Heart Rate and Rhythm Changes in Dogs Treated in a Hyperbaric Oxygen Chamber

Abstract

In veterinary medicine, hyperbaric oxygen chamber treatment (HBOT) is gaining popularity. Therefore, an increasing number of patients referred for this therapy are being recorded, mainly due to ischemic events, wound healing support, and a high risk of reperfusion damage. During the HBOT procedure, several changes occur in the body’s micro- and macroenvironments. This study involved 34 dogs of various ages and health statuses. The atmospheric conditions in the test hyperbaric chamber included a pressure of 1.5 atmosphere absolute (ATA) at 100% oxygenation. The individuals were divided into three groups: (1) individuals with degenerative mitral valve disease (DMVD), (2) individuals with diseases other than cardiac issues, and (3) healthy individuals who qualified for the HBOT procedure. The period of measurement using the Holter apparatus was divided into four stages: 30 min before the subject’s placement in the hyperbaric chamber; a 4-min compression period (setting chamber conditions); a 90 min HBOT period; and a 1 min decompression period of the hyperbaric chamber. During the HBOT, there was a statistically significant decrease in heart rate in groups 2 and 3 compared to group 1. The heart rate in group 1 remained unchanged through every period of the study. In addition, some of the dogs developed respiratory arrhythmia; in two dogs, premature ventricle beats occurred. The changes observed during the ventricular period indicate that the HBOT procedure causes a significant change in heart rate in dogs without cardiac diseases.

1. Introduction

Studies on dogs in a hyperbaric oxygen chamber date from the 1960s, when the effects of short-term treatment on the cardiovascular system or cases of cerebral ischemia were examined [1,2,3,4]. While these were laboratory models, it was already known that they were important counterparts in the context of human medicine. However, they were not the only models used in research as others focused on procedures using rats, cats, pigs, and other animal species [5]. Furthermore, with the development of veterinary medicine services, it was recognized that hyperbaric oxygen chamber treatment (HBOT) would also work well to treat certain diseases in dogs. Among the indications for HBOT treatment in dogs are wound healing, diseases related to the nervous system, or ischemic events [6,7]. Therefore, the growing popularity of HBOT services has attracted the interest of researchers and clinicians, as reflected in retrospective studies and case reports [8,9,10,11,12,13,14]. Given the possibilities and benefits of HBOT, its use is gaining popularity in veterinary medicine, especially since this procedure carries a low risk of severe complications [11,12].

Such sustained conditions of hyperoxia result in vasoconstriction and hypertension due to stimulation of the baroreflex. Furthermore, by decreasing the activity of the sympathetic nervous system and increasing the activity of the parasympathetic system, bradycardia occurs with a decrease in cardiac output [15]. These mechanisms have been described in a number of papers on the physiology and pathophysiology of the cardiovascular system under hyperbaric chamber conditions [5]. However, apart from a composite human patient group [16], these changes have not been described in dogs under veterinary care. It is indicated that heart rate reductions are largely dependent on oxygen; so, pressure conditions should not have as strong an effect as that of high oxygen saturation [17]. Nevertheless, it is known that acquired heart disease and the changes it causes, especially those associated with degenerative mitral valve disease, lead to hyperactivation of the sympathetic nervous system, with a decrease in vagus nerve tone [18]. This leads to an increased heart rate, often resulting in tachycardia, as noted particularly in mitral valve degenerative disease [18]. In addition, available studies in dogs subjected to HBOT describe repeated sessions at different intervals. One of these described one or at least two targeted therapies within a 24 h period in dogs with acute clinical signs [11]. In contrast, another study indicated the tolerability of the chamber and its effect on wound healing in dogs subjected to a single HBOT for 7 consecutive days [14]. However, in nervous system diseases, the number of treatments can reach more than 70 sessions, making the procedure associated with high safety and tolerability [12]. Furthermore, HBOT did not show any negative effects on females after neutering 6 and 18 h after surgery [13]. All procedures from the mentioned studies took place under 100% oxygen saturation with different atmospheric pressures. This varied between 1.5 and 3 ATA, depending on the case being undertaken. Apart from studies on laboratory animals including healthy dogs, no studies of heart rate variability using veterinary patients of different health statuses have been carried out to date. It has not yet been described how dogs with degenerative diseases of the spine, joints, or acquired heart disease respond to changes associated with hyperbaric chamber conditions. Furthermore, the risks associated with potential arrhythmias that could be generated by HBOT have not been described.

Therefore, the present study aimed to evaluate the effect of a single hyperbaric chamber treatment at 1.5 ATA under 100% oxygenation on the heart rate and rhythm in dogs of different health statuses.

2. Materials and Methods

2.1. Patient Selection

The procedures performed in the hyperbaric chamber were performed at a certified external facility not affiliated with the Institute of Veterinary Medicine in Torun. In the first stage, patients were referred to HBOT by independent veterinarians as a result of an initial examination and diagnosis. Healthy individuals subjected to this study were included according to the owners’ wishes. Before the study, the owners gave their full consent to the procedures. Furthermore, an extensive interview about the patient’s history, medications taken, and past illnesses or procedures was conducted with the animal’s owners before the HBOT procedure. In addition, each patient underwent a complete clinical examination, including the measurement of their temperature, an examination of the mucous membranes, auscultation of the heart, lungs, and trachea, and palpation of the lymph nodes and abdomen. Full echocardiography and electrocardiography were also performed on all animals prior to the study. Patient randomization was made possible due to the referral of patients by independent veterinarians from different parts of the country. Therefore, the formation of study groups was executed on the basis of the individuals sent for the HBOT procedure. Criteria for excluding patients from the study included pneumothorax, mediastinal emphysema, tracheal collapse, and epilepsy or other conditions causing uncontrolled seizures; patients with cardiac arrhythmias, individuals with implanted pacemakers, and those requiring constant inhalation were also excluded.

The consent of the ethical committee for animal welfare was not required to perform the procedures because the HBOT procedures were performed as part of activities in the field of veterinary medicine, following applicable Polish law; according to paragraph 2 of the Law on the Protection of Animals Used for Scientific or Education, this Act does not apply to veterinary services within the meaning of the Act of 18 December 2003 on animal treatment establishments.

2.2. Treatment Procedure

The patients underwent HBOT in a single-place chamber with 100% oxygen saturation in a hyperbaric chamber at a pressure of 1.5 ATA. Holter monitoring was performed using a Holter device Aspel AsPEKT 712 v. 301 (Aspel, Poland). Due to the high randomization in terms of reported cases, it was decided to group individuals during data analysis. The individuals were divided into three groups: (1) individuals with degenerative mitral valve disease (DMVD), (2) individuals with diseases other than cardiac issues, and (3) healthy individuals qualified for the HBOT procedure. The ECG recording was divided into four periods: (1) a 30 min recording before the start of the procedure, (2) 4 min of pressure increase in the chamber (compression), i.e., achieving appropriate atmospheric and oxygen conditions, (3) HBOT treatment lasting 90 min, and (4) 1 min decompression of the hyperbaric chamber. Heart rate and potential arrhythmias were assessed. Due to the possibility of an explosion inside the chamber [19], metal collars and leashes were removed from the patients. Additionally, no electrical appliances or toys were allowed during the treatment except for dog beds to ensure psychological comfort. Seven electrodes were placed in the area from the sternum to the posterior wall of the scapula, three on the left side and four on the right side, following the oblique position standards [20]. The device and electrodes were carefully secured with veterinary adhesive bandages to prevent the risk of sparks and fire.

The chamber had a built-in window that allowed the patient’s condition and reactions to be monitored in the event of adverse reactions. The dogs were observed continuously during compression and decompression, during which the risk of adverse effects is greatest [11]. During a 90 min session in the hyperbaric chamber, the dogs were observed five times (every 15 min) for 30 s.

The hyperbaric chamber was operated by a person properly trained to use the entire system following the current guidelines.

Analysis of the Holter recordings was performed by an experienced veterinarian specializing in veterinary cardiology using licensed ASPEL HTL-712 v.301 Holter ECG recorder with ASPEL HLT HOLCARD-712 v.301ALFA software. The heart rate, morphology of individual ECG waves, and the occurrence of possible arrhythmias before and after the start of the HBOT procedure were evaluated. Comparisons were also made between the voltages of selected waves in terms of the change in amplitude of each QRS complex as well as the P and T waves. Comparisons were made at each stage of the procedure and were made possible by the software mentioned above.

Side effects that occurred during 90 min of hyperbaric chamber therapy at a pressure of 1.4 ATA were also observed. Possible side effects were divided into minor and major adverse effects according to the formula used in another study [11].

2.3. Statistical Analysis

Statistical software 13.3 (TIBCO Software Inc., Palo Alto, CA, USA) was used to perform the analysis. Continuous variables were presented with medians and interquartile ranges (IQRs). Discrete variables are presented as numbers and percentages. The Shapiro–Wilk test was used to assess the distribution of variables. Levene’s test was used to check the homogeneity of the variables’ variance. Comparisons between any groups of animals were made using the Student’s T test for normally distributed variables and the Mann–Whitney U test for variables with non-normal distribution. A comparison of the variables between the studied periods was performed using a repeated-measures ANOVA and the NIR post hoc test for normally distributed variables with homogenous variance; for variables with a non-normal distribution or non-homogeneous variance, the Kruskal–Wallis test was used, and repeated measurements were compared using a Friedman ANOVA and the Kendall concordance test. p values < 0.05 were considered significant in all analyses.

3. Results

Thirty-four dogs with different health statuses were enrolled in the study. Among them were 11 completely healthy individuals undergoing prophylactic therapy at the request of their owners and 25 individuals with various medical conditions, including neurological problems, skeletal-related conditions, and cardiovascular diseases, as shown in

Table 1. Demographic and health variables of the animals included in groups 1 (degenerative mitral valve disease), 2 (non-cardiac diseases), and 3 (healthy individuals).

	Group 1	Group 2	Group 3
N	6	15	11
Number of different breeds	2	7	6
Age (years)	13 (4–22)	9.58 (3–14)	9.6 (4–14)
Body weight (kg)	14.8 (4–27)	26.2 (4.4–44)	17.3 (8–23)
Temperature	38.1 (37.5–38.7)	38.6 (37.8–39.5)	38.5 (37.9–39.2)
Sex (female/male)	3:3	8:7	6:5
Health condition	Degenerative mitral valve disease (6)	Elbow joint dysplasia n = 1 Hip joint dysplasia n = 2 Hip joint degeneration n = 2 Cruciate ligament rupture n = 2 Spondylarthrosis n = 5 Ischemic stroke n = 3	Healthy

The age and weight of the patients are presented as averages and their minimum and maximum ranges.

. The largest group of dogs comprised a mix of breeds (12 dogs): American Staffordshire Terriers (3), Border Collies (3), and Huskies (3). Fifteen males and seventeen females were included in the study. After the qualification examinations, 32 dogs were qualified to participate in the procedure due to the exclusion criteria. Two patients were excluded from the study due to severe arrhythmias. The first involved a Tosa Inu dog with advanced DMVD and tricuspid regurgitation, ascites, and grade III atrioventricular block with slow ventricular action. The second patient, a mixed-breed dog, had fixed bigeminy alternating with trigeminy with premature supraventricular and ventricular origination.

The median values and interquartile ranges of the heart rates for the different stages of the procedure are provided in

Table 2. Representation of heart rate variables at each part of the hyperbaric oxygen treatment procedure.

Variable	Group 1 n = 6	Group 2 n = 15	Group 3 n = 11
Number of QRS bases (median, IQR)	4061.5 (3588–4350)	3603 (2796–4559)	3973 (3444–4358)
Min HR base (median, IQR)	87 (75–100)	62 (51–94)	66 (55–88)
Max HR (median, IQR)	206.5 (204–210)	205 (154–215)	201 (195–213)
Average HR (median, IQR)	134.5 (112–145)	121 (101–153)	128 (104–135)
Number of QRS compressions (median, IQR)	484 (410–602)	483 (379–547)	603 (448–624)
Min HR compression (median, IQR)	90.5 (73–114)	84 (68–110)	83 (72–97)
Max HR compression (median, IQR)	188 (181–198)	173 (157–196)	195 (167–220)
Average HR compression (median, IQR)	130 (103–151)	131 (107–140)	127 (119–157)
Number of QRS HBOTs (median, IQR)	1025.5 (7504–12,586)	8379 (7553–9224)	8983 (8041–11,236)
Min HR HBOT (median, IQR)	65.5 (61–77)	50 (42–62)	53 (39–65)
Max HR HBOT (median, IQR)	208.5 (204–211)	204 (160–220)	194 (168–215)
Average HR HBOT (median, IQR)	127 (996–148)	96 * (86–107)	107 * (90–134)
Number of QRS decompressions (median, IQR)	127 (96–148)	131 (71–109)	147 (136–163)
Min HR decompression (median, IQR)	109 (97–124)	89 (71–109)	101 (59–112)
Max HR decompression (median, IQR)	197 (181–209)	155 (143–191)	173 (169–191)
Average HR decompression (median, IQR)	157 ^ (149–168)	123 (115–141)	142 (118–163)

The values provided in the table are shown in beats per minute (bpm). Group 1—DMVD; Group 2—diseases other than cardiac diseases; 3—healthy; IQR—interquartile range; bpm—beats per minute; HR—heart rate; Min—minimum; Max—maximum; HBOT—hyperbaric oxygen treatment; base—30 min before procedure HBOT started. * within groups, values between the HBOT period and pre-study period, chamber compression period, and decompression period differ significantly (p < 0.05). ^ Within groups, values between decompression period and pre-study period, chamber compression period, and HBOT period differ significantly (p < 0.05).

. During the procedure, a significant decrease in heart rate was observed in group 2 and group 3 (p < 0.05) in a period of 1.5 h of the HBOT period compared to the rest of the periods included in the procedure (30 to the 30-min recording before the start of the procedure, chamber compression, and the decompression period of the chamber). This significance was not observed in group 1. In the same group, there was a significant increase in heart rate during the chamber decompression period (p < 0.05) compared to the rest of the periods of the HBOT procedure (30-min recording before the start of the procedure, HBOT, and chamber compression period). These data are shown in Table 2 and Figure 1.

In two patients (groups B and C), at the end of the HBOT period, after a decrease in heart rate, premature ventricular beats appeared (Figure 2), which were not observed in the remaining phases of the study. During the rest of the study period, a regular sinus rhythm remained unchanged. In 16 sick dogs, during the HBOT procedure, respiratory rhythm disturbances were found, which were not observed in the 30 min examination before the procedure or during the periods of chamber compression and decompression. In all dogs, changes in the voltages of individual P-waves, ST segments, and QRS complexes were observed during all stages of the study. In addition, no changes related to the PQ segment and QT segment prolongation were observed in any of the dogs studied. Furthermore, there were no side effects associated with the time spent in the hyperbaric chamber and the changes in barium pressures or high oxygen saturation.

4. Discussion

Hyperbaric chamber therapy has found application in many fields of veterinary and human medicine. However, in order to assess the safety and validity of this therapy in humans, different animal species have been used for studies. They describe the body’s response in cerebral ischemic conditions in rats [21], cats [22], or pigs [23]. Furthermore, they show the benefits of HBOT in different animal species in the healing of burn wounds in guinea pigs [24] as well as mechanical trauma in pigs [25,26], mice [27], rats [28], or dogs, being one of the most important clinical indications used in veterinary practice [29,30]. They also describe ototoxicity reactions in guinea pigs [31] or induced endotoxemia in horses [32]. Finally, they describe the hemodynamic mechanisms associated with high oxygen exposure under high-pressure conditions. Admittedly, the detailed mechanism is not known; however, it was indicated that bradycardia induction occurs through two mechanisms: (1) an oxygen-dependent mechanism and (2) a non-oxygen-dependent mechanism. In the oxygen-dependent mechanisms, rapid vasoconstriction of the peripheral vasculature occurs due to the direct effect of oxygen on the vessel wall. The mechanisms of vasoconstriction are not fully understood; however, high plasma oxygen concentrations lead to the displacement of nitric oxide from the vascular wall, one of the best known vasodilators [33]. Additional hypotheses are based on the inhibition of prostaglandin synthesis [34] or the enhancement of the activity of endothelin 1 [35,36]. Furthermore, this results in hypertension and activation of the baroreceptor reflex of the aortic arch and carotid sinuses. This leads to a decrease in sympathetic nervous system activity in favor of increased vagal nerve tone. The result is bradycardia, reduced myocardial contractility, and, consequently, reduced cardiac output [37,38,39]. Furthermore, it has been shown that 100% oxygen saturation, regardless of the prevailing pressures, leads to bradycardia mainly due to the direct effect of high oxygen pressure on the myocardium [40]. The oxygen-independent mechanism is mainly due to a reduction in sympathetic nervous system tone associated with lower levels of circulating catecholamines, which further enhances the bradycardic effect [41,42]. Nevertheless, due to the high concentration of pure oxygen dissolved in blood serum, tissue oxygenation remains at a similar level despite severe peripheral vasoconstriction [5]. Furthermore, blood distribution in the parenchymal organs increases in favor of peripheral circulation [43], sustaining vital organs with sufficient blood perfusion; thus, it is a safe and well-tolerated procedure. Standard conditions in the HBOT involve pressures ranging between 2 and 3 ATA with 100% oxygen saturation as they bring numerous benefits without much risk of adverse effects [44]. Some protocols are based on sessions at conditions above 3 ATA; their main aim is to rapidly counteract gas embolism. For this reason, treatment sessions can even take place under 6 ATA conditions [45]. Nevertheless, many studies prove that HBOT therapy with a pressure equal to or greater than 1.4 ATA with 100% oxygen saturation leads to similar changes in the body as in the case of the standard procedures. These are associated with a significant increase in the production of ROS and antioxidant reactions, as well as with the cardiovascular system [37,46]. They are mainly attributed to conditions associated with 100% oxygen saturation, but it has been indicated that hyperbaric conditions with a normal physiological oxygen concentration mobilize proangiogenic and hematopoietic stem progenitor cells, which may be another therapeutic target [47]. Positive aspects of modified hyperbaric chamber therapy are also indicated. During sessions under conditions of 30% oxygen saturation at a pressure of 1.3 ATA, no changes in heart rate, blood gas parameters, or heart rate variability were observed; however, significantly higher glutathione peroxidase activity and lower skin conductance and cortisol serum levels were indicated [48]. Therefore, conditions described as low-HBOT may be useful in animals exposed to oxygen toxicity, but further studies are needed to establish potential therapeutic targets for these conditions. In the present study, animals were subjected to HBOT monotherapy under conditions of 100% oxygen saturation at 1.5 ATA to determine its effect on electrocardiographic parameters and heart rate in dogs with different health statuses. Our study confirmed previous reports in other animal species, showing a significant reduction in heart rate in dogs undergoing the HBOT procedure compared to the conditions before the procedure, the compression period, or the decompression period in individuals with non-cardiovascular disease as well in healthy individuals. There was a significant decrease in heart rate in these dogs, which may suggest the induction of a bradycardia mechanism due to exposure to the set chamber conditions, especially 100% oxygen saturation.

Interestingly, no significant differences in heart rate were observed during the HBOT procedure in the group of individuals with DMVD. However, this may be related to the low group of animals in this group. Nevertheless, it is worth noting the interesting role of the sympathetic nervous system in heart failure [49]. In the early stages of the disease, the regional influence of the sympathetic nervous system plays an important role, especially in the area of the heart and kidneys. This increases heart rate and blood pressure, as well as norepinephrine levels, leading to systemic hypertension [50]. In the chronic course, cardiac diseases such as left ventricular myocardial hypertrophy, arrhythmias, or ischemia occur [51]. In addition, these changes can be exacerbated in patients with degenerative mitral valve disease. This results in volume overload of the left atrium and left ventricle, tachycardia, and decreased cardiac output, which, together with the increased afterload, can contribute to the development of heart failure [52]. This condition is expressed by overactivation of the sympathetic nervous system tone and decreased vagal tone, the alteration of which is more pronounced with the development of DMVD [18,53]. In the present study, all the patients in the DMVD group showed changes related to cardiac remodeling, which could indicate a chronic course of the disease. Therefore, it can be assumed that there was a significant override on the sympathetic/parasympathetic system axis with a significant predominance of the former. Its strong activation led to the silencing of the vagus nerve, which was also not overcome by the stimulus of the hyperbaric chamber session. This indicates a very strong involvement of the sympathetic nervous system in DMVD patients. However, this hypothesis requires further research on a larger group of animals.

It is also worth emphasizing the fact that in subjects with sinus tachycardia (most likely caused by high agitation) recorded throughout the entire 30 min study period, a decrease in heart rate was also noted after being exposed to hyperbaric chamber conditions, which indicates the independent impact of high oxygen saturation on the above-described mechanisms, especially the inhibition of the sympathetic nervous system. In a study conducted by Ishibashi et al., 2015 [48], it was indicated that HBOT at a lower pressure (1.3 ATA) and 30% oxygen saturation in beagle dogs may significantly affect antioxidant processes, but it does not affect heart rate variability. Nevertheless, the study was conducted using a small number of patients and, more importantly, a low percentage of oxygen saturation, which may not have been sufficient to induce oxygen-dependent bradycardia mechanisms [48].

The patients in the present study varied widely in age, which would seem, especially in older individuals, to significantly affect the results of the study. Nevertheless, it has been indicated that with age, the heart rate tends to change less abruptly due to the establishment of a balance between decreasing sympathetic nervous system activity and unchanged parasympathetic system activity under physiological conditions [54,55]. However, under hyperbaric hyperoxia, the response of the parasympathetic system to ventricular conditions in aged individuals is preserved and fully safe [56].

In two patients, at the end of the HBOT procedure, additional premature ventricular beats occurred, induced by bradycardia and prolongation of the R-R interval. Similar observations were made by Stuhr et al., 1994 [57], where arrhythmia occurred, as a result of bradycardia in conscious rats. Furthermore, Simon and Tobrati, 1982 [58] also observed dysrhythmic events in rats, including atrial extrasystoles, wandering pacemakers, and second-degree atrioventricular block after 2 h of exposure to 3 ATA HBOT. Although these were isolated cases, it should be kept in mind that prolonged exposure to this condition may provoke syncope [59] or lead to life-threatening arrhythmias [60]. Nevertheless, in our study, no changes in the length of the PQ segment were observed, which excludes the occurrence of atrioventricular blocks or pre-excitation syndromes. Moreover, no prolongation of the QT interval was observed, which significantly reduces the risk of life-threatening attacks of ventricular tachycardia (torsade de point). This fact has been confirmed in human studies [61,62]. Voltage changes associated with an increase or decrease in the amplitude of individual components of the electrocardiogram appeared during changes in heart rate. A similar phenomenon has been observed in humans during paroxysmal supraventricular tachycardias, where it was indicated that the changes associated with an increase in amplitude were probably due to reduced blood flow to the ventricles, thereby reducing the distance of the left ventricle from the precordial electrodes [63,64]. This theory could be reflected in the case of the patients in this study due to the characteristic arrangement of the electrodes of the Holter device on the side of the chest at the level of the heart. Specifically, when the heart rate slows down, there would be an increased inflow to the ventricles and a shortening in the distance of the left ventricle from the electrodes of the Holter device, thus increasing the voltage of the waves of individual leads and vice versa. In states of increased heart rate, blood flow to the ventricles would decrease, leading to an extension of the distance of the left ventricle from the electrodes of the Holter device, thus decreasing the voltage of individual waves of the electrocardiogram. Additionally, in some patients, respiratory arrhythmia was observed during the HBOT procedure and during the 30 min examination before the start of the procedure, as well as during the compression and decompression phases. These changes have also been observed in humans, where a significant exacerbation of respiratory arrhythmia occurred [56,62]. In the present study, we observed similar changes where dogs developed or exacerbated characteristics associated with respiratory arrhythmia as a result of the HBOT procedure.

During the chamber compression period, the HBOT procedure, and the decompression period, none of the side effects noted in the previous procedures were observed [11,12]. However, it should be borne in mind that the procedures performed in this study were performed under lower pressure and performed as monotherapy. Nevertheless, this fact confirms the safety of the dogs and their tolerance to the therapeutic procedures performed in a hyperbaric chamber despite different health statuses.

5. Conclusions

This study showed that HBOT treatment leads to significant changes in heart rate, as manifested by bradycardia in healthy individuals as well as in those with non-cardiovascular diseases. Interestingly, due to the slowing of the heart rate and a prolongation of the interval between QRS complexes, premature ventricular beats can occur. Considering the observed changes, one of the contraindications for HBOT should be bradyarrhythmia, due to the risk of arrhythmias being worsened and exacerbated during the procedure. Nevertheless, the lack of difference in the PQ and QT intervals indicates that in dogs unaffected by cardiac arrhythmias, the procedure is completely safe. Moreover, the changes in the voltages of the individual waveforms in all dogs indicate that it is probably due to changes related to the distance of the left ventricle, which depends on the heart rate, from the heart wall and the electrodes attached to it.

In addition, the lack of side effects observed in this study associated with the HBOT procedure that are known from previous reports indicates that hyperbaric chamber therapy is harmless and well tolerated by dogs.

6. Limitations and Future Perspectives

The main and most important limitation of the present study was the number of animals studied, especially in the group associated with degenerative mitral valve disease. Due to the under-representation of animals in this group, it is not entirely possible to accept these results as valid. Another limitation of this study was its heterogeneous patient group. The subjects tested were of different ages, health statuses, and breeds, which could have influenced the results of this study; additionally, the use of a single therapy and the lack of determination of the impact of several HBOT sessions on changes in heart rhythm and the potential for cardiac arrhythmias.

Due to the heterogeneous group of animals, it will be important to determine changes in cardiac performance in a large group of healthy dogs not affected by any disease entity that could affect the obtained results. In addition, due to the lack of significant changes during the HBOT procedure in individuals with DMVD, it will be important to validate the obtained results with a larger group of animals. In addition, it will be crucial to determine the activity of the vegetative system, pressure, and also important biomarkers responsible for vasocontraction-related processes both in monotherapies and multiple sessions. It will also be important to assess the systolic and diastolic function of the heart in response to given conditions.