糖尿病大鼠视网膜微血管变化与血液流变学的关系

Relationship between the changes of retinal microvessel and hemorrheology in diabetic rats

目的:观察糖尿病大鼠早期视网膜毛细血管的变化情况及其与血液流变学的关系。方法:实验于2003-03/2004-03在中山大学医学动物实验中心完成。选取无菌级成年纯系雄性Wistar大鼠30只,随机分为糖尿病组和对照组,15只/组。糖尿病组腹腔注射链脲佐菌素65mg/kg(用0.1mol/L、pH4.5柠檬酸盐缓冲液配制)诱发糖尿病,对照组腹腔注射等量柠檬酸盐缓冲液。于糖尿病发病后4,6,12周,将两组大鼠空腹麻醉后尾静脉采血进行糖化血红蛋白、全血黏度、血浆黏度、红细胞压积、纤维蛋白原、红细胞聚集性及血液流变触性等检测,制备视网膜血管铺片,常规过碘酸-Schiff染色法染色后,光学显微镜观察,计数10个高倍镜视野的视网膜微血管壁的细胞数。空腹血糖检测采用葡萄糖氧化法,糖化血红蛋白检测用微柱层析法,全血黏度、血浆黏度、红细胞压积、纤维蛋白原的测定用SDZ-3型自动电子计时黏度计,红细胞聚集性和血液流变触性的测定采用LG-B-190型红细胞聚集仪。结果:实验共纳入30只大鼠,全部进入结果分析。①两组大鼠腹腔注药前后血糖变化:腹腔注药前,两组大鼠空腹血糖均低于6.5mmol/L(t=1.6149,P>0.05),尿糖定性均为阴性。腹腔注药后第1,3,7天,糖尿病组大鼠血糖均持续高于19mmol/L,且尿糖持续阳性,而对照组大鼠血糖均低于6.5mmol/L以下,尿糖阴性。注药后两组大鼠血糖具有显著性差异(t=5.6482～5.6694,P<0.01)。②糖尿病发病后不同时间两组大鼠视网膜血管形态观察结果:糖尿病组发病后4周,毛细血管行走尚规则,血管壁细胞数目及血管形态无明显异常改变;发病后6周,血管壁细胞数目减少;发病后12周,血管壁细胞数进一步减少,毛细血管迂曲,管径粗细不一,毛细血管呈囊样扩张。对照组在以上各实验时点毛细血管行走规则,管径粗细均一,视网膜毛细血管无异常改变。③糖尿病发病后不同时间两组大鼠糖化血红蛋白与视网膜微血管壁细胞数目的变化:发病后4,6,12周糖尿病组糖化血红蛋白均明显高于对照组犤(7.9±1.3),(6.1±1.2)%,P<0.05;(10.3±2.1),(6.2±1.1)%,P<0.01;(10.4±2.5),(6.2±1.1)%,P<0.01犦。发病后4周糖尿病组视网膜微血管壁细胞数与对照组基本相近犤(56±6.5),(58±6.2)个,P>0.05犦,而发病后6,12周糖尿病组视网膜微血管壁细胞数均显著低于对照组犤(42±5.3),(59±6.1);(28±4.5),(58±5.9)个,P均<0.01犦。④血液流变学各指标间的相关性分析:双变量相关分析表明,糖尿病大鼠视网膜微血管变化与糖化血红蛋白水平、全血黏度、血浆黏度、红细胞压积、血浆纤维蛋白原含量、红细胞聚集指数及糖尿病病程呈高度负相关(r值分别为-0.8216,-0.7518,-0.7422,-0.7651,-0.7366,-0.7537,P均<0.01),而与血液流变触性呈正相关(r=0.7682,P<0.01)。结论:糖尿病大鼠发病后4周即可检测到血液流变学的异常,且随病程的延长而逐渐加重,发病后6周开始发生视网膜微血管形态的改变。视网膜微血管的变化与糖化血红蛋白水平、血液流变学各指标的异常和糖尿病病程密切相关。因此,对糖尿病患者早期纠正其血液流变学的异常糖代谢紊乱对降低糖尿病视网膜病变的发生率有一定帮助。

AIM： To observe the relationship of the changes of retina blood capillary in the earlier period with hemorrheology in diabetic rats. METHODS： The experiment was carried out in the animal experimental center of Sun Yat-sen University from March 2003 to March 2004. Thirty germ-free adult pure male Wistar rats were selected, and divided randomly into two groups： diabetes group and control group with 15 in each group. The rats in the diabetes group were injected with 65 mg/kg streptozotocin through abdominal cavity （dispensed by 0.1 mol/L, pH 4.5 citrate buffer） to induce diabetes, and those in the control group were injected with ,the same dosage of citrate buffer through abdominal cavity. At the 4, 6 and 12 weeks after the onset of diabetes, the blood was gained from caudal vein after anesthesia in rats with an empty stomach in the two groups to perform the detection of glycosylation hemoglobin （HbAlc）, blood viscosity, plasma viscosity, hematocrit, fibrinogen, erythrocyte aggregation and blood thixotropy etc. The stretched preparation of blood vessel of retina was made, and after routine iodic acid-Schiff staining, observed by the light microscope, under 10 high magnification fields the number of cells of microvessel of retina was counted. The fasting blood glucose （FBG） was detected by glucose oxidase detection; HbAle was detected by microcylinder chromatography; . The blood viscosity, plasma viscosity, hematocrit and fibrinogen were measured by SDZ-3 auto-electron viscosimeter. The erythrocyte aggregation and blood thixotropy were measured by LD-B-190 aggregatimeter. RESULTS： Thirty rats were involved in the experiment and were all involved in the result analysis. ① Changes of blood sugar in rats in the two groups before and after abdominal cavity injection： Before injection, the blood sugar in the rats with an empty stomach in the two groups was lower than 6.5 mmol/L（t=.614 9,P 〉 0.05）, and the urine sugar was negative. At the 1^th, 3^rd and 7^th days after abdominal cavity injection, the blood sugar in the rats of diabetes group was higher continuously than 19 mmol/L, and the urine sugar was positive, while that in rats of the control group was lower than 6.5 mmol/L, and showed negative. There were significant differences of the blood sugar in the rats of the two groups after injection （t=5.648 2- 5.669 4,P〈 0.01 ）. ② Observational result of formation of blood vessel of retina in rats of the two groups at the different time after onset of the diabetes： At the 4 weeks after episode, the ambulation of blood capillary was regular, and there was insignificant change of the number of cell in the vessel wall and the formation of blood vessel; At 6 weeks after episode, the number of cells in the vessel wall decreased; At 12 weeks after episode, the number of cells in the vessel wall reduced continuously with winding blood capillary and different diameter of caliber, and the blood capillary showed bladder-like dilatahion. The ambulation of blood capillary at every experimental time point in the control group was regular with different diameter of caliber and without abnormal changes of blood capillary of retina, ③ Changes of the level of HbAlc and the number of cells of microvessel wall of retina in different time after the onset of diabetes： At 4, 6 and 12 weeks after episode, the level of HbAlc in the diabetes group was significantly higher than that in the control group [（7.9±1.3）, （6.1 ±1.2）%, P〈 0.05; （10.3±2.1）, （6.2±1.1）%, P〈 0.01;（10.4±2.5）, （6.2±1.1）%,P 〈 0.01]. At 4 weeks after the episode, the number of cells on microvessel wall of retina in the diabetes group was close to that in the control group [（56±6.5）, （58±6.2） each, P 〉 0.05], while at the 6 and 12 weeks after episode, the number of cells on microvessel wall of retina in the diabetes group was remarkably lower than that in the control group [（42±5.3）,（59±6.1）; （28±4.5）, （58±5.9） each, P〈0.01]. ④ Correlation analysis among every index of hemorheology： The double variable related analysis showed that the changes of the microvessel of retina in diabetic rats had highly negative correlation with level of HbAlc, content of blood viscosity, plasma viscosity, hematocrit and fibrin0gen, the erythrocyte aggregation index and the progress of diabetes （r value were -0.821 6, -0.751 8, -0.742 2,-0.765 1, -0.736 6, -0.753 7, respectively,P 〈 0.01）, while had positive correlation with blood thixotropy （r =0.7682, P 〈 0.01）. CONCLUSION： The hemorheologic abnormity can be detected in 4 weeks after the onset of diabetes in the diabetic rats, and become more pronounced with the prolongation of diabetic course. The microvessel formation of retina changes at the 6 weeks after episode. The changes of microvessel of retina have closed correlation with the level of HbAlc, the abnormity of every index of hemorheology and the progress of diabetes. Therefore, it has certain assistance on the correction of abnormal carbohydrate metabolism of hemorheology in an earlier period and the decrease of the incidence of diabetic retinopathy in diabetic patients.