| Part One: Establish the method to test dysfunction of skin vasodilation and study on the microcirculation function in individuals with normal glucose toleranceObjective: To investigate the reproducibility of the capillary recruitment and the influence of occlusion time on postocclusive skin reactive hyperemia using computerized videophotometric capillaroscopy and to measure microcirculation function in individuals with normal glucose tolerance.Methods: We estimated baseline capillary density by counting the number of continuously erythrocyte-perfused capillaries and used postocclusive reactive hyperemia to estimate the functional reserve. Postocclusive capillary recruitment was calculated by dividing the increase in density by the baseline density. 53 individuals with normal glucose tolerance were enrolled and analyzed. Nailfold capillaries in finger skin were recorded before and after 1, 4 or 8 minutes of arterial occlusion with a digital cuff in 9 individuals to investigate the influence of different occlusion time. Duplicate measurements were performed in 9 individuals on 2 separate days to determine the day-to-day coefficient of variation of postocclusive capillary recruitment.Results: The percent increase of capillary density was only (21.3±7.0) % when the occlusion time was 1 minute, lowed than those with the occlusion time of 4 and 8 minutes. There was no difference in the percent increase between 4 or 8 minutes occlusion. The average age of non-diabetic subjects was (65.6±6.7) years old. The average baseline capillary density was (37.5±8.2) /mm~2, with an average peak capillary density of (50.3±9.6) /mm~2. The average percent increase was (37.50±6.8) %, and there was no difference between male and female subjects. Age was statistically associated with percent increase(r=0.413, P＜0.05). The day-to-day coefficient of variation of postocclusive capillary recruitment was 18.7±2.0%.Conclusion: Capillary recruiment is an ideal method to measure the microcirculation dysfunction because of high reproducibility and stability. We selected 4 minutes as the occlusion time for its convenience and precise. We performed Capillary recruitment in individuals with normal glucose tolerance and found that age was associated with the percent increase of capillary density. Part Two: Study on the microcirculation dysfunction in type 2 diabetic patients and its relation with diabetic complicationsObjective: To measure microcirculation function in type 2 diabetic patients and to analyze its relation with diabetic complications.Methods: 367 type 2 diabetic patients in Shanghai downtown were enrolled and categorized into several groups, those with diabetic retinopathy or not, those with albuminuria or not, those with diabetic peripheral neuropathy or not, and those with carotid artery plaque or not. We examined the capillary recruitment, the randomized morning urinary albumin creatinine ratio, the digitally stored fundus images, the carotid artery intima media thickness and carotid plaque by the Doppler, and compared the percentage increase of capillary density among these groups.Results: The average age of diabetic patients was (66.0±10.9) years old. The average percentage increase of capillary density was (29.6±12.3) %. Age, diabetes duration, HbAlc, and systolic blood pressure were statistically associated with the percent increase of capillary density (P＜0.05). The percentage increases of capillary density in those with diabetic retinopathy, albuminuria, and peripheral neuropathy were significantly decreased as compared of those of controls. There was no significant difference between patients with diabetic macrovascular complications and controls. The percentage increase of capillary density of 20-30% or＜20% was an independent risk factor of diabetic retinopathy (adjusted OR: 1.013, 95%CI: 1.006-1.020; adjusted OR: 1.023, 95%CI: 1.015-1.030) and albuminuria (adjusted OR: 1.007; 95%CI: 1.003-1.011; adjusted OR: 1.015; 95%CI: 1.010-1.020). Also, the percent increase of＜20% was an independent risk factor of diabetic peripheral neuropathy (adjusted OR: 5.188; 95%CI: 2.201 -12.231).Conclusion: The percentage increase of capillary density was an independent risk factor of diabetic retinopathy, albuminuria, and diabetic peripheral neuropathy. There was no significant relation between dysfunction of microcirculation and diabetic macrovascular complications. Measurement of capillary recruitment might help us to observe the occurrence and progression of diabetic microvascular complications.Part Three: Study on the changes of mesentery microcirculation in type 2 diabetic rats and the possible mechanismObjective: To study the dysfunction of microvessels and micro-blood-flow in mesentery microcirculation of type 2 diabetic rats.Methods: Thirty-three OLETF (otsuka long-evans tokushima fatty) type 2 diabetic rats and seven LETO (long-evans tokushima otsuka) non-diabetic control rats were used in the study. After general anesthesia, a video microscope with image analysis system was used to observe the mesentery microcirculation in diabetic rats and controls. The microvascular number, diameter of arterioles and venules, number of rolling and sticking leukocytes on the surface of venules endothelium, width of marginal flow and axial flow in the arterioles, and thickness of endothelium were measured. The results were analysed quantitatively with the video microscopic image analysis system. Semi-quantitative RT-PCR and Western blotting were performed to verify the differential expression of some important antioxidative enzymes.Results: Compared with control rats, the microvascular number in the mesentery of diabetic rats was reduced by 24.5 % (P＜0.01), which was inversely correlated to blood glucose level (r=-0.44, P＜0.05). The width of marginal flow in diabetic rats was decreased, and the ratio of width of marginal flow to width of arteriole reduced significantly (P＜0.01). The rolling and sticking leukocyte number adhered to the endothelium in the venule was increased significantly (P＜0.01). Results of RT-PCR and Western blotting showed no significantly different expression of SOD2 and catalase between OLETF and LETO rats. Meanwhile, the level of urine 8-iso-PGF2a was significantly increased in OLETF rats(P＜0.05).Conclusions: The overt microvascular abnormality and micro-blood-flow dysfunction was shown in mesentery microcirculation of diabetic rats. Meanwhile, the level of oxidative stress in diabetic rats was increased, acting with microcirculation dysfunction to make a vacious cycle, which together might contribute to the development of diabetes and its complications. They were the pathogenetic basis of microangiopathy and various complications in diabetic rats. |
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