Hematopoietic Endothelial Progenitor cells enhance motor function and cortical motor map integrity following cerebral ischemia

Abstract

Hematopoietic stem cells (HSC) are recruited to ischemic areas in the brain and contribute to improved functional outcome in animals. However, little is known regarding the mechanisms of improvement following HSC administration post cerebral ischemia. To better understand how HSC effect post-stroke improvement, we examined the effect of HSC in ameliorating motor impairment and cortical dysfunction following cerebral ischemia.

Methods:

Baseline motor performance of male adult rats was established on validated motor tests. Animals were assigned to one of three experimental cohorts: control, stroke, stroke + HSC. One, three and five weeks following a unilateral stroke all animals were tested on motor skills after which intracortical microstimulation was used to derive maps of forelimb movement representations within the motor cortex ipsilateral to the ischemic injury.

Results:

Stroke + HSC animals significantly outperformed stroke animals on single pellet reaching at weeks 3 and 5 (28±3% and 33±3% versus 11±4% and 17±3%, respectively, p < 0.05 at both time points). Control animals scored 44±1% and 47±1%, respectively. Sunflower seed opening task was significantly improved in the stroke + HSC cohort versus the stroke cohort at week five-post stroke (79±4 and 48±5, respectively, p < 0.05). Furthermore, Stroke + HSC animals had significantly larger forelimb motor maps than animals in the stroke cohort. Overall infarct size did not significantly differ between the two stroked cohorts.

Conclusion:

These data suggest that post stroke treatment of HSC enhances the functional integrity of residual cortical tissue, which in turn supports improved behavioral outcome, despite no observed reduction in infarct size.

Keywords

Stroke stem cells motor map behavior

Abbreviations

HSC

Hematopoietic stem cells

TPA

greaterthan Tissue Plasminogen Activator

BMSC

greaterthan bone marrow derived stem cell

PBS

greaterthan Phosphate Buffered Saline

ET-1

greaterthan Endothelin-1

MCAO

greaterthan Middle Cerebral Artery Occlusion

MCA

greaterthan Middle Cerebral Artery

greaterthan Intravenous

ICMS

greaterthan Intracortical Microstimulation

CFA

greaterthan Caudal forelimb area

RFA

greaterthan Rostral Forelimb Area

MRI

greaterthan Magnetic Resonance Imaging

1 Introduction

Stroke is the leading cause of permanent disability in industrialized nations (Donkor, 2018; Lloyd-Jones et al., 2010). Thrombolytic and thrombectomy therapies have revolutionized stroke outcomes, and yet these therapies only aid a small minority of stroke patients (Gopurappilly et al., 2011; Goyal et al., 2015; Jauch et al., 2013; Jovin et al., 2015; Shafie & Yu, 2021). Alternative therapies using stem cells are being examined to extend potential therapy benefits in the subacute and chronic phase of recovery (Kawabori et al., 2020). The benefits of stem cell therapy may include cell replacement, but recent studies suggest that neuromodulatory effects of stem cells, such as secretion of neurotrophic factors, may attenuate inflammation and facilitate adaptive reorganization of neural connectivity (J. Chen & Chopp, 2018; Kawabori et al., 2020). Post ischemic intravascular administration of exogenous bone marrow derived stem cells (BMSC) ameliorates ischemic stroke in rodents, (Schwarting et al., 2008) suggesting a potentially critical role for the BMSC in limiting stroke injury and/or facilitating recovery (Taguchi et al., 2009; Yip et al., 2008). Methods of BMSC delivery evaluated have included direct injection into the ischemic core (J. Chen et al., 2001), delivery through lumbar puncture (Lim et al., 2011), and intravenous administration. Intravenous administration of BMSC has been shown to improve neurological function (Li et al., 2002; Lu et al., 2001). BMSC’s have the potential to enhance angiogenesis (J. Chen et al., 2001) and neurogenesis (Zhao et al., 2002) and contribute to neuro-functional recovery (Tsai et al., 2014). We sought to evaluate a similarly promising stem cell population, bone marrow derived Hematopoietic Stem Cells (HSC). HSC’s are an attractive candidate for cerebrovascular regeneration since they can proliferate and differentiate into mature endothelial cells (Asahara et al., 1997; Hristov et al., 2003). We sought to evaluate the potential benefit of systemically administered HSC on improving motor outcome following stroke and to determine whether this effect correlated with altered cortical motor representations using intracortical microstimulation techniques (ICMS) in a rat model of stroke. The capacity of the cortical areas to functionally reorganize after injury (cortical plasticity) has been correlated with the extent of motor recovery after stroke as seen with functional magnetic resonance imaging (fMRI) (Ward et al., 2003) and transcranial magnetic stimulation (TMS) (Liepert et al., 2000). Rat models of stroke using ICMS are well established and have shown a positive correlation between recovery of motor function and increases in motor map size (Kleim et al., 2004; Nishibe et al., 2015; Okabe et al., 2016).

2 Methods

A total of 50 male nine-week-old Long Evans hooded rats (300-350 g) were obtained from Jackson laboratories (Bar Harbor, Maine). The animals were kept under specific pathogen free conditions according to protocols approved by the institutional animal care and usage committee (IACUC). All aspects of this study were approved by IACUC guidelines for the use and care of experimental animals. Post stroke, animals were placed on a restricted food regimen where they received 20 g of food per day. Their body weights were monitored and maintained at 90% for the duration of the study; water was administered ad libitum.

2.1 Cohorts

Animals were assigned to one of three experimental cohorts. Animals in the control group (n = 8) received no stroke or HSC infusion but did receive a Phosphate Buffered Saline (PBS) injection into the tail vein; stroke cohort (n = 12) received infusion of Endothelin-1 (ET-1) onto the middle cerebral artery contralateral to the preferred/dominant paw as well as a PBS injection into the tail vein immediately post stroke. Animals in the stroke + Hematopoietic Stem Cell (HSC) cohort (n = 14) received the same infusion of ET-1 but also received ten million Lin-/CD90 + HSC injected into the tail vein immediately after ET-1. HSC were isolated from the remaining animals (n = 16) as described (Jun et al., 2012; Zubcevic et al., 2014). The number of HSC to be injected was determined empirically. It has been established that HSC have no effect in the absence of an injury and are removed from the circulation, (Massberg et al., 2007; Masterson et al., 2019; Wang et al., 2023) therefore, a separate group of control animals+HSC was not included in the cohorts.

2.2 Motor training

After a 7-day acclimation period, rats were assessed for baseline activity on the cylinder paw placement, skilled reaching, and sunflower seed test (Gonzalez & Kolb, 2003; Kleim et al., 2007). Baseline and post-stroke behavior was video recorded for offline assessment. Following the ET-1 induced MCAO stroke no additional rehabilitative training was given. The rats were assessed for natural or spontaneous recovery.

2.2.1 Cylinder paw placement

Animals were placed inside a specially designed cylinder (Fig. 1A) where they naturally use the walls for upright support and vertical exploration. Non-injured rats use both forelimbs for support and exploration. Following a unilateral cortical injury, rats will rely on their forepaw ipsilateral to the ischemic cortical hemisphere while in the cylinder. The number of times an animal uses the forelimb contralateral to the injury (affected limb) or ipsilateral to the injury (less affected limb), alone or simultaneously were recorded for each animal. The results were confirmed by slow motion playback.

Fig. 1

Motor testing and performance. Animals were assessed at baseline and post injury weeks 1, 3 & 5 for cylinder paw placement, skilled reaching, and sunflower seed opening (A) Cylinder paw placement did not show any improvements with Hematopoietic Stem Cells (HSC). (B) Skilled reaching significantly improved at weeks one, three and five with HSC and (C) Sunflower seed opening time (sec.) significantly reduced at weeks three and five with HSC. Data are presented as mean±S.E.M; ^*p < 0.05 ^**p < 0.005 (stroke group compared to control group).

2.2.2 Skilled single pellet reaching

Animals were pre trained in cages with food pellet trays mounted to the front of the cages. The animals were encouraged to reach outside the cage and retrieve pellets from the food tray to eat (Fig. 1B). Ten successful reaches were needed to conclude the pre-training period for each animal. The animal was then transferred to a clear Plexiglas box, which had a 1 cm slot located at the front of the box. The animal was trained daily to reach thru the slot and retrieve the food pellet from a shelf placed outside the slot. Animals were allowed to use either limb and were videorecorded to assess performance and their preferred limb for each animal noted. Once hand preference was established, a barrier was placed in the Plexiglas box to ensure that the rat only used his preferred forelimb throughout the experiment. A successful reach consisted of grasping the food pellet, bringing it inside the box and to the mouth and finally eating the pellet. The percentage of successful reaches was calculated as follows: [# of successful retrievals/total # of reaches]x100. Each animal was trained for 2 weeks to establish a baseline (average accuracy across 3 final days of training) measure of motor performance. A baseline of at least 40% was required to be included into the final experimental cohort.

2.2.3 Sunflower seed task

Animals were placed in a clear plastic arena (Fig. 1C) with an angled mirror under the box to allow ventral videorecording of animal activity. This is a species-typical behavior that requires fine manipulation of the sunflower seed with both distal forelimbs (digits and wrist) to break the shell and consume the contents (Whishaw et al., 1998). Each animal received 5 seeds in one corner of the arena and the time (seconds) it took each animal to open and eat the 5 seeds was recorded. A timer was started as soon as the animal touched the first seed, the timer was stopped if the animal stopped or was distracted.

2.3 Endothelial-1 induced middle cerebral artery occlusion

The ET-1 induced MCAO procedure used was as described (Mecca et al., 2009). Briefly, anesthetized animals were placed in a stereotaxic frame and maintained under anesthesia with an oxygen/isoflurane (2%) mixture for the duration of the surgery. The skull was exposed, and a small hole drilled using stereotaxic co-ordinates of 1.6 mm anterior and 5.2 mm lateral to bregma. A 26-gauge needle was attached to a Hamilton syringe and lowered 8.7 mm ventral to the cortical surface and 3 ul of 80uM ET-1 delivered to the MCA at 1ul/min. The needle was withdrawn 3 minutes after the injection and the wound closed using nylon sutures and the animal allowed to recover.

2.4 Hematopoietic stem cells

A separate group of 16 animals were used as bone marrow donors. The bone marrow was flushed and enriched for Lineage negative, CD90 + endothelial progenitor cells per manufacturers protocol (Stem Cell Technologies, Vancouver, CA) (Zubcevic et al., 2014). Briefly, intact hind leg femur and tibia were removed and collected in PBS + 2% FBS + 1 mM EDTA buffer. Following removal of any residual muscle/fat, the epiphysis was removed from both ends and bone marrow cells flushed out of the diaphysis with PBS + 2% FBS + 1 mM EDTA and spun down at 1200 RPM for 15 minutes. Residual red blood cells were removed by adding ammonium chloride (Stem Cell Technologies, inc. Vancouver, BC) for 10 minutes on ice, followed by 2 washes with buffer. Enrichment for CD90 + /CD4.5.8^- HSC was performed using Stem Cell Technologies’ negative and positive selection kits. A negative selection was first performed to exclude all CD 4.5.8⁺ cells and any remaining RBC from the cell suspensions. A positive selection was then performed by adding a mouse anti-CD90 antibody (1 : 10) for 15 minutes, followed by the Easy Sep Immunomagnetic selection (repeated three times) (Jun et al., 2012; Zubcevic et al., 2014). The resulting CD90 + cells were washed with PBS and 10 million cells/animal injected IV at reperfusion.

2.5 Electrophysiological mapping

Microstimulation techniques were used to derive high-resolution maps within the motor cortex contralateral to the trained/impaired paw as described (Kleim et al., 1998). Briefly, animals were anesthetized with a mixture of ketamine hydrochloride (70 mg/kg i.p.) and xylazine (5 mg/kg i.p.). A craniotomy was performed over the motor cortex in the injured hemisphere, contralateral to the rat’s preferred forelimb and the cisterna magna punctured to reduce edema before retracting the dura. Warm silicone oil was placed on the exposed cortex and a hydraulic Microdrive controlled microelectrode used to penetrate to cortical layer V (depth of approximately 1550μm). The motor cortex was stimulated at 350 Hz with thirteen 200μs cathodal pulses. The animals were maintained in the prone position and their limb supported in a consistent position. The minimal threshold required to elicit movement was recorded at each penetration site. If no movement was detected at a site at ≤60μA, it was recorded as a non-responsive area. Forelimb movements were classified as distal forelimb (wrist/digit) or proximal (elbow/shoulder). Representational maps were generated from a pattern of electrode penetrations. The rat forelimb motor cortex has a caudal and rostral division. The caudal forelimb area (CFA) is considered the primary motor cortex and the rostral forelimb area (RFA) is considered premotor cortex (Fig. 3A, B) (Neafsey et al., 1986). The proportion of the distal and proximal movement categories that occupied the CFA and RFA were calculated with an image analysis program (CANVAS v. 3.5) (Fig. 3C-E) and the mean stimulation threshold for each movement category calculated. Motor maps are presented for CFA and RFA in mm². The CFA and RFA were then combined to generate the total forelimb area.

2.6 Infarct volume

Animals were perfused with 0.1M Phosphate buffered saline, followed by 4% paraformaldehyde. Brain tissue was immediately removed and post fixed in 4% paraformaldehyde for 24 hours, followed by 30% sucrose at 4°C for 5 days. Brain tissue was then embedded in Tissue Tek and cryosectioned at 40μm thickness. Sections were mounted onto Poly-L lysine coated slides and stained with 0.1% Cresyl Violet dissolved in water. The sections were then dehydrated in 100% ethanol, cleared in xylene and mounted with DPX and a coverslip (Pilati et al., 2008). Mounted slides were scanned using a flatbed scanner and analyzed for infarct volumes (% intact hemisphere) using Image J, as described (Boychuk et al., 2016).

2.7 Analysis

Surgical procedures and all subsequent analysis were completed with total blinding to experimental cohorts across all experiments. All data are presented as mean±S.E.M and all pairwise comparisons were made using a one- or two-way ANOVA with a Tukey’s post hoc Multiple Comparison test (GraphPad Software, Inc. La Jolla, CA, USA). A Tukey’s corrected p value less than 0.05 was considered to be significant and is indicated on subsequent graphs with an asterisk.

3 Results

3.1 Behavior

Cylinder Paw Placement. Injured animals showed a tendency toward increased use of uninjured forelimb for paw placement while exploring along the cylinder wall, but this was not significant for either the stroke or stroke + HSC groups (p > 0.5; Fig. 1A). Skilled Single Pellet Reaching. Control animals exhibited successful reaching performance throughout the five weeks of behavioral assessments corresponding to post-injury week1, week3 and week5. The stroke cohort experienced significantly reduced reaching success at each of these timepoints (p < 0.05). Administration of HSC significantly improved reaching performance in the stroke + HSC group at post-injury weeks 3 and 5 relative to the stroke group (p < 0.05; Fig. 1B). Sunflower Seed Task. Control animals remained consistent throughout the assessment period compared to baseline (p > 0.05). Injury significantly increased the time required to open the sunflower seeds compared to pre-injury baseline for the stroke (p < 0.05) and stroke + HSC (p < 0.05) groups. Administration of HSC significantly improved function for the sunflower seed-opening task at post-injury week 5 compared to the stroke cohort (p < 0.05; Fig. 1C).

3.2 ICMS

ICMS demonstrated a decreased motor area (black dots, Fig. 2C) in both the stroke animals and the stroke + HSC animals. However, treatment with HSC showed a significant increase in spared forelimb motor map area compared to the non-treated rats (Fig. 2D). Rodents have two distinct frontal cortical regions: RFA and CFA (Neafsey et al., 1986) which evoke contralateral forelimb movement (Morandell & Huber, 2017). Even though the CFA extends over a larger region than the RFA, (Neafsey et al., 1986) both areas are essential for arm and forelimb movements and learning new motor skills (Deffeyes et al., 2015). Because the RFA and CFA ICMS motor maps have been well studied in rodents post stroke, (Barbay et al., 2013) we used the standard ICMS methods to derive motor maps to assess whether HSC play a role in rescuing these lesions. Figure 3A shows the extent of the sensory and motor cortex and Fig. 3B indicates the delineation of the RFA and the CFA. Figure 3C, 3D and 3E show representative motor maps from a pre-trained control animal, a pre-trained stroke animal and a pre-trained stroke + HSC animal, respectively. These motor maps were used to derive the extent of the RFA and the CFA as described above. The CFA in the stroke + HSC group was significantly increased compared to stroke animals (Fig. 4A: control = 5±0.1, stroke = 1±0.2 and stroke + HSC=3±0.2 mm²), and the RFA also significantly increased in the stroke + HSC animals compared to stroke (Fig. 4B: control = 1±0.05, stroke = 0.2±0.06 and stroke + HSC=0.5±0.04 mm²). The total forelimb area (Fig. 4C) also showed a significant increase in the stroke + HSC group (control = 5.3±0.1, stroke = 1.3±0.2 and stroke + HSC=3±0.2 mm²).

Fig. 2

Intracortical Microstimulation (ICMS) was used to derive motor maps of forelimb movement representations. Shown are representative images of ICMS evoked responses in the motor cortex relative to Bregma coordinates from (A) An untrained animal showing the extent of the motor cortex. (B) Trained control animal. (C) Trained Stroke animal. (D) Trained Stroke + Hematopoietic Stem Cells (HSC) animal.

Fig. 3

Derivation of Rostral Forelimb Area (RFA) and Caudal Forelimb Area (CFA). (A) An untrained animal showing the extent of the sensory and motor cortex. (B) Image indicating the delineation of the RFA and CFA with color decoder for the motor maps. Intracortical Microstimulation (ICMS) was used to derive motor maps of animals; shown are representative images from (C) A trained control animal., (D) A trained stroke animal and (E) A trained stroke + Hematopoietic Stem cell (HSC) animal. Gridlines (250 μm X 250μm) were superimposed over a photomicrograph of the cortex to guide microelectrode penetration for ICMS.

Fig. 4

Intracortical Microstimulation (ICMS) was used to derive the Rostral Forelimb Area (RFA), Caudal Forelimb Area (CFA) and total forelimb area. (A) Compared to non-injured control rats, CFA maps were significantly reduced in both the stroke groups but was significantly increased in the stroke + HSC group compared to the stroke group. (B) RFA was also significantly reduced in both the stroke groups, but significantly increased in the stroke + HSC group compared to control and stroke group. (C) Combined total forelimb area was significantly reduced in both stroke groups, but larger in the stroke + HSC group, compared to the stroke group. Data are presented as mean±S.E.M; ^*p < 0.05 ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001.

A histological Infarct lesion verification and volume analysis was performed on all the animals to determine the extent of the intact hemisphere (Fig. 5). Animals in the stroke cohort had significantly less spared subcortical tissue (70.7±4.2%) in comparison to uninjured controls (90±2.8%). Interestingly, however, the stroke + HSC cohort did not have a significantly higher spared subcortical tissue compared to stroke animals (67±3.6%).

Fig. 5

Infarct Volume. Cryosectioned brain tissue was stained with cresyl violet, and the percentage of intact hemisphere calculated. The percentage of intact hemisphere was significantly lower in the stroke and stroke + Hematopoietic Stem Cell (HSC) group compared to the control group. No significance difference was found between the stroke and stroke + HSC groups. Data are presented as mean±S.E.M; ^*p < 0.05 ^**p < 0.01.

4 Discussion

These data demonstrate that systemically administered HSC to rats immediately following stroke preserved more functional forelimb representation in the motor cortex compared to nontreated injured rats and enhanced behavioral outcome on tasks requiring skilled use of distal forelimb. However, there was no significant difference in infarct size between the two stroke cohorts. While somewhat counterintuitive, several studies have previously shown improved behavioral outcomes without a corresponding improvement in histology (Johansson, 1996; Johansson & Ohlsson, 1996; Yamamoto et al., 1991). It may be that the enhanced behavioral and physiological effects are due to increased functional integrity of residual tissues as seen within spared motor maps.

Timing of HSC administration after injury influences recruitment of these cells and phenotypic conversion at the site of injury. Yan et al. (Yan et al., 2007) showed that cells administered within 4 hours after lung injury engrafted as epithelial or vascular endothelial cells. However, if the HSC’s were administered at later time points following the injury then the cells contributed to fibrosis development (Epperly et al., 2003; Yan et al., 2007). We chose to inject the HSC’s immediately following reperfusion, although the practical application of this approach is limited. Further studies are necessary to evaluate whether more delayed administration would maintain the observed effects. Another factor contributing to recovery with HSC’s is the number of cells injected; studies have shown that HSC tend to get sequestered in the lungs or the spleen and may never make it to the target organ (Everaert et al., 2012; Fischer et al., 2009; Harting et al., 2009). To overcome potential sequestration, we chose to inject 10 million HSC to ensure that a subset of the HSC home to the brain.

Of the motor tasks evaluated, the Cylinder test, which evaluates rodent forelimb use for postural support, did not show any significant difference. Healthy animals typically use both limbs for upright support and post stroke, rodents have an asymmetric reliance on their ipsilateral (less affected) limb. This test is very sensitive in the early stages of motor damage; however, rodents return to symmetrical use of forelimbs with time depending on severity of stroke. In addition, the cylinder test is an evaluation of the gross motor movement for which the rats are not pre-trained, therefore, may not be as sensitive a test for moderate injury. Others have also reported no significant difference when using this test (T. Chen et al., 2014).

Rats receiving HSC, demonstrated significant improvement at weeks 3 and 5 in single pellet reaching performance for which they were trained. This motor test is highly sensitive to the lesion size (Kleim et al., 2007) and is widely used to assess motor impairments in rodents. The sunflower seed test is an index of fine motor function, and therefore may be more sensitive to stroke effects. Rodents are inherently adept at opening seed to obtain food and therefore this task is an effective measure of bilateral object manipulation (Whishaw et al., 1998) and motor impairments post stroke (Gonzalez & Kolb, 2003). Encouragingly, rats that received the HSC had a significantly improved time 5 weeks post stroke. However, no significant differences were found in the earlier time points. This may be secondary to the benefit of HSC resulting in their facilitation of recovery, rather than in any immediate reduction in the degree of ischemic injury.

Redistribution of movement representations within motor cortex occurs in response to various experimental manipulations requiring development of new motor skills or compensatory motor skills after injury and these have been well documented in the rodent motor cortex (Kleim et al., 2004). It has also been shown that motor map re-organization cannot be induced by simple repetitive training (Kleim et al., 1998). Figure 2A shows the extent of the sensory and motor cortex of a non-injured, untrained animal. The topography of this region within the CFA and RFA significantly changes after motor training (Fig. 2B). The motor cortex has significant changes post stroke + HSC compared to stroke alone (Fig. 2C and 2D). This suggests, although further confirmation is certainly needed, that the administration of the HSC induced motor map re-organization. The total forelimb area was significantly enhanced in the stroke + HSC group compared to the stroke animals. This suggests that the HSC can affect functional organization of the motor cortex post stroke, despite our inability to identify a significant change in observed stroke volume. Therefore, these data may suggest that the beneficial effects of post stroke HSC result, at least partially, from facilitating physiologic recovery, rather than being exclusively a neuroprotective effect during the initial ischemic and immediately post-ischemic effect.

Previous studies using similar behavioral and physiological measurements to assess a unilateral MCAo in female rats have found effects comparable to those reported in this study (Gharbawie, Gonzalez, & Whishaw, 2005; Gharbawie, Gonzalez, et al., 2005). There was substantial necrotic injury to lateral cortex but not to CFA and RFA in dorsal motor cortex. As in this study, even though the forelimb motor cortex was spared from necrotic damage, skilled volitional use of the contralateral forelimb was not spared and ICMS derived motor maps of distal forelimb were substantially diminished. Using the neuroanatomical anterograde tracer BDA (10k MW) to label corticofugal fibers from forelimb motor cortex and the neuroanatomical retrograde tracer True Blue to label descending pyramidal track fibers from motor cortex, Gharbawie et al (Gharbawie, Gonzalez, et al., 2005) found these cortical efferent fibers were also spared. Reduction in the size of forelimb motor maps in CFA and RFA was attributed to deafferentation from necrotic damage to the lateral cortex reducing input from face and secondary somatosensory areas. Major reductions in sensory input to the forelimb motor cortex may diminish motor maps by suppressing neural activity (diaschisis) and destabilizing synaptic efficacy associated with sensorimotor integration (Kleim et al., 2004; Monfils et al., 2005; Plautz et al., 2023). Rehabilitative therapies implemented for restoration of motor skills or acquisition of compensatory motor skills can enhance synaptic efficacy and have a stabilizing impact on map topography as skills are re-established (Kleim et al., 2002; Kleim et al., 2004; Nishibe et al., 2015). Use of the impaired limb as seen prior to rehabilitative training returns prior to the reappearance or expansion of movement representation maps in spared motor cortices (Eisner-Janowicz et al., 2008; Nishibe et al., 2015; Plautz et al., 2023). This suggests that during the early stages of motor recovery, other regenerative or restorative molecular processes are initiated (Carmichael, 2006; Kleim et al., 2004; Urban et al., 2012). The similarity in treatment effects between the Gharbawie studies (Gharbawie, Gonzalez, & Whishaw, 2005; Gharbawie, Gonzalez, et al., 2005) that implemented rehabilitative forelimb training and the present study that allowed spontaneous recovery may be related to the increase in these early molecular processes since both motor activity (Klintsova et al., 2004; Mang et al., 2013; Vaynman & Gomez-Pinilla, 2005) and stem cells treatments (J. Chen & Chopp, 2018; Kawabori et al., 2020) can increase neurotropic factors associated with improvements in motor function (Adkins et al., 2006). An adjunctive treatment with rehabilitative training and HSC should be examined for a synergistic effect in further studies. These future studies should also include sex as a biological factor. Even though similar behavioral and physiological consequences of MCAo have been shown for male and female Long Evans rats (Gharbawie, Gonzalez, & Whishaw, 2005; Gharbawie, Gonzalez, et al., 2005; Gonzalez et al., 2004), our previously published work has shown the impact and recruitment of exogenously implanted progenitors in female animals post stroke (Mocco et al., 2014). Certainly, these data are not conclusive, but rather, they provide a strong suggestion of effect, and will hopefully represent an early step towards a better understanding of how HSC contribute to post-stroke recovery.

Footnotes

Acknowledgments

The authors would like to thank Dr. Jeff Kleim, School of Biological & Health systems engineering, Arizona State University, Tempe AZ, for his contribution to this manuscript.

Funding

The authors have no funding to report.

Conflicts of interests/disclosures

The authors have no conflicts of interest to declare.

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