Participants included recovered COVID-19 patients, male or female aged 18 and above with and without neurological symptoms. Participants with long COVID, with symptom persistence over 12 weeks from infection were also recruited. Candidates were excluded if they had a history of a neurological disorder that may better explain the results of the study such as epilepsy, brain trauma, neuropsychiatric disorder, or mild cognitive impairment. Suitable candidates proceeded to assessment with DCE-MRI imaging, Q-SIT olfactory testing and a review of pulmonary imaging and haematological parameters at the time of COVID-19 diagnosis. The Joint Research Ethics Committee (JREC) of St James’s and Tallaght Hospital’s approved the study and informed consent was obtained from all participants. Research was performed according to the principles of the Declaration of Helsinki. The legal basis for the Study was consent according to GDPR principles.
Participants olfactory function was assessed using the quick smell identification test (Q-SIT). The Q-SIT is a standardised and validated three item odour identification screen60. A score of 2 or more is a normal test and cut-off score of 1 or less is an abnormal test for anosmia. Q-SIT has displayed high positive and negative predictive value in detecting olfactory dysfunction in COVID-19 patients. In addition, the Q-SIT is a tear-off card test is disposable so there is no concern about contamination and transmission of disease form COVID-19 patients61.
Dynamic contrast-enhanced magnetic resonance imaging
BBB permeability maps were created using the slope of contrast agent concentration in each voxel over time, calculated by a linear fit model as previously described62,63. Thresholds of high permeability was defined by the 95th percentile of all slopes in a previously examined control group. Imaging was performed with a 3T Philips Achieva scanner. Sequences included a T1- weighted anatomical scan (3D gradient echo, TE/TR =3/6.7 ms, acquisition matrix 268x266, voxel size: 0.83x0.83x.9mm), T2-weighted imaging (TE/TR =80/3000 ms, voxel size: 0.45x0.45x.4mm), FLAIR (TE/TR =125/11000 ms, voxel size:0.45x0.45x4mm). For the calculation of pre-contrast longitudinal relaxation time (T10), the variable flip angle (VFA) method was used (3D T1w-FFE, TE/TR = 2.78/5.67 ms, acquisition matrix: 240x184, voxel size: 0.68x0.68x5 mm, flip angles: 2, 10, 16 and 24°). Dynamic contrast enhanced (DCE) sequence was then acquired (Axial, 3D T1w-FFE, TE/TR = 2.78/5.6 ms, acquisition matrix: 240x184, voxel size: 0.68x0.68x5 mm, flip angle: 6°, Tt = 6.5 Sec, temporal repetitions: 61, total scan length: 22.6 minutes). An intravenous bolus injection of the contrast agent gadobenate dimeglumine (Gd-BOPTA, Bracco Diagnostics Inc., Milan, Italy) was administered using an automatic injector after the first three DCE repetitions. To control for interindividual variabilities due to heart rate, blood flow or rate of contrast injection, each voxel’s leakage rate was normalised to that of the superior sagittal sinus. The percent of suprathreshold voxels was used as a measure reflecting global BBB leakage.
Volumetric and thickness measurements
T1-weighted anatomical images were uploaded to the VolBrain online brain volumetry software (https://volbrain.upv.es)64, and analysed with vol2brain 1.0 which is an online pipeline that registers images to the Montreal Neurological Institute (MNI) space, and reports the volumes of expert-labelled anatomical structures as percentage of total intracranial volume. We analysed the volume of the right/left cerebral and cerebellar grey/white matter, frontal, temporal, parietal and occipital and CSF along with thickness of the frontal, parietal, occipital and temporal lobes. All volume data was normalised to total intracranial volume (TIV) which is the sum of grey matter, white matter and CSF. Volumes were expressed as a percentage of TIV. 60 age and sex matched healthy control scans were randomly selected from the IXI dataset (https://brain-development.org/ixi-dataset/) which represents 10 % of the entire dataset. All scans were performed on the same Philips 3T system at Hammersmith Hospital. Volumetric maps for comparisons between COVID positive and negative groups were generated in xjview following automatic brain segmentation in the CAT12 toolbox with default parameters and subsequent smoothing with an 8 mm kernel. Thickness maps for comparisons between COVID positive and negative groups were generated in CAT12 toolbox run in SPM12 in MATLAB R2021a following brain segmentation as above and smoothing with a 15 mm kernel. Two-sample t-test was used for statistical analysis with age, sex and TIV as covariates.
Blood samples were collected into serum separator tubes and EDTA-coated tubes for serum and PBMC isolation respectively. Serum was separated by centrifugation at 2000 rpm for 10 min at room temperature. PBMCs were separated via layering of blood samples diluted twofold in PBS (ThermoFisher, #14190) over LymphoprepTM density gradient medium (Stemcell Technologies, #07851) followed by centrifugation at 400 rcf for 25 min at room temperature at 0 break and 0 acceleration. Plasma was collected and PBMC layer was collected into a new 50 ml falcon tube, resuspended to 50 ml with PBS and centrifuged at 2000 rpm for 5 min at room temperature. PBMCs were resuspended in 50 ml PBS and centrifuged at 1000 rpm for 10 min at room temperature. PBMCs were resuspended to 2 x 106 cells/ml in RPMI 1640 media with L-glutamine (Lonza, #LZBE12-702F) supplemented with 50 % fetal bovine serum (Merck, #F7524) and 10 % DMSO (Merck, #D5879) and frozen at -800C overnight before being moved to liquid nitrogen.
A 10-plex Luminex assay (R&D Systems, #LXSAHM-10) was used for cytokine profiling. Serum samples were diluted twofold in sample dilution buffer. Then, 50 µl of sample or standard were pipetted in duplicate into each wall of an assay 96 well plate. 50 µl of diluted Microparticle Cocktail were added to each well, the plate was covered and incubated for 2 hours at room temperature on a shaker at 300 rpm. Wells were washed 3 times with Wash Buffer before addition of 50 µl of diluted Biotin-Antibody Cocktail. The plate was covered and incubated for 1 hour at room temperature on a shaker at 300 rpm. Wells were washed as above before addition of 50 µl of diluted Streptavidin-PE to each well. The plate was covered and incubated for 30 min at room temperature on a shaker at 300 rpm. Wells were washed as above before microparticles were resuspended in 100 µl of Wash Buffer. The plate was incubated for 2 min at room temperature on a shaker at 300 rpm and was read on a MAGPIX plate reader (Luminex).
Plasma samples were spotted (2 µl) onto 0.2 µm nitrocellulose membrane (Whatman, #10401391) and allowed to dry for 30 minutes. Membranes were blocked in 5 % bovine serum albumin (BSA, Merck, #A7906) in phosphate buffered saline supplemented with 0.1 % Triton X-100 (PBST) for 1 hour at room temperature. Membranes were incubated overnight in primary antibody in blocking buffer. Membranes were washed three times for five minutes each in PBST, followed by incubation in secondary HRP-conjugated antibodies. Membranes were washed three times for five minutes each in PBST and incubated with strong ECL substrate (Advansta, #K-12045-D50) for 2 min before being developed on a C-Digit (LiCor). Protein bands were quantified in ImageJ (National Institutes of Health, Rockville, MD, USA). Primary antibodies used were mouse anti-GFAP (1/500, Merck, #G3893), rabbit anti-TGFβ (1/500, Abcam, #ab92486) and mouse anti-Phospho-Tau (1/500, Fisher Scientific, #10599853). Secondary antibodies used were anti-mouse HRP (1/5000, Merck, #A4416) and anti-rabbit HRP (1/5000, Merck, #A6154).
RNA was isolated from PBMCs and the human brain endothelial cell line hCMEC/d3 (Millipore, #SCC066) with the Omega RNA isolation kit (Omega, #R6834-02) according to manufacturer’s instructions. cDNA was reverse transcribed from 500 ng RNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, # 4368814). Transcript levels were quantified on a StepOne Plus instrument (Applied Biosystems) with FastStart Universal SYBR Green Master (ROX) master mix (Roche, #04913914001). RT-PCR was performed with the following conditions: 950C x 2 min, (950C x 5s, 600C x 30s) x40, 950C x 15s, 600C x 1 min, 950C x 15s, 600C x 15s. Primer sequences for RT-PCR experiments are supplied in Supplementary Table 4. Relative gene expression levels were quantified using the comparative CT method (ΔΔCT). Expression levels of target genes were normalised to β-actin.
hCMEC/d3 cells were cultured in EGM2-MV growth medium (Lonza, #CC-3202) and were stimulated with 10 ng/ml recombinant human TNF-a (Peprotech, #300-01A) for 4 hours and incubated with 1x105 MitoTracker Orange (ThermoFisher, # M7510) labelled PBMCs for 1 hour at 37oC. Cells were washed three times in PBS to remove unbound PBMCs and fixed in 4 % formaldehyde (Merck, #F1635) for 10 min at room temperature. The number of adhered PBMCs was counted with the ImageJ cell counter plugin. Images were imported and converted to 8-bit and thresholded. Noise was removed with the despeckle function, and the images were converted to binary. The cell counter plugin was then used for counting adhered PBMCs. Counts were averaged from 5 images per treatment.
Serum and spike protein treatment
hCMEC/d3 cells were seeded in 12-well plates at 2x105 cells/well and grown to confluence. Media was replaced with media containing 10 % serum from COVID and unaffected controls and incubated for up to 72 hours followed by RNA isolation. hCMEC/d3 cells were cultured in12-well plates as described above and stimulated with 4, 40 and 400 nM Recombinant SARS-CoV-2 Spike S1 subunit protein (R&D Systems, #BT10569) for up to 72 hours and RNA was isolated as described above.