Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Neuroscience. 2021 Mar 2;461:57–71. doi: 10.1016/j.neuroscience.2021.02.023

Delayed exercise-induced upregulation of angiogenic proteins and recovery of motor function after photothrombotic stroke in mice

Abdullah Al Shoyaib 1, Faisal F Alamri 1, Abbie Biggers 1, Serob T Karamyan 2, Thiruma V Arumugam 3, Fakhrul Ahsan 1, Constantinos M Mikelis 1, Taslim A Al-Hilal 4, Vardan T Karamyan 5
PMCID: PMC8044041  NIHMSID: NIHMS1679046  PMID: 33667592

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: Post-stroke recovery, angiogenesis, pericyte, infarct core, physical rehabilitation, neural repair, physiotherapy, wheel running

Introduction

A substantial proportion of stroke survivors suffer from functional disabilities for the rest of their life due to the lack of effective therapies that promote post-stroke recovery (Virani et al. 2020). This unmet need for treatments has led to an increased research focus on understanding the neural repair and recovery mechanisms to develop restorative therapies for stroke (Corbett et al. 2017; Bernhardt et al. 2019; Bernhardt et al. 2017b). Among a number of therapeutic strategies, physical rehabilitation has resulted in most reproduced positive outcomes in preclinical and clinical studies (Krakauer et al. 2012; Bernhardt et al. 2016) and is the current standard of care for stroke survivors (Winstein et al. 2016). There is a large heterogeneity in how physical rehabilitation is carried out in various clinical and outpatient settings, but it is generally agreed that this intervention is mostly effective at high intensity and early after stroke (Krakauer et al. 2012; Winstein et al. 2016). Unfortunately, most stroke survivors have great difficulty to meet the requirement of high-intensity and early rehabilitation, because of which their recovery is usually limited (Nicholson et al. 2013; Krakauer et al. 2012). This reality necessitates better understanding of physical rehabilitation-induced recovery mechanisms to explore new therapeutic strategies for achieving better outcomes from delayed and/or suboptimal physiotherapy.

Although, delayed physical rehabilitation strategies are less explored and established (Hacke et al. 2004; Quinn et al. 2009), it is generally understood that physiotherapy modulates and/or enhances the cellular and molecular mechanisms of neural plasticity (Murphy and Corbett 2009; Carmichael 2016; Hatakeyama et al. 2020; Caleo 2015; Christie et al. 2008). In the post-stroke rodent brain, these mechanisms are progressively activated after the acute phase, peak within the first two weeks and then gradually dissipate (Krakauer et al. 2012; Carmichael 2016; Karamyan 2021). Notably, it is deemed that angiogenesis and neurogenesis (neurovascular remodeling) and axonal sprouting and re-myelination are central among post-stroke recovery mechanisms (Carmichael 2016). Several studies have shown that physical exercise-mediated angiogenesis in the brain promotes neurogenesis and upregulates neurotropic factors which facilitate recovery of the lost function(s) (P. Zhang et al. 2013; Ruan et al. 2015; Xiong et al. 2010). A number of other studies have shown that physical exercise promotes the angiogenic capacity of ischemic brain and thus leads to functional recovery (Ding et al. 2004; Gertz et al. 2006; Matsuda et al. 2011; Gao et al. 2014; X. Wang et al. 2014). Importantly, the vast majority of these studies were performed in a middle cerebral artery occlusion (MCAO) model of stroke, which according to experts, is most suitable for neuroprotective but not post-stroke recovery studies (Corbett et al. 2017). On the contrary, little has been done to link post-stroke angiogenesis, physical rehabilitation and functional recovery in the expert-recommended mouse model of stroke, i.e. photothrombotic model. To this end, the aim of this study was to conduct initial characterization of the angiogenic potential of brain in the mouse photothrombotic stroke model while correlating it with the extent of delayed physiotherapy and recovery of motor function. Our study is the first to directly evaluate and compare the dose-dependent effect of delayed physical rehabilitation (daily 5 h or overnight voluntary wheel running; post-stroke days 7 to 21) on recovery of motor function in the mouse photothrombotic model of ischemic stroke. Our observations indicate that overnight but not 5 h access to running wheels, when initiated 7 days after stroke, facilitates recovery of motor function in mice in grid-walking test. Western blotting and immunofluorescence experiments evaluating the expression of angiogenesis-associated proteins VEGF receptor 2 (VEGFR2), doppel and PDGFR receptor-β (PDGFRβ) in the peri-infarct and corresponding contralateral motor cortices of these mice indicate substantial upregulation of these proteins in the infarct core and surrounding cerebral cortex in the overnight running group on post-stroke day 21. These findings confirm the occurrence of angiogenesis in the mouse photothrombotic model of stroke and indicate that there is a dose-dependent relationship between the extent of voluntary exercise, motor recovery and expression of angiogenesis-associated proteins. Furthermore, our observations point out to enhanced angiogenic potential and presence of pericytes within the infarct core during the chronic phase of stroke, warranting future studies to understand their contribution in the mechanisms governing post-stroke functional recovery.

Methods

Animals and study design

In this study, which was approved by the Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee, 3 – 4 month old male CD-1 mice (purchased from Charles River Laboratories) and male B6;129 (purchased from the Mutant Mouse Regional Resource Center and bred in-house) were used. The animals were kept in 12-h light/dark cycle with ad libitum access to chaw and water. All mice were handled for õne week (~2 min, twice daily) by experimenters to reduce handling stress and were acclimated to the experimental room before behavioral tests. Before stroke surgeries, the mice were randomly divided into sham and stroke groups (https://www.random.org/lists/). Assignment of stroke animals to 5 h or overnight running groups as well as their brain processing on post-stroke day 21 (fresh tissue collection or fixing) was also done randomly. Following stroke surgery, all animals were housed individually in a new home cage. Note that many of the mice in running groups were so called vehicle-treated animals, from experimental studies testing pharmacological agents, which received control vehicle intraperitoneally without an active agent (Al Shoyaib et al. 2019). Our observations indicate that these treatments do not affect post-stroke recovery outcomes (F.F. Alamri et al. 2021; Al Shoyaib et al. 2021).

Motor function of the experimental animals was evaluated during the light cycle (8 to 11 am), 3 days before and 7, 14 and 21 after stroke. Experimenters were blinded from assignment of mice to a specific experimental group.

Voluntary wheel running

Running wheels (Innowheel, Inno Vive), custom equipped with an odometer to measure running distance, were used in each animal’s home cage to initiate physical rehabilitation. In both 5 h and overnight running groups mice gained access to the running wheels starting from post-stroke day 7 through the end of the study for 6 days a week (no running on the evening/night before the behavioral test). For both groups access to the wheels started at 6 pm (one hour before the dark cycle) and lasted until 11 pm or 8 am, respectively. There was no animal that had access to a wheel but did not use it to run. Animals in the sham group had no access to the running wheels.

Photothrombotic stroke model

Photothrombosis was induced three days after baseline evaluation of motor function according to our previously published protocol (Vijayan et al. 2019; F. F. Alamri et al. 2018). In brief, mice received Rose Bengal solution intraperitoneally (80 mg/kg) under isoflurane anesthesia at 36.9 ± 0.5 °C and 5 min later the skull was illuminated with cold light through intact skull for 15 min (2 mm diameter, fiber optic illuminator light source with halogen lamp; in the right hemisphere, 1.5 mm lateral from Bregma 0). Following the surgery mice were transferred to a recovery chamber (~37°C for 1.5 h) and then housed individually in a new cage. Sham-operated mice were subjected to the same procedure with the exception of light illumination. Throughout this manuscript, the left (contralateral) forelimb is referred as ‘affected forelimb’ and right (ipsilateral) forelimb is referred as ‘unaffected forelimb’, for all experimental mice.

Grid-walking test

Motor function of experimental animals was assessed in grid-walking test, which is one of the most sensitive tests recommended for post-stroke recovery studies in mice and focused on spontaneous motor behavior of the forelimb during gait (Zhou et al. 2016; Corbett et al. 2017). The test was carried out as detailed in our earlier publications (F. F. Alamri et al. 2018; Syeara et al. 2020). In brief, each mouse was placed on a 12 mm square wire grid (33 cm × 20 cm total surface) and allowed to explore for 5 minutes while being video-recorded. The video recordings were used to count footfaults for each forelimb and total number of normal steps, followed by calculation of footfault index by: [(#affected forelimb footfaults – #unaffected forelimb footfaults) / (#affected forelimb footfaults + #unaffected forelimb footfaults + #normal steps)].

Brain collection and infarct size assessment

After completion of the grid walking test on post-stroke day 21, the animals were deeply anesthetized to either cardially perfuse with 4% paraformaldehyde (PFA) solution in phosphate-buffered saline for fixing and cryosectioning (infarct evaluation and immunostaining, n = 8/group), or to decapitate and dissect the brain for isolation of peri-infarct cortex (1–2 mm surrounding the core) and corresponding contralateral cerebral cortex (n = 6 for overnight runners, n = 5 for all other groups). PFA-fixed brains were further overnight incubated in 4% PFA solution followed by cryopreservation (20 and 30% sucrose solutions) and cryosectioning (40 μm thickness at coronal plane). To evaluate the size of infarction, the cryopreserved sections were stained with Cresyl Violet followed by digitization and volumetric assessment of infarction using ImageJ software (Syeara et al. 2020; Jayaraman et al. 2020).

Brain tissue homogenization and Western blotting

Peri-infarct and contralateral cerebral cortical samples were homogenized in RIPA buffer (Boston BioProducts, catalog #BP-115–5X) supplemented with protease and phosphatase inhibitors (products 78430 and 78428, HALT Inhibitor Cocktails, Thermo Fisher Scientific) using a micro-pestle with micro-tube (product 199222, Research Products International) (Jayaraman et al. 2020). Homogenized samples were intermittently vortexed while kept on ice for 30 min, followed by centrifugation (17,000× g for 20 min at 4 °C) and storage of the supernatant at −80 °C. Protein quantification was done using BCA assay kit (product 23227, Thermo Fisher Scientific). Western blotting was carried out using 5% (for VEGFR2 and PDGFRβ), 6.5% (for β-actin of VEGFR2 and PDGFRβ gels) and 7.5% (for doppel and its β-actin) SDS PAGE gels following conventional procedures as described in our earlier publications (Rashid et al. 2014; Wangler et al. 2012). Primary antibodies used were anti-VEGFR2 and anti-PDGFRβ (products 2479S and 3169S, Cell Signaling Technology), anti-doppel (product sc-16863, Santa Cruz Biotechnology), and anti-beta-actin (product A5441, Sigma). The secondary antibodies used were HRP-conjugated goat anti-rabbit and goat anti-mouse immunoglobulins (products 170–6515 and 170–6516, Bio-Rad Laboratories). Immunoreactive bands were quantified using Quantity One software (Bio-Rad Laboratories), β-actin was used as loading control to normalize the relative density of bands for measured proteins. To account for variability, samples from all experimental groups were included in each SDS PAGE gel run (2 gels with 8 samples for each target protein: gel #1 included 3 sham, 2 stroke and 3 overnight running samples, gel #2 included 2 sham, 3 stroke and 3 overnight running samples). For each gel, the recorded density of bands representing the ‘sham’ group was arbitrarily set to 100% and the density values of other experimental groups was compared to ‘sham’ values.

Immunofluorescence staining and imaging

For this purpose, we used free floating cryopreserved coronal sections to double label for CD31 and VEGFR2, CD31 and PDGFRβ or CD31 and Doppel in separate, infarct-containing sections of the same brain. The sections (3 per brain for each marker, 240 μm apart in rostral-caudal axis) were washed, permeabilized (2 h in 200 mM glycine in TBS with 1% Triton-X 100), blocked (2 h in 1% BSA in TBS with 1% Triton-X 100) and incubated overnight with the same primary antibodies noted for Western blotting, except for VEGFR2, for which Alexa Fluor 488-conjugated anti-VEGFR2 antibody was used (product 12687S, Cell Signaling Technology). The primary antibody for CD31 was R-phycoerythrin-conjugated anti-CD31 antibody (product 561073, BD Biosciences), whereas the secondary antibody used for detection of the other antigens was anti-rabbit or anti-goat Alexa Fluor 488 (products A11008 and A11055, Invitrogen).

Fluorescence microscopic images were acquired on a Nikon A1R MP confocal microscope using a 20x water immersion lens and FITC and TRITC filters for the green and red channel acquisition, respectively. Throughout this manuscript ‘infarct core’ is referred to a 100 μm-wide outmost edge of the infarcted cortical tissue, whereas the continuing 500 μm-wide “healthy” tissue is referred to as ‘peri-infarct cortex’. To capture an image of the infarct core and peri-infarct cortex together in each coronal brain section, 3 to 4 microscopy fields per X and Y axes were selected to include the entire area of interest. Then, automatic image capturing option of Nikon NIS Elements AR software was used to acquire Z stack images of each field (in green and red channels separately), followed by automatic stitching of Z stack images at 3% overlap. The same approach was used to acquire an image of contralateral cerebral cortex of each brain section. The stitched Z stack images were maximally projected using the same software, followed by measurement of CD31-associated intensity of VEGFR2, Doppel or PDGFRβ signal in 6 successive regions of interest (1 for infarct core and 5 for peri-infarct cortex). Each region of interest was a 100 × 1000 μm2 area starting from the edge of the infarct core and extending through the peri-infarct cortex as illustrated in Fig. 7.

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

Figure 7.

Figure 7.

Open in a new tab

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.

Data Analysis

Data from grid-walking test were analyzed using two-way repeated measures ANOVA with Dunnett’s post hoc test for multiple comparisons (Prism 7.05, GraphPad). One-way ANOVA followed by Dunnett’s multiple comparisons was used for comparing all other experimental data expect running and body weight data, for which two-tailed, unpaired and paired t-tests were used, respectively. A p-value of less than 0.05 was considered as statistically significant. Throughout the manuscript the data is presented in in box-plot graphs, where the box extends from the 25th to 75th percentiles, whiskers range from 5 to 95 percentiles and the line in the box is plotted at the median.

Results

Running distance and body weights

To mimic delayed physical rehabilitation the animals received access to a running wheel in their home cage for 5 or 14 (i.e. overnight) hours daily, from post-stroke day 7 through completion of the study. Because of voluntary running, on average it took about one week for overnight runners to gradually increase their covered distance and consistently run ~10 kilometers per session/day (Fig. 1). In the meantime, it took about a day or two longer for 5 h runners to reach consistent running and cover ~4 kilometers per session/day (Fig. 1). Compared to baseline body weight values, both regimens of wheel running resulted in loss of ~2 g by post-stroke day 21, whereas “sham” and “stroke” groups recorded ~2 g weight gain during the same period (Fig. 1).

Figure 1. Voluntary running.

Figure 1.

Open in a new tab

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).

Recovery of motor function

To evaluate the effect of wheel running on recovery of motor function after stroke, spontaneous motor behavior of the forelimb during gait was monitored in grid-walking test. As expected, focal cerebral stroke caused a significant and sustained deficit in the contralateral, i.e. affected, forelimb function of mice in the grid-walking test (Fig. 2; group x day interaction F (9, 138) = 14.68, p < 0.001; n = 14 for overnight runners, n = 10 for 5 h runners, n = 13 for stroke and sham groups). Within each stroke-affected experimental group, post hoc analyses with Dunnett’s correction indicated statistically significant differences in the affected forelimb function between baseline and post-stroke days 7, 14 and 21 (p < 0.001). Accordingly, within sham group, no statistically significant difference was documented in the affected forelimb function between baseline and post-stroke evaluation days (p > 0.05). Within day comparisons of experimental groups (stroke vs others; with Dunnett’s post hoc analyses) revealed statistically significant difference of the forelimb function between stroke and overnight running groups on post-stroke days 14 and 21 (Fig. 2a and b; p = 0.03 and 0.001 for day 14 and 21, respectively). As expected, within day comparison of stroke and sham-operated mice also showed statistically significant difference of the forelimb function on all evaluation days (p < 0.001) except the baseline (p > 0.05).

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

Figure 2.

Open in a new tab

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).

Cortical expression of angiogenic proteins after overnight or 5-hour voluntary wheel running

In addition to motor function, we evaluated expression levels of three angiogenesis-associated molecular markers as potential outcome measures of post-stroke recovery in overnight running experimental animals on day 21 after stroke. For this, expression levels of VEGFR2, doppel and PDGFRβ were quantified in peri-infarct and corresponding contralateral cerebral cortices using Western blotting (Figs. 3 and 4). The results of these experiments revealed statistically significant (≥ 2-fold) increase in the relative density of all three proteins in the peri-infarct but not contralateral cerebral cortex of overnight running animals (Fig. 3).

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

Figure 3.

Open in a new tab

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.

Figure 4.

Open in a new tab

Representative films from Western blotting experiments summarized in Figure 3.

To verify if these angiogenesis-associated markers may serve as outcome measures of post-stroke recovery, the same evaluation was carried in 5 hour running group, which as presented in Fig. 5, did not show recovery of motor function in the grid-walking test. These additional Western blotting experiments revealed a lack of statistically significant alteration in expression levels of VEGFR2, doppel and PDGFRβ in peri-infarct or corresponding contralateral cerebral cortices of the experimental groups (Fig. 5).

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

Figure 5.

Open in a new tab

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).

Overnight voluntary running induces CD31-associated expression of VEGFR2, doppel and PDGFRβ in the post-stroke brain

To confirm our observations associating upregulation of the noted angiogenic proteins with overnight voluntary running and motor recovery, we used immunofluorescence microscopy to evaluate endothelial cell-associated, i.e. CD31-associated, expression of VEGFR2, doppel and PDGFRβ in peri-infarct and corresponding contralateral cerebral cortex of the experimental animals. Similar to Western blotting experiments summarized in Fig. 3, the results of these experiments revealed statistically significant increase in the relative expression of all three proteins in the peri-infarct but not contralateral cerebral cortex of the overnight running animals (Figs. 6 and 7). Contrary to this, we did not observe statistically significant alteration of CD31 immunofluorescence signal among experimental groups (Fig. 7). Notably, we also documented profound upregulation of these proteins in the infarct core of stroke and overnight running animals.

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

Figure 6.

Open in a new tab

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 1st, 2nd, 3rd, 4th and 5th 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).

Infarct location and volume

To evaluate the location of cerebral infarction and its volume on post-stroke day 21, PFA-fixed brain sections of all experimental groups were stained with Cresyl violet and histologically examined (Fig. 8). As expected, infarction of cerebral cortex involved the primary motor area in all stroke-affected experimental groups. Volumetric evaluation revealed no statistically significant differences in the infarct volume among stroke-affected experimental groups (Fig. 8; p > 0.05, 0.58 to 1.3 mm3 average stroke volume), despite the apparent reduction in the running groups.

Figure 8. Infarct location and volume.

Figure 8.

Open in a new tab

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.

Discussion

The practice of physical rehabilitation greatly varies among different clinical and outpatient settings, however, it is generally accepted that higher intensity and earlier physiotherapy is correlated with better functional outcomes after stroke (Krakauer et al. 2012; Winstein et al. 2016). The primary reason that higher intensity and earlier physiotherapy is more efficacious for functional recovery is because physical activity/training substantially enhances spontaneous endogenous plasticity after brain injury and facilitates neural repair (Bernhardt et al. 2017a; Krakauer et al. 2012). Unfortunately, a complicating reality is that most stroke survivors have difficulty to engage in high-intensity physical rehabilitation for a long period of time, especially within days after stroke (Fini et al. 2017; West and Bernhardt 2012), because of which their recovery is usually very limited (Nicholson et al. 2013; Krakauer et al. 2012).

Among physical rehabilitation-induced recovery mechanisms in the post-stroke brain, angiogenesis is deemed to be causally associated with functional recovery. Numerous experimental studies have linked physical exercise to the increased angiogenic capacity of ischemic brain and subsequent functional recovery (Ding et al. 2004; Matsuda et al. 2011; Gao et al. 2014; Ruan et al. 2015; Xiong et al. 2010). Importantly, most of these observations were carried out in transient MCAO model of stroke but not in stroke models recommended for studying recovery mechanisms and therapeutic interventions for promoting functional recovery (Corbett et al. 2017). Furthermore, little has been done to study post-stroke angiogenesis in association with physical rehabilitation and functional recovery in the mouse photothrombotic model of stroke, which is the expert-recommended mouse model for recovery studies (Corbett et al. 2017). To this end, the aim of this study was to conduct initial characterization of the angiogenic potential of brain in the mouse photothrombotic stroke model while correlating it with the extent of delayed physiotherapy and recovery of motor function.

In our experiments, physiotherapy consisted of daily voluntary wheel running at two “doses” (5 h and overnight access to wheels) in animal’s home cage, starting on post-stroke day 7 and lasting until the end of the study. The rationale for delayed start of wheel running was because post-stroke day 7 is comparable with the time when most stroke survivors start some degree of more advanced, regular physical rehabilitation (second week), which however is usually suboptimal (Lay et al. 2016; Fini et al. 2017). Functional recovery of motor control was determined in a task of spontaneous motor behaviors of the forelimb during gait known as grid-walking test, which is a gold standard, expert-recommended motor test for post-stroke recovery studies (Corbett et al. 2017). The daily average distance ran by animals in our study was ~3 km during the 5 h rehabilitation period and ~8 km when mice had overnight access to the running wheels (Fig. 1). The shorter duration of running was insufficient to promote motor recovery in mice by post-stroke day 21, however the overnight running animals showed significantly improved function of the forelimb on days 14 and 21 after stroke. Importantly, the documented recovery did not reach to baseline values indicating presence of substantial impairment of the function in the stroke-affected forelimb (Fig. 1). This observation is in agreement with earlier studies in photothrombotic and MCAO models of stroke where delayed, high-intensity skill-training (Ng et al. 2015), treadmill exercise (Yang et al. 2003) or environmental enrichment (Tang et al. 2019) promoted functional recovery only to a limited extent. Notably, neither our, nor the mentioned studies extended the observations several weeks or months after stroke, with or without the rehabilitative intervention, and hence it is unknown whether the recovery continues further, plateaus or worsens over time. These questions are of great interest, and future studies should address them in more detail and provide insight about associated molecular mechanisms.

Another important consideration is the relevance of our observations to stroke survivors. At the moment, we are unaware of any equivalency scale for comparing physical exercise between rodents and humans, and hence, it is difficult to tell what the human equivalent of overnight voluntary running would be. However, it is likely that daily ~10 km running (daily average in the second week of running) is a highly demanding and intense physical activity for mice, the equivalent of which would be unrealistic to expect from majority of stroke survivors. Because consistent and structured physical rehabilitation is not practiced well, i.e. at sufficient intensity and earlier time points, in post-stroke patients (Lay et al. 2016; Fini et al. 2017) perhaps another key take-away from our study is that add-on pharmacotherapy or other interventions together with delayed and/or limited physiotherapy is the most practical way to promote functional recovery in post-stroke patients. This question also is a subject of our ongoing studies (F.F. Alamri et al. 2021; Al Shoyaib et al. 2021) and has been recognized after the recent failed clinical trials focusing on pharmacotherapy of post-stroke functional recovery (Kwakkel et al. 2020).

In this study, we also evaluated the expression levels of angiogenesis-associated proteins VEGFR2, doppel and PDGFRβ in the peri-infarct cortical samples of experimental animals collected on post-stroke day 21. Our observations indicate substantial upregulation of all three proteins in the peri-infarct but not corresponding contralateral cerebral cortex of overnight running animals (Figs. 3 and 6). Notably, the expression of these proteins was not substantially changed in 5 h running animals (Fig. 5), indicating that these proteins may serve as molecular markers to evaluate post-stroke functional recovery. Among various factors involved in angiogenesis, vascular endothelial growth factor A (VEGF) is likely the most studied and potent mediator of angiogenesis, whereas VEGFR2 is deemed the primary receptor in transducing angiogenesis-associated effects of VEGF (Shibuya and Claesson-Welsh 2006). VEGFR2 is likely the most widely used angiogenesis marker, since its increased expression has been directly correlated with various angiogenesis-promoting conditions (Shibuya 2011). Notably, both VEGF and VEGFR2 have been a focus of multiple experimental stroke studies related to both neuroprotection and neurorestoration (Sun et al. 2003; Z. G. Zhang et al. 2000; Reeson et al. 2015; Y. Wang et al. 2005; Pianta et al. 2019). Doppel, also known as prion protein two and encoded by PRND gene, is a protein with polypeptide domains similar to those of cellular prions (Qin et al. 2006). Doppel is recognized for its role in peripheral angiogenesis via modulation of VEGFR2 internalization (Al-Hilal et al. 2016), and brain angiogenesis and blood-brain barrier development via modulation of PDGFRβ and recruitment of pericytes (Chen et al. 2020; Li et al. 2000). Doppel is considered to be a marker for active angiogenesis in both physiological and pathological conditions (Al-Hilal et al. 2016; Chen et al. 2020), however it was not studied in a stroke setting before. Lastly, when it comes to brain angiogenesis and stable, properly functioning capillaries and blood-brain barrier, pericytes are increasingly recognized as important cells carrying a number of critical functions (Brown et al. 2019; Filosa et al. 2016). Pericytes are recruited to the brain endothelial cells via platelet derived growth factor B (PDGFB)/PDGFRβ signaling axis (Daneman et al. 2010) and should be taken into consideration when judging about beneficial or detrimental angiogenesis flowing CNS injury (Shen et al. 2012; Liu 2015). Similar to VEGFR2 and doppel, the role of PDGF/PDGFRβ signaling pathway in angiogenesis has been well established and it is considered an important vascular maturation marker and a target for anti-angiogenic therapy (Raica and Cimpean 2010; Hosaka et al. 2020). Collectively, the expression or functions of VEGFR2, doppel and PDGFRβ and associated molecular pathways has been linked to various disorders related to CNS angiogenesis, including stroke, and that is why we selected these molecules as potential molecular outcome measures for post-stroke functional recovery.

To confirm our Western blotting observations, upregulation of VEGFR2, doppel and PDGFRβ in overnight running animals was verified by another, independent technique. These confocal fluorescence microscopic evaluations confirmed our Western blotting observations indicating CD31-associated upregulation of all three proteins in the peri-infarct cortical region of the overnight running but not control animals (Fig. 6). Upregulation of these proteins was not accompanied by significant alteration of CD31 expression itself, indicating that microvascular density was comparable in the experimental groups on day 21 after stroke. This is a phenomenon that has been studied in normal physiological conditions (Imoukhuede and Popel 2012; Sabbah et al. 2019) and more extensively in the setting of cancer (Orre and Rogers 1999; Nakayama et al. 2013), indicating that expression of angiogenic proteins, e.g. VEGFR2, in endothelial cells does not directly or poorly correlates with microvascular density. Perhaps the closest example supporting our observations is a recent study by Reeson and colleagues (2015) exploring VEGFR2 expression in peri-infarct vascular networks in naïve and diabetic mice on days 3, 7 and 28 after photothrombotic stroke. The authors documented upregulation of VEGFR2 (~3-fold) by Western blotting and immunofluorescence microscopy and observed amplification of the receptor upregulation in diabetic post-stroke animals. Notably, vascular branching density (a surrogate measure of angiogenesis) was not significantly affected in the same experimental groups throughout the time-course of the study (Reeson et al. 2015). Similarly, another recent study utilizing a rehabilitation paradigm following photothrombotic stroke in mice documented only ~0.36-fold increase in blood vessel density (stroke+rehabilitation group vs. stroke only) in regions proximal to the stroke core on post-stroke day 30 (Allegra Mascaro et al. 2019). Unfortunately, the expression of angiogenic markers was not evaluated in this study, however the noted difference in blood vessel density was no longer observed in regions distal from the stroke core (Allegra Mascaro et al. 2019). It is important to note, other experimental studies using transient MCAO model correlated increased vascular density with improved functional outcomes after stroke. For examples, in two successive studies utilizing cell therapy approaches in rats after transient MCAO (Kanazawa et al. 2017; Hatakeyama et al. 2019), Kanazawa and colleagues have documented increased (~3-fold) CD31 immunoreactivity in the border of the ischemic core on day 28 after stroke. These observations do not contradict our and other research findings noted above, but rather point out to the well-recognized phenomenon of smaller penumbra and lower degree of post-stroke angiogenesis in the photothrombotic model of stroke in comparison to the transient MCAO model (Carmichael 2005). Thus, our collective results suggest that peri-infarct VEGFR2, doppel and PDGFRβ may have a potential to serve as molecular outcome measures to assess post-stroke functional recovery. However, to fully adopt this, independent confirmation of our observations is required, together with more detailed analysis of angiogenesis in the peri-infarct region and its causal association with post-stroke functional recovery.

Our unexpected observation of potential angiogenesis occurring in the infarct core itself is also noteworthy. The vast majority of experimental studies exploring the molecular mechanisms of post-stroke recovery have focused on the peri-infarct and/or contralateral cortex but not the infarct core (Murphy and Corbett 2009; Carmichael 2016). The latter has rarely been considered to be involved in the recovery process, however, our data indicate that the infarct core is likely an actively evolving region in the chronic phase of stroke. Notably, the phenomenon of enhanced angiogenesis in the infarct core has been documented by two other research groups (Jiang et al. 2017; Kanazawa et al. 2017; Hatakeyama et al. 2019), and was elegantly summarized in a recent review article (Kanazawa et al. 2019). These observations warrant future detailed studies which should shed more light on our understanding of the infarct core in the chronic phase of stroke and its contribution in the recovery processes.

Lastly, it is important to point out that our study is not without shortcomings. Among notable limitations are use of adult male but not female and older animals (Kluge et al. 2018; Rahimian et al. 2019), lack of information on whether induced functional recovery and angiogenesis reverse once rehabilitation is halted (Cahill et al. 2018), and our focus on expression of antigenic markers but not respective signaling pathways (Zan et al. 2014). Future studies should explore these questions in more detail to better integrate our findings with biology of neural repair and post-stroke functional recovery. Another important point of consideration is the potential influence of circadian rhythm on the efficacy of rehabilitative interventions and biology of neural repair, in general. This is based on a recent study by Esposito and colleagues (2020) who, in a series of elegantly designed experiments, revealed that time of the day influences the therapeutic impact of neuroprotective interventions. At the moment, it is unclear whether neurorestorative interventions are prone to influence of the circadian rhythm, however, given the intricate association of neuroprotective and neurorestoraive mechanisms in the post-stroke brain (Iadecola and Anrather 2011; Carmichael 2016) it is most likely that findings of Esposito and colleagues (2020) may also be applicable in post-stroke recovery. Future studies should clarify this question with physical rehabilitation being among top interventions to study, since it is the standard of care for post-stroke patients.

In summary, our study is the first to directly evaluate and compare the dose-dependent effect of delayed physical rehabilitation on recovery of motor function in the mouse photothrombotic model of ischemic stroke. Our observations indicate that overnight but not 5 h access to running wheels, when initiated 7 days after stroke, facilitates recovery of motor function in mice in the grid-walking test. Western blotting and immunofluorescence microscopy experiments evaluating the expression of angiogenesis-associated proteins VEGFR2, doppel and PDGFRβ indicate strong association between upregulation of these proteins in the peri-infarct cerebral cortex and motor recovery of the overnight running animals on post-stroke day 21. Furthermore, our observations point out to enhanced angiogenesis and presence of pericytes within the infarct core during the chronic phase of stroke, warranting future studies to understand their contribution in the mechanisms governing post-stroke functional recovery.

Highlights.

  • Dose-dependent effect of delayed physiotherapy on post-stroke recovery was studied.

  • Overnight but not 5 h voluntary wheel running promoted post-stroke motor recovery.

  • Recovery of motor function was accompanied by induction of angiogenic proteins.

  • Induction of angiogenic proteins was observed in peri-infract region and infarct core.

  • Infarct core maybe involved in mechanisms governing post-stroke functional recovery.

Acknowledgments:

This work was partly supported by a NIH research grant (1R01NS106879). Breeding pairs of B6;129 mouse strain used for this research project were obtained from the Mutant Mouse Regional Resource Center, a NIH funded strain repository at the University of California, Davis.

Abbreviations:

VEGFR2

VEGF receptor 2

PDGFRβ

PDGF receptor-β

PFA

paraformaldehyde

RIPA buffer

radioimmunoprecipitation assay buffer

CD31

cluster of differentiation 31

HRP

horseradish peroxidase

MCAO

middle cerebral artery occlusion

Footnotes

Declarations of interest: None.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Al-Hilal TA, Chung SW, Choi JU, Alam F, Park J, Kim SW, et al. (2016). Targeting prion-like protein doppel selectively suppresses tumor angiogenesis. J Clin Invest, 126(4), 1251–1266, doi: 10.1172/JCI83427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Al Shoyaib A, Alamri FF, Syeara N, Jayaraman S, Karamyan ST, Arumugam TV, et al. (2021). The effect of histone deacetylase inhibitors panobinostat or entinostat on motor recovery in mice after ischemic stroke. Neuromolecular Med, (accepted for publication on January 18, 2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Al Shoyaib A, Archie SR, & Karamyan VT (2019). Intraperitoneal Route of Drug Administration: Should it Be Used in Experimental Animal Studies? Pharm Res, 37(1), 12, doi: 10.1007/s11095-019-2745-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alamri FF, Al Shoyaib A, Syeara N, Paul A, Jayaraman S, Karamyan ST, et al. (2021). Delayed atomoxetine or fluoxetine treatment coupled with limited voluntary running promotes motor recovery in mice after ischemic stroke. Neural Regen Res, 16(7), 1244–1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Alamri FF, Shoyaib AA, Biggers A, Jayaraman S, Guindon J, & Karamyan VT (2018). Applicability of the grip strength and automated von Frey tactile sensitivity tests in the mouse photothrombotic model of stroke. Behavioural brain research, 336, 250–255, doi: 10.1016/j.bbr.2017.09.008. [DOI] [PubMed] [Google Scholar]
  6. Allegra Mascaro AL, Conti E, Lai S, Di Giovanna AP, Spalletti C, Alia C, et al. (2019). Combined Rehabilitation Promotes the Recovery of Structural and Functional Features of Healthy Neuronal Networks after Stroke. Cell Rep, 28(13), 3474–3485 e3476, doi: 10.1016/j.celrep.2019.08.062. [DOI] [PubMed] [Google Scholar]
  7. Bernhardt J, Borschmann K, Boyd L, Thomas Carmichael S, Corbett D, Cramer SC, et al. (2016). Moving rehabilitation research forward: Developing consensus statements for rehabilitation and recovery research. International journal of stroke : official journal of the International Stroke Society, 11(4), 454–458, doi: 10.1177/1747493016643851. [DOI] [PubMed] [Google Scholar]
  8. Bernhardt J, Godecke E, Johnson L, & Langhorne P (2017a). Early rehabilitation after stroke. Curr Opin Neurol, 30(1), 48–54, doi: 10.1097/WCO.0000000000000404. [DOI] [PubMed] [Google Scholar]
  9. Bernhardt J, Hayward KS, Dancause N, Lannin NA, Ward NS, Nudo RJ, et al. (2019). A stroke recovery trial development framework: Consensus-based core recommendations from the Second Stroke Recovery and Rehabilitation Roundtable. International journal of stroke : official journal of the International Stroke Society, 14(8), 792–802, doi: 10.1177/1747493019879657. [DOI] [PubMed] [Google Scholar]
  10. Bernhardt J, Hayward KS, Kwakkel G, Ward NS, Wolf SL, Borschmann K, et al. (2017b). Agreed definitions and a shared vision for new standards in stroke recovery research: The Stroke Recovery and Rehabilitation Roundtable taskforce. International journal of stroke : official journal of the International Stroke Society, 12(5), 444–450, doi: 10.1177/1747493017711816. [DOI] [PubMed] [Google Scholar]
  11. Brown LS, Foster CG, Courtney J-M, King NE, Howells DW, & Sutherland BA (2019). Pericytes and Neurovascular Function in the Healthy and Diseased Brain. Frontiers in Cellular Neuroscience, 13, 282. doi: 10.3389/fncel.2019.00282 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cahill SP, Cole JD, Yu RQ, Clemans-Gibbon J, & Snyder JS (2018). Differential Effects of Extended Exercise and Memantine Treatment on Adult Neurogenesis in Male and Female Rats. Neuroscience, 390, 241–255, doi: 10.1016/j.neuroscience.2018.08.028. [DOI] [PubMed] [Google Scholar]
  13. Caleo M (2015). Rehabilitation and plasticity following stroke: Insights from rodent models. Neuroscience, 311, 180–194, doi: 10.1016/j.neuroscience.2015.10.029. [DOI] [PubMed] [Google Scholar]
  14. Carmichael ST (2005). Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics, 2(3), 396–409, doi: 10.1602/neurorx.2.3.396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Carmichael ST (2016). Emergent properties of neural repair: elemental biology to therapeutic concepts. Ann Neurol, 79(6), 895–906, doi: 10.1002/ana.24653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chen Z, Morales JE, Avci N, Guerrero PA, Rao G, Seo JH, et al. (2020). The vascular endothelial cell-expressed prion protein Prnd/Doppel promotes angiogenesis and blood-brain barrier development. Development, doi: 10.1242/dev.193094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Christie BR, Eadie BD, Kannangara TS, Robillard JM, Shin J, & Titterness AK (2008). Exercising our brains: how physical activity impacts synaptic plasticity in the dentate gyrus. Neuromolecular Med, 10(2), 47–58, doi: 10.1007/s12017-008-8033-2. [DOI] [PubMed] [Google Scholar]
  18. Corbett D, Carmichael ST, Murphy TH, Jones TA, Schwab ME, Jolkkonen J, et al. (2017). Enhancing the alignment of the preclinical and clinical stroke recovery research pipeline: Consensus-based core recommendations from the Stroke Recovery and Rehabilitation Roundtable translational working group. International journal of stroke : official journal of the International Stroke Society, 12(5), 462–471, doi: 10.1177/1747493017711814. [DOI] [PubMed] [Google Scholar]
  19. Daneman R, Zhou L, Kebede AA, & Barres BA (2010). Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature, 468(7323), 562–566, doi: 10.1038/nature09513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ding YH, Luan XD, Li J, Rafols JA, Guthinkonda M, Diaz FG, et al. (2004). Exercise-induced overexpression of angiogenic factors and reduction of ischemia/reperfusion injury in stroke. Curr Neurovasc Res, 1(5), 411–420. [DOI] [PubMed] [Google Scholar]
  21. Esposito E, Li W, E TM, Park JH, Sencan I, Guo S, et al. (2020). Potential circadian effects on translational failure for neuroprotection. Nature, 582(7812), 395–398, doi: 10.1038/s41586-020-2348-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Filosa JA, Morrison HW, Iddings JA, Du W, & Kim KJ (2016). Beyond neurovascular coupling, role of astrocytes in the regulation of vascular tone. Neuroscience, 323, 96–109, doi: 10.1016/j.neuroscience.2015.03.064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fini NA, Holland AE, Keating J, Simek J, & Bernhardt J (2017). How Physically Active Are People Following Stroke? Systematic Review and Quantitative Synthesis. Phys Ther, 97(7), 707–717, doi: 10.1093/ptj/pzx038. [DOI] [PubMed] [Google Scholar]
  24. Gao Y, Zhao Y, Pan J, Yang L, Huang T, Feng X, et al. (2014). Treadmill exercise promotes angiogenesis in the ischemic penumbra of rat brains through caveolin-1/VEGF signaling pathways. Brain Res, 1585, 83–90, doi: 10.1016/j.brainres.2014.08.032. [DOI] [PubMed] [Google Scholar]
  25. Gertz K, Priller J, Kronenberg G, Fink KB, Winter B, Schrock H, et al. (2006). Physical activity improves long-term stroke outcome via endothelial nitric oxide synthase-dependent augmentation of neovascularization and cerebral blood flow. Circ Res, 99(10), 1132–1140, doi: 10.1161/01.RES.0000250175.14861.77. [DOI] [PubMed] [Google Scholar]
  26. Hacke W, Donnan G, Fieschi C, Kaste M, von Kummer R, Broderick JP, et al. (2004). Association of outcome with early stroke treatment: pooled analysis of ATLANTIS, ECASS, and NINDS rt-PA stroke trials. Lancet, 363(9411), 768–774, doi: 10.1016/S0140-6736(04)15692-4. [DOI] [PubMed] [Google Scholar]
  27. Hatakeyama M, Kanazawa M, Ninomiya I, Omae K, Kimura Y, Takahashi T, et al. (2019). A novel therapeutic approach using peripheral blood mononuclear cells preconditioned by oxygen-glucose deprivation. Scientific reports, 9(1), 16819, doi: 10.1038/s41598-019-53418-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hatakeyama M, Ninomiya I, & Kanazawa M (2020). Angiogenesis and neuronal remodeling after ischemic stroke. Neural Regen Res, 15(1), 16–19, doi: 10.4103/1673-5374.264442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hosaka K, Yang Y, Seki T, Du Q, Jing X, He X, et al. (2020). Therapeutic paradigm of dual targeting VEGF and PDGF for effectively treating FGF-2 off-target tumors. Nature communications, 11(1), 3704, doi: 10.1038/s41467-020-17525-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Iadecola C, & Anrather J (2011). Stroke research at a crossroad: asking the brain for directions. [Research Support, N.I.H., Extramural]. Nature neuroscience, 14(11), 1363–1368, doi: 10.1038/nn.2953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Imoukhuede PI, & Popel AS (2012). Expression of VEGF receptors on endothelial cells in mouse skeletal muscle. PLoS One, 7(9), e44791, doi: 10.1371/journal.pone.0044791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jayaraman S, Al Shoyaib A, Kocot J, Villalba H, Alamri FF, Rashid M, et al. (2020). Peptidase neurolysin functions to preserve the brain after ischemic stroke in male mice. J Neurochem, 153(1), 120–137, doi: 10.1111/jnc.14864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jiang MQ, Zhao YY, Cao W, Wei ZZ, Gu X, Wei L, et al. (2017). Long-term survival and regeneration of neuronal and vasculature cells inside the core region after ischemic stroke in adult mice. Brain Pathol, 27(4), 480–498, doi: 10.1111/bpa.12425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kanazawa M, Miura M, Toriyabe M, Koyama M, Hatakeyama M, Ishikawa M, et al. (2017). Microglia preconditioned by oxygen-glucose deprivation promote functional recovery in ischemic rats. Scientific reports, 7, 42582, doi: 10.1038/srep42582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kanazawa M, Takahashi T, Ishikawa M, Onodera O, Shimohata T, & Del Zoppo GJ (2019). Angiogenesis in the ischemic core: A potential treatment target? J Cereb Blood Flow Metab, 39(5), 753–769, doi: 10.1177/0271678X19834158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Karamyan VT (2021). The role of peptidase neurolysin in neuroprotection and neural repair after stroke. Neural Regen Res, 16(1), 21–25, doi: 10.4103/1673-5374.284904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kluge MG, Jones K, Kooi Ong L, Gowing EK, Nilsson M, Clarkson AN, et al. (2018). Age-dependent Disturbances of Neuronal and Glial Protein Expression Profiles in Areas of Secondary Neurodegeneration Post-stroke. Neuroscience, 393, 185–195, doi: 10.1016/j.neuroscience.2018.07.034. [DOI] [PubMed] [Google Scholar]
  38. Krakauer JW, Carmichael ST, Corbett D, & Wittenberg GF (2012). Getting neurorehabilitation right: what can be learned from animal models? Neurorehabil Neural Repair, 26(8), 923–931, doi: 10.1177/1545968312440745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kwakkel G, Meskers C, & Ward NS (2020). Time for the next stage of stroke recovery trials. Lancet neurology, 19(8), 636–637, doi: 10.1016/S1474-4422(20)30218-0. [DOI] [PubMed] [Google Scholar]
  40. Lay S, Bernhardt J, West T, Churilov L, Dart A, Hayes K, et al. (2016). Is early rehabilitation a myth? Physical inactivity in the first week after myocardial infarction and stroke. Disabil Rehabil, 38(15), 1493–1499, doi: 10.3109/09638288.2015.1106598. [DOI] [PubMed] [Google Scholar]
  41. Li A, Sakaguchi S, Shigematsu K, Atarashi R, Roy BC, Nakaoke R, et al. (2000). Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration. Am J Pathol, 157(5), 1447–1452, doi: 10.1016/s0002-9440(10)64782-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Liu J (2015). Poststroke angiogenesis: blood, bloom, or brood? Stroke, 46(5), e105–106, doi: 10.1161/strokeaha.115.007643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Matsuda F, Sakakima H, & Yoshida Y (2011). The effects of early exercise on brain damage and recovery after focal cerebral infarction in rats. Acta Physiol (Oxf), 201(2), 275–287, doi: 10.1111/j.1748-1708.2010.02174.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Murphy TH, & Corbett D (2009). Plasticity during stroke recovery: from synapse to behaviour. Nat Rev Neurosci, 10(12), 861–872, doi: 10.1038/nrn2735. [DOI] [PubMed] [Google Scholar]
  45. Nakayama M, Nakayama A, van Lessen M, Yamamoto H, Hoffmann S, Drexler HC, et al. (2013). Spatial regulation of VEGF receptor endocytosis in angiogenesis. Nat Cell Biol, 15(3), 249–260, doi: 10.1038/ncb2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ng KL, Gibson EM, Hubbard R, Yang J, Caffo B, O’Brien RJ, et al. (2015). Fluoxetine Maintains a State of Heightened Responsiveness to Motor Training Early After Stroke in a Mouse Model. Stroke, 46(10), 2951–2960, doi: 10.1161/strokeaha.115.010471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nicholson S, Sniehotta FF, van Wijck F, Greig CA, Johnston M, McMurdo ME, et al. (2013). A systematic review of perceived barriers and motivators to physical activity after stroke. Int J Stroke, 8(5), 357–364, doi: 10.1111/j.1747-4949.2012.00880.x. [DOI] [PubMed] [Google Scholar]
  48. Orre M, & Rogers PA (1999). VEGF, VEGFR-1, VEGFR-2, microvessel density and endothelial cell proliferation in tumours of the ovary. Int J Cancer, 84(2), 101–108, doi:. [DOI] [PubMed] [Google Scholar]
  49. Pianta S, Lee JY, Tuazon JP, Castelli V, Mantohac LM, Tajiri N, et al. (2019). A Short Bout of Exercise Prior to Stroke Improves Functional Outcomes by Enhancing Angiogenesis. Neuromolecular Med, 21(4), 517–528, doi: 10.1007/s12017-019-08533-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Qin K, O’Donnell M, & Zhao RY (2006). Doppel: more rival than double to prion. Neuroscience, 141(1), 1–8, doi: 10.1016/j.neuroscience.2006.04.057. [DOI] [PubMed] [Google Scholar]
  51. Quinn TJ, Paolucci S, Sunnerhagen KS, Sivenius J, Walker MF, Toni D, et al. (2009). Evidence-based stroke r-ehabilitation: an expanded guidance document from the european stroke organisation (ESO) guidelines for management of ischaemic stroke and transient ischaemic attack 2008. J Rehabil Med, 41(2), 99–111, doi: 10.2340/16501977-0301. [DOI] [PubMed] [Google Scholar]
  52. Rahimian R, Cordeau P Jr., & Kriz J (2019). Brain Response to Injuries: When Microglia Go Sexist. Neuroscience, 405, 14–23, doi: 10.1016/j.neuroscience.2018.02.048. [DOI] [PubMed] [Google Scholar]
  53. Raica M, & Cimpean AM (2010). Platelet-Derived Growth Factor (PDGF)/PDGF Receptors (PDGFR) Axis as Target for Antitumor and Antiangiogenic Therapy. Pharmaceuticals (Basel), 3(3), 572–599, doi: 10.3390/ph3030572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rashid M, Wangler NJ, Yang L, Shah K, Arumugam TV, Abbruscato TJ, et al. (2014). Functional up-regulation of endopeptidase neurolysin during post-acute and early recovery phases of experimental stroke in mouse brain. J Neurochem, 129(1), 179–189, doi: 10.1111/jnc.12513. [DOI] [PubMed] [Google Scholar]
  55. Reeson P, Tennant KA, Gerrow K, Wang J, Weiser Novak S, Thompson K, et al. (2015). Delayed inhibition of VEGF signaling after stroke attenuates blood-brain barrier breakdown and improves functional recovery in a comorbidity-dependent manner. J Neurosci, 35(13), 5128–5143, doi: 10.1523/JNEUROSCI.2810-14.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Ruan L, Wang B, ZhuGe Q, & Jin K (2015). Coupling of neurogenesis and angiogenesis after ischemic stroke. Brain Res, 1623, 166–173, doi: 10.1016/j.brainres.2015.02.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sabbah N, Tamari T, Elimelech R, Doppelt O, Rudich U, & Zigdon-Giladi H (2019). Predicting Angiogenesis by Endothelial Progenitor Cells Relying on In-Vitro Function Assays and VEGFR-2 Expression Levels. Biomolecules, 9(11), doi: 10.3390/biom9110717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Shen J, Ishii Y, Xu G, Dang TC, Hamashima T, Matsushima T, et al. (2012). PDGFR-β as a positive regulator of tissue repair in a mouse model of focal cerebral ischemia. J Cereb Blood Flow Metab, 32(2), 353–367, doi: 10.1038/jcbfm.2011.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Shibuya M (2011). Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies. Genes Cancer, 2(12), 1097–1105, doi: 10.1177/1947601911423031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Shibuya M, & Claesson-Welsh L (2006). Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis. Exp Cell Res, 312(5), 549–560, doi: 10.1016/j.yexcr.2005.11.012. [DOI] [PubMed] [Google Scholar]
  61. Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, et al. (2003). VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest, 111(12), 1843–1851, doi: 10.1172/JCI17977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Syeara N, Alamri FF, Jayaraman S, Lee P, Karamyan ST, Arumugam TV, et al. (2020). Motor deficit in the mouse ferric chloride-induced distal middle cerebral artery occlusion model of stroke. Behavioural brain research, 380, 112418, doi: 10.1016/j.bbr.2019.112418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tang Y, Li M-Y, Zhang X, Jin X, Liu J, & Wei P-H (2019). Delayed exposure to environmental enrichment improves functional outcome after stroke. Journal of Pharmacological Sciences, 140(2), 137–143, doi: 10.1016/j.jphs.2019.05.002. [DOI] [PubMed] [Google Scholar]
  64. Vijayan M, Alamri FF, Al Shoyaib A, Karamyan VT, & Reddy PH (2019). Novel miRNA PC-5P-12969 in Ischemic Stroke. Mol Neurobiol, 56(10), 6976–6985, doi: 10.1007/s12035-019-1562-x. [DOI] [PubMed] [Google Scholar]
  65. Virani SS, Alonso A, Benjamin EJ, Bittencourt MS, Callaway CW, Carson AP, et al. (2020). Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association. Circulation, 141(9), e139–e596, doi: 10.1161/cir.0000000000000757. [DOI] [PubMed] [Google Scholar]
  66. Wang X, Zhang M, Feng R, Li WB, Ren SQ, Zhang J, et al. (2014). Physical exercise training and neurovascular unit in ischemic stroke. Neuroscience, 271, 99–107, doi: 10.1016/j.neuroscience.2014.04.030. [DOI] [PubMed] [Google Scholar]
  67. Wang Y, Kilic E, Kilic U, Weber B, Bassetti CL, Marti HH, et al. (2005). VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena. Brain : a journal of neurology, 128(Pt 1), 52–63, doi: 10.1093/brain/awh325. [DOI] [PubMed] [Google Scholar]
  68. Wangler NJ, Santos KL, Schadock I, Hagen FK, Escher E, Bader M, et al. (2012). Identification of Membrane-bound Variant of Metalloendopeptidase Neurolysin (EC 3.4.24.16) as the Non-angiotensin Type 1 (Non-AT1), Non-AT2 Angiotensin Binding Site. J Biol Chem, 287(1), 114–122, doi:M111.273052 [pii] 10.1074/jbc.M111.273052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. West T, & Bernhardt J (2012). Physical activity in hospitalised stroke patients. Stroke Res Treat, 2012, 813765, doi: 10.1155/2012/813765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Winstein CJ, Stein J, Arena R, Bates B, Cherney LR, Cramer SC, et al. (2016). Guidelines for Adult Stroke Rehabilitation and Recovery: A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke, 47(6), e98–e169, doi: 10.1161/STR.0000000000000098. [DOI] [PubMed] [Google Scholar]
  71. Xiong Y, Mahmood A, & Chopp M (2010). Angiogenesis, neurogenesis and brain recovery of function following injury. Curr Opin Investig Drugs, 11(3), 298–308. [PMC free article] [PubMed] [Google Scholar]
  72. Yang YR, Wang RY, & Wang PS (2003). Early and late treadmill training after focal brain ischemia in rats. Neuroscience letters, 339(2), 91–94, doi: 10.1016/s0304-3940(03)00010-7. [DOI] [PubMed] [Google Scholar]
  73. Zan L, Zhang X, Xi Y, Wu H, Song Y, Teng G, et al. (2014). Src regulates angiogenic factors and vascular permeability after focal cerebral ischemia-reperfusion. Neuroscience, 262, 118–128, doi: 10.1016/j.neuroscience.2013.12.060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Zhang P, Yu H, Zhou N, Zhang J, Wu Y, Zhang Y, et al. (2013). Early exercise improves cerebral blood flow through increased angiogenesis in experimental stroke rat model. J Neuroeng Rehabil, 10, 43, doi: 10.1186/1743-0003-10-43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Zhang ZG, Zhang L, Jiang Q, Zhang R, Davies K, Powers C, et al. (2000). VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest, 106(7), 829–838, doi: 10.1172/JCI9369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Zhou LY, Wright TE, & Clarkson AN (2016). Prefrontal cortex stroke induces delayed impairment in spatial memory. [Research Support, Non-U.S. Gov’t]. Behavioural brain research, 296, 373–378, doi: 10.1016/j.bbr.2015.08.022. [DOI] [PubMed] [Google Scholar]

RESOURCES