Hyperbaric Oxygen Therapy Counters Oxidative Stress/Inflammation-Driven Symptoms in Long COVID-19 Patients: Preliminary Outcomes

Long COVID-19 patients show systemic inflammation and persistent symptoms such as fatigue and malaise, profoundly affecting their quality of life. Since improving oxygenation can oppose inflammation at multiple tissue levels, we hypothesized that hyperbaric oxygen therapy (HBOT) could arrest inflammation progression and thus relieve symptoms of COVID-19. We evaluated oxy-inflammation biomarkers in long COVID-19 subjects treated with HBOT and monitored with non-invasive methods. Five subjects (two athletes and three patients with other comorbidities) were assigned to receive HBOT: 100% inspired O2 at 2.4 ATA in a multiplace hyperbaric chamber for 90 min (three athletes: 15 HBOT × 5 days/wk for 3 weeks; two patients affected by Idiopathic Sudden Sensorineural Hearing Loss: 30 HBOT × 5 days/wk for 6 weeks; and one patient with osteomyelitis: 30 HBOT × 5 days/wk for week for 6 weeks and, after a 30-day break, followed by a second cycle of 20 HBOT). Using saliva and/or urine samples, reactive oxygen species (ROS), antioxidant capacity, cytokines, lipids peroxidation, DNA damage, and renal status were assessed at T1_pre (basal level) and at T2_pre (basal level after treatment), and the results showed attenuated ROS production, lipid peroxidation, DNA damage, NO metabolites, and inflammation biomarker levels, especially in the athletes post-treatment. Thus, HBOT may represent an alternative non-invasive method for treating long COVID-19-induced long-lasting manifestations of oxy-inflammation.

Hyperbaric oxygen therapy (HBOT) is used in pathologies where tissues are starved for oxygen.The combined action of hyperoxia and hyperbaric pressure leads to a significant improvement in tissue oxygenation while targeting both O 2 and pressure-sensitive genes [17,18].The Undersea and Hyperbaric Medical Society (UHMS) describes HBOT as an intervention whereby patients breath near 100% oxygen while being pressurized to at least 1.4 to 2.5 atmosphere absolute (ATA) in a hyperbaric chamber [19].Fourteen medical applications have been accepted by UHMS [20].Recent clinical and preclinical results have suggested the potential usefulness of HBOT in treating long COVID-19 [21][22][23][24][25][26][27][28][29][30][31].In particular, in neurological tissues, Zilberman-Itskovich et al. (2022) [32] provided some interesting observations about the effects of hyperbaric therapy on perfusion; in myocardial function, Leitman et al. (2023) [27] suggested that HBOT can indicate subclinical left ventricular dysfunction.HBOT has also yielded improvements in health, performance parameters, and in specific blood gas parameters, as reported by Kitala et al. (2023) [32].
The current pilot study aimed to investigate the effects of HBOT on oxy-inflammation biomarkers in non-invasively obtainable biofluids (saliva and urine) for the first time in the literature by investigating five subjects suffering from long COVID-19 (with or without other morbidities) with ongoing symptoms three months after confirmed infection.

Subjects
This pilot study involved five subjects who were diagnosed with COVID-19 in January-February 2021 (two professional athletes and three patients).Table 1 describes the included subjects' features (evaluated at the time of the first visit, before the HBOT sessions).The athletes and ISHHL patients did not receive any medications or supplements from 2 weeks before and during the HBOT sessions, while a patient with osteomyelitis to the thumb received only nonsteroidal anti-inflammatory drugs when necessary.All subjects had no history of allergic rhinitis and did not report any drug or food allergies.No alcohol consumption or smoking was reported.Based on the criteria for classifying the post-COVID-19 syndrome [31,[33][34][35], three expert clinicians classified the five included subjects, as shown in Table 2.This proposed criterion is based on the initial symptoms, duration of symptoms, period of quiescence, and time of onset of symptoms.Five categories of post-COVID-19s: Type 1 includes duration of recovery (relates to the severity of infection), organ damage, and underlying medical conditions.Type 2 is characterized by symptoms persisting for six weeks from the onset of illness.Type 3 shows a period of quiescence or nearly full recovery after initial infection, followed by a recurrence of symptoms that persist for at least three months (Type 3A) or at least six months (Type 3B).Type 4 includes subjects who are initially asymptomatic at the time of a positive COVID-19 test but develop symptoms one to three months (Type 4A) or at least three months later (Type 4B) that persist for varying lengths of time.Type 5 includes those who have no or few symptoms at the time of a positive COVID-19 test and experience sudden death within the next 12 months [35].The protocol of the study was approved by the Human Ethical Committee (HEC-DSB/04-19) of the Department of Biomedical Science of the University of Padova (Italy), and all subjects provided informed consent.The study was carried out according to the Declaration of Helsinki.

HBOT Protocol
The five examined subjects were exposed to 100% inspired oxygen at 2.4 ATA in a multiplace hyperbaric chamber for 90 min using an overboard demand regulator and oral-nasal mask.The two athletes received 15 treatments of HBOT (5 days per week for 3 weeks).The two patients affected by ISSHL received 30 HBO treatments (5 days per week for 6 weeks); the patient with osteomyelitis received 30 HBO treatments (5 days per week for 6 weeks), and after a 30-day break, a second cycle of 20 HBOT sessions was administered [17] (Figure 1).

Fatigue Severity Scale
Fatigue was assessed according to a numeric rating scale (NRS) [36,37].Subjects were asked to measure their average severity of fatigue over the last 24 h on a scale from 0 to 10 (0 = none; 10 = worst).

Saliva and Urine Samples Collection
Saliva and urine were collected on the 1st day of the HBOT session (T1_pre = basal value; T1_post = value after the 1st session) and on the final day of HBOT (T2_pre = basal value last day; T2_post = value after the last session HBOT) (see Figure 1).For saliva sampling, the subjects were instructed to refrain from drinking, eating, smoking, brushing their teeth, and using a mouthwash during the 30 min before salivary collection.They were instructed on the correct use of Salivette devices (Sarstedt, Nümbrecht, Germany).The samples were spun down, and approximately 1 mL of saliva was obtained [38][39][40][41].Urine samples were collected via voluntary voiding in a sterile container provided to the subjects.All samples were stored in multiple aliquots at −80 • C until assayed and thawed only once before analysis.

Fatigue Severity Scale
Fatigue was assessed according to a numeric rating scale (NRS) [36,37].Subjects were asked to measure their average severity of fatigue over the last 24 h on a scale from 0 to 10 (0 = none; 10 = worst).

Saliva and Urine Samples Collection
Saliva and urine were collected on the 1st day of the HBOT session (T1_pre = basal value; T1_post = value after the 1st session) and on the final day of HBOT (T2_pre = basal value last day; T2_post = value after the last session HBOT) (see Figure 1).For saliva sampling, the subjects were instructed to refrain from drinking, eating, smoking, brushing their teeth, and using a mouthwash during the 30 min before salivary collection.They were instructed on the correct use of Salivette devices (Sarstedt, Nümbrecht, Germany).The samples were spun down, and approximately 1 mL of saliva was obtained [38][39][40][41].Urine samples were collected via voluntary voiding in a sterile container provided to the subjects.All samples were stored in multiple aliquots at −80 °C until assayed and thawed only once before analysis.
2.8.8-Hydroxy-2 -deoxyguanosine (8-OH-dG) 8-OH-dG has been established as a marker of nuclear oxidative DNA damage.A commercial ELISA kit (Cayman Chemical, Ann Arbor, MI, USA, Item No. 89320) was utilized to measure urinary concentrations of 8-OH-dG.The EIA employed an anti-mouse IgG-coated plate and a tracer consisting of an 8-OH-dG-enzyme conjugate, and the sample 8-OH-dG concentration was determined.The samples were read at a wavelength 412 nm, and the sample 8-OH-dG concentration was determined using an 8-OH-dG standard curve as previously described [45,46,49,51].

NO Metabolites (Nitrite and Nitrate)
Nitrite plus nitrate (NO 2 + NO 3 = NOx) concentration was assessed in urine via the use of a colorimetric method based on the Griess reaction [40,51,52] using a commercial kit (Cayman Chemical, Ann Arbor, MI, USA; Item No. 780001) that provided an accurate and convenient method for the measurement of nitrite and nitrate concentration.NO metabolites were determined at 545 nm.A linear calibration curve was computed from pure nitrate standard.

Quantification of Inflammatory Markers Levels in Saliva
IL-6, TNF-α, and IL-1β, saliva levels were determined by using ultrasensitive ELISA immunoassays (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions [53][54][55].The assays were based on a double-antibody sandwich technique.The levels of inflammatory markers (in pg/mL) in the saliva samples were calculated.Sample concentrations were determined for IL-6 at 450 nm and for TNF-α and IL-1β at 412 nm.
All samples were determined in duplicate using a microplate reader spectrophotometer (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland).

Creatinine, Neopterin, and Uric Acid Concentration in Urine
Creatinine, neopterin, and uric acid concentrations were measured via the use of an isocratic high-pressure liquid chromatography (HPLC) method that has been previously described [39,40,47,50,56].The concentration levels were measured using a Varian pump (240, auto sampler ProStar 410) coupled to a specific fluorometric detector.The calibration curves were linear over the range of 0.125-1 µmol/L for neopterin, 0.625-20 mmol/L for uric acid, and 1.25-10 mmol/L for the creatinine levels.The inter-assay and intra-assay coefficients of variation were <5%.

Secondary Outcomes
In all subjects, standardized clinical hematological analyses were conducted by using an automated hematology analyzer according to the standard analysis methods of Azienda Ospedaliera of Padua laboratories.

Spirometry
We verified the impact of long COVID-19 on daily activity, focusing on lung function, via the use of traditional spirometry.Each subject underwent a clinical evaluation before HBOT.Furthermore, the 2 athletes repeated the spirometry at the end of the treatments.The spirometry parameters were recorded by using the turbine sensor of a portable spirometer (Pony FX; Cosmed; Rome, Italy) [49] with international standard: flowmeter, bidirectional digital turbine Ø 28 mm; flow range 0.08-20 L/s; volume range 12 L; accuracy of reading ±2%; resistance <0.6 cmH 2 O/L/s; temperature sensor 0-50 • C. We measured forced expiratory volume (FVC) in the first second (FEV1) FEV1/FVC%; peak expiratory flow (PEF); forced expiratory flow (FEF25-75%); and maximal expiratory flow (MEF) in the usual intervals (MEF75%, MEF50%, MEF25%).The equipment received the flow data in real-time at a frequency of 100 Hz before recording and exporting the data to a computer.The spirometry data were compared with predicted values, which were estimated on the basis of recent standards corrected for age and height.

Statistical Analysis
Data are presented as mean ± standard deviation (SD).Considering the low number of subjects reported, we performed a detailed statistical analysis.Percentage changes (∆%), defined as [(postHBOT − preHBOT)/pre-HBOT) × 100], were used for the analysis to assess the effects of HBOT on the examined biomarkers.Also, an ANOVA with repeated measures and Dunn's multiple comparison test were performed to further check the inter-group significance.dCohen was used to calculate the size effect, and a Confidence Interval 95% for dCohen was calculated.A p < 0.05 was considered statistically significant.Our statistical analysis was performed using SPSS statistics software (Version 25; IBM Corporation, Armonk, NY, USA) and the GraphPad Prism package for Mac (GraphPad Prism 9.5.1,GraphPad Software Inc., San Diego, CA, USA).

Results
All the included subjects completed the study without reporting HBOT-related complications or discomfort.

Hematological and Biochemical Analysis
The results derived from conducting hematological and biochemical tests on both athletes showed critical immunological profile changes in total lymphocytes and T cell compartment alterations, along with an increased expression of CD57+ in CD8 T cells.Moreover, high levels of mycoplasma pneumoniae (IgG, indicating a possible co/sub-infection between COVID-19 and atypical bacteria such as Mycoplasma pneumoniae) and liver enzyme abnormalities with aspartate aminotransferase (AST) were found.The IS-SHL patients had hematological and biochemical results within the normal range, while in the patient affected by osteomyelitis, our hematological biochemical analysis results showed an increase in velocity sedimentation rate (VES) and C-reactive proteins (CRPs); these values remained altered even after HBOT.

Spirometry
The subjects were divided into athletes and patients.At basal examination, the athletes showed impaired lung function, specifically showing a reduction in lung volume (FVC-forced vital capacity: ~3.3; FEV1-forced expiratory volume in the first second: about 81%; FEV1/FVC% ~76.5%).After HBOT, improved lung function was recorded in both athletes, with a return to baseline values.In contrast, the patients showed normal basal spirometry, with no changes occurring after HBOT.

Discussion
To the best of our knowledge, this is the first study to monitor the oxidative stress, inflammation status, and immunological levels of subjects affected by long COVID-19, treated with HBOT, and examined via non-invasive methods (only saliva and urine were The fatigue scores on the numerical rating scale (NRS) at T1_Pre were 7 ± 2.8 in athletes and 4.6 ± 3.3 in patients; at T2_Pre 2.0, the same metrics were ±1.4 and 4.1 ± 4.1, respectively.

Hematological and Biochemical Analysis
The results derived from conducting hematological and biochemical tests on both athletes showed critical immunological profile changes in total lymphocytes and T cell compartment alterations, along with an increased expression of CD57+ in CD8 T cells.Moreover, high levels of mycoplasma pneumoniae (IgG, indicating a possible co/subinfection between COVID-19 and atypical bacteria such as Mycoplasma pneumoniae) and liver enzyme abnormalities with aspartate aminotransferase (AST) were found.The ISSHL patients had hematological and biochemical results within the normal range, while in the patient affected by osteomyelitis, our hematological biochemical analysis results showed an increase in velocity sedimentation rate (VES) and C-reactive proteins (CRPs); these values remained altered even after HBOT.

Spirometry
The subjects were divided into athletes and patients.At basal examination, the athletes showed impaired lung function, specifically showing a reduction in lung volume (FVCforced vital capacity: ~3.3; FEV1-forced expiratory volume in the first second: about 81%; FEV1/FVC% ~76.5%).After HBOT, improved lung function was recorded in both athletes, with a return to baseline values.In contrast, the patients showed normal basal spirometry, with no changes occurring after HBOT.

Discussion
To the best of our knowledge, this is the first study to monitor the oxidative stress, inflammation status, and immunological levels of subjects affected by long COVID-19, treated with HBOT, and examined via non-invasive methods (only saliva and urine were collected for the measurements).From this study, we aimed to gain a detailed insight into the physiological mechanisms involved in the above.None of the patients experienced any adverse reactions or complained of HBOT-related discomfort.
Recently, it has been reported that oxidative stress is likely implicated in the pathophysiology of all factors causing long COVID-19 and its symptoms [57] and that inflammation and oxidative stress mutually reinforce one another, thus contributing to the systemic hyperinflammatory state [58].Furthermore, it is also worth mentioning how these responses might result in some alterations in physiological parameters, such as altered brain perfusion and metabolism [59,60], as a result of possible autonomic dysregulation and vascular damage [61].
The findings of this study show that ROS and oxidative damage to lipids and DNA are significant factors in long COVID-19 patients.Also, our data confirm that the evolution of long COVID-19 is driven by cytokines produced in the body due to inflammation [62], which are often generated in response to viral infections and lead to oxidative stress, with immune status and altered lung function measures.
Experimental evidence shows that HBOT reduces the inflammation in animal models of sepsis (i.e., characterized by an overwhelming inflammatory response), with significant improvement in survival after a single treatment (2.4 ATA × 60 min) [63].Additionally, HBOT could potentially mitigate oxidative stress [16,64] and inflammatory responses by reducing inflammatory cytokines through several transcriptional factors (i.e HIF-1 and NfKb) [65,66] and/or directly affecting the innate immune system [67].
HBOT stimulates the modulation of oxygen-sensitive transcription factors and ROSmediated signaling pathways; in fact, our results show an increase in ROS production levels immediately after hyperbaric oxygen treatment (T1_post and T2_post) due to the hyperoxia exposition.Despite this, this increase in ROS subsided after subsequent treatment sessions, and at the end of HBOT, ROS levels decreased.One possible explanation for this is that the first HBOT cycle exerts a preconditioning activity by enhancing cellular protection against subsequent oxidative stress damage.In fact, we observed the same behavior with respect to the membrane lipids and DNA oxidation.
Recent studies show that an abnormal diffuse inflammatory cytokine profile can persist in long COVID-19 subjects for at least 8 months [68], along with the persistent deregulation of IL-1β, IL-6, and TNF-α [69].Recent evidence links elevated cytokines to various symptoms in long COVID-19, including the peripheral immune response, neuroinflammation and microglial cell dysregulation, autonomic nervous system dysfunction, neuropathic pain, extreme fatigue, gastrointestinal tract symptoms, and arthralgia [70].Furthermore, oxidative stress is another factor that contributes to cytokine release syndrome, and the signaling link between oxidative stress (OxS) and cytokines involves the p38 MAPK [71].
Therefore, HBOT assists in returning cytokines to homeostatic levels by modulating the interaction of IL-6 and/or TNFα, as our data proves.The response to hyperbaric oxygen therapy had an evident effect in the two young athletes in our study, both of whom had no comorbidities, and in the two patients in our patients, among whom the pathology underlying long COVID-19 was idiopathic sudden sensorineural hearing loss, a pathology that is considered to be an otolaryngologic emergency at rapid onset [72].Unfortunately, the patient with severe osteomyelitis associated with long COVID-19 did not obtain positive results even after HBOT, showing high levels of oxidative stress (ROS, lipid peroxidation, DNA damage) and inflammation biomarkers associated with low levels of antioxidant capacity and nitric oxide metabolites.
In a study on long COVID-19 in England, Kim and colleagues [73] described the condition's strong similarities to post-traumatic stress disorder.This could be related to the fatigue state recorded in our subjects, especially in the two young athletes, which could be triggered by an infection occurring during a time of increased mental or physical stress.
Furthermore, as reported in the literature, activated immune-inflammatory and oxidative/nitrosative stress pathways may underpin the somatic symptoms of long COVID-19 due to chronic fatigue syndrome [74].In support of this, after the complete HBOT protocol, the NRS fatigue decreased from 5.6 to 2.8, considering all of the subjects in our study together.According to several authors, long COVID-19 fatigue might be characterized by altered cortical excitability and neurotransmission [75][76][77]; HBOT might have an effect on some brain excitability and neurotransmission parameters, as some authors have discussed [78][79][80].Furthermore, some therapeutic approaches, such as nutraceuticals, might improve symptoms through this modulation [80,81] in adjuvant to HBOT.Despite the differences in age, physical fitness, individual susceptibility, and number of HBOT sessions, among the studied subjects, the responses were pretty homogeneous (except in the patient suffering from osteomyelitis associated with long COVID- 19).
Limitations: The current study has some limitations, namely its small sample size, the heterogeneity among the subjects' characteristics, and the absence of a pre-COVID-19 evaluation.Therefore, further verification in additional studies is necessary.

Metabolites 2023 , 16 Figure 1 .
Figure 1.HBOT Experimental study design of working protocol with timeline of sample collection.

Figure 1 .
Figure 1.HBOT Experimental study design of working protocol with timeline of sample collection.

Figure 3 .
Figure 3.Effect of HBOT on NO metabolites (NOx) in (A) athletes (white and blue bars) and (B) patients (white and grey bars).Data are mean ± SD.

Figure 4 .
Figure 4. Effect of HBOT on inflammation in athletes and patients.Histogram plot (mean ± SD) and single plot of (A) IL-6, (C) TNF-α, and (E) IL-1β in athletes (white and blue bars) and (B,D,F) patients (white and gray bars).

Figure 3 . 16 Figure 3 .
Figure 3.Effect of HBOT on NO metabolites (NOx) in (A) athletes (white and blue bars) and (B) patients (white and grey bars).Data are mean ± SD.

Figure 4 .
Figure 4. Effect of HBOT on inflammation in athletes and patients.Histogram plot (mean ± SD) and single plot of (A) IL-6, (C) TNF-α, and (E) IL-1β in athletes (white and blue bars) and (B,D,F) patients (white and gray bars).

Figure 4 .
Figure 4. Effect of HBOT on inflammation in athletes and patients.Histogram plot (mean ± SD) and single plot of (A) IL-6, (C) TNF-α, and (E) IL-1β in athletes (white and blue bars) and (B,D,F) patients (white and gray bars).

Figure 5 .
Figure 5.Effect of HBOT on renal function in athletes and patients.Histogram plot (mean ± SD) and single plot of (A) creatinine, (C) neopterin, and (E) uric Acid in athletes (white and blue bars) and (B,D,F) patients (white and gray bars).

Figure 5 .
Figure 5.Effect of HBOT on renal function in athletes and patients.Histogram plot (mean ± SD) and single plot of (A) creatinine, (C) neopterin, and (E) uric Acid in athletes (white and blue bars) and (B,D,F) patients (white and gray bars).

Table 1 .
Anthropometric and physiological parameters of all subjects.Parameters collected before HBOT.BMI: Body Mass Index; HR: Heart Rate; SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure; T: Tympanic Temperature; ISSHL: Idiopathic Sudden Sensorineural Hearing Loss.

Table 2 .
Characteristics and symptoms of subjects with long COVID-19.In three cases, an association with another pathology was found (ISSHL and osteomyelitis).