Hyperbaric Oxygen Therapy in Patients with COVID-19

Для цитирования: С.С. Петриков, А.К. Евсеев, О.А. Левина, А.К. Шабанов, В.В. Кулабухов, Н.Ю. Кутровская, Н.В. Боровкова, Е.В. Клычникова, И.В. Горончаровская, Е.В. Тазина, К.А. Попугаев, Д.А. Косолапов, Д.С. Слободенюк. Гипербарическая оксигенация в терапии пациентов с COVID-19. Общая реаниматология. 2020; 16 (6): 4–18. https://doi.org/10.15360/1813-9779-2020-6-4-18. [На русск. и англ.] For citation: Sergey S. Petrikov, Anatoly K. Evseev, Olga A. Levina, Aslan K. Shabanov, Vladimir V. Kulabukhov, Natalia Yu. Kutrovskaya, Natalia V. Borovkova, Elena V. Klychnikova, Irina V. Goroncharovskaya, Elizaveta V. Tazina, Konstantin A. Popugaev, Denis A. Kosolapov, Dariya S. Slobodeniuk. Hyperbaric Oxygen Therapy in Patients with COVID-19. Obshchaya Reanimatologiya = General Reanimatology. 2020; 16 (6): 4–18. https://doi.org/10.15360/1813-9779-2020-6-4-18. [In Russ. and Engl.]


Introduction
First identified in December 2019 in Wuhan City, Hubei Province, China, the new acute respiratory infection COVID-19 caused by the coronavirus SARS-CoV-2 (2019-nCov) has now spread to all continents [1].At the time of writing, the number of cases of infection exceeded 21 million people, of which more than 770,000 were fatal.
However, despite the wide range of organs affected by coronavirus infection, the leading factor in the pathogenesis of disease and its complications is hypoxia [2,9], which follows several pathways [9]: -resulting from inflammatory process in the lung tissue, causing disruption of oxygen diffusion through the alveolar-capillary barrier; -due to impaired hemoglobin oxygen transport function caused by direct exposure to viral proteins; -because of alteration of microcirculation arising from microthrombus formation in capillaries.This process is multi-stage and involves several mechanisms [2].
One of the options for addressing this issue is to bypass the lungs using extracorporeal membrane oxygenation (ECMO) technology, i.e. direct oxygen saturation of hemoglobin using special equipment.The utilization of ECMO, however, is associated with several significant shortcomings and is usually used as a last resort.
On the other hand, hyperbaric oxygen therapy allows to increase oxygen content in tissues and overcome diffusion barriers.Moreover, unlike ECMO, which normalizes only the oxygen content in plasma and red blood cells, hyperbaric oxygen therapy (HBOT) is safe, has a minimum of contraindications and side effects and has a multifactorial effect by increasing: -diffusion of oxygen through the alveolarcapillary barrier, -oxygen solubility in plasma, -hemoglobin oxygen saturation, -delivery of oxygen to the microcirculation system and tissues [10].
The efficacy of HBOT has already been shown in patients with respiratory dysfunction, for example, as an adjuvant to the treatment of patients with chronic obstructive pulmonary disease [11].Importantly for COVID-19 treatment, HBOT has shown high efficacy in infectious complications when other methods have proved ineffective or inappropriate, as has been demonstrated in patients with anaerobic pleural pulmonary infection [12] and after lung transplantation [13,14].
In addition, a positive effect of HBOT on improvement of capillary proliferation and density of capillaries in microthrombosis has been described [20].HBOT increases production of vascular endothelial growth factor (VEGF) [21], as well as accelerates the induction of collateral blood flow in thrombosis [22].
At the same time, failure to understand the mechanism of action of hyperbaric oxygen therapy leads to misconceptions about its ineffectiveness and limits its implementation in clinical practice.Thus, there is a widespread opinion about the activation of free-radical processes and the depletion of the body's antioxidant protection system by HBOT, although, on the contrary, under therapeutic modes (no more than 2 ATA) HBOT does not cause oxidative stress [18,19], and, moreover, reduces lipid peroxidation processes [23,24] and stimulates antioxidant production [25].
The aim of this study was to examine the efficacy of hyperbaric oxygen therapy and its effect on oxidative stress and apoptosis in patients with new coronavirus infection COVID-19.

Materials and Methods
We examined 90 patients (44 men, 46 women, mean age 60.7±13.5 years) diagnosed with new coronavirus infection caused by SARS-CoV-2 virus addmited to N. V. Sklifosovsky Research Institute for Emergency Medicine.All patients received standard therapy in accordance with the guidelines of the Ministry of Health of Russian Federation and internal protocols.Patients were divided into 2 groups using the envelope method in the ratio of 1:2 (control:study group).Three patients were excluded from the study group due to their refusal from procedures.
Out of 87 patients, 57 (who underwent HBOT sessions) comprised the study group, and 30 patients who were not subjected to HBOT sessions represented the control group (table 1).
In the study group, 19 (33.3%) patients were assessed as having moderate severity disease (CT 1-2) and 38 (66.7%) were recognized as patients with severe disease (CT 3-4), 43 (75.4%)participants needed respiratory support (oxygen through the nasal cannula or face mask with a flow of 3-6 l/min, in severe cases using high-flow oxygen therapy (HFOT) or non-invasive lung ventilation (NILV).The control group patients were distributed according to the severity of condition, 24 (80.0%) of them needed respiratory support.
The HBOT procedures were performed in the resuscitation chamber Sechrist 2800 (USA) in 1.4-1.6ATA mode for 40 minutes.Depending on the timing of HBOT with respect to the moment of patients' admission, the study group was further divided into two additional subgroups (table 2).In total, the patients received 247 sessions of HBOT.Before and after each session of HBOT, hemoglobin oxygen saturation (SpO 2 ) was measured.
The markers of oxidative stress and apoptosis of blood lymphocytes were analyzed in 18 patients of the study group.Blood was drawn using a vacuum system (Vacutainer ® Hemogard™, Vacutainer ® SSTTM II Advance and Vacutainer ® EDTA (BD, UK)).
To assess the severity of oxidative stress, the products of lipid peroxidation (LPO), the antioxidant system status before the HBOT session and the platinum electrode open circuit potential (OCP) before and immediately after the HBOT session were measured.The content of LPO products was estimated based on serum malone dialdehyde (MDA) [27].The antioxidant system status was assessed by the serum total antioxidant activity (TAA) measured by the spectrophotometry using AU 2700 biochemical analyzer (Beckman Coulter, USA) with the TAS kit (Randox, UK).Measurements of OCP of platinum electrode in blood serum were carried out with IPC Compact potentiostat (NTF «Volta» Ltd., Russia) according to the predefined technique [28].
Statistical data analysis was performed using the Statistica 10 software package (StatSoft, Inc., USA).Descriptive statistics of quantitative features were presented as M±m.The groups under study were compared using the Mann-Whitney U-criterion and the Wilcoxon crite-

Results and Discussion
All examined patients with new coronavirus infection COVID-19 had positive progress manifested as reduced shortness of breath and improved overall health.
As might be expected, the inclusion of HBOT as a part of comprehensive treatment plan led to a significant increase in SpO 2 both during the session and over the HBOT course (fig. 1, a).Thus, after the 1st session of HBOT, the hemoglobin oxygen saturation increased from 91.3±5.9% to 98.4±3.0%, while after the 4 th session further increase was recorded, from 93.4±4.6% to 98.8±2.1%.At the same time, a significant increase in SpO 2 (above 90%) was observed even in patients with SpO 2 values below 80% prior to the HBOT session when breathing ambient air.Positive changes in hemoglobin oxygen saturation were noted even after the end of the HBOT sessions, as evidenced by the values of SpO 2 , which averaged 95% and more.The graphs reflecting changes in values for the study group were limited to 7 sessions of HBOT, which corresponded to the course completion in 86,5% of patients.
In the control group and study subgroup 1 we compared the changes in hemoglobin oxygen saturation (table 3), the severity of condition and the required type of respiratory support (table 4).To assess the severity of the condition in the control group, days 4 and 10 were chosen as reference points correlated with the beginning and end of HBOT course in the study subgroup 1 (table 4).
From the presented data (table 3) we can see that in the control group the hemoglobin oxygen saturation remained practically unchanged for 2 weeks.According to Henry's law, the concentration of oxygen in the lung blood is proportional to the partial oxygen pressure.The oxygen absorption and its binding by hemoglobin in the pulmonary capillaries depends on the diffusion of dissolved oxygen following the pathway «alveolar wall pulmonary interstitium capillary wall blood plasma erythrocyte membrane erythrocyte cytoplasm hemoglobin».Inhibiting the pathway at any stage leads to reduced rate of oxygen diffusion through the alveolar-capillary barrier.During normobaric therapy, even by increasing the oxygen concentration in the alveoli, it is not possible to overcome the diffusion barriers in the lungs [10], which is reflected in the SpO 2 level.
The incorporation of HBOT in the comprehensive therapy plan in patients with COVID-19 allowed for the transfer of all patients receiving respiratory support in NILV mode or high flow oxygen therapy to conventional oxygen insufflation through the nasal cannula or face mask with a flow rate of 3-6 l/min.Later, 19 (67.9%) patients were returned to spontaneous respiration and did not require further oxygenation therapy.While in the control group only 2 (6.7%) patients were transferred from «hard» oxygen therapy modes and 3 (10%) patients were transferred to spontaneous breathing.Thus, the use of HBOT significantly enhances the efficacy of treatment, which helps to withdraw respiratory support in a shorter time, but our data require a separate, more detailed analysis.
While studying oxidative stress markers in patients, it was found that in the serum MDA level prior to HBOT was 4.34±0.52µmol/l, which did not exceed normal references for persons over 60 years of age (5.02±1.31µmol/l).During the first 3 sessions there was a slight increase in MDA level up to 4.51±0.76µmol/l, and then, along with the other HBOT sessions, MDA level was constantly decreasing and by the 7 th session reached 3.98±0.48µmol/l (fig.2).In the study of total antioxidant activity, the opposite picture was observed, when prior to HBOT this value was 1.26±0.28mmol/l (with normal reference 1.58±0.12mmol/l), dropped by the 4 th session to 1.13±0.11mmol/l and then grew again by the 7 th session to reach 1.21±0.05mmol/l (fig. 1, b).
When studying the balance of pro-and antioxidants by measuring OCP of platinum electrode in blood serum during the first 3 sessions, this value did not change prior to HBOT session, whereas after the 3 rd session, there was a shift toward a more negative potential region as the sessions of HBOT were held (fig.1, c), which, in general, is in the line with MDA and OAA changes.
As with the MDA and OAA, it was noticed that the values of OCP of platinum electrode in patients prior to 1 session of HBOT were -22.78±24.58mV, which slightly differed from the range of values typical for apparently healthy people (-33.7±22.5 mV [33]).However, after the 3 rd session the changes in OCP became more intensive and by the 4th session the values of OCP were -30.45±15.32mV, which practically corresponded to the normal references.
Как и в случае МДА и ОАА, обращало на себя внимание то, что величины ПРЦ у пациентов до 1-го сеанса ГБО составляли -22,78±24,58 мВ, что несколько отличалось от диапазона значений ПРЦ, характерных для практически здоровых людей (-33,7±22,5 мВ [33]).Однако после 3-го сеанса динамика ПРЦ стала интенсивней и уже к 4-му сеансу величины ПРЦ составили negative potential region (fig.2, 3), which may indicate a shift of the balance towards antioxidants.Moreover, the magnitude of the shift during a HBOT session, which can be interpreted as a criterion of efficiency, increased with the number of sessions.Thus, if after the first sessions OCP did not exceed 8 mV, by the 7 th session OCP already reached more than 20 mV.The observed increase in OCP change coincided with MDA reduction and OAA increase, i.e., the use of hyperbaric oxygen therapy did not cause the activation of free-radical processes and exhaustion of the body's antioxidant protection system.Thus, at the level of redox processes, the prominent effect of HBOT starts to develop after 3-4 sessions.This effect fits into the adaptive-metabolic concept of therapeutic action of HBOT proposed by A. N. Leonov [34], according to which during the evolution the body developed stereotypic reactions of cells and functional-metabolic systems to oxygen hypersaturation, which not only ensure its safe presence in a hyperoxic environment, but also increase its resistance to noxious factors [34,35].
A study of intravascular lymphocyte apoptosis (fig. 1, d) showed moderate increase in early apoptotic cell counts in patients with coronavirus-induced pneumonia before HBOT sessions.On average, the apoptotic lymphocyte count in patients with COVID-19 was 10.6±1.2%, while the norm was 3.4±0.8%.The variability of changes in the count of apoptotic lymphocytes in patients should be noted.Thus, in 30% of patients the apoptotic lymphocyte content in the venous blood corresponded to the values seen in healthy donors, and in another 30% of patients apoptotic lymphocyte numbers were significantly increased (15-23.4%).In the remaining 40% of patients there was a moderate increase in this parameter, typical for inflammatory process.After the HBOT sessions, the apoptotic lymphocyte count in blood did not change significantly.In all patients with initially low level of apoptotic lymphocytes after HBOT sessions the level of apoptotic cells in the venous blood increased.The content of cells already dead through apoptosis (AnnexinV+/7AAD+), initially increased in the studied patients, decreased after HBOT sessions and corresponded to the normal reference ranges.
The percentage of venous blood lymphocytes expressing Fas-receptor prior to HBOT initiation was also reduced in half of the patients examined and ranged from 22 to 34% with the normal range 37-47%, and only in 20% of the patients it was elevated.The HBOT sessions helped to increase the number of CD95+ lymphocytes in the first day after the session and to normalize this value on further observation (fig. 1, e).
Induction of apoptosis in viral diseases is usually regarded as a protective mechanism to prevent the spread of infection [36].In influenza, apoptosis induction has been shown to be the main factor inhibiting the spread of infection and aims to remove infected cells without developing a local inflammatory response.The progression of infectious mononucleosis is associated with reduced Fas-receptor expression on T-lymphocytes and their spontaneous apoptosis.At the same time, extensive intravascular lymphocyte apoptosis in patients with viral infections, including COVID-19 coronavirus infection, is considered to be the main factor causing immunosuppression and lymphopenia in severe and extremely severe disease [37,38].
However, our study has demonstrated multidirectional changes in lymphocyte apoptosis intensity.In patients with severe respiratory failure, the concentration of apoptotic lymphocytes was the lowest.It should be noted that the examination of patients was performed as late as on days 10-12 after the first symptoms of the disease, so we can assume that in the late period, an increase in percentage of lymphocytes undergoing programmed cell death is physiological and aims at the elimination of activated lymphocytes.In this case, normalization of Fas-receptor expression on the lymphocyte surface and induction of their apoptosis under HBOT exposure indicates restoration of immune system function.
The favorable effect of HBOT on the restoration of normal activity of the immune system is also evidenced by the recovery of monocytes' ability to express HLA-DR on their surface.The IL-6 is known to inhibit the expression of HLA-DR, and the immune system studies of patients with new coronavirus infection COVID-19 revealed an inverse correlation between the serum level of IL-6 and the count of HLA-DR molecules on CD14 monocytes [39].Specific IL-6 blocker tocilizumab was also found to be able to partially restore HLA-DR expression on monocytes of patients with immune dysregulation.Most likely, HBOT inhibits the production of proinflammatory cytokines and the development of «cytokine storm».
In general, based on our data, we can assume that HBOT has a multifactorial effect on the body in COVID-19.Specifically, it causes an increase in hemoglobin oxygen saturation, reduces the intensity of lipid peroxidation processes, activates the body antioxidant system, restores the balance of pro-and antioxidants, helps normalize apoptosis processes, etc. (fig.2).
with new coronavirus infection COVID-19 has demonstrated its effectiveness in increasing the hemoglobin oxygen saturation, as well as in improving subjective indicators of patients' condition.
The use of HBOT does not cause activation of free-radical processes and exhaustion of the body antioxidant system.
Apoptotic parameters during HBOT were brought back to the physiological normal ranges, which manifested in a decrease in the number of cells that died through apoptosis, restoration of the monocyte ability to express HLA-DR on their surface.
The marked effect of HBOT manifesting as normalization of redox and apoptotic processes in the body begins to appear after 3-4 sessions.