The Expression Of Nerve Growth Factor In Lumbar Dorsal Root Ganglia And Bladder Of Diabetic Rats After Being Treated

Objective: The purpose of this study was to investigate the expression of nerve growth factor(NGF) in lumbar dorsal root ganglia (dorsal root ganglia, DRG) and bladder of streptozotocin- induced diabetic rats which were treated by insulin and NGF,and to analysize the pathogenesis of diabetic bladder disease ( diabetic cystopathy, DC) and the treatment of NGF and insulin. furthermore,that was to provide adequate experimental basis for us to cure DC by NGF.Methods: 35 adult male Wistar rats whose weight is 180～220g were randomly selected 7 rats which were intraperitoneal injection of 50mg/kg sodium citrate - citric acid buffer to make a control group (NC group), when they were at 1, 4 and 8 weeks we test the tail vein blood glucose to eliminate diabetic rats(BG> 16.7mmo1/L ).According Schleicher methods, the remaining rats can drink water but not eat anything at dusk one day before ,then they were singly injected with 1% of the streptozotocin (50mg/kg, by 0.1mol / L of sodium citrate - citric acid buffer, PH4.2～4.5)to induce diabetes, measured the blood glucose of tail vein by blood glucose device after 72 hours. BG> 16.7mmo1 / L was identified as a diabetic rat model of success. As a result there were 26 rats which were successful model. The diabetic model rats were feed for common forage and taken food and water freely.There were 24 rats which were remaining after 4 weeks as that, then they were randomly divided into 4 groups, named: DC group 6 rats, DC only NGF-injected group (NGF group) 6 rats, DC only insulin-injected group (RI group) 6 rats, DC combining injection of insulin and NGF group (NGF + RI group, subcutaneous injection of insulin administered through the neck, NGF through intra-abdominal injection) 6 rats. The rats were given different treatments: one time one day, a total of 4 weeks.Experiment was carried out when 8 weeks after diabetic model was built and the rats'tail vein blood glucose concentrations would be re-tested except NC group and the ones whose BG≤16.7mmol/L would be removed. One day before the experiment the rats were putted into the metabolism cage to measure urine volume in 24 hours.On the experimental day ,the weight of rats were measured ,the whole urinary bladder and both sides of L6S1 DRG were cut off,urinary bladder wet weight was measured. A part of bladder and one side of L6S1 DRG were put into 4% glutaraldehyde liquid for being observed under electron microscope.The surplus of bladder and L6S1 DRG were put into 4% paraformaldehyde liquid,using hematoxylineosin (HE) dyeing,morphological changes of diabetic rats urinary bladder detrusor and L6S1 DRG was observed under microscope.The expression of NGF in the bladder detrusor cell and the neurons of L6S1 DRG were measured by Immunohistochemical staining.Under the microscope the results were observed ,it was positive in the urinary bladder destusor cells or the neurons of L6S1 DRG when yellowish brown pellet emerges. the average optical density value (OD value) represented NGF expression.Using Motic Med 6.0 digital medical image analysis system the results of immunohistochemistry was semi-quantitative analysis under the same magnifying power .Under high power field (×200) 6 random access locations were elected in corresponding location but not repeat from each slice (3 slices each rat, to observe 2 high power field each slice). The average value of 6 fields was the average optical density value. The average value of two slices was the experimental results of the sample rats.Statistical analysis: All measurement data was demonstrated by mean±standard ( (x|-)±s) deviation. we tested the two means using t-test or t'-test and tested multiple means using one way analysis of variance(one way ANOVA), and Q-test was used to conduct group comparison with SAS 8.0 statistical software. Statistical significance: Set P> 0.05 for no significant difference; P <0.05 said that the difference was significant; P <0.01, said there was very significant difference.Results:1 Comparisons of weight, blood glucose, urine volume in 24h, urinary bladder wet weight of DC group and NC groupThe model after 8 weeks was successfully set up that is to say 4 weeks after treatment, weight, blood glucose, a 24h urine volume, urinary bladder wet weight of DC group compard with NC group: (219.5±11.06g)vs(309.7±8.38g)(t=-16.73 P<0.01) ;(30.9±2.33mmol/L)vs(4.3±0.18mmol/L)(t=30.33 P<0.01) ;(90.63±5.00ml)vs(21.33±2.08ml)(t=33.65 P<0.01) ;(171.97±4.32mg)vs(136.19±3.47mg)(t=16.57 P<0.01)were significantly higher than NC group.2 The results of transmission electron microscopyControl group: detrusor cells were well-distributed, similar in configuration, neat in arrangement, close in connection between the cells; the muscle cell membrane vesicle was a lot; the size and shape of mitochondria in cells was normal, the stroma density was normal and dense bodys were well-distributed; the size and shape of myelin sheath of L6S1 DRG nerve was similar, arranged in concentric circles, cytoplasm was uniform, neuroglia was normal.Experimental groups: detrusor cells were clutter in distribution, diverse in form, disorder in arrangement, connections between the cells reduced and the gap was filled by collagen fibers and amorphous components, the muscle cell membrane vesicle reduced, mitochondria in cells was oncotic, the ridge reduced, breaked and even disappeared, the stroma density reduced and dense bodys were clutter in distribution; the nerve myelin sheath of L6S1 DRG was dropsical, degenerated, breaked and deciduous. The nerve cytoplasm was vacuolar degeneration.The nerve cytoplasm was vacuolar degeneration. Above performance was worst in DC group, was relative lighter in RI and NGF group, which was similar with the normal control group. 3 Immunohistochemical analysis of the resultsThe structure of the bladder detrusor and L6S1 DRG of experimental group rats was observed under light microscope. (1)The normal control group: After HE staining the transverse and longitudinal muscle bundle which was cut off was arranged neatly, the structure was tight, the gap was filled by connective tissue. The lamina propria was integrity.The nerve bundle could be seen under mucous membrane and intermuscular. After Immunohistochemical staining the size and shape of detrusor cell was uniform, muscle fiber was arranged in order, the connection was tight between cells.The size and shape of nerve neurons cell was arranged in order, the connection was tight between cells. (2)The experimental group: After HE staining the muscle budle of detrusor muscle was arranged in disorder,the space between the muscle budle was enlarged obviously,the gap were not filled by connective tissue,the collagen fiber was reduced between muscle budle. Small vessels between muscle budles hyperemia. The nerve bundle could be seen. After Immunohistochemical staining detrusor muscle cell was hypertrophic, the form was diverse and arranged in disorder, the structure was fluffy. The nerve neurons cell was arranged in disorder, the cell was dropsical, the shape was variform, interstitial tissue hyperplasia, the structure was fluffy. Above performance was worst in DC group, was relative lighter in RI and NGF group, which was similar with the normal control group.4 The comparison of NGF expression in the bladder wallUsing immunohistochemical staining we tested the NGF expression of NC group, DC group, RI group, NGF and NGF + RI group's rats in the detrusor muscle cells.Under light microscope the rat detrusor muscle cells containing yellowish brown particles were positive cells.The NGF expression of detrusor muscle cells of diabetic rats decreased obviously than the normal control group; The NGF expression of detrusor muscle cells of NGF and NGF + RI group's rats were significantly higher than the DC group (P < 0.05); At the same time,the NGF expression between NGF group RI group and NGF + RI group were significantly different (P <0.05). 5 The comparison of NGF expression in the L6S1 DRG neuronsUsing immunohistochemical staining we tested the NGF expression of NC group, DC group, RI group, NGF and NGF + RI group's rats in the L6S1 DRG neurons. Under light microscope the rat L6S1 DRG neurons containing yellowish brown particles were positive cells. The NGF expression of L6S1 DRG neurons of diabetic rats decreased obviously than the normal control group; The NGF expression of L6S1 DRG neurons of NGF and NGF + RI rats were significantly higher than the DC group (P < 0.05); At the same time, the NGF expression between NGF group , RI group and NGF + RI group were significantly different (P <0.05).Conclusion: 1 The bladder lesion of diabetic cystopathy rats treated by NGF could significantly improve.2 The NGF levels in bladder and L6S1 DRG of diabetic cystopathy rats treated by NGF could significantly increase.3 When diabetic cystopathy rats were treated by NGF and insulin, there was synergistic interaction.