Study Of IGF-1Expression In The Bladder Of Diabetic Rats

Objective:The diabetic bladder is a urinary complication of diabetes, manifested as bladder dysfunction that is mainly caused by diabetic peripheral neuropathy. Diabetic bladder is related to the course of disease, whether experienced systematic treatments and the degree of neuropathy, while has nothing to do with sex and age. Diabetic bladder neuropathy mainly involves the parasympathetic (sacral2,3,4) and sympathetic (chest11,12and lumbar1,2) of urinary bladder, causing abnormal micturition reflex and weak bladder contractility, and ultimately leading to urinary retention or Urine dripping endless, even incontinence. The symptoms of the patient are lower abdominal pain, urinary frequency, urgency, odynuria and unable to urinate and so on. If the bladder of the patient is always with residual urine, urinary tract infection is easy to be caused, then leading to renal damage, and even renal failure. Therefore, early diagnosis and treatment of diabetic bladder disease is very important, and the research of its mechanism is paid more and more attention. In recent years, the insulin-like growth factor-1is found widely distribute in neurons and muscles, and its chemical structure is similar to insulin. Moreover, IGF-1is combined with its corresponding receptors so as to play a role, promoting reproduction of nerve and muscle cells, inhibiting apoptosis,and playing an important role in the growth and differentiation of neurons, energy metabolism and nerve protection.This study tested the expression changes of insulin-like growth factor1(IGF-1) in the bladder and lumbosacral dorsal root ganglion of diabetic rats by immunohistochemical methods, in order to indicate the role of IGF-1in the development of diabetic bladder diseases.Methods:1The experimental animals and grouping: 35male Sprague-Dawley (SD) rats were from Hebei Medical University Experimental Animal Center, weighing200-250g. Divided into two groups randomly, including15in control group,20in diabetic group. SD rats were fasted for more than12h (preferably overnight).Diabetic rats were injected STZ according to60mg/Kg by intraperitoneal, while control rats were injected sodium citrate buffers according to60mg/kg by intraperitoneal. Measured the concentration of fasting blood glucose (BG)after72h. If BG>16.7mmol/L, there was the successful modeling. if the fasting blood glucose concentration (BG) of diabetic rats was more than16.7mmol after11weeks/L, the diabetes rats suffered from diabetic bladder disease.2The production of the specimens and immunohistochemistry:Rats were anesthetized, we were taken to the bladder and lumbosacral dorsal root ganglia of rats at the experimental and control group, we were washed them by the normal saline and placed them in the bottle with the4%formaldehyde (0.1MPBS, PH7.0～7.6, containing0.1%of DEPC), we took0.5cm×0.1cm tissue block into the dehydration process, the solid paraffin was melted at the high temperature,we took the tissue block to embedd in the paraffin mold within the organization block;Immunohistochemical reagents were from Hebei Bohai Biological Engineering Development limited company packing products of SANTA CRUZ company in the United States, in accordance with the manual operation, the specific steps as follows:(1) Put5um thickness tissue sections onto glass slides which covered by polylysine forming the membrane, and placed in60℃overnight;(2) Immersed a slice in xylene for5minutes twice,100%ethanol for5minutes twice, and95%ethanol for5minutes twice,90%ethanol for5minutes twice,85%ethanol for5minutes twice,75%ethanol for5minutes twice, then rinsed by tap water, and washed by the PBS twice;(3) Put the slices into a hot solution of fixing antigen citric acid,it was warm through the microwave oven,it cooled to room temperature (100%fire one minute,20%of the fire one minute, stop for a minute, a total of four),we rinse them with distilled water;(4) Plus3%H2O2in it, incubating for10minutes to eliminate endogenous peroxidase activity, and washed by the distilled water once,0.1MPBS two times for5minutes;(5) Added ormal goat serum blocking solution onto sections and placed at room temperature for20minutes. Threw off the excess liquid and did not wash;(6) Dropped the first antibody onto sections and placed in4℃overnight, washed by0.1MPBS two times for5minutes;(7) Dropped biotin of the second antibody (I g, G) onto sections and placed in37℃for20minutes, washed by0.1MPBS two times for5minutes;(8) Dropped horseradish peroxidase-labeled streptavidin solution (SA/HRP) onto sections and placed in37℃for20minutes, washed by0.1MPBS two times for5minutes;(9) DAB coloring:used DAB coloration kit to mix1ml distilled water and each drop of the color reagent A, B, C, and mixed evenly, added to the specimen, colored for1minutes, the fully washed;(10) Counterstain the cell nucleus by hematoxylin for1minute, fully washed by water, differentiated by1%hydrochloric acid alcohol, blued by1%ammonium water, fully washed by water, with70%ethanol for5minutes,80%ethanol for5minutes,90%ethanol for5minutes twice, and95%ethanol for5minutes twice, dehydrated by100%ethanol for5minutes twice, xylene for5minutes twice, sealed sheet by neutral resin;(11) chose positive and negative tissue phases of experimental and control groups to take100×and400×photomicrography;chose meaningful tissue phases to login ID, number, collect, analyse, read data, and the last save.3Statistical analysis:statistical analysis is according to the percentage of IGF-1immunohistochemistry, with statistical significance of P<0.05, and completed by SPSS13.0software.Results:1Changes of blood sugarThe blood glucose was significantly increased (BG>16.7mmol/L) after72h of peritoneal injection of streptozotocin in rats.The blood sugar of the diabetic group (n=19) was significantly higher than that in the control group (n=15) when drawing materials. These two groups belonged to the measurement data, and applied two independent samples t-test. Compared control group and diabetic group (4.7333±0.61721vs29.52±1.62241), P=0.000. there was statistically significant differences when P value was less than0.05, indicating that this experiment STZ model was successful, and results were shown in Table.1and Fig.1.2HE staining and immunohistochemical resultsThe rat bladder detrusor structure under light microscope is as follows: the rat bladder transitional epithelium of control arranged in neat rows, submucosal and myenteric plexus were common, muscle fibers were arranged neatly, muscle bundles packed tightly, connective tissues were within the muscle gap, and the size of muscle cells were identical. The inflammatory cells infiltrated in the mucosa of diabetic rats, muscle bundles were deranged and loosened structure, muscle gap widened significantly, vascular congestion were between the muscle bundles, detrusor muscle cells were hypertrophy and with irregular shape. Based on these results, the diabetic bladder disease rat model was successful.Immunohistochemical results:compared with the normal group, IGF-1which was distributed in bladder mucosa and detrusor cell of diabetic group was significantly decreased. The results of IGF-1expression in bladder and DRG belonged to ranked data, using Wilcoxon test and the statistical results showed in Figure1:Mann-whitneyU statistic was49.000; WilcoxonW statistic was239.000; Z=-3.354, two-sided test P=0.001, as the standard of a=0.05, indicating that the IGF-1expression in the bladder of diabetic group markedly decreased with statistical significance. In the lumbosacral dorsal root ganglia, IGF-1mainly distributed in cytoplasm. Compared with the normal group, the expression in diabetic group were significantly reduced, and statistical results showed in Figure2:Mann-whitneyU statistic was9.000; WilcoxonW statistic was54.000; Z=-2.911, two-sided test P=0.004, as the standard of a=0.05, indicating that the IGF-1expression in lumbosacral dorsal root ganglia of diabetic rats was significantly lowered with statistically significance.Conclusion:This study observed the expression changes of insulin-like growth factor1(IGF-1) in the bladder of diabetic rats by immunohistochemical methods, and conclusions are as follows:IGF-1expression in bladder tissue of diabetic rats significantly reduced, and participated in the occurrence and development of the diabetic bladder disease.