| Background: Type 2 diabetes,as a chronic disease,is prone to complications of cerebral vascular and central nerve injury.The study is little that type 2 diabetes result in cerebrovascular remodeling and nerve damage in brain neurons form structure and synaptic changes.Its pathologic process is still not very clear,and the intervention in this area is still no breakthrough,lacking of effective treatment measures.Therefore,clear brain damage mechanism of type 2 diabetes and look for effective diabetes neuroprotective drugs,intervent cerebrovascular and brain injury caused by diabetes,still are the research hotspot and difficulty in medicine today.Some studies have found that aminoguanidine showed good effects on delaying all kinds of complication caused by type 2 diabetes,so we speculate that it has potential therapeutic value in brain protection in type 2 diabetes,but its protection mechanism has not yet been elucidated.Objective: This experiment aims to study the pathological process and mechanism of neurologic dysfunction with cerebral injure caused by type 2 diabetes,observe the cerebral vascular remodeling of rats with type 2 diabetes,the changes of dendrites and dendritic spines,and the regulation effect of related factors.This experiment aims to study the effects of aminoguanidine intervention on cerebral injury in type 2 diabetic rats,investigate the neuropathological changes of brain injury and the possible neuroprotective effects and mechanism of aminoguanidine intervention in type 2 diabetic rats,provide some experimental basis for clinical research and drug therapy.Part I: Behavioral changes of type 2 diabetic rats and the effects of aminoguanidine interventionMethods: SD rats were divided into three groups randomly: Control group? model group(diabetes group)? treatment group(aminoguanidine intervention group).The model group and treatment group were modeled Type 2 diabetic rats successfully(high sugar and high fat diet + STZ(30 mg/kg)by intraperitoneal injection of one-time),the treatment group were received aminoguanidine oral gavaging(at a dose of 150 mg.kg-1.min-1).Respectively in 12 weeks,16 weeks,observe the neurological symptoms of rats in each group,calculate the score of stroke index,and evaluate the behavioral changes of learning and memory ability in rats by Morris water maze.Results: 1.Stroke index of rats Compared with the control group,the index of stroke in the model group and the treatment group was significantly increased(P < 0.05 or P < 0.01).Compared with the model group,the index of stroke in the treatment group was significantly decreased(P < 0.05).Over time,the index of stroke in the 16-week model group increased significantly(P < 0.05).It was suggested that cerebral vascular injury increased with the course of type 2 disease.After aminoguanidine intervention,the cerebral vascular injury and neurological function of rats improved significantly.2.Ethology assessment of rats Compared with the control group,model group and treatment group rats showed significantly increased escape latency and reduced number crossing the platform(P < 0.05 or P < 0.01),it showed that the learning and memory ability of rats in the model group decreased,and there was a certain cognitive dysfunction.Over time,the alteration was significantly aggravated at 16-weeks group.Compared with model group,treatment group at each time point showed significantly improved latency and number crossing the platform(P < 0.05),indicating that aminoguanidine intervention ameliorated the learning and memory deficits,improved neural function.Part II: The cerebral vascular remodeling and protective effect of aminoguanidine intervention in rats with type 2 diabetes mellitus.Methods: SD rats were divided into three groups randomly: Control group? model group (diabetes group)? treatment group(aminoguanidine intervention group).Respectively in 12 weeks.16 weeks,we observed the changes of cerebrovascular endothelial cells in the hippocampus of rats under transmission electron microscope,determined the expression of CD34 and EGFL7 by immunohistochemistry,detected the ROS content using flow cytometry instrument LSRII,determined the level of TNF-?and VEGF taking ELISA method.Aim to investigate the pathologic mechanism of cerebrovascular remodeling in type 2 diabetes and the protective effect of aminoguanidine intervention.Results: 1.Cerebrovascular remodeling under transmission electron microscope Control group: The cerebrovascular cavity was normal.The endothelial cells had normal nucleolus.The chromatin distribution was uniform and the basement membrane was clear.Model group: In 12 weeks,the cerebrovascular cavity was deformed,vascular endothelial cells were swollen and the nucleolus were rare,the intracellular chromatin was concentrated,apoptotic bodies appeared,mitochondrial swelling was found.And the basement membrane structure is not clear.In 16 weeks,the cerebrovascular cavity had different degrees of deformation and collapse,the endothelial cells were more swollen,the nucleus disappeared,the chromatin was large gathering in cells,the apoptotic bodies was more,vesicular degeneration can be seen in the cytoplasm.The basement membrane was swollen and thickened.Treatment group: In each time point of the treatment group,the vascular cavity was irregular in parts of cerebrovascular,the endothelial cells was less swollen,the intracellular dyeing tend to be even,nucleolus was visible or invisible.The basement membrane less swollen and the structure was clear.2.Expression of CD34 Compared with the control group,the expression of CD34 in the model group and the treatment group increased significantly(P < 0.05 or P < 0.01).In the model group,the expression of CD34 was significantly increased in 16 weeks(P < 0.05).Compared with the model group,the expression of CD34 was significantly reduced in the treatment group(P < 0.05). 3.Expression of EGFL7 The expression of EGFL7 was not seen in the cerebrovascular endothelial cells in the control group,and the expression of EGFL7 in the model group and treatment group was significantly increased compared with the control group(P < 0.05 or P < 0.01).In the model group,the expression of EGFL7 was significantly increased in 16 weeks(P < 0.05)with disease progression.Compared with the model group,the expression of EGFL7 was significantly reduced in the treatment group(P < 0.05).4.ROS content Compared with the control group,both the model group and the treatment group showed a significant increase in ROS content(P < 0.05 or P < 0.01).Comparison in the model group,with the progress of the disease,the ROS content increased significantly in 16 weeks(P < 0.05).Compared with the model group,the ROS content was significantly reduced at each time point in the treatment group(P < 0.05).5.Level of TNF-?/VEGF Compared with the control group,the levels of TNF-?/VEGF in both the model group and the treatment group were significantly increased(P < 0.01).In the model group,the level of TNF-?/VEGF was significantly increased in the 16 weeks with the progress of the disease(P < 0.05 / P < 0.01).Compared with the model group,the levels of TNF-?/VEGF were significantly reduced in the treatment group(P < 0.01/P < 0.001).Part III: The mechanism of dendritic and dendritic spines changes and protective effect of aminoguanidine intervention in type 2 diabetic rats.Methods: SD rats were divided into three groups randomly: Control group? model group(diabetes group)? treatment group(aminoguanidine intervention group).Respectively in the 12 weeks,16 weeks,the brains were processed for rapid Golgi staining.And the changes of dendritic branch and length and spine density in hippocampal neuron were observed under optical microscope.The expression of Spinophilin were determined by immunohistochemistry.Aim to explore the pathogenesis of cerebral injury in type 2 diabetes mellitus and the neuroprotective effect and mechanism of aminoguanidine intervention.Results: 1.Results of dendritic branches and length.Compared with the control group,the dendritic branches and length in the model group and treatment group decreased significantly(P < 0.05).In the model group,the dendritic branches and length in the 16 weeks was significantly reduced(P < 0.05).Compared with the model group,the dendritic branches and length in the treatment group increased significantly(P < 0.05).2.Determination of dendritic spine.Compared with the control group,the density of dendritic spines in the model group and treatment group decreased significantly(P < 0.05).The density of dendritic spine was significantly reduced in 16 weeks in the model group(P < 0.05),indicating that the density of dendritic spine decreased with the progression of diabetes.Compared with the model group,the density of dendritic spine increased significantly(P < 0.05).3.Expression of Spinophilin.Compared with the control group,the expression of Spinophilin in the model group and treatment group showed significantly reduced(P < 0.05).The expression of Spinophilin was significantly reduced in 16 weeks in the model group(P < 0.05).The expression of Spinophilin in treatment group increased significantly than that in model group(P < 0.05).Conclusion 1.Type 2 diabetes can result in different levels of stroke and behavioral impairment in learning and memory loss in rats,and it progressed with the progression of the disease.2.Aminoguanidine intervention can improve the stroke index and the learning and memory ability of rats,and improve the neural function of type 2 diabetic rats.3.Cerebrovascular remodeling was showed in Type 2 diabetes mellitus rats.The cerebrovascular endothelial cells were swollen,the cavity was deformed,the basement membrane thickened,the chromatin gathered,and the hyperplasia capillaries were present,but the proliferating blood vessels were pathologic.4.The process of cerebrovascular remodeling in type 2 diabetes rats was a chronic pathological process involving multiple factors,including EGFL7,ROS,TNF-?,VEGF and CD34.5.Type 2 diabetes could cause traumatic changes in dendritic morphology and dendritic spines,and lower expression of Spinophilin,which was one of the pathogenesis of diabetes complicated cerebral injury.6.It was suggested that the neuroprotection of Aminoguanidine intervention in type 2 diabetes rats might be attributed to inhibition of EGFL7 expression and VEGF level,decrease of ROS content and TNF-? level,so as to improve the vascular endothelial cells injury and angiopathologic hyperplasia in cerebrovascular remodeling process.7.The neuroprotection of Aminoguanidine intervention in type 2 diabetes rats appeared to be associated with the improvement of the cerebral dendritic morphology,increase the expression of Spinophilin to regulate the density of dendritic spines,improving the function of synaptic. |
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