Chronic Remote Ischemic Conditioning Is Cerebroprotective and Induces Vascular Remodeling in a VCID Model

The animals were housed at Augusta University’s animal care facility, which is approved by the American Association for Accreditation of Laboratory Animal Care. This study was conducted in accordance with the National Institute of Health guidelines for the care and use of animals in research, and all protocols were approved by the institutional animal care and use committee. All the STAIR (Stroke Therapy Academic Industry Roundtable) and RIGOR recommendations and guidelines regarding randomization, blinding, and statistical analysis were followed in this study [16, 17].

Forty male mice were randomized into four groups: (1) sham (operated group for procedures of BCAS), (2) BCAS and sham RIC, (3) BCAS treated with daily RIC for 1 month post BCAS surgery (BCAS + RIC-1MO, N = 10), and (4) BCAS treated with daily RIC for 4 months post BCAS surgery (BCAS + RIC-4MO, N = 10). The outcomes were assessed by a blinded investigator. Cognitive function, functional outcomes, and CBF changes were considered as the primary outcomes and were determined at 4 and 6 months after BCAS, with either 1MO or 4MO RIC. Thereafter, the animals were sacrificed, the blood was collected for plasma nitrite estimation, and the brains were isolated and dissected for IHC (Schematic representation of the study plan in Supplemental Fig. 1).

BCAS was performed as previously described [10, 15]. In brief, animals were anesthetized with 2% isofluorane and both common carotid arteries (CCAs) were exposed by a midline cervical incision. Customized microcoils, specially designed to mimic a VCID model in the mice which was made of steel wire with an inner diameter of 0.18 mm, was twined by rotating around both right and left CCA at 30 min interval.

Non-invasive RIC therapy method was performed as published elsewhere [18, 19] with a programmable cuff or its sham procedure with a cuff that did not inflate or deflate (see detail in “ Supplemental Methods ”) [15]. Bilateral RIC therapy was performed in both hind limbs simultaneously (4 cycles × 5 min duration of each cycle inflate and deflate × 5 min interval between each cycle) daily for 1 month (BCAS+RIC-1MO) or 4 months (BCAS+RIC-4MO) after 1 week from BCAS surgery.

Cerebral blood flow (CBF) was measured using high-resolution Laser Speckle Contrast Imager (LSCI) (PSI system, Perimed Inc.) at different time points as indicated in the figure and as previously reported by us [15]. Mice were placed on a warming pad and thermostatically controlled at around 37 °C to avoid the effect of body temperature during the measurement of CBF.

Plasma NO _x (NO₂ ⁻ + NO₃ ⁻) levels were measured by NO-specific chemiluminescence, as described previously [20] with slight modification. Briefly, 100 ul of plasma were mixed with twice volumes of ethanol (100%) and kept at −20 C for 40–60 min followed by centrifuged at 13000 rpm for 10 min to remove protein as pellet. The supernatant (100 ul) was taken and injected to measure nitrite. The nitrite levels were measured by NO Analyzer 280i (GE Analytical Instruments, CO, USA). The level of nitrite was expressed in nanomolar.

An investigator who was blinded to the experimental design and behavioral test or treatment including NOR test, [21] Y-maze test, [22] beam walk, [23] and wire hanging test [24] (Detailed procedures are explained in the “Supplemental Methods.”). All groups were examined at 4 and 6 months after sham surgery, BCAS surgery, and with RIC (1MO or 4MO). NOR and Y-maze test were performed for cognitive test where beam walk test required mice to balance on a wooden beam to evaluate any gait abnormality and wire hanging test for muscular or motor function.

Immunohistochemistry was performed according to the protocol previously described [15] with slight modification. Both paraffin and cryo-sections were used for immunohistochemistry and immunofluorescence with similar anatomic features (see the “Supplemental Methods” for details). The primary antibodies were anti-myelin basic protein (MBP) (C-16 clone, SC-13914, Santa Cruz, CA, USA; 1:100 dilution); rat anti-platelet endothelial cell adhesion molecule [PECAME-1 (CD31), BD no. 550274, USA,1:200 dilution], with mouse monoclonal anti-α-smooth muscle actin [α-SMA, SC-53142, Santa Cruz, CA, USA; 1:50 dilution]; and anti-platelet-derived growth factor [PDGFR-β (958): SC-432, Santa Cruz, CA, USA; 1:200 dilution], isolectin B4 conjugates (Invitrogen, molecular probe Life Technology, IB4 no. 121411). For biotinylated immunostaining, the brain sections were incubated in the anti-MBP primary antibodies and using the avidin–biotin–peroxidase complex method with diaminobenzidine (DAB) as the chromogen. The immunostaining was carried out using the ABC kit system (Vector, Burlingame, CA, USA). After staining, the sections were counterstained with Harris hematoxylin (cat. no. HHS35-1L; Sigma) for few seconds. The sections were then dehydrated rapidly through ethanol and xylene and mounted with VectaMount medium (Vector).

Twenty-seven male mice were randomized into three groups: (1) sham, (2) BCAS and sham RIC (BCAS), and (3) BCAS treated with daily RIC for 3 weeks post BCAS surgery (BCAS+RIC). Three weeks after BCAS, blood was collected either from the eye (retro orbital) for flow cytometry analysis of endothelial progenitor cells (EPCs) and macrophages, or from the heart plasma nitrite estimation. Animals were transcardially perfused with heparinized saline to flush out the blood, followed immediately by freshly prepared BriteVu™ solution (Scarlet Imaging, LCC; Murray, UT, USA) according to manufacturer instruction. Carcasses were kept on ice at 4 °C overnight to ensure solidification of BriteVu dye prior to imaging. After 24 h, brains were isolated, fixed with 4% PFA for 48 h, and then saved in 70% alcohol till the time of imaging. SkyScan 1174 (Bruker micro-CT/formerly known as SkyScan, USA) is used for imaging. SkyScan is associated with a full-range software that provides fast volumetric reconstruction of 2D/3D quantitative analysis and realistic 3D visualization of cerebrovasculature. Outcomes: (1) full 3D picture of cerebrovasculature and (2) quantification of vascular number, vascular volume, vascular density, lumen diameter, and formation of new collaterals.

Whole blood (WB) was collected using heparinated microtubes as described previously with slight modification [25, 26]. One hundred fifty milliliters of WB were then incubated with antibodies for EPC markers including CD31, CD34, VEGFR2, and surface markers for M1/M2 macrophages, CD11b, F4/80, and CD206 (eBioscience, USA) for 20 min on ice in the dark. After washing was completed, cells were fixed and permeabilized using fix/perm concentrate (eBioscience, USA) before incubation with antibodies for intracellular staining of TNFα (for M1 macrophages) and IL10 (for M2 macrophages). Samples were then washed and run through a four-color flow cytometer (FACSCalibur, BD Biosciences), and data were collected using the CellQuest software. Samples were double-stained with control IgG and cell markers to assess any spillover signal of fluorochromes. Proper compensation was set to ensure that the median fluorescence intensities of negative and positive cells were identical and then was used to gate the population. Gating excluded dead cells and debris using forward and side scatter plots. To confirm the specificity of primary antibody binding and rule out non-specific Fc receptor binding to cells or other cellular protein interactions, negative control experiments were conducted using isotype controls matched to each primary antibody’s host species, isotype, and conjugation format.

All statistical analysis was performed using SAS 9.4, and statistical significance was assessed using a significance level of 0.05. To examine differences between the groups (sham, BCAS, BCAS+RIC-1MO, BCAS+RIC-4MO) for nitrites, a one-way ANOVA was used. If the overall test for the one-way ANOVA was statistically significant a Tukey–Kramer multiple comparison procedure was used to examine differences between the four groups. For cerebral blood flow, beam walk, hanging wire, NOR, and Y-maze measures, a repeated measures mixed model was used to examine differences between the four groups over time (CBF measurement times: baseline, post surgery, 4 months, 6 months; all other outcomes measurement times: 4 months, 6 months). Main effects of group and time as well as the two-factor interaction between group and time were included in the model. For CBF, an auto-regressive order 1 correlation structure fit the data best and was used to account for the correlation between measurement times. For the beam walk, hanging wire, NOR discrimination index, and Y-maze measures, an unstructured correlation structure was used as there were only two measurement times. Of statistical interest was the F-test for the two-factor interaction between group and time, and if it is statistically significant, it indicates that the change in the outcome over time is different for the four groups. To examine differences between groups within measurement time and between measurement times within group, a Bonferroni adjustment to the overall alpha level was used to control for the multiple post hoc pair-wise tests as not all pair-wise tests were of interest. For CBF, the Bonferroni adjusted alpha is 0.0010 and for the other outcomes, the Bonferroni adjusted alpha is 0.0031.

C-RIC therapy after BCAS increased CBF compared to sham RIC (Fig. 1a–e; Supplemental Fig. 2). CBF was significantly increased in both 1MO and 4MO RIC therapy as compared to BCAS-sham RIC. Moreover, 1MO of RIC was as effective as 4MO RIC in improving CBF at 6 months.

Both 1MO and 4MO RIC improved cognition. Results from NOR test (for spatial memory) (Fig. 2a–d) and Y-maze test for working memory (Supplemental Fig. 3A–D) showed that the sham group is more attracted toward novel objects compared to BCAS. The BCAS group had significantly shorter exploration time for novel object (TN) and less discrimination index (DI). This indicates that BCAS causes impairment of discriminative ability in mice. RIC therapy for either 1-MO or 4-MO significantly restored the TN and DI scores. Moreover, RIC therapy for either 1MO or 4-MO significantly increased the entries’ alternations in the arms of Y-maze compared to the sham group tested at 4 and 6 months after BCAS.

Animals subjected to BCAS spent more time to cross a beam in comparison to the sham control animals (Fig. 3a, b). RIC therapy for either 1MO or 4MO significantly reduced the time spent by the animals to cross the beam as compared to BCAS groups. Moreover, muscular impairment or motor impairment was assayed by wire hanging test (Fig. 3c, d). BCAS groups spent significantly less time suspending their body to the hanging wire compared to sham. RIC therapy for 1MO or 4MO significantly increased the hanging wire time compared to the BCAS group.

RIC therapy significantly increased capillary density, angiogenesis, and arteriogenesis as indicated by increased expression of CD31 and α-SMA, compared to the BCAS group (Fig. 4a–d; Supplemental Figs. 4 and 5). However, there was no significant difference between RIC-1MO and RIC-4MO therapy groups. Moreover, RIC therapy increased the expression and colocalization of pericytes with cerebral blood vessels as indicated by increased expression of PDGFR-B and IB4, respectively (Supplemental Fig. 6A). Chronic treatment also showed to increase the capillary diameter (Supplemental Fig. 6B).

The white matter degeneration in the corpus callosum was tested by Klüver–Barrera staining. The intensity in Klüver–Barrera staining was significantly reduced after BCAS compared to sham animals. RIC therapy for 1 or 4 months significantly reversed the BCAS-induced white matter damage (Supplemental Fig. 7A–B). MBP staining significantly decreased in cortical and hippocampal CA1 field region (Supplemental Fig. 8A–B), and this was reversed with RIC therapy in both BCAS+RIC1-MO and BCAS+RIC4-MO groups.

The use of BriteVu staining enabled the 3D visualization of the complete tree of the cerebrovasculature. BCAS caused a significant decline in number and volume of cerebral vessels. RIC significantly induced angiogenesis and collaterals formation as indicated by the increase in the vessels number and volume (Fig. 5a–c; Supplemental Fig. 9A–C). In support with this, results from the flow cytometry studies showed a corresponding increase in the EPC count (as indicated by increased expression of CD31 and VEGF-R2) and in macrophage expression and polarization (as indicated by increased expression of CD11b, F4/80, and CD206) with RIC therapy (Fig. 6a, b).

RIC therapy for 3 weeks post BCAS significantly increased the plasma nitrite levels compared to BCAS without RIC. However, RIC therapy for 1-MO and 4-MO showed a trend but insignificant increase in plasma nitrite levels at 6 months compared to the BCAS group (Fig. 6c, d). However, plasma nitrite level in the BCAS group was significantly decreased as compared to the sham group. Probably the short half-life of NO and long delay after RIC was finished caused NO levels to decrease.