PROPOSED TREATMENT PROTOCOL WITH HYPERBARIC OXYGEN THERAPY FOR COVID- 19 PATIENTS

Scientific Committee

Principal investigators:

Prof. Gerardo Bosco1 Prof. Nazareno Paolocci1

Co-investigators:

Prof. Franco Bassetto2
Dr. Giacomo Garetto3
Dr. Eva Kohlscheen 2
Prof. Vincenzo Vindigni2 Prof. Giampiero Avruscio4 Dr. Annamaria Cattelan6 Dr. Carlotta Scarpa2

Dr. Daniele Bonvicini5

Collaborators:

Dr. Antonio Amabile2 Dr. Federico Facchin 2 Dr. Regina Sonda 2
Dr. Leonardo Scortecci 2 Dr. Bernardo Biffoli 2

Dr. Filippo Perozzo 2 Dr. Matteo Paganini1 Dr. Iva Sabovic1

  1. Department of Biomedical Sciences, University of Padua

  2. Plastic and Reconstructive Surgery, Padua Hospital

  3. A.t.i.p. Centre of Hyperbaric Medicine, Padua

  4. Angiology Department, Padua Hospital

  5. Anaesthesia and Intensive Care Department, Padua Hospital

  6. Department of Infectious and Tropical Diseases, Padua Hospital

Consultant and external advisor :

Enrico Camporesi, MD
Anesthesia Research Director, THRI TEAMHealth Anesthesia, TGH
Emeritus Professor of Surgery, USF, Tampa

Steven Thom MD PhD,
Director Of Research and Professor of Emergency Medicine, University of Maryland, Baltimore

Project Budget

Estimated Budget ($K) Labor
Travel Material Indirect
TOTAL

LABOR

  • Direct Investigators labor

    Two research fellow position TRAVEL

  • Co-I and Fellow Exchange visiting program

  • Two meetings for dissemination MATERIAL

• Laboratory materials INDIRECT

  • Office expenses, audit and legal fees

  • Rent (materials and medical instruments)

  • Supervisor salaries

  • Insurance

    Background

Year 1

50 10 35 60 155

Year 2

40 15 20 35 110

Hyperbaric oxygen (HBO) therapy is an exposure of pure oxygen at more than 1 ATA. This treatment is used to treat several medical conditions [Moon 2019]. Your doctor may suggest hyperbaric oxygen therapy if you have one of the following conditions:

  • Anemia, severe

  • Brain abscess

  • Bubbles of air in your blood vessels (arterial gas embolism)

  • Burn

  • Decompression sickness

  • Carbon monoxide poisoning

  • Crushing injury

  • Deafness, sudden

  • Gangrene

  • Infection of skin or bone that causes tissue death

  • Nonhealing wounds, such as a diabetic foot ulcer

  • Radiation injury

  • Skin graft or skin flap at risk of tissue death

  • Vision loss, sudden and painless

    Why HBO therapy to treat patients with COVID-19?

    There are, at least, primary reasons that led us to think that HBO could help patients with COVID-19, either with latent or manifest infection.

1) HBO as therapy has several advantages over other treatments. HBOT is based on the laws of gas physics related to pressure. The differences and advantages of HBOT from atmospheric oxygen absorption are: a) Improve the diffusion efficiency of oxygen; therefore, it overcomes the gas diffusion obstacle caused by interstitial lesions as long as there is a certain amount of ventilation; in essence, we can obtain better gas exchange efficiency than in normal people while breathing; b) The physically dissolved oxygen content in the blood is much larger than the combined hemoglobin transport capacity; c) The diffusion distance of oxygen is increased, and this property meets the need for aerobic metabolism of deep tissues or hypoperfused regions of the body [Thom 2011; Camporesi, Bosco 2014].

2) HBO has documented anti-inflammatory properties. The anti-inflammatory potential of HBO has already been validated, both in animal models to limit the reperfusion ischemia damage and in orthopedic and vascular patients. HBO makes it possible to redistribute blood to hypoxic tissues and reactivate oxygenation, therefore exerting an antibacterial action, promoting neoangiogenesis, reducing post-traumatic and/or post- surgical oedema, and fostering reparative processes and tissue protection [Camporesi, Bosco 2014]. During treatment, hemoglobin reaches 100% saturation, and also more oxygen is dissolved in the plasma than normal environmental pressure. For these reasons, a high amount of oxygen is delivered to all tissues, enhancing blood and tissue oxygenation. This therapy could be used as an adjunct to antiviral treatment of patients with viral pneumonia or, in general, respiratory infections to reduce morbidity and mortality. A wealth of literature is available concerning the impact of HBO therapy on inflammation in the context of several clinical disease conditions. Yet the available reports on HBO in COVID-19 patients (mainly from China) are missing critical details, such as the modalities of administering O2 in the chamber. Further to this, it is

unclear whether the clinical course of those patients, when improved, was due to HBO or only time. In essence, whether there may be some anti-viral effect, or whether Hyperbaric Oxygen Therapy (HBO) may attenuate the inflammation, is unknown in patients COVID-19. And this intriguing and pragmatically relevant question will be addressed here. More specifically, here, we aim at demonstrating that HBO COVID- 19 positive paucisymptomatic patients limit the inflammatory cascade responsible for the typical pulmonary and respiratory evolution of this infection. [Bosco 2007,Yang 2001; Harch 2016]

3) HBO increases reactive oxygen species (ROS) burden that can directly affect viral assembly and replication. The role played by ROS in the pathogenesis and progression of infective disorders remains partially understood. On the one hand, studies have suggested that oxidative stress may play a role in the HIV disease in that HIV-infected patients are under chronic oxidative stress (OS) as a result of perturbations in their antioxidant defense system [Budiarti R, 2018]. Further to this, Israel and coworkers and Peterhans and colleagues have concluded that oxidative stress (OS) can contribute to increased viral replication, transcription and/or reactivation of latent infection [ Pace GW, 1995]. However, the vast majority, if not all, these conclusions are based on a “guilty by association” type of evidence, and not on a clear factual (cause- effect) nexus. When it comes to viruses, we cannot exclude that ROS may represent of front-line defense against latent virus infections remains a partially explored one. Studies have shown that oxidative stress can play a role in the progression of the HIV disease in that HIV- infected patients are under chronic OS as a result of perturbations in their antioxidant defense system [Baugh MA, 2000 ]. Further to this, Israel and coworkers and Peterhans and colleagues have suggested that oxidative stress can contribute to increased viral replication, transcription and/or reactivation of latent infection[Peterhans E, 1995]. However, the conclusions reported above are based on a “guilty by association” type of evidence, and not on factual (cause- effect) nexus. Moreover, there is a consensus in the literature that a viral infection per se does not trigger OS. Instead, it is in the host defense armamentarium to induce ROS to counter viral effects. For instance, it often goes unnocited that viruses use phospholipids and proteins taken from the host membranes to make their own capsid (envelop), and ROS avidly react with phospholipids, modifying their structure, thus function. A therapy based on the exposure to high levels of O2, such as the hyperbaric one can increase the ROS burden while boosting antioxidant defenses, at the same time, thus potentially limiting the consequences of oxidative stress itself (Fig.1). More in detail, HBO treatment can increase the concentration of “environmental “ superoxide. This rise may enhance the expression of major antioxidant enzymatic activities/transcription factors, such as that of heme oxygenase-1 (HO-1) and hypoxia-inducible factor (HIFα). Indeed, previous evidence by Speit and colleagues has demonstrated that the expression of HO-1 increases 24 hours after hyperbaric oxygen exposure. [Rothfuss A, 2001]. Altogether, the evidence enunciated above provides a strong conceptual rationale for testing whether it’s in HBO therapy possibility to counter viral invasion and infection of the host tissue by directly attacking and modifying the viral capsid (envelope), including the one of the nowadays widely spread COVID-19. In the present proposal, we will evaluate if HBO

therapy increases ROS levels (mainly superoxide) in the blood of patients with COVID-19 and if this effect correlates with the outcome of the disease.

4) HBO, stem cell and cytokine storm
HBO increases the recruitment of SPC; therefore, the presence of HIF-1 and Trx-1; The platelet activity of eNOS in diabetics is stimulated by HBO [Thom 2006-2009-2011]. Recently intermittent hyperoxia or different oxygen partial pressures have an impact on stem cell, cytokine expression and neuroprotection. [MacLaughlin 2019; Schulze 2017]. Coronavirus-induced respiratory syndrome also appears to be related to the release of pro-inflammatory cytokines such as IL1 beta and IL6. [Conti et al. 2020]. In general, the correlation between pulmonary fibrosis and IL6 is known and already described in the literature. However, the use of monoclonal antibodies against IL6 in an animal study has demonstrated a bidirectional role of this cytokine in the pathogenesis of pulmonary fibrosis, confirming once again the complex homeostasis of cytokines. [Kobayashi et al. 2015]. On the contrary, elevated blood levels of IL6 and IL8 in patients with pulmonary fibrosis correlate with faster progress of pulmonary fibrosis. Conversely, IL10, TGF beta, IL4 and IL 13 have not shown statistically significant differences. [Papiris et al. 2018]

As we learned before, one of the most critical aspect of HBOT is its ability to increase ROS and Reactive Nitrogen Species (RNS) production, which serve as signaling molecules for multiple intracellular cascades [Camporesi, Bosco 2014; Thom 2011]. The resulting free radicals are a result of hyperoxia, and are widely recognized for their beneficial and harmful effects [Thom 2006]. It is essential to note the difference between oxygen toxicity and oxidative stress; the former is an excessive amount of free radicals that causes damaging effects, while the latter has been proven to be well-tolerated by the body’s antioxidants. A balance between free radicals and antioxidants is necessary for protecting the physiologic well-being of the cell and organism. For instance, ROS can initiate downstream changes involved in stem/progenitor cell mobilization from bone marrow and lowering monocyte chemokine synthesis. These events which ultimately lead to wound neovascularization and improved post-ischemic tissue survival, respectively as shown in Fig.1 (Thom 2011, Yang 2001).

Fig.1 Mechanism of action of Hyperbaric oxygenation

5) HBO, platelet function and ROS production.
Nitrogen monoxide has multiple and important actions: vasodilator (antihypertensive), antiplatelet, anti-

inflammatory (reduces the adhesion of leukocytes to the wall and the consequent diapedesis). [Bosco, 2001- 2006; Thom 2009]. However, whether HBO prevents platelet aggregation in patients with COVID-19 is currently unknown. In addition, activated platelets can be involved in prothrombotic and pro-inflammatory processes and can be induced by microbubbles and environmental stress. Flow cytometry has been widely used as an assay for platelet function. Platelet activation is at the basis of several pathological conditions with vascular impairment, ranging from microcirculation dysfunction (sepsis) to thrombotic ischemia (e.g., stroke, myocardial infarction). Modifications in platelet membrane receptors allow the detection of their activated form, and expression and release of osteocalcin (OCN) [Fusaro M, 2019]. Pretreatment with hyperbaric oxygenation can reduce vascular risk [Landolfi 2006]. Data show that pre-breathing oxygen, more effective with HBO than with NBO, reduces air bubbles and platelet activation and, therefore, can be useful in reducing the development of decompression sickness [Bosco 2010]. Pretreatment with oxygen, particularly at 12 msw, can also improve the antioxidant activity of lymphocytes and reduce the levels of reactive oxygen species. Pre-respiratory oxygen in water can also preserve homeostasis of calcium, suggesting a protective role in the physiological functions of lymphocytic cells [Morabito 2011]. Variations in partial oxygen pressures can induce the production of reactive oxygen species (ROS), which aggravate oxidative stress and, consequently, influence endothelial function. Nitric oxide metabolites, inducible nitric oxide synthase (iNOS), aminothiols, and renal function were also evaluated as markers of redox status and

kidney damage. The overproduction of ROS and the consequent oxidative damage to the lipids of the membrane and the reduction of the antioxidant capacity also reflect a hypoxic condition [Bosco 2018; Makric 2019].

In Fig.2, we have summarized the proven and hypothetical effects of HBO therapy that led us to suspect that this therapy can be beneficial in COVID-19 patients and possibly in other forms of viral infection.

Efficient O2 delivery to hypoperfused tissues

Stem cell and Citokines stimulation

Anti- Inflammatory Properties

COVID-19

Respiratory and other symptoms

HBO THERAPY

ROS-induced reduction in viral load

Antiplatelet/ Antithrombotic Effects

Significance

Coronavirus infection represents the current health emergency that is saturating the care and receptive capacities of our national health system. While in many cases (estimated at about 81% of the total) the COVID-19 disease starts asymptomatically or paucisymptomatically, in others this infection can lead to severe respiratory compromise due to interstitial pneumonia with consequent hospitalization and possible orotracheal intubation. Given the significant mortality and morbidity associated with this COVID 19 pandemic and the risks inherent to the transfer of critically-ill patients, especially in the current worldwide situation, the beneficial potential of adjunctive treatments cannot be dismissed. Our current objectives are to review the evidence concerning the use of HBO as an adjunctive treatment for patients with viral respiratory infections, especially those affected by diseases of the lower trait of the airways. Further to this, we aim at testing the following specific questions: 1) Does the administration of HBOT reduce mortality or morbidity associated with viral pneumonia (VP)?; 2) What adverse effects are related to the use of HBOT in the treatment of individuals with VP? Therefore, the questions we are posing here are highly significant, both conceptually and pragmatically.

Innovation

Considering that there is no current approved treatment that has been probed unequivocally effective in treating patients with later or manifest COVID-19, the present proposal is highly innovative and timely.

Feasibility

All the necessary equipment is already in place at our Institution (the University of Padova, Department of Biomedical Sciences) and at Atip Hyperbaric Medical centre in Via Cornaro 1, Padova, nearby the University Hospital of Padova to address the intriguing yet-to-be tested hypotheses reported above. Therefore, we do not anticipate any barrier, either technical or related to patient recruitment, that would impede the successful completion of the proposed studies. Thus, the present project is highly feasible.

Preliminary Data in Support of the Current Specific Aims

Rationale. Hyperbaric oxygen (HBO) therapy involves the intermittent inhalation of 100% oxygen in chambers pressurized between 1.5 and 3.0 atmosphere absolute (ATA). HBO increases both dissolved oxygen and the partial pressure of oxygen in plasma [Moon 2019; Camporesi 2014]. HBO is commonly used in the treatment of decompression sickness, carbon monoxide intoxication, arterial gas embolism, necrotizing soft

tissue infections, chronic skin ulcers, severe multiple trauma with ischemia and ischemic diabetic foot ulcers [Thom 2011; Moon 2019]. A possible mechanism of HBO mediating beneficial effects has been described as attenuation of the production of pro-inflammatory cytokines in response to an inflammatory stimulus such as surgery [Bosco 2007- 2014 ] and modulation of the immune response [Thom 2011].

Hyperbaric oxygen therapy has beneficial effects in reducing the inflammatory state by modulating oxidative stress, including lipoperoxidation, with the increase of antioxidant enzymes. [Bosco et al. 2018; Thom 2011]. This evidence is incisive enough to provide a strong rationale for testing the impact of HBO therapy in coronavirus patients. Accordingly, hyperbaric therapy can modulate the inflammatory response and cytokine level in animal models of bladder, lung, and sepsis fibrosis. [Halbach et al. 2019; Marmo et al. 2017; Pedoto et al. 2003]. Studies in humans have confirmed this experimental evidence concerning the benefit emanating from HBO during different inflammatory states [Li et al. 2011]. Of relevance, in cases of pneumonia with interstitial involvement, reports have probed the ability of oxygen therapy to decrease fibrosis [Sahin et al. 2011]. In the diabetic patient, whose peripheral arterial vascularization is compromised, OTI is indicated for its ability to increase tissue oxygenation by limiting ischemic damage. Considering the high percentage of diabetic subjects with more compromised clinical pictures affected by COVID-19, hyperbaric oxygen therapy could improve the perfusion of peripheral systems but not only (see kidneys) by reducing the risk of MOF [Bosco et al. 2014; Rinaldi et al. 2011; Yang et al. 2006]. Besides, hyperbaric therapy should not have effects on viral reproduction; a study of the literature shows some work on the virus-static capacity of therapy (also on RNA viruses). The scientific studies are attached [Gabrilovich et al. 1990; Hosokawa et al. 2014; Peng et al. 2012; Savva-Bordalo et al. 2012; Wong et al. 2008]. HIF-1a and NF-kB crosstalk regulates essential inflammatory functions in myeloid cells. HIF-1a increases macrophage aggregation, invasion, and motility and drives the expression of proinflammatory cytokines. HIF-1a enhances intracellular bacterial killing by macrophages and also promotes granule protease production and release of nitric oxide (NO.) and TNF-a, which in turn further contribute to antimicrobial control. HIF-1a in myeloid cells increases the transcription of key glycolytic enzymes, resulting on increased glucose uptake and glycolytic rate. HBO increases the recruitment of SPC; therefore, the presence of HIF-1 and Trx-1. [Thom SR et al. 2006-2011]; The platelet activity of eNOS in diabetics is stimulated by HBO [Thom SR 2006]. HBO Stimulates the growth of new blood vessels in places with reduced circulation.

Preliminary evidence. We have previously reported on how HBO modulates inflammatory markers and reactive oxygen species (ROS) in patients. To date, only a few studies have investigated the preconditioning effects of HBO in the human brain and myocardium [Hentia 2018]. In 2004 Sharifi et al. described the use of HBO to inhibit restenosis after PTCI in acute myocardial infarction [Sharifi 2004]. In 2005, Alex et al.observed that repetitive pre-treatment with three sessions of HBO at 2.4 ATA before on-pump coronary artery bypass graft (CABG) surgery reduced neuropsychometric dysfunction and modulated favorably the inflammatory response after CPB [Alex 2005]. Yogaratnam et al. reported that preconditioning with a single session of HBO

at 2.5 ATA before on-pump CABG surgery improved left ventricular stroke work post-CABG surgery while reducing intraoperative blood loss, intensive care unit (ICU) length of stay, and postoperative complications [Yogaratnam 2010].

Recently, Li et al. aimed to determine whether HBO preconditioning could decrease the release of cerebral and myocardial biochemical markers. Endpoints of this study included serum troponin I, inotrope usage, ventilator hours, length of ICU stay, postoperative length of hospital stay, hemodynamic parameters, and serum CAT activity [Hentia 2018]. In one pilot randomized study we found that a single preoperative HBO session the day before pancreatic surgery should modulate the inflammatory response, especially for

] The results in patients with avascular femoral necrosis showed a significant reduction in TNF-α and IL-6 plasma levels over time HBO treatments, with an increase of ROS up to 15 treatments. This decrease in inflammatory markers mirrored the observed reductions in bone marrow edema and reductions in self-reported patient pain.(Bosco 2018) Congruent with our findings, Gardin and colleagues recently suggested that the exposure of osteogenic differentiating MSCs to HBO in simulated inflammatory conditions in vitro increases the differentiation towards the osteogenic phenotype, providing evidence of the potential application of HBO in all those processes that require bone regeneration. The evidence presented above, from the one hand, lends further support to our central hypothesis that HBO can benefit COVID-19 patients by stimulating tissue regeneration.

On the other, it testifies that we are highly qualified to conduct the proposed studies.

Central Hypothesis and Specific Aims

Based on the strong rationale and promising preliminary data reported above, here we advance the following Central Hypothesis: HBO therapy limits respiratory distress and reduces the risk of cardiovascular accidents in patients with latent or manifest COVID-19 infection .
To do so, we propose the following two aims:

AIM 1): To assess pulmonary mild interstitial edema with pulmonary ultrasound, Covid patients related hypoxia due to atelectasis using arterial blood gas analysis and pulmonary ultrasound and assess the impacts on brain blood oxygenation, cerebral blood volume change, SpO2 and heart rate.

AIM 2) To test the hypothesis that progenitor cells, inflammatory cascade and platelet function are linked and impaired in Covid-19 patients by HBO and related to cardiovascular risk.
Endpoints.

1. EPCs: specific subpopulation of SCs that contribute to repair of damaged endothelial. • Cell-based therapy for damaged blood vessels;
• Biomarkers of cardiovascular diseases;

IL-6 and IL-10 with a decrease in postoperative pneumonia. [

Bosco et al. 2014

 
  1. Endothelial progenitor cells bind and inhibit platelet function and thrombus formation. Platelet activation is at the basis of several pathological conditions with vascular impairment, ranging from microcirculation dysfunction (sepsis) to thrombotic ischemia (e.g., stroke, myocardial infarction). Modifications in platelet membrane receptors allow the detection of their activated form and expression and release of osteocalcin (OCN) [Fusaro 2019].

  2. ROS and RNS production and metabolites.

Research Design

We aim at identifying 40 subjects meeting the inclusion criteria reported below. We will divide this patient population into four cohorts of 10 persons (3 case group - 1 control group).

Patient Population Criteria

Inclusion Criteria

Ascertained COVID 19 infection
Signature of the Informed Consent (according to law 648/96) Detection of early-stage interstitial pneumonia at chest CT/RX/ECO Patients who do not need intubation
Age between 30-70 years old
Patients with no additional comorbidities (except hypertension) Patients able to perform Valsalva Manoeuvre in ambient air Mobile patients

Exclusion Criteria

  • Patients suffering from diseases for which hyperbaric oxygen therapy cannot be administered

  • Patients under 30 and over 70 years old

  • Patients with a radiological pattern of severe interstitial pneumonia

  • Extubated patients

  • Smoking patients
    Transport and monitoring of patients: Patients will be picked up from the hospital wards by appropriate and

    will be accompanied by doctors related to plastic surgery, infective and pulmonology Units.

    Treatment chambers and Study Protocol

    -We have available to us two multispace hyperbaric chambers (Vecom, 14 seats) at the ATIP Hyperbaric Medical Center (Padova).
    Workflow

The overall HBO protocol will consist of 10 sessions in total. Each session will be 60 minutes per day. The O2 regimen will be as follows: 1,5 (-5 m); 2 Atm (-10 m) and 2,5 Atm (-15 m) to reduce related risks.

Parameters Monitored/Specific Endpoints
Outcome monitored: Basal (T0) is the day before the first OTI session (T1), fifth OTI session

(T2), tenth OTI session (T3) and 2 mounths later (T4).

  • Radiological picture TC: T0 - T5 - T10

  • Radiological picture RX or ECO: T1-T2-T3-T4

  • Breathing function (T0 - and then daily):

  • EGA (B-pH- B-pCO2 - B-pO2 - B-HCO3 - B-TCO2 total CO2, lactates)

  • Respiratory frequency

  • Oxygenotherapy requirements (l/m)

  • Body Temperature

  • Hemochrome with formula + renal function + PCR + IL-6 + TNF alpha + FERRITIN

  • PBMC, ROS detection, osteocalcin

  • Questionnaire administered pre- and post-therapy to patients to indicate subjective

    ability to breathe

  • Length of hospital stay

    Expected Outcomes/Data Interpretation, Pitfalls, and Possible Side-Effects

    Expected Outcomes and Data Interpretation. We anticipate that increasing the amount of oxygen in the plasma stimulate stem cell, decreasing the inflammatory cascade, thus slowing down the interstitial fibrosis. In turn, this chain of events would delay the onset of ARDS, that is one of the ultimate goals of the present proposal. We predict the proposed HBO protocol by increasing peripheral oxygenation will reduce the risk of multiorgan failure (MOF) due to an overall abated COVID-19 viral load. Conventional HBO has no noticeable side effects and has been recommended for healthy people's oxygen therapy. Usually, once a day for ten days as a course of treatment. In view of the limitations of hyperbaric oxygen therapy technology (large equipment such as an oxygen cabin and a limited number of patients at a time), based on the comprehensive clinical evaluation after treatment, the exposure dose (medication dose) of hyperbaric oxygen is flexibly controlled. Limiting oxygen toxicity, we recommend using a depth among 5 to 15 meters for 60 minutes to detect the outcome and the compliance of COVID-19 patients. In addition , these results could be usefull to suggest respiratory recovery and rehabilitation.

Pitfalls. HBOT requires special large-scale equipment, complex structure, and a limited number of one-time treatments. At ATIP Hyperbaric medical center in Padova is a well-equipped facility to treat critically-ill and infectious patients [Bosco 2018]. In the same vein, although there are systematic hygienic management regulations (cabin sterilization, etc.), given the high infectivity of the new virus, we will further strengthen the sensory control management, along with even more strict disinfection measures can be further strengthened. At this regards, we will follow the disinfection and medical protection of the treatment process suggested by international HBO scientific societies (UHMS, ECHM, SIAARTI, SIMSI).

Side effects of HBO therapy. Studies (mainly conducted in animals) have shown interactions between hyperbaric oxygen and other medicines, in particular dexamethasone, epinephrine, estrogen, amphetamines, thyroid hormones, and bleomycin, which may therefore increase the risk of lung toxic effects. Therefore, we will collect a thorough history of the medicaments taken by the subject before he/she enters the hyperbaric treatment [Clark et al. 1971; Jackson 1985; Yam et al. 1979], and, if it is case, we will exclude him/her from the protocol. The main known side effects are limited to the pulmonary and neurological areas. Pulmonary toxicity usually manifests itself with initial retrosternal or pleural pain due to tracheobronchial irritation. Today's application protocols, reducing ATA, and exposure time minimize this toxicity [Thorsen et al. 1998; Heyboer et al. 2017; Clark et al. 1999]. Thanks to the work of the group of Hadanny et al 2019, it is now consolidated that HBOT (according to the most recent canons) does not cause toxic effects on lung function. Actually, it may turn beneficial (as we anticipated in the present proposal) due to ROS-mediated attenuation of pro-inflammatory processes/signaling cascades. In this regard, studies have already shown that this eventuality could be real in a group of patients with chronic lung disease [Hadanny et al. 2019]. Randomized studies on animal models report damage to the complex phospholipidic system of the lung surfactant: hyperbaric oxygen would increase alveolar surface tension causing hyperoxic toxicity atelectasis [Webb WR et al. 1966]. Changes in protein and phospholipid complexes in the surfactant after prolonged periods of oxygen exposure have also been reported by Prokof’ev et al. 1995 and Bergren et al. 1975. Treatment protocols aimed at limiting O2 exposure to high ATA with increased exposure ranges have shown that this effect on the surfactant has no clinical relevance [Fife and Piantadosi 1991]. Clinical signs of neurological toxicity are visual impairment, tinnitus, nausea, facial spasms, dizziness, and disorientation. In the worst-case scenario, some these adversities may be followed by clonic tonic seizures and loss of consciousness.

We are perfectly aware of these eventualities. However, more recent hyperbaric therapy protocols have allowed to overcome (or minimize) these issues, to a great extent, mainly through air respiration pauses, the reduction of each session duration (<2hrs), and the use of pressure below the threshold of nerve toxicity (see Naval Manual 2008). Oxygen toxicity has also been reported in the retina, particularly in preterm newborns. In adults, on the other hand, cases of hyperoxic myopia have been reported, due to an increase in the

refractive power of the crystalline lens, which is usually reversible over time [Anderson et al. 1978]. Oxidative damage can still affect any cell in the body: red blood cells, myocardium, endocrine system, and renal parenchyma. However, the dosage and timing of hyperbaric oxygen employed here follows the most updated national and international guidelines. Should a problem intervene, such as signs and symptoms of worsening lung function or other organs, we would discontinue the treatment immediately.

In essence, what we are proposing here does not depart – in any manner – from the most recent guidelines both in terms of conditions of HBO therapy administration and patient selection criteria. Finally, our publication record is a testament to our expertise and long-standing acquaintance with the benefits and possible side-effects of hyperbaric medicine. Therefore, we feel that, through a careful monitoring of the patient during the HBO treatment, we will always be in the position to avoiding major side-effects.

Detailed Methods

Quantification of plasma levels of inflammatory markersIL-1β, IL-6 and TNF-α plasma levels were determined by ultrasensitive ELISA immunoassays (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. A nine well micro-plate was pre-coated with monoclonal antigen specific antibodies designed to target our inflammatory markers of interest (IL-1β, IL-6 or TNF-α), three plates per antigen for a positive and negative control standards to ensure there was no cross contamination. Standards and samples (∼200 μL) were pipetted into the wells and the immobilized antibody bound any antigen of interest present. Following the washing procedure, an enzyme-linked, antibody specific, polyclonal antibody was added to the wells. After subsequent washing, a substrate solution was added to the wells and color developed in proportion to the amount of cytokine bound at the initial step. The signal was then spectrophotometrically measured at a wavelength of 450 nm. Plasma levels of inflammatory markers in pg/mL were then calculated according to optical density of each well. This process was repeated for the plasma isolated from each blood sample taken and aggregated for each specific point of measure (T0, T1, T2, etc.) for comparison.

Oxygen free radical detection via electron paramagnetic resonance (EPR).
A X-band electron paramagnetic resonance (EPR) instrument (E-scan-Bruker BioSpin, GmbH, MA) will be used for determination of ROS. The instrument is designed to function with very low concentrations of paramagnetic species in small (50 μL) samples. For each recruited participant, the ROS production rate was determined by means of a recently implemented EPR method [Mrakic 2018]. Determination involved analyse 50 μL plasma samples treated with a CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) probe solution (1:1), in order to transform ROS into more stable radical species that are EPR detectable. 50 μL of the obtained solution was then put in a glass EPR capillary tube (Noxygen Science Transfer & Diagnostics, Germany) that was placed inside the cavity of the E-scan spectrometer for data acquisition. Acquisition parameters were inclusive of a microwave frequency of 9.652 GHz; modulation frequency of 86 kHz; modulation amplitude of 2.28 G; sweep width of 60 G, microwave power of 21.90 mW, number of scans was 10; and receiver gain was equivalent to 3.17·10. Sample temperature was first stabilized and then kept at 37 °C by the temperature and gas controller Bio III unit, interfaced with the spectrometer. Spectra were recorded and analysed using the Win EPR software (2.11 version) supplied by Bruker. EPR measurements allowed us to obtain a relative quantitative determination of ROS production rate in samples. All data were, in turn, converted into absolute concentration (μmol·min−1) by adopting CP• (3-Carboxy-2,2,5,5-tetramethyl- 1-pyrrolidinyloxy) stable radical as an external reference. This process was also repeated for the plasma isolated from each blood sample taken and aggregated for each specific point of measure (T0, T1, T2, etc.) for comparison.

For serum OCN levels, blood (3 ml) will be collected in serum collection tubes, centrifuged at 2300 rpm for 10 minutes, and then divided equally into two aliquots to be frozen and stored at −80 °C. Serum OCN levels will be assessed by ELISA (Takara, Basel, Switzerland) according to the manufacturer’s instructions.

Statistical analysis

Data were analysed using repeated Shapiro-Wilk-s tests. Experimental data were compared using ANOVA repeated measures with Tukey-s multiple comparison test to further check the among-groups significance. Data are presented as means ± SD All p values were two sided and a p value < .05 was considered statistically significant.

An outlook of the proposal and Long-Terms Goal

This proposal aims to improve the clinical outcome in COVID-19 patients at an early stage or asymptomatic pulmonary distress. Evaluation of different oxygen partial pressure treatments could clarify the mechanism of action of HBO and suggesting new respiratory training in pulmonary rehabilitation (long-term goal).

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Hyperbaric oxygen pretreatment and preconditioning. Undersea and

Hyperbaric Medicine, 41(3

Gu, G. J. , Li, Y. P. , Peng, Z. Y. , Xu, J. J. , Kang, Z. M. , Xu, W. G. , ... Sun, X. J. (2008). Mechanism of ischemic tolerance induced by hyperbaric oxygen preconditioning involves upregulation of hypoxia‐inducible factor‐ 1alpha and erythropoietin in rats. Journal of Applied Physiology, 104, 1185–1191

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Padova, 17/04/2020

Gerardo Bosco

Associate Professor

Director: Master II level in Diving and Hyperbaric Medicine
Course in Technical and health management in the hyperbaric chamber Dept. of Biomedical Sciences, University of Padova

Camporesi, E. M. , & Bosco, G. (2014).

), 259–263