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RESEARCH PAPER VOLUME 12, ISSUE 22 PP 22445—22456


HYPERBARIC OXYGEN THERAPY INCREASES TELOMERE LENGTH AND DECREASES
IMMUNOSENESCENCE IN ISOLATED BLOOD CELLS: A PROSPECTIVE TRIAL

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 EFRATI1,2,3,5, ,

 * 1 Research and Development Unit, Shamir Medical Center, Zerifin, Israel
 * 2 The Sagol Center for Hyperbaric Medicine and Research, Shamir
   (Assaf-Harofeh) Medical Center, Zerifin, Israel
 * 3 Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
 * 4 Bar Ilan University, Ramat-Gan, Israel
 * 5 Sagol School of Neuroscience, Tel-Aviv University, Tel-Aviv, Israel

* Equal contribution

RECEIVED: SEPTEMBER 3, 2020       ACCEPTED: OCTOBER 22, 2020       PUBLISHED:
NOVEMBER 18, 2020      

https://doi.org/10.18632/aging.202188
How to Cite


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HOW TO CITE

Hachmo Y, Hadanny A, Abu Hamed R, Daniel-Kotovsky M, Catalogna M, Fishlev G,
Lang E, Polak N, Doenyas K, Friedman M, Zemel Y, Bechor Y, Efrati S, .
Hyperbaric oxygen therapy increases telomere length and decreases
immunosenescence in isolated blood cells: a prospective trial. Aging (Albany
NY). 2020 Nov 1812:22445-22456. https://doi.org/10.18632/aging.202188


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Citation | Citation & Abstract

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 the
original author and source are credited.


ABSTRACT

Introduction: Aging is characterized by the progressive loss of physiological
capacity. At the cellular level, two key hallmarks of the aging process include
telomere length (TL) shortening and cellular senescence. Repeated intermittent
hyperoxic exposures, using certain hyperbaric oxygen therapy (HBOT) protocols,
can induce regenerative effects which normally occur during hypoxia. The aim of
the current study was to evaluate whether HBOT affects TL and senescent cell
concentrations in a normal, non-pathological, aging adult population.

Methods: Thirty-five healthy independently living adults, aged 64 and older,
were enrolled to receive 60 daily HBOT exposures. Whole blood samples were
collected at baseline, at the 30th and 60th session, and 1-2 weeks following the
last HBOT session. Peripheral blood mononuclear cells (PBMCs) telomeres length
and senescence were assessed.

Results: Telomeres length of T helper, T cytotoxic, natural killer and B cells
increased significantly by over 20% following HBOT. The most significant change
was noticed in B cells which increased at the 30th session, 60th session and
post HBOT by 25.68%±40.42 (p=0.007), 29.39%±23.39 (p=0.0001) and 37.63%±52.73
(p=0.007), respectively.

There was a significant decrease in the number of senescent T helpers by
-37.30%±33.04 post-HBOT (P<0.0001). T-cytotoxic senescent cell percentages
decreased significantly by -10.96%±12.59 (p=0.0004) post-HBOT.

In conclusion, the study indicates that HBOT may induce significant senolytic
effects including significantly increasing telomere length and clearance of
senescent cells in the aging populations.




INTRODUCTION

Aging can be characterized by the progressive loss of physiological integrity,
resulting in impaired functions and susceptibility for diseases and death. This
biological deterioration is considered a major risk factor for cancer,
cardiovascular diseases, diabetes and Alzheimer’s disease among others. At the
cellular level, there are two key hallmarks of the aging process: shortening of
telomere length and cellular senescence [1].

Telomeres are tandem nucleotide repeats located at the end of the chromosomes
which maintain genomic stability. Telomeres shorten during replication (mitosis)
due to the inherent inability to fully replicate the end part of the lagging DNA
strand [2]. Telomere length (TL), measuring between 4 to 15 kilobases, gradually
shorten by ~20-40 bases per year and is associated with different diseases, low
physical performance and cortical thinning of the brain [3–5]. When TL reaches a
critical length, cells cannot replicate and progress to senescence or programmed
cell death [6]. Goglin et al. demonstrated that adults with shorter TLs have
increased mortality rates [7]. Shortened TLs can be a direct inherited trait,
but several environmental factors have also been associated with shortening TL
including stress, lack of physical endurance activity, excess body mass index,
smoking, chronic inflammation, vitamins deficiency and oxidative stress [2, 8,
9].

Cellular senescence is an arrest of the cell cycle which can be caused by
telomere shortening [10], as well as other aging associated stimuli independent
of TL such as non-telomeric DNA damage [1]. The primary purpose of senescence is
to prevent propagation of damaged cells by triggering their elimination via the
immune system. The accumulation of senescent cells with aging reflects either an
increase in the generation of these cells and/or a decrease in their clearance,
which in turn aggravates the damage and contributes to aging [1].

A growing body of research has found several pharmacological agents that can
reduce the telomere shortening rate [11, 12]. Several lifestyle interventions
including endurance training, diets and supplements targeting cell metabolism
and oxidative stress have reported relatively small effects (2-5%) on TL3, [2,
8, 9].

Hyperbaric oxygen therapy (HBOT) utilizes 100% oxygen in an environmental
pressure higher than one absolute atmospheres (ATA) to enhance the amount of
oxygen dissolved in body’s tissues. Repeated intermittent hyperoxic exposures,
using certain HBOT protocols, can induce physiological effects which normally
occur during hypoxia in a hyperoxic environment, the so called hyperoxic-hypoxic
paradox [13–16]. In addition, it was recently demonstrated that HBOT can induce
cognitive enhancements in healthy aging adults via mechanisms involving regional
changes in cerebral blood flow [17]. On the cellular level, it was demonstrated
that HBOT can induce the expression of hypoxia induced factor (HIF), vascular
endothelial growth factor (VEGF) and sirtuin (SIRT), stem cell proliferation,
mitochondrial biogenesis, angiogenesis and neurogenesis [18]. However, no study
to date has examined HBOT’s effects on TL and senescent cell accumulation.

The aim of the current study was to evaluate whether HBOT affects TL and
senescence-like T-cells population in aging adults.


RESULTS

Thirty-five individuals were assigned to HBOT. Five patients did not complete
baseline assessments and were excluded. All 30 patients who completed baseline
evaluations completed the interventions. Due to the low quality of blood samples
(low number of cells or technician error), four patients were excluded from the
telomere analysis and 10 patients from senescent cell analysis (Figure 1). The
baseline characteristics and comparison of the cohorts following exclusion of
the patients are provided in Table 1. There were no significant differences
between the three groups (Table 1).

Figure 1. Patient flowchart.


TABLE 1. BASELINE CHARACTERISTICS.

HBOTTelomere analysisSenescent analysisP-valueN3025 (83.3%)20 (66.6%)Age
(years)68.41±13.267.56±14.3566.70±16.000.917BMI26.77±3.2026.89±3.3427.14±3.810.946Males16
(53.3%)13 (52.0%)10 (50.0%)0.987Females14 (47.7%)12 (48.0%)10
(50.0%)0.987Complete blood countHemoglobin6.33±1.256.57±1.156.58±1.290.707White
blood
cells14.02±1.4013.92±1.3513.97±1.490.969%PBMC39.96±6.7539.25±6.6438.59±6.630.774Platelets239.87±1.39244.08±43.0254.05±41.40.559Chronic
medical conditionsAtrial fibrillation4 (13.3%)4 (16.0%)2
(10.0%)0.841Hypothyroidism4 (13.3%)4 (16.0%)3 (15.8%)0.956Obstructive sleep
apnea4 (13.3%)4 (16.0%)3 (15.0%)0.961Asthma1 (3.3%)1 (4.0%)00.680BPH7 (23.3%)5
(20.0%)6 (30.0%)0.733GERD3 (10%)2 (8.0%)2 (10.0%)0.961Osteoporosis5 (16.7%)5
(20.0%)4 (20.0%)0.936Rheumatic arthritis1 (3.3%)01 (5.0%)0.561Osteoarthritis7
(23.3%)4 (16.0%)5 (25.0%)0.755Diabetes mellitus3 (10%)3 (12.0%)2
(10.0%)0.966Hypertension7 (23.3%)5 (20.0%)5 (25.0%)0.918Dyslipidemia16 (53.3%)14
(56.0%)12 (60.0%)0.897Ischemic heart disease2 (6.7%)1 (4.0%)2
(10.0%)0.725History of smoking10 (33.3%)8 (32.0%)7 (35.0%)0.978Chronic
medicationsAnti-aggregation8 (26.7%)6 (24.0%)5 (25.0%)0.974ACE-Inhibitors/ARB
blockers6 (20%)6 (24.0%)6 (30.0%)0.720Beta blockers5 (16.7%)5 (20.0%)3
(15.0%)0.901Calcium blockers3 (10%)3 (12.0%)2 (10.0%)0.966Alpha blockers7
(23.3%)5 (20.0%)6 (30.0%)0.733Diuretics2 (6.7%)1 (4.0%) 1 (5.0%)0.906Statins10
(33.3%)9 (36.0%)7 (35.0%)0.978Oral hypoglycemic1 (3.3%)1 (4.0%)1
(5.0%)0.958Bisphosphonates1 (3.3%)1 (4.0%)1 (5.0%)0.958Proton pump inhibitors3
(10%)3 (12.0%)3 (15.0%)0.726Hormones3 (10%)3 (12.0%)2
(10.0%)0.966Benzodiazepines3 (10%)2 (8.0%)1 (5.0%)0.816SSRI5 (16.7%)5 (20.0%)3
(15.0%)0.990


TELOMERE LENGTH

Compared to the baseline, the T-helper telomere lengths were significantly
increased at the 30th session and post-HBOT by 21.70±40.05 (p=0.042),
23.69%±39.54 (p=0.012) and 29.30±38.51 (p=0.005), respectively (Figure 2).
However, repeated measures analysis shows a non-significant trend (F=4.663,
p=0.06, Table 2 and Figure 2).

Figure 2. Telomere length changes with HBOT. Mean+SEM *p<0.05, **p<0.01,
***p<0.001.


TABLE 2. TELOMERE LENGTH AND SENESCENT CELL CHANGES POST-HBOT.

Absolute changesRelative changes (%)Repeated measures F (p)PBMCBaseline30th
Session60th SessionPost HBOT30th session60th sessionPost-HBOTPBMC
((N=25)2.55±0.53-0.15±0.40-4.91±16.701.987 (t) 0.09PBMC
(N=20)2.50±0.53-0.13±0.31-4.21±11.991.810 (t) 0.07Relative telomeres length
(N=25)Natural killer9.27±1.9111.77±5.14 (0.045)10.73±2.73 (0.013)11.75±4.22
(0.06)25.02±51.4220.56±33.3522.16±44.810.812 (0.391)B-cells8.36±2.0210.22±3.04
(0.007)11.23±3.58 (0.0001)11.17±2.98
(0.007)25.68±40.4229.39±23.3937.63±52.737.390 (0.017)T Helper8.04±1.829.92±3.68
(0.042)9.63±2.17 (0.012)10.20±2.77 (0.005)21.70±40.0523.69±39.5429.30±38.514.663
(0.063)T Cytotoxic8.26±1.549.83±4.08 (0.11)10.08±3.33 (0.019)10.15±2.74
(0.023)18.29±45.6224.13±40.8819.59±33.981.159 (0.310)Senescent cells (% of T
cells) (N=20)T Helper10.29±5.427.84±7.09 (0.09)8.51±7.45 (0.20)6.22±4.88
(<0.0001)-19.66±80.03-11.67±94.30-37.30±33.048.548 (0.01)T
Cytotoxic52.19±21.0745.53±19.91 (<0.0001)45.45±18.81 (0.002)46.59±21.91
(0.0004)-12.21±8.74-9.81±9.50-10.96±12.596.916 (0.018) P-values shown in ()
compared to baseline.P-values in bold <0.05.

Compared to baseline, telomere lengths of B cells increased significantly at the
30th session, 60th session and post-HBOT by 25.68%±40.42 (p=0.007), 29.39%±23.39
(p=0.0001) and 37.63%±52.73 (p=0.007), respectively (Figure 2). Repeated
measures analysis shows a significant within-group effect (F=0.390, p=0.017,
Table 2 and Figure 2).

Compared to baseline, natural killer cells telomer lengths significantly
increased at the 30th session (p=0.045) and at the 60th session by 20.56% ±33.35
(p=0.013). Post-HBOT, telomere lengths increased by 22.16%±44.81 post-HBOT
(p=0.06, Table 2 and Figure 2). Repeated measures analysis indicates that there
was no additional significant effect after the 30th session (F=0.812, p=0.391).

Compared to baseline, cytotoxic T-cells had a non-significant increase at the
30th session by 18.29%±45.62 (p=0.11), followed by a significant increase of
24.13%±40.88 at the 60th session (p=0.0019) and 19.59%±33.98 post-HBOT
(p=0.023). Repeated measures analysis indicates that there was no additional
significant effect after the 30th session (F=1.159, p=0.310, Table 2 and Figure
2).


SENESCENT CELLS

There was a non-significant decrease in the number of senescent T-helpers at the
30th session and 60th session by -19.66%±80.03 (p=0.09) and -11.67%±94.30
(p=0.20) respectively. However, there was a significant drop in the number of
senescent T helpers by -37.30%±33.04 post-HBOT (P<0.0001, Figure 3). Repeated
measures analysis showed a significant continuous effect even after the 30th
session, with a within-group effect (F=8.547, p=0.01, Table 2 and Figure 3).

Figure 3. Senescent cell changes with HBOT. Mean+SEM *p<0.05, **p<0.01,
***p<0.001.


T-cytotoxic senescent cell percentages decreased significantly by -12.21%±8.74
(P<0.0001) at the 30th HBOT session, -9.81%±9.50 at the 60th HBOT session
(0.002) and -10.96%±12.59 (p=0.0004) post-HBOT (Table 2 and Figure 3). Repeated
measures analysis shows a significant continuous effect even after the 30th
session, with a within-group effect (F=6.916, p=0.018, Table 2).


HIF-1ALPHA

HIF-1alpha levels were increased from 10.54±3.39 to 19.71±3.39 at the 60th
session (p=0.006) where 2 weeks post HBOT levels of 16.81±7.65 were not
significantly different from baseline (p=0.16).


DISCUSSION

In this study, for the first time in humans, it was found that repeated daily
HBOT sessions can increase PBMC telomere length by more than 20% in an aging
population, with B cells having the most striking change. In addition, HBOT
decreased the number of senescent cells by 10-37%, with T helper senescent cells
being the most effected.

A substantial number of associations between telomere length and lifestyle
modifications have been observed. This has led to several interventional studies
which included diet, supplements (such as omega-3, and walnuts among others),
physical activity, stress management and social support. A two year trial
conducted on cognitively healthy elderly adults, using a diet rich in walnuts,
showed a non-significant trend to preserve telomere length when compared to a
control diet [19]. In another study which evaluated the effect of a twelve week
low frequency explosive-type resistance training in elderly people, telomere
length was better preserved in the intervention group without a significant
increase [20]. A recent study found that aerobic endurance training or high
intensity interval training for six month increased telomere length up to 5%
[21]. Additional weight loss, yoga and stress management techniques failed to
show significant telomere length changes [22–25]. However, most of these studies
have shown significant correlations between antioxidant activity and telomerase
activity [22–25].

While many genetic and environmental factors are associated with telomere
shortening, the most common suggest mechanism is oxidative stress. Oxidative
stress can occur from imbalances between the production of reactive oxygen
species (ROS) and cellular scavengers. Telomeres are highly sensitive to
oxidative DNA damage, which can induce telomere shortening and dysfunction [26].
The association between oxygen and/or oxidative stress and telomere length has
been debated for the past several decades. Human cell culture studies
consistently show that mild oxidative stress accelerates telomere shortening,
whereas antioxidants and free radical scavengers decrease shortening rates and
increase the cellular proliferative lifespan [27]. Several clinical studies on
pathological conditions (such as diabetes, inflammatory diseases, Parkinson’s
disease) have shown correlations between oxidative stress markers, reactive
oxygen species scavengers levels and telomere length [28]. However, healthy
individuals did not show similar results [29].

Exposing cell cultures to a hyperbaric environment has been previously suggested
to induce significant oxidative stress and premature cells senescence [30].
However, this was based on isolated cells grown in a hyperbaric incubator and
not on the complex biological system of humans as in this study. Similar to the
current study, a previous prospective one-year observational study in divers
exposed to intense hyperbaric oxygen, showed significant telomere elongation in
leukocytes [31]. As used in the current study, the HBOT protocol utilizes the
effects induced by repeated intermittent hyperoxic exposures, the so called
hyperoxic hypoxic paradox [13, 18]. These intermittent hyperoxic exposures
induce an adaptive response which includes increased upregulation of
antioxidants genes [32] and production of antioxidants/scavengers that adjust to
the increased ROS generation causing the ROS/scavenger ratio to gradually
becomes similar to the ratio under a normal oxygen environment. However, because
the scavenger elimination half-life (T1/2) is significantly longer than the T1/2
of ROS, upon return to normoxia, following repeated hyperoxic exposures, there
are significantly higher levels of scavengers and increased antioxidant activity
[13, 18]. Thus, similar to physical exercise and caloric restriction, a daily
repeated HBOT protocol can induce the hormesis phenomenon. Single exposures
increase ROS generation acutely, triggering the antioxidant response, and with
repeated exposures, the response becomes protective [13, 18].

Additionally, intermittent hyperoxic exposures induce many of the physiological
responses that occur during hypoxia [13]. HBOT induces the release of
transcription factors called hypoxic induced factors (HIF) and increase their
stability and activity [14]. In turn, HIF induces a cellular cascade including
vascular endothelial growth factor and angiogenesis induction, mitochondria
biogenesis, stem cells mobilization and SIRT1 increased activity [18]. Our study
confirms increased HIF expression is induced by repetitive HBOT exposures, which
gradually decreases towards normalization of HIF levels at nonmonic environment.

Currently, many interventions that genetically or pharmacologically (senolytic
drugs) remove senescent cells have been developed in animal models and are
waiting for safety and efficacy evaluations in humans [33]. The current study
suggests a non-pharmacological method, clinically available with
well-established safety profile, for senescent cells populations decrease. Our
protocol included 60 sessions of 100% oxygen at 2 ATA including three air breaks
during each session to utilize the hyperoxic hypoxic paradox and minimize the
risk of oxygen toxicity. Interestingly, both TL and senescent cell reduction
peaked at the 30th session. However, the dose response curve related to the
applied pressure, time and number of HBOT exposures and its relation to HIF
expression and its related regenerative effects are still not fully understood
and further studies are needed to find the optimal HBOT protocols.

Hyperbaric oxygen therapy is a well-established treatment modality for
non-healing wounds, radiation injuries as well as different hypoxic or ischemic
events (such as carbon monoxide toxicity, infections, etc). In recent years, a
growing evidence from pre-clinical as well as clinical trials demonstrate the
efficacy of HBOT for neurological indications including idiopathic sudden
sensorineural hearing loss [34], post stroke and post traumatic brain injury
[35–41], central sensitization syndrome such as fibromyalgia syndrome [42, 43]
and age related cognitive decline [17] and animal models of Alzheimer’s disease
[44]. For the first time, the current study aimed to evaluate the physiological
effect on the cellular level in aging humans without any functional limiting
disease.


STUDY LIMITATIONS

The current study has several limitations and strengths to consider. First, the
limited sample size has to be taken into account. Second, the lack of control
group. However, the study suggests impressive results on TL and senescent cell
clearance, which weren't observed in other interventions. Moreover, the baseline
telomere length values of our cohort match the expected values for the aging
population [45–47]. Third, the duration of the effect has yet to be determined
in long-term follow-ups. Fourth, telomerase activity was not evaluated due to
the method chosen for blood preservation and evaluation. Nevertheless, several
strengths should be stressed. In this study, CD28 was used as a biomarker for
senescent cells whereas CD57 was not available as a confirmatory marker for T
cell senescence. Biomarkers were assessed on specific leukocytes populations
rather than using the entire PBMCs as one group. The isolated HBOT effect was
measured and participants were monitored for not making any lifestyle changes
(such as nutrition and exercise), medications or any other intervention that may
have acted as possible confounders.

In summary, the study indicates that HBOT can induce significant senolytic
effects, including significant increased telomere length and clearance of
senescent cells in aging populations.


MATERIALS AND METHODS


SUBJECTS

Thirty-five adults without pathological cognitive declines, aged 64 and older,
who lived independently in good functional and cognitive status, were enrolled.
The study was performed between 2016-2020 in the Shamir (Assaf-Harofeh) Medical
Center, Israel. Included patients did not have cardiac or cerebrovascular
ischemia histories for the last year prior to inclusion. Exclusion criteria
included: previous treatment with HBOT for any reason during the last three
months, any history of malignancy during the last year, any pathological
cognitive decline, severe chronic renal failure (GFR <30), uncontrolled diabetes
mellitus (HbA1C>8, fasting glucose>200), immunosuppressants, MRI
contraindications (including BMI>35), active smoking or pulmonary diseases.


STUDY DESIGN

The study protocol was approved by Institutional Review Board of the Shamir
Medical Center, Israel. The study was performed as a prospective clinical trial.
After signing an informed consent and undergoing a baseline evaluation, the
subjects were assigned to HBOT. Measurement points were evaluated at baseline,
half-point of the treatment protocol (30th session), the day of the last HBOT
session and 1-2 weeks after the HBOT.

The study cohort included only patients treated by HBOT, which is part of a
larger cohort of normal ageing population studied at the Shamir medical center,
Israel (NCT02790541 [17]).


INTERVENTIONS

The HBOT protocol was administrated in a Multiplace Starmed-2700 chamber (HAUX,
Germany). The protocol comprised of 60 daily sessions, five sessions per week
within a three-month period. Each session included breathing 100% oxygen by mask
at 2ATA for 90 minutes with 5-minute air breaks every 20 minutes.
Compression/decompression rates were 1 meter/minute. During the trial, neither
lifestyle and diet changes, nor medications adjustments were allowed.


BLOOD SAMPLES

Whole blood samples were collected into EDTA tubes using a standard technique,
at baseline, at the half-point of the HBOT protocol (30th session), the day of
the last HBOT session (60th session) and 1-2 weeks following the last HBOT
session.


PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) ISOLATION

Whole blood was diluted using phosphate buffered saline (PBS). Density gradient
separation was performed using Leucosep tubes filled with Lymphoprep. The tubes
were then centrifuged at 1000×g for 10 min at 25° C degrees. Following
centrifugation, the cell layers (buffy coat) were immediately collected via
pipette and transferred to 50 mL conical centrifuge tubes, resuspended with
sufficient 1X PBS to a volume of 50 mL and centrifuged at 300×g for 10 min at
25° C degrees. Following removal of the supernatant, each sample was labeled.


TELOMERE LENGTH

Telomeres were labelled according to the Dako PNA/FITC kit protocol (Code
K5327). On a single cell suspension consisting of a mixture of PBMCs (sample
cells) and TCL 1301 cell line (control cells), the DNA was denatured for 10
minutes at 82° C in a microcentrifuge tube either in the presence of
hybridization solution without probe or in hybridization solution containing the
fluorescein-conjugated PNA telomere probe. The hybridization took place in the
dark at room temperature (RT) overnight. The hybridization was followed by two
10-minute post-hybridization washes with a wash solution at 40° C. The sample
was then labeled with CD4+, CD8+, CD3+, CD19+ and CD56+ conjugated antibodies in
an appropriate buffer for further flow cytometric analysis [48, 49]. Each sample
was run in duplicate. Following flow cytometric analysis, the relative telomere
length (RTL) was calculated for CD3+/CD4+ (T-helper), CD3+/CD8+ (T-cytotoxic),
CD3+/CD56+ (natural killer) and CD19+ (B-cells). The RTL value was calculated as
the ratio between the telomere signal of each sample and the control cell (TCL
1301 cell line) with correction for the DNA index of G0/1 cells. Sample cells
and control cells were analyzed separately for DNA ploidy using propidium iodide
staining to standardize the number of telomere ends per cell and thereby
telomere length per chromosome. See Figure 4 for FACS analysis example.

Figure 4. Example of Flow Fish data analysis of T helper subpopulation. Each
blood sample was either stained with PNA probe (b) or without (a), following by
antibodies staining (CD3, CD4, CD8, CD16, CD19), before data acquisition.



IMMUNOPHENOTYPING

Percentages of CD3+CD4+CD28-null T cells (senescent T helpers) and
CD3+CD8+CD28-null T cells (senescent T cytotoxics) were determined by
flow-cytometric analysis. PBMC were stained with VioBlue conjugated anti-CD3,
Viogreen conjugated anti-CD8, PE-VIO 770A conjugated anti-CD4 and APC-VIO 770A
anti-CD28 antibodies (Miltenyi Biotec). Cells were analyzed with a MACSQuant
Flow Cytometer (Miltenyi Biotec). The percentage of CD28null T cells within the
CD4+ or CD8+ T cell population was then calculated.


HYPOXIA INDUCED FACTOR (HIF-1ALPHA)

Intracellular HIF1a staining was performed with APC conjugated anti-HIF1a
antibody or corresponding Isotype Control (R&D systems) following fixation and
permeabilization (Life Technologies). Cells were analyzed with a MACSQuant Flow
Cytometer (Miltenyi Biotec) and the percentage of HIF1a expressing PBMCs, was
determined.


STATISTICAL ANALYSIS

Unless otherwise specified, continuous data were expressed as means ±
standard-deviation. The normal distribution for all variables was tested using
the Kolmogorov-Smirnov test. One-way ANOVA was performed to compare variables
between and within the three groups at baseline.

Categorical data is expressed in numbers and percentages and compared by
chi-square tests. Univariate analyses were performed using Chi-Square/Fisher’s
exact test to identify significant variables (P<0.05).

To evaluate HBOT’s effects, a repeated measures ANOVA model was used to test the
main within-subject effect. Post hoc tests on the means was conducted to test
for time differences using t tests with a Bonferroni correction.


AUTHOR CONTRIBUTIONS

All authors contributed substantially to the preparation of this manuscript. HY,
HA, ES were responsible for protocol design. HA, ZY, BY, ES, DKM were
responsible for patients' recruitment. YH, AHR, SM, YY, SM, ZR, ESW, HA, DKM,
SG, BGR, DG, HY, AHR, FG, LE, PN, DK, FM, ZY, BY were responsible for data
acquisition. HY, HA. and ES were responsible for data analysis. All authors
interpreted the data. HY, HA, CM, and ES wrote the manuscript. All authors
revised and finalized the manuscript.


ACKNOWLEDGMENTS

We would like to thank Dr. Mechael Kanovsky for his editing of this manuscript.


CONFLICTS OF INTEREST

AH, BY, ZY work for AVIV Scientific LTD. ES is a shareholder at AVIV Scientific
LTD.


FUNDING

The study was funded by a research grant from the Sagol network for neuroscience
established by Mr. Sami Sagol.


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CORRESPONDING AUTHORS

Amir Hadanny
hadannya@shamir.gov.il


Shai Efrati
efratishai@outlook.com
https://orcid.org/0000-0001-5523-999X


KEYWORDS

telomere senescence aging hyperbaric oxygen length

TABLE OF CONTENTS

 * Abstract
 * Introduction
 * Results
 * Discussion
 * Materials and Methods
 * Author Contributions
 * Acknowledgments
 * Conflicts of Interest
 * Funding
 * References

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