Establishment Of Neurogenic OAB Rat Model Caused For Cerebral Ischemia And The Role Of ICC In This Model

Background and objectiveNeurogenic bladder(NB)is a bladder or urethral dysfunction caused by damage to the nervous system.Common causes include central nerve injury such as spinal cord injury,brain injury,and peripheral nerve injury such as pelvic nerve injury and retroperitoneal nerve injury.The bladder dysfunction caused by NB can be divided into detrusor underactivity(DU)and detrusor overactivity(DO)according to the results of urodynamics.The clinical symptoms of NB are dysuria or overactive bladder symptom(OAB,characterized by increasing urination frequency,feelings of urgency to urinate with or without incontinence).Ischemic stroke is a neurological injury caused by insufficient blood supply to the brain due to a vascular disease that supplies blood to the brain.Ischemic stroke can also cause NB complications.Because the spinal cord urination center loses the inhibition of the cerebral urination center after the injury of the cerebral urination center,it will lead to DO,which will lead to OAB.Therefore,patients with cerebral ischemia neurogenic bladder are mostly manifested as OAB.Neurogenic OAB in ischemic stroke leads to a severe decline in quality of life and can lead to recurrent urinary tract infections that eventually damage kidney function.In recent years,due to the extension of life expectancy and changes in lifestyle,more and more patients with cerebral ischemic neurogenic OAB have been diagnosed.However,the specific mechanism of cerebral ischemic neurogenic OAB is unclear,which makes clinical treatment very difficult.It is important to study the mechanism of neurogenic OAB symptoms of cerebral ischemia from animal experiments and to develop high-quality drugs according to the mechanism,so we need to make a stable animal model of neurogenic OAB of cerebral ischemia.Rats are easy to feed,cheap,90%of the same gene as human,and the familiar urinary system as human,so they are the best model animals to make cerebral ischemia neurogenic OAB.To establish the neurogenic OAB rat model of cerebral ischemia,the OAB status of the rat should be determined by the awake cystometry.To carry out cystometry in conscious rat,cystostomy must be performed.However,cystostomy can also cause OAB state in rats because the bladder injury caused for cystostomy,so it is necessary to carry out cystometry in conscious cerebral ischemia rats in the time window when the bladder function returns to normal after cystostomy to accurately reflect the bladder changes after cerebral ischemia.However,the time window when the bladder function returns to normal after cystostomy has not been reported.One of reason caused for cerebral ischemia neurogenic OAB symptoms is micturition nervous center of brain injury after cerebral ischemia,but is there has bladder tissue reason,such as the bladder tissue spontaneous contraction function and reactivity to neurotransmitter acetylcholine changed,involved in the formation of OAB needs further research.Bladder interstitial cell of cajal(ICC)recently been identified as a type of interstitial cell with spontaneous excitability in bladder tissue,and the current research results showed that they were involved in the regulation of bladder detrusor contractile function,which is more obvious in pathological bladder.Previous studies have found a significant increase in the number of them on the human non-specific OAB bladder,and a significant increase in the number of them in the bladder tissues of OAB rats caused for bladder outlet obstruction with a significant increase in spontaneous excitability.However,whether the number and excitability of ICC cells on the bladder of cerebral ischemia neurogenic OAB rats also changed remains to be investigated.C-kit is a landmark receptor on the cell membrane of bladder ICC.Studies have found that its specific antagonist,imatinib mesylate(Glivec),can down-regulate the spontaneous excitability of bladder ICC cells and thus reduce it function.The role of bladder ICC cells in the formation of neurogenic OAB from cerebral ischemia can be further clarified by applying Glivec to the bladder muscle strips and bladder in vivo of cerebral ischemia neurogenic OAB rat then observe the changes of muscle strips spontaneous contraction and bladder function.Therefore,the purpose of this study includes:?,To find the optimal time window for cystometry in conscious rats after cystostomy.?,Make the neurogenic OAB rat model caused for cerebral ischemia.?,To identify the changes of the spontaneous contraction frequency and amplitude and of the contractile amplitude response to the neurotransmitter acetylcholine in isolated bladder muscle strips of cerebral ischemia neurogenic OAB rats.?,To investigate the changes in the number of ICC cells in the bladder of cerebral ischemia neurogenic OAB rats.?,To investigate the spontaneous excitatory changes of bladder ICC cells in cerebral ischemia neurogenic OAB rats.?,To investigate the spontaneous contractile amplitude,frequency and contractile amplitude response to the neurotransmitter acetylcholine of bladder muscle strips and the changes in bladder function in vivo in cerebral ischemia neurogenic OAB rats after Glivec intervention.Methods1,Carry out cystometry in conscious rats in deferent time points postcystostomy and perform HE and Masson staining,then make the results of cystometry and HE and Masson staining compared with normal rats,to found the best time window for cystometry in conscious rats.2,use homemade nylon thread bolt block the right middle cerebral artery of rats for 3 hours through the right internal carotid to cause the right hemisphere ischemic injury,then perform cystometry in conscious cerebral ischemia rats in the best time window,postcystometry bladder tissues were collected for HE staining,then the results of cystometry and HE staining been compared with control group.If the cerebral ischemia rats had decreased bladder capacity,shortened voiding interval,no obvious postvoiding residual urine and no obvious inflammation of bladder tissue,the modeling was successful.3,The bladder tissues of cerebral ischemia neurogenic OAB rats were collected and prepared into 10mm×3mm muscle strips.The frequency,amplitude of spontaneous contraction and the contraction amplitude response to acetylcholine of the bladder muscle strips in cerebral ischemia neurogenic OAB rats were detected by muscle strip tension test and compared with the control group to investigate the changes of bladder myogenic factors in forming cerebral ischemia neurogenic OAB.4,Immunofluorescence staining,real-time quantitative PCR and western blotting were used to analyze the number of ICC cells in the bladder of cerebral ischemia neurogenic OAB rats and compare them with the control group.5,The ICC cells of cerebral ischemia neurogenic OAB rats and the control group rats were isolated and cultured,then the ICC cell morphology and C-kit immunofluorescence staining results been observed,then calcium probe Fluo4 incubated with bladder ICC cells of cerebral ischemia neurogenic OAB rats,after that the bladder ICC's spontaneous excitability been compared with the control group.6,Different concentrations Glivec was used to intervene the bladder muscle strips and bladder in vivo then observe the changes of spontaneous contraction amplitude,frequency and contraction amplitude responsed to acetylcholine chloride of bladder muscle strip and changes in bladder function in cerebral ischemia neurogenic OAB rats and control group rats.Results1,The optimal time window for cystometry in conscious rats was 5-15 days postcystostomy,when the bladder function of cystostomy rats was consistent with that of normal rats,and the pathophysiological status of bladder tissues was closest to that of normal rats.2,The cerebral ischemia neurogenic OAB rat model was successfully produced by blocking of the right middle cerebral artery for 3 hours.The model rats showed OAB symptoms of shortened voiding interval and voided volume.3,The spontaneous contractile amplitude,frequency and response contractile amplitude to acetylcholine of bladder muscle strips in cerebral ischemia neurogenic OAB rats were significantly higher than those of the control group.4,The number of ICC cells in the bladder of cerebral ischemia neurogenic OAB rats was significantly higher than that of the control group.5,In vitro cultured bladder ICC cells were fusiform or stellate,positive for C-kit receptor,and their spontaneous excitability was significantly higher in cerebral ischemia neurogenic OAB rats than in the control group.6,Glivec can dose-dependent inhibit the spontaneous contraction amplitude,frequency and response contraction amplitude to acetylcholine of bladder muscle strips in cerebral ischemia neurogenic OAB rats,and can dose-dependent prolong voiding interval,voided volume,bladder capacity and without increasing the postvoided residual urine volume,thus improve cerebral ischemia neurogenic OAB symptoms.Conclusions1,A stable neurogenic OAB rat model caused for cerebral ischemia could be made after 3 hours of right middle cerebral artery blocked by nylon thread bolt.2,In cerebral ischemia neurogenic OAB rats,the upregulation of ICC cells number and spontaneous excitability in the bladder leads to the instability of bladder detrusor muscle,which is involved in the generation of neurogenic OAB symptoms caused for cerebral ischemia.3,Glivec,a C-kit receptor specific inhibitor,can improve neurogenic OAB symptoms caused for cerebral ischemia.