Effects Of Constraint-induced Movement Therapy On Endogenous Angiogenesis And Neurogenesis Following Cerebral Ischemia-Reperfusion And Its Relationship To Nogo-A—RhoA—ROCK Pathway In Rats

Objectives:Spontaneous axonal plasticity and function restoration after stroke is thought to be limited by myelin-associated neurite growth inhibitory proteins in the adult CNS which includes Nogo-A,myelin-associated neurite growth inhibitory proteins and oligodendrocyte myelin glycoprotein,particularly Nogo-A.Studies have shown that anti-Nogo-A immunotherapy improves functional recovery,neuroregeneration and neuroplasticity after central nervous system lesions in adult or aged rats.The constraint-induced movement therapy?CIMT?can improve the motor and mnemonic function of rats with cerebral ischemia,and the mechanism may have relation with axonal regeneration by down-regulating the expression of Nogo-A—RhoA—ROCK.Exercise therapy increases the bioavailability of EPCs,VEGF and BDNF,all of which promote cerebral perfusion mediated by collateral recruitment and revascularization.Using in vitro and in vivo approaches,further study showed that Nogo-A is a negative regulator of angiogenesis in the developmental CNS after birth.The Nogo-A inhibits spreading,adhesion and migration of microvascular endothelial cells?MVECs?,in which Nogo-A—RhoA—ROCK plays a roll probably.Adult Nogo-A^-/-mice showed no significant changes in the blood vessel densities in the examined brain areas.It is unclear whether there are some associations between Nogo-A—RhoA—ROCK and angiogenesis in pathological processes such as stroke.Researchs are necessary to further address the effects of CIMT on angiogenesis and its relationship to Nogo-A—RhoA—ROCK.An increasing number of studies have identified a close relationship between neurogenesis and angiogenesis,and they play an important role in improving functional recovery after stroke.Neural and vascular cells not only are anatomically closely tied to each other,but they are also guided by similar signals to establish correct connectivity and crosstalk.An angiogenic niche may provide a novel interface where mesenchyme-derived cells and circulating factors influence plasticity in the adult central nervous system.The generation of new vasculature can stimulate cell proliferation within SVZ and SGZ,migration and differentiation in the peri-infarct areas.In the long term,formation of new blood vessels is necessary to fully supply tissue metabolic and functional requirements.A recent research showed that CIMT decreased the expressions of Nogo-A,RhoA and ROCK in peri-infarct areas and enhanced axon growth.But the relationship between Nogo-A—RhoA—ROCK signal pathway and endogenous neurogenesis is not clear.We used middle cerebral artery occlusion?MCAO?model to measure angiogenesis,proliferation of neural precursor cells in SVZ,neural differentiation in the peri-infarct areas and function restoration by fasudil.Next,comparing with the effects of the fasudil,we investigated whether CIMT might facilitate angiogenesis and neurogenesis by overcoming the Nogo-A—RhoA—ROCK pathway.Within the framework of neurovascular units?NVUs?,neurotransmitter is regulated and blood–brain barrier stability is maintained through cell-cell signaling between neuronal,glial and vascular elements.Blood–brain barrier?BBB?,an an important part of NVU,plays key roles in maintaining homeostasis of NVU.BBB properties are mainly determined by endothelial TJs.Some studies have shown that inhibition of Rho kinase could improve the BBB integrity by mediating tight junction protein.CNS ECs and perivascular support cells,including pericytes and perivascular astrocytes are involved in BBB formation,maintenance,and stability.contribute to vascular maturation and arteriogenesis.Vascular smooth muscle cells??-SMA?,one of the primary pericyte markers,contribute to vascular maturation and arteriogenesis.From a NVU perspective,we speculate that CIMT may protect the BBB from ischemic injury,which is regulated by pericytes.Nogo-A—RhoA—ROCK is necessary for functional recovery after stroke,and its expression could be affected by CIMT.To determine whether CIMT could influence the Nogo-A—RhoA—ROCK pathway to induce endogenous angiogenesis and neurogenesis,we used an inhibitor of Nogo-A receptor?NgR?,NEP1-40,to accomplish this goal.In addition,we chosed to determine VEGFR2 in the VEGF-A–VEGFR2–Dll4–Notch signaling pathway,the main regulatory pathway of angiogenesis in the central nervous system.We made comparative analysis to explore whether there were other potential mechanisms participating in CIMT-induced neuroprotection.Materials and Methods:Forty-one adult male Sprague–Dawley rats?280–320g?were used for this research.We selected 5 rats randomly for triphenyltetrazolium chloride?TTC?staining to assess the size and distribution of infarcts at 24h after cerebral ischemia reperfusion.The other 36 rats were randomly assigned into a Sham-operated control group?n=6?,an IR group?n=10?,an Fasudil group?n=10?and a CIMT group?n=10?.The rats were subjected to right middle cerebral artery occlusion?MCAO?and reperfusion except the Sham-operated control group.The constraint-induced movement model was induced in rats of CIMT group by wrapping the ipsilateral forelimbs on the bodys with plaster 7d postsurgery.Fasudil?10mg/Kg?was injected intraperitoneally to rats in Fasudil group 7d after MCAO once a day.BrdU?50mg/Kg?was given to all rats 7d after MCAO every other day.Rats were tested for modified neurological severity scores?mNSS?,tapered/ledged beam-walking test and Morris water maze test at 4 weeks after stroke.Immunofluorescence stain was performed to evaluate the number of BrdU/Nestin,BrdU/DCX positive cells in the ipsilateral SVZ.BrdU/NeuN and BrdU/GFAP positive cells in the ischemic boundary zone?IBZ?were also evaluated.Immunohistochemistry was applied to determine the expression of CD31,claudin-5??-SMA and VEGFR2 in the IBZ.Western blot analysis was applied to determine the expression of CD31 in the SVZ and expression of CD31 in the IBZ.The other experiment was conducted to determine the extent of the impact of CIMT and NEP1-40?a specific antagonist of NOGO-66 action at NgR?on the Nogo-A—RhoA—ROCK pathway.In this experiment,the rats were randomly separated into 2 groups and then sacrificed at 2 weeks or 4 weeks after stroke.The animals in each group were randomly assigned to the following treatment and control groups:?1?Sham?no lesion,vehicle;n=3?;?2?IR?lesion,vehicle;n=5?;?3?NEP1-40?lesion,drug treatment;n=5?;and?4?CIMT?ischemia,vehicle and CIMT;n=5?.NEP1-40 was continuous infused into the lateral ventricle in Fasudil group 7d after MCAO via osmotic minipumps for 7 days or 21days?1.25ug/h?.Immunohistochemistry was performed to confirm the expression and distribution of Nogo-A,RhoA and ROCK.All data were analyzed by the software of SAS 9.1.Results:1.The TTC staining of rat MCAO model:The results showed that the normal tissue was stained red,while the infarct tissue was unstained.The cerebral infarction areas were restricted to the frontal,parietal,tempora cortex and outside parts of striatum supplied by ipsilateral MCA.2.Effects of CIMT and fasudil on proliferation of vascular endothelial cells after cerebral ischemia reperfusion in rats:At the 4^st week postsurgery,quantitative determination of BrdU/CD31-positive vasculars in the SVZ suggested that compared with the IR group,the CIMT and Fasudil groups exhibited markedly enhanced vascular density.Moreover,CIMT-treated rats had more BrdU/CD31-positive microvessels in the SVZ than that fasudil-treated rats?P<0.05?.Western blots confirmed a robust upregulation of the expression of CD31 after CIMT and fasudil treatment.The IBZ exhibited similar outcomes.3.Behavioral outcomes of rats in each group:All rats showed similar mNSS scores,and no differences were achieved among the groups at day 1 following MCAO?P>0.05?.However,all rats showed improvement in behavioral outcomes over time.At week 4after MCAO,there was significant statistical difference in the mNSS,foot fault scores in the tapered beam-walking test and escape latency in the water maze test among the groups.The foot faults for the impaired forelimbs of fasudil-treated rats were significantly lower than those of IR rats?P<0.01?.Furthermore,the CIMT group had lower foot fault scores than the Fasudil group?P<0.05?.The CIMT and Fasudil groups demonstrated better spatial learning function than the IR group.The CIMT group had shorter escape latency than the Fasudil group?P<0.05?.The results demonstrated that CIMT treatment improved sensorimotor and cognitive outcomes in rats after stroke significantly.4.Effects of CIMT and fasudil on the proliferation of neural precursors in the SVZ after cerebral ischemia reperfusion in rats:The neural precursor cells were mainly found in the frontal horn and lateral wall of lateral ventricle on the ipsilateral side of infarct,and some located in contralateral wall.At the 4^st week postsurgery,quantitative analysis indicated that the CIMT and Fasudil groups markedly elevated BrdU/Nestin and BrdU/DCX expression levels in the SVZ compared with the IR group?P<0.05?.Moreover,compared with the administration of fasudil,the CIMT treatment significantly increased the number of neural precursor cells?P<0.05?.5.Effects of CIMT and fasudil on neurogenesis in the IBZ in rats:Enhanced BrdU/NeuN and BrdU/GFAP expression was detected in the IBZ.As expected,the IR appeared to show an increase in neurogenesis,whereas fasudil further increased the number of BrdU/NeuN positive cells?new adult neurons?in the cortex ipsilateral to the infarct?P<0.05?.After 3 weeks of CIMT,the number of BrdU/NeuN-positive cells was markedly increased compared with that after 3 weeks of fasudil?P<0.05?.A similar result was found for BrdU/GFAP-positive cells in the IBZ,which indicated gliogenesis,but there was no significant difference between the Fasudil and CIMT groups?P>0.05?.6.Effect of CIMT on the expression of tight junction protein claudin-5 in the IBZ in rats:At 4 weeks after MCAO,the tight junctions?TJs?claudin-5 was detected to characterize the barrier properties of BBB.The immunohistochemistry showed that claudin-5 was located to cerebral microvessels in rats.We found that cerebral ischemia significantly decreased the mean optical density?OD?values of claudin-5.Fasudil and CIMT treatment significantly increased the mean OD values of claudin-5.However,the expression of claudin-5 in the CIMT group was significantly higher than that in Fasudil group?P<0.05?.7.Effect of CIMT on the expression of?-SMA in the IBZ in rats:Fasudil and CIMT significantly increased the number of?-SMA-positive vessels after cerebral ischemia reperfusion.Moreover,CIMT-treated rats presented more?-SMA-positive vessels in the IBZ than fasudil-treated rats?P<0.05?.8.Effect of CIMT on the expressions of Nogo-A-RhoA-ROCK pathway in the IBZ in rats:The numbers of Nogo-A,RhoA and ROCK positive cells were significantly increased in the IBZ,compared with that in the Sham cortex.The expression of Nogo-A was not affected by NEP1-40,while the expression levels of RhoA and ROCK were significantly reduced by NEP1-40?P<0.05?.At 2 weeks after MCAO,rats treated with NEP1-40 had lower expression levels of RhoA and ROCK than rats treated with CIMT.However,at 4 weeks after MCAO,significantly decreased expression levels of RhoA and ROCK were observed in the NEP1-40 and CIMT groups,but there these were not significantly different between them?P>0.05?.9.Effect of CIMT on the expression of vascular endothelial growth factor receptor 2?VEGFR2?in the IBZ in rats:Enhanced VEGFR2 expression was detected in the IBZ of the impaired cortex after stroke.The numbers of VEGFR2-positive cells were not significantly changed in rats treated with fasudil.By contrast,the CIMT group showed a significant increase in VEGFR2-positive cells?P<0.05?.Conclusions:1.Spontaneous angiogenesis and neurogenesis after cerebral ischemia reperfusion are not enough to enhance brain self-repair because of limited reparative capability of mature central neurons.2.The inhibition of Nogo-A—RhoA—ROCK signaling pathway by fasudil after cerebral ischemia reperfusion in adult rats can significantly promote endogenous angiogenesis,which is accompanied by the proliferation,migration and differentiation of endogenous neural precursor cells into nerve cells in the ischemic border zone,and at the same time promotes the recovery of behavior functions.3.CIMT can also significantly promote endogenous angiogenesis after cerebral ischemia reperfusion in adult rats,which is accompanied by the proliferation,migration and differentiation of endogenous neural precursor cells into nerve cells in the ischemic border zone,and at the same time promotes the recovery of behavior functions.The neuroprotective regenerative repair induced by CIMT was stronger than fasudil 4 weeks after stroke.4.CIMT is an effective treatment to improve BBB integrity damaged by cerebral ischemia,which is regulated by the preservation of pericytes and a reduction in TJ damage.CIMT may offer a novel neurovascular strategy for protecting the NVU after focal cerebral ischemia.5.At 4 weeks after MCAO,the inhibition degree of RhoA and ROCK by CIMT was similar to that by NEP1-40.CIMT promotes neuroprotective regenerative repair after stroke partially by overcoming Nogo-A—RhoA—ROCK signaling with a time-dependent mechanism.6.CIMT's positive regulatory effect occurs through a pathway other than the Nogo-A–RhoA–ROCK pathway,such as upregulation of VEGFR2.