| Background Diabetic nephropathy and acral gangrene are severe microvascular changes in Diabetes mellitus (DM), as well as the common causes of mortality and morbidity. Early microvascular changes are functional and usually reversible. As early intervention can prevent or delay the progression of kidney impairment, early vascular investigation may provide clue for early diagnosis and treatment, so studies on microvascular pathologic changes has become an importan subject.The development and progression of kidney impairment and acral gangrene often lead to changes in blood perfusion or prefusion-related changes. It is therefore important to evaluate tissue blood perfusion correctly because it is of great significance for judging the status of tissue function, severity of impairment and reversion.Studies have confirmed that contrast-enhanced ultrasonography (CEUS) is a new technique for evaluating blood perfusion. As the new contrast agent SonoVue contains microbubbles that are smaller than red blood cells, they can reach the capillary network of renal parenchyma, thus enhancing and displaying blood flow signals of intra-renal microvessels, and facilitating blood perfusion in the kidneys. CEUS is characterized by real time, non-ionizing radiation, repeatability, low cost, and high space and time resolution. To some extent, we could learn the characteristics of renal blood perfusion by studying perfusion and elimination of the microbubbles in the kidneys, and by analysing contrast time-intensity curves (TIC).e-Flow technique is a new blood flow detection scheme based on conventional energy Doppler technique. It has a significantly higher sensitivity to extremely low blood flow and better imaging, thus able to truely display blood filling of peripheral microvessels and providing more sensitive blood flow information as compared with the colored Doppler and energry Doppler techniques.The present study was divided in five parts,the first three parts are animal experiments designed to observe the changes of renal blood perfusion in GK rats and pathologic changes by comparing CEUS and histomorphologic findings.The fourth and fifth part are the clinical sdudies to investigate facts in the occurence and progression of microvessels lesions of kidney and acra in DM patients. Objective To study early pathologic changes in kidney tissue of Goto-Kakizaki (GK) diabetes prone rats and facts of dynamic change with progression of the disease so as to provide a morphologic basis for early diagnosis and intervention of early renal blood perfusion and impairment in type 2 DM patients.Methods①Based on ages,30 GK male rats were equally divided to 4-week,12-and 20-week groups. Thirty age-matched Wistar male rats were used as the control.②Systolic pressure was measured by tail volume photoelectric manometry.③Blood from the cardiac cavity was measured for glucose, blood urea nitrogen (BUN), creatinine (CR), total cholesterol (TC) and triglyceride (TG).④Urine taken by bladder puncture was measured for protein concentration by BCA, and protein composition by SDS-PAGE electrophoresis.⑤Kideny specimens were paraffin-embedded routinely, prepared electromicroscopically, and study under optical microscopy and electronic microscopy.Results①The blood glucose levels of GK rats in each group are higher than their corresponding control groups (P<0.01).②Urine protein, kidney weight, and length of 12-and 20-week GK rats were higher than those of the corresponding control groups (P<0.05), and this tendency increased gradually while the age in increased in each group. There was statistical differences between the three DM groups (P<0.01).③The thickness of the renal parencyma and cortex of the 12-week GK rats was significantly higher than that of the corresponding control group (P<0.01), The differences between the three DM groups were significant (P<0.01).④Optical microscopy in the 12-week GK rats revealed hyalinization, sclerosis, inflammatory cell infiltration. In the 20-week GK rats, there are local or complete hyalinization and sclerotic atrophy of multiple glomerula, and absence of glomerula in some visual fields; increased composition of glomerular mesangial matrix and collagen; thickening of the basement membrane; hardening, hyalinization and luminal stenosis of interstitial small blood vessels; atresia of afferent glomerular arterioles.⑤Electronic microscopy in the 12-week GK rats revealed absence and abnormal morphology of some podocytes, with fuzzy fusion of inter-cellular space. In the 20-week GK rats, we can see collapse of renal glomerula, swelling of epothelial cells, decrease in organelles, swelling of mitochondria, and absence of crests; expansion, proliferation and swelling of mesangial cells, and stenosis or obstruction of lumens; swelling of podocytes, partial or extensive fusion or disappearance of foot processes, and uneven thickening of the basement membrane.Conclusion Hyperglycemia occurred in 4-week GK rats, and typical pathologic changes of kidney impairment occurred in 12-week group. The impairments include: enlargement and focal sclerosis of renal glomerula, expansion of mesangial mesangial areas, thickening of glomerular capillary basement membranes which deteriorated with the course of disease prolonging. These morphologic abnormalities may be the pathogenic basis of early glomerular hyperfiltration. GK rats is an ideal animal model for studying kidney impairment of tyep 2 DM.Objective To investigate changes in microvascular density in the kidney of GK rats and its correlation with the development and progression of type 2 DM in order to provide the morphological information for study in kidney perfusion.Methods The experimental animals and grouping criteria are the same as of part one. Kideny specimens were paraffin-embedded and sliced routinely and stained by immunohistochemistry.5 specimen sections from each rat, eighteen visual fields from each section were selected randomly in the cortex and medulla areas of each section. Microvascular density in these areas was analyzed quantitatively using an image analysis system. Five glomerula were selected randomly from the cortex in each section, separated irregularly, and analyzed for the density of glomerular capillaries, and the area and circumference of the glomerula.Results①The medulla microvascular density of the three GK groups was significantly lower than that of the three corresponding control groups (P<0.05-P<0.01). There was significant difference between the three GK groups (P<0.01), while there was no significant difference between the three control groups (P>0.05).②The glomerula microvascular density of the 20-week GK rats was significantly lower than that of the corresponding control group (P<0.01). There was significant difference between both the GK group and the control group (P<0.01-P<0.05).③The mean circumference of glomerulus of 4-week GK rats was significantly longer than that of the corresponding control group (P=0.01). There was significant difference both between the GK groups and between the control groups (P<0.01-P<0.05).Conclusion Renal microvascular sparsity was present in GK rats. As this phenomenon occurred after DM and became gradually apparent along with the progress of diabetes, we assume that it is a secondary change caused by DM. Medulla microvascular sparsity in young GK rats and may also be related to hereditary factors.Objective To explore changes of blood perfusion in renal parenchyma of spontaneously diabetic GK rats in an attempt to provide experimental clues for early diagnosis and intervention of diabetic kidney impairment in diabetic patients.Methods The experimental animals and grouping criteria are the same as of part one. Ultrasound contrast agent SonoVue was administered at a dose of 0.028ml/100g body weight by quick intravenous bolus injection through femoral vein cannulation. Imaging changes in the rat renal parenchyma were observed in real time, and dynamic images within 0-5min of contrast injection were observed and stored. The characteristics of blood perfusion in the renal parenchyma were analyzed using the time-intensity curve (TIC).Results①The kidney was quickly enhanced after injection of the contrast agent in all groups, and the renal artery, cortex, renal vein were displayed in sequence;②TIC of the renal parenchyma was a single-peak type with a steep ascending stair and a flat descending stair. The overall shape of TIC of the DM groups was wider than that of the control groups. With increase in age, TIC became progressively wider;③The slope rate of the ascending curve in 4-,12-and 20-week GK rats became smaller, the time to peak (TTP) was prolonged, and the area under the ascending curve and the total area under the curve were increased. The differences were statistically significant (P<0.05). The total area under the curve increased with the age of the diabetic rats increasing. There were significant differences between the groups (P<0.05).Conclusion Real-time contrast-enhanced ultrasonography can effectively display changes of blood perfusion in renal parenchyma of spontaneously diabetic GK rats, indicating that abnormal blood perfusion of renal parenchymal microcirculation already exists in the early stage of spontaneous diabetes.Objective To investigate the clinical importance of real-time gray-scale contrast-enhanced ultrasonography and quantitative analysis in assessing diabetes complicated kidney perfusion impairment.Methods 21 healthy adults were selected as the control.19patients with early kidney impairment (Chronic Kidney Disease, CKD 1-2) and 14 patients with late kidney impairment (CKD 4-5) were included in the study groups. Cr and BUN were detected one day before ultrasonographic examination in all subjects. SonoVue (0.03 ml/kg) was injected by intravenous bolus injection through the cubital vein for real-time gray-scale contrast-enhanced ultrasonographic examination. Dynamic images within 0-5min of contrast injection were observed and stored. The perfusion process of the renal cortex was observed. Using the quantitative analysis software, the outage time-intensity curves (TIC) with more than 85% goodness of fitness index (GOF) were chosen for analysis for basic intensity (BI), intensity increment (A1), slope rate of descending curve (A2), slope rate of ascending curve (A3), arriving time (AT), time to peak (TTP), mean transit time (MTT), peak intensity (PI), and area under the curve (AUC). The above results were statistically analyzed, and correlations between these parameters of TTP,AUC and GFR were also analyzed.Results①The renal artery-cortex-pyramid-renal vein was displayed in sequence after intravenous bolus injection of the contrast agent. AT of the renal cortex in the early and late kidney impairment groups was slower than that in the control group;②TIC of renal cortex perfusion presented as an asymmetric single-peak curve. Compared with the control group, the ascending curve of the early and late kidney impairment group was flat, and the peak was low.③Compared with the control group, AT and TTP of the early kidney impairment group were prolonged (P<0.05-P<0.01); AUC1, AUC2 and AUC were increased, though the differences were not statistically significant (P>0.05); Al and PI were decreased (P<0.05). Compared with the control group, AT and TTP of the late kidney impairment group were prolonged (P<0.05-P<0.01); AUC1, AUC2 and AUC were decreased (P<0.05); Al and PI were reduced (P<0.05-P<0.01). Compared with the early kidney impairment group, AUC1, AUC2 and AUC of the late kidney impairment group were increased significantly (P<0.01); AT was prolonged significantly (P<0.01); TTP was prolonged. AUC were positively correlated with GFR(r= 0.472, P=0.01),and TTP was not correlated with GFR((r=0.262, P=0.177).Conclusion Contrast-enhanced ultrasonography is sensitive to blood perfusion changes in diabetes-induced kidney impairment, and could possibly be a clinically practicable technique for the reflecting changes in haemodynamics and blood perfusion of kidney, due to early kidney impairment, evaluating the degree of affection; estimating filterability of renal glomerulm and providing grading standard for kidney impairment.Objective To study the anatomic structures and the hemodynamics parameters of small artery of digit tip or arteriole on nail bed; To evaluate the microcirculation in digit tip of 2 type diabetic. Methods The small arteries in digit tip and arteriole on nail bed of 73 case of diabetic divided into three groups according to the course of disease and 40 normal adults setup as a control group were imaged and their hemodynamics parameters were measured by e-flow imaging. The amount of arteries was assessed by calculating their pixel.Results①The way of most arteriole on nail bed were parallel with finger and toe and there are transverse branch among longitudinal arteries.②In the diabetes groupⅡ,Ⅲthere are less arteriole on nail bed of finger and toe and the blood flow frequency spectrum present a high block waveform..③In the diabetes group I,contrasted with control group,the EDV of small arteries in finger tip and toe tip and arteriole on nail bed of toe decreased (P<0.05) and RI increased remarkably (P<0.05/P<0.01);The average pixel of arteriole on nail bed of toe decreased (P<0.05).④In the diabetes groupⅡ,Ⅲ, the PSV,EDV,average pixel of small arteries in finger tip and toe tip and arteriole on nail bed are lower (P<0.01/P<0.05) and RI are higher (P<0.01)than that of control group.⑤PSV,EDV,average pixel of small artery and arteriole on nail bed of toe tip were lower (P<0.01)and RI were higher (P<0.01/P<0.05)than same mark of finger significantly in every groups of diabetes and control group.Conclusions The changes of homodynamic parameters of arteriole of digit tip can occur within 5 years after the diagnosis of diabetes. The amount of arteries reduced in advanced stage. The arteriole injury of the end of extremity happens earlier than ischemia symptom. The arteriole damage in toe tip occurs earlier and more severe than that in finger. E-flow can be used as an efficient method to evaluate early arteriole pathological changes in the end of extremity.
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