Limb stimulation modulates blood-brain barrier permeability

a. Timelines of the experimental protocols for stimulations and imaging with 30 min stimulation (left) and without (right). b. Image of the cortical window over the rat sensorimotor cortex, followed by maps of the change in HbT concentration showing the evolution of the hemodynamic response during and after stimulation. Red rectangle marks the responding region magnified in c. c. Images of the responding arteriole in the rats’ cortex before (left) during (middle) and after stimulation (right). Red arrows mark the responding arteriole. d. The responding arterioles’ diameter during 1 min stimulation. Gray area corresponds to time of stimulation e. Top: local field potential (LFP) trace from the corresponding region in L2/3 sensorimotor cortex before during (greyed area) and after 30 min limb stimulation. Bottom: 5 s excerpts of the above trace (left-to-right: before during and after stimulation). f. NaFlu permeability maps before (left) and after 30 min stimulation (right) showing tracer accumulation around a responding arteriole. g. Permeability index is higher after stimulation compared to baseline. Wilcoxon, n=13 rats, mean ± SEM. h-i. Fluorescence images of cortical sections of stimulated rats injected with the albumin-binding dye Evans blue (h) and Alexa-488-Alb (i). j-k. Total fluorescence of EB (j) and Alexa488-Alb (k) after stimulation in the contralateral hemisphere compared to the ipsilateral hemisphere. Wilcoxon, EB (n=5 rats, 30 sections), Alexa488-Alb (n=5 rats, 15 sections), mean ± SEM. l. Albumin concentration in the contralateral hemisphere relative to the ipsilateral in different timepoints after stimulation, compared to sham stimulation. Kruskal-Wallis with FDR correction, 0.5 and 4 h post-stimulation n=8, sham n=7, mean ± SEM. m. Cortical sections of the area of limb representation from both hemispheres of a stimulated rat (left), and higher magnification images (right) n. Fluorescence image of a cortical small vessel in the stimulated hemisphere showing extravascular accumulation of EB and NaFlu. *p<0.05, ***p<0.001.

Stimulation and cortical perfusion of serum albumin induce LTP

a. Top: LFP trace from the rat L2/3 sensorimotor cortex before (left) during (middle) and after (right) 1 min test stimulation (6 Hz, 2 mA). Bottom: LFP trace from the same rat during 1 min test stimulation, following a 30 min stimulation. b. The somatosensory evoked potential (SEP) of 1 min test stimulation at baseline (BL) and after 30 and 60 minutes (blue, red, black, respectively, each averaged over 360 stimuli). c. SEP in response to 1 min test stimulation at baseline (blue) and following a 30 min stimulation (red). d. Maximum amplitude of the SEP (absolute values) following a 30 min stimulation compared to baseline (n=6). e. Area under curve (AUC) of the SEP following a 30 min stimulation compared to baseline. f. Top: 1 hour LFP trace from a representative rat. Bottom: 5 s magnifications of the above trace at selected time points (noted by asterisks). Left to right: baseline activity; during 1 min test stimulation; following cortical application of 0.1 mM albumin (Alb); during 1 min test stimulation post-Alb. g. SEP amplitude during 1 min stimulation at baseline (normal aCSF, blue) and following 0.1 mM Alb (red). h. Maximum amplitude of the SEP during 1 min stimulation post-Alb compared to baseline (n=5). i. AUC of the SEP post-Alb compared to baseline. j. Power spectrum density of 10 min spontaneous activity before (blue) and post-Alb (red). *p<0.05, Wilcoxon, data are mean ± SEM.

Stimulation-induced plasticity is associated with BBB modulation

a. Timeline of the experimental protocol for stimulations and imaging with blockers application. b. NaFlu permeability maps of the cortical window before (control, left), and after CNQX/AP5 + 30 min stimulation (stim, right). c. PI following CNQX and AP5 compared to a 30 min stimulation, presented as % change from baseline (30 min stim n=6, AP5 n=4, CNQX n=5). d. SEP amplitude in response to 1 min test stimulation. baseline (blue); following cortical application of CNQX (left, red) or AP5 (right, red); following CNQX + 30 min stimulation (left, black); and following AP5 + 30 min stimulation (right, black); e. Left: Max SEP amplitude to 1 min test stimulation following CNQX compared to baseline. Right: SEP amplitude following AP5 compared to baseline. f. SEP amplitude following 30 min stimulation or albumin application compared to AP5, AP5 + 30 min stimulation, CNQX and CNQX + 30 min stimulation. (% change from baseline). g. Permeability maps before (control, left), and after SJN + 30 min stimulation (right). h. PI following SJN application compared to 30 min stimulation (n=5). i. PI following mβCD compared to a 30 min stimulation (n=6). j. SEP amplitude: baseline (blue); following SJN (black); following SJN + 30 min stimulation (red); and following mβCD (right). k-l. SEP amplitude following 30 min stimulation or albumin application compared to SJN and SJN + 30 min stimulation (k), and mβCD, mβCD + 30 min stimulation (l). *p<0.05, **p<0.001, Kruskal-Wallis with FDR correction; data are mean ± SEM. (c-f, h-l).

Neuronal activity regulates BBB transport and synaptic plasticity genes

a. Scatter plot of gene expression from RNA-seq in the contralateral cortex 24 vs. 1 h after stimulation. The x axis represents the log fold change and the y axis represents the mean expression levels. Blue dots indicate statistically significant differentially expressed genes (DEGs) by Wald Test (n=8 rats per group). b. Top Gene Ontologies (GO) enriched terms in the contralateral cortices of rats 24 vs. 1 h after stimulation. c. Vascular cell specific DEGs to pericytes (PC), arterial smooth muscle cells (aSMC), and venous smooth muscle cells (vSMC). d. Scatter plot of DEGs divided by groups of interest: BBB properties, NVU properties, Synaptic plasticity, and inflammatory related genes in the contralateral (red) vs. ipsilateral (black) cortices of stimulated rats. Upward and downward triangles indicate up- and down-regulated genes respectively.

Cortical activation in fMRI co-localizes with BBB modulation

a. Subjects were given an elastic stress-ball to squeeze continuously for the length of the session (30 min). b. Timeline of the experimental protocol for task performance and MRI sequences. c. Activation map for the localizer task displayed over the inflated brain of an exemplary subject. (p<0.05, FWE corrected, neurologic convention). d. Activation maps for the localizer task displayed over anatomical axial slices of an exemplary subject. (p<0.05, FWE corrected, radiologic convention). e. Left: Superimposed masks of BBB modulated voxels (red) and fMRI activation (yellow). Right: Co-localized voxels map on the same slice (cyan). f. BBB modulation in M1 (precentral gyrus, PrG) and S1 (postcentral gyrus, PoG) for subjects performing the task compared to controls (% area). g. Heatmap of BBB modulated voxels percentage in motor/sensory related areas of task vs. controls. *p<0.05, ***p<0.001, two-way ANOVA with FDR correction, task n=6, controls n=10, (i–ipsi, c-contra). All data are mean ± SEM.