Detection Of Changes Of Intracellular Ca²⁺ In The Ureter Smooth Muscle Cell Of The Rats With Neuropathic Urinary Tract Dysfunction

Objective: Neurogenic urinary tract dysfunction（NUTD）refers to the control of urinary central nervous system or the peripheral nervous system caused by various causes of injury followed by the occurrence of urinary system voiding dysfunction of a class of diseases.Improving the quality of life and the protection of upper urinary tract function is the treatment principles of urinary tract dysfunction.Preliminary studies had found that NUTD primary ureteral dysfunction in the early pathological changes,which had a relationship with the L-type voltage-dependent calcium channels reduced expression and calcium activated potassium channels increased expression and its ultrastructure change in ureteral smooth muscle cells.As a result,Ca²⁺ concentration in USMC of NUTD would changed.Studies have shown that Ca²⁺ is able to adjust the excitability of ureteral smooth muscle cells,which played a key role in normal peristalsis of the ureter,maintaining a constant pressure gradient and the resistance to the flow mechanism.This study investigated the changes of intracellular Ca²⁺ in the ureter smooth muscle cell（USMC）of the rats with neuropathic urinary tract dysfunction（NUTD）by laser scanning confocal microscope.We believe that the study may provide a basis for the development of more effective therapeutic strategies to prevent upper urinary tract damage in NUTD.Method: 1.To establish the model of animal:Provided by the Experimental Animal Center of Henan province 45 only SD rats,female,250± 20 g,adaptability of the conventional breeding a week or so.Random number table method was applied to 45 rats,which were divided into experimental group（NUTD group）and control group（EC）,blank control group（BC）groups.（1）the BC group: do not do any processing.（2）NUTD groups: area located in L1 spine surgery,vascular clamp bite to L1 completely severed L1 spinal cord after spine,and completely undermine L1 segment of the spinal cord below,step by step after close operation area and closing a wound.Special nursing postoperatively was offered to the rats of NUTD group.（3）EC group: only remove the L1 spines of rats,and don’t do other processing.2.At the sixth week the rats of three groups underwent the test of urine dynamics,and ureter smooth muscle cells（USMC）were obtained by collagenase digestion at last.3.Intracellular Ca²⁺ in the USMC of three groups were observed by laser scanning confocal microscope..4.We detected that the effects of Bay K8644（10-8mol/L,10-7mol/L,10-6mol/L）on cytosolic Ca²⁺ concentrations([Ca²⁺]i)in NUTD group were studied by calculating the fluorescence intensity under the laser scanning confocal microscope.Result:1.The test of urine dynamics showed the rats of in NUTD groups were showed no detrusor overactivity and vesicoureteral reflux,which met standards of the experimental animal models.2.The cells have been adherent growth after culturing for 24 hours,most of the cells were long spindle,purified cells were consistent,which presented "peak and valley" structure.Using immunofluorescence method confirmed that the cells were USMC.3..We observed under the laser scanning confocal microscope that compared with BC group（31.44±2.82）and EC group（32.06±3.67）,the fluorescence intensity of intracellular Ca²⁺ in USMC was much lower in the NUTD group（9.80±1.11,P<0.05）.4.We observed under the laser scanning confocal microscope that Bay K8644（10-8mol/L,10-7mol/L,10-6mol/L）increased the FI of [Ca²⁺]i in a concentration-dependent manner,which were 3.80±1.30,10.04±2.15,19.89±2.06 respectively（P<0.05）.Conclusion: 1.The relatively pure ureteral smooth muscle cells were effectively obtained by enzyme digestion.2.The decrease of Ca²⁺ concentration in ureter smooth muscle cells may be one of the important factors for the primary ureteral dysfunction of NUTD.3.Special calcium channel agonist can be meaningful for adjustment of abnormal Ca²⁺ concentration in USMC of NUTD,which may bring hope to the treatment of NUTD.