Activation of cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 promotes post-ischemic stroke functional recovery in aged mice

Fan Bu ,Jia-Wei Min ,Md Abdur Razzaque ,Ahmad El Hamamy ,Anthony Patrizz ,Li QiAkihiko UrayamaJun Li

Abstract Brain functional impairment after stroke is common;however,the molecular mechanisms of post-stroke recovery remain unclear.It is well-recognized that age is the most important independent predictor of poor outcomes after stroke as older patients show poorer functional outcomes following stroke.Mounting evidence suggests that axonal regeneration and angiogenesis,the major forms of brain plasticity responsible for post-stroke recovery,diminished with advanced age.Previous studies suggest that Ras-related C3 botulinum toxin substrate (Rac) 1 enhances stroke recovery as activation of Rac1 improved behavior recovery in a young mice stroke model.Here,we investigated the role of Rac1 signaling in long-term functional recovery and brain plasticity in an aged (male,18 to 22 months old C57BL/6J) brain after ischemic stroke.We found that as mice aged,Rac1 expression declined in the brain.Delayed overexpression of Rac1,using lentivirus encoding Rac1 injected day 1 after ischemic stroke,promoted cognitive (assessed using novel object recognition test) and sensorimotor (assessed using adhesive removal tests) recovery on days 14-28.This was accompanied by the increase of neurite and proliferative endothelial cells in the periinfarct zone assessed by immunostaining.In a reverse approach,pharmacological inhibition of Rac1 by intraperitoneal injection of Rac1 inhibitor NSC23766 for 14 successive days after ischemic stroke worsened the outcome with the reduction of neurite and proliferative endothelial cells.Furthermore,Rac1 inhibition reduced the activation of p21-activated kinase 1,the protein level of brain-derived neurotrophic factor,and increased the protein level of glial fibrillary acidic protein in the ischemic brain on day 28 after stroke.Our work provided insight into the mechanisms behind the diminished plasticity after cerebral ischemia in aged brains and identified Rac1 as a potential therapeutic target for improving functional recovery in the older adults after stroke.

Key Words: aging;angiogenesis;brain-derived neurotrophic factor (BDNF);cerebral ischemia;cognitive recovery;neurite;Pak1;Rac1;sensorimotor recovery;stroke

Introduction

Stroke is the primary cause of long-term disability among adults in the USA and worldwide,and the annual costs for stroke care are estimated at 70 billion dollars in the USA (Ovbiagele et al.,2013;Thayabaranathan et al.,2022).Mounting evidence suggests that brain plasticity and neuronal remapping are critical aspects of this recovery because it is essential for establishing new neural connections and neurovascular units to compensate for the strokeinduced loss of function (Lopez-Valdes et al.,2014;Liu et al.,2018).However,this regrowth and remodeling in the adult mammalian central nervous system (CNS) is limited after injury.

Ras-related C3 botulinum toxin substrate (Rac) 1 is a Rho-related small guanosine triphosphatase.Rac1 is ubiquitously expressed (Stankiewicz and Linseman,2014).Rac1 becomes inactivated when bound to guanosine diphosphate and activated when bound to guanosine triphosphate.The activation of Rac1 is catalyzed by guanine nucleotide exchange factors,which stimulate the release of guanosine diphosphate to allow guanosine triphosphate to bind (Stankiewicz and Linseman,2014).Upon activation,Rac1 promotes allosteric regulation of p21-activated kinase (Pak) in a manner that facilitates their phosphorylation and,therefore,their activation.Our previous work suggested that Rac1 enhanced stroke recovery as neuronal activation of Rac1 improved behavior recovery in a young mouse ischemic stroke model (Liu et al.,2018;Bu et al.,2019,2020).

Age is the most important independent predictor of poor outcomes after stroke,and older patients show both higher in-hospital mortality and poorer functional outcomes following stroke (Cafferty et al.,2008).As our population ages,the incidence of stroke is expected to rise.However,the vast majority of experimental stroke studies are performed almost exclusively in young animals,as mortality rates,expenses,and technical difficulties are much greater in aged mouse models.Older animals invariably have significantly higher mortality rates and worse functional recovery after stroke (Ding et al.,2011;Furlan et al.,2021).Diminished recovery from stroke in aged animals implies that repair processes are affected by advanced age.Indeed,after stroke,gene expression profiles that promote axonal sprouting and the formation of new neuronal connections in adjacent brain regions differ significantly between young and aged brains (Li et al.,2010).Moreover,it was demonstrated that reactive gliosis and the formation of a glial scar,the major inhibitory factors for regeneration,in aged subjects after stroke are higher than young subjects (Manwani et al.,2011).Therefore,the inhibitory signal is stronger while the intrinsic growth potential is weaker in aged brains for axonal regeneration after stroke.Increased angiogenesis was reported in the ischemic border zone of human brain autopsy sections.However,this was decreased in elderly patients (Szpak et al.,1999).Experimental studies revealed more diminished expression of cluster of differentiation (CD) 31 after stroke in aged braind compare to young brains after stroke (Tang et al.,2014).It can be reasonably speculated that age-dependent reduction in ischemiainduced angiogenesis might contribute to a worse functional outcome (Jin et al.,2004;Pradillo et al.,2021).New paradigms for conducting translational research,including preclinical testing of novel therapies that examine aging as a major factor,are urgently needed.We and others have shown that Rac1 signaling declines in aged mouse brains.Additionally,phosphorylation of extracellular signal-regulated kinase (Erk) 1/2,another downstream target of Rac1 for neuronal regenerative potential,is also reduced in the cerebral cortex in aged rats (Zhen et al.,1999).In this study,we investigated the role of Rac1 signaling in long-term functional recovery and brain plasticity,including axonal regeneration and angiogenesis in the aged brain after stroke.

Methods

Animals and study design

Our animal protocols were approved by the Center for Laboratory Animal Medicine and Care at University of Texas Health Science Center in Houston (protocol and approval No.AWC-18-0123,approval date April 6,2018),and experiments were performed in accordance with the National Institutes of Health guidelines for the Care and Use of Laboratory Animals.All experiments were designed and reported according to the Animal Research: Reporting ofIn VivoExperiments (ARRIVE) guidelines (Percie du Sert et al.,2020).We kept 5 mice per cage in a 12-hour light/dark cycle,and they were fed with food and waterad libitumat 18-22°C and 50-60% humidity (Liu et al.,2014,2016).Mice (C57BL/6J,specific-pathogen-free grade) used were young (10-12 weeks) and aged (18-22 months) males.A total of 58 mice purchased from The Jackson Laboratories (Bar Harbor,ME,USA) were used in this study.Fourteen mice were removed from the experiments because of death,no behavior impairment 24 hours after reperfusion (Liu et al.,2014,2016),or failure to complete the pre-stroke functional assessment (Bu et al.,2020).A detailed description of the experimental design is presented inFigure 1.

Figure 1｜Schematic representation of the study design.

Middle cerebral artery occlusion

Transient cerebral ischemia (60 minutes of middle cerebral artery occlusion [MCAO]) was induced in aged wild-type mice followed by reperfusion as described previously (Bu et al.,2020).Briefly,inhalation (route of administration) anesthesia was conducted with air containing isoflurane (4% induction,around 1.5% during operation,Henry Schein,Dublin,OH,USA,Cat# 029405;inhalation solution 99.9% bottle 250 mL/bottle).We also used a heating pad (TC-1000 System,CWE Incorporated,Ardmore,PA,USA) to keep mouse body temperatures at the physiological range of 37 ± 0.5°C.An intraluminal filament (Doccol Corporation,Sharon,MA,USA,Cat# 602323PK5Re) was inserted in the internal carotid artery from the external carotid artery to reach the origin of the MCA of the right side,thus blocking blood flow into the MCA territory.The filament was withdrawn to allow reperfusion after 60 minutes of occlusion.Same surgery without inserting filament was performed in the sham group.We placed softened chow in the cage and administered saline intraperitoneally for 7 days to keep mice hydrated.Therefore only males are used in the present study as we did not want to intermix males and females in the same cohort to avoid the potential effect of sex and subsequent difficulties in data interpretation.

Rac1 intervention in vivo

For delayed inhibition of Rac1,intraperitoneal (i.p.) injection of Rac1 inhibitor NSC23766 (Sigma-Aldrich,St.Louis,MO,USA,Cat# SML0952) was performed once daily from day 1 to day 14 (4.0 mg/kg in 200 µL saline per day) after stroke.The same volume of saline was applied in the vehicle control group.The dosing regimen was used previously in our lab (Liu et al.,2018).

In experiments that require delayed overexpression of Rac1,we stereotaxically injected lentivirus (LV) that carries GFP-Rac1 with CMV promoter (LV-Rac1,accession number: NM_009007,ABM,Richmond,Canada,Cat# 38393064) into mice cortex and striatum 1 day after the onset of stroke (Bu et al.,2020).Briefly,a four-point injection was carried out at the following coordinates: 0.5 mm anterior to the bregma,2.0 or 3.0 mm lateral (right) to the sagittal suture,and 1.0 or 2.8 mm from the surface of the skull (Paxinos and Franklin,2013).We previously injected the same dose of LV into the young mouse brain and found that 7 days is sufficient to see Rac1 overexpression (Liu et al.,2018).That was done using a four-point approach with each point receiving 1 µL of LV at 1×109transducing units/mL.Control mice received vectors that carry GFP only (accession number: NM_009007,ABM,Richmond,Canada,Cat# 38393064).

Behavior measurements

Stroke survivors continue to suffer cognitive and sensorimotor impairment,and assessing these key behavior domains is important for animal models of stroke (Smania et al.,2008).We thus used a novel object recognition test to assess cognitive memory function in mice at 28 days after stroke (Bu et al.,2021).We used an adhesive removal test to examine sensorimotor impairment on days 7,14,21,and 28 after stroke.

Novel object recognition test

We conducted this assessment as previously described in (Antunes and Biala,2012).Mice were placed in a cage with two identical objects for 5 minutes (trial 1).After that,mice were placed back into their home feeding cage for a 5-minute break.Furthermore,one of the objects in the testing cage was then replaced with a novel one.Mice were placed back into the same testing cage for an additional 5 minutes (trial 2).We recorded the time spent in seconds on each object.Then we calculated a discrimination index (DI) with the equation: DI=(TNtrial2/TFtrial2)/(TFtrial1/TFtrial1).TN=time spent with the novel object.TF=time spent with familiar objects.We performed pre-tests to eliminate mice if they did not show exploration activity.It is common to see stroke-induced impairment in animal locomotion in this model due to ischemic injury in cerebral motor and sensory areas.To minimize the influence of locomotion on the cognitive measurement,we used a DI but not absolute value to calculate our data,in which the time spent on the novel object is divided by that on the familiar object.This way,any potential changes in locomotion are controlled.

Adhesive removal test

We used one piece (25 mm2) of adhesive-backed paper dots as unilateral tactile stimuli.It was placed on the distal-radial region on the wrist of the contralateral forelimb of stroke.We recorded the time spent removing each paper dot for three trials and pre-trained mice for 3 days.Pre-tests were done to eliminate mice if they could not remove the dots within 10 seconds (Bouet et al.,2009;Ruan and Yao,2020).

Immunohistochemistry

Animals were perfused with 4% paraformaldehyde 28 days after stroke after being anesthetized with 5% isoflurane as described above.The brain was then removed and post-fixed in 4% PFA overnight followed by 30% sucrose solution for 48 hours.After that,the brain was sliced into 30 µm thick coronal sections using a cryostat (Thermo Fisher Scientific,Waltham,MA,USA,Cat# CM1950).The slices were incubated using 0.1% Triton X-100 for 15 minutes and were blocked for 60 minutes using blocking buffer 1% BSA at room temperature (RT).We used the following primary antibodies: rabbit anti-Rac1 (1:100,Thermo Fisher Scientific,Cat# PA1-091,RRID: AB_2539856),mouse anti-neurofilament-L (NFL,1:250,Thermo Fisher Scientific,Cat# MA1-2010,RRID: AB_347003),rat anti-bromodeoxyuridine (BrdU 1:500,NOVUS,Centennial,CO,USA,Cat# NB500-169,RRID: AB_10002608) and goat anti-CD105 (1:100,R&D Systems,Minneapolis,MN,USA,Cat# AF1320,RRID: AB_354735) 30 minutes at RT and incubated with the fluorescently-labeled secondary antibodies: anti-rabbit 594 (1:500,Thermo Fisher Scientific,Cat# A11072,RRID: AB_2534116),anti-mouse 594 (1:500,Thermo Fisher Scientific,Cat# A11005,RRID: AB_2534073),anti-rat 594 (1:500,Abcam,Cambridge,UK,Cat# ab150160,RRID: AB_2756445) and anti-goat 488 (1:500,Abcam,Cat# ab150129,RRID: AB_2687506) for 60 minutes at RT.Cell nuclei were stained using 4′,6-diamidino-2-phenylindole (VectorLabs,Newark,CA,USA,Cat# H-1500).I.p.injection of BrdU (Sigma,Cat# B5002) at 50 mg/kg was performed on days 3,5,7,9,11,and 13 after MCAO surgery for cell proliferation assay.

We used a Zeiss Axiovert 200M microscope (Carl Zeiss,Göttingen,Germany) with an X-Cite 120Q fluorescence illumination system (Lumen Dynamics Group Inc.,Mississauga,Canada) for brain slices imaging and analyzed data using ImageJ (v1.52o,National Institutes of Health,Bethesda,MD,USA).Three images were randomly acquired in a blinded manner in the penumbra regions of the ischemic hemisphere using consistent parameters.40× objective was used.We used the integrated optical density for NFL+and CD105+semiquantification and normalized the data by dividing each value by the mean integrated optical density of the control groups.For BrdU+/CD105+and Rac1+,we counted the positive cells per mm2(Bu et al.,2020).We considered the tissue area within 1.5 mm around the infarct core as the peri-infarct area in mice (Agulla et al.,2013).

Tissue loss analysis

We assessed brain damage/tissue loss 28 days after MCAO by cresyl violet (CV,Cambridge,UK,Cat# ab246816) staining (Liu et al.,2018).Coronal sections at 30 µm-thick (total 8 per brain) were used for CV staining in accordance with previously published procedures (Liu et al.,2018).Brain tissue loss was determined using the following equation: % brain tissue lost=[(contralateral hemisphere area -contralateral ventricular area) -(ipsilateral hemisphere area -ipsilateral ventricular area)]/(contralateral hemisphere area -contralateral ventricular area)×100 (Bu et al.,2020).

Western blot analysis

We performed Western blots to determine the protein levels of interest quantitatively 28 days after MCAO using stroke hemispheres.Equal amounts of protein (30 µg) were loaded onto 4-15% precast protein gels (Bio-Rad,Hercules,CA,USA,Cat# 4561084) for electrophoresis.After running gels,they were transferred to a polyvinylidene fluoride membrane (Bio-Rad,Cat# 1620177).Blocking was done using skim milk for 1 hour at RT.We then added primary antibodies to the membranes for incubation.The primary antibodies used are anti-Rac1 (1:500,Thermo Fisher Scientific,Cat# PA1-091,RRID: AB_2539856),anti-brain-derived neurotrophic factor (BDNF,1:1500,Abcam,Cat# ab108319,RRID: AB_10862052),anti-phospho T212 Pak1 (P-Pak1,1:500,Thermo Fisher Scientific,Cat# PA5-104983,RRID: AB_2816456),anti-Pak1 (1:1000,Abcam,Cat# ab131522,RRID: AB_11156726),anti-glial fibrillary acidic protein (GFAP,1:1000,CST,Danvers,MA,USA,Cat# 12389,RRID: AB_2631098) and β-actin (1:1000,CST,Cat# 4970,RRID: AB_2223172) for overnight at 4°C.After primary antibody incubation,the membranes were incubated with anti-rabbit horseradish peroxidase-linked secondary antibody (1:1000,CST,Cat# 7074,RRID: AB_2099233) for 1 hour at RT.The loading control used was β-actin (Bu et al.,2020).The signal was visualized by enhanced chemiluminescence Western Blotting Substrate (Thermo Fisher Scientific,Cat# 32106).Optical density was captured by Bio-Rad imaging system (ChemlDoc,Hercules,CA,USA) and analyzed by ImageJ software.

Statistical analyses

No statistical methods were used to predetermine sample sizes;however,our sample sizes are similar to those reported in a previous publication (Bu et al.,2021).Our data were presented in the format of mean ± SEM.GraphPad Prism 7.0 software was used to perform statistical analysis.We used Mann-WhitneyUtest to compare the differences between two independent groups including immunohistochemistry,CV staining,and protein levels in Western blots.For multiple comparisons across groups (functional outcome tests),we used two-way analysis of variance withpost hocBonferonni test.P<0.05 was considered statically significant.

Results

Brain Rac1 declines with age

To determine Rac1 levels,cerebral hemispheres were collected from young (8-10 weeks) and aged (18-22 months) mice for western blot assay.Rac1 levels were significantly lower in older mice compared with young mice (P<0.05;Figure 2).

Figure 2｜The protein level of Ras-related C3 botulinum toxin substrate (Rac) 1 in the cerebral hemisphere of aged mice.

Delayed overexpression of Rac1 improves behavior recovery after stroke

To assess whether overexpression of cerebral Rac1 could improve poststroke functional recovery in aged mice,lentiviral vectors carrying Rac1 were administrated into mice brain on day 1 after MCAO.We confirmed the effect of the vector by demonstrating an increased number of Rac1-positive cells 27 days after injection compared with the control group (P<0.05;Figure 3A).Cerebral ischemia produced the impairment of cognitive function and sensorimotor function (Bu et al.,2020).Herein,delayed overexpression of Rac1 improved cognitive recovery on day 28 (P<0.05;Figure 3B).In addition,the sensorimotor function was assayed.MCAO caused a marked impairment assessed by adhesive removal test (pre-strokevs.7 days after stroke in the control group,P<0.05).Importantly,LV-Rac1 also produced an improved performance in sensorimotor function recovery compared with the control group (P<0.05 on days 14 and 21 after stroke;Figure 3C).

Figure 3｜The effects of delayed overexpression of cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 on functional recovery after ischemic stroke in aged mice.

Delayed inhibition of Rac1 leads to poorer functional recovery

We further confirm the contribution of Rac1 to functional recovery after stroke by i.p.injection of Rac1 inhibitor NSC23766.As expected,we found that delayed inhibition of Rac1 worsened cognitive recovery (P<0.05 on day 28;Figure 4A),and sensorimotor recovery (P<0.05 on day 21;Figure 4B) after stroke compared to the control group.

Figure 4｜The effect of delayed inhibition of Ras-related C3 botulinum toxin substrate 1 on functional recovery after ischemic stroke in aged mice.

Delayed overexpression of Rac1 promotes axonal regeneration and angiogenesis without altering tissue loss

Our previous study showed that cell-specific overexpression of neuronal Rac1 promoted axonal regeneration and that overexpression of endothelial Rac1 promoted angiogenesis in the young mouse brain after stroke (Bu et al.,2019,2020).In the present study,we examined the role of cerebral Rac1 in these two major forms of plasticity in aged mice.We found that delayed overexpression of cerebral Rac1 improved neurite outgrowth (P<0.05;Figure 5A) in the peri-infarct zone compared with the control group on day 28 after stroke.In addition,endothelial proliferation was also improved by delayed overexpression of cerebral Rac1 as evidenced by the increase of either CD105 staining intensity (P<0.05;Figure 5B) or the number of CD105 positive cells with BrdU (P<0.05;Figure 5B) in the peri-infarct zone compared with the control group on day 28 after stroke.We further performed CV staining to evaluate the tissue loss.Compared with the control group,no differences in cavity sizes were seen after treatment (P>0.05;Figure 5C).

Figure 5｜The effect of delayed overexpression of cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 on neurite density,endothelial proliferation,and tissue loss on day 28 after stroke in aged mice.

Delayed inhibition of Rac1 reduces axonal density and endothelial proliferation without altering tissue loss

The contribution of cerebral Rac1 to axonal density and endothelial proliferation was confirmed using Rac1 inhibitor NSC23766.We found that delayed inhibition of Rac1 reduced NFL intensity (P<0.05;Figure 6A),CD105 intensity (P<0.05;Figure 6B),and the number of proliferative endothelial cells (P<0.05;Figure 6B) compared with the control group on day 28 after stroke.No differences in tissue loss assessed by CV staining were observed after treatment (P>0.05;Figure 6C).

Figure 6｜The effect of delayed inhibition of Rasrelated C3 botulinum toxin substrate 1 on neurite density,endothelial proliferation,and tissue loss on day 28 after stroke in aged mice.

Rac1 inhibition reduces pro-regenerative responses and exacerbates GFAP signaling

We assessed the molecular basis of Rac1 in promoting axonal regeneration and endothelial proliferation on day 28 after stroke.Pak1 is a key downstream molecule of Rac1 (Kumar et al.,2006;Shutes et al.,2007).It has been demonstrated that Pak1 can promote BDNF secretion and thus stimulate cell proliferation (Liu et al.,2006;Kichina et al.,2010;Bu et al.,2019).We thus assessed Pak1 activation after Rac1 inhibition and demonstrated that Rac1 inhibition reduced the phosphorylation of Pak1 (P<0.05;Figure 7) but not the total level of Pak1 (P>0.05;Figure 7) in the ipsilateral hemisphere of stroke compared with the control group.In addition,the total level of BDNF was also reduced (P<0.05;Figure 7),which is in line with our previous studies (Bu et al.,2019,2021) performed in young mice,suggesting that Rac1 activates similar pro-regenerative pathways in aged mice.In addition,inhibition of Rac1 led to an increase of the GFAP in the ipsilateral hemisphere of stroke (P<0.05;Figure 7).

Figure 7｜The effect of delayed inhibition of Rasrelated C3 botulinum toxin substrate 1 on the expression of brain-derived neurotrophic factor (BDNF),phosphorylated p21-activated kinase (Pak) 1 (T212),and glial fibrillary acidic protein (GFAP) in the ipsilateral hemisphere on day 28 after stroke.

Discussion

We revealed several significant findings in the present study.First,we found that the baseline levels of Rac1 in aged mice were significantly lower than those in young mice.Second,our present study is the first to report that Rac1 plays a critical role in long-term functional recovery and CNS plasticity in the aged stroke model.We demonstrated that delayed overexpression of Rac1 promoted functional recovery while pharmacological inhibition exacerbated behavior outcomes following 4 weeks of survival.Third,improved axonal regeneration and angiogenesis were observed in mice with Rac1 overexpression after stroke.In contrast,we found reduced axonal density and diminished angiogenic response in Rac1 inhibitor-treated stroke mice.Finally,at the molecular level,we demonstrated that pharmacological Rac1 inhibition reduced pro-regenerative responses and exacerbated GFAP signaling after stroke.

One of the major downstream regulators of Rac1 is Pak1 (Yao et al.,2020;Erasmus et al.,2021),Multiple isoforms of Paks have been identified;of these,Pak1 is highly expressed in the brain,muscle,and spleen (Arias-Romero and Chernoff,2008).Pak1 is known to further activate mitogen-activated protein kinase (Mek)1/2 and Erk1/2 signaling,enhance BDNF production,and stimulate cell proliferation (Bu et al.,2019,2020).In young mice,Rac1 overexpression in mice neurons activates Pak1,Mek1/2,and Erk1/2,and the elevation of BDNF on day 14 after stroke (Bu et al.,2020).The delayed intervention of neuronal Rac1 affects the expression of GFAP after stroke in young mice (Bu et al.,2020).These earlier studies performed in young animals suggested a key role of Rac1 in axonal regeneration by targeting the BDNF pathway in young mice models,consistent with what we found in the aged model.Additionally,in young mice,delayed overexpression of endothelial Rac1 increased endothelial cell proliferation after ischemic stroke (Bu et al.,2019) and promoted post-stroke recovery of cognition and accelerated sensorimotor recovery.In cell cultures,delayed inhibition of Rac1 reduced endothelial cell viability,tube formation,and migration after transient oxygen and glucose deprivation (Bu et al.,2019).Our present work showed Rac1 activation improves endothelial cell proliferation while pharmacological inhibition reduces endothelial cell proliferation in an aged stroke model.We found the tissue loss caused by stroke was not altered by post-stroke Rac1 overexpression/inhibition,indicating that the pro-regenerative effect is not related to the size of the injury.

Aging is the most important independent predictor of poor outcomes after stroke (Liu et al.,2016;Finger et al.,2022).We have demonstrated worse functional recovery after stroke in aged mice.However,the mechanisms of age-related loss of brain plasticity after stroke are unclear,but the loss of intrinsic potential and increased glial inhibition could be major causes.Older animals invariably have significantly higher mortality rates and worse functional recovery after stroke (Manwani et al.,2011;Jiao et al.,2021).Diminished recovery from stroke in aged animals implies that repair processes are affected by advanced age.Indeed,after stroke,gene expression profiles that promote axonal spouting and the formation of new neuronal connections in adjacent brain regions differ significantly between young and aged brains (Li et al.,2010).Furthermore,it was demonstrated that reactive gliosis and the formation of a glial scar in aged subjects after stroke are higher than those in young subjects (Manwani et al.,2011).Therefore,the inhibitory signal is stronger while the intrinsic growth potential is weaker in aged brains for axonal regeneration after stroke.New paradigms for conducting translational research,including preclinical testing of novel therapies that examine aging as a major factor,are urgently needed.Phosphorylation of Erk1/2,another downstream target of Rac1 for neuronal regenerative potential,is also reduced in the cerebral cortex in aged animals (Zhen et al.,1999;Iroegbu et al.,2021).In our present study,we showed aging led to a decline in Rac1 protein levels,which is consistent with reduced intrinsic regeneration capacity and increased astrocytic inhibition in aged after stroke.We will study the effect of aging on Rac1 and its downstream pathways,including Pak1 and BDNF et al.,comprehensively after stroke with young controls in the future.

NSC is highly selective for Rac1 (Liang et al.,2021).Our previous studies using lentiviral overexpression also suggested Rac1 is important for functional recovery and potentially for CNS regeneration and reorganization after stroke;therefore,we focused on this isoform of Rac.However,as all pharmacological approaches may have off-target effects,NSC may also inhibit the activities of the other Rac isoforms (Rac2 and Rac3),at least in blood cells (Mizukawa et al.,2011).However,Rac2 is exclusively expressed in hematopoietic cells,and it is less likely to be a critical contributor to axonal outgrowth and angiogenesis (Mizukawa et al.,2011).Levay showed that NSC23766 is not only an inhibitor of Rac1,but also a competitive antagonist at M1,M2,and M3 mAChRs within the same concentration range (Levay et al.,2013).Microglial M3 mAChRs are protective to ischemic brain injury (Levay et al.,2013),but the contribution of M1 and M2 mAChR2 to stroke is not studied yet.In this case,i.p.injection of NSC23766 might worsen ischemic brain injury through inhibition of M3 mAChRs.To address the potential non-specificity issues of pharmacology,we designed genetic approaches using LV to overexpress Rac1 in the aged model after stroke.Our data collectively showed that Rac1 isoform plays a critical role in brain cellular regeneration and remapping after ischemic stroke in the aged.

Our vector/drug treatment does not specifically target cells of certain types in the brain.Systemic/CNS manipulation of Rac1 signaling allowed us to test whether this pathway stimulates the CNS capacity for growth and counteracts glial inhibition simultaneously to maximize axonal and endothelial regeneration after stroke.This approach is the most appropriate choice,as our focus was to test if enhancing the Rac1 pathway will improve recovery in the aged after stroke.Further studies are warranted to test the cell-specific function of Rac1 in the aged stroke model.

This study has several limitations that should be noted.First,assessment of NFL density is difficult to count neurite numbers and measure the length of axons in brain slices.Typically,this is done in cell culture using microfluidic chambers in which the axons and cell bodies are grown in separate compartments.Using such an approach,we found that inhibition of Rac1 reduced axonal length and numbers after transient oxygen and glucose deprivation,suggesting a critical role of Rac1 in neurite outgrowth (Liu et al.,2018).This was done in cells cultured from peri-natal brains.As aging neurons long-term in a microfluidic chamber is technically challenging,NFL assessment remains the feasible and valuable approach for neurite examinationin vivowith the limitations mentioned above.

The use of GFAP protein as a measure of the glial scar has limitations as well.Astrocytes are just one component of the glial scar,and only a small number of astrocytes contribute to the compact scar at the lesion border,meaning that it is likely that most GFAP protein is from reactive but not compact scarforming astrocytes.Reducing the complex process of astrogliosis to the amount of GFAP could be an oversimplification.An increase in GFAP protein might not directly mean the glial scar is bigger/denser/etc.Moreover,other work has shown that reactive astrocytes facilitate vascular plasticity after CNS injury (Williamson et al.,2021;Puebla et al.,2022),although this is not clear in axonal regeneration in a stroke model.The role of glial scar formation in stroke recovery and its assessment warrants future investigation in stroke models,especially in aged models.

Our study helped reveal the cellular processes behind the diminished axonal regeneration and angiogenesis after cerebral ischemia in the aged.Very few/no studies have investigated the role of Rac1 in axonal regeneration in the aged brain at baseline or after stroke.Axonal loss and synaptic impairment,diminished angiogenesis may be the leading causes of cognitive dysfunction in brain aging.Using a translationally relevant aging model,we herein conclude that overexpression of Rac1 improved functional outcomes in aged mice after stroke and revealed the underpinning cellular and molecular mechanisms.Augmenting this signaling pathway in the aged brain could potentially reduce disability and enhance recovery in the population at the greatest risk for stroke,the elderly.

Acknowledgments:We would like to thank Mai N.Le (Department of Neurology,University of Texas Health Science Center,USA) for proofreading the manuscript.

Author contributions:FB performed the majority of the experiments,

analyzed data,and drafted manuscript;JWM,MAR,AEH,AP,and LQ provided technical support;AU edited the manuscript and helped with data analysis;JL conceived the research project,helped design the experiments and finalized the paper.All authors approved the final version of this paper.

Conflicts of interest:The authors declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Data availability statement:All relevant data are within the paper.

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