| WTO defines “cerebral ischemic stroke” as a kinds of clinical syndromes.Its characteristic is the ischemia of blood vessel,which would lead focal outbreak of cerebral injury over 24 hours,or even death.The rate of disability and death over patients might be increasing due to the reducement of oxygen and glucose,which caused by brain’s blood interruption.As we all known,cerebral ischemic stroke usually combined with high rate of incidence,disease recurrence,death,as well as disability,which may bring heavy burden towards society and family.Cerebral ischemic stroke’s risk factors includes age,smoking,diabetes,hypertension and obesity.Besides,diseases with high possibility of embolism may also increase the risk of incidence,such as valvulopathy and auricular fibrillation.Along with the change of life style and the aggravating of aging population,the morbidity of angiocardiopathy includes cerebral ischemic stroke tends to raise year by year,and has yet became the major death cause of our country’s inhabitants.That’s why the prevention of cerebral ischemic stroke deserves no delay.Although recent research has achieved some progressions,cerebral ischemic stroke remains a global troublesome issue with great influence over economy of society.At present,several studies show the self-healing ability of central nervous system is quite limited.However,the self-healing ability of organism notably surpass that of central nervous system after the outbreak of cerebral ischemic stroke.The ultimate purpose of neurorestoration mainly achieved through neuranagenesis,neural plasticity and revascularization.More and more studies indicate that revascularization’s effect on neurorestoration is increasing.Unfortunately,so far there’s no highly effective means that could make clear observation of these newborn blood vessels’ particular changes and trends.A variety of methods have been used to label the changes of structure and quantify density of brain vasculature surrounding peri-infarct tissue in histological sections of experimental animals.With the advances of the multi-labeling technique of confocal microscopy,immunohistochemistry for vascular associated antigens including platelet endothelial cell adhesion molecule(PECAM-1)also known as cluster of differentiation 31(CD31),and von Willebrand factor on endothelial cells;collagen IV,laminin and fibronectin in basal lamina have been widely used for detection and quantification of vascular changes in both animal models and humans.There are also protein antigens on endothelial cells which had been exploited to distinguish microvascular development by use of glucose transporter 1(GLUT1)and integrin αvβ3.However,all of these methods only showed clear staining of blood vessel walls under microscope.As the limitation of absent display of lumens by these protein antigens,it makes errors for quantification of vascular density by stereological methods.Moreover,most cerebral blood vessels in paraformaldehye fixed tissue stain poorly for these antigens.In living transgenic animals,the brain capillary network has also been labeled by fluorescent proteins,making it available for long term observation of the structural and hemodynamic changes of the same microvessel at a particular location.However,long-term investigation of vascular structure changes in vivo is usually dependent on two-photon laser scanning microscopy and the new technology of genetic engineering,which makes it not suitable for use in most of the labs due to the expensive cost.Gelatin-ink perfusion is an effective method for detecting microvessels.Gelatin can be dissolved in water at temperatures above 40°C and then coagulated at temperatures below 30°C.We found that gelatin-ink perfusion made a complete filling of peripheral retinal vessel lumen with a satisfactory vascular continuity.The number of retinal microvessels labeled by gelatin-ink was more than by vWf immunostaining.Moreover,gelatin-ink perfusion caused no contamination to the following immunostaining of neurons or glial cells in the same tissue.Except ink perfusion,gelatin can also be conjugated with different fluorescent substances for co-staining with other antigens in the brain.Adult male C57BL/6 mice were used and subjected to occlusion of distal branches of middle cerebral artery(dMCAO).In this study,we used gelatin-conjugated perfusion of mice ischemic brain to test whether the labeling was an alternative method to quantify angiogenesis in penumbra as well as in infarct core region.We also investigated whether this labeling technique can be used to detect formation of leptomeningeal anastomoses after stroke on the same brain slice.By using Image J analyzing software,we presented the detailed operation approach for quantification.This study is divided into three parts,each part is summarized as follows.Part one To observe the ethology and hemodynamics of the experimental cerebral infarction miceObjective: The neural function defect and cerebral infarction volume of experimental cerebral infarction mice were observed by ethology and hemodynamic methods to verify the effectiveness of d MCAO.Methods: Adult male C57BL/6 mice were used and subjected to dMCAO.Experiment 1: All the mice were randomly assigned to the following groups: Sham group(Sham): animals received sham operation.3 days after stroke group(3 d): three days after animals received operation.7 days after stroke group(7 d): seven days after animals received operation.14 days after stroke group(14 d): fourteen days after animals received operation.We assessed neurologic deficits with Rota-Rod test and modified neurological severity score(mNSS)at base,3,7 and 14 days after stroke.Experiment 2: All the mice were randomly assigned to the following groups: Sham group(Sham): animals received sham operation.3 days after stroke group(3 d): three days after animals received operation.7 days after stroke group(7 d): seven days after animals received operation.14 days after stroke group(14 d): fourteen days after animals received operation.We calculated infarct volume with TTC staining in each group.Experiment 3: All the mice were randomly assigned to the following groups: Sham group(Sham): animals received sham operation.3 days after stroke group(3 d): three days after animals received operation.7 days after stroke group(7 d): seven days after animals received operation.14 days after stroke group(14 d): fourteen days after animals received operation.We examined cerebral blood flow(CBF)using a laser speckle imaging at baseline,0,3,7 and 14 days after stroke.Results:1.In the Rota-Rod test,Compared with the sham group without middle cerebral artery occlusion,the neurological function of the 3 d,7 d and 14 d groups decreased significantly after middle cerebral artery occlusion(P<0.05).Compared with 3 d group,the neurological function of 14 d group was improved significantly(P<0.05).mNSS results showed that the neurological deficit score in other group was significantly higher than that in sham group(P < 0.05).The neurological deficit score of 14 d group was significantly lower than that of 3 d group(P<0.05).2.After TTC staining,The volume of cerebral infarction in mice immediately after MCA occlusion,3 days after MCA occlusion,7 days after MCA occlusion and 14 days after MCA occlusion was significantly increased(P < 0.05).3.CBF measurements showed that cerebral blood flow was significantly reduced in 3 d,7 d and 14 d groups compared with the sham group without MCA(P<0.05).Part two The optimal concentration was determined by perfusing gelatin-ink at different concentrations,and microvessels in different regions of the cerebral hemisphere of healthy mice were observedObjective: By perfusing healthy mice with different concentrations of gelatin-ink,the microvessels perfused at which concentrations were most clear and which sites were the best suited for subsequent observation.Methods: Adult male C57BL/6 mice were used as the research object.Experiment 1: All the mice were randomly assigned to the two groups: 20% group: mice were infused with 20% gelatin-ink;3% group: mice were perfused with 3% gelatin-ink.The microvessels of the two groups were compared to observe the concentration of gelatin-ink perfusion can show more clear microvessels.Experiment 2: mice were perfused with 3 % gelatin-ink to observe the density and filling status of the microvessels in each region.Results:1.We found 20% gelatin-ink showed a satisfactory filling and vascular continuity of cortical arteries and proximal anastomoses at the circle of Willis including basilar artery,MCA,anterior and posterior cerebral arteries.The potential secondary collateral pathways provided by leptomeningeal vessels also achieved a complete filling with low background.However,due to the high viscosity of 20% gelatin-ink,a less filling effect was observed in micro capillaries when compared to that in the condition using 3% gelatin-ink perfusion.2.After 3% gelatin-ink perfusion in mice,we observed the microvasculature in the anterior cerebral artery(ACA),middle cerebral artery(MCA),posterior cerebral artery(PCA),striatum and hippocampus,which had the largest number of perforators and capillaries and the best filling performance.We found that the number and size of these perforating and capillary vessels are greatest in MCA territory.Compared with MCA,the vascular density of the remaining four regions decreased significantly(P<0.05).The microvessel density in the hippocampus was also significantly reduced compared with that in the striatum and the anterior cerebral artery(P<0.05).Part three Different methods were used to observe the angiogenesis of experimental cerebral infarction mice in different time periodsObjective: The vascular regeneration of experimental cerebral infarction mice in different time and different areas was observed by gelatin-ink perfusion and HE staining.Methods: Adult male C57BL/6 mice were used as the research object.Experiment 1: mice 3 days after cerebral infarction were perfused with 3% gelatin-ink.The blood vessel density of the infarct,the contralateral side of the infarct,the superior side of the infarct,the contralateral side of the superior side of the infarct,the inferior side of the infarct,the contralateral side of the inferior side of the infarct,the basal ganglia and the contralateral side of the basal ganglia were observed under a microscope and analyzed statisti-cally.Experiment 2: mice 7 days after cerebral infarction were perfused with 3% gelatin-ink.The blood vessel density of the infarct,the contralateral side of the infarct,the superior side of the infarct,the contralateral side of the superior side of the infarct,the inferior side of the infarct,the contralateral side of the inferior side of the infarct,the basal ganglia and the contralateral side of the basal ganglia were observed under a microscope and analyzed statistically.Experiment 3: mice 14 days after cerebral infarction were perfused with 3% gelatin-ink.The blood vessel density of the infarct,the contralateral side of the infarct,the superior side of the infarct,the contralateral side of the superior side of the infarct,the inferior side of the infarct,the contralateral side of the inferior side of the infarct,the basal ganglia and the contralateral side of the basal ganglia were observed under a microscope and analyzed statistically.Experiment 4: The mice were randomly assigned to the two groups: Sham group(Sham): animals received sham operation,the contours of the cells and capillaries were observed by HE staining and gelatin-ink perfusion double labeling.14 days after stroke group(14 d): fourteen days after animals received operation,the contours of the cells and capillaries were observed by HE staining and gelatin-ink perfusion double labeling.Results:1.The results of experiment 1 show that by 3% gelatin-ink perfusion,we observed a significant reduction in the number of capillaries in the center of the infarct and in the ischemic penumbra,whereas almost no intact filled capillaries were seen in the center of the infarct.In contrast,the capillaries on the opposite side of the infarct were well filled and did not decrease significantly.According to the statistical analysis of the capillary density in different areas,it can be seen that the density of blood vessels in the infarct has a significant decrease compared with the contralateral side of the infarct,the superior side of the infarct,the inferior side of the infarct and the basal ganglia(P<0.05).The density of blood vessels on the superior side of the infarct and its opposite side,the inferior side of the infarct and its opposite side,the basal ganglia and its opposite side also decreased significantly(P < 0.05).2.The results of experiment 2 show that by 3% gelatin-ink perfusion,the density of blood vessels in the infarct has a significant decrease compared with the contralateral side of the infarct,the superior side of the infarct,the inferior side of the infarct and the basal ganglia(P<0.05).The density of blood vessels on the superior side of the infarct and its opposite side also decreased significantly(P<0.05).In contrast,no difference of the density of blood vessels on the inferior side of the infarct and its opposite side,the basal ganglia and its opposite side(P>0.05).3.The results of experiment 3 show that by 3% gelatin-ink perfusion,we observed a significant increase in vascular density in the infarct center and ischemic penumbra on day 14 after surgery.The new perforating vessels and capillaries outgrew aberrantly in necrotic tissue in infarct core came from leptomeningeal branches,and presented as more larger,tortuous and engorged vessels.According to the statistical analysis of the capillary density in different areas,it can be seen that the density of blood vessels in the infarct has a significant decrease compared with the contralateral side of the infarct,the superior side of the infarct,the inferior side of the infarct and the basal ganglia(P<0.05).In contrast,no difference of the density of blood vessels on the superior side of the infarct and its opposite side,the inferior side of the infarct and its opposite side,the basal ganglia and its opposite side(P>0.05).4.The results of experiment 3 show that during the recovery stage following stroke,the number of intensely HE labeled nuclei were significantly increased in infarct core which was companied by increased new born vessels originating from leptomeningeal anastomoses.Degenerating necrotic plaque presented as even eosinophilic staining without nuclear components were also shown in the infarct core.These observation in gelatin-ink infused slice showed three major events occurring in infarct core region during the recovery stage of stroke: increased glial cell proliferation;enhanced angiogenesis from secondary leptomeningeal collaterals;degeneration of glial cells with formation of necrotic plaque.Conclusions:1.Adult male C57BL/6 mice were successfully established by dMCAO.We found that the neural function of experimental mice in acute stage of ischemic stroke was seriously damaged and CBF was significantly reduced.In the convalescent period,the neural function of the experimental mice was improved obviously,the volume of cerebral infarction was decreased obviously,and the CBF was increased obviously.2.In this experiment,different concentrations of gelatin-ink were used to perfuse the experimental mice,and 3% gelatin-ink was used to perfuse the mice for the first time,which solved the key problem of how to observe the microvascular regeneration after ischemic stroke,and provided a new method for microvascular perfusion and observation in the future.3.In this study,3% gelatin-ink was used to perfuse mice 3,7,14 days after cerebral infarction.We found in the acute phase of ischemic stroke,the perforators and microvessels in the central area of the infarct are fragmented and hardened.During the convalescence of ischemic stroke,cerebral cortical branches present a large number of tortuous,swollen meningeal vascular hyperplasia,which can cross the cortical surface in the center of the infarct.At the same time,by using HE staining and gelatin-ink perfusion double labeling,we found that increased glial cell proliferation;enhanced angiogenesis from secondary leptomeningeal collaterals;degeneration of glial cells with formation of necrotic plaque during the recovery stage of stroke. |
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