Patients were ≥ 18 years old with reported post-COVID-19 cognitive symptoms that affected their quality of life and persisted for more than three months following an RT-PCR test confirming a symptomatic SARS-CoV-2 infection. Patients were excluded if they had a history of pathological cognitive decline, traumatic brain injury or any other known non-COVID-19 brain pathology. The inclusion and exclusion criteria are listed Supplementary information.
A prospective randomized, double blind, sham-controlled, phase II exploratory study was conducted from December 14, 2020, to December 27, 2021, at Shamir Medical Center (SMC), Israel. After signing an informed consent, patients were randomized to either HBOT or sham-control groups in a 1:1 ratio according to a computerized randomization table, supervised by a blinded researcher. To evaluate participant masking, patients were questioned after the first session on their perception regarding the treatment they received. Evaluation procedure was done at baseline and 1–3 weeks after the last HBOT/control session. All evaluators were blinded to the patients’ group allocation. The study was approved by SMC’s Institutional Review Board (IRB) (No. 332-20-ASF) and all participants signed an informed consent prior to their inclusion. All research was performed according to the relevant guidelines and regulations. This study was registered with ClinicalTrials.gov, number NCT04647656 on 01/12/2020.
Both HBOT and sham protocols were administered in a multi-place Starmed-2700 chamber (HAUX, Germany). The protocol comprised of 40 daily sessions, five sessions per week within a two-month period. The HBOT protocol included breathing 100% oxygen by mask at 2ATA for 90 min with five-minute air breaks every 20 min. Compression/decompression rates were 1.0 m/min. The sham protocol included breathing 21% oxygen by mask at 1.03 ATA for 90 min. To mask the controls, the chamber pressure was raised up to 1.2 ATA during the first five minutes of the session along with circulating air noise followed by decompression (0.4 m/min) to 1.03 ATA during the next five minutes.
Primary and secondary outcomes
The primary outcome of the study was the cognitive assessment as evaluated by the Mindstreams computerized cognitive testing battery (NeuroTrax Corporation, Bellaire, TX). This assessment evaluates various cognitive domains including: memory, executive function, attention, information processing speed, and motor skills. Cognitive scores were normalized for age, gender and educational levels. The testing methods are described in the Supplementary information.
The secondary outcomes include the following measures:
Brain imaging MRI scans were performed on a MAGNETOM VIDA 3 T scanner, configured with 64-channel receiver head coils (Siemens Healthcare, Erlangen, Germany). The MRI protocol included T2-weighted, 3D fluid attenuated inversion recovery (FLAIR), susceptibility weighted imaging (SWI), pre- and post-contrast high-resolution MPRAGE 3D T1-weighted, dynamic susceptibility conSupplementary informationtrast (DSC) for calculating whole- brain quantitative perfusion maps, and diffusion tensor imaging (DTI) for microstructure changes in gray and white matter determination. A detailed description is found in the . Briefly, preprocessing of DSC and DTI images was performed using the SPM software (version 12, UCL, London, UK) and included motion correction, co-registration with MPRAGE T1 images, spatial normalization, and spatial smoothing with a kernel size of 6 mm full width half maximum (FWHM). Whole-brain quantitative perfusion analysis was performed as described in previous studies39.40. 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 brain perfusion maps.
Diffusion brain volumes denoising was performed using Joint Anisotropic LMMSE Filter for Stationary Rician noise removal41 and calculation of DTI-FA (fractional anisotropy) and MD (mean diffusivity) maps were performed using an in-house software written in Matlab R2021b (Mathworks, Natick, MA).
Included self-reported questionnaires were the short form-36 (SF-36) to assess quality of life, the Pittsburgh Sleep Quality Index (PSQI) to assess sleep quality, the Brief Symptom Inventory (BSI-18) to evaluate psychological distress, based on three subscales: depression, anxiety, and somatization, and the Brief Pain Inventory (BPI) to measure pain intensity and impact.
The sense of smell was evaluated by the Sniffin’ Sticks Test (Burghardt, Wedel, Germany). The kit is standardized for age and gender. Taste was evaluated by a Taste Strip Test (Burghardt, Wedel, Germany), including four tastes: bitter, sour, salt and sweet.
Pulmonary function measurements were performed by a KoKo Sx1000 spirometer (Nspire health, USA). Blood samples were collected for complete blood count, chemistry and inflammatory markers. Participants were monitored for adverse events including barotraumas (either ear or sinuses), and oxygen toxicity (pulmonary and central nervous system). This article discusses cognitive and behavioral aspects of post-COVID-19 condition. Additional secondary outcomes including neuro-physical evaluation, cardiopulmonary exercise test, echocardiography, and functional brain imaging will be presented in future manuscripts.
Continuous data are expressed as means ± standard deviations (SD). Two-tailed independent t-tests with were performed to compare variables between groups when a normality assumption held according to a Kolmogorov–Smirnov test. Net effect sizes were evaluated using Cohen’s d method, defined as the improvement from baseline after sham intervention was subtracted from the improvement after HBOT, divided by the pooled standard deviation of the composite score. Categorical data were expressed in numbers and percentages, compared by chi-square/Fisher’s exact tests. To evaluate HBOT’s effect, a mixed-model repeated-measure ANOVA model was used to compare post-treatment and pre-treatment data. The model included time, group and the group-by-time interaction. A Bonferroni correction was used for the multiple comparisons. A value of p < 0.05 was considered significant. Pearson's correlations were performed between perfusion and diffusion changes and the change in questionnaire scores before and after HBOT and sham. Imaging data analysis was performed on the normalized CBF, FA and MD maps, using the voxel-based method to generate statistical parametric maps. A gray matter mask was applied on the CBF and MD maps, and a white matter mask on the FA maps (using a threshold of 0.2). A within-subject repeated measure ANOVA model was used to test the main interaction effect between time and group implemented in SPM software (version 12, UCL, London, UK). A sequential Hochberg correction was used to correct for multiple comparisons (P < 0.05)42. Data analysis was performed using Matlab R2021b (Mathworks, Natick, MA) Statistics Toolbox.
The estimated sample size was calculated based on our recent study in healthy adults19. A Mindstreams-NeuroTrax global cognitive score improvement of 5.2 and 0.8 points, with a standard deviation of 6.7 points was found in the HBOT and control groups respectively. Assuming a power of 80%, and 5% two-sided level of significance, a total of 74 participants would be required, 37 participants in each arm. Considering a dropout rate of 15% the total sample size required is 85.