| Ischemic stroke is a cerebral dysfunction caused by the stenosis or occlusion of cerebral arteries induced by multiple factors.With the characteristics of high incidence,high disability and high mortality,ischemic stroke has become one of the major diseases that endanger human health.Currently,its therapy methods in acute phase,such as thrombolysis and neuroprotection,are still limited.Until recently,recombinant tissue plasminogen activator(rt-PA)was the only FDA approved therapy for patients with acute ischemic stroke presenting within 0 to 4.5 hours after stroke onset.Unfortunately,the narrow therapeutic time window and the risk of subsequent hemorrhage limit the use of thrombolysis with rt-PA.Neuroprotective agents developed in preclinical studies have failed miserably in clinical trials.Therefore,there is a urgent need for novel strategies or drugs to come up to treat ischemic stroke.Recently,the vessels-networks theory of traditional Chinese medicine(TCM)suggested by Professor Wu Yiling for guidance for the prevention and treatment of vascular diseases has drawn increasing attention,especially that he has put forward the novel viewpoint "cerebral microvascular protection in ischemic area after ischemic stroke" from the perspective of the correlation between brain minute collaterals in TCM and microvessels in Western medicine.Previous studies have preliminarily shown that Tongxinluo capsule(TXL),a representative of collateral-unblocking medicines,has a microvascular mediated-brain protection against ischemic stroke.Thus,this study mainly focused on the role of brain "minute collateral-microvascular" injury in the pathogenesis of ischemic stroke from the viewpoint of the vessels-networks theory and the protective mechanisms of TXL on cerebral microvessels after cerebral ischemia,respectively.Part I:An theoretical investigation of the role of brain "minute collateral-microvascular"injury in the pathogenesis of ischemic stroke and its intervention strategies1 Objectives:The aim of this study was to investigate the role of brain "minute collateral-microvascular"injury in the pathogenesis of ischemic stroke from the viewpoint of the vessels-networks theory of TCM and further provide the theoretical support for the novel viewpoint cerebral "minute collateral-microvascular protection in ischemic area after ischemic stroke".As the same time,the study could be used as a theoretical instruction for exploring the protective mechanisms of TXL on cerebral microvascular in ischemic stroke model in mice.2 Methods:This study summarized the researches on minute collaterals in the vessels-networks theory,proposed the view "a close correlation between brain minute collaterals in TCM and microvessels in Western medicine",and further explored the role of brain "minute collateral-microvascular" injury in the pathogenesis of ischemic stroke to clarify the importance of brain"minute collateral-micro vascular" protection in the periinfarct area after stroke.In addition,we reviewed the advances of TXL on cerebral microvascular protection after cerebral ischemia.3 Results:The role of brain "minute collateral-microvascular" injury was elucidated in the pathogenesis of ischemic stroke.The brain "minute collateral-microvascular" injury caused the dysfunction of Qi-blood perfusion,nutrition and metabolism,and exchange of body fluid and blood,which led to the dystrophy of brain.Meanwhile,the pathogenic toxin was endogenously produced and further damaged the brain tissue and minute collateral-vessels.The collateral-unblocking therapy could protect cerebral minute collateral-microvessels in the ischemic area after ischemic stroke and thus exert a protective effect on ischemic brain.These provided the theoretical support for the novel viewpoint "cerebral minute collateral-microvascular protection in ischemic area after ischemic stroke".Previous research preliminarily showed that TXL had a cerebral microvascular protection against ischemic stroke.4 Conclusions:Under the guidance of vessels-networks theory of TCM,the critical role of brain "minute collateral-mierovascular" injury has been revealed in the pathogenesis of ischemic stroke based on a close correlation between brain minute collaterals in TCM and microvessels in Western medicine.The collateral-unblocking therapy could protect cerebral minute collateral-microvessels in the ischemic area after ischemic stroke and thus exert a protective effect on ischemic brain.These two points provided the theoretical support for the novel viewpoint"cerebral minute collateral-microvascular protection in the ischemic area after ischemic stroke".Meanwhile,this study could be used as a theoretical instruction for exploring the protective mechanisms of TXL on cerebral microvessels in ischemic stroke animal model.Part ? The protective effect and underlying mechanisms of Tongxinluo capsule on cerebral microvessels in the periinfarct area after ischemic stroke in mice Chapter ? The induction and evaluation of ischemic stroke model in mcie1 Methods:The ischemic stroke model was induced by permanent middle cerebral artery occlusion(pMCAO)using the intraluminal suture method in adult male C57BL/6J mice.Mice were randomly divided into sham group(Sham)and pMCAO group(pMCAO).The stability and reliability of the model were evaluated by neurological tests and brain TTC(2,3,5-Triphenyltetrazolium chloride)staining at 6 hours and 24 hours after pMCAO,respectively.2 Results:The mice in Sham group moved about freely,exhibited no neurological deficits and brain infarct lesions by TTC staining at 6 and 24 hours after pMCAO.However,the mice in pMCAO group showed significant neurological deficits such as inability to walk straight,circling toward the side,hypalgesia of forelimb skin,and absence of pinna reflex.In addition,the mice in pMCAO group clearly displayed brain infarct lesions.These results demonstrated the pMCAO model was successful.3 Conclusion:The pMCAO induced by an intraluminal filament is an ideal model of ischemic stroke due to its reliability,easy operation and high successful rate,which has set a foundation for further study.Chapter ? The protective effect of Tongxinluo capsule on ischemic brain in mice 1 Methods:After subjected to pMCAO,adult male C57BL/6J mice were randomly divided into 6 groups:Sham,pMCAO group,NBP group(NBP),TXL high dose group(TXL-H),TXL medium dose group(TXL-M),and TXL low dose group(TXL-L).The mice were orally administered with TXL(3.0,1.5 and 0.75 g/kg)or NBP(123 mg/kg)at 1,3,and 21 hours after pMCAO in the corresponding groups.The following parameters were examined at 6 and 24 hours after pMCAO:neurological deficit scores,brain infarct volume and water content.2 Results:(1)Neurological deficit scores:Compared with pMCAO group at 6 and 24 hours,TXL(6 hours:TXL-H,TXL-M,and TXL-L;24 hours:TXL-H and TXL-M)and NBP significantly reduced neurological deficit scores(P<0.01).Moreover,TXL-H and TXL-M had a similar efficacy to NBP statistically and were better than TXL-L in terms of improving neurological deficits(P<0.05).There were no significant differences between TXL-H,TXL-M and NBP(P>0.05).(2)Brain infarct volume:At 6 hours,TXL-H reduced significantly brain infarct volume with the same efficacy to NBP statistically compared with pMCAO group(P<0.05 or P<0.01),while both TXL-M and TXL-L failed to do so(P>0.05).At 24 hours,3 doses of TXL and NBP all remarkedly decreased brain infarct volume compared with pMCAO group(P<0.05 or P<0.01),and TXL-H worked better than TXL-L in reducing brain infarct volume statistically(P<0.01).(3)Water content:The water content in pMCAO group was higher than that in Sham at 6 and 24 hours(P<0.05)indicating that brain edema existed in pMCAO mice.However,none of different doses of TXL and NBP reduced brain water content(P>0.05).3 Conclusion:TXL reduces brain infarct volume,improves neurological deficits,and thus exerts a protective effect on ischemic brain in mice.Chapter III The protective effect and underlying mechanisms of Tongxinluo capsule on cerebral microvessels after ischemic stroke in miceSection I The protective effect of Tongxinluo capsule on cerebral microvessels after ischemic stroke in mice1 Methods:pMCAO mice were grouped and administrated as the Chapter ?.Brain microvessel structure was examined by immunostaining with Alexa FluorTM 488 Conjugate isolectin B4 from Griffonia simplicifolia(IB4 Alexa 488)at 6 and 24 hours after pMCAO.In addition,BBB permeability was evaluated by Evans blue at 6 and 24 hours after pMCAO.2 Results:(1)Brain microvessel structure:IB4 Alexa 488 regularly exhibited a uniform staining pattern for endothelial cells in Sham at 6 and 24 hours after pMCAO,while ischemia-affected brain microvessels in pMCAO group frequently appeared to be irregular and discontinuous,or even loss of the IB4 Alexa 488 staining,which was interpreted as endothelial structural alterations.However,compared with those in pMCAO group,microvessels in TXL groups and NBP group remained relatively complete,especially in TXL-H group,indicating its cerebral microvascular protection.(2)BBB permeability:? EB fluorescence showed that the dye was largely confined to the vasculature in the nonischemic contralateral hemisphere or in both hemispheres of Sham at 6 and 24 hours,while the mice in pMCAO group exhibited significantly greater dye extravasation in the periinfarct regions.However,EB leakage in TXL and NBP group was obviously alleviated at 6 and 24 hours,especially TXL-H group,when compared with that in pMCAO group.? Our quantitative results showed that EB content in pMCAO group increased significantly compared with that in Sham at 6 and 24 hours indicating BBB disruption after pMCAO(P<0.01).However.after treatment of TXL(TXL-H and TXL-M)or NBP,mice had a significantly lower EB content in the ipsilateral hemisphere compared with pMCAO group at 6 and 24 hours(P<0.05 or P<0.01).while TXL-L did not reduce EB content in the ipsilateral hemisphere at 6 and 24 hours(P>0.05).Moreover,there were no significant differences between TXL-H,TXL-M,and NBP at 6 hours(P>0.05),while TXL-H reduced EB content significantly in the ipsilateral hemisphere compared with TXL-M.TXL-L,and NBP groups at 24 hours(P<0.05 or P<0.01).These results suggested that all of TXL-H.TXL-M and NBP could protect against BBB disruption after pMCAO,and that TXL-H exhibited the best efficacy in protecting against BBB disruption.3 Conclusion:TXL protects cerebral microvessels against ischemic stroke in mice.Section ? The underlying mechanisms of Tongxinluo capsule on cerebral microvascular protection after ischemic stroke in miceExperiment I The sonic hedgehog pathway mediates Tongxinluo capsule-induced protection against blood-brain barrier disruption after ischemic stroke in mice1 Methods:In the following mechanism research part,we chose the best efficacy of three doses(TXL-H)and still used the same stroke model(pMCAO).Mice were randomly divided into 5 groups:Sham,pMCAO group.TXL group(TXL),cyclopamine group(CP)and TXL combined with cyclopamine group(TXL+CP).The mice in both TXL group and TXL+CP group were orally administered with TXL(3.0 g/kg)at 1,3 and 21 hours after pMCAO,while other mice were given the same volume solvent in the corresponding time.Meanwhile,the mice in both CP group and TXL+CP group were intraperitoneally injected with cyclopamine(10 mg/kg)in 8%DMSO + 30%PEG 300 + 5%Tween 80 + ddH20 at 1 and 21 hours after pMCAO,while other mice were intraperitoneally administered with the same volume solvent in the corresponding time.BBB permeability was evaluated by Evans blue in the ischemic penumbra of the ipsilateral hemisphere at 6 and 24 hours after pMCAO.Shh immunofluorescence was performed in the ischemic penumbra of the ipsilateral hemisphere at 6 and 24 hours after pMCAO.In addition,Western blot assay was performed for measuring the expression of tight junction(TJ)proteins[occludin,claudin-5 and zonula occludens-1(ZO-1)],and Shh signaling molecules[Shh,Patched,Smoothened(Smo)and Gli-1]in the ischemic boundary cortex of the ipsilateral hemisphere at 6 and 24 hours after pMCAO,2 Results:(1)Both EB fluorescence area and intensity in pMCAO group were significantly greater than those in Sham at 6 and 24 hours(P<0.01).TXL obviously reduced the fluorescence area and intensity at 6 and 24 hours(P<0.01).However,treatment with TXL combined with cyclopamine or cyclopamine alone exhibited greater fluorescence area and intensity compared with that with TXL alone at 6 and 24 hours(P<0.05 or P<0.01).These data demonstrated that TXL could protect against BBB disruption after ischemic stroke,and the protective effect could be reversed by the Shh pathway inhibitor cyclopamine.(2)Western blot data showed that the expression of these TJ proteins(Occludin,Claudin-5 and ZO-1)in pMCAO group was significantly downregulated compared with that in Sham at 6 and 24 hours(P<0.01),which indicated cerebral ischemia caused the damage of TJs.After TXL treatment,the expression of these TJ proteins was obviously upregulated when compared with that in pMCAO group at 6 and 24 hours indicating that TXL could upregulate the expression of these TJ proteins(P<0.01).However,the upregulating effect of TXL on these TJ proteins was inhibited when TXL combined with cyclopamine or cyclopamine alone was administrated at 6 and 24 hours(P<0.05 or P<0.01).(3)Double immunostaining of Shh and GFAP positive cells suggested that some of Shh positive cells colocalized with GFAP positive astrocytes,while the others surrounded remaining EB indicating that some of Shh positive cells also colocalized with endothelial cells.Therefore,the immunofluorescence analysis combined with previous reports about Shh has demonstrated that Shh is mainly derived from astrocytes and then acts upon brain endothelial cells in the central nervous system.Western blot analysis exhibited that the expression of Shh,Patched,Smo and Gli-1 in pMCAO group was significantly downregulated compared with that in Sham at 6 and 24 hours(P<0.01),which indicated that cerebral ischemia caused the inactivation of Shh pathway.After TXL treatment,the expression of these signaling molecules was remarkedly upregulated compared with that in pMCAO group at 6 and 24 hours(P<0.05 or P<0.01).Moreover,the Western blot analysis of Shh expression was further confirmed in immunofluorescence assay.These results suggest that TXL activates the Shh pathway after ischemic stroke.When TXL combined with cyclopamine or cyclopamine alone was administrated at 6 and 24 hours,the expression of Patched,Smo and Gli-1(only at 24 hours)was reduced than that in TXL group in Western blot assay(P<0.05 or P<0.01).However,there was no statistical difference between TXL group and TXL+CP group in the Shh expression(P>0.05).Meanwhile,the Shh expression in TXL+CP group was increased when compared with that in CP group(P<0.01).These data show that cyclopamine mainly blocks the downstream molecules of the Shh pathway such as Patched,Smo and Gli-1,but doesn't inhibit the Shh protein.Taken together,these results demonstrate that TXL could activate the Shh pathway after ischemic stroke,and the effect could be reversed by the Shh pathway inhibitor cyclopamine.3 Conclusion:The Shh pathway mediates Tongxinluo capsule-induced protection against blood-brain barrier disruption after ischemic stroke in mice.Experiment II Tongxinluo capsule protects against blood-brain barrier disruption after ischemic stroke through downregulating the low-density lipoprotein receptor-related protein-1 in mice1 Methods:The stroke model(pMCAO)was still used.Mice were randomly divided into 5 groups:Sham,pMCAO group,TXL group,receptor-associated protein(RAP)group(RAP)and TXL combined with RAP group(TXL+RAP).The mice in both TXL group and TXL+RAP group were orally administered with TXL(3.0 g/kg)at 1,3 and 21 hours after pMCAO,while other mice were given the same volume solvent(normal saline)in the corresponding time.Meanwhile,the mice in both RAP group and TXL+RAP group were intracerebroventricularly injected with RAP(5 ?L,9 ?M),while other mice were intracerebroventricularly administered with the same volume solvent(steril PBS)in the corresponding time.BBB permeability was examined by Evans blue in the ischemic penumbra of ipsilateral hemisphere at 6 and 24 hours after pMCAO.LRP-1 immunofluorescence was performed in the the ischemic penumbra of ipsilateral hemisphere at 6 and 24 hours after pMCAO.In addition,Western blot assay was performed for measuring the expression of TJ proteins[occludin,claudin-5 and zonula occludens-1(ZO-1)],and LRP-1 in the ischemic boundary cortex of ipsilateral hemisphere at 6 and 24 hours after pMCAO.2 Results:(1)Both EB fluorescence area and intensity in pMCAO group were significantly greater than those in Sham at 6 and 24 hours(P<0.01).Treatment with TXL,RAP or TXL+RAP reduced the fluorescence area and intensity at 6 and 24 hours compared with those in pMCAO group(P<0.05 or P<0.01).However,there were no statistical differences between TXL group and TXL+RAP group at 6 and 24 hours(P>0.05).These results suggest that both TXL and RAP could protect against BBB disruption after ischemic stroke,but the protective effect of TXL on BBB was not strengthened by RAP.(2)Western blot data showed that the expression of these TJ proteins(Occludin,Claudin-5 and ZO-1)in pMCAO group was significantly downregulated compared with that in Sham at 6 and 24 hours(P<0.01),which indicated cerebral ischemia caused the damage of TJs.After treatment with TXL,RAP or TXL+RAP,the expression of these TJ proteins was obviously upregulated when compared with that in pMCAO group at 6 and 24 hours(P<0.05 or P<0.01)indicating that TXL and RAP could upregulate the expression of these TJ proteins.(3)At 6 and 24 hours,the expression of LRP-1 in pMCAO group was significantly upregulated when compared with that in Sham(P<0.01).However,TXL and RAP downregulated the expression of LRP-1 in the periinfarct regions compared with that in pMCAO group(P<0.05 or P<0.01).3 Conclusion:TXL protects against BBB disruption after ischemic stroke through downregulating the expression of LRP-1 in mice.Chapter IV The effect and underlying mechanisms of Tongxinluo capsule on cerebral microcirculatory disturbances after ischemic stroke in miceSection I The effect of Tongxinluo capsule on cerebral microcirculatory disturbances after ischemic stroke in mice1 Methods:The ischemic stroke model(pMCAO)was still used.pMCAO mice were grouped and administrated as the Chapter ?.A cranial window of 3 mm in diameter was implemented on the top of the left parietal cortex(craniotomy window center:bregma 2.5 mm,left 2.5 mm).The angioarchitecture of brain microvessels in periinfarcted zones of different groups at 6 and 24 hours were mapped by the two-photon laser scanning microscopy(TPLSM)imaging after the mice were injected with RITC-Dextran(100 mg/mL,0.1 mL)for labeling plasma.TPLSM line scanning was recorded along the central axis of brain capillary in periinfarcted zones to measure capillary blood flow velocity(red blood cell velocity).Meanwhile,the volume flux through capillary cross sections per unit time Was calculated according to the formula F = V ×?×D2/4(F,flux;V,velocity of capillary blood flow;D,diameters of the scanning capillary).In addition,leukocytes in cortex microvenules labeled with Rhodamine 6G(0.04%,0.1 mL)were imaged by TPLSM at 6 and 24 hours.The number of leukocytes adhesion to venular walls per mm2 in periinfarcted zones was calculated with the software Image J.2 Results:(1)The velocity and volume flux in pMCAO group were si gnificantly decreased compared with those in Sham at 6 and 24 hours(P<0.01)indicating that ischemic stroke led to cerebral microcirculatory disturbances.However,different doses of TXL and NBP all increased the velocity and volume flux in periinfarcted cortex at 6 hours(P<0.01),while only TXL-H and NBP did at 24 hours(P<0.01).TXL-H was the best dose in increasing the velocity and volume flux.(2)The number of adherent leukocytes in pMCAO group was significantly increased compared with that in Sham at 6 and 24 hours(P<0.01).However,at 6 hours,different doses of TXL and NBP all decreased the number of adherent leukocytes in cortex venules after pMCAO,while only TXL-H and NBP did so at 24 hours(P<0.05 or P<0.01).Judging from the trend of bar graph,TXL-H was the best dose in decreasing the number of adherent leukocytes.3 Conclusion:TXL alleviates cerebral microcirculatory disturbances after ischemic stroke in association with inhibiting leukocyte-endothelial cell interactions in mice.Section II The underlying mechanisms of Tongxinluo capsule on improving cerebral microcirculatory disturbances after ischemic stroke in mice1 Methods:In the following mechanism research part,we chose the best efficacy of three doses(TXL-H)and still used the same stroke model(pMCAO).The mice were randomly divided into 3 groups:Sham,pMCAO group and TXL group(TXL).The mice in TXL group were orally administered with TXL(3.0 g/kg)at 1,3 and 21 hours after pMCAO,while other mice were given the same volume solvent(normal saline)in the corresponding time.6-keto-PGF1? and TXB2(stable metabolite of PGI2 and TXA2),and ET-1 in serum were measured by ELISA at 6 and 24 hours after pMCAO.In addition,adhesion molecules[intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VACM-1),P-selectin and E-selectin]in the ischemic cerebral cortex were examined by Western blot at 6 and 24 hours after pMCAO.2 Results:(1)The level of 6-keto-PGF1? in pMCAO group was significantly higher than that in Sham at 24 hours(P<0.01),while TXL reduced the level of 6-keto-PGFla at 24 hours(P<0.05).There were no significant differences between Sham,pMCAO group,and TXL group in 6-keto-PGF1?(6 hours)and TXB2(6 and 24 hours)levels(P>0.05).Unexpectedly,the ratio of 6-keto-PGF1? to TXB2 serum levels in pMCAO group was greater than that in Sham at 6 and 24 hours(P<0.01),whereas TXL lowered the ratio at 6 and 24 hours compared with that of pMCAO group(P<0.05).ET-1 level in pMCAO group was lower than that in Sham at 6 and 24 hours(P<0.01),while TXL increased ET-1 level compared with that in pMCAO group only at 6 hours(P<0.05).These interesting results suggest that the body has a compensatory adjustment against cerebral ischemia by increasing vasodilators such as 6-keto-PGF1? and the ratio of 6-keto-PGF1? and TXB2 levels,and reducing vasoconstrictors such as ET-1.(2)The expression of ICAM-1 in pMCAO group was significantly higher than that in Sham at 6 and 24 hours(P<0.01),while TXL downregulated ICAM-1 expression(P<0.05 or P<0.01).TXL downregulated P-selectin expression at 24 hours(P<0.01),but did not change the level of P-selectin at 6 hours compared with that in pMCAO group(P>0.05).There were no significant differences between pMCAO group and TXL group in the levels of VCAM-1 and E-selectin at 6 and 24 hours(P>0.05).3 Conclusion:TXL alleviates cerebral microcirculatory disturbances after ischemic stroke in association with modulated vascular endothelial function and inhibited leukocyte-endothelial cell interactions by regulating the expression of PGI2,TXA2,and ET-1,and suppressing adhesion molecules ICAM-1 and P-selectin.Total conclusions:1 Theoretical investigation:Under the guidance of vessels-networks theory of TCM,the critical role of brain "minute collateral-microvascular" injury has been revealed in the pathogenesis of ischemic stroke based on a close correlation between brain minute collaterals in TCM and microvessels in Western medicine.The collateral-unblocking therapy could protect cerebral minute collateral-microvessels in the ischemic zones after ischemic stroke and thus exert a protective effect on ischemic brain.These two points provide the theoretical support for the novel viewpoint"cerebral minute collateral-microvascular protection in the ischemic area after ischemic stroke”.Meanwhile,this study is used as a theoretical instruction for exploring the protective mechanisms of TXL on cerebral microvessels in ischemic stroke model.2 Experimental exploration:This study has confirm the protective effect of TXL on ischemic brain mediated by cerebral microvascular protection.Further study found that TXL could protect against BBB disruption after ischemic stroke through activating the Shh pathway and inhibiting LRP-1.In addition,TXL alleviates cerebral microcirculatory disturbances after ischemic stroke in association with modulated vascular endothelial function and inhibited leukocyte-endothelial cell interactions by regulating the expression of PGI2,TXA2,and ET-1,and suppressing adhesion molecules ICAM-1 and P-selectin. |
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