Nitric Oxide Pathway Of Vascular Sympathetic Nerve And Its Effect On VSMC Proliferation

Objective: To investigate nitric oxide pathway of vascular sympathetic nerve and its effect on VSMC proliferation.Methods: Model establishment and grouping:30 SD rats, male and weight 250g±20g, were randomly divided into 4 groups: sham group, model 1d group, model 5d group and LNNA group, 12 rats for model 5d group, 6 rats for every other group. Right carotid artery of the rat was brought into exposure, then a slip of filter paper(2mm×1mm) dipped with 1μL FeCl3 (2.16mol/L) was covered above the carotid artery for 20 min. Sewed up the incision after the paper slip was removed, the model was successfully established. The sham group was substituted with the same amount normal saline solution compared with model group. LNNA (NOS inhibitor) group had additional intraperitoneal injection with LNNA15mg/kg twice a day for six days from the day before operation. 6 rats of model 5d group were selected to had FG retrograde tracing, 3 rats were injected at carotid artery, 3 rats were injected into superior cervical ganglion (SCG).FG retrograde tracing: 10 SD rats, weight 250g±20g, were randomly divided into 2 groups. After anesthesia, rats were cut into a 3 cm long incision in the median line of cervical part in supine position. One group was slowly injected 1μl 1%FG and retained for 15 min between Tunica adventitia and Tunica media of right carotid artery by glass micropipe conjucted with microsyringe; the other group was slowly injected 1μl 1%FG and retained for 15 min into right superior cervical ganglion exposed in the crotch of internal carotid artery and external carotid artery. Samples were collected after rats which survived one week from operation had perfusion with formalin. NADPH-d enzymohistochemistry staining: Slices of spinal cord and ganglions were incubated 1h(incubation solution content:0.3% TritonX-100; 0.1g/L NBT;1.0g/Lβ-NADPH), then were rinsed 5 min with 0.01M PBS for three times. Slices had mounting with neutral resin after mounted to glass slides from PBS, air drying, alcohol gradient dehydration, vitrification by dimethylbenzene. Slices injected with FG had mounting with liquid paraffin instead and were observed by fluorescence microscope.Results:Pathway of NO nerve:FG labeled cells were found in SCG, Median cervical ganglion (MCG) and inferior cervical ganglion (ICG),but labeled cells in SCG were more than that of MCG and ICG; FG labeled cells were also found in intermediolateral nucleus of T1-T3 in spinal cord, NADPH-d enzymohistochemistry staining of slices injected with FG suggested that FG labeled cells found in SCG and spinal cordT1-T3 were NADPH-d positive cells, SCG that innervated carotid artery had NO positive neurons.Change of NADPH-d positive neurons: NADPH-d positive neurons in spinal cord of model 5d group were remarkably more than that of sham group(P<0.05), while model 1d group betrayed no statistically significance vs sham group(P>0.05); NADPH-d positive cells in intermediolateral nucleus of LNNA group prominently decreased compared with that of model 5d group. NADPH-d positive neurons were expressed in small amount in cervical sympathetic ganglion(CSG) of sham group, NADPH-d positive neurons in model 1d group and model 5d group increased compared with that of sham group, model 5d group especially showed growth in number(P<0.05), NADPH-d positive cells in CSG of LNNA group prominently decreased compared with that of model 5d group.Change of smooth muscle cell proliferation: HE staining of carotid artery indicated that the tendency smooth cells started to proliferate and showed apparent proliferation on day 5 was at equal pace with the tendency of NADPH-d positive cells in CSG and cornu laterale medullae spinalis. NADPH-d positive cells in CSG and cornu laterale medullae spinalis of LNNA group decreased remarkably, and VSMC proliferation of LNNA group showed upregulated than that of non-LNNA group. Conclusion:1. Carotid artery was innervated by CSG, especially by SCG from sympathetic nerve.2. Sympathetic nerve innervated carotid artery and contained transmitter NO, and T1-T3 intermediolateral nucleus also contained NO transmitter.3. NO nerve that innervated carotid artery showed low expression in normal state but high expression in the state VSMC proliferation after vascular injury. This indicated that NO nerve played a role in the regulation of VSMC proliferation.