Observation Of Basilar Membrane In The Choroid Plexus Of Lateral Ventricle And Cerebral Small Blood Vessel And Investigation Of The Expressions Of α-SMA And CD31 In The Cerebral Small Blood Vessel From Diabetic Rats

With an aging population and changing lifestyles, the life span was significantly prolonged in patients with diabetes. Then the chronic complications of diabetes are increasing. Autonomic and peripheral neuropathies are well-described complications in diabetes. Diabetes is also associated to central nervous system damage. But diabetic encephalopathy appears to be less considered than coronary and peripheral disease because of failure to find available distinctive diagnostic tools for its early identification. This little-known complication is characterized by impairment of brain functions associated with neurochemical and structural abnormalities.Microangiopathy as one of the distinctive complication of diabetes, its pathologic changes mainly show that small blood vessel basement membrane thickening, perithelial cell loss, and vascular endothelial cell proliferation and functional disorder. And then microangiopathy barrier function is destroyed and progressingly obstruction, tissue oxygen deficiency and so forth. The injury of the microangiopathy is correlation with the cognition functional disturbance in diabetic encephalopathy. These changes of function are accompanied with the abnormalities of the structural and neurochemical which is resulted from chronic high blood glucose concentration intra-cellular of the brain tissue.PartⅠObservation of basilar membrane in the choroid plexus of lateral ventricle and cerebral small blood vessel from diabetic ratsObjective: To observe the basilar membrane in the choroid plexus of lateral ventricle and cerebral small blood vessel from an experimental diabetic model in rats was established successfully induced by streptozotocin (STZ). The purpose of this study was to approach the pathologic progression of the damage in the brain small blood vessel after diabetes and provide laboratory basis for clinical early identification and therapy.Methods:⑴Experimental group: Healthy adult male Sprague-Dawley rats were randomly divided into 2 groups: STZ induced diabetic group (STZ group) and the control group (CON group). After the model was established successfully, each group was divided into 3 subgroups: 2 week group, 6 week group and 10 week group.⑵Model preparation: After fasting for 12 hours, the STZ group rats were intraperitoneal injection of STZ by i.p.at 55mg·kg-1. After monitoring plasma glucose consecutive for 3 days, rats having a non-fasting plasma glucose level of 16.7 mmol/L were considered to be diabetic.⑶Materials and samples preparation: Preparation for Paraffin block: At the corresponding time point, the body weight and the plasma glucose of rats in each group were both measured. After the perfusion with the 4% paraformaldehyde solution with intraperitoneal injection of 10% chloral hydrate, the rats were sacrificed. Pieces of selected brain tissue were immediately fixed in the same paraformaldehyde solution, then the samples were embedded in paraffin to make up solid paraffin block. Thereafter, cerebrum was obtained for optical microscopy studies with HE and trichrome staining. Preparation of samples for electron microscopy: At the corresponding time point, the body weight and the plasma glucose of rats in each group were both measured. After perfusion with the 4% paraformaldehyde-2.5% glutaraldehyde solution with intraperitoneal injection of 10% chloral hydrate, the rats were sacrificed. Pieces of selected cerebral cortex were immediately fixed in the 4% glutaraldehyde solution, chopped to get pieces of approximately 1 mm2 , and then post-fixed in 1% osmium tetroxide. They were then dehydrated in acetonum series, soaked, embedded in epoxy resin. Semithin sections were stained with toluidine for orientation and identification of the small blood vessel. Thin sections were stained with uranyl acetate and lead citrate. The sections were viewed and photographed with a OLYMPUS JEM-1230 electron microscope.⑷Observation of basilar membrane in the choroid plexus of lateral ventricle and cerebral small blood vessel: Using trichrome staining, changes of basilar membrane in the choroid plexus of lateral ventricle and cerebral small blood vessel were observed by optical microscopy. Alteration of the cerebral small blood vessel was observed by transmission electron microscopy.Results: Observation of basilar membrane in the choroid plexus of lateral ventricle and cerebral small blood vessel:Optical microscopy: No destruction of the basilar membrane in the choroid plexus of lateral ventricle and cerebral small blood vessel was observed in CON rats. Ambiguity, dissolve and the lacune widen of the vascular basilar membrane outside the choroid plexus of lateral ventricle were observed in STZ rats. Additionally the basilar membrane ambiguity, dissolve and even fracture from the cerebral small blood vessel were observed in STZ rats.Transmission electron microscopy: No destruction of the cerebral small blood vessel was observed in CON rats. Vascular endothelial cell and periphery astrocyte swelling, the basilar membrane thickening, ambiguity, dissolve and even fracture were observed in STZ rats.Conclusion: With the progression of diabetes, the vascular basilar membrane ambiguity, dissolve and the lacune widen were observed from outside of the choroid plexus of lateral ventricle, and basilar membrane thickening, dissolve and even fracture were observed from the cerebral small blood vessel in an experimental diabetic model. Additionally, vascular endothelial cell and periphery astrocyte swelling were observed in STZ rats. It indicates that the blood brain barrier may be damaged after diabetes. It may be one of the pathologic basis for the changes of the brain structural. PartⅡInvestigation of the expressions ofα-SMA and CD31 in the cerebral small blood vessel from diabetic ratsObjective: To detect the expression ofα-SMA and CD31 in the cerebral small blood vessel from an experimental diabetic model in rats was established successfully induced by streptozotocin(STZ). The purpose of this study was to approach the damage of the VSMC and VEC after diabetes and provide laboratory basis for clinical early identification and therapy.Methods:⑴Experimental group:Healthy adult male Sprague-Dawle -y rats were randomly divided into 2 groups: STZ induced diabetic group (STZ group) and the control group (CON group). After the model was established successfully, each group was divided into 3 subgroups: 2 week group, 6 week group and 10 week group.⑵Model preparation: After fasting for 12 hours, the STZ group rats were intraperitoneal injection of STZ by i.p.at 55mg·kg-1. After monitoring plasma glucose consecutive for 3 days, rats having a non-fasting plasma glucose level of 16.7 mmol / L were considered to be diabetic.⑶Materials and samples preparation: Preparation for Paraffin block: At the corresponding time point, the body weight and the plasma glucose of rats in each group were both measured. After the perfusion with the 4% paraformaldehyde solution with intraperitoneal injection of 10% chloral hydrate, the rats were sacrificed. Pieces of selected brain tissue were immediately fixed in the same paraformaldehyde solution, then the samples were embedded in paraffin to make up solid paraffin block. Thereafter, cerebrum was obtained for optical microscopy studies with immunohistochemical staining.⑷Immunohistochemical staining: Taking the paraffin block, processing relatively thin sections (5 um) on slides were made by paraffin machine. Thereafter, blocking with blood serum, treating respectively with anti-α-SMA and anti-CD31, incubatting by secondary and third antibodies, coloring with DAB, afterstainning with hematoxylin, dehydrating through alcohols and clearing in xylene, mounting with neutral gum,the expressions ofα-SMA and CD31 in the selected sections were detected by optical microscopy.(5)Statistical analysis: Data are expressed as mean±standard deviation ( x±s), statistical analysis was used SPSS 13.0 software. Group comparison was determined by one-way ANOVA. Differences with P < 0.05 values were considered statistically significant.Results:⑴Significantly decreased weight gain was observed in each STZ subgroup rats when compared to the corresponding controls(P<0.05). Significantly decreased weight gain was observed in 10 week group of STZ rats when compared to the 2 week group of STZ subgroup(P<0.05). Significantly increased weight gain was observed along with the increasing feeding time in CON group rats(P<0.05).⑵Significantly increased plasma glucose was observed in each STZ subgroup rats when compared to the corresponding controls(P<0.05). No significantly change in CON group rats. Significantly increased plasma glucose were observed in 6 week group and 10 week group of STZ subgroup rats when compared respectively to the 2 week group of STZ subgroup(P<0.05).⑶No significantly change of the expression ofα-SMA was observed in 2 week group of STZ subgroup rats when compared to the corresponding control. Significantly decreased the expressions ofα-SMA were observed in 6 week group and 10 week group of STZ subgroup rats when compared to the corresponding control(P<0.05). Significantly decreased the expressions ofα-SMA were observed in 6 week group and 10 week group of STZ subgroup rats when compared respectively to the 2 week group of STZ subgroup(P<0.05). No significantly change was observed in CON group rats.⑷No significantly change of the expression of CD31 was observed in 2 week group of STZ subgroup rats when compared to the corresponding control. Significantly increased the expressions of CD31 were observed in 6 week group and 10 week group of STZ subgroup rats when compared to the corresponding control(P<0.05). Significantly increased of the expressions of CD31 were observed in 6 week group and 10 week group of STZ subgroup rats when compared respectively to the 2 week group of STZ subgroup(P<0.05). No significantly change was observed in CON group rats.Conclusion:With the progression of diabetes, significantly increased the expressions of CD31 were observed in STZ rats when compared to the corresponding control at 6 week group and 10 week group subgroups were detected in an experimental diabetic model. While significantly decreased the expressions ofα-SMA were observed in STZ rats when compared to the corresponding control at 6 week group and 10 week group subgroups. It indicates that the phenotype conversion of VSMC may be involved in leading to diabetic cerebrovascular disease. Meanwhile, the damage of VSMC and VEC after diabetes seems to influence each other.