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.

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.

		HBOT	Telomere analysis	Senescent analysis	P-value
N		30	25 (83.3%)	20 (66.6%)
Age (years)		68.41±13.2	67.56±14.35	66.70±16.00	0.917
BMI		26.77±3.20	26.89±3.34	27.14±3.81	0.946
Males		16 (53.3%)	13 (52.0%)	10 (50.0%)	0.987
Females		14 (47.7%)	12 (48.0%)	10 (50.0%)	0.987
Complete blood count
	Hemoglobin	6.33±1.25	6.57±1.15	6.58±1.29	0.707
	White blood cells	14.02±1.40	13.92±1.35	13.97±1.49	0.969
	%PBMC	39.96±6.75	39.25±6.64	38.59±6.63	0.774
	Platelets	239.87±1.39	244.08±43.0	254.05±41.4	0.559
Chronic medical conditions
	Atrial fibrillation	4 (13.3%)	4 (16.0%)	2 (10.0%)	0.841
	Hypothyroidism	4 (13.3%)	4 (16.0%)	3 (15.8%)	0.956
	Obstructive sleep apnea	4 (13.3%)	4 (16.0%)	3 (15.0%)	0.961
	Asthma	1 (3.3%)	1 (4.0%)	0	0.680
	BPH	7 (23.3%)	5 (20.0%)	6 (30.0%)	0.733
	GERD	3 (10%)	2 (8.0%)	2 (10.0%)	0.961
	Osteoporosis	5 (16.7%)	5 (20.0%)	4 (20.0%)	0.936
	Rheumatic arthritis	1 (3.3%)	0	1 (5.0%)	0.561
	Osteoarthritis	7 (23.3%)	4 (16.0%)	5 (25.0%)	0.755
	Diabetes mellitus	3 (10%)	3 (12.0%)	2 (10.0%)	0.966
	Hypertension	7 (23.3%)	5 (20.0%)	5 (25.0%)	0.918
	Dyslipidemia	16 (53.3%)	14 (56.0%)	12 (60.0%)	0.897
	Ischemic heart disease	2 (6.7%)	1 (4.0%)	2 (10.0%)	0.725
	History of smoking	10 (33.3%)	8 (32.0%)	7 (35.0%)	0.978
Chronic medications
	Anti-aggregation	8 (26.7%)	6 (24.0%)	5 (25.0%)	0.974
	ACE-Inhibitors/ARB blockers	6 (20%)	6 (24.0%)	6 (30.0%)	0.720
	Beta blockers	5 (16.7%)	5 (20.0%)	3 (15.0%)	0.901
	Calcium blockers	3 (10%)	3 (12.0%)	2 (10.0%)	0.966
	Alpha blockers	7 (23.3%)	5 (20.0%)	6 (30.0%)	0.733
	Diuretics	2 (6.7%)	1 (4.0%)	1 (5.0%)	0.906
	Statins	10 (33.3%)	9 (36.0%)	7 (35.0%)	0.978
	Oral hypoglycemic	1 (3.3%)	1 (4.0%)	1 (5.0%)	0.958
	Bisphosphonates	1 (3.3%)	1 (4.0%)	1 (5.0%)	0.958
	Proton pump inhibitors	3 (10%)	3 (12.0%)	3 (15.0%)	0.726
	Hormones	3 (10%)	3 (12.0%)	2 (10.0%)	0.966
	Benzodiazepines	3 (10%)	2 (8.0%)	1 (5.0%)	0.816
	SSRI	5 (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 changes

Relative changes (%)

Repeated measures F (p)

PBMC

Baseline

30th Session

60th Session

Post HBOT

30th session

60thsession

Post-HBOT

PBMC ((N=25)

2.55±0.53

-0.15±0.40

-4.91±16.70

1.987 (t) 0.09

PBMC (N=20)

2.50±0.53

-0.13±0.31

-4.21±11.99

1.810 (t) 0.07

Relative telomeres length (N=25)

Natural killer

9.27±1.91

11.77±5.14 (0.045)

10.73±2.73 (0.013)

11.75±4.22 (0.06)

25.02±51.42

20.56±33.35

22.16±44.81

0.812 (0.391)

B-cells

8.36±2.02

10.22±3.04 (0.007)

11.23±3.58 (0.0001)

11.17±2.98 (0.007)

25.68±40.42

29.39±23.39

37.63±52.73

7.390 (0.017)

T Helper

8.04±1.82

9.92±3.68 (0.042)

9.63±2.17 (0.012)

10.20±2.77 (0.005)

21.70±40.05

23.69±39.54

29.30±38.51

4.663 (0.063)

T Cytotoxic

8.26±1.54

9.83±4.08 (0.11)

10.08±3.33 (0.019)

10.15±2.74 (0.023)

18.29±45.62

24.13±40.88

19.59±33.98

1.159 (0.310)

Senescent cells (% of T cells) (N=20)

T Helper

10.29±5.42

7.84±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±33.04

8.548 (0.01)

T Cytotoxic

52.19±21.07

45.53±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±12.59

6.916 (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 30thsession, 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).

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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