Establishment Of Experimental Diabetic Rat Model And Surveying Indexes

Objective To establish an animal model similar to the metabolic abnormalities of human type 2 diabetes mellitus with insulin resistance,hyperglycemia,hyperlipemia and hyperinsulinism which could induce complication of angioneuropathy.Methods Eight-week-old SD male rats(n=120) were radomly divided into control group and model group.The model group male SD rats(n=90) were fed with high-fat diet that contained unsaturated fatty acid for 4 weeks and then streptozotion(STZ, one-step,25mg/kg) was injected intraperitoneally,then streptozotion(STZ,40 mg/kg) was injected intraperitoneally again 4 weeks later to result in insulin-secretion defect and hyperglycemia.The control group(n=30) were fed with normal diet.Blood lipid was detected by automatic biochemical equipments.Results Biochemical parameters proved that the two-step-treatment methods could develop the type 2 diabetic rat model,the successfully executive rate was 76%.The model was similar to the metabolic abnormalities of human type 2 diabetes mellitus with insulin resistance,hyperglycemia,hyperlipemia and hyperinsulinism.In the type 2 diabetic group,LDL was 0.75±0.32mmol/L,TC was 1.43±1.07 mmol/L,TG was 1.72±0.6 mmol/L,insulin was 42.36±9.57 mIU/L,GHb was 6.71±1.37%.There were statistical significance of these indexes between type 2 diabetic group and control group(P value＜0.05).Conclusion1.Relatively convenient and easily got experimental type 2 diabetic rat model which reproduced human type 2 diabetic pathogenic mechanism was successfully induced by high-fat diet and twice different dose of STZ injection.2.The model exhibited type 2 diabetic characters of hyperglycemia,hyperlipemia and insulin resistance.It was the ideal animal model for investigating mechanism of type 2 diabetic pathogenic mechanism and medicine research.3.The model presented lipoidosis metabolic obstruction,hyperglycemia and insulin resistance which were the key factors of vasoneuropathy complication.This model was of long term stability and suitable for pathogenesy research of diabetic chronic complications which established a good foundation for observing vasoneuropathy of diabetic experimental animals. Objective:Diabetic neuropathy is an important complication of type two diabetes,but its mechanism is still unclear.We have establish a type 2 diabetic rat model in order to investigate the relationship of aldose reductase(AR),O₂^—,catalase(CAT) and glutathion peroxidase(GPx) with brain tissue damage,and observe the impact of blood glucose concentration on each observed index to illuminate internal association between hyperglycosemia,oxidative stress,vasculitides and brain pathology,and to reveal close factors of diabetic vasoneuropathy in order to provide reliable laboratory proof for prevention and treatment of diabetic brain neuropathy.Methods:203 healthy adult male SD rats were randomly divided into control group(n=60) and diabetic group(n=143).The diabetic group was further divided into L group(13.8mmol/L＜blood glucose＜16.7mmol/L) and H group(blood glucose＞16.7 mmol/L).Blood and brain tissues were taken at 8th week,12th week and 16th week and have been made into pathological sections.Pathological injurey of brain tissue was observed through optical microscope and electron microscope.Expressions of hyperoxide,glutathion peroxidase,catalase and aldose reductase in brain were detected by immunohistochemistry and immunoblot essay.Messenger ribonucleic acid expressions of hyperoxide,glutathion peroxidase and aldose reductase were tested by reverse transcription polymerase chain reaction.The serum O₂^—,H₂O₂,SOD,CAT levels were measured by ultraviolet spectrophotometry and activity of aldose reductase was tested by NADPH decreasing method.Antineutrophil cytoplasmic antibody was detected by immunofluorescence and enzymic method.Results:1.There were brain tissue vascular endothelial cell swelling,dim nucleus, sparseness of endochylema,perithelial cell's degeneration,glassy degeneration of capillary vessel wall,crinkle collapse of vessel wall,focal hyperplasia of gliocyte and attenuation of GRL under optical microscope at the 16th week.Injury of brain blood vessel and neuron was gradually aggravated following course of disease.Pathology induced by different blood glucose concentration exhibited diversely in different groups. Pathology of H group was more severe than that of L group.2.The O₂^- activity in diabetic groups were significantly lower than that in control group(P＜0.05).There was no statistical difference of O₂^- activity between diabetic group L and diabetic group H(P＞0.05).The H₂O₂ activity in diabetic groups were significantly higher than that in control group(P＜0.01).There was statistical difference of H₂O₂ activity between diabetic group L and diabetic group H(P＜0.05).3.The serum messenger ribonucleic acid expressions of GP_x,Cu-Zn-SOD,Mn-SOD in diabetic group were all higher than that in control group(p＜0.05).There was no statistical difference of mRNA expressions between diabetic group L and diabetic group H(P＞0.05).Aldose reductase mRNA expression of serum and brain tissue bomogenate in diabetic group was obviously higher than that in control group(p＜0.05),but there was no statistical difference of aldose reductase mRNA expression between diabetic group L and diabetic group H(P＞0.05) both in serum and in brain tissue bomogenate.4.Both AR mRNA expression and AR activity of serum and brain tissue in diabetic group were significantly higher than that in control group.The increased AR expression of brain tissue had close correlation with pathological changes in brain,and it indicated high AR expression was a main reason of brain tissue damage.AR expression of brain tissue in diabetic H group was lower than in L group,which hinted it was not a simple linear relationship between AR and blood glucose.5.The expressions of SOD,GPX and CAT in brain tissue grew downwards gradually with the course of disease.AR expressions rised at the 8th and 12th week,and declined at the 16th week.There were both expressions of GP_x,CAT,SOD and AR in diabetic rats and control group rats'pallium,cornu ammonis and blood vessels,SOD expression mainly located in kytoplasm and cellular membrane,CAT and GP_x expressed in kytoplasm,and expression of AR concentrated in nucleus and nuclear membrane. 6.Antineutrophil cytoplasmic antibody(ANCA) was detected neither in human nor in rat control group,but there was one probable positive case diabetic rat group.and four positive case in diabetic human group.Conclusion1.There were pathological changes in brain,blood vessels,and nerve tissue of diabetic rats to different extents,and which turned to be more obvious with the developmental course of disease.Pathological changes of H group with higher blood glucose took place more early and quickly than in L group,which indicated apparent brain tissue damages correlated intimately with course of disease and blood glucose level.2.In diabetic rats,oxygen free radical level rised,gene expression of antioxidase increased but activity of antioxidase decreased,and the difference had statistical significance compared with control group,which offered a proof of strengthened oxidative stress in the whole body of diabetic rats.SOD,GP_x and CAT expressions in brain tissue were raised up at initial stage and decreased gradually with development of course of disease,which indicated obviously strengthened oxidative stress in brain tissue.3.There was statistical difference of H₂O₂ contents and SOD activity in rats serum between diabetic groups with different blood glucose concentration,and it indicated a close correlation between activity of H₂O₂ and SOD with blood glucose concentration. There were no statistical difference of O₂^—,GPx,CAT activity and gene expressions of SOD and GPx between diabetic groups with different blood glucose concentration. Blood glucose concentration didn't have obvious effects on these factors,and it might be one of the reasons for bad preventive and curing effects on diabetic complications just merely by blood glucose control.4.Both AR mRNA expression and AR activity of serum and brain tissue in diabetic group were significantly higher than that in control group.The increased AR expression of brain tissue homogenate had close correlation with pathological changes in brain,and it indicated high AR expression was a main reason of brain tissue damage. AR expression of brain tissue in diabetic H group was lower than in L group,which hinted AR didn't have linear relationship with rising of blood glucose.5.Antineutrophil cytoplasmic antibody(ANCA) was detected neither in human nor in rat control group,but there was one probable positive case diabetic rat group.and four positive case in diabetic human group,and it manifested that ANCA did not take the principal role in capillary inflammation,but it might be one of the etiological factors of diabetes.There were evident oxidative stress and aldose reductase alteration in diabetic rat brain tissue,and they had close correlation with vasculopathy and neuropathy in brain tissue.Oxidative stress and aldose reductase promoted diabetic brain damage in common.The relationship of oxyradical,antioxidase and aldose reductase with blood glucose concentration showed different regularities,and it might be the key reasons for altered curing effects of anti-oxidized therapia and aldose reductase inhibitor on diabetic neuropathy.