外周神经损伤对中枢核团的影响及骨髓间充质干细胞治疗外周神经损伤后中枢逆行性病变的可行性研究
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摘要
第一部分外周神经损伤后中枢核团的逆行性病变
目的
建立大鼠单侧迷走神经离断模型,观察建立模型后不同时间点迷走神经中枢背核神经元的变化,包括神经元形态学和数量的变化；观察建立模型后不同时间点迷走神经中枢背核诱导型一氧化氮合成酶(iNOS)和烟酰胺腺嘌呤二核苷酸脱氢酶(NADPH)的表达；观察建立模型后不同时间点迷走神经中枢背核单核细胞趋化蛋白一1(MCP-1)的表达,以此来观察外周神经损伤后除了神经损伤部位的功能和生化发生改变外,损伤神经的更高位的通路比如脊髓和中枢核团是不是也会发生逆行性的改变,并初步探讨神经递质NO在迷走神经损伤后中枢背核逆行性病变中的意义及炎性细胞因子MCP-1在中枢背核逆行性病变中所起的作用。
方法
1.选择健康雄性SD大鼠,体重220-280g,10%水合氯醛(0.3ml/kg),腹腔内注射麻醉,固定大鼠,在颈部分离出左、右迷走神经,离断右侧迷走神经,左侧迷走神经则不做处理,随后缝合切口。对照组大鼠只分离出左、右迷走神经,但均不予切断,随后缝合切口。
2.选择健康雄性SD大鼠,按方法1建立迷走神经离断模型,分别于术后1d、5d、10d做进一步研究,每组3只大鼠,每只大鼠5片切片。用10%水合氯醛深度麻醉大鼠后,即刻打开胸腔,显露心脏,经左心室插入灌注管,快速肝素化,剪开右心耳,快速灌注4℃冰生理盐水150m1,再以40g/L多聚甲醛行心脏灌注,灌注结束后开颅取脑,解剖出脑干,标本收集于40g/L多聚甲醛液中过夜,然后置20%蔗糖多聚甲醛浸泡3.0-4.0h,再置30%蔗糖磷酸缓冲液浸泡沉底,4℃保持12h,随之取出用冰冻组织包埋剂包埋后置于液氮冷冻固定,随后用Leica全自动冷冻切片机切片,片厚20μim,切片放置室温下1h干燥固定后置于-20℃恒温冰箱保存。
3.取右侧迷走神经离断后1d、5d以及对照组大鼠左、右侧冰冻脑干切片,置于甲酚紫染色液(CFV)中行尼氏染色,观察建立模型后不同时间点背核区神经元细胞的组织形态学改变并行神经元计数。
4.取右侧迷走神经离断后5d、10d以及对照组大鼠左、右侧冰冻脑干切片,行一步法TUNEL荧光染色,观察建立模型后不同时间点背核区神经元有无凋亡发生。
5.取右侧迷走神经离断后5d、10d以及对照组大鼠左、右侧冰冻脑干切片,行免疫组织化学染色,观察建立模型后不同时间点背核区iNOS和NADPH的表达变化。
6.取右侧迷走神经离断后5d、10d以及对照组大鼠左、右侧冰冻脑干切片,行免疫组织化学染色,观察建立模型后不同时间点背核区MCP-1的表达变化。
结果
1.1背核神经元的免疫组化观察
对照组大鼠左、右侧背核神经元形态完整,以大中型细胞为主,分布均匀,形态规则,大多数神经元呈椭圆形或锥体形,部分神经元可见突起分支,胞质轻微染色,胞核未见明显显色；有少量较小的神经元细胞,形态规则,结构完整。
迷走神经离断后1d左侧背核神经元形态和分布与对照组相比,除染色稍微深之外无明显差异；但在右侧背核区,大多数神经元染色明显加深,不均匀分布,其中较大的神经元大多数明显皱缩,突起缩短或消失,胞质深染,部分神经元的胞核消失,部分较小神经元也表现出同样的变化,但神经元细胞的数量与对照组相比未见明显减少。
迷走神经离断后5d左侧背核区神经元与对照组相比无明显变化,但在右侧背核区,神经元数量较左侧及对照组相比则明显减少,并有较多深染、胞膜皱缩的较小的神经元出现。
1.2背核神经元计数
为确认迷走神经离断后背核区神经元的死亡情况,模型组和对照组大鼠每只选5个切片,计数神经元数量。对照组大鼠左、右侧背核神经元计数分别为53±4/片和51±5/片；建立模型后1d大鼠左侧背核神经元数量为52±4,与对照组比无统计学意义,而右侧背核神经元数量为46±3,与对照组及同时间点左侧相比数量明显减少(p<0.005)；建立模型后5d左侧背核神经元计数为51±6/片,与对照组比无统计学意义,而右侧背核神经元数量为35±5/片,与对照组及同时间点左侧相比数量明显减少(p<0.01)。
2 TUNEL染色结果
对照组大鼠右侧背核区未见TUNEL阳性神经元,荧光强度深浅一致。
右侧迷走神经离断后5d大鼠右侧背核区,可见少数TUNEL染色阳性神经元出现,表现为染色明显增强,荧光更为明亮,均匀一致的团块样结构。
右侧迷走神经离断后10d的大鼠右侧背核区未观察到TUNEL染色阳性神经元,同时神经元细胞的致密程度较对照组相比有所减弱。
3 iNOS免疫组织化学染色结果
对照组大鼠左、右侧背核区均未见iNOS染色阳性神经元。
右侧迷走神经离断后5d大鼠右侧背核区均可见大量iNOS染色阳性神经元,分布均匀,但在左侧背核区未见iNOS染色阳性神经元细胞。
右侧迷走神经离断后10d大鼠左侧未见iNOS染色阳性神经元,右侧背核区仍可见iNOS染色阳性神经元细胞,但在染色深度及细胞数量上与迷走神经离断后5d大鼠右侧背核相比明显减弱及减少。
4 NADPH免疫组织化学染色结果
对照组大鼠左、右侧背核区均未见NADPH染色阳性神经元。
右侧迷走神经离断后5d大鼠右侧背核区均可见较多NADPH染色阳性神经元细胞,呈散在分布,但在左侧背核区未见NADPH染色阳性神经元细胞。
5右侧迷走神经离断后10d大鼠右侧背核区可见大量的NADPH染色阳性神经元细胞,并且在染色深度及阳性细胞数量上与迷走神经离断后5d大鼠右侧背核相比明显增强及增多,但在左侧背核区未见NADPH染色阳性神经元细胞。
6右侧迷走神经离断后背核区单核细胞趋化蛋白—1(MCP=1)免疫组织化学染色的结果
对照组左、右侧背核区未见单核细胞趋化蛋白—1阳性细胞。
右侧迷走神经离断后5d大鼠右侧背核区可见较多散在分布的单核细胞趋化蛋白—1阳性细胞,左侧背核区未见单核细胞趋化蛋白—1免疫反应阳性细胞。右侧迷走神经离断后10d右侧背核区可见散在分布的单核细胞趋化蛋白—1阳性细胞,但阳性细胞数量较右侧迷走神经离断后5d大鼠右侧背核相比要明显减少,左侧背核区未见单核细胞趋化蛋白—1免疫反应阳性细胞。
结论
1、大鼠单侧迷走神经离断后可导致同侧迷走中枢背核发生逆行性改变,具体表现在神经切断后早期迷走中枢背核神经元数目减少,神经元形态发生改变,并且神经元细胞形态学的变化要早于数量的变化,表明大鼠单侧迷走神经离断后其同侧的中枢核团存在明显的神经元变性或凋亡,而对侧迷走中枢背核则无此变化。
2、大鼠单侧迷走神经离断后5d时同侧迷走中枢背核iNOS和NADPH蛋白表达明显上调,而离断后10d时iNOS免疫反应性则逐渐减弱,NADPH免疫反应性则逐渐增强,提示迷走神经离断后同侧迷走神经中枢核团有内源性NO形成,从而可能导致迷走神经离断后同侧迷走神经中枢背核神经元发生凋亡,并且不同时间断有不同的NOS生成。
3、大鼠单侧迷走神经离断后可导致同侧迷走中枢背核MCP-1表达上调,提示迷走神经离断后同侧迷走中枢背核MCP-1表达增多,表达增多的MCP-1作为炎性细胞因子可以通过聚集并活化吞噬细胞而对中枢造成进一步损伤。
第二部分骨髓基质干细胞治疗外周神经损伤所致的中枢核团逆行性病变的可行性研究
目的
1.本实验拟在体外分离,纯化及扩增大鼠骨髓间充质干细胞,免疫细胞化学染色观察第3代大鼠骨髓间充质干细胞CXCR4的表达情况。
2.同本实验第一部分建立单侧大鼠迷走神经离断模型,用组织化学细胞染色观察大鼠右侧迷走神经离断后不同时间点迷走神经背核基质细胞衍生因子—1表达的变化。
3.同本实验第一部分建立单侧大鼠迷走神经离断模型,用免疫组织化学法观察大鼠右侧迷走神经离断后经尾静脉移植的细胞增值示踪荧光探针标记的骨髓间充质干细胞在背核分布的变化。
方法
1.根据文献,采用全骨髓法。取220～280 g SD大鼠,10%水合氯醛腹腔注射麻醉(3ml/Kg),15min后断颈处死,碘伏消毒大鼠双下肢,收集双侧胫、股骨中全部骨髓。按1×106细胞/ml接种于75cm2培养瓶,24～72h后换液,去除未贴壁的细胞。以后每3～4天完全换液一次,12-14天后细胞密度达80%～90%,用0.25%的胰酶消化后1：3传代,记为P1代MSC(P1-MSC)。随后按上述方法传代培养。收集P3代的MSC(P3-MSC),制成2×106/ml细胞悬液,以备迁移实验。细胞形态在倒置显微镜下观察,拍照。
2.取生长旺盛的第3代细胞,选择兔抗CXCR4多克隆抗体并按照相应的试剂盒说明进行SP3法免疫组织化学染色,观察3代骨髓基质干细胞对CXCR4的表达情况。
3.选择健康雄性SD大鼠,体重220～280g,10%水合氯醛(0.3ml/kg),腹腔内注射麻醉,固定大鼠,在颈部分离出左、右迷走神经,离断右侧迷走神经,左侧迷走神经则不做处理,随后缝合切口。对照组大鼠只分离出左、右迷走神经,但均不予切断,随后缝合切口。取右侧迷走神经离断后10d及对照组大鼠,深度麻醉后,以4℃生理盐水,40g/L多聚甲醛依次行心脏灌注。灌注结后开颅取脑,脱水后行冰冻切片,片厚约20μm,以免抗基质细胞衍生因子-1多克隆抗体检测迷走神经背核基质细胞衍生因子—1的表达情况,二抗为CY3结合的羊抗免抗体,反应结束后用激光共聚显微镜观察、拍照。
4.取3代大鼠MSCs,用细胞增殖示踪荧光探针(CFDA-SE)标记后,于大鼠右侧迷走神经离断后24h经尾静脉将1ml标记的MSCs (2×106/ml)注入实验组和对照组大鼠体内,分别于移植后5、10d,将大鼠灌注、固定、取脑、切片,在荧光显微镜下观察MSCs在迷走神经背核的分布。
结果
1.大鼠骨髓基质干细胞(第3代)形态变得比较均匀一致,其中大部分细胞为细长梭形,也有小部分细胞为扁平形带突起的细胞,大部分细胞似成纤维样细胞,大部分梭形细胞集落明显呈克隆样生长,细胞呈极性排列,集落呈漩涡状,也有细胞呈散在生长。
2.第3代骨髓基质干细胞大量表达CXCR4, CXCR4主要表达于大鼠骨髓基质干细胞的胞膜和胞浆,对照组未见阳性染色。
3.右侧迷走神经离断后10d大鼠右侧迷走神经背核可见较多基质细胞衍生因-1阳性细胞,而对照组左、右侧背核及迷走神经离断后10d大鼠左侧背核均未见SDF-1阳性细胞。
4.骨髓基质干细胞移植后5、10d,右侧迷走神经背核可见大量CFDA-SE阳性细胞,而左侧背核及对照组左、右侧背核均未见CFDA-SE阳性细胞。
结论：
1.免疫细胞化学染色发现,体外培养的大鼠骨髓基质干细胞可表达CXCR4,而CXCR4主要表达于MSCs的胞膜和胞浆。
2.大鼠迷走神经离断后,同侧迷走神经背核可表达基质细胞衍生因子-1,而对侧背核及对照组左、右侧背核均未见明显SDF-1表达。
3.经尾静脉移植的骨髓基质干细胞可以迁移至迷走神经离断后的同侧迷走神经背核,其机制可能与同侧背核大量表达SDF-1以及SDF-1/CXCR4的相互作用有关。
Part 1 Retrograde changes in dorsal motor nuclei of vagus nerve after peripheral nerve iujury in rats
Objective
Neurodegeneration 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.
Methods
1.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.
Results
1.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 rats
3.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.
Conclusion
1.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 vagotomy
Part 2 Feasibility study on the treatment for retrograde lesions in nuclei with bone marrow mesenchymal stem cells after peripheral nerve injury
Objective
In 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.
Methods
1.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.
Results
1.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.
Conclusion
1. 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.
目的
建立大鼠单侧迷走神经离断模型,观察建立模型后不同时间点迷走神经中枢背核神经元的变化,包括神经元形态学和数量的变化；观察建立模型后不同时间点迷走神经中枢背核诱导型一氧化氮合成酶(iNOS)和烟酰胺腺嘌呤二核苷酸脱氢酶(NADPH)的表达；观察建立模型后不同时间点迷走神经中枢背核单核细胞趋化蛋白一1(MCP-1)的表达,以此来观察外周神经损伤后除了神经损伤部位的功能和生化发生改变外,损伤神经的更高位的通路比如脊髓和中枢核团是不是也会发生逆行性的改变,并初步探讨神经递质NO在迷走神经损伤后中枢背核逆行性病变中的意义及炎性细胞因子MCP-1在中枢背核逆行性病变中所起的作用。
方法
1.选择健康雄性SD大鼠,体重220-280g,10%水合氯醛(0.3ml/kg),腹腔内注射麻醉,固定大鼠,在颈部分离出左、右迷走神经,离断右侧迷走神经,左侧迷走神经则不做处理,随后缝合切口。对照组大鼠只分离出左、右迷走神经,但均不予切断,随后缝合切口。
2.选择健康雄性SD大鼠,按方法1建立迷走神经离断模型,分别于术后1d、5d、10d做进一步研究,每组3只大鼠,每只大鼠5片切片。用10%水合氯醛深度麻醉大鼠后,即刻打开胸腔,显露心脏,经左心室插入灌注管,快速肝素化,剪开右心耳,快速灌注4℃冰生理盐水150m1,再以40g/L多聚甲醛行心脏灌注,灌注结束后开颅取脑,解剖出脑干,标本收集于40g/L多聚甲醛液中过夜,然后置20%蔗糖多聚甲醛浸泡3.0-4.0h,再置30%蔗糖磷酸缓冲液浸泡沉底,4℃保持12h,随之取出用冰冻组织包埋剂包埋后置于液氮冷冻固定,随后用Leica全自动冷冻切片机切片,片厚20μim,切片放置室温下1h干燥固定后置于-20℃恒温冰箱保存。
3.取右侧迷走神经离断后1d、5d以及对照组大鼠左、右侧冰冻脑干切片,置于甲酚紫染色液(CFV)中行尼氏染色,观察建立模型后不同时间点背核区神经元细胞的组织形态学改变并行神经元计数。
4.取右侧迷走神经离断后5d、10d以及对照组大鼠左、右侧冰冻脑干切片,行一步法TUNEL荧光染色,观察建立模型后不同时间点背核区神经元有无凋亡发生。
5.取右侧迷走神经离断后5d、10d以及对照组大鼠左、右侧冰冻脑干切片,行免疫组织化学染色,观察建立模型后不同时间点背核区iNOS和NADPH的表达变化。
6.取右侧迷走神经离断后5d、10d以及对照组大鼠左、右侧冰冻脑干切片,行免疫组织化学染色,观察建立模型后不同时间点背核区MCP-1的表达变化。
结果
1.1背核神经元的免疫组化观察
对照组大鼠左、右侧背核神经元形态完整,以大中型细胞为主,分布均匀,形态规则,大多数神经元呈椭圆形或锥体形,部分神经元可见突起分支,胞质轻微染色,胞核未见明显显色；有少量较小的神经元细胞,形态规则,结构完整。
迷走神经离断后1d左侧背核神经元形态和分布与对照组相比,除染色稍微深之外无明显差异；但在右侧背核区,大多数神经元染色明显加深,不均匀分布,其中较大的神经元大多数明显皱缩,突起缩短或消失,胞质深染,部分神经元的胞核消失,部分较小神经元也表现出同样的变化,但神经元细胞的数量与对照组相比未见明显减少。
迷走神经离断后5d左侧背核区神经元与对照组相比无明显变化,但在右侧背核区,神经元数量较左侧及对照组相比则明显减少,并有较多深染、胞膜皱缩的较小的神经元出现。
1.2背核神经元计数
为确认迷走神经离断后背核区神经元的死亡情况,模型组和对照组大鼠每只选5个切片,计数神经元数量。对照组大鼠左、右侧背核神经元计数分别为53±4/片和51±5/片；建立模型后1d大鼠左侧背核神经元数量为52±4,与对照组比无统计学意义,而右侧背核神经元数量为46±3,与对照组及同时间点左侧相比数量明显减少(p<0.005)；建立模型后5d左侧背核神经元计数为51±6/片,与对照组比无统计学意义,而右侧背核神经元数量为35±5/片,与对照组及同时间点左侧相比数量明显减少(p<0.01)。
2 TUNEL染色结果
对照组大鼠右侧背核区未见TUNEL阳性神经元,荧光强度深浅一致。
右侧迷走神经离断后5d大鼠右侧背核区,可见少数TUNEL染色阳性神经元出现,表现为染色明显增强,荧光更为明亮,均匀一致的团块样结构。
右侧迷走神经离断后10d的大鼠右侧背核区未观察到TUNEL染色阳性神经元,同时神经元细胞的致密程度较对照组相比有所减弱。
3 iNOS免疫组织化学染色结果
对照组大鼠左、右侧背核区均未见iNOS染色阳性神经元。
右侧迷走神经离断后5d大鼠右侧背核区均可见大量iNOS染色阳性神经元,分布均匀,但在左侧背核区未见iNOS染色阳性神经元细胞。
右侧迷走神经离断后10d大鼠左侧未见iNOS染色阳性神经元,右侧背核区仍可见iNOS染色阳性神经元细胞,但在染色深度及细胞数量上与迷走神经离断后5d大鼠右侧背核相比明显减弱及减少。
4 NADPH免疫组织化学染色结果
对照组大鼠左、右侧背核区均未见NADPH染色阳性神经元。
右侧迷走神经离断后5d大鼠右侧背核区均可见较多NADPH染色阳性神经元细胞,呈散在分布,但在左侧背核区未见NADPH染色阳性神经元细胞。
5右侧迷走神经离断后10d大鼠右侧背核区可见大量的NADPH染色阳性神经元细胞,并且在染色深度及阳性细胞数量上与迷走神经离断后5d大鼠右侧背核相比明显增强及增多,但在左侧背核区未见NADPH染色阳性神经元细胞。
6右侧迷走神经离断后背核区单核细胞趋化蛋白—1(MCP=1)免疫组织化学染色的结果
对照组左、右侧背核区未见单核细胞趋化蛋白—1阳性细胞。
右侧迷走神经离断后5d大鼠右侧背核区可见较多散在分布的单核细胞趋化蛋白—1阳性细胞,左侧背核区未见单核细胞趋化蛋白—1免疫反应阳性细胞。右侧迷走神经离断后10d右侧背核区可见散在分布的单核细胞趋化蛋白—1阳性细胞,但阳性细胞数量较右侧迷走神经离断后5d大鼠右侧背核相比要明显减少,左侧背核区未见单核细胞趋化蛋白—1免疫反应阳性细胞。
结论
1、大鼠单侧迷走神经离断后可导致同侧迷走中枢背核发生逆行性改变,具体表现在神经切断后早期迷走中枢背核神经元数目减少,神经元形态发生改变,并且神经元细胞形态学的变化要早于数量的变化,表明大鼠单侧迷走神经离断后其同侧的中枢核团存在明显的神经元变性或凋亡,而对侧迷走中枢背核则无此变化。
2、大鼠单侧迷走神经离断后5d时同侧迷走中枢背核iNOS和NADPH蛋白表达明显上调,而离断后10d时iNOS免疫反应性则逐渐减弱,NADPH免疫反应性则逐渐增强,提示迷走神经离断后同侧迷走神经中枢核团有内源性NO形成,从而可能导致迷走神经离断后同侧迷走神经中枢背核神经元发生凋亡,并且不同时间断有不同的NOS生成。
3、大鼠单侧迷走神经离断后可导致同侧迷走中枢背核MCP-1表达上调,提示迷走神经离断后同侧迷走中枢背核MCP-1表达增多,表达增多的MCP-1作为炎性细胞因子可以通过聚集并活化吞噬细胞而对中枢造成进一步损伤。
第二部分骨髓基质干细胞治疗外周神经损伤所致的中枢核团逆行性病变的可行性研究
目的
1.本实验拟在体外分离,纯化及扩增大鼠骨髓间充质干细胞,免疫细胞化学染色观察第3代大鼠骨髓间充质干细胞CXCR4的表达情况。
2.同本实验第一部分建立单侧大鼠迷走神经离断模型,用组织化学细胞染色观察大鼠右侧迷走神经离断后不同时间点迷走神经背核基质细胞衍生因子—1表达的变化。
3.同本实验第一部分建立单侧大鼠迷走神经离断模型,用免疫组织化学法观察大鼠右侧迷走神经离断后经尾静脉移植的细胞增值示踪荧光探针标记的骨髓间充质干细胞在背核分布的变化。
方法
1.根据文献,采用全骨髓法。取220～280 g SD大鼠,10%水合氯醛腹腔注射麻醉(3ml/Kg),15min后断颈处死,碘伏消毒大鼠双下肢,收集双侧胫、股骨中全部骨髓。按1×106细胞/ml接种于75cm2培养瓶,24～72h后换液,去除未贴壁的细胞。以后每3～4天完全换液一次,12-14天后细胞密度达80%～90%,用0.25%的胰酶消化后1：3传代,记为P1代MSC(P1-MSC)。随后按上述方法传代培养。收集P3代的MSC(P3-MSC),制成2×106/ml细胞悬液,以备迁移实验。细胞形态在倒置显微镜下观察,拍照。
2.取生长旺盛的第3代细胞,选择兔抗CXCR4多克隆抗体并按照相应的试剂盒说明进行SP3法免疫组织化学染色,观察3代骨髓基质干细胞对CXCR4的表达情况。
3.选择健康雄性SD大鼠,体重220～280g,10%水合氯醛(0.3ml/kg),腹腔内注射麻醉,固定大鼠,在颈部分离出左、右迷走神经,离断右侧迷走神经,左侧迷走神经则不做处理,随后缝合切口。对照组大鼠只分离出左、右迷走神经,但均不予切断,随后缝合切口。取右侧迷走神经离断后10d及对照组大鼠,深度麻醉后,以4℃生理盐水,40g/L多聚甲醛依次行心脏灌注。灌注结后开颅取脑,脱水后行冰冻切片,片厚约20μm,以免抗基质细胞衍生因子-1多克隆抗体检测迷走神经背核基质细胞衍生因子—1的表达情况,二抗为CY3结合的羊抗免抗体,反应结束后用激光共聚显微镜观察、拍照。
4.取3代大鼠MSCs,用细胞增殖示踪荧光探针(CFDA-SE)标记后,于大鼠右侧迷走神经离断后24h经尾静脉将1ml标记的MSCs (2×106/ml)注入实验组和对照组大鼠体内,分别于移植后5、10d,将大鼠灌注、固定、取脑、切片,在荧光显微镜下观察MSCs在迷走神经背核的分布。
结果
1.大鼠骨髓基质干细胞(第3代)形态变得比较均匀一致,其中大部分细胞为细长梭形,也有小部分细胞为扁平形带突起的细胞,大部分细胞似成纤维样细胞,大部分梭形细胞集落明显呈克隆样生长,细胞呈极性排列,集落呈漩涡状,也有细胞呈散在生长。
2.第3代骨髓基质干细胞大量表达CXCR4, CXCR4主要表达于大鼠骨髓基质干细胞的胞膜和胞浆,对照组未见阳性染色。
3.右侧迷走神经离断后10d大鼠右侧迷走神经背核可见较多基质细胞衍生因-1阳性细胞,而对照组左、右侧背核及迷走神经离断后10d大鼠左侧背核均未见SDF-1阳性细胞。
4.骨髓基质干细胞移植后5、10d,右侧迷走神经背核可见大量CFDA-SE阳性细胞,而左侧背核及对照组左、右侧背核均未见CFDA-SE阳性细胞。
结论：
1.免疫细胞化学染色发现,体外培养的大鼠骨髓基质干细胞可表达CXCR4,而CXCR4主要表达于MSCs的胞膜和胞浆。
2.大鼠迷走神经离断后,同侧迷走神经背核可表达基质细胞衍生因子-1,而对侧背核及对照组左、右侧背核均未见明显SDF-1表达。
3.经尾静脉移植的骨髓基质干细胞可以迁移至迷走神经离断后的同侧迷走神经背核,其机制可能与同侧背核大量表达SDF-1以及SDF-1/CXCR4的相互作用有关。
Part 1 Retrograde changes in dorsal motor nuclei of vagus nerve after peripheral nerve iujury in rats
Objective
Neurodegeneration 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.
Methods
1.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.
Results
1.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 rats
3.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.
Conclusion
1.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 vagotomy
Part 2 Feasibility study on the treatment for retrograde lesions in nuclei with bone marrow mesenchymal stem cells after peripheral nerve injury
Objective
In 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.
Methods
1.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.
Results
1.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.
Conclusion
1. 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.
引文
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