The Experimental Study About The Effects And Mechanism Of Serotonin (5-HT)2A Receptors In Lumbosacral Spinal Cord And Spinal Nerves On Micturition In Rats

OBJECTIVETo investigate the effects of ketanserin on micturition mediated via the 5-HT_2A receptor in the motoneuron nucleus of the Lumbosacral cord in anesthetized male rats.To evaluate the electrical stimulation effect of spinal nerve roots on micturition reflex in normal rats.MATERIALS AND METHODSThis research included two major experiments.Section one:pharmacological research.Male Sprague-Dawley rats were used.Route of intrathecal administration of medicine at the spinal L6-S1 segment was established.Normal saline was administered intrathecally in the control group and while ketanserin?0.003,0.01,0.03,and 0.1mg/kg?was given in the experimental group.The 5-HT_2A/2CA/2C receptor agonist DOI,0.1 mg/kg was administered intrathecally after a dose-response study for ketanserin.Cystometry and EUS-EMG were performed in all rats under urethane anesthesia to examine the variations after successive intrathecal administration of various doses of ketanserin into the lumbosacral cord.The number of HFOs?high-frequency oscillations,HFOs?in the cystometrograms was also calculated.Immunofluorescence histochemical staining and Western blot analyses were made to observe the distribution of 5-HT_2A and-_2C receptors in the lumbosacral cord motor neurons.Section two:electrical stimulation research.Female Sprague-Dawley rats were used.Unilateral L5-S2 dorsal roots?DRTs?and ventral roots?VRTs?were dissected under the operating microscope.Hooklike electrode was placed on the spinal nerve roots and bladder reflex contractions?BRCs?were recorded under isovolumetric condition while unilateral L5-S2 dorsal roots?DRTs?or ventral roots?VRTs?were electrically stimulated in a certain order.PRV?encoding for green fluorescent protein?was injected into the bladder to performe retrograde tracing.Spinal cord and the dorsal root ganglia?DRGs?from L5-S2 were removed and all PRV-positive cells displaying typical neuronal morphology in sacral parasympathetic nucleus?SPN?and DRG were counted separately under fluorescence microscope.RESULTSSection one:In the control group the rats presented rhythmic micturition with high voiding efficiency.The HFOs were in alignment and in accordance with the variation of EUS-EMG.Compared to the controls,ketanserin-treated rats showed a significant dose-dependent decrease in the HFOs,in accordance with the variation of EUS-EMG,while decreased micturition volume,voiding efficiency and increased post-void residual volume were only observed at the dose of 0.1mg/kg.The effects of ketanserin on the HFO and EUS-EMG activity were partially or completely reversed by the 5-HT_2A/2C receptor agonist,DOI.Meanwhile,immunohistochemical staining and Western blot analysis showed 5-HT_2A receptor immunoreactivity density ratio was higher than that of 5-HT_2C,labeling in the lumbosacral cord motoneurons.Section two:Repeated stimulation of L6 and S1 DRTs not only abolished BRC but also induced a post-stimulation inhibitory effect,whereas repeated stimulation of L5 and S2 DRTs had no influence.The prolonged time interval of repeated stimulation of S1 DRTs was significant longer than that of repeated stimulation of L6 DRTs.Only L6VRT directly caused bladder contraction when the VRTs of L5-S2 were stimulated in sequence.Pseudorabies virus?PRV?retrograde tracing demonstrated that the sacral parasympathetic nucleus?SPN?of L6segment was more strongly PRV-positive compared with the other segments,followed by S1,L5 and S2.On the other hand,the S1 dorsal root ganglion?DRG?was found to have the highest density of PRV-positive neurons,followed by L6,S2 and L5.CONCLUSIONS5-HT_2A receptors were detected in the motoneuron nucleus of the lumbosacral cord.The intrathecally administrated 5-HT_2A receptor antagonist ketanserin can weaken the EUS bursting activity,decrease the number of HFOs and reduce voiding efficiency dose dependently.Various afferent and efferent nerves innervate the bladder and are involved in micturition reflex,but the L6 VRT could be the most efficient in producing detrusor muscle contraction,and the S1 DRT could have the superiority of inhibiting micturition reflex.