| Diabetes mellitus (DM) is one of the predominant diseases threatening human health in the world at present .Up to now, its pathogeney is still unclear. DM, with long-term chronic hyperglycemia, lead an increased risk of cerebral/cardiovascular diseases such as heart attack, stroke, vascular cognitive impairment and peripheral artery diseases(mainly occurs in the arms and legs). DM has been considered as one of the risk factors of cerebral vascular diseases. DM increase the risk of cerebral vascular diseases and may do so by the effects of insulin resistance, hyperglycemia-related increases in advanced glycation end products, and oxidative stress, inflammatory stimulus, and macrovascular or microvascular injured. Long-term hyperglycemia may be associated with cerebral microvascular instructural integrity and functions. At present the effect to cerebral microvascular by diseases, especially to the BBB appears to be less considered.P-gp(P-glycoprotein), a member of the ATP-binding cassette(ABC)-transporter family, is a 170 kDa membrane protein that is constitutively expressed on the BBB. P-gp plays a critical role on maintainning the homeostasis between the central nervous system and the blood circulation.Objective :To observe the structure of the blood-brain-barrier(BBB), we use the diabetic rat model caused by Streptozotocin (STZ) .The structure of blood capillary and the expression of P-gp on BBB were observed at various time points by HE staining and electron microscope. The purpose of this study was to approach the pathologic of the damage in central nervous system after diabetes and to provide science basis for furthe study about its damage to brainMethods and materials1 Experimental group: A total of 75 healthy adult male Sprague-Dawley rats with body weighing between 180～200g 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 5 subgroups: 2 week, 4 week, 6 week, 8 week and 10 week.There are 15 rats for each control subgroup and 15 rats for each diabetic subgroup.2 Model 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.3 Samples preparation for histopathology study: 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. The structure of blood capillary was observed by optical microscopy studies with HE staining.4 Preparation of samples for electron microscopy: 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 and hippocampus were immediately fixed in the 4%glutaraldehyde solution, chopped to get pieces of approximately 1mm×1mm×2mm , 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 capillary. Thin sections were stained with uranyl acetate and lead citrate. The sections were viewed and photographed with electron microscope.5 Immunohistochemical staining: 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.Taking the paraffin block, processing relatively thin sections (5 um) on slides were made by paraffin machine. Thereafter, blocking with blood serum, treating with anti-P-gp, incubatting by secondary and third antibodys, coloring with DAB, afterstainning with hematoxylin, dehydrating through alcohols and clearing in xylene, mounting with neutral gum,the expressions of P-gp on the selected sections were detected by optical microscopy.Results1 Histopathology and electron microscope observation1.1 Histopathology: No destruction in the cerebral blood capillary was observed both in every control group and diabetic group1.2 Electron microscope observation: Vascular endothelial cell swelling was observed at week 2 STZ group. Discontinuity of the vascular endothelium was observed in STZ rats with the progression of diabetes.1.3 Immunohistochemical staining: Significantly decreased the expressions of P-gp were observed in STZ subgroup rats when compared to the corresponding control(P<0.01).Conclusion1 Our findings were manifested diabete mellitus indeed can injure the formation and constructure of BBB,even in its early stage.Although there is no significent change in HE pictures between control and experimental groups, but many apparent impairments can be found by electron microscope. The hazards of hyperglycaemia should arouse our attention.The effective measures were taken to prevent or reverse the vascular damage caused by hyperglycaemia.2 The expression of P-gp locates in microvessel endothelium. Through the experiment we found DM can down-regulate the expression of P-gp.It might have some connection with DM complications. |
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