shown in a prospective study conducted by Trytko & Bennett, which assessed mean blood glucose change across up to 10 consecutive HBOT sessions per participant [9]. This study analysed 226 HBOT sessions across 27 patients, and reported that there was a decrease in blood glucose levels in 80 of the 102 sessions which were in patients with type 2 diabetes mellitus. A prospective cohort study in 23 patients with diabetes mellitus by Al-Waili et al. also demonstrated significant reduction in blood glucose levels as a mean across 15–30 HBOT sessions per participant [19]. Peleg et al. also showed statistically significant decrease in blood glucose levels after a HBOT session in patients with type 2 diabetes mellitus [11], while no significant reduction in blood glucose levels was noted in healthy volunteers without diabetes following HBOT, agreeing with the earlier findings by Ekanayake & Doolette. Moreover, the study by Peleg et al. did not find any significant reduction in blood glucose levels in patients with type 1 diabetes mellitus following exposure to both hyperbaric and normobaric conditions. A retrospective review by Heyboer et al. also found a greater impact of HBOT in patients with type 2 diabetes mellitus as opposed to those with type 1 diabetes mellitus [8]. This retrospective review of prospectively collected data showed that blood glucose levels in patients with diabetes mellitus decreased in 75.4% of 1825 HBOT cycles surveyed. However, on further analysis, a statistically significant greater percentage of treatments of patients with type 2 diabetes mellitus resulted in a decrease in blood glucose levels (77.5%) compared to treatments of patients with type 1 diabetes mellitus (51.5%).

Contrary, the study by Stevens et al. does not support this general finding of a reduction of blood glucose in patients with type 2 diabetes mellitus following HBOT [17]. This retrospective review of prospectively collected data from 190 patients with diabetes mellitus found that in-chamber glucose was higher than pre-HBOT glucose in 54% of sessions. However, there is no evidence in this study of statistical analysis of change in blood glucose levels following HBOT.

The potential mechanism for a reduction in blood glucose levels caused by HBOT appears to be mediated by increased insulin sensitivity, as opposed to enhanced insulin secretion. The study by Ekanayake & Doolette measured insulin levels in patients with diabetes mellitus during both a single session under hyperbaric and normobaric conditions, and found no change in insulin levels following treatment in either condition [12]. This finding was also seen in the study by Wilkinson, Chapman & Heilbronn; demonstrating that there was no change in fasting insulin levels measured in five patients with type 2 diabetes mellitus even after 30 sessions of HBOT performed over five weeks [18]. This study also demonstrated a statistically significant increase in peripheral insulin sensitivity after both 3 and 30 sessions of HBOT measured using a hyperinsulinaemic clamp in those patients with type 2 diabetes mellitus. A further study by Xu et al. subjected 23 patients with type 2 diabetes mellitus to 30 sessions of either hyperbaric or normobaric conditions and assessed peripheral insulin sensitivity using hyperinsulinaemic-euglycaemic clamps [15]. This study

also demonstrated a significant increase in peripheral insulin sensitivity in patients with type 2 diabetes mellitus after 30 HBOT sessions, which was not seen in those exposed to normobaric conditions. However, this study also showed a significant decrease in insulin levels after 30 sessions in both HBOT and normobaric condition groups. This evidence further supports HBOT-induced increased insulin sensitivity as the proposed mechanism for reducing blood glucose levels in patients with type 2 diabetes mellitus.

The reduction in blood glucose levels in patients with type 2 diabetes mellitus attributed to HBOT appears to be longitudinal. The study by Xu et al. demonstrated a significant reduction in fasting plasma glucose after 30 sessions of HBOT [15]. A study by Vera-Cruz et al., which measured fasting plasma glucose and performed an oral glucose tolerance test (OGTT) at baseline and after 20 sessions of HBOT over four weeks, found that whilst fasting plasma glucose did not significantly decrease, there was a significant decrease in glycaemia following an OGTT after 20 sessions of HBOT in patients with type 2 diabetes mellitus [16]. Similarly, Wilkinson, Chapman & Heilbronn also showed no significant reduction in fasting plasma glucose after 30 sessions of HBOT [18].

HbA1c was used as an outcome measure in three studies following up participants after multiple sessions of HBOT. The study by Trytko & Bennett found that in 17 patients with type 2 diabetes mellitus who completed 10 sessions of HBOT, there was a small, non-significant reduction in HbA1c [9]. The study by Wilkinson, Chapman & Heilbronn also found no significant change in HbA1c after 30 sessions of HBOT [18]. However, the study by Xu et al. demonstrated a significant reduction in HbA1c after 30 sessions of HBOT, which was not seen in those exposed to normobaric conditions [16]. The study by Irawan et al. corroborates this finding of a reduction in HbA1c, with a significant reduction after 10 HBOT sessions [14].

Some studies suggest that the reduction in blood glucose levels in patients with type 2 diabetes mellitus following HBOT may be independent of the hyperbaric conditions. Peleg et al. found that there was a significant decrease in blood glucose for patients with type 2 diabetes after a session under normobaric control conditions [11]. Ekanayake & Doolette also identified a decrease in blood glucose levels following a session under normobaric conditions. However, this decrease did not reach significance [12]. Both of these studies had relatively small participant numbers and only considered the change in blood glucose after a single session. On the contrary, Xu et al. did not find any change in fasting plasma glucose or HbA1c after 30 sessions under control normobaric conditions [15]. This study by Xu et al. had a larger number of participants and considered the longitudinal impact on glycaemia after multiple sessions. The outcomes measured by Xu et al. could therefore be considered more reliable when considering the clinical utility of HBOT in type 2 diabetes mellitus as a treatment adjunct. However, these discrepancies highlight the need for further controlled trials with larger participant numbers to ascertain the true impact of HBOT on glycaemia in patients with type 2 diabetes mellitus compared to normobaric conditions.

Moreover, four of the studies do have a consideration for the incidence of hypoglycaemic events during or immediately after HBOT. The retrospective review by Heyboer et al. found that none of the patients with diabetes mellitus experienced a hypoglycaemic episode following a HBOT session [8]. However, the retrospective review by Stevens et al. found an incidence of 1.5% for hypoglycaemia during or immediately after HBOT in the 3136 sessions reviewed, but noted that severe or symptomatic hypoglycaemic events were rare [17]. The prospective study by Trytko & Bennett had symptomatic hypoglycaemia occur in 11 out of 237 HBOT treatments in patients with diabetes mellitus; only two of these occurring in patients not requiring insulin treatment [9]. Al-Waili et al. also reported occurrences of symptomatic hypoglycaemic episodes in two of the 41 study participants undergoing HBOT; one of these being an insulin-treated diabetes mellitus patient [19]. Patients with type 1 diabetes mellitus are at an increased risk of hypoglycaemic episodes with HBOT, as found in the study by Stevens et al. and suggested by the results of the Trytko & Bennett study [9,17]. A suggestion is made for a threshold blood glucose level below which the

risk of hypoglycaemia during HBOT is increased, with Al-Waili et al. noting this threshold level to be 120 mg/dL, whilst the data from Stevens et al. suggesting that this threshold blood glucose level is 150 mg/dL [17,19].

There are a number of sources of bias to consider when interpreting these studies. The most significant would be the presence of sampling bias. Moreover, most of the studies included for analysis in this review had relatively small sample sizes. Even those with large numbers of HBOT sessions often sourced these from a small number of participants. The small sample sizes used may reduce the reliability of the obtained results and the external validity of the reported findings. These small sample sizes may have also impacted the power of the studies to identify significant changes in glycaemic variables, such as HbA1c, which HBOT may impact in the longer-term.

A number of the studies included patients with both type 1 and type 2 diabetes mellitus that were analysed together in a single diabetes mellitus group. Evidence presented suggests that the effects of HBOT on blood glucose differ between type 1 and type 2 diabetes mellitus when the subgroups are analysed. Therefore, including both patients with type 1 and type 2 diabetes together may impact the demonstrated effects of HBOT.

The patients included in the majority of these studies received HBOT for treatment of diverse conditions. Common indications for HBOT included non-healing ulcers and diabetic foot ulcers. These indications for HBOT can be associated with poorly controlled diabetes mellitus or long-standing disease, with most studies also having an older age range of participants. These factors could also have an effect on the impact of HBOT on glycaemia.

Another source of bias to consider is that the identified studies have included different HBOT protocols. This included different hyperbaric conditions, different lengths of treatments, and different numbers of treatment sessions per patient. Heyboer et al. have adjusted for this by using each treatment as a unit of analysis as opposed to each participant [8]. Al-Waili et al. and Trytko & Bennet have taken this into account by measuring the mean for each patient as the unit of analysis [9,19]. Trytko & Bennet also found that mean blood glucose reduction following treatment did not significantly alter with treatment number during the course. However, the methodology used by Peleg et al. suggests that the number of treatment sessions is an important factor. Peleg et al. suggest that factors such as anxiety when first introduced to the chamber environment may act as a confounding factor, and so recruited only patients who had already received at least 10 sessions of HBOT to limit this [11]. Ekanayake & Doolette incorporated a similar principle into their methodology for the same reason, only sampling blood glucose and insulin on the third to fifth day of HBOT [12]. Indeed, the findings from the study by Xu et al. also suggest that the number of treatment sessions is influential, with significant changes in insulin sensitivity, HbA1c and fasting plasma glucose only after 30 session of HBOT [15]. Whilst an increase in insulin sensitivity and decreases in fasting plasma glucose and HbA1c were also observed after 10 sessions of HBOT, these changes were not significant.

Finally, two of the prospective studies were cross-over in design [11,12]. Ekanayake & Doolette had all participants exposed to control normobaric conditions on the day of their HBOT session. Peleg et al. had all participants receive their HBOT session before their normobaric session between 1–14 days later. Randomising the sequence of exposures for participants may have helped to ensure that the sequence of exposures is not influencing the results seen.

#### **4. Discussion**

The impact of HBOT on glycaemia in people with diabetes mellitus is an area of contention as demonstrated by several published studies [8–12]. This review represents the first systematic review conducted on the published research literature to explore the potential impact of HBOT on glycaemic control in people with diabetes. A total of 10 studies were eligible to be included in this systematic review which comprised of seven prospective cross-over and cohort studies; two retrospective reviews of prospectively collected quality data; and one randomised, prospective, placebo-controlled trial.

The majority of the studies demonstrated a reduction in blood glucose levels following HBOT in patients with diabetes, mainly for people with type 2 diabetes mellitus [8,9,15]. The nine original studies reviewed showcased various results involving different methodologies. Variables observed included the use of HBOT and normobaric conditions in people with diabetes mellitus (mainly those with type 2 diabetics mellitus) with assessment of changes in insulin levels, insulin sensitivity, OGTT, and HbA1c (Table 4). Whilst most of the studies support the hypothesis that HBOT reduces blood glucose levels in patients with type 2 diabetes mellitus, there was one study that demonstrated high in-chamber glucose levels contrasting the other study findings [17]. Blood glucose levels, both basal and following an OGTT, were also reduced in people with type 2 diabetes mellitus after several sessions of HBOT [18,19]. Whilst some studies did show a significant reduction in HbA1c following HBOT, the impact seen was not consistent [9,14,15,18]. This highlights the need for large prospective trials to ascertain the precise longer-term effects of HBOT on glycaemia in people with type 2 diabetes mellitus.

**Table 4.** Summary of the eligible studies reviewed with their outcomes for this systematic review.


\* Indicates statistical significance. † There is no evidence in this study of statistical analysis of change in blood glucose levels following HBOT. OGTT = Oral Glucose Tolerance Test, HbA1c = Glycated haemoglobin. Green background colour indicates a positive impact, red background colour indicates a negative impact and an orange background colour indicates no impact.

> The mechanism responsible for the reduction in blood glucose levels caused by HBOT appears to be, at least in part, attributed to increased insulin sensitivity as opposed to enhanced insulin secretion, with two studies demonstrating a significant increase in peripheral insulin sensitivity [15,18]. This finding was particularly noted for people with type 2 diabetes mellitus.

> This review has several strengths. These include prospective Prospero registration, independent two author identification, and extraction, a rigorous PRISMA-based approach to reporting, an individualised approach to quality assessment of each paper, no restriction to publication date.

> However, there are also certain limitations to consider regarding this systematic review. Indeed, this research only considers the literature published in the English language, thus excluding relevant studies that may have been published in other languages. Accordingly, only 10 studies published in the English language were eligible for inclusion, whilst the small sample sizes included in these studies may reduce the reliability and generalisability of the relevant findings. Finally, due to the small sample sizes and diverse methodology involved in the eligible studies, a meta-analysis was not possible, and rather, an individualised approach to conducting this systematic review was considered.

#### **5. Conclusions**

This systematic review suggests that HBOT can impact glycaemia for people with diabetes. Indeed, this systematic review brings together articles demonstrating the impact of HBOT in lowering blood glucose and improving insulin sensitivity in people with type 2 diabetes mellitus. Despite these findings, there remains uncertainty as to the clinical significance of these HBOT-induced effects on glycaemic control. There is, therefore, a need for further research to consider the longer-term clinical impact of HBOT on glycaemia for people with type 2 diabetes mellitus, which could be considered as a potential adjunctive therapy to potentially improve glycaemic control in selected cases.

**Author Contributions:** Conceptualization, H.S.R. and T.R.; methodology, S.B., A.H.P. and T.R.; formal analysis, S.B. and A.H.P.; data curation, S.B., A.H.P. and T.R.; writing—original draft preparation, S.B. and A.H.P.; writing—review and editing, N.M., S.S., I.K., A.A., H.S.R. and T.R.; supervision, H.S.R. and T.R.; S.B. and A.H.P. are joint first authors. All authors have read and agreed to the published version of the manuscript.

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

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

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** Clinical Evidence Based Information Service at University Hospitals Coventry and Warwickshire NHS Trust for assisting in developing the search terms.

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

#### **Appendix A**

**Table A1.** Demonstrating the search strategy used on the PubMed database.



**Table A2.** Demonstrating the search strategy used on the Medline database.

#### **Table A3.** Demonstrating the search strategy used on the Embase database.


#### **References**


## *Review* **Critical Flicker Fusion Frequency: A Narrative Review**

**Natalia D. Mankowska 1,\* , Anna B. Marcinkowska 1,2,3 , Monika Waskow <sup>3</sup> , Rita I. Sharma 4,5 , Jacek Kot <sup>6</sup> and Pawel J. Winklewski 2,3,4**


**Abstract:** This review presents the current knowledge of the usage of critical flicker fusion frequency (CFF) in human and animal model studies. CFF has a wide application in different fields, especially as an indicator of cortical arousal and visual processing. In medicine, CFF may be helpful for diagnostic purposes, for example in epilepsy or minimal hepatic encephalopathy. Given the environmental studies and a limited number of other methods, it is applicable in diving and hyperbaric medicine. Current research also shows the relationship between CFF and other electrophysiological methods, such as electroencephalography. The human eye can detect flicker at 50–90 Hz but reports are showing the possibility to distinguish between steady and modulated light up to 500 Hz. Future research with the use of CFF is needed to better understand its utility and application.

**Keywords:** critical flicker fusion frequency; threshold of flicker fusion; neuropsychology; diving and hyperbaric medicine; minimal hepatic encephalopathy

## **1. Introduction**

Critical flicker fusion frequency (CFF or CFFF) is defined as the frequency at which flickering light can be perceived as continuous and it is used to assess the processing of temporal vision. The upper level of one's abilities in visual processing is described as the critical flicker fusion threshold (or threshold for flicker fusion, TFF), which represents the maximum speed of flickering light that can be perceived by the visual system [1,2]. Because of its efficiency in detecting rapid changes, it is used as an index of cerebral nervous system (CNS) function that is described as alertness and cortical arousal in humans [3,4].

The ability to detect flicker fusion is dependent on: (1) frequency of the modulation, (2) the amplitude of the modulation, (3) the average illumination intensity, (4) the position on the retina at which the stimulus occurs, (5) the wavelength or colour of the LED, (6) the intensity of ambient light [3,5,6] or (7) the viewing distance and (8) size of the stimulus [7]. Moreover, there are also internal factors of individuals that can affect CFF measures: age, sex, personality traits, fatigue, circadian variation in brain activity [4] and cognitive functions like visual integration, visuomotor skills and decision-making processes [3]. The performance of CFF in humans and predators alike is dependent on these factors. Umeton et al. also describe preys' features like a pattern or even the way they move as relevant in perceiving the flicker fusion effect [2].

**Citation:** Mankowska, N.D.; Marcinkowska, A.B.; Waskow, M.; Sharma, R.I.; Kot, J.; Winklewski, P.J. Critical Flicker Fusion Frequency: A Narrative Review. *Medicina* **2021**, *57*, 1096. https://doi.org/10.3390/ medicina57101096

Academic Editor: Akira Monji

Received: 22 August 2021 Accepted: 5 October 2021 Published: 13 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/).

It is believed that the human eye cannot detect flicker above 50 to 90 Hz and it depends on intensity and contrast, but some reports indicate people can distinguish between modulated and steady light at up to 500 Hz [8].

In recent years, there have been many studies with animal models. The pioneers of these studies were Shure and Halstead who investigated the influence of brain lesions in monkeys on TFF [9]. The reason for this decision was Halstead's finding that the removal of the specific brain localisation relates to CFF performance. Nowadays, not only monkeys are of interest to scientists. Lisney and colleagues used electroretinograms and indicated that flicker from fluorescent lamps may be a stress factor for hens [10,11]. The limitation of these studies is the number of included chickens (4 to 15), thus future research is needed.

A high CFF threshold is crucial for flying animals (like pigeons—143 Hz or peregrine falcons—129 Hz) that need an efficient visual system, e.g., to detect rapidly approaching objects to avoid colliding with them, but there are some studies with shrimps that need similar skills. The shrimp TFF is about 160 Hz, although there are individuals with a threshold of 200 Hz [12]. These are just a few examples of the use of CFF in animal studies; in the literature, reports may be found on its usage in dogs, mice, rats or snakes and more. Researchers typically use two types of method to determine the CFF of an animal—electroretinograms and behavioural methods, like two-alternative forced-choice procedure [13,14], e.g., pecking at the lit panel by a bird [15].

Flicker has its countertype in a different sensory modality, i.e., hearing. A few researchers have done a comparison of visual and auditory stimuli. Shipley found that in healthy and young individuals, critical flicker frequency is worse than a critical flutter frequency (which is "the frequency at which a clicking sound appears steady") [16]. Shams et al. point out that the perception of visual stimulus intensity may be modulated by the occurrence of sound [5]. Moreover, the frequency of flickering light is prone to change under the influence of the frequency of fluttering sound; multiple audio signals with a single flash result in perceiving this as multiple flashes.

The CFF test may be used in different forms, but the instruction is usually the same; a subject has to focus their vision on a light-emitting diode when the light frequency is increased at a constant rate (e.g., 1 Hz) and has to press a button when it seems to be continuous light in their opinion. It is also possible to reverse the order of the task to one in which the light frequency decreases and the subject has to report when they see flicker.

In some studies, the CFF test is used as a computer program where participants have to report altered vision by pressing a button [17]; in others, it is a device that can be described as similar to virtual reality glasses, for example, the HEPAtonorm Analyzer [18] or with the addition of other methods like electroretinograms or functional magnetic image resonance, among others [10]. The choice of (1) the device to measure TFF and (2) additional methods should depend on the research hypotheses and the research group.

#### **2. Arousal as an Indicator of Cognitive Performance**

The relationship between arousal and cognitive performance has been known since 1908, when two psychologists, Robert M. Yerkes and John D. Dodson, did a series of experiments with mice and described the Yerkes-Dodson Law, which states that cognitive performance increases with both physiological and mental arousal, but when it becomes too high, the performance decreases. This law could be illustrated in a bell-shaped curve [19]. The critical flicker fusion frequency test can be used as a tool to monitor these changes in brain function and to assess cortical arousal in various environmental conditions [3,20,21]. Additionally, arousal can be modulated by physical activity. Based on this knowledge, several experiments investigated the impact of physical exercise on the CFF performance and found that arousal increases directly after exercise and returns to the output level during recovery [21].

Tomporowski et al. compared results from the Paced Auditory Serial Addition Test (PASAT) before and after exercise. In Experiment 1, nine men completed two sessions of a 40-min bout of cycling, and in Experiment 2, 10 women completed four 120-min sessions of cycling [22]. Researchers have proven that arousal is exercise-induced and may induce better PASAT performance, which means that acute aerobic exercise may affect working memory and attention.

Lambourne and colleagues assessed the influence of aerobic exercises (on cycle ergometer) on two mental processes: sensory discrimination and executive functions in 19 young adults [21]. They used the CFF test as a visual sensory-discrimination task. The measures of the CFF performance were made five times during 40 min of cycling at a moderate level and three times during the 30-min post-exercise period. The results showed an improvement in cognitive performance after physical exercise. However, the CFF results rapidly decreased after the termination of exercise. This means that the increasing level of CFF may result from enhancing receptiveness to sensory stimulation involved in stimulus detection. The arousal probably does not influence executive processing, but the effect of acute exercise on higher-level processes is unclear.

#### **3. Use of the Critical Flicker Fusion Test in Neuropsychology**

The use of CFF for CNS function assessment is postulated by Casey et al. [23]. In experimental procedures with electroretinogram and functional magnetic resonance imaging researchers found that subjects report fusion much longer after the retina and visual cortex responded to flicker [24,25] which means that the human perception of flicker is associated with the activity of higher cortical regions [26,27].

The Halstead-Reitan Battery of Neuropsychological Tests was initially used as a method to assess cognitive functioning in individuals with brain lesions above 15 years old. It was created in 1947 [28] and included two critical flicker fusion measures that were dropped later because of insufficient discrimination between groups of patients with or without brain lesions [29].

CFF as a method to assess cognitive functions is objective, simple, quick, low-cost and it is not affected by factors such as a level of education or language [30–33]. A significant advantage of CFF performance is its resistance to the learning effect [34–36].

Studies with CFF can be performed in 3-month-old infants [37]. They estimate infant TFF by a two-alternative forced-choice preferential looking technique. The researcher (observer) estimates the location of the flickering target (left or right side of the visual field) based on cues given by the infant (e.g., gaze direction or length of looking time to each side). If 75% or more trials were well estimated, then it was inferred that the infant was able to detect flicker at that frequency. The location of light was semi-randomised, and the first trials began at 20 Hz. The findings indicate a significant improvement in development is occurring between 3 to 4.5 months of age, which then may slow or even plateau. The forcedchoice preferential looking procedure is not the only method used in research involving infants; researchers also use visual evoked potentials and electroretinograms [38].

The differences in CFF performance (as an index of processing speed) in children might be a predictor of future cognitive functioning. Visual processing speed, however, is not the only predictor of cognitive abilities in the future [1]. Saint and colleagues examined 54 children with CFF and the Woodcock-Johnson III Tests of Cognitive Abilities and observed that psychomotor coordination became better with age (younger children had longer reaction time latency) [1].

The CFF test in children or adolescents has also been used in studies with groups with brain injuries [18], diabetes [39] and reading disabilities [40].

#### **4. The Diagnostic Values of CFF**

Several studies have investigated the effect of dietary supplementation, especially with compounds that are naturally found throughout the CNS (e.g., lutein, fatty acids or plant pigments, xanthophylls, found in the brain and retina in particular), with the threshold of CFF [41–43]. Bovier et al. have indicated improvements in reaction times and increasing CFF thresholds in adults after lutein and zeaxanthin supplementation [41,42]. Other research projects support this hypothesis [44,45] and even use CFF to find drug effects on psychomotor performance, attention and concentration [46].

Lauridsen et al. compared a continuous reaction times test (CRT) with CFF for the diagnosis of minimal hepatic encephalopathy [47]. The CRT test was a measure of the subject's ability to perform motor reactions adequately and repeatedly. The CFF test reflected biological activity in retinal cells and provided information about visual processing, arousal and attention. The patient's CFF threshold was the average of the nine measurements at 60 Hz, and if it was lower than 39 Hz, then it was considered cerebral dysfunction. In the trial with CRT, participants were asked to press the button as soon as they heard the signal at 500 Hz and 90 dB. A reaction time above 2 s was registered as a lack of response. Both CRT and CFF tests gave false positives and inconsistent results. These two measures describe different aspects of minimal hepatic encephalopathy so the choice between the CRT or CFF test should be made carefully.

The CFF threshold may be a useful measure to follow cognitive processing abilities in patients with implanted vagus nerve stimulators (VNSs) for epilepsy treatment and Alzheimer's disease [17,48]. After a 12-month treatment with a VNS, all epilepsy patients showed significant improvement in CFF compared to baseline (*p* < 0.05).

Some research has suggested that a reduced CFF threshold could be used to detect individuals with Alzheimer's disease [27,48–51]. In these patients, impairment of shortterm memory is observed, which could be enhanced by the 10 Hz flicker. This result confirms previous ones that found memory dysfunction correlated with the loss of the 10 Hz alpha rhythm [52,53]. The EEG frequency and amplitude fall with age, especially in those with mild memory problems [54]. These problems may be partially solved by cholinesterase inhibitors that enhance alpha rhythms as flicker probably does [51,55]. There is a possibility that activity induced by flicker enhances mouse hippocampus activity and the human cortico-cortical and cortico-thalamic loops [56–58].

The use of the CFF was considered in subjects with obstructive sleep apnoea syndrome who may have cognitive impairment. This syndrome may affect attention, memory and executive functions that are associated with brain changes, especially in frontal regions [59]. Depending on the existence and size of brain lesions, CFF performance may vary, but the results of previous studies were inconclusive, and there is a need for future research with a larger sample size [60,61].

#### **5. Diving and Hyperbaric Medicine**

Neuropsychological assessment may be difficult in the underwater environment. The number of available tests for these conditions is limited so CFF seems to be a good solution due to its advantages. In recent years, many studies have focused on examining the impact of both recreational and professional diving on cognitive functions and take into consideration both acute and chronic effects.

CFF is widely used in experiments involving divers [34,62] and provides a reliable assessment that can be compared to psychometric testing (e.g., trail-making tasks or math processing) under normobaric—10 min of breathing air or oxygen in a quiet room at a constant temperature of 22 ◦C [35]—and hyperbaric-dry chamber—breathing air or enriched air nitrox for 20 min at 4 ATA—conditions [63]. A decrease in cerebral performance was reported in association with a decreasing level of CFF and vice versa [3,63]. Balestra and colleagues highlighted that breathing pure oxygen in normobaric conditions has an impact on CFF performance and proved that CFF results are dependent on cortical arousal because of increased brain blood flow in occipital regions and modification of pupil size induced by scopolamine, a muscarinic antagonist [64].

While breathing hyperbaric oxygen, it seems that neuronal excitability measured by CFF depends on the oxygen dose. During a study on professional military divers from the Special Forces, the CFF was increased while breathing a PPO<sup>2</sup> of 2.8 ATA, which represents augmented neuronal excitability, while CFF was decreased at a PPO<sup>2</sup> of 1.4 ATA, which represents attention and alertness deterioration [65]. Interestingly results at the

lower PPO<sup>2</sup> (1.4 ATA) in this group seem contradictory to those observed in recreational divers [35]. Differences in performance might be explained by investigated populations (elite, experienced, combat vs. occasional, recreational divers). Hyperbaric oxygen induces neuromuscular hyperexcitability in normal volunteers, while attenuates such an effect in elite military divers frequently exposed to oxygen and pressure [66].

Recreational diving (generally to 40 m of seawater, msw; in the mentioned literature the depth is 33 msw) may result in changes in the CFF performance even at 30 min postdive [35,36,67].

Another risk in diving is nitrogen narcosis, which in the course of the hyperbaric exposure can be compared to alcohol intoxication and may cause a neurologic syndrome characterised by an impairment of cerebral performance or increased arousal [3]. The influence of the depth narcosis can be compared to the effect of a glass of Martini for every 15 m of depth [68]. Actually, the inert gas (nitrogen) penetration in the lipids of the brain's nerve cells and interference with nerve cells' signal transmission (according to the Meyer-Overton rule) might be further strengthened by the pressure effect [69]. Consequently, it is likely that the depth interval between subsequent "glasses of Martini" would be possibly presented with a smaller value and 10 m is probably more realistically correlated to the increase in the narcotic risk in diving. Impaired cerebral performance includes dysfunction of time perception, reaction speed and ability to think, calculate and react [70].

High-pressure nervous syndrome (HPNS) may appear especially in professional divers who dive deeper than 150 msw due to rapidly increasing pressure in the CNS during compression [20,71]. Nowadays, advanced diving equipment with closed-circuit breathing apparatuses allows recreational divers to also reach depths previously unreachable. Therefore, HPNS becomes a real hazard also for sport and recreational underwater activities [72].

The negative effects of HPNS (e.g., impairment of motor, sensory, behavioural and cognitive function) are known, but there is still a need to conduct future research to understand better its influence on humans. A significant decrease of CFF was observed in divers exposed to high pressures while breathing heliox at 62 ATA, which is equivalent to a depth of 610 m [73].

Quite recently, Ardestani and colleagues asked two groups of divers (reported or non-reported HPNS symptoms) to complete a few tests from the Physiopad package to measure their working memory, vigilance and decision making at 180 to 207 msw [20]. The Physiopad package includes HPNS questionnaires, a hand dynamometry test, a CFF test, an adaptive visual analog scale (AVAS), a simple math process (MathProc test), a perceptual vigilance task (PVT) and a time estimation task (time-wall). The CFF was performed daily and showed no differences between these two groups of divers, which means that the association between psychometric tests and subjective measurements may not exist. These results may arise from one of the limitations of this study; namely, none of the participants was medically diagnosed with HPNS.

Interestingly, the CFF correlates with hypoxemia, as shown during experimental exposures in hypobaric chambers for aviation purposes [74].

#### **6. CFF and Its Connection with Brainwaves**

CFF performance is related to other measures of brain activity. Some electrophysiological experiments have shown that the human visual cortex is sensitive to the frequency of flickering light, which induces neural activity changes in electroencephalogram (EEG) at the same level. Adrian and Matthews discovered " . . . that regular potential waves at frequencies other than 10 a second can be induced by flicker" [75] (p. 377). Authors recorded the EEG activity from the occipital lobe of subjects exposed to flickering light with frequencies up to 25 Hz, and that was the first time when steady-state visually evoked potentials were recorded [76,77].

In healthy older people, alpha-like EEG (8–12 Hz) can be induced by flicker and may even strengthen memory [78]. This relationship is highly specific for frequency. Williams

et al. asked participants to identify the real word in 10 pairs of trigrams (three-letter words). After the practice task, the learning phase ensued (48 pairs of trigrams) [51]. Two minutes later the participants had to choose the "old" word in a pair of "old"-"new" trigrams (this time all of the words were real). Flickering light occurred in the learning phase (1000 ms in the beginning when the screen was blank); then the flicker was continued for 500 ms before and after the trigrams appeared. The frequency and intensity of flickers were randomised and unique for each participant. Six pairs of trigrams occurred after flickering at each frequency (two at each frequency-intensity combination). Results showed that flicker frequencies close to 10.0 Hz have a positive effect on recognition, unlike 8.7 and 11.7 Hz, which were ineffective.

Similar conclusions were made by Herrmann, who observed that 10 Hz visual stimulation has the strongest impact on the visual cortex and induces the strongest neural entrainment (brainwave frequency synchronisation) [79].

Sauseng et al. also showed the connection of alpha-frequency (around 10 Hz) stimulation with memory [80]. They concluded that repetitive transcranial magnetic stimulation might increase the capacity of short-term memory by enhancing the ability to ignore distractors. They emphasised that memory capacity also relied on a larger number of skills such as successful retention of the most important information as well as attention and executive functions.

#### **7. Conclusions**

To conclude, critical flicker fusion frequency has been a widely used method to assess cortical arousal and visual system parameters for many years. It may be successfully used in research involving both human and animal subjects. The CFF test may measure cognitive functioning just as well as other psychometric methods, although it must be considered carefully. As a simple and quick method, resistant to the learning effect, it may be used in many groups, from new-borns to the elderly, from healthy people to people with various diseases, such as epilepsy, dementia, minimal hepatic encephalopathy, etc. Despite these advantages, there is a need to conduct future research to compare the CFF test with other measures, especially neuropsychological ones, to verify its reliability and to better understand its advantages and limitations.

**Author Contributions:** Conceptualization, A.B.M. and P.J.W.; methodology, N.D.M.; validation, A.B.M., R.I.S. and M.W.; formal analysis, N.D.M. and A.B.M.; investigation, N.D.M.; resources, M.W., J.K. and P.J.W.; writing—original draft preparation, N.D.M.; writing—review and editing, all authors; supervision, A.B.M., J.K. and P.J.W.; project administration, N.D.M.; funding acquisition, M.W., J.K. and P.J.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Pomeranian University in Slupsk, 76-200 Slupsk, Poland and the Medical University of Gdansk, 80-210 Gdansk, Poland.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

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

#### **References**


## *Comment* **Comment on Mankowska et al. Critical Flicker Fusion Frequency: A Narrative Review.** *Medicina* **2021,** *57***, 1096**

**Xavier C. E. Vrijdag 1,\* , Hanna van Waart <sup>1</sup> , Jamie W. Sleigh 1,2 and Simon J. Mitchell 1,3**

	- jamie.sleigh@waikatodhb.health.nz (J.W.S.); sj.mitchell@auckland.ac.nz (S.J.M.)

We have read with great interest the review by Mankowska et al. and congratulate the authors on providing an overview of the use of critical flicker fusion frequency (CFFF) in medicine and particularly in diving medicine [1]. In their diving and hyperbaric medicine section they discuss CFFF as a measure of cognitive performance, and how this could be influenced by hyperbaric oxygen, nitrogen narcosis, and high-pressure neurologic syndrome. We would like to comment specifically on the diving medicine section of the article and point out some missing literature that provides important context for the use of CFFF in measuring nitrogen narcosis.

First, the paragraph describing nitrogen narcosis mentions the penetration of nitrogen into the lipids of neurons acting to interfere with signal transmission. This is an outdated view on the cellular mechanism of narcosis. Research on anaesthetic gases suggest an effect on ligand-gated ion-channels in the postsynaptic membrane of excitable neurones [2]. Specifically, the GABAA-receptor is known for binding sedative anaesthetics with consequent opening of the ionophore for chloride-ions, causing hyperpolarization of the cell membrane and thereby signal inhibition [2]. Nitrogen is also known to bind to this GABAAreceptor [3]. This indicates that nitrogen narcosis is not a phenomenon of a gas-lipid reaction in the bilayer but is more likely to be a gas-protein reaction within the receptors in the synapses [4].

Second, an incomplete description is given of the effects of air breathing at different diving depths on CFFF. The authors describe several studies that found a reduction in CFFF in divers breathing air at 405 kPa (the equivalent of 30 m of seawater (msw)) either inside a hyperbaric chamber or underwater [5–7]. This reduction in CFFF was interpreted as a reduction in cognitive performance due to nitrogen narcosis. Accordingly, it would be expected that CFFF would be further reduced when diving to 608 kPa (50 msw). However, three uncited studies where divers breathed air at 608 kPa (50 msw) inside a hyperbaric chamber or underwater did not show a further reduction in CFFF as one would expect. They found either no change [8] or an increase in CFFF [9,10]. This would indicate that there are possibly other factors influencing the CFFF measurement at 608 kPa, which casts considerable doubt on the suitability of CFFF to measure nitrogen narcosis across the plausible range of air diving exposures. A more elaborative overview of the diving CFFF literature is given elsewhere [8].

Third, in the section about "CFFF and its connection with brainwaves," there seems to be a semantic discrepancy between CFFF, defined as 'critical flicker fusion frequency' and 'flickering light.' The description of the influence of flickering light on the electroencephalogram (EEG) has little or nothing to do with CFFF. It therefore seems out of place in a narrative review about CFFF.

**Citation:** Vrijdag, X.C.E.; van Waart, H.; Sleigh, J.W.; Mitchell, S.J. Comment on Mankowska et al. Critical Flicker Fusion Frequency: A Narrative Review. *Medicina* 2021, *57*, 1096. *Medicina* **2022**, *58*, 739. https:// doi.org/10.3390/medicina58060739

Academic Editor: Enrico Camporesi

Received: 26 April 2022 Accepted: 18 May 2022 Published: 30 May 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* **Author Contributions:** Conceptualization, X.C.E.V.; writing—original draft preparation, X.C.E.V.; writing—review and editing, all authors; supervision, H.v.W., J.W.S. and S.J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by funding from the Office for Naval Research Global (ONRG), United States Navy (N62909-18-1-2007).

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

#### **References**


## *Reply* **Reply to Vrijdag et al. Comment on "Mankowska et al. Critical Flicker Fusion Frequency: A Narrative Review.** *Medicina* **2021,** *57***, 1096"**

**Natalia D. Mankowska 1,\* , Anna B. Marcinkowska 1,2,3 , Monika Waskow <sup>3</sup> , Rita I. Sharma 4,5 , Jacek Kot <sup>6</sup> and Pawel J. Winklewski 2,3,4**


**Citation:** Mankowska, N.D.; Marcinkowska, A.B.; Waskow, M.; Sharma, R.I.; Kot, J.; Winklewski, P.J. Reply to Vrijdag et al. Comment on "Mankowska et al. Critical Flicker Fusion Frequency: A Narrative Review. *Medicina* 2021, *57*, 1096". *Medicina* **2022**, *58*, 765. https:// doi.org/10.3390/medicina58060765 4.0/). *medicina*

Academic Editor: Enrico Camporesi

Received: 27 May 2022 Accepted: 31 May 2022 Published: 6 June 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/

Thank you very much for your interest and comments [1] on the review by Mankowska et al. [2], aiming at providing an overview of the use of critical flicker fusion frequency (CFFF) to investigate cognitive functions.

We agree with the authors of the Commentary [1] that the GABAA-receptor might be involved in nitrogen narcosis [3,4]. Yet, the precise molecular mechanisms of the adaptation of lipid bilayers to pressure are unknown and require further investigation [5]. The traditional view is that the lipid bilayer of the cellular membrane is the main target for anesthesia and pressure, while newer theories stress the role of transmembrane proteins. It is, however, likely that nitrogen may exert a pluripotent activity, targeting lipids and transmembrane proteins and implicitly affect water molecules at the lipid–solvent interface [5]. Consequently, the membrane theory and the GABA<sup>A</sup> theory do not need to exclude each other [5,6]. Most importantly, presenting a discussion of the physiological mechanisms underlying anesthesiologic and pressure effects, although fascinating, was not the aim of the review. Rather, we strived to summarize the existing knowledge regarding the reliability of CFFF in the assessment of cognitive functioning versus other psychometric methods.

We have never implied that a reduction in CFFF while diving should be interpreted as a decline in cognitive performance solely due to nitrogen narcosis. On the contrary, we stressed that it is a multifactorial phenomenon and, particularly when diving below 50 msw (more than 608 kPa), there might be other variables such as oxygen toxicity. The dose–reaction relations between oxygen and cognitive functions is not clear and actually it is not known whether the increased excitability, and which forms of neuronal excitability, should be considered a part of the learning process or, rather, cellular manifestation of neuronal oxygen poisoning [7]. Consequently, it is not surprising that below 50 msw a further reduction in CFFF is not seen.

Indeed, "critical flicker fusion frequency" is not the same as "flickering light". However, to the best of our knowledge, there are no studies yet that conclusively explain the mechanisms underlying the processing of flickering light, so we do not know how exactly decisions to perceive flicker or light continuity are made, and thus how the CFFF threshold is determined. We believe that it is impossible to understand CFFF without understanding

these mechanisms, so describing CFFF in the context of flickering light was intended to suggest the need for further research using neuroimaging (e.g., electroencephalography), which could explain what dependencies and interactions we might expect when using the CFFF test. If we want to use the CFFF test as a measure of an individual's arousal [8–10] or cognitive ability [11–13], including in pathological conditions such as epilepsy [14] or Alzheimer's disease [15,16], we must understand how it interacts with the individual's brain. In diving medicine, the use of electroencephalography to investigate the mechanisms underlying processes measured by CFFF seems particularly interesting in the light of the theory focused on the depth-related "effect on ligand-gated ion-channels in the postsynaptic membrane of excitable neurons".

**Author Contributions:** Conceptualization, N.D.M., J.K. and P.J.W.; methodology N.D.M.; validation, A.B.M., R.I.S. and M.W.; formal analysis, N.D.M. and A.B.M.; resources, M.W., J.K. and P.J.W.; writing—original draft preparation, N.D.M. and P.J.W.; writing—review and editing, all authors; supervision, A.B.M., J.K. and P.J.W.; project administration, N.D.M. All authors have read and agreed to the published version of the manuscript.

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

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

#### **References**


## *Article* **Effect of SCUBA Diving on Ophthalmic Parameters**

**Laurent Deleu 1,\* , Janet Catherine <sup>2</sup> , Laurence Postelmans <sup>2</sup> and Costantino Balestra 3,4,\***


**Abstract:** *Background and Objective*: Several cases of central serous chorioretinopathy (CSC) in divers have been reported in our medical retina center over the past few years. This study was designed to evaluate possible changes induced by SCUBA diving in ophthalmic parameters and especially subfoveal choroidal thickness (SFCT), since the choroid seems to play a crucial role in physiopathology of CSC. *Materials and Methods*: Intraocular pressure (IOP), SFCT, pachymetry, flow-mediated dilation (FMD), blood pressure, and heart rate were measured in 15 healthy volunteer divers before diving, 30 and 60 min after a standard deep dive of 25 m depth for 25 min in a dedicated diving pool (NEMO 33). *Results*: SFCT reduces significantly to 96.63 ± 13.89% of pre-dive values (*p* = 0.016) 30 min after diving. It recovers after 60 min reaching control values. IOP decreases to 88.05 ± 10.04% of pre-dive value at 30 min, then increases to 91.42 ± 10.35% of its pre-dive value (both *p* < 0.0001). Pachymetry shows a slight variation, but is significantly increased to 101.63 ± 1.01% (*p* = 0.0159) of the pre-dive value, and returns to control level after 60 min. FMD pre-dive was 107 ± 6.7% (*p* < 0.0001), but post-dive showed a diminished increase to 103 ± 6.5% (*p* = 0.0132). The pre-post difference was significant (*p* = 0.03). *Conclusion*: Endothelial dysfunction leading to arterial stiffness after diving may explain the reduced SFCT observed, but SCUBA diving seems to have miscellaneous consequences on eye parameters. Despite this clear influence on SFCT, no clear relationship between CSC and SCUBA diving can be drawn.

**Keywords:** subfoveal choroidal thickness; intraocular pressure; central corneal thickness; central serous chorioretinopathy; flow-mediated dilation; arterial stiffness; endothelial dysfunction

#### **1. Introduction**

Diving is a recreational and professional activity with many potential risks. Effects of SCUBA diving on the eye have been often reported, including retinal complications (ocular barotrauma, decompression sickness syndrome, arterial gas embolism, ultraviolet keratitis, choroidal ischemia, retinal vein occlusion, central serous chorioretinopathy, variation in color/contrast sensibility, etc.) [1–5].

Central serous chorioretinopathy (CSC) is a potentially severe ocular disease of the retina characterized by recurrent and/or persistent subretinal fluid, causing severe retinal pigment epithelial alterations and a variable degree of visual loss. In our medical retina center, we noticed the presence of common history of SCUBA diving among CSC patients representing around 4% of cases during a two-year period. It is known that CSC is commonly associated with choroidal hyperpermeability and increased subfoveal choroidal thickness (SFCT), but its physiopathology is still poorly understood [6,7].

**Citation:** Deleu, L.; Catherine, J.; Postelmans, L.; Balestra, C. Effect of SCUBA Diving on Ophthalmic Parameters. *Medicina* **2022**, *58*, 408. https://doi.org/10.3390/ medicina58030408

Academic Editor: Enrico Camporesi

Received: 6 February 2022 Accepted: 7 March 2022 Published: 9 March 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*

Considering the importance of SFCT in the diagnostic of CSC, we studied the effect of diving on SFCT and other parameters before and after a deep dive.

#### **2. Methods**

#### *2.1. Patients and Timing of the Study*

For this study, 15 volunteer healthy divers were first interrogated about their medical and diving history, and signed an inform consent. Divers were otherwise healthy Caucasian males between 28 and 72 years old (median 48.93 years). Body mass index 20–25, good general health, nonsmoking (except one), and certified as "advanced divers" with at least 50 logged dives. Of these, three presented treated arterial hypertension, and one with diabetes mellitus without related ophthalmic impairment. Exclusion criteria included previous eye diseases. The study protocol was approved by the Local Ethic Committee Brussels (Academic Bioethical Committee, Brussels, Belgium. Reference Number: B200- 2020-088. Date: 10 October 2020), and each subject gave written informed consent before participation. All studies were performed in accordance with the Declaration of Helsinki [8]. The study is part of a series of ongoing field studies on vascular gas emboli (VGE).

All divers received one drop of tropicamide 0.5% (5 mg/mL) in both eyes. Then, 20 min after instillation, they were examined for intraocular pressure (IOP), pachymetry (PACHY), keratometry (KR), subfoveal choroidal thickness (SFCT), and retinal autofluorescence. They were all tested for post-ischemic flow-mediated dilation (FMD), blood pressure (BP), and heart rate (HR). They also underwent transthoracic heart echography (TTE). After pre-dive measures, all divers went for 25 m dive for 25 min in Nemo 33 diving pool in Brussels. Half of the divers underwent measures during the morning, and the other half in the afternoon. All divers respected published decompression tables while coming back to the surface.

Respectively 30 and 60 min after regaining surface, the patients were retested for the same ocular tests. FMD of brachial artery was measured 30 min after regaining surface. BP, HR, and TTE were also controlled after diving looking for post-dive vascular gas emboli (VGE). All divers drank 500 mL water 60 min after the dive.

#### *2.2. Data Acquisition*

#### 2.2.1. Ophthalmic Measurements

Nidek Tonoref III (NIDEK Co., Ltd., Tokyo, Japan) was used to measure IOP, PACHY, and KR. Pachymetry is here defined by the central corneal thickness, and keratometry is defined by the corneal curvature of the main corneal meridians. Results were calculated using mean of three values for each eye. IOP was not corrected by pachymetry. Nidek Tonoref III provides accurate and reliable measurements of IOP, compared with Goldmann applanation tonometry, considered as the gold-standard for IOP measurements [9]. Even older devices of air tonometers show low variability ranging from −2 mmHg to +2 mmHg, principally due to the cardiac cycle, explaining why three measures should be taken in regular practice [10].

SFCT was measured with enhanced depth imaging (EDI) modality using a Spectral Domain-Optical Coherence Tomography (SD-OCT) (Spectralis, wavelength: 870 nm; Heidelberg Engineering Co. Manufacturer: Heidelberg Engineering GmbH, Heidelberg, Germany). The same device was used for retinal autofluorescence imaging. SD-OCT gives two or three-dimensional images of the retina with near-cellular resolution, allowing ophthalmologists to analyze histologic-like images. SD-OCT uses near-infrared wavelength, and thus does not expose patients to radiation. The procedure for SFCT measurement that we used was previously described by Spaide et al. [11], and is defined as the vertical distance from the hyperreflective line of Bruch's membrane to the hyperreflective line of the inner surface of the sclera. All images were taken by one clinician, and were assessed by three different clinicians. Clinicians were blinded, as they did not know the patient's identification and timing of the measure (pre or post dive). Personal keratometry was encoded in Heidelberg OCT to improve each diver's measurements.

#### 2.2.2. Flow-Mediated Dilation (FMD)

FMD, an established measure of the endothelium-dependent vasodilation mediated by nitric oxide (NO) [12], was used to assess the effect of diving on main conduit arteries. Subjects were at rest for 15-min in a supine position before the measurements were taken. They were asked not to drink caffeinated beverages for the 6 h preceding measurements. Subjects were instructed not to perform strenuous physical exercise 24 h before, or stay in altitude up to 2 weeks before and during the entire study protocol. Brachial artery diameter was measured by means of a 5.0–10.0 MHz linear transducer using a Mindray DP-30 digital diagnostic ultrasound system immediately before and 1-min after a 5-min ischemia induced by inflating a cuff placed on the forearm to 180 mmHg as previously described [13].

All ultrasound assessments were performed by an experienced operator, with more than 100 scans/year, which is recommended to maintain competency with the FMD method [14].

When the images were chosen for analysis, the boundaries for diameter measurement were identified manually with an electronic caliper (provided by the ultrasonography software) in a threefold repetition pattern to calculate the mean value. In our laboratory, the mean intra-observer variability for FMD measurement for the operator (CB) recorded the same day, on the same site, and on the same subject was 1.2 ± 0.2%.

Post-dive values were obtained 20–30 min after surfacing. The divers were given a specific time to enter into the water with their companion (buddy) in order to make it possible to respect the tight timing after the dive for the measurements to be taken. FMD were calculated as the percent increase in arterial diameter from the resting state to maximal dilation.

#### 2.2.3. Post Diving Vascular Gas Emboli (VGE)

Post-dive vascular gas emboli (VGE) (decompression bubbles) were observed using transthoracic echocardiography and a "frame-based" counting method for VGE recently described [15], allowing for continuous values and parametric statistical approaches. Echocardiography was done with a Vivid-I portable echocardiograph (Manufacturer: GE Healthcare, Chalfont Saint Giles, UK) used at the poolside; echocardiography loops were recorded on hard disk for offline analysis by three blinded evaluators. VGE numbers were counted at 30 min and 60 min post dive.

#### **3. Statistical Analysis**

The normality of data was performed by means of Shapiro–Wilk or D'Agostino-Pearson tests. When a Gaussian distribution was assumed, they were analyzed with a one-way ANOVA for repeated measures with Dunnett's post-hoc test; when comparisons were limited to two samples, paired or non-paired *t*-test were applied. If the Gaussian distribution was not assumed, the analysis was performed by means of a non-parametric multiple comparisons using Dunn's test or, if limited to two samples, a Wilcoxon test. Taking the baseline measures as 100%, percentage changes were calculated for each diver, allowing for an appreciation of the magnitude of change rather than the absolute values. All statistical tests were performed using a standard computer statistical package, GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA). A threshold of *p* < 0.05 was considered statistically significant. All data are presented as mean ± standard deviation (SD). Sample size was calculated setting the power of the study at 95%, and assuming that variables associated to diving would have been affected on a similar extent than that observed in our previous studies [16–18].

#### **4. Results**

#### *4.1. Generalities*

All results are expressed in a percentage of pre-dive values (relative values) rather than absolute values. Studied parameters and especially SFCT have high interindividual variability. As each diver acts as his/her own control, results were expressed in percentage of pre-dive values so that this proportion could be compared and analyzed with others. As explained in statistical analysis section, taking the baseline measures as 100%, percentage changes were calculated for each diver, allowing an appreciation of the magnitude of change rather than the absolute values.

#### *4.2. Diving Related*

FMD comparison between pre/post dive situation and control values is shown in Figure 1. FMD in our divers is increased in pre-dive situation (107.15 ± 6.6% (*p* < 0.0001)). This dilatation is significantly reduced after the dive (103 ± 6.5%, *p* = 0.026). This reduction between the two conditions shows a difference consistent with previously published data and is significant (*p* = 0.027) [19].

**Figure 1.** Bar graph illustrating the reduction of FMD 30 min post dive (black bar) compared to predive values (Blue Bar) (mean ± SD). (*N* = 15).

All divers underwent trans-thoracic echocardiography (TTE) 30 min and 60 min after the dive. Figure 2 shows that divers had, on average, eight bubbles per heartbeat (8.0 ± 9.3; mean ± SD) in the right heart (ventricle or atrium) 30 min after the dive. This number is reduced to a mean of 5.25 ± 8.8 Bubbles per HB. The number of circulating decompression bubbles per heartbeat after an hour post the dive was significantly reduced. No participant had symptoms of decompression disease, and the number of VGE found and its decrease is consistent with previously published data [20,21].

**Figure 2.** Number of bubbles (BBS = Bubbles) per heartbeat 30 min (blue bar) and 60 min (black bar) after diving. (Mean ± SD).

#### *4.3. Ophthalmological*

Comparisons between pre and post-dive values appear in Table 1, and are expressed as a percentage relative to pre-dive values. SFCT and IOP both decrease significantly 30 min after the dive. SFCT recovers pre-dive thickness after 60 min, but IOP is still diminished after an hour. Corneal thickness tends to slightly increase, but the change is no longer significant after 60 min. Table 2 presents mean ± SD of measurements. Ophthalmic data post-dive are significantly altered 30 min post-dive, most of which returned to basal levels 60 min post diving, except IOP, which stayed still reduced 60 min post dive. Figure 3 shows OCT imaging of normal posterior pole of the eye, with color code to distinguish retina from choroid and sclera. This figure helps better understanding of Figure 4, which shows comparison between SFCT before and 30 min after diving in a same eye. High magnification was used to highlight the 11 µm difference in this case.

**Table 1.** Comparison (in percentage of pre-dive values) of SFCT, IOP, and pachymetry between preand post-dive values at 30 min and 60 min post dive.


(ns = not significant).

**Table 2.** Mean measures ± SD of SFCT (expressed in µm), IOP (expressed in mmHg) and pachymetry (expressed in µm).


(ns = not significant).

**Figure 3.** Enhanced depth imaging-optical coherence tomography (EDI-OCT) showing different structures. From anterior to posterior: Vitreous body (green), neurosensory retina and the retinal pigment epithelium (red), choroid (SFCT in blue), and sclera (yellow).

**Figure 4.** Comparison of SFCT in the same diver before the dive ((**A**), SFCT estimated at 243 µm by one of the investigators), and 30 min after the dive ((**B**), SFCT estimated at 232 µm).

#### **5. Discussion**

#### *5.1. Subfoveal Choroidal Thickness*

The choroid is a vascular layer situated between the sclera and the retina. It is composed of several layers: the choriocapillaris, 10 µm-thick capillary network; the Sattler's layer, composed of arterioles, small arteries, and veins; Haller's layer, composed of larger blood vessels; the suprachoroid, which is non-vascular, composed of melanocytes, fibroblasts and collagen; and the lamina fusca, separating the choroid from the sclera [22]. It is a highly vascularized space, as the flow per perfused volume is the highest of any other human tissue [23]. While it is well-known that myopic eyes (with greater axial length) tend to have thinner choroid, myopic shift can induce thickening of SFCT in animals, while hyperopic shift induces thinning of SFCT [24]. Mechanisms remain hypothetic, but Wallman et al. observed that, at least in birds, SFCT variation is linked to expansion or compression of lacunae present in the outer choroid [25]. Liquid redistribution seems to be the key of SFCT variation by different suggested mechanisms [24]. However, people with myopic eyes tend to have thinner choroid than emmetropic or hyperopic eyes. These factors make it difficult to know what abnormal SFCT is or not. Studies may give different normal values. Akhtar et al. concluded that subfoveal choroidal thickness was 307 ± 79 µm in an Indian population of any age [26]. Entezari et al. concluded 363 ± 84 µm in an Iranian population [27]. Karapetyan et al. compared SFCT in Caucasians, Africans, and Asians populations, and concluded no significant differences between those ethnics. The mean SFCT in Caucasians was 403.62 ± 37.4 µm. The literature presents sometimes normal SFCT ranges that may be different from ours [28]. Moreover, SFCT seems to decrease over day time [11]. All of these considerations show the importance of measuring variations taking the baseline measures as 100%, for each diver, allowing an appreciation of the magnitude of change rather than the absolute values.

To the best of our knowledge, acute effects of diving on SFCT have never been studied. Our study shows transient and significant SFCT decrease in the 30 first minutes following a deep dive. After 60 min, SFCT returns to its initial thickness. Different hypotheses were investigated to explain those results.

Our preferred hypothesis is that SFCT decreases due to vascular phenomena. As previously said and confirmed by other studies, FMD decreases after a deep dive due to endothelial dysfunction [29,30]. Considering the density of blood vessels in the choroid, reduced dilation of this vascular meshwork could explain by itself the reduction in SFCT. Moreover, diving involves an increase in oxygen partial pressure leading to oxidative stress by increased free radical concentration in blood. This contributes to endothelial dysfunction and, ultimately, arterial stiffness. It seems likely that vascular smooth muscles also have a role in FMD reduction, but various results are found in studies [29,31]. Implications of smooth-muscle cells might be important, as it has been shown that non-vascular smoothmuscle cells are present in primates in the suprachoroid (just next to lacunae), and in a single layer just beneath Bruch's membrane [32]. It has been suggested that contraction of those smooth-muscle cells oppose the tendency of lacunae to gain fluid [24]. Insufficient relaxation of these cells might prevent swelling of the lacunae, inducing vascular shift from the choroid to general circulation, decreasing SFCT. Thus, both arterial stiffness due to endothelial dysfunction and insufficient relaxation in choroidal smooth-muscle cells reduce the choroidal intravascular fluid and are an explanation to reduced SFCT post diving.

In the literature, SFCT tends to be negatively correlated to IOP [33–38]. As both IOP and SFCT decrease in our study, IOP variation does not explain the SFCT decreases. It is also interesting to notice that pachymetry increases in our study and would be expected to falsely elevate the IOP. We can confidently conclude that changes in IOP and pachymetry are not responsible for the diminution of SFCT after the dive. It is a good argument to suggest that there is an extra-ophthalmological phenomenon explaining SFCT reduction.

Other hypotheses were also considered as being unlikely with our experimental setting: Effects of physical exercise on SFCT have been reported but results are various and not well established [39–41].

Tropicamide was instilled before pre-dive measures, therefore there was no influence in comparison between pre- and post-dive values. Iovino et al. published in 2020 that there was no significant difference in SFCT before or after mydriatic instillation [42].

SFCT presents diurnal variation and tends to decrease over the day. Some divers underwent measures in the morning, other in the afternoon. Both are periods of decreasing SFCT, so we do not expect any difference between those two cohorts for relative measurements. Moreover, this circadian cycle does not explain the re-increase of SFCT 60 min after the dive. This points out an extra-physiological phenomenon allowing us to exclude this hypothesis.

#### *5.2. Flow-Mediated Dilation (FMD) and Vascular Gas Emboli*

A nitric oxide (NO) mediated change in the surface properties of the vascular endothelium favoring the elimination of gas micronuclei has previously been suggested to explain this protection against bubble formation [43]. It was shown that NO synthase activity increases following 45 min of exercise, and that NO administration immediately before a dive reduces VGE [44]. Nevertheless, bubble production is increased by NO blockade in sedentary but not in exercised rats [45], suggesting other biochemical pathways such HSPs, antioxidant defenses or blood rheology.

Vascular gas emboli are probably involved in the post dive reduction of FMD. Nevertheless the available literature refrains us to draw a direct link between FMD reduction and VGE, since micro and macro vascularization react differently [30], and different preconditioning procedures before diving have specific actions independently on FMD and VGE, while others interfere with both [20].

It appears that FMD seems more linked to oxygen partial pressure changes during diving, whereas VGE are more depending on preexisting gas micronuclei population in the tissues and vascular system and coping with inflammatory responses [20,46,47].

FMD is a marker of endothelial function and is reduced in the brachial artery of healthy divers after single or repetitive dives [48,49]. This effect does not seem to be related to the amount of VGE, and was partially reversed by acute and long-term pre-dive supplementation of antioxidants, implicating oxidative stress as an important contributor to post-dive endothelial dysfunction [29]. Decreased nitroglycerin-mediated dilation after diving highlights dysfunction in vascular smooth-muscle cells as possible etiology of those results [29]. Very recent data show that the FMD reduction encountered after a single dive without presence of VGE, is comparable to the reduction encountered with the presence of VGE [19].

Our results about FMD are in tune with what has been previously described in literature on the subject and, with its consequences, is the most likely explanation to decreased SFCT observed in this study.

#### *5.3. Central Serous Chorioretinopathy*

CSC is characterized by localized serous retinal detachment associated with focal altered retinal pigment epithelium. Known risk factors are genetics, male gender, cardiovascular diseases and arterial hypertension, increased corticosteroids blood concentration by any income, pregnancy, psychopathology (type A personality), peptic ulcer and Helicobacter Pylori, some drugs (including phosphodiesterase-5 inhibitors), and sleep disturbances. Despite being a common chorioretinal disease, pathophysiology of CSC remains ambiguous. Advances in imaging techniques have shown that CSC is associated with localized areas of delayed choriocapillaris perfusion, congestion of choroidal vessels, choroidal hyperpermeability, and increased SFCT, causing damage to the retinal pigmented epithelium. Imbalance in mineralocorticoid pathway has also been suggested as potential cause. Sympathetic overaction and a decreased parasympathetic tone might also play a role [7,50].

In our department of ophthalmology, there have been several cases of CSC in SCUBA divers in the past years. However, CSC in divers was rarely described in literature [4]. The relationship between hyperbaric environment and CSC could be easily overlooked.

A hypobaric environment might also have influence on SFCT. CSC was reported in at least four air pilots [51–54] and a case during hypobaric chamber exposure [55] together with a small but significant CCT increase was described in high-altitude exposure [56].

Diving has been suspected to cause macular damages since 1988 by the study of Polkinghorne et al. [57]. They highlighted that divers had significantly more retinal pigment epithelial defects, and the prevalence of defects increased with years of diving experience and history of decompression sickness. However, many other studies revealed no significant differences with control groups regarding retinal pigment epithelial alterations [58–60]. A total of three eyes (10%) were found to present retinal epithelium alterations in our study. It is difficult to know if those are the results of diving practice or if it is just incidental finding similar to general population. Decrease in SFCT has been demonstrated in our study, while CSC is typically described with the pachychoroid, which is the opposite.

Regarding those elements, it seems uncertain if SCUBA diving is a risk factor for CSC. Transient decreased SFCT would not explain increased CSC incidence. We did not observe more macular damage, significant retinal pigment alterations nor increased SFCT in our divers compared with general population seen in our daily practice. It seems more likely that cases of CSC in divers reported in our center are just coincidental, as patients had other risk factors of CSC.

#### *5.4. Intraocular Pressure*

Our results demonstrated decrease of 88.05% (*p* < 0.0001%) in IOP 30 min after diving. Lowered IOP is widely described following physical exercise. However, it still remains a poorly understood phenomenon [61]. A total of three theories of its etiology involve decreased blood pH, elevated blood plasma osmolarity, and elevated blood lactate [62]. Increases in trabecular meshwork thickness, area, and perimeter of Schlemm's canal have also been observed after physical activity, and are thought to be a consequence of sympathetic response to exercise. It was not significantly correlated with the decrease in IOP [63]. Similar observations are expected in SCUBA diving, and it is the most likely hypothesis to explain our results. Other hypotheses were also considered as being unlikely with our experimental setting:

Goenadi et al. suggest that in contrast with swimming goggles, diving masks can induce small decrease of 0.43 mmHg in IOP after diving [64]. All divers wore diving masks (different from swimming goggles), respected mask pressure normalization during diving, and no mask squeeze was observed.

Corneal parameters as central corneal thickness and external curvature radius have influence on IOP measurements [65]. Increased pachymetry is associated with overestimated IOP measures, which also does not explain the results. Intraocular bubbles might block trabecular outflow, increasing IOP.

Fadini et al. interestingly showed that patients without any cardiovascular risk factors but suffering from ocular hypertension and primary open-angle glaucoma (POAG) had both FMD and endothelial progenitor cell (EPC) reduced [66]. It seems likely that chronic reduced FMD and endothelial dysfunction increase IOP. However, it was not demonstrated in transient FMD variation. No other description of decreased IOP after SCUBA diving was found on Pubmed. In contrast to our results, Maverick et al. showed increase IOP post-dive negatively correlated to pachymetry [67].

Instillation of tropicamide was made before pre-dive measures, and so does not explain our results. Effects of tropicamide on IOP vary in literature [68–72].

Our results on IOP are in tune with previous papers studying influence of sports on IOP [61]. Also, as IOP is negatively correlated to SFCT, we can formally exclude the role of IOP in the diminution of SFCT after the dive. It is a good argument to suggest that there is an extra-ophthalmological phenomenon explaining SFCT reduction.

#### *5.5. Pachymetry*

This study shows increase in pachymetry 30 min after diving (101.6 ± 1.0%; *p* = 0.015). Results are not significant anymore at 60 min.

Maverick et al. described no significant change of pachymetry in 24 eyes after diving from 34 to 100 feet of depth [67]. However, in a major review, Butler et al. explained how the use of diving mask may, if the divers do compensate air compression in the mask exhaling gas through the nose into the mask, cause a negative pressure around the eye [1]. In severe cases, this can lead to ocular barotrauma. We can easily imagine that this negative pressure may be responsible for increased pachymetry after the dive.

Increased pachymetry also does not explain the SCFT decrease nor IOP decrease.

#### **6. Conclusions**

SCUBA diving appears to have miscellaneous consequences on ophthalmic parameters. We postulate that SFCT is transiently reduced as a consequence of vascular changes, involving increased arterial stiffness and insufficient relaxation in vascular smooth-cells due to oxidative stress and endothelial dysfunction. IOP showed transient decrease until 60 min after the dive, and was not correlated with changes of SFCT or pachymetry. It is a strong argument to point out an extra-ophthalmic phenomenon to explain our results. The results brought us no argument to conclude in a relationship between CSC and SCUBA diving.

**Author Contributions:** All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication Conceptualization, C.B., J.C. and L.P.; writing, L.D. and C.B.; review and editing, C.B., J.C. and L.P. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and received ethical approval from Local Ethic Committee Brussels (Academic Bioethical Committee, Brussels, Belgium. Reference Number: B200-2020-088). Date: 10 October 2020.

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

**Data Availability Statement:** Data are available at request from the authors.

**Acknowledgments:** Authors are grateful to volunteer participants and John Beernaerts, owner of NEMO 33 (https://www.nemo33.com/en/ accessed on 8 March 2022), for letting us use their facilities. **Conflicts of Interest:** The authors declare no conflict of interest.

## **Abbreviations**


## **References**


## *Article* **Pulmonary Effects of One Week of Repeated Recreational Closed-Circuit Rebreather Dives in Cold Water**

**Emmanuel Gouin 1,2,† , Costantino Balestra 3,4,5,6,† , Jeremy Orsat <sup>2</sup> , Emmanuel Dugrenot 1,7 and Erwan L'Her 8,9,\***


**Abstract:** *Background and Objectives*: The use of closed-circuit rebreathers (CCRs) in recreational diving is gaining interest. However, data regarding its physiological effects are still scarce. Immersion, cold water, hyperoxia, exercise or the equipment itself could challenge the cardiopulmonary system. The purpose of this study was to examine the impact of CCR diving on lung function and autonomous cardiac activity after a series of CCR dives in cold water. *Materials and Methods*: Eight CCR divers performed a diving trip (one week) in the Baltic Sea. Spirometry parameters, SpO<sup>2</sup> , and the lung ultrasonography score (LUS) associated with hydration monitoring by bioelectrical impedance were assessed at the end of the week. Heart rate variability (HRV) was recorded during the dives. *Results*: No diver declared pulmonary symptoms. The LUS increased after dives combined with a slight non-pathological decrease in SpO<sup>2</sup> . Spirometry was not altered, and all body water compartments were increased. Global HRV decreased during diving with a predominant increase in sympathetic tone while the parasympathetic tone decreased. All parameters returned to baseline 24 h after the last dive. *Conclusions*: The lung aeration disorders observed seem to be transient and not associated with functional spirometry alteration. The HRV dynamics highlighted physiological constraints during the dive as well as environmental-stress-related stimulation that may influence pulmonary changes. The impact of these impairments is unknown but should be taken into account, especially when considering long and repetitive CCR dives.

**Keywords:** adverse effects; autonomic nervous system; decompression; lung ultrasound; mixed gas diving; pulmonary function; technical diving

#### **1. Introduction**

The use of closed-circuit rebreathers (CCRs) has become increasingly common in the recreational scuba diving community over the past two decades. Their use allows longer and deeper dives than classical open-circuit (OC) scuba equipment. CCRs bring major advantages in terms of gas consumption, an optimal oxygen mix, and warm humidified breathing gas [1]. Conversely, since the breathing system is much more complicated to use, it exposes the diver to technical failures or specific emergencies [2].

During a dive, the cardio-pulmonary system is challenged by various combinations of stressors and adaptive mechanisms such as blood shift, thermal strain, exercise, gas density,

**Citation:** Gouin, E.; Balestra, C.; Orsat, J.; Dugrenot, E.; L'Her, E. Pulmonary Effects of One Week of Repeated Recreational Closed-Circuit Rebreather Dives in Cold Water. *Medicina* **2023**, *59*, 81. https:// doi.org/10.3390/medicina59010081

Academic Editor: Enrico Mario Camporesi

Received: 23 November 2022 Revised: 21 December 2022 Accepted: 28 December 2022 Published: 30 December 2022

**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*

hypercapnia, narcosis and hyperoxia [3–5]. In addition, the breathing apparatus by itself may add to the respiratory workload (work of breathing) that could be increased by the negative transpulmonary pressure gradient in the prone position with back-mounted counterlungs on the CCR used [6]. A number of studies have investigated pulmonary function following OC diving under varying conditions, but the results remain contradictory [4]. Many did not show any spirometric alteration after a single OC dive for a maximum depth of 65 m [7,8]. However, changes in pulmonary function have been found to be associated with depth, cold temperatures, oxidative or decompression stress and duration. These post-dive obstructive pattern changes appeared to be limited and transient [9,10]. Exposure to pure oxygen, even at shallow depths (5 msw), leads to a lung diffusing capacity alteration [11]. CCR diving exposes to high, and potentially prolonged, PpO<sup>2</sup> and specific mechanical constraints [1,6]. There is a lack of data about cardio-pulmonary effects during CCR diving. A CCR deep diving study has shown an almost 30% decrease in forced vital capacity (FVC) after bounce dives at 100 msw [12]. Conversely, CCR use did not affect the spirometry despite the long duration at a maximum depth of 20 msw [13,14]. The impact of CCR repeated dives has not been evaluated in cold conditions (<10 ◦C) and no data are available about the evolution of such abnormalities over time. More recently, asymptomatic lung aeration defects were assessed by ultrasound with B-line detection after dives. These artefacts may suggest extravascular lung water accumulation [15,16]. Most studies have shown an accumulation of B-lines with incomplete resolution between each in repetitive deep OC dives to 60–80 msw, which is not observed at a 33 msw depth [3,8,17]. With CCR, a lung aeration loss was detected, even in shallow water, between 1 and 10 msw, and was substantially amplified by exercise and negative-pressure breathing. The right-to-left heart imbalance and increase in pulmonary vasoconstriction seem to be related to these impairments [5,6]. This phenomenon was already described during breath-hold diving and was related to diaphragmatic spasms with a closed glottis adding to the negative pressure gradient explanation [18].

All immersion constraints, such as blood centralization, pulmonary workload, or hyperoxia, also modulate the autonomic nervous system (ANS). Heart rate variability (HRV) reflects the constant fluctuation of the interaction between pulmonary ventilation, blood pressure, and cardiac output to maintain homeostasis [19]. It can be used to indirectly study changes in parasympathetic (PNS) and sympathetic (SNS) nervous system activity, which express the level of intensity in physiological adaptation. There are marked changes in autonomic cardiac activity during and after scuba diving with a predominance of PNS activity [20,21]. There are complex and sometimes conflicting additional ANS modulations, and CCR diving seems to provoke a different HRV response in divers as compared to OC diving [22].

We hypothesize that in-water breathing constraints may have a negative impact on the lung after CCR dives, especially in case of repetitive exposures. Better knowledge of the physiological impacts of CCR appears essential given the growing diving community and technical developments. Data regarding the cardio-pulmonary effects of repetitive CCR diving are still needed for different depths and environments. The aim of this study was to examine the impact of CCR diving on lung function together with autonomous cardiac activity in asymptomatic healthy volunteers after a recurrent diving exposure in cold water.

#### **2. Methods**

#### *2.1. Diving Sites*

The "Vräk diving expedition" took place in the Stockholm archipelago (Sweden) in September 2022. This study was approved by the Bio-Ethical Committee for Research and Higher Education, Brussels (No B200-2020-088), and adhered to the principles of the Declaration of Helsinki [23].

#### *2.2. Study Population*

This observational study was an intrasubject experimental design with repeated measures. A total of eight male divers were included. Table 1 summarizes the anthropometric data. None were smokers and none were taking medication. All of the subjects were at least recreational rebreather mixed-gas divers. The median CCR experience was 4.5 (2.9–8.3) years. Two divers (25%) have DCS history (one musculoskeletal and one lymphatic manifestations). All divers were fit to dive and had a valid medical certificate for diving. None dived in the previous week. They were all informed about the physiological study and its implications. All participants gave written consent prior to the program.

**Table 1.** Participants' anthropometric parameters (*n* = 8).


BMI, body mass index.

#### *2.3. Diving Procedures*

The dives were performed in accordance with usual CCR dive planning and were not modified for the study requirements. All dives were performed from a rigid inflatable boat. The dive sites were 30 min to 2 h away from the harbor. One to two dives were performed daily, depending on the maximal planned depth. A surface interval of 2 to 3 h between the first and second dive was met. Helium mixed-gases were used for dives below 40 m. Day 2 was taken off. The surface and bottom temperatures were 16.5 (16–17) and 8.5 (6–15.5) ◦C, respectively, with a thermocline at approximately 15 m. The dive parameters are shown in Table 2. Five divers performed five more comparable consecutive diving days in a second week.

**Table 2.** Diving description during the monitoring week. The third dive on the first day were performed by 5 divers to test equipment. Cumulative central nervous system oxygen toxicity (CNS) represents the time spent at a given oxygen partial pressure (PpO<sup>2</sup> ) and dividing by the NOAA time limit for that PpO<sup>2</sup> (corresponding to the cumulative oxygen exposition).


Divers used back-mounted counter lung electronic controlled rebreathers (rEvo™ Rebreathers, Brugge, Belgium; *n* = 5 or JJ-CCR DiveCAN®, Presto, Denmark; *n* = 3). Decompression (Buhlmann ZHL-16C algorithm) was conducted using a connected Petrel 2 computer (Shearwater, Richmond, BC, Canada). The gradient factors were set to 30% (low) and 70% (high) for all dives. The oxygen partial pressure (PpO2) was maintained at 130 kPa during the entire dive. Subjects each wore a dry-suit with dry-gloves and an active heating system [24].

#### *2.4. Measurements 2.4. Measurements*

heating system [24].

Divers were monitored prior to the first dive, after the dive during the first five diving days, and 24 h after their last dive (i.e., after five diving days for three divers and ten days for five divers) in a dry and heated room (temperature 18–20 ◦C). The study flowchart is shown in Figure 1. Divers were monitored prior to the first dive, after the dive during the first five diving days, and 24 h after their last dive (i.e., after five diving days for three divers and ten days for five divers) in a dry and heated room (temperature 18–20 °C). The study flowchart is shown in Figure 1.

(low) and 70% (high) for all dives. The oxygen partial pressure (PpO2) was maintained at 130 kPa during the entire dive. Subjects each wore a dry-suit with dry-gloves and an active

*Medicina* **2023**, *59*, x FOR PEER REVIEW 4 of 14

2.4.1. Functional and Anatomical Pulmonary Monitoring 2.4.1. Functional and Anatomical Pulmonary Monitoring

Measurement of pulmonary parameters was performed daily, 150 to 180 *min* after the dive. All measurements were performed in the sitting position and at rest. Pulse oxygen saturation (SpO2) and heart rate (HR) were recorded for 30 *s*, using a dedicated oximetry module connected to the spirometer (Spirobank II Smart; MIR Medical International Research Srl, Rome, Italy). The mean value for SpO<sup>2</sup> and HR was considered. Spirometric parameters were collected including the forced vital capacity (FVC), forced expiratory volume in one second (FEV1), FEV1/FVC ratio, peak expiratory flow (PEF), and forced expiratory flow (FEF25-75) following GLI (Global Lung Initiative) 2017 for Caucasian adults [25]. The device used for measurements meets the ISO26782:2009 international standards technical characteristics and is CE marked [26]. Flow data were recorded in real time in a dedicated computer, using the manufacturer WinspiroPRO v8.1.0 software. Three repeated loops were performed to assess the reproducibility under the investigator's supervision. The highest FVC and FEV1 values observed over the measurement se-Measurement of pulmonary parameters was performed daily, 150 to 180 min after the dive. All measurements were performed in the sitting position and at rest. Pulse oxygen saturation (SpO2) and heart rate (HR) were recorded for 30 s, using a dedicated oximetry module connected to the spirometer (Spirobank II Smart; MIR Medical International Research Srl, Rome, Italy). The mean value for SpO<sup>2</sup> and HR was considered. Spirometric parameters were collected including the forced vital capacity (FVC), forced expiratory volume in one second (FEV1), FEV1/FVC ratio, peak expiratory flow (PEF), and forced expiratory flow (FEF25-75) following GLI (Global Lung Initiative) 2017 for Caucasian adults [25]. The device used for measurements meets the ISO26782:2009 international standards technical characteristics and is CE marked [26]. Flow data were recorded in real time in a dedicated computer, using the manufacturer WinspiroPRO v8.1.0 software. Three repeated loops were performed to assess the reproducibility under the investigator's supervision. The highest FVC and FEV1 values observed over the measurement series were reported [26].

ries were reported [26]. The anatomical pulmonary aeration was evaluated by lung ultrasonography with a 1.1–4.7 MHz phased array probe (Venue Go™, General Electric Healthcare, Buc, France). Six areas were longitudinally scanned on each hemithorax (anterosuperior, anteroinferior, laterosuperior, lateroinferior, posterosuperior and posteroinferior) to count the total number of B-lines [27]. The exam was performed simultaneously by two trained operators to assess consistent scoring after comparison. A B-line is defined as an echogenic, coherent, wedgeshaped signal with a narrow origin arising from the hyperechogenic pleural line and extending to the far edge of the viewing area [15]. The amount of lung aeration loss was calculated in a semi-quantitative approach using the validated lung ultrasound score (LUS). For each explored region, the worst finding was reported according to the following rating: normal: 0; well-separated B-lines: 1; coalescent B-lines: 2; and consolidation: 3 [27]. The LUS corresponded to the sum of each scanning site score (range 0 to 36). An increase in score indicates a decrease in lung aeration without necessarily reaching pathology levels [28].

#### 2.4.2. Hydration Status

All divers had unrestricted access to drinking water. Bioelectrical impedance analysis (BIA) is a safe and fast method to evaluate the body composition or hydration status [29–31]. These changes were estimated by a multifrequency tetrapolar impedancemetry Biody XPertZM (Aminogram, La Ciotat, France) in order to evaluate the total body hydration status and its related changes after diving. It is presented as a hand–foot analyzer and the measuring time is within the minute. Data were measured according to the manufacturer's instructions in a seated position before the first dive, 150 to 180 min after the first-day dive and at the end of the fifth-day dive, and 24 h after the last-day dive. The data were directly transferred via Bluetooth using the proprietary Aminogram Biodymanager app for Android. The device is accredited to the ISO13485:2016 standard and is CE marked. The response of different body tissues to the application of a weak alternating current at five different frequencies (range 5 to 200 KHz) determines the resistant indices (IRs) and the phase angle at 50 kHz (PA◦ ). It allows an estimate of the total body (TBW), intracellular (ICW) and extracellular (ECW) water. The phase angle expresses both changes in the amount as well as the quality of soft tissue mass (i.e., cell membrane permeability and soft tissue hydration) [32,33].

#### 2.4.3. Heart Rate Variability

Heart rate variability (HRV) is a non-invasive assessment of the variation in time between consecutive inter-beat intervals (R–R intervals) that results in a dynamic relationship between PNS and SNS [19]. It represents the ability of the heart to respond to a variety of physiological and environmental stimuli. Each diver wore a chest elastic belt sensor Polar H10 connected to the Polar Unite watch (Polar Electro Oy, Kempele, Finland) to record the R-to-R interval at a 1000 Hz sampling frequency. The validity of this device for HRV measurement has already been demonstrated in several studies [22,34]. The resting baseline was recorded in a sitting position, after ten minutes at rest, before the first dive and 24 h after the last day for ten minutes. During diving, the Polar watches were placed on the shoulder strap of the undergarment inside the dry suit after starting recording. Full data were extracted daily and analyzed using the Kubios HRV Premium Analysis Software 3.5.0 (UKU, Kuopio, Finland). The automatic artifact correction function of the program was used to correct data corruption for each subject before analysis. Time–domain results with mean HR, standard deviation of normal-to-normal R waves (SDNN), root mean square of the successive difference (RMSSD) of the R–R intervals, and integral of the density of the R–R interval histogram divided by the maximum of its weight (RR triangular index) were calculated. RMSSD mainly reflects the parasympathetic tone while the SDNN and triangular index are indicators of the overall ANS activity frequency-domain measures including the very-low-frequency (VLF), low-frequency (LF), and high-frequency (HF) spectral absolute power and LF/HF ratio. These estimate the distribution of absolute or relative power in different frequency bands [35]. Quantitative analyses of Poincare plot features (SD1, SD2, and SD2/SD1 ratio), Shannon entropy (ShanEn), and Multi-scale entropy (MSE) were computed. This non-linear approach is less dependent on the respiratory sinusal arrythmia variations in the R–R intervals [22]. Furthermore, two composite indexes were calculated. These indexes are based on known HRV parameters that reflect PNS and SNS activity. PNS and SNS indexes were based on the mean R-to-R interval, RMSSD and

SD1, and the mean HR interval, the stress index, and SD2, respectively [36]. An index value of zero reflected that the PNS or SNS activity is equal to the normal population average [37].

#### **3. Statistical Analysis**

Statistical analysis was performed with GraphPad Prism v9.0.2 (GraphPad Software Inc., San Diego, CA, USA). All data are presented as the median (first and third quartile). The normality of distribution was assessed by Shapiro–Wilk test. ANOVA for repeated measures was used to analyze more than two related groups followed by multiple comparison Tukey's post hoc test. A two-way ANOVA for repeated measures was used to assess the effects of the dive day and chest site on the LUS. If non-normality was found, a non-parametric Friedman test was used followed by multiple comparison Dunn test. Statistical significance was set at a *p*-value < 0.05.

The sample size was calculated setting the power of the study at 95% and assuming that variables associated with diving would have been affected to a similar extent as observed in our previous studies where our sample reached 98% [12,30].

#### **4. Results**

#### *4.1. Respiratory and Pulmonary Parameters*

Divers performed the repetitive diving program without any pulmonary symptoms or any other disturbance. No modification of spirometry parameters was observed (Table 3). The mean SpO<sup>2</sup> significatively decreased during all diving days by −1.4 [−2.6; −0.8] % (F = 5.02, *p* = 0.02). Few B-lines were observed on the baseline for several divers, with a median LUS at 1.5 [0.3; 2.8] and a basal predominance.

**Table 3.** Spirometry parameters. Data were assessed at baseline and 150–180 min after each day dive. FCV, forced vital capacity; FEV1, forced expiratory volume in one second; PEF, peak expiratory flow; FEF2575, forced expiratory flow; SpO<sup>2</sup> , pulse oxygen saturation; HR, heart rate. Data are expressed in absolute value and the percentage of expected values according to the GLI (% pred). *p*-value for the ANOVA. Tukey's multiple comparisons test is expressed versus baseline with \* *p* < 0.05. Differences versus post-24 h are expressed with † *p* < 0.05 and †† *p* < 0.01.


There was significative B-line accumulation after diving days 3, 4, 5 and 6 for all participants (Figure 2, F = 8.50, *p* = 0.003) with a return to baseline 24 h after the last dive (*p* > 0.99). There was no difference in chest site repartition (F = 1.02, *p* = 0.4) between lung territories or interaction with the day effect (F = 0.67, *p* = 0.9).

ritories or interaction with the day effect (F = 0.67, *p* = 0.9).

*Medicina* **2023**, *59*, x FOR PEER REVIEW 7 of 14

ritories or interaction with the day effect (F = 0.67, *p* = 0.9).

**Figure 2.** Evolution of individual lung ultrasound score (LUS) throughout repetition of day dives. All divers developed B-lines after several dive days and a return to baseline 24 h after the last one. *p*-value for the ANOVA. Tukey's multiple comparisons test is expressed versus baseline with \* *p* < 0.05, \*\* *p* < 0.01. **Figure 2.** Evolution of individual lung ultrasound score (LUS) throughout repetition of day dives. All divers developed B-lines after several dive days and a return to baseline 24 h after the last one. *p*value for the ANOVA. Tukey's multiple comparisons test is expressed versus baseline with \* *p* < 0.05, \*\* *p* < 0.01. **Figure 2.** Evolution of individual lung ultrasound score (LUS) throughout repetition of day dives. All divers developed B-lines after several dive days and a return to baseline 24 h after the last one. *p*-value for the ANOVA. Tukey's multiple comparisons test is expressed versus baseline with \* *p* < 0.05, \*\* *p* < 0.01.

0.99). There was no difference in chest site repartition (F = 1.02, *p* = 0.4) between lung ter-

0.99). There was no difference in chest site repartition (F = 1.02, *p* = 0.4) between lung ter-

#### *4.2. Impedancemetry 4.2. Impedancemetry 4.2. Impedancemetry*

All body water compartments and TBW increased after dives. Figure 3 depicts variation as compared to the baseline. No significative variation was found 24 *h* after dives in each compartment. A trend towards an IR (impedance ratio) decrease between baseline and after dives was observed at D1 and D6 (*−*1.5 [*−*2.3; *−*0.2] % and *−*1.85 [*−*3.4; *−*0.6] % F = 4.35, *p* = 0.06, respectively). There was no change in the angle phase (F = 0.20, *p* = 0.8). All body water compartments and TBW increased after dives. Figure 3 depicts variation as compared to the baseline. No significative variation was found 24 h after dives in each compartment. A trend towards an IR (impedance ratio) decrease between baseline and after dives was observed at D1 and D6 (−1.5 [−2.3; −0.2] % and −1.85 [−3.4; −0.6] % F = 4.35, *p* = 0.06, respectively). There was no change in the angle phase (F = 0.20, *p* = 0.8). All body water compartments and TBW increased after dives. Figure 3 depicts variation as compared to the baseline. No significative variation was found 24 *h* after dives in each compartment. A trend towards an IR (impedance ratio) decrease between baseline and after dives was observed at D1 and D6 (*−*1.5 [*−*2.3; *−*0.2] % and *−*1.85 [*−*3.4; *−*0.6] % F = 4.35, *p* = 0.06, respectively). There was no change in the angle phase (F = 0.20, *p* = 0.8).

**Figure 3.** Evolution of hydration status measured by impedancemetry. Results are expressed in percentage of baseline variation. TBW, total body water; ECW, extra-cellular water; ICW, intra-cellular water. The *p*-value for the ANOVA. Tukey's multiple comparisons test is expressed versus baseline with \* *p* < 0.05. **Figure 3.** Evolution of hydration status measured by impedancemetry. Results are expressed in percentage of baseline variation. TBW, total body water; ECW, extra-cellular water; ICW, intra-cellular water. The *p*-value for the ANOVA. Tukey's multiple comparisons test is expressed versus baseline with \* *p* < 0.05. **Figure 3.** Evolution of hydration status measured by impedancemetry. Results are expressed in percentage of baseline variation. TBW, total body water; ECW, extra-cellular water; ICW, intra-cellular water. The *p*-value for the ANOVA. Tukey's multiple comparisons test is expressed versus baseline with \* *p* < 0.05.

#### *4.3. Heart Rate Variability* Only 23 measurements of HRV were accurately recorded during dives due to meth-*4.3. Heart Rate Variability* Only 23 measurements of HRV were accurately recorded during dives due to meth-*4.3. Heart Rate Variability*

odological issues. HRV data are shown in Table 4. No change was observed between baseline and 24 *h* post-dive. RMSSD and HF were significatively lower in dive versus rest odological issues. HRV data are shown in Table 4. No change was observed between baseline and 24 *h* post-dive. RMSSD and HF were significatively lower in dive versus rest Only 23 measurements of HRV were accurately recorded during dives due to methodological issues. HRV data are shown in Table 4. No change was observed between baseline and 24 h post-dive. RMSSD and HF were significatively lower in dive versus rest measurements. Similarly, the global activity showed a decrease in variability with lower triangular index, SD2/SD1 and LF/HF ratios. The SDNN decrease was not significative (F = 5.83, *p* = 0.05).

At baseline, the PNS index was −1.92 [−2.17; −0.69] and decreased to −3.26 [−2.17; −2.8] on immersion (*p* < 0.001). Conversely, the SNS index varied to 5.570 [4.97; 6.62] at 13.5 [9.38; 19.72] (*p* = 0.02). The PNS index decreased on immersion (F = 35.83, *p* < 0.0001) while the SNS increased (F = 23.74, *p* < 0.0001), as shown in Figure 4.

**Table 4.** HRV parameters. Data were recorded at rest, in sitting position at baseline and 24 h after last day dive. Dive measurements were recorded in immersion during the diving program (*n* = 23). HR, heart rate; SDNN, standard deviation of normal-to-normal R waves; RMSSD, root mean square of the successive difference; RR triangular index, R–R intervals, and integral of the density of the R–R interval histogram divided by the maximum of its weight; VLF, very low-frequency; LF, low frequency; HF, high frequency; SD1, beat-to-beat HR variability; SD2, global HR variability; ShanEn, Shannon entropy; MSE, multi-scale entropy. *p*-value for the Friedman test. Dunn's multiple comparisons test is expressed versus dive with \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 and \*\*\*\* *p* < 0.0001. Differences versus baseline are expressed with † *p* < 0.05 and †† *p* < 0.01. triangular index, SD2/SD1 and LF/HF ratios. The SDNN decrease was not significative (F = 5.83, *p* = 0.05). **Table 4.** HRV parameters. Data were recorded at rest, in sitting position at baseline and 24 h after last day dive. Dive measurements were recorded in immersion during the diving program (*n* = 23). HR, heart rate; SDNN, standard deviation of normal-to-normal R waves; RMSSD, root mean square of the successive difference; RR triangular index, R–R intervals, and integral of the density of the R– R interval histogram divided by the maximum of its weight; VLF, very low-frequency; LF, low frequency; HF, high frequency; SD1, beat-to-beat HR variability; SD2, global HR variability; ShanEn, Shannon entropy; MSE, multi-scale entropy. *p*-value for the Friedman test. Dunn's multiple comparisons test is expressed versus dive with \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 and \*\*\*\* *p* < 0.0001.

measurements. Similarly, the global activity showed a decrease in variability with lower


*Medicina* **2023**, *59*, x FOR PEER REVIEW 8 of 14

**Figure 4.** Autonomic nervous system (ANS) composite index measured by HRV. HRV parameters were performed at rest, in sitting position at baseline and 24 h after last day dive. Dive measurements were recorded in immersion during the diving program (*n* = 23). PNS, in parasympathetic nervous system; SNS, sympathetic nervous system; SD, standard deviation of measurement to the normal population average. Dunn's multiple comparisons test is expressed versus dive with \* *p* < 0.05, \*\*\* *p* < 0.001 and \*\*\*\* *p* < 0.0001.

#### **5. Discussion**

The present study depicts that lung aeration disorders are observed during repeated CCR dives. However, these abnormalities, associated with a slight but significant SpO<sup>2</sup> decrease, may be transient and not associated with lung spirometry modifications.

A long-term FVC increase has already been reported in the experienced diving community, in relation to the chest muscular workload at depth. This change suggests a distension of the alveoli wall that may cause narrowing of small airways [4]. It could explain the moderate but not significant basal FEF 25–75 alteration observed in our study. However, the absence of any significant spirometric alteration after such shallow dives is similar to previous studies on CCR diving. There is a general consensus in pulmonary medicine and anesthesiology that breathing oxygen at an oxygen partial pressure (PpO2) higher than 50 kPa causes acute pulmonary injury, which can result in atelectasis, interstitial oedema, and inflammation [38]. In such diving conditions, there was no significant clinically relevant impairment of clinical airway physiology. After breathing PpO<sup>2</sup> at 140 kPa for 20 min at 15 msw, an increase in oxidative stress urinary markers has been described but was not considered sufficient to affect the spirometry [14]. The pulmonary function also remained unchanged either after a prolonged 3- and 12 h exposure at 5 msw and 20 msw, respectively, with a PpO<sup>2</sup> of 150 kPa or after repeated dives (20 dives within 11 days) at an average depth of 69 msw during 112 min with a PpO<sup>2</sup> set at 130–140 kPa. It should be noted that the recommended maximal repetition excursion oxygen exposure (REPEX) threshold was approached in these studies [11,13,39]. In contrast, for deeper dives (90–120 msw) with a duration of 2 or 3 h, we previously found a gradual FVC decrease from 109 to 73% of the predicted value after a second dive, without returning to baseline between dives. No alteration of pulmonary resistance was observed, which might suggest other physiological mechanisms than hyperoxia. Considering all these arguments, one might consider that the alteration of spirometry data seems more likely to result from the effects of prolonged and deeper immersion at depth than from oxygen toxicity by itself [12].

A loss of lung aeration was observed after dives, as shown by the accumulation of B-lines without the alteration of spirometry. B-lines are an index of extravascular congestive lung fluid, which has been previously validated with high sensitivity and intrapatient reliability, allowing good interrater consistency of pulmonary fluid assessment using radiographic imaging [16,28]. Some authors have reported up to 75% of divers showing extravascular lung water detected as B-line accumulation. Many factors seem to be associated with asymptomatic changes in cardiovascular and pulmonary physiology in diving, therefore linked to the development of extravascular lung water [40]. However, B-lines are not specific, and their occurrence may also reflect any interstitial disorder or ventilation impairment. Some studies indicate a good correlation between their number and the intensity of damages [41]. An aeronautic study has shown that hyperoxia and hypergravity are independent risk factors of pulmonary atelectasis formation in healthy humans after a long arm centrifuge session. The increase in B-lines has been reported to reflect the onset of hyperoxic atelectasis [42]. Our study does not enable us to distinguish extravascular lung water or atelectasis contribution. It is interesting to note, while not significant, that a higher LUS number was observed during the first two days. Dives were shallower but the total immersion time and oxygen exposure were longer. Moreover, helium mixed gases were used for the deepest following dives, thus inducing a lesser gas density and a decrease in breathing workload. Two or three hours after surfacing from a deep Trimix dive, the B-lines were already largely resolved, similar to what has been reported by a previous Croatian study with similar dives [8]. Our results suggested that most of the pulmonary changes including loss of aeration lasted only for a short time after dives with a return to baseline 24 h post-dive.

A reduction in pulmonary diffusing capacity was shown only after a wet shallow oxygen dive as compared to a dry similar dive that suggested the implication of cardiopulmonary changes in immersion [11]. This impairment was inconsistent and was not correlated with the presence of B-lines [8]. In our study, SpO<sup>2</sup> slightly decreased after dives

but remained within physiological values considered to be normal [43]. This oxygenation decrease may be related to the lung aeration loss and a potential alteration of alveolocapillary gas exchange, even though it persists while LUS values have decreased and/or returned to baseline values. Atelectasis could lead to a pulmonary ventilation/perfusion mismatch and shunt opening [42]. Although non-pathological, this may interfere with gas elimination and decompression. Similar results were found after CCR dives at 10 msw [5] but not after deep dives despite spirometric alteration [12]. This SpO<sup>2</sup> decrease might be compensated by a prolonged high PpO<sup>2</sup> up to 150 kPa during long decompression stop after deep dives. Several artefacts can interfere with SpO<sup>2</sup> monitoring. However, data were always recorded after rewarming, in dry conditions, and after hydration in order to reduce these methodological artefacts [43].

It is well known that immersion induces hyperdiuresis, which in turn alters the hydration status with a loss of body weight of up to 3% and a potential impact on the cardio-pulmonary system [12,44]. Conversely, our results showed an increase in body water after diving. Considering that dehydration plays a role in decompression stress and that water intake could provide a decrease in the risk of decompression sickness (DCS), no specific instruction was given to fluid management for the team [45,46]. Technical divers were aware of this problem, and they probably rehydrated themself effectively during hours prior to measurements. There is no direct evidence within the literature that immersion pulmonary oedema is related to hydration in healthy divers [47,48]. In our study, there is no clear evidence that the observed state of hyperhydration could have contributed to lung ultrasound abnormalities.

HRV monitoring during scuba diving is only available from a limited number of studies, and CCR diving seems to induce a different HRV response than OC diving [22,49]. Immersion stimulates both the PNS and SNS branches. A predominant PNS is usually observed during descent and bottom stay, in accordance with human diving responses [49]. After emersion and continuation of atmospheric air breathing, the SNS takes over the PNS. A PNS tone increase in dive, related to dive length and depth, has been demonstrated [50]. Nitrox diving seems to induce a higher PNS activation [51] but also to be the principal dynamic component of SNS [50]. Short-term HRV is in fact influenced by many other factors, including PNS/SNS balance, as well as respiration via the respiratory sinus arrhythmia, heart and vascular tone via baroreceptor and cardiac stretch receptor activity, the central nervous system (CNS), the endocrine system, and chemoreceptors [21]. The PNS activity is a major contributor to the HF component (which reflects the power of vagally modulated respiratory sinus arrhythmia) [19]. Non-linear analysis revealed complexity in heart-rate patterns, which could not be perceived from time–domain [52]. Compared to OC diving, no variation in HF power or SDNN at depth was found in CCR diving, while the non-linear analysis increased. This suggests a lower PNS dynamic and variability in CCR dives [22]. In CCR cold diving (2 to 4 ◦C), the PNS index significatively decreased at submersion and increased gradually throughout the dive. At the same time, the SNS index sharply increased during immersion and then slowly declined back to the pre-dive rest levels [21]. Our results demonstrate a similar PNS dynamic. However, the SNS index appeared mostly predominant while global HRV activity and PNS decreased, which is contradictory to previous observations during OC diving. The SNS activity can be stimulated by many factors including physical activity or psychologically stressful situations [53]. The divers wore heavy equipment and were not necessarily previously familiar with the diving conditions that they experienced in this dynamic. Our HRV analysis has some limitations as we were only able to record 23 measurements due to interference between the heating system and the sensor. Moreover, the dive profiles varied over time, and we are not able to compare the responses at these different depths.

#### **6. Limitations**

The findings of this study have to be viewed with caution due to the small number of subjects and lack of daily pre-dive measurements due to logistical constraints. All measurements are compared to the first-day dive baseline, which reduces the accuracy in the assessment of physiological variations throughout the program. That may have particularly affected our impedancemetry analyses. In addition, the lack of monitoring fluid intake makes the interpretation difficult.

The mandatory boat travel-back time led to many hours of delay before measurements. Thus, we are aware of the risk of missing potential transient changes in the measured parameters. Our study was not conducted in controlled laboratory-like conditions. Essential parameters that could interfere with our results such as temperature, visibility, current, depth, or dive duration could not be controlled. Unfortunately, due to the small number of dives, the effect of the dive profile and breathing mixture cannot be evaluated.

#### **7. Conclusions**

The present observation represents the first original data regarding the pulmonary effects of repetitive CCR dives, combining spirometry, oxygenation evaluation and lung ultrasound imaging. Despite no detectable change in pulmonary function, we observed a significative loss of lung aeration. The impact of these impairments is unknown but should be taken into account, especially when considering long and repetitive dives. The marked changes that were also observed in autonomic cardiac activity highlight the important physiological and environmental constraints in CCR diving. All cardiac and pulmonary function changes were, however, transient, without the negative effect of dive repetition, and returned to baseline within 24 h after the last dive. Further research on this topic is encouraged to gain better knowledge about cardiopulmonary constraints during CCR diving.

**Author Contributions:** All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Conceptualization, E.G., C.B., J.O., E.D. and E.L.; methodology, C.B., E.D. and E.L.; validation, C.B. and E.L.; formal analysis, E.G., C.B. and E.L.; investigation, E.G., E.D. and E.L.; resources, E.G., C.B., J.O., E.D. and E.L.; data curation, E.G., E.D. and E.L.; writing—original draft preparation, E.G.; writing—review and editing, E.G., C.B. and E.L.; visualization, E.G.; supervision, C.B. and E.L.; funding acquisition, C.B. and E.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Divers Alert Network (DAN), (Grant 10-25-2022) and Brest University Foundation.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Bio-Ethical Committee for Research and Higher Education, Brussels (No B200-2020-088, Date: 22 August 2020).

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

**Data Availability Statement:** Data are available on request from the authors.

**Acknowledgments:** The authors wish to thank General Electric Healthcare France and Sweden for their technical support. We acknowledge all divers and film crew for their kind collaboration and taking for a genuine interest in the research with special mention to Emmy Ahlén and Björn Lindoff from Vrakdykarpensionatet (Dalarö) for their logistical support and warm hospitality.

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

#### **Abbreviations**


#### **References**


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## *Article* **Vascular Function Recovery Following Saturation Diving**

**Jean-Pierre Imbert 1,2, Salih-Murat Egi 1,3 and Costantino Balestra 1,4,5,6,\***


**Abstract:** *Background and Objectives*: Saturation diving is a technique used in commercial diving. Decompression sickness (DCS) was the main concern of saturation safety, but procedures have evolved over the last 50 years and DCS has become a rare event. New needs have evolved to evaluate the diving and decompression stress to improve the flexibility of the operations (minimum interval between dives, optimal oxygen levels, etc.). We monitored this stress in saturation divers during actual operations. *Materials and Methods*: The monitoring included the detection of vascular gas emboli (VGE) and the changes in the vascular function measured by flow mediated dilatation (FMD) after final decompression to surface. Monitoring was performed onboard a diving support vessel operating in the North Sea at typical storage depths of 120 and 136 msw. A total of 49 divers signed an informed consent form and participated to the study. Data were collected on divers at surface, before the saturation and during the 9 h following the end of the final decompression. *Results*: VGE were detected in three divers at very low levels (insignificant), confirming the improvements achieved on saturation decompression procedures. As expected, the FMD showed an impairment of vascular function immediately at the end of the saturation in all divers but the divers fully recovered from these vascular changes in the next 9 following hours, regardless of the initial decompression starting depth. *Conclusion*: These changes suggest an oxidative/inflammatory dimension to the diving/decompression stress during saturation that will require further monitoring investigations even if the vascular impairement is found to recover fast.

**Keywords:** flow-mediated dilation; FMD; decompression; arterial stiffness; endothelial dysfunction; underwater; hyperbaric; commercial diver; off-shore energy operation; human

#### **1. Introduction**

Saturation diving is a standard technique of divers' intervention in the North Sea because of its depth (average 100 to 150 msw). Saturation is conducted from large diving support vessel that employs around 80 divers in multiple rotations during a working season. The contractors have developed saturation procedures empirically over the last 40 years and reached a mature level of technology and safety. On the other hand, the need for evaluating the diving and decompression stress to improve flexibility of the operations (minimum interval between dives, optimal oxygen levels, etc.) arose.

An increasing number of research reports have been published to document procedures, diver's subjective evaluations [1], hematological changes [2], high pressure nervous syndrome [3], divers hydration status [4] or oxidative stress [5]. Saturation permits divers to live under pressure in chambers onboard of a vessel and to be deployed directly to the seabed by a diving bell. Historically, commercial saturation diving was developed

**Citation:** Imbert, J.-P.; Egi, S.-M.; Balestra, C. Vascular Function Recovery Following Saturation Diving. *Medicina* **2022**, *58*, 1476. https://doi.org/10.3390/ medicina58101476

Academic Editor: Marcus Daniel Lancé

Received: 29 September 2022 Accepted: 13 October 2022 Published: 17 October 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*

during the 1970s for the North Sea oil platform installations. At the time, the concern was decompression sickness (DCS) that was associated with bubbles in the divers' blood. During these "early days" when diving in such a harsh environment was not as safe, and still difficult today, alarming reports were available:

*"Records show that since 1966 seventy-seven diving personnel tragically have lost their lives in the quest for, depending on your perspective "Black Gold" or "Devil's excrement" in the North Sea Basin. By nationality: 53 British/Commonwealth subjects, 9 American, 7 Norwegian, 4 Dutch, 3 French, and 1 Italian. Prior to 1971/1974, applicable laws & regulations (if any) required no accurate fatal accident statistics. One can conclude that the actual combined number of deaths is higher. However, it is known that several divers received severe injuries from which they never recovered."*

(https://the-norwegian.com/north-sea-diving-fatalities (accessed on 20 September 2022))

Fifty years later, saturation procedures have improved a lot and decompression sickness has become a rare event. Official safety records published in Norway on the Website of the PSA (Petroleum safety Authority) indicate an incidence of less than one case per 2000 exposure over the last 10 years (https://www.ptil.no/en/technical-competence/exploretechnical-subjects (accessed on 20 September 2022)). As a result, the diving companies have the duty to evaluate the performances of their procedures such as the minimal permitted interval between two saturations. This minimum interval has been arbitrarily defined for a long time by industry guidelines or diving regulations but the divers' recovery between saturations has never been studied scientifically. This recovery period remains important for companies to optimize their crew changes and divers to manage their professional career.

Saturation diving is obviously associated with multiple stressors that may be organized along three dimensions for simplicity. The first dimension is characterized by the diving work and includes stresses such as the physical, mental, or thermal.

The second dimension is associated with the vascular gas emboli (VGE) produced during decompression. Although there is no clear relation between the number of VGE measured and the risk of DCS, it is recognized that the smaller the number of VGE detected, the safer is the decompression [6]. The number of circulating VGE was therefore taken as the principal measurement of the decompression stress.

The third dimension covers several biological processes recently identified in the literature [7]. New insight demonstrate that bubbles tear the vessel inner layer away and create microparticles of endothelial debris when detaching from the endothelium during decompression [8–10]. Bubbles and oxygen partial pressure increase trigger defense mechanisms like platelets and neutrophil activation that will also elicit some microparticles [11,12]. In this study, the vascular function assessed by means of Flow Mediated Dilation (FMD) was considered as the third dimension representing the oxidative and or inflammatory stress [10,13,14].

The objective of the study was to define a monitoring package and use it on board a vessel to monitor saturation divers at surface, before the saturation and after exiting saturation (after decompression), in order to evaluate their recovery during the 9 h following the end of their final decompression.

#### **2. Methods**

#### *2.1. Worksites*

A leading diving company provided access to one of their diving support vessels (DSV) operating in the North Sea for this study. Two monitoring sessions were conducted onboard the DSV Deep Arctic (The vessel DEEP ARCTIC is an Offshore Support Vessel built in 2009 with particulars of Gross Tonnage 18,640 t; Summer Deadweight 13,000 t; Length Overall 157 m; Beam 31 m.) in April and October 2016, during two different projects, one in the Norwegian sector at 121 m of sea water (msw) and the other in the UK sector at 155 msw working depth. The two projects, performed on the same vessel, corresponded

to a well intervention on the seabed; the divers used the same breathing gasses, the same diving equipment and performed the same tasks (see Figure 1). corresponded to a well intervention on the seabed; the divers used the same breathing gasses, the same diving equipment and performed the same tasks (see Figure 1).

**Figure 1.** A typical saturation worksite. The divers are deployed from the diving support vessel inside a diving bell. Once on site, the bell's door opens, and the divers lock out in the water using an umbilical attached to the bell to breathe and being supplied with hot water in their suit for thermal comfort. The working depth corresponds to the maximum depth reached by the divers. The working depth defines the chamber storage depth from excursion tables prepared in the company diving manual. The bell depth is usually set at 5 msw deeper than the storage depth to clear from subsea structures when opened. The "storage" and the "bell" are almost at the same pressure allowing for getting back to storage after work without decompression needed. The excursion of the diver out of the diving bell is limited to some meters not to add additional decompression time. The breathing gas is Heliox (Helium-Oxygen) to limit the density of the breathed gas (significant at such pressures) to reduce the work of breathing as well as Oxygen toxicity and Nitrogen narcosis. **Figure 1.** A typical saturation worksite. The divers are deployed from the diving support vessel inside a diving bell. Once on site, the bell's door opens, and the divers lock out in the water using an umbilical attached to the bell to breathe and being supplied with hot water in their suit for thermal comfort. The working depth corresponds to the maximum depth reached by the divers. The working depth defines the chamber storage depth from excursion tables prepared in the company diving manual. The bell depth is usually set at 5 msw deeper than the storage depth to clear from subsea structures when opened. The "storage" and the "bell" are almost at the same pressure allowing for getting back to storage after work without decompression needed. The excursion of the diver out of the diving bell is limited to some meters not to add additional decompression time. The breathing gas is Heliox (Helium-Oxygen) to limit the density of the breathed gas (significant at such pressures) to reduce the work of breathing as well as Oxygen toxicity and Nitrogen narcosis.

#### *2.2. Saturation Procedures*

The two projects were conducted with saturations according to the Company procedures defined in their diving manuals. However, specific requirements are defined in the Norwegian diving regulations that introduced slight variations.

The chambers were initially compressed to 10 msw in 10 min for a 20 min leaks check. Compression then proceeded to the "storage" depth at 1 msw/min.

The chamber PO<sup>2</sup> at storage depth was controlled at 40 hPa. The storage depth was selected from the working depth using the standard excursion tables (110 msw storage depth for 121 msw working depth in the Norwegian sector, 136 msw storage depth for 155 msw working depth in the UK sector). During the bottom phase, divers performed one bell dive of 8 h per day but may sometime skip a dive due to weather conditions or vessel transit. During the dives, the divers' breathing mixture was Heliox with a PO<sup>2</sup> ranging from 60 to 80 hPa.

The final decompression can only start after an 8 h period following the last excursion dive.

The decompression is performed in two phases. It starts with constant chamber PO<sup>2</sup> (50 hPa in the UK, 48 hPa in Norway) until 15 msw and finishes with a chamber oxygen percentage maintained between 23.1 and 23% to limit the fire hazard and optimize inert gas exhalation.

Despite the difference between sectors, the total decompression durations were very similar (5 days 5 h in the UK sector and 5 days 11 h in the Norwegian sector, a difference of less than 3%).

The divers were organized in three men teams (two divers and the bellman). Teams worked in shifts (12:00 p.m. to midnight and midnight to 12:00 p.m.). Each team was involved in one bell excursion dive per day during their shift. The divers' in-water time was limited to 6 h with a mandatory break at mid-excursion.

#### *2.3. Participant Eligibility and Enrollment*

The study group consisted of volunteer, male, certified commercial saturation divers. These divers were declared fit for the saturation by the vessel hyperbaric nurse after a mandatory pre-dive medical examination.

All experimental procedures were conducted in accordance with the Declaration of Helsinki [8] and were approved by the Academic Ethical Committee of Brussels (B200-2009- 039). The methods and potential risks were explained in detail to the participants. Each subject gave written informed consent before participation.

A total of 49 divers accepted to participate to the study.

The group anthropometric parameters were obtained after a confidential interview in the vessel hospital. (See Table 1).


**Table 1.** Participants anthropometric parameters (*n* = 49).

As expected from saturation divers, all were very experienced divers with a long diving career. (See Table 2).

**Table 2.** Participants diving experience (commercial experience includes Saturation experience).


Part of the group freely took of antioxidant supplements (commercially available products containing as vitamin C, D, or E) before and during the saturation. (See Table 3).

**Table 3.** Group antioxidant supplement intake (free administration).


Saturation divers generally spend a lot of time maintaining a high level of physical fitness and are involved in all sorts of sports. Every diver in the group except one had a daily or at least weekly physical activity when at home. (See Table 4).

**Table 4.** Participants' usual physical activities. **Type of Physical Activity Percentage** 

**Table 4.** Participants' usual physical activities.

*Medicina* **2022**, *58*, 1476 5 of 13


The participants were divided as follows: 37 divers in saturation in the Norwegian project (75%) and 12 divers in saturation in the UK project (25%). The saturation duration depended on the sector regulations. It is 14 days maximum bottom time in Norway and 28 days total saturation time in the UK. The mean saturation duration was 19.70 ± 6.5 days (minimum 10 days, maximum 28 days) (see Figure 2). The participants were divided as follows: 37 divers in saturation in the Norwegian project (75%) and 12 divers in saturation in the UK project (25%). The saturation duration depended on the sector regulations. It is 14 days maximum bottom time in Norway and 28 days total saturation time in the UK. The mean saturation duration was 19.70 ± 6.5 days (minimum 10 days, maximum 28 days) (see Figure 2).

**Figure 2.** Description of the saturation in the UK sector: depth profile (compression, storage depth, bell dives, decompression) and associated PO2 profile. **Figure 2.** Description of the saturation in the UK sector: depth profile (compression, storage depth, bell dives, decompression) and associated PO<sup>2</sup> profile.

#### *2.4. Organizational Constraints 2.4. Organizational Constraints*

The voluntary divers were first involved in the study in the few hours after arriving onboard, after their pre-saturation medical examination, just before entering the saturation chambers. Baseline (control) measurements (FMD and Questionnaires) were recorded. The group of divers were then monitored during the next 12 h following the end of the decompression to surface. The voluntary divers were first involved in the study in the few hours after arriving onboard, after their pre-saturation medical examination, just before entering the saturation chambers. Baseline (control) measurements (FMD and Questionnaires) were recorded. The group of divers were then monitored during the next 12 h following the end of the decompression to surface.

The questionnaires and measurements were run in the vessel hospital room that warranted confidentiality. The questionnaires and measurements were run in the vessel hospital room that warranted confidentiality.

It is admitted that after the decompression, due to operational constraints, it was difficult to "catch" the divers at regular times and some subjects (30%) only performed one or two sessions of the four initially planned (see Figure 3). It is admitted that after the decompression, due to operational constraints, it was difficult to "catch" the divers at regular times and some subjects (30%) only performed one or two sessions of the four initially planned (see Figure 3).

**Figure 3.** Experimental flowchart. **Figure 3.** Experimental flowchart.

#### *2.5. Data Acquisition*

#### *2.5. Data Acquisition*  2.5.1. Flow-Mediated Dilation (FMD)

2.5.1. Flow-Mediated Dilation (FMD) FMD, an established measure of the endothelium-dependent vasodilation mediated by nitric oxide (NO) [15], was used to assess the effect of diving on main conduit arteries. Subjects were at rest for 15-min in a supine position before the measurements were taken. Brachial artery diameter was measured by means of a 5.0–10.0 MHz linear transducer M-Turbo portable echocardiograph (Sonosite M-Turbo, FUJIFILM Sonosite Inc., Amsterdam, The Netherlands) immediately before and 1-min after a 5-min ischemia (induced by inflating a sphygmomanometer cuff placed on the forearm to 180 mmHg as previously de-FMD, an established measure of the endothelium-dependent vasodilation mediated by nitric oxide (NO) [15], was used to assess the effect of diving on main conduit arteries. Subjects were at rest for 15-min in a supine position before the measurements were taken. Brachial artery diameter was measured by means of a 5.0–10.0 MHz linear transducer M-Turbo portable echocardiograph (Sonosite M-Turbo, FUJIFILM Sonosite Inc., Amsterdam, The Netherlands) immediately before and 1-min after a 5-min ischemia (induced by inflating a sphygmomanometer cuff placed on the forearm to 180 mmHg as previously described [16].

scribed [16]. All ultrasound assessments were performed by an experienced operator, with more All ultrasound assessments were performed by an experienced operator, with more than 100 scans/year, which is recommended to maintain competency with the FMD method [17].

than 100 scans/year, which is recommended to maintain competency with the FMD method [17]. When the images were chosen for analysis, the boundaries for diameter measurement were identified manually with an electronic caliper (provided by the ultrasonography proprietary software) in a threefold repetition pattern to calculate the mean value. In When the images were chosen for analysis, the boundaries for diameter measurement were identified manually with an electronic caliper (provided by the ultrasonography proprietary software) in a threefold repetition pattern to calculate the mean value. In our laboratory, the mean intra observer variability for FMD measurement for the operator recorded the same day, on the same site and on the same subject was 1.2 ± 0.2%.

our laboratory, the mean intra observer variability for FMD measurement for the operator recorded the same day, on the same site and on the same subject was 1.2 ± 0.2%. FMD were calculated as the percent increase in arterial diameter from the resting state to maximal dilation.

#### 2.5.2. Post Saturation Diving Decompression Vascular Gas Emboli (VGE)

The echocardiographic VGE signals over the 1 min recording were evaluated by framebased bubble counting as described by Germonpré et al. [18], but also scored according to the Eftedal-Brubakk categorical score [19].

Echocardiography was performed with a M-Turbo portable echocardiograph (Sonosite M-Turbo, FUJIFILM Sonosite Inc, Amsterdam, The Netherlands) used in a medical clinic included in the vessel while the patient was comfortably lying in a medical bed (Left Lateral Decubitus); four chamber view echocardiography loops were recorded on hard disk for offline analysis by three blinded evaluators. VGE numbers were counted at 30 min and 60 min post saturation decompression.

Evaluation of decompression stress and of the potential benefit of preventive measures has been done historically based on the presence or absence of clinical symptoms of DCS. However, for obvious ethical reasons, this is not acceptable in the field of recreational or professional diving [20]. Although imperfect, it is now accepted that research projects can use VGE data as a surrogate endpoint [6,21]. Different methods of detection of VGE are possible, such as Doppler ultrasonic bubble detectors or 2D cardiac echography [22]. During field studies, bubbles are usually detected in the right atrium, ventricle (right heart), and pulmonary artery. Then, the amount of detected VGE is graded according to different systems, either, categorical [19], semi-quantitatively [18] or continuous [21,22].

#### **3. Statistical Analysis**

The normality of data was performed by means of Shapiro–Wilk or D'Agostino-Pearson tests.

When a Gaussian distribution was assumed, and when comparisons were limited to two samples, paired or non-paired t-test were applied. If the Gaussian distribution was not assumed, the analysis was performed by means of a non-parametric Mann-Whitney U test or, a Wilcoxon paired test. Taking the baseline measures as 100%, percentage changes were calculated for each diver, allowing for an appreciation of the magnitude of change rather than the absolute values (one sample t-test). All statistical tests were performed using a standard computer statistical package, GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA).

A threshold of *p* < 0.05 was considered statistically significant. All data are presented as mean ± standard deviation (SD).

Sample size was calculated setting the power of the study at 95%, and assuming that variables associated with diving would have been affected to a similar extent as that observed in our previous studies [16–18] our sample reached 99%.

The linear regression line was performed using the least squares method and the lateral bands represented are in the 95% predictivity range.

#### **4. Results**

#### *4.1. Vascular Gas Emboli*

A very low number of bubbles were found in the participants after their decompression during their "bend-watch" period (the first 9 h).

Among all divers (*n* = 49), only three showed circulating gas emboli according to the EB scale that represented 0.2 ± 0.05 (mean ± SD) bubbles per heartbeat, which represents less than grade 1 on the EB grading scale in three divers. This is extremely low and doesn't allow statistical analysis. To allow the reader to compare with other diving situations this grading is 10 times lower than an average number of bubbles after a simple dive of 25 min at 25 m considered within safety limits [23].

#### *4.2. Flow Mediated Dilation*

FMD comparison between pre/post dive situation and control values is shown in Figure 4. Flow Mediated Dilation is calculated as the percentage increase of arterial diameter after an occlusion period (5 min); this post occlusion dilation was normal in our divers in

pre-dive situations (107.15 ± 6.6%). After vascular occlusion, the dilation provoked by the imposed shear stress was around 7–10%. Taking the individual FMD of each diver as the baseline, the percentual mean reduction reaches 94.7 ± 0.9 % (*p* < 0.0001) during the first two hours after decompression and quickly recovers reaching 98.75 ± 0.91 (*p* < 0.0001) in the last two hours (6–8 h after decompression) (see Figure 4). pre-dive situations (107.15 ± 6.6%). After vascular occlusion, the dilation provoked by the imposed shear stress was around 7–10%. Taking the individual FMD of each diver as the baseline, the percentual mean reduction reaches 94.7 ± 0.9 % (*p* < 0.0001) during the first two hours after decompression and quickly recovers reaching 98.75 ± 0.91 (*p* < 0.0001) in the last two hours (6–8 h after decompression) (see Figure 4).

FMD comparison between pre/post dive situation and control values is shown in Figure 4. Flow Mediated Dilation is calculated as the percentage increase of arterial diameter after an occlusion period (5 min); this post occlusion dilation was normal in our divers in

*Medicina* **2022**, *58*, 1476 8 of 13

*4.2. Flow Mediated Dilation* 

**Figure 4.** Bar graph illustrating FMD changes during the first 2 h (First 120 min.) (*n* = 23) and last 2 h (*n* = 29) (7–9 h) after saturation decompression (\*\*\*\* = *p* < 0.0001) (One sample t-test). (FMD **Figure 4.** Bar graph illustrating FMD changes during the first 2 h (First 120 min.) (*n* = 23) and last 2 h (*n* = 29) (7–9 h) after saturation decompression (\*\*\*\* = *p* < 0.0001) (One sample t-test). (FMD Changes are presented compared to predive values represented by the dotted line at 100%).

Changes are presented compared to predive values represented by the dotted line at 100%). Our data suggest that total vascular function recovery has not yet reached 8 h after the end of decompression. We then computed a best fit equation to extrapolate the time Our data suggest that total vascular function recovery has not yet reached 8 h after the end of decompression. We then computed a best fit equation to extrapolate the time needed to achieve recovery. The linear regression line and the equation are shown in Figure 5. *Medicina* **2022**, *58*, 1476 9 of 13

needed to achieve recovery. The linear regression line and the equation are shown in Fig-**FMD Recovery Time**

**Figure 5.** FMD evolution after exiting saturation the linear solution has been selected as the best fit approach, and the dotted lateral bands represent the 95% prediction bands. **Figure 5.** FMD evolution after exiting saturation the linear solution has been selected as the best fit approach, and the dotted lateral bands represent the 95% prediction bands.

Few scientific studies have been performed in real commercial saturation conditions

Our goal in this experiment was to document vascular recovery post saturation diving (after decompression). Vascular gas emboli are probably involved in the post dive reduction of FMD. Nevertheless, the available literature refrains us to draw a direct link between FMD reduction and VGE, since micro and macro vascularization react differently [28], and different preconditioning procedures before diving have specific actions inde-

In a recent experiment, a similar reduction in FMD was found in a setting excluding bubble formation, but a significant change in FMD was demonstrated depending on the

Moreover, in this experimental setting, we only saw minimal levels of bubbles allowing for neglecting this stressor in such saturation decompression procedures. Decompression bubbles are very likely not to be found post decompression after saturation diving. Further investigations are needed to monitor bubbles production after excursions while

A nitric oxide (NO) mediated change in the surface properties of the vascular endothelium favoring the elimination of gas micronuclei has previously been suggested to explain this protection against bubble formation [31]. It was shown that NO synthase activity increases following 45 min of exercise, and, if done before a dive, it reduces VGE [32]. In saturation, although work can be considered as an exercise, it should be considered that

cost of accommodating the scientific team onboard. Available studies are related to the subjective evaluation of saturation operations by the divers themselves [1]. More advanced studies such as evolution of plasma or blood derived measurements have been conducted [2,24]. Given the difficulties to achieve blood sampling, other studies are conducted based on salivary, urine, epithelial, or other minimally invasive sampling tech-

pendently on FMD and VGE, while others interfere with both [29].

oxygen partial pressure of the breathed gas [30].

the divers are otherwise sedentary.

being in saturation or during the decompression phase.

**5. Discussion** 

niques [24–27].

#### **5. Discussion**

Few scientific studies have been performed in real commercial saturation conditions during the last ten years. These studies are difficult because of the offshore constraints and project planning that do not allow much time for scientific testin—not to mention the cost of accommodating the scientific team onboard. Available studies are related to the subjective evaluation of saturation operations by the divers themselves [1]. More advanced studies such as evolution of plasma or blood derived measurements have been conducted [2,24]. Given the difficulties to achieve blood sampling, other studies are conducted based on salivary, urine, epithelial, or other minimally invasive sampling techniques [24–27].

Our goal in this experiment was to document vascular recovery post saturation diving (after decompression). Vascular gas emboli are probably involved in the post dive reduction of FMD. Nevertheless, the available literature refrains us to draw a direct link between FMD reduction and VGE, since micro and macro vascularization react differently [28], and different preconditioning procedures before diving have specific actions independently on FMD and VGE, while others interfere with both [29].

In a recent experiment, a similar reduction in FMD was found in a setting excluding bubble formation, but a significant change in FMD was demonstrated depending on the oxygen partial pressure of the breathed gas [30].

Moreover, in this experimental setting, we only saw minimal levels of bubbles allowing for neglecting this stressor in such saturation decompression procedures. Decompression bubbles are very likely not to be found post decompression after saturation diving. Further investigations are needed to monitor bubbles production after excursions while being in saturation or during the decompression phase.

A nitric oxide (NO) mediated change in the surface properties of the vascular endothelium favoring the elimination of gas micronuclei has previously been suggested to explain this protection against bubble formation [31]. It was shown that NO synthase activity increases following 45 min of exercise, and, if done before a dive, it reduces VGE [32]. In saturation, although work can be considered as an exercise, it should be considered that the divers are otherwise sedentary.

It appears that FMD seems more linked to oxygen partial pressure changes during diving, whereas VGE are more depending on preexisting gas micronuclei population [33,34] in the tissues and vascular system and coping with inflammatory responses [29,35].

FMD is a marker of endothelial function and is reduced in the brachial artery of healthy divers after single or repetitive dives [29,35]. This effect does not seem to be related to the amount of VGE and was partially reversed by acute and long-term pre-dive supplementation of antioxidants, implicating oxidative stress as an important contributor to post-dive endothelial dysfunction [36,37].

Decreased nitroglycerin-mediated dilation after diving highlights dysfunction in vascular smooth-muscle cells as possible etiology of those results [37].

Very recent data show that the FMD reduction encountered after a single dive without the presence of VGE, is comparable to the reduction found with the presence of VGE [30].

The divers that volunteered in our saturation experiment were taking some antioxidant "medication" (see Table 3) as a protective measure, the trend of our data doesn't show a clear inflexion for some participants that could be explained by antioxidants intake, although 60% of the divers declared doing so.

A recent manuscript [25] shows very interesting results allowing for following the oxidative defenses status post saturation. Although the depth and duration differ from our setting, the recovery time for NOx is around 24 h.

Our data are in tune with the NOx returning to baseline, since FMD is closely related to the availability of nitric oxide (NO), and we can see from our results that FMD almost fully recovers after 8 h. If we apply the formula extracted from our data the mean time needed to reach 100% recovery would be around 540 min (9 h) and in the least predictive range (−95%) around 600 min (10 h) would be needed to fully recover, which is confirmed by Mrakic-Sposta et al. (2020) results. In fact, their results show that 24 h post saturation, the ROS

(Reactive Oxygen Species) are still significantly higher than baseline, but concomitantly TAC (Total Antioxidant Capacity) is also still high. From our results we can consider that the vascular dysfunction has already recovered and that the balance between antioxydants and prooxydants is clearly efficient and therefore fostering recovery. Another parameter measured by Mrakic-Sposta et al. [25] was IL-6 (Interleukin-6), this citokine reflects pro/antiinflammatory response, and was increased during saturation but it was not significantly different than baseline 24 h post saturation.

#### **6. Limitations**

Strengths:


Weaknesses:


#### **7. Conclusions**

This monitoring session has no equivalent in the commercial diving industry because of its duration (6 month), conditions (a working diving support vessel) and the large number of divers who volunteered for the study. It was the first time that the possibility for assessing onsite the vascular function of divers was offered during actual saturation diving operations. The study not only confirmed the role of inflammation and oxidative stress in saturation diving but it also permitted to obtain an estimation of the recovery time needed.

The lessons learnt from this experiment were that (1) scientific studies are possible even on a diving support vessel during operations under extreme environmental conditions. (2) Both national safety rules seem to provide health of the divers. (3) The equipment selected for the study was too heavy to be easily mobilized, and it could only work at ambient pressure and required a specific expertise. The future monitoring sessions, if any, should aim at using simpler equipment, which could be operated by the divers themselves inside the chamber, under pressure. Future experiments should include pressure resistance bubble measuring devices such as the O'Dive system tool to ascertain a minimal bubble number in the sub-clavian vein during excursion dives and during decompression [38].

**Author Contributions:** All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Conceptualization, J.-P.I., S.-M.E. and C.B. Onboard monitoring J.-P.I.; Writing, J.-P.I., S.-M.E. and C.B.; Review and editing, J.-P.I., S.-M.E. and C.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** Technip FMC, generously hosted the study and the research team on one of their diving support vessels which is a major contribution. The work is also supported with a grant by The Scientific and Technological Research Council of Turkey (TUBITAK) (for S.-M.E.) The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and the decision to submit the manuscript for publication.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and received ethical approval from Local Ethic Committee Brussels (Academic Bioethical Committee, Brussels, Belgium. Reference Number: B200-2009-039). Date: 10 October 2015.

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

**Data Availability Statement:** Data are available at request from the authors.

**Acknowledgments:** The authors are grateful to Technip FMC that supported the study and, in particular to Andy Butler, Diving Technical Authority & Lead; Technip FMC who actively promoted the study within the Company and organized the venue of the team onboard the Deep Arctic DSV. We also want to express special acknowledgements to the divers who volunteered to participate to study and all the surface operational personnel who made the monitoring sessions possible.

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

#### **Abbreviations**


#### **References**


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