Delayed Exercise-induced Upregulation of Angiogenic Proteins and Recovery of Motor Function after Photothrombotic Stroke in Mice

Abstract

Treatments promoting post-stroke functional recovery continue to be an unmet therapeutic problem with physical rehabilitation being the most reproduced intervention in preclinical and clinical studies. Unfortunately, physiotherapy is typically effective at high intensity and early after stroke - requirements that are hardly attainable by stroke survivors. The aim of this study was to directly evaluate and compare the dose-dependent effect of delayed physical rehabilitation (daily 5 h or overnight voluntary wheel running; initiated on post-stroke day 7 and continuing through day 21) on recovery of motor function in the mouse photothrombotic model of ischemic stroke and correlate it with angiogenic potential of the brain. Our observations indicate that overnight but not 5 h access to running wheels facilitates recovery of motor function in mice in grid-walking test. Western blotting and immunofluorescence microscopy experiments evaluating the expression of angiogenesis-associated proteins VEGFR2, doppel and PDGFRβ in the peri-infarct and corresponding contralateral motor cortices indicate substantial upregulation of these proteins (≥2-fold) in the infarct core and surrounding cerebral cortex in the overnight running mice on post-stroke day 21. These findings indicate that there is a dose-dependent relationship between the extent of voluntary exercise, motor recovery and expression of angiogenesis-associated proteins in this expert-recommended mouse ischemic stroke model. Notably, our observations also point out to enhanced angiogenesis and presence of pericytes within the infarct core region during the chronic phase of stroke, suggesting a potential contribution of this tissue area in the mechanisms governing post-stroke functional recovery.

Keywords: angiogenesis; infarct core; neural repair; pericyte; physical rehabilitation; post-stroke recovery.

Figure 1.. Voluntary running.

Daily running distance (panel A) and total average running (panel B) of mice in experimental groups. Mice with overnight (o/n) access to running wheels covered significantly longer distance of running compared to animals that accessed the running wheels for 5 h daily (***, p<0.001; n = 14 for overnight runners and n = 10 for 5 h runners). Panel c, compared to their baseline body weights sham and stroke animals gained 1 – 2 g weight by completion of the study (p < 0.01), whereas 5 h and overnight (o/n) runners lost 1.5 – 2 g during the same period (p < 0.05).

Figure 2.. Overnight running enhances motor recovery in grid-walking test.

Following photothrombotic stroke mice with overnight (o/n) access to running wheels showed improvement in the affected forelimb motor function (i.e., decreased number of footfaults) on days 14 and 21 after stroke (*, p < 0.05; ***, p < 0.001 in comparison to stroke group). Mice with 5 h access to the running wheels did not exhibit improvement in the affected forelimb function (p > 0.05; n = 14 for overnight runners, n = 10 for 5 h runners, n = 13 for stroke and sham groups).

Figure 3.. Overnight voluntary running induces expression of angiogenic proteins in the post-stroke brain measured by Western blotting.

Following photothrombotic stroke, mice with overnight (o/n) access to running wheels had statistically significantly increased expression of VEGFR2 (panel A), doppel (panel C) and PDGFRβ (panel E) in the peri-infarct cortical tissue on day 21 after stroke (**, p < 0.01 in comparison to sham group). No significant difference was documented in the expression levels of VEGFR2 (panel B), doppel (panel D) and PDGFRβ (panel F) in the corresponding contralateral cortical tissue of the same experimental animals (p > 0.05; n = 6 for overnight runners and n = 5 for the other groups). Note, relative density of measured proteins should not be compared between different panels (variations in film exposure times and loaded protein amounts).

Figure 4.. Western blotting evaluation of angiogenic proteins (VEGFR2, doppel and PDGFRβ) after overnight voluntary running.

Representative films from Western blotting experiments summarized in Figure 3.

Figure 5.. Wheel running for 5 hours does not induce expression of angiogenic proteins in the post-stroke brain.

Similar to Western blotting experiments summarized in Figure 3, expression levels of VEGFR2 (panels A and B), doppel (panels C and D) and PDGFRβ (panel E and F) were evaluated in the peri-infarct and corresponding contralateral cortical tissue of sham, stroke and 5 h running groups on day 21 after stroke (n = 5 for all groups). No statistically significant difference was documented in the expression levels of these proteins in peri-infarct or contralateral cerebral cortices. Note, relative density of measured proteins should not be compared between different panels (variations in film exposure times and loaded protein amounts).

Figure 6.. Overnight voluntary running induces expression of CD31-associated angiogenic proteins in the peri-infarct cerebral cortex measured by immunofluorescence microscopy.

On day 21 after photothrombotic stroke, mice with overnight (o/n) access to running wheels had statistically significantly increased expression of VEGFR2 (panel A), doppel (panel C) and PDGFRβ (panel E) in the peri-infarct cortex and infarct core (*, p < 0.05, **, p < 0.01 in comparison to sham group; n = 8 for all groups). As described in the methods section and Fig. 7, 100 μm, 200 μm, 300 μm, 400 μm and 500 μm correspond to the 1^st, 2^nd, 3^rd, 4^th and 5^th 100 μm segment of the peri-infarct cortex, i.e. regions of interest, in which fluorescence intensity was measured, whereas ‘Core’ donates to the edge of the infarct core in the same brain sections. Note, the relative fluorescence signal should not be compared between the noted proteins (variations in reactivity of primary and secondary antibodies). Contrary to these angiogenic proteins, the recorded fluorescence signal from CD31 immunolabeling was not statistically significantly different between the experimental groups (panels G and H).

Figure 7.. Representative confocal immunofluorescence microscopic images of brains from experimental animals.

Panel A, for each angiogenic protein three separate, infarct-containing sections were used (~240 μm apart in rostral-caudal axis) to double label with CD31, followed by image acquisition and quantification of the fluorescence signal as detailed in the methods section. Panel B, schematic presentation of image acquisition area for subsequent quantitative analysis. The coronal brain section shows the approximate area of image acquisition in ipsilateral and contralateral cerebral cortex. The inserts demonstrate six areas of interest (100 × 1000 μm² starting from the edge of the infarct core and extending through peri-infarct cortex: core, 100 μm, 200 μm, 300 μm, 400 μm and 500 μm) in which intensity of CD31-associated fluorescence signal for VEGFR2, doppel or PDGFRβ was measured individually.

Figure 7.. Representative confocal immunofluorescence microscopic images of brains from experimental animals.

Panel A, for each angiogenic protein three separate, infarct-containing sections were used (~240 μm apart in rostral-caudal axis) to double label with CD31, followed by image acquisition and quantification of the fluorescence signal as detailed in the methods section. Panel B, schematic presentation of image acquisition area for subsequent quantitative analysis. The coronal brain section shows the approximate area of image acquisition in ipsilateral and contralateral cerebral cortex. The inserts demonstrate six areas of interest (100 × 1000 μm² starting from the edge of the infarct core and extending through peri-infarct cortex: core, 100 μm, 200 μm, 300 μm, 400 μm and 500 μm) in which intensity of CD31-associated fluorescence signal for VEGFR2, doppel or PDGFRβ was measured individually.

Figure 8.. Infarct location and volume.

Top panel, despite evident reduction of average infarction volume in the running groups, no statistically significant difference was observed among stroke-affected animals on post-stroke day 21 (^#p = 0.051, ***p < 0.001 in comparison to stroke group; n = 5 for 5 h runners and n = 8 for all other groups; o/n, overnight). Lower panel, representative Cresyl violet-stained mouse brains on day 21 after stroke, indicating location of infarction in the primary motor cortex.