Hyperbaric oxygen therapy can increase telomere length and reduce immune senescence in ex vivo blood cells: A prospective trial

——Hyperbaric oxygen therapy (HBOT) can slow down cellular aging in the human body and extend human lifespan.

Researchers: Yafit Hachmo1,*, Amir Hadanny2,3,4,*, Ramzia Abu Hamed1, Malka Daniel-Kotovsky2, Merav Catalogna2, Gregory Fishlev2, Erez Lang2, Nir Polak2, Keren Doenyas2, Mony Friedman2, Yonatan Zemel2, Yair Bechor2, Shai Efra2,3,5

1. Research and Development Department, Zerifin Shamir Medical Center, Israel
2. Sagol Center for Hyperbaric Medicine and Research at the Zerifin Shamir (Assaf-Harofeh) Medical Center in Israel
3. Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
4. Bar-Ilan University, Ramat Gan, Israel
5. Sagol School of Neuroscience, Tel Aviv University, Israel
*All of the above contributors are equally credited.*

Contact: Shai Efrati Amir Hadanny; Email: hadannya@shamir.gov.il; efratishai@outlook.com, https://orcid.org/0000-0001-5523-999X
Keywords: telomere, aging, senescence, hyperbaric oxygen, length
Received: September 3, 2020 Accepted: October 22, 2020 Published: November 18, 2020

Copyright: ©2020 Yafit et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided that the author and original source are properly credited.

Abstract

Introduction: Aging is characterized by the gradual loss of physiological functions. At the cellular level, two key hallmarks of the aging process include shortening of telomere length (TL) and cellular senescence. Repeated intermittent hyperoxia administered according to certain hyperbaric oxygen therapy (HBOT) protocols can induce a regenerative effect, typically occurring during periods of hypoxia. The aim of this study is to evaluate whether HBOT influences TL and the concentration of senescent cells in a population of healthy, non-pathological, older adults.

Methods: Thirty-five healthy, independently living adults aged 64 and older were recruited and received 60 sessions of HBOT daily. Whole blood samples were collected at baseline, after the 30th session, after the 60th session, and 1–2 weeks following the final HBOT session. Telomere length and cellular aging in peripheral blood mononuclear cells (PBMCs) were assessed.

Results: Following HBOT, the telomere lengths of T helper cells, cytotoxic T cells, natural killer cells, and B cells increased significantly by more than 20%. The most pronounced changes were observed in B cells at the 30th and 60th sessions following HBOT, with increases of 25.68% ± 40.42 (p = 0.007), 29.39% ± 23.39 (p = 0.0001), and 37.63% ± 52.73 (p = 0.007), respectively.

The number of senescent T helper cells significantly decreased after HBOT—by 37.30% ± 33.04 (P < 0.0001). The percentage of senescent cytotoxic T cells also significantly declined after HBOT—by 10.96% ± 12.59 (p = 0.0004).

In summary, this study suggests that HBOT may induce pronounced anti-aging effects, including a significant increase in telomere length and the clearance of senescent cells in aging populations.

I. Introduction
The hallmark of aging is the gradual loss of physiological integrity, leading to impaired function and increased susceptibility to disease and death. This biological decline is considered a major risk factor for conditions such as cancer, cardiovascular disease, diabetes, and Alzheimer’s disease. At the cellular level, the aging process is characterized by two key hallmarks: shortening of telomere length and cellular senescence [1].
Telomeres are tandem repeats of nucleotide sequences located at the ends of chromosomes, and they play a crucial role in maintaining genomic stability. During replication (mitosis), telomeres tend to shorten due to the inherent inability to fully replicate the lagging DNA strand’s terminal region [2]. Telomere length (TL) typically ranges from 4 to 15 kilobases, gradually shortening by approximately 20–40 bases each year. Shortened telomeres have been associated with various diseases, reduced physical fitness, and thinning of the cerebral cortex [3–5]. When TL reaches a critical length, cells can no longer divide and will either enter senescence or undergo programmed cell death [6]. Studies by Goglin et al. have shown that adults with shorter TLs exhibit an increased mortality rate [7]. While telomere shortening may be directly influenced by genetic factors, it is also associated with several environmental factors, including stress, lack of physical activity, high body mass index, smoking, chronic inflammation, vitamin deficiencies, and oxidative stress [2, 8, 9].
Cellular senescence represents a state of cell cycle arrest, which may be triggered by telomere shortening [10] as well as other aging-related stimuli unrelated to telomere length (TL), such as non-telomeric DNA damage [1]. The primary purpose of cellular senescence is to prevent the proliferation of damaged cells by prompting their clearance via the immune system. The accumulation of senescent cells with age reflects an increase in their production and/or a decrease in their clearance rate, which in turn exacerbates damage and contributes to aging [1].
An increasing number of studies have identified several pharmacological agents that can reduce the rate of telomere shortening [11, 12]. Several lifestyle interventions—including endurance training, diets and supplements targeting cellular metabolism and oxidative stress—have relatively modest effects on TL3 (2–5%) [2, 8, 9].
Hyperbaric oxygen therapy (HBOT) involves the administration of 100% oxygen at an environmental pressure exceeding one absolute atmosphere (ATA), thereby increasing the amount of dissolved oxygen in body tissues. Repeated intermittent exposures to high oxygen levels under certain HBOT protocols can elicit physiological effects that typically occur during periods of lower oxygen levels within the hyperoxic environment—a phenomenon known as the hyperoxia-hypoxia paradox [13-16]. Moreover, recent studies have demonstrated that HBOT can induce cognitive enhancement in healthy, aging adults through mechanisms involving changes in cerebral blood flow regions [17]. At the cellular level, HBOT has been shown to upregulate the expression of hypoxia-inducible factor (HIF), vascular endothelial growth factor (VEGF), and sirtuins (SIRT), promote stem cell proliferation, enhance mitochondrial biogenesis, stimulate angiogenesis, and facilitate neurogenesis [18]. However, to date, no studies have examined the effects of HBOT on telomere length and the accumulation of senescent cells.
The aim of the current study is to evaluate whether HBOT affects telomere length (TL) and senescent-like T cells in adults.

II. Results
Thirty-five individuals were assigned to the HBOT group. Five patients did not complete the baseline assessment and were therefore excluded. All 30 patients who completed the baseline assessment also completed the intervention. Due to poor blood sample quality (low cell count or technician error), four patients were excluded from telomere analysis, and ten patients were excluded from senescent cell analysis (Figure 1). Table 1 lists the baseline characteristics and cohort comparisons after exclusion of these patients. There were no significant differences among the three groups (Table 1).

Telomere length
Compared to the baseline, the telomere length of T helper cells significantly increased by 21.70 ± 40.05 (p = 0.042), 23.69% ± 39.54 (p = 0.012), and 29.30 ± 38.51 (p = 0.005) after the 30th treatment session and HBOT, respectively (Figure 2). However, repeated-measures analysis revealed a non-significant trend (F = 4.663, p = 0.06; Table 2 and Figure 2).
Compared to the baseline, the telomere length of B cells significantly increased by 25.68% ± 40.42 (p = 0.007), 29.39% ± 23.39 (p = 0.0001), and 37.63% ± 52.73 (p = 0.007) after the 30th treatment session, the 60th treatment session, and following HBOT, respectively (Figure 2). Repeated-measures analysis revealed a significant within-group effect (F = 0.390, p = 0.017; Table 2 and Figure 2).
Compared to the baseline, the telomere length of natural killer cells significantly increased by 20.56% ± 33.35 (p = 0.045) after the 30th treatment session and by 22.16% ± 44.81 (p = 0.06) after HBOT, as shown in Table 2 and Figure 2. Repeated-measures analysis indicated no other significant effects following the 30th treatment session (F = 0.812, p = 0.391).
Compared to the baseline, there was no significant increase in cytotoxic T cells at the 30th treatment session, with an increase of 18.29% ± 45.62 (p = 0.11). Subsequently, a significant increase of 24.13% ± 40.88 (p = 0.0019) and 19.59% was observed at the 60th treatment session following HBOT, with a further increase of 33.98% ± 33.98 (p = 0.023). Repeated-measures analysis indicated that no other significant effects were observed after the 30th treatment session (F = 1.159, p = 0.310; Table 2 and Figure 2).

Senescent cells
At the 30th and 60th treatment sessions, the number of senescent T helper cells decreased by -19.66% ± 80.03 (p = 0.09) and -11.67% ± 94.30 (p = 0.20), respectively. However, following HBOT, the number of senescent T helper cells showed a significant decrease of -37.30% ± 33.04 (P < 0.0001; Figure 3). Repeated-measures analysis revealed that this effect remained statistically significant even after the 30th treatment session, with a consistent within-group effect (F = 8.547, p = 0.01; Table 2 and Figure 3).
The percentage of T-cell-mediated cytotoxic senescent cells significantly decreased after the 30th HBOT session—by 12.21% ± 8.74 (P < 0.0001); by 9.81% ± 9.50 after the 60th HBOT session (P = 0.002); and by 10.96% ± 12.59 after HBOT (1–2 weeks) (Table 2 and Figure 3). Repeated-measures analysis revealed that even after the 30th session, a significant continuous effect persisted, with a within-group effect (F = 6.916, P = 0.018; Table 2).
HIF-1α
At the 60th treatment session (p = 0.006), the HIF-1alpha level increased from 10.54 ± 3.39 to 19.71 ± 3.39 (p = 0.006). Specifically, the level 2 weeks after HBOT was 16.81 ± 7.65, which showed no significant difference compared to the baseline (p = 0.16).

3. Discussion
In this study, for the first time in humans, it was found that repeated daily hyperbaric oxygen therapy (HBOT) sessions in aging individuals can increase PBMC telomere length by more than 20%, with the most significant changes observed in B cells. Furthermore, HBOT reduced the number of senescent cells by 10–37%, with T-helper senescent cells being the most affected.
A substantial body of evidence has already demonstrated associations between telomere length and lifestyle changes. This has prompted several intervention studies focusing on diet, supplements (such as omega-3 fatty acids and walnuts), physical exercise, stress management, and social support. In a two-year trial involving cognitively healthy older adults, a diet enriched with walnuts showed no significant trend toward preserving telomere length when compared to a control diet [19]. In another study evaluating the effects of twelve weeks of low-frequency explosive resistance training in older adults, the intervention group exhibited better preservation of telomere length without any significant increase [20]. A recent study found that aerobic endurance training or six months of high-intensity interval training could lead to a 5% increase in telomere length [21]. Additional interventions, including weight loss, yoga, and stress-management techniques, failed to show significant changes in telomere length [22-25]. However, most studies have indicated a significant correlation between antioxidant activity and telomerase activity [22-25].
Although numerous genetic and environmental factors are associated with telomere shortening, the most commonly implicated mechanism is oxidative stress. Oxidative stress may arise from an imbalance between the production of reactive oxygen species (ROS) and the cell’s capacity to neutralize them. Telomeres are highly sensitive to oxidative DNA damage, which can lead to telomere shortening and functional impairment [26]. The relationship between oxygen and/or oxidative stress and telomere length has been a subject of debate over the past several decades. Consistent findings from human cell-culture studies indicate that mild oxidative stress accelerates telomere shortening, whereas antioxidants and free-radical scavengers can slow down the rate of shortening and extend cellular proliferative lifespan [27]. Some clinical studies on pathological conditions—such as diabetes, inflammatory diseases, and Parkinson’s disease—have demonstrated correlations among oxidative-stress markers, levels of reactive oxygen species scavengers, and telomere length [28]. However, healthy individuals have not shown similar results [29].
Previously, it has been suggested that exposing cell cultures to high-pressure environments can induce significant oxidative stress and premature cellular senescence [30]. However, these findings were based on isolated cells grown in high-pressure chambers, rather than on complex human biological systems as in the present study. Similar to the current research, a previous one-year prospective observational study of divers exposed to hyperbaric oxygen at high pressure showed a significant lengthening of telomeres in leukocytes [31]. As used in the current study, the HBOT protocol leverages the effects induced by repeated, intermittent exposures to high oxygen—what is known as the “hyperoxia-hypoxia paradox” [13, 18]. These intermittent high-oxygen exposures elicit adaptive responses, including upregulation of antioxidant genes [32] and the production of antioxidants and scavengers that are better adapted to cope with the increased generation of reactive oxygen species (ROS). As a result, the ratio of ROS to scavengers gradually becomes similar to that observed under normal oxygen conditions. However, since the half-life (T1/2) of scavengers is significantly longer than that of ROS, after repeated cycles of normal high-oxygen exposure followed by return to baseline oxygen levels, scavenger levels rise markedly, leading to enhanced antioxidant activity [13, 18]. Thus, much like physical exercise and caloric restriction, daily repetition of the HBOT protocol may trigger an "excitatory effect." A single exposure sharply increases ROS production, thereby triggering an antioxidant response; whereas repeated exposures progressively enhance protective effects [13, 18].
In addition, intermittent exposure to hyperbaric oxygen triggers many physiological responses that normally occur during hypoxic conditions [13]. HBOT induces the release of a transcription factor known as hypoxia-inducible factor (HIF) and enhances its stability and activity [14]. In turn, HIF initiates a cascade of cellular responses, including the induction of vascular endothelial growth factor and angiogenesis, mitochondrial biogenesis, stem cell mobilization, and increased SIRT1 activity [18]. Our study confirms that repeated exposure to HBOT can induce an increase in HIF expression; however, under non-monochamber conditions, HBOT-induced HIF expression gradually declines until HIF levels return to baseline.
Currently, numerous interventions aimed at eliminating senescent cells—either through genetic or pharmacological approaches (senolytics)—have been developed in animal models and are awaiting evaluation for safety and efficacy in humans [33]. Recent studies have proposed a non-pharmacological approach that is clinically applicable, boasts well-established safety profiles, and can be used to reduce senescent cells. Our protocol involves 60 sessions of 100% oxygen therapy at 2 ATA, each session interspersed with three air breaks, thereby leveraging the hyperoxia-hypoxia paradox while minimizing the risk of oxygen toxicity. Interestingly, both TL and the reduction in senescent cells reached their peak by the 30th session. However, the dose-response curves associated with the applied pressure, duration, and number of HBOT sessions—and their relationship to HIF expression and the associated regenerative effects—remain incompletely understood. Further research is needed to identify the optimal HBOT regimen.
Hyperbaric oxygen therapy is a well-established treatment for non-healing wounds, radiation injuries, and various hypoxic or ischemic conditions (such as carbon monoxide poisoning and infections). In recent years, an increasing body of preclinical and clinical evidence has demonstrated the efficacy of HBOT in neurological indications, including idiopathic sudden sensorineural hearing loss [34], post-stroke and post-traumatic brain injury conditions [35-41], central sensitization syndromes such as fibromyalgia syndrome [42, 43], age-related cognitive decline [17], and Alzheimer’s disease animal models [44]. This study aims, for the first time, to evaluate the physiological effects at the cellular level in elderly humans without any functional limitations.

Study limitations:
The current study has several limitations that need to be taken into account. First, the limited sample size must be considered. Second, there is a lack of a control group. However, this study demonstrates that impressive results in terms of TL and senescent cell clearance have not been observed with any other interventions. Moreover, the baseline telomere length values in our cohort are consistent with expected values for an aging population [45-47]. Third, the duration of the effect remains to be determined through long-term follow-up. Fourth, since the methods chosen for blood preservation and assessment were used, telomerase activity was not evaluated. Nevertheless, some advantages should be highlighted. In this study, CD28 was used as a biomarker for senescent cells, whereas CD57 cannot serve as a definitive marker for T-cell senescence. Biomarkers were assessed specifically in particular subsets of white blood cells rather than using the entire PBMC population as a single group. The effects of isolated HBOT were measured, and participants were monitored closely to ensure that they did not alter their lifestyle (e.g., diet and exercise), take medications, or undergo any other interventions that could potentially confound the results.
In summary, this study demonstrates that HBOT can induce pronounced (anti-)aging effects, including a significant increase in telomere length and the clearance of senescent cells in aging populations.

4. Materials and Methods
Project:
Thirty-five adults aged 64 and older who had not yet experienced pathological cognitive decline were recruited. These participants lived independently and were in good physical and cognitive condition. The study was conducted between 2016 and 2020 at the Shamir (Assaf-Harofeh) Medical Center in Israel. Prior to enrollment, the selected patients had no history of cardiac or cerebrovascular ischemia in the year preceding their inclusion. Exclusion criteria included: prior hyperbaric oxygen therapy for any reason within the past three months; a history of malignancy within the past year; any evidence of pathological cognitive decline; severe chronic renal failure (GFR <30); uncontrolled diabetes (HbA1C >8, fasting blood glucose >200); use of immunosuppressive agents; contraindications to MRI (including BMI >35); smoking addiction or pulmonary disease.
Study design:
The study protocol has been approved by the Institutional Review Board of the Shamir Medical Center in Israel. This study is a prospective clinical trial. After signing the informed consent form and undergoing baseline assessment, participants will be assigned to HBOT. Measurement points will be assessed at baseline, halfway through the treatment regimen (at the 30th session), on the day of the last HBOT session, and 1–2 weeks after completion of HBOT.
This study cohort included only patients who received HBOT treatment and was part of a larger, normally aging population studied at the Shamir Medical Center in Israel (NCT02790541 [17]).

Intervention measures:
The HBOT protocol was conducted in the German HAUX multi-person chamber (model: Starmed-2700). The treatment course lasted 60 days, with five sessions per week, completed within three months. Each session involved breathing 100% oxygen through a mask at a pressure of 2 ATA for 90 minutes, during which the mask was removed every 20 minutes to allow breathing air for 5 minutes. The compression/decompression rate was 1 meter per minute. During the trial period, no changes were permitted in lifestyle, dietary habits, or medication regimens.

Blood sample:
Using standard techniques, whole blood samples will be collected into EDTA tubes at baseline, halfway through the HBOT regimen (at the 30th treatment session), one day after the last HBOT treatment session (at the 60th treatment session), and 1–2 weeks after the final HBOT treatment session.
Peripheral Blood Mononuclear Cell (PBMC) Isolation:
Dilute whole blood with phosphate-buffered saline (PBS). Perform density gradient separation using Leucosep tubes pre-filled with Lymphoprep. Then, centrifuge the tubes at 1000×g for 10 minutes at 25°C. Immediately after centrifugation, collect the cell layer (the buffy coat) using a pipette and transfer it into a 50-mL conical centrifuge tube. Resuspend the cells in sufficient 1X PBS to bring the final volume to 50 mL, and centrifuge again at 300×g for 10 minutes at 25°C. After removing the supernatant, label each sample accordingly.

Telomere length:
Telomeres were labeled according to the Dako PNA/FITC Kit protocol (code K5327). In a single-cell suspension composed of a mixture of PBMCs (sample cells) and the TCL 1301 cell line (control cells), DNA was denatured at 82°C for 10 minutes in microcentrifuge tubes in the absence of probes, using a hybridization solution without any probes. Hybridization was then carried out overnight at room temperature (RT) in the dark, using a hybridization solution containing fluorescein-conjugated PNA telomere probes. Following hybridization, two additional 10-minute washes were performed at 40°C using a washing solution. Subsequently, the samples were stained with antibodies conjugated to CD4+, CD8+, CD3+, CD19+, and CD56+ in an appropriate buffer for further flow cytometric analysis [48, 49]. Each sample was analyzed in duplicate. After flow cytometric analysis, the relative telomere length (RTL) was calculated for CD3+/CD4+ (T-helper cells), CD3+/CD8+ (cytotoxic T cells), CD3+/CD56+ (natural killer cells), and CD19+ (B cells). The RTL values were determined as the ratio of the telomere signal from each sample to that of the control cells (the TCL 1301 cell line), with correction for the DNA index of G0/G1-phase cells. DNA ploidy of both sample cells and control cells was analyzed separately using propidium iodide staining to normalize the number of telomeric ends per cell and thereby standardize the telomere length per chromosome. For an example of FACS analysis, see Figure 4.

Immunophenotyping:
The percentages of CD3+CD4+CD28-negative effector T cells (senescent T helper cells) and CD3+CD8+CD28-negative effector T cells (senescent cytotoxic T cells) were determined by flow cytometric analysis. PBMCs were stained with VioBlue-conjugated anti-CD3, Viogreen-conjugated anti-CD8, PE-VIO 770A-conjugated anti-CD4, and APC-VIO 770A-conjugated anti-CD28 antibodies (Miltenyi Biotec). Cells were analyzed using the MACSQuant flow cytometer (Miltenyi Biotec). The percentage of CD28-null T cells within the CD4+ or CD8+ T cell populations was then calculated.

Hypoxia-inducible factor (HIF-1alpha):
After fixation and permeabilization (Life Technologies), intracellular HIF1α staining was performed using an APC-conjugated anti-HIF1α antibody or the corresponding isotype control (R&D Systems). Cells were analyzed using a MACSQuant flow cytometer (Miltenyi Biotec), and the percentage of PBMCs expressing HIF1α was determined.

Statistical analysis:
Unless otherwise specified, continuous data are presented as mean ± standard deviation. The Kolmogorov-Smirnov test was used to assess the normal distribution of all variables. One-way analysis of variance was conducted to compare variables among and within the three groups at baseline.
Categorical data are presented as numbers and percentages and compared using the chi-square test. Univariate analysis is conducted using the Chi-Square/Fisher’s exact test to identify significant variables (P < 0.05).
To evaluate the effects of HBOT, a repeated-measures ANOVA model was used to test the main within-subject effects. Post-hoc pairwise comparisons were conducted using t-tests with Bonferroni correction to examine differences across time points.

        This is a groundbreaking, forward-looking research study—and the first-ever experimental data to demonstrate the positive effects of hyperbaric oxygen therapy in slowing down the human aging process.