| Part 1 Retrograde changes in dorsal motor nuclei of vagus nerve after peripheral nerve iujury in ratsObjectiveNeurodegeneration resulting from peripheral nerve injury is seriously impairing the living quality of people.Understanding the mechanism of neuropathic pain would lead to the development of therapeutic approaches,which aims to interfere with the processes of neuropathic pain.The present study aims to investigate the retrograde changes in dorsal motor nucleus of vagus nerve after the ligation of vagus nerve. We investigate the morphological and quantitative changes of the vagal motorneurons in the DMV after right vagatomy and study the activation of apoptosis pathaway in the degeneratain of the axotomized motorneurons in the DMV following right vagotomy involving the expression of some apoptosis molecules like iNOS and NADPH and the expression of MCP-1 in the DMV after right vagatomy.Methods1.Adult male Wistar Strain rats weighing about 220～280g were used in the present study. The rats were purchased from the laboratory animal center of Xiangya Medical college.The rats were housed in clean cages in a temperature-controlled room with 12 hours light and 12 hours dark schedule.All animals were deeply anaesthesized by an intraperitonal injection of 10% chloralhydrate (3ml/kg body weight) administered in the lower abdomen.After 10～15 minutes,one of the foot pads of the animals was pinched with a pair of toothed forceps to ensure the animals were under full anesthesia.Following anesthesia,a vertical midline incision was made at the cervical level,the neck muscles were separated by pulling the suture aside,the right vagus nerve was exposed by carefully clearing the carotid sheath and a segment(5mm) of the vagus nerve was ligated and excised. The left vagus nerve was similarly exposed but kept intact as internal sham-control.The wound was sutured and 3 animals were studied at1,5,10 or 20 days after operation,A group of 3 normal rats was assigned to the control group.2.Frozen coronal sections(20μm)of the brainstems of the vagotomized rats at 1 or 5 days following operation were cut from +2.5mm to-2.5mm from obex aneltvery,5th section was then immersed in CFU staining solution.After Nissl Staining,they were then viewed under a nikon light microscope.The neurons in the DMV of four sections for cach rat (three rats for each group)were counted.3.The brainstem sections of the rats at 5 and 10 days after right vagatomy were processed as above. The sections were incubated in the NADPH histochemistry medium,after the incubation, the sections were then air dried and viewed under a Nikon light microscope. For control group,β-NADPH was omitted from the incubation solution.4.The braistem sections of the rats at 5 and 10 days after righ vagotomy were processed as above, According to the recommended protocol,the sections were incubated with the TUNEL reaction mixture solution containing Tdt and fluorescein-dUTP.Apoptosis cells were detected and studied using a confocal microscope.5.The brainstem sections of the rats at 5 and 10 days vagatomized rats ware processed as above.iNOS immune staining was performed according the recommeded protocol.Tissue sections were incubated with the primary antibody and biotinylated secondary antibody. Control sections were incubated as above but the primary antibody was omitted. 6.The brainstem sections of the rats at 5 and 10 days after right vagotomy were processed as above.MCP-1 immunohistochemistry staining was performed according to the recommeded protocol.Tissue sections were incubated with the primary MCP-1 antibody and biotinylated secondany antibody.Control sections were incubated as above but the primary antibody was omitted.Results1.In both left and right DMV of the control rats,motorneurons with different sizes and morphologies were revealed,indicating the heterogenity of vagal motorneurons in the nuclei,most of neurons were round and regular in shape and displayed light staining in their cytoplasm,whereas their nuclei were not stained. Some large motorneurons were spindled-shape.No obvious morphological changes in the left DMV of rats at 1 day followng right vagotomy.However,in the right DMV at 1 day vagotomized rats,the majority of the vagal motorneurons were observed to be darkly stained,mostof large neurons became shrunked and intensive staining in the whole cytoplasm,some nuclei in these neurons were even not visible,moreover,the motorneurons were not evenly distributed in the right DMV at 1 day followoy right vagotomy.But at 5 days after right vagotomy,no remarkable changes in Nissl staining were observed in the left DMV compared to that in the control.However,in the right DMV,a few small darkly-stained and shrunken neurons were still detectable,many motorneurons appeared to be lost in the right DWV at 5 days after operation.2.To confirm the death of the vagotomized motorneurons,theneurons in the DMV of the Nissl-stained sections were counted,averaged and analyzed using t-test.In the right and left DMV of control rats,the mean numbers of neurons were 53±2/setion and 51±6/section respectively;At 1 day after right vagotomy,the number was significantly decreased to 46±3/section (P<0.05)in the right DWV,whereas thre was no significant change in the left DMV;At 5 days after right vagotomy,the number was significantly decreased to 35±5/section (P<0.01) in the right DMV,however,no siginficant change in the number of motorneunons was observed in the left DMV.Average number of motomeurons per section in the DMV (x±S) of normal and vagotomized rats3.NO TUNEL positive neurons were observed in the right DMV of the normal rats;At 5 days after right vagotomy, some TUNEL pcsitive neurons were detected in the right DMV,However,no TUNEL positive neurons were observed in the right DMV of rats at 10 days after operation.4.In the right brainstem of normal rats, the immunostaining of MCP-1 was hardly detectable in the bilateral DMV of the normal rats.However,at 10 days after operation upregulated immunoreactivity of MCP-1 was shown and many positively stained cells were detected in the right DMV,At 20 days following operation,the immunostaining appeared to be decreased and only a few weakly stained cells were still detectable in the right DMV. The immunostaining of MCP-1 was also hardly detectable in the left DMV at 10 and 20 days in operation rats.5.The immunohistochemistry staining of iNOS was undetectable in the bilateral DMV of the normal rats.However, at 5 days following operation,the immunoreactivity of iNOS was upregulated and many iNOS immunopositve cells appeared in the right DMV. At 10 days after right vogotomy,the iNOS immunoreactivity and the number of iNOS immunopositive cells appeared to be decreased in the right DMV. And no obvious change in the immunoreactivity of iNOS was observed in the left DMV at 5 day and 10 days after operation.6.No NADPH positive staining cell was detected in the right DMV of the normal rats,a few motorneurons had increased NADPH staining in the right DMV at 5 days after right vagotomy;At 10 days more intensive NADPH staining was detected in the right DMV with neurons varying in size and staining intensity.Conclusion1.The retrograde changes of rats in the right DMV will take place after right vagal nerve was ligated and excised involving the siginficant derease in the mumber of motorneurons and obvious morphologically changes of motorneurons,whereas no significant change in the left DMV was detected.Above foundings indicate an apparent neural degeneration or apoptosis in right DMV after right vagotomy in rats.2.The immunoreactivity of iNOS and NADPH was upreguated in the right DMV at 5 days after right vagtomy. The immunoreactivity of iNOS appeared to be decreased in the right DMV at 10 days after right vagotomy,whereas the immunoreactivity of NAPPH appeared to be more strongerly increased at 10 days after right vagotomy.Above observation indicates endogenious NO may be the main fact which leading to the happening of neuron apoptosis in the right DMV after right vogotovwy.3.The significant upregulation level of inflammatory cytokine MCP-1 in the right DMV after right vagotomy indicates MCP-1 may mediate the retrograde changes in DMV by assembling and activating inflammtory cells after right vagotomyPart 2 Feasibility study on the treatment for retrograde lesions in nuclei with bone marrow mesenchymal stem cells after peripheral nerve injuryObjectiveIn this experiment,MSCs were isolated,depurated, amplificated and labelled and were transplanted into the vagotomized rats via vein;The effects of stromal cells derived factor-1(SDF-1)and its receptor CXCR4 were studied;The distribution of transplanted CFDA-SE labelled MSCs in right DMV were also observed in right vagotomized rats.Through this experiment, we hope to provide experiment basis for treating the central nuclei damage induced by the injury of peripheral nerve and the possible mechanism about this phenomenon.Methods1.According to the character of sticking to the wall rapidly,MSCs were isolated and amplified by combination of gradient centrifugation and different adherent time methed.The third generation MSCs growth and morphology characteristics was observed through the inverted microscope.2.Take some 3 generation MSCs,CXCR4 expression of MSCs was verified by immunohistochemistry under a confocal microscope. 3.The vagotomized rat models were established as before,the immunohistochemistry of SDF-1 in DMV was observed at 10 days after the right vagus was ligated and excised under a confocal microscope.4. Some 3 generation MSCs were washed with PBS and then incubated in CFDE-SE solution.24hs after right vagotomized,1 ml CFDE-SE labelled MSCs at the concentration of 2×109/L or lml PBS were transplanted via vein. Rats were sacrificed at 5 and 10 days after transplatation.The labelled MSCs in DMV were observed under a fluorescence microscope.Results1.MSCs were isolated from the femurs and tibiae of adult rats and propagated in vitro.As shown by the image under phase contrast microscope,the 3 generation MSCs grew as colonies,most of cells were spindle-shaped,large flat and small round.2.Immunohistochemistry revealed that rat MSCs were positive for CXCR4 and CXCR4 mainly expressed in cell membrane and cytoplasm of MSCs.3.Immuohistochemistry revealed that upregulated immunoreactivity of SDF-1 was undetectable in bilateral DMV in control group.However a few SDF-1 immunoreactivity positive cells were observed in the right DMV at 10 days following operation compared to that in bilateral DMV in control group and the lelf DMV in experimental group.4.Examination of frozen sections by fluorescence microscopy revealed that there were no CFDA-SE labelled cells detected in the bilateral DMV of the control rats. CFDA-SE labelled MSCs were observed in the right DMV at 5 and 10 days with cells displaying brightgreen fluorescence, whereas no labelled MSCs were detectabe in the left DMV at 5 and 10 days after right vagotomy. Conclusion1. The upregulated expression of SDF-1 in the impaired nuclei after peripheral nerve injugy revealed in this experinent suggests that SDF-1 could be involved in the inflammatory process in the DMV after right vagotomy.The increased temporal expression of SDF-1 paralling the number of migrated CXCR4-expressing MSCs in this study suggests that the chemotactic interaction of CXCR4-SDF-1 could facilitate the homing of MSCs to the impaired nuclei in the brain.2.Transplanted MSCs could migrate into the impaired nuclei after peripheral nerve injury. This study provides an important insight into the understanding of the mechanisms governing the trafficking of transplanted MSCs and provides cues to the development of therapeutic strategies to interfere with the neurodegenerative processes and also expands our knowledge on the potential roles of MSCs in cell theraply for neuropathic pain therapy. |
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