Cognitive enhancement of healthy older adults using hyperbaric oxygen: a randomized controlled trial

Cognitive function

Results of the cognitive function evaluations are summarized in Tables 2, 3.

Primary endpoint

Both groups had similar global cognitive scores at baseline which was higher than the average score normalized to age and education level (>100). There was a significant group by time interaction in the primary endpoint of global cognitive function post HBOT compared to the control group (F=10.811, p=0.0017 with a net effect size of 0.849 (Tables 2, 3 and Figure 2).

Figure 2. NeuroTrax parameters significant changes. The NeuroTrax significant domains, shown in boxplots, with each line representing a patient flow from baseline to post intervention. The central mark indicates the median, and the bottom and top edges of the box indicate the 25^th and 75^th percentiles, respectively. Red symbols indicate outliers.

Secondary endpoints

Both groups had similar cognitive scores in all domains measured by Neurotrax at baseline, The most striking improvements were in attention (net effect size=0.745, F=8.445, p=0.005) and information processing speed (net effect size=0.788, F=9.142, p=0.003) (Figure 2). Both overall memory domain score, the immediate and the delayed nonverbal memory scores were all improved post-HBOT compared to the control group (Tables 2, 3 and Figure 2). However, they were statistically insignificant following multi-comparisons corrections.

In the CANTAB battery, at baseline, the HBOT group had shorter response times. However, they were insignificant following multiple comparison corrections (Table 2). The HBOT group showed significant improvement in the set shifting task performance (a subset of executive function) (Tables 2, 3 and Figure 3). There was a significant reduction in median response time in both congruent (net effect size=-0.861, F=10.702, p=0.002) and incongruent trials (net effect size=-0.840, F=11.254, p=0.001) following HBOT (Tables 2, 3 and Figure 3). There was an increased performance in the visual memory task post-HBOT, which was statistically insignificant following multiple comparisons corrections. The results of covariate-adjusted (age and education) analyses were similar.

Figure 3. CANTAB parameters significant changes. The set shifting parameters, shown in boxplots, with each line representing a patient flow from baseline to post intervention. The central mark indicates the median, and the bottom and top edges of the box indicate the 25^th and 75^th percentiles, respectively. Red symbols indicate outlie.

In the traditional paper and pencil tasks, at baseline, both groups showed similar cognitive functions in all tasks. Both verbal fluency (FAS semantic, net effect size=0.566, F=4.646, p=0.03) and verbal memory (RAVLT total, net effect size=0.603, F=5.439, p=0.02) improved in the HBOT group, compared to the control group. However, they were statically insignificant following multiple comparisons corrections (Table 2, 3). (See all cognitive results in Supplementary Table 1, Supplementary Table 2).

Brain perfusion

Ten subjects were excluded due to excessive head motion (>1mm or 1°) and ten subjects were excluded due to low AIF peak or wide AIF. A total of 20 subjects from the control group and 19 subjects from the HBOT group were included in the analysis.

There was an insignificant increase in whole brain CBF (p=0.054) and whole grey matter CBF (p=0.057) and no significant group by time interaction in whole brain, grey matter and white matter CBF (p>0.05) (Supplementary Table 3).

Voxel-based analysis revealed significant CBF increases in the HBOT group compared to the control group in the following regions: right superior medial frontal gyrus (BA 10), right and left supplementary motor area (BA 6), right middle frontal gyrus (BA 6), left middle frontal gyrus (BA9), left superior frontal gyrus (BA8) and the right superior parietal gyrus (BA 7) (Table 4 and Figure 4).

Figure 4. Brain regions with significant post hyperbaric oxygen therapy changes in cerebral blood flow.

There were moderate correlations between cognitive score changes and these regional perfusion changes. The Neurotrax memory score change correlated to the left middle frontal gyrus CBF change (BA8), (r=0.379, p=0.023) and the attention score change correlated to the right middle frontal gyrus (BA6) (r=0.339, p=0.043). CANTAB set switching parameter changes correlated with the right superior frontal gyrus and supplementary motor area (BA6) (r=0.38-0.45, p<0.05, Supplementary Table 4). The RAVLT total score correlated with both the right superior medial frontal gyrus (r=0.393, p=0.016), right supplementary motor area (r=0.394, p=0.016) and the right superior parietal gyrus (r=0.380, p=0.002) (Table 4). The FAS semantic score change correlated with the right medial frontal gyrus (r=0.353, p=0.03) (Supplementary Table 4).

Quality of life

At baseline, there were no significant differences in all quality of life domains as calculated from the SF-36 questionnaire. All but the energy domain, had high normal values (>75) in both groups. There was a significant increase in the HBOT group’s energy levels, compared to none in the control group (Supplementary Tables 5, 6).

Safety

Four participants (13.3%) experienced mild middle ear barotrauma (TEED 1-2) in the HBOT group compared to none (0%) in the control group. All events were treated conservatively and all participants completed their protocol. Fifteen participants (62% of those without intraocular lens implantation) had visual acuity changes in the HBOT group, compared to ten (37% of those without intraocular lens implantation). No changes were found in participants who had intraocular lens surgery prior to the study. In the HBOT group, nine patients (31.0%) had far sight acuity deterioration while six patients (20.6%) had improvement in their far sight acuity. Six patients (20.6%) had improved near sight acuity and three (10.3%) had near sight acuity deterioration. Four participants (16.7%) in the HBOT group, compared to nine (33.3%) in the control group had cataract level acceleration.

Study limitations

The current study has several limitations and strengths to consider. First, the limited sample size has to be taken into account, possibly causing decreased sensitivity and false negative changes. However, the presence of significant changes following strict statistical analyses in a small group is indicative for the relatively high potency of the intervention. Second, the control group was a non-intervention rather than a sham-intervention. Although the outcome assessors were blinded, the participants were unblinded. Third, the duration of the effect is yet to be determined in long-term follow-ups. Nevertheless, several strengths should be stressed. The isolated HBOT effect was measured as both groups were monitored for any lifestyle changes (such as nutrition and exercise), medications or any other intervention that may have acted as possible confounders. Patients did not perform any cognitive training tasks during the trial, thereby excluding training effects. Both computerized cognitive batteries had alternate forms with test-retest validity as well as the brain perfusion sequence and analysis. Moreover, the improvements in the cognitive domains correlated with the significant changes in perfusion MRIs.

In summary, the study indicates that HBOT can induce cognitive enhancement in healthy aging populations. The main improvements include attention, information processing speed and executive functions, which are known to decline with normal aging. In correlation with the cognitive improvements, HBOT induced a significant brain perfusion increase in specific brain regions with high cognitive roles.

Subjects

Seventy 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, active smoking and pulmonary diseases.

Included patients who were diagnosed with pathological cognitive decline based on their cognitive tests were excluded. Pathological cognitive decline was diagnosed by a certified neuropsychologist.

Study design

The study protocol was approved by Institutional Review Board of Shamir medical center, Israel. The study was performed as a randomized, prospective controlled clinical trial. After signing an informed consent, the subjects were assigned either to HBOT or control (no intervention) arms. Assessors were blinded to the participants’ intervention assignment. Measurement points were evaluated at baseline and 1-2 weeks after the HBOT or control period.

Interventions

The HBOT protocol was administrated in a multiplace Starmed-2700 chamber (HAUX, Germany). The protocol comprised of 60 daily sessions, 5 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. The control arm received no active intervention as a no-contact group. During the trial, neither lifestyle and diet changes, nor medications adjustments were allowed for either group.

Cognitive measures

Cognitive functions were assessed using two computerized batteries and one traditional paper-based battery, given by a certified neuropsychologist.

NeuroTrax computerized cognitive testing battery (NeuroTrax Corporation, Bellaire, TX). The NeuroTrax system and a detailed description of the tests included were detailed in previous publications [45–47] and are also available on the NeuroTrax website (http://www.neurotrax.com/).

In brief, the NeuroTrax tests evaluate multiple aspects of brain cognitive functions including: memory, executive function (EF), attention, information processing speed (IPS), motor skills (MS) visuospatial skills (VS) and verbal function (VF). Cognitive domain scores were normalized for age, gender and education-specific levels.

The participants completed validated alternate test forms of the NeuroTrax test battery at baseline and post-HBOT, to allow for iterative administrations with minimal learning effects. Test-retest reliability of the tests were found to be high in both normal and injured populations, without significant learning effects except in the VF and VS domains that were not evaluated in the current study [48, 49].

CANTAB computerized cognitive tests (Cambridge cognition, England) [50]. CANTAB is a semiautomated test battery which can be administered on a handheld tablet. The battery included: attention switching tasks (AST) for executive function and set shifting testing, pair associates learning (PAL) for evaluating visual memory and new learning, reaction time (RTI), rapid visual information processing (RVP) for assessing sustained attention, spatial span (SSP) for assessing visuospatial working memory and spatial working memory (SWM) [51, 52]. To combine accuracy and reaction time, the inverse efficiency score (IES) was calculated by the following formula:

Notably, the patients were given different test versions of the CANTAB test battery at baseline and after the control/HBOT period, to allow repeated administrations with minimal learning effects. The current version of CANTAB has no population norms for either parameter.

Traditional paper and pencil based neuropsychological tests included: the Rey-Osterrieth complex figure test (ROCFT), a popular measure of visuoconstructive skills and visual memory [53]; the Rey auditory verbal learning test (RAVLT), a neuropsychological assessment designed to evaluate verbal memory in patients [54]; the digit symbol substitution test (DSST) offers high sensitivity to detect overall cognitive impairment rather than a specific domain [55]; a digit-span (DS) task, used to measure working memory’s number storage capacity [56]; the five points test (5PT) is a structured and standardized test that assesses figural fluency functions which are associated with executive functioning [57]; the trails making test (TMT), a widely used test that assesses organized visual search, planning, attention, set shifting, cognitive flexibility, and divided attention, all capacities thought to be executive in nature [58]; the FAS test, which measures phonemic word fluency, which is a type of verbal fluency [59]; the bells test, a cancellation test, which permits qualitative and quantitative evaluation of visual neglect [60]. Scores were normalized to age, gender and education as suggested in the manuals and presented as Z-scores. In case of diagnosed pathological cognitive decline using both the cognitive scores and MMSE, the patients were excluded from the study.

Brain MRIs

MRI scans were performed on a MAGNETOM Skyra 3T scanner, configured with20-channel receiver head coils (Siemens Healthcare, Erlangen, Germany). The MRI protocol included dynamic susceptibility contrast (DSC), and post-contrast high-resolution MPRAGE 3D T1-weighted images.

MRI sequences parameters:

DSC: Fifty T2*-weighted gradient-echo echo planar imaging (EPI) volumes were acquired, two repetitions before a bolus injection of gadolinium-DTPA (Gd-DTPA, 0.2 ml/kg, administered at 5 ml/sec), 48 repetitions after injection of Gd-DTPA. Sequence parameters: TR: 2,300 ms, TE: 40ms, flip angle: 30°, voxel size :1.8 x1.8, matrix: 128x128, number of slices: 25, and slice thickness = 3.9 mm.

MPRAGE3D: was acquired in sagittal orientation with 0.9 mm isotropic resolution. Sequence parameters: TR: 2,000 ms, TE: 2.41 ms, flip angle: 8°, TI: 928 ms, FOV: 245 x 245, and 192 contiguous slices.

DSC-MRI analysis

The preprocessing of the perfusion MRI data was preformed using the SPM software (version 12, UCL, London, UK) and included motion correction, and co-registration with MPRAGE T1 images. Individual gray matter (GM) and white matter (WM) segmentation of T1 anatomy was also performed to extract mean perfusion values. Whole-brain quantitative perfusion analysis was performed as described in previous studies [61, 62]. Detailed description is found in the supplementary material (SI-1). Briefly, MR signal intensity was converted to Gd concentrations, AIF was determined automatically, fitted to the gamma variate function and deconvolved on a voxel-by-voxel basis to calculate the CBF, CBV, and MTT maps. Following normalization to MNI space, WM and GM masking and smoothing using a 6 mm full-width at half-maximum Gaussian kernel, statistical analysis was performed on the normalized CBF maps, using the voxel-based method.

Quality of life measures

The RAND health status survey, short form-36 (SF-36) was used to assess quality of life. SF-36 is a self-report measure that evaluates physical functioning, bodily pain, role limitations due to physical health problems, role limitations due to personal or emotional health, general mental health, social functioning, energy/fatigue, and general health perception [63–65]. Each scale generates a score from 0 to 100, with a high score indicating better health and less body pain.

Safety

Participants were monitored for adverse events including: barotraumas (either ear or sinuses), oxygen toxicity (pulmonary and central nervous system). Participants were examined by a certified blinded ophthalmologist before and after the control/HBOT term to monitor visual acuity and cataracts.

Statistical analysis

Continuous data were expressed as means ± standard-deviation. The normal distribution for all variables was tested using the Kolmogorov-Smirnov test. unpaired and paired t-tests were performed to compare variables between and within the two groups. Net effect sizes were evaluated using Cohen’s d method.

Continuous parameters correlations were performed using Pearson and Spearman’s as appropriate.

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 on cognitive scores, a within-subject repeated measures ANOVA model was used to test the main interaction effect between time and group. The false discovery rate (FDR) method was used for multiple comparisons correction. In addition, covariate-adjusted effects were examined, with covariates of age, sex and years of education.

To evaluate HBOT’s effects on CBF, statistical analysis was performed on the normalized CBF maps, using the voxel-based method implemented in SPM12 (Wellcome Trust Centre for Neuroimaging, London, England). A within-subject repeated measure ANOVA model was used to test the main interaction effect between time and group, using the SPM factorial model. The statistical significance level was set to a voxel-wise P-value of 0.05 corrected for multiple comparisons using the sequential Hochberg correction [66], with a minimum cluster size of 25 (200 mm3) contiguous significant voxels. CBF values in each cluster were extracted and averaged.

Statistical significance threshold was set to 0.05. Data were statistically analyzed using MatLAB 2018b (Mathworks, Natick, MA).

Sample size

Based on previous studies on cognitive improvements following HBOT, assuming a five-point improvement in the global cognitive score in NeuroTrax following HBOT, compared to two points in the control group, with a four point standard deviation of the change, with a power of 80% and an alpha of 5%, 29 participants would be required in each arm. Adding a 15% dropout rate would require 70 patients in total.