嗅神经髓鞘细胞移植促进大鼠横断脊髓轴突再生的研究

目的;观察嗅神经髓鞘细胞（OECs)对脊髓轴突再生的促进作用。方法：将分离,纯化与体外培养2周的SD大鼠OECs,移植于12只成年SD大鼠第10胸椎横断模型的脊髓两断端;8只对照动物注入DMEM／F12培养基,移植后2周和8周进行神经嗜银染色与髓磷脂碱性蛋白（MBP）组织化学和神经生长因子受体（NGFR）免疫组织化学研究。结果：OECs移植组2周后脊髓两端在形态学上呈现初步愈合现象,组织学观察显示,移植OECs与损伤脊髓组织整合良好,再生轴突长入断端组织,8周时再生轴安全检查明显增多,OECs与再生轴突均呈MBP和NGFR阳性表达。对照组两断端液化坏死,未见再生轴突。结论：OECs移植可保护损伤的脊髓并促进宿主脊髓轴突再生。     

Influences of olfactory ensheathing cells transplantation on axonal regeneration in spinal cord of adult rats

Objective: To observe whether offactory ensheathing cells could be used to promote axonal regeneration in a slmntaneously nonregenerating system. Methods: After laminectomy at the lower thoracic level, the spinal cords of adult rats were exposed and completely transected at T10. A suspension of ensheathing cells was injected into the lesion site in 12 adult rats, and control D/F-12 (1:1 mixture of DMEM and Ham‘s F-12) was injected in 12 adult rats. Six weeks and ten weeks after cell transplantation, the rats were evaluated by climbing test and motor evoked potentials (MEPs) monitoring. The samples were procured and studied with histologiel and immounohistochemical methods. Results: At the 6th week after cell transplantation,d the rats in both the transplanted and control groups were paraplegic and the MEPs could not be recorded. At the 10th week after cell transplantation, of 7 rats in the control group, 2 rats had muscles‘ contraction of the lower extremities, 2 rats had hips and/or knees‘ active movement; and 5 rats‘ MEPs could be recorded in the hind limbs in the transplanted group ( n = 7). None of the rats in the control group had functional improvement and no MEPs recorded ( n = 7 ). Numerous regenerating axons were observed through the transplantation and continued to regenerate into the denervated host tract. Cell labelling using anti-Myelin Basic Protein (MBP) and anti-Nerve Growth Factor Receptor (anti-NGFR) indicated that the regenerated axons were derived from the appropriate neuronal source and that donor cells migrated into the denervated host tract. But axonal degeneration existed and regenerating axons were not observed within the spinalcords of the adult rats with only D/F-12 injection. Conclusions: The axonal regeneration in the transected adult rat spinal cord is possible after eusheathing cells transplantation.     

嗅鞘细胞经低能激光照射后移植对大鼠脊髓损伤神经功能修复的影响

目的 探讨嗅鞘细胞(OECs)经低能激光照射(LPLI)后移植对大鼠脊髓横断损伤后功能修复的影响. 方法 24只SD大鼠T<,12>脊髓完全横断伤模型建模4周后,随机分为OECs移植组、经LPLI的OECs的移植组和空白对照组(DMEM培养液),应用BBB评分法、组织学评价以及Flurogold逆行示踪评价各组脊髓损伤的修复情况. 结果三组大鼠不同移植方式以及评分时间点对移植后下肢功能BBB评分结果的影响差异均有统计学意义(P=0.000),且组间差异均有统计学意义(P=0.000).经LPLI的OECs移植组大鼠头尾侧均可见NGFRp75阳性及GFAP阳性的OECs,OECs移植组则仅可见GFAP阳性的OECs,空白对照组未见NGFRp75阳性及GFAP阳性的OECs.OECs移植组和经LPLI的OECs移植组大鼠头尾侧及瘢痕区均可见Flurogold标记的神经纤维穿行,空白对照组未见Flurogold标记的神经纤维穿行. 结论 OECs及经LPLI的OECs均可促进损伤脊髓的功能恢复,OECs经LPLI后可能更具有促进损伤脊髓功能恢复的作用.     

嗅鞘细胞移植对大鼠损伤脊髓组织中BDNF表达的影响及意义

目的 观察嗅鞘细胞(OEC)移植治疗大鼠脊髓损伤的作用以及脊髓损伤组织中脑源性神经营养因子(BDNF)表达的变化,从神经营养因子角度探讨OEC移植修复脊髓损伤的机制.方法 分离和培养绿色荧光蛋白转基因大鼠的OEC,备移植用.取SD大鼠制备脊髓损伤模型,用随机数字表法将建模成功后的SD大鼠分为2组.实验组:建模成功后立即进行OEC移植,移植部位为脊髓损伤处及其首尾正中血管的左右两侧;对照组:用DMEM/F12培养液替代OEC悬液,操作同实验组.移植后对各组大鼠的运动功能进行评分,每周1次.采用实时逆转录聚合酶链反应检测BDNF mRNA的表达,采用免疫组织化学法检测BDNF蛋白的表达,取正常SD大鼠BDNF mRNA和蛋白的检测水平作为正常对照.结果 移植后两组大鼠的运动功能均逐渐改善,移植后21 d时,实验组大鼠运动功能评分明显高于对照组(P＜0.05).移植后OEC存活良好,实验组损伤的脊髓组织周围分布有大量的呈绿色荧光的OEC.移植后21 d时,实验组BDNF mRNA和蛋白的表达水平显著高于对照组和正常对照组(P<0.05),而对照组也显著高于正常对照组(P<0.05).结论 OEC移植可明显修复大鼠的脊髓损伤,其机制可能与OEC通过增强损伤脊髓组织中BDNF mRNA和蛋白的表达,改善局部微环境有关.

Abstract：

Objective To observe the expression of brain-derived neurotrophical factor (BDNF) in injury spinal cord after transplantation olfactory ensheathing cells (OECs), and to investigate the mechanism of OECs repairing spinal cord injury.Methods OECs from GFP transgenic rats were separated and cultured for transplantation. Spinal cord injury rats were separated two groups by random digits table. In experimental group, OECs suspension were transplanted into injured spinal cord following spinal cord injury. In control group, DMEM was transplanted into the injured spinal cord after spinal cord injury. Motor function was evaluated per week after transplantation. The expression levels of BDNF mRNA and protein were detected by using RT-PCR and immunohistochemistry respectively, and compared with those from normal SD rats.Results Motor function of two groups was improved gradually after transplantation. The motor function scores in experimental group was obviously higher than in control group at 21st day after transplantation (P<0.05). A lot of survival GFP OECs distributed around impaired myeloid tissue. At 21st day after transplantation, BDNF mRNA and protein expression in experimental group were strongest (P<0.05), and stronger in control group than in normal group (P<0.05).Conclusion The transplantation of OECs can repair the injured spinal cord by increasing the expression of BDNF mRNA and protein to improve local microenvironment.     

低温保存的嗅鞘细胞移植治疗脊髓损伤的实验研究

目的:观察低温保存复苏后嗅鞘细胞(OECs)局部移植治疗脊髓损伤(SCI)的疗效,探讨低温保存对嗅鞘细胞活性的影响。方法:建立大鼠T10节段脊髓半切损伤模型56只,随机分为四组,每组12只:新鲜OECs移植组(A组),冻存OECs移植组(B组),D/F12培养液移植组(C组)和空白对照组(D组)。A组损失伤局部行新鲜OECs移植,B组将处于对数生长期的OECs冻存3个月,复苏后(用双苯亚甲胺标记)移植到脊髓半切模型大鼠脊髓损伤区。术后第1天、1、2、3、4、6、8及10周时进行联合行为学(CBS)综合评分,术后5周和10周时取材观察移植细胞存活及迁移情况;HE染色及嗜银染色观察脊髓组织病理及轴突情况;NGFRp75免疫组织化学染色情况。结果:术后第1天,2、10周时CBS评分,A组分别为85.78±1.07、58.80±5.00、6.52±2.37;B组分别为86.12±1.29、60.06±6.51、8.15±2.26;C组分别为86.4±1.03、66.28±7.00、30.65±5.60;D组分别为86.75±1.37、69.85±6.61、34.13±5.38。A、B两组间无明显差别(P>0.05);C组与D组间比较无差异(P>0.05);A组及B组较C组和D组在2周后各时段差别有显著意义(P<0.05)。组织学方面,HE染色和嗜银染色A、B两组在术后5周可见多量突起经近侧断端向损伤区域生长,10周时可见神经纤维跨越损伤区域,而C、D组未见有神经纤维跨越损伤区;NGFRp75免疫组化染色,无论5周还是10周,A、B两组在损伤部位均可见阳性染色区域,C、D组未见有阳性着色。术后5周时A、B两组可检测到荧光标记细胞,且可见细胞发生迁移。结论:低温保存的OECs脊髓局部移植治疗SCI与新鲜OECs移植治疗效果无差别。     

嗅球组织块和细胞悬液移植对脊髓损伤的修复

目的探讨嗅球组织块与嗅球细胞悬液髓内移植修复脊髓损伤的可行性和疗效。方法将Wistar大鼠做成脊髓半切模型,随机分成3组,髓内分别移植嗅球组织块、嗅球细胞悬液及DMEM培养液,分期进行功能联合行为学评分(CBS)及组织学检查,评价对脊髓修复情况。结果嗅球组织块与嗅球细胞悬液髓内移植3周后各时段CBS评分明显高于DMEM组(P<0.05),组织学显示前两组有嗅鞘细胞修复的轴突再生。结论嗅球组织块与嗅球细胞悬液髓内移植修复脊髓损伤不但简化了操作而且有较好的疗效,为临床应用提供了实验依据。     

NGF、BDNF基因修饰嗅鞘细胞脊髓内移植对损伤脊髓细胞的保护作用

目的：观察神经生长因子(nerve growth factor，NGF)和脑源性神经营养因子(brain-derived neurotmphic fac-tor，BDNF)基因修饰的嗅神经鞘细胞(Olfactory ensheathing cells，OECs)移植对损伤脊髓组织的保护作用。方法：将脊髓半横断伤SD大鼠模型，随机分为：NGF、BDNF基因修饰的OECs移植组(A组)、OECs移植组(B组)、损伤对照组(C组)和正常对照组(D组)。24h后每组8只动物取伤段标本，测水离子含量。其余动物第6周和12周每组8只动物爬坡试验，评价下肢运动功能及运动诱发电位(MEP)检测。结果：脊髓损伤(SCI)后组织水肿，Na^ 、Ca^2 离子浓度升高，K^ 、Mg^2 离子浓度降低。NGF、BDNF、基因修饰的OECs脊髓内移植后显著改善这些变化，且使SCI后神经功能有显著恢复。结论：NGF、BDNF基因修饰的OECs脊髓内移植对SCI有保护作用。其机制可能与减少神经细胞离子失衡，改善细胞内环境有关。     

GDNF基因修饰的嗅鞘细胞移植联合IN-1注射修复大鼠急性脊髓损伤

目的 研究胶质细胞源性神经营养因子(GDNF)基因修饰的嗅鞘细胞(OECs)移植联合轴突生长抑制蛋白抗体(IN-1)局部持续注射对大鼠急性横断性脊髓损伤(SCI)的修复作用.方法 构建载有GDNF基因的慢病毒(Lentivirus)载体并体外转染OECs,Western Blot检测GDNF的表达.用50只成年雌性SD大鼠建立胸脊髓全横断损伤模型,随机分为A(对照组)、B(IN-1微泵注射组)、C(OECs组)、D(GDNF-OECs组)和E(GDNF-OECs+IN-1组)5组各10只.应用神经丝蛋白200(NF200)单抗免疫组化、生物素化的葡聚糖胺(BDA),顺行神经追踪对SCI区神经纤维再生进行形态学观察.采用BBB评分评估大鼠后肢功能恢复情况.结果 术后共有13只大鼠死亡.术后8周可观察到Hoechst标记的OECs在体内存活并在脊髓内迁移;E组和D组可见SCI区杂乱无序的再生轴突,有连续性神经纤维通过损伤区;C组可见少量无序的再生轴突,可疑连续性神经纤维通过损伤区;B组和A组脊髓残端萎缩,未见轴突再生.A、B、C、D和E组后肢功能运动平均BBB评分分别为7.70±0.24、7.89±0.15、10.50±0.25、11.43±0.23和12.81±0.40.结论 GDNF-OECs移植联合IN-1抗体注射可有效促进损伤脊髓神经轴突的存活、再生,促进损伤脊髓的修复.     

Adult olfactory ensheathing cell transplantation for acute spinal cord injury

Cellular transplantation strategies have been explored for the treatment of spinal cord injury. In particular, olfactory nerve ensheathing cell (OEC) transplantation has been reported to improve functional outcome following injury. We investigated the effect of OEC transplantation using cells derived from adult animals on the restoration of function following a contusion injury to the spinal cord. The NYU impactor was used to create a moderate to severe spinal cord injury in 17 rats. Hoescht stained cultured OECs derived from adult rats (n = 7) or culture medium alone (n = 10) were injected into the injury site immediately following injury. Histological and functional outcomes were measured using immunohistochemistry and the Basso, Beattie, and Bresnahan (BBB) scale. All animals transplanted with OECs were found to have surviving Hoescht positive cells within the spinal cord when sacrificed 6 weeks following injury. Immunohistochemical staining of the explanted cords revealed that the surviving cells stained positively for nerve growth factor receptor. Functional outcomes were not different between the transplanted and control groups. OECs transplanted immediately following a contusion injury to the spinal cord survive during the first 6 weeks following injury. These cells do not appear to influence functional outcome during the first 6 weeks following injury. Additional studies are required in order to definitively determine the utility of this type of cellular transplantation for spinal cord injury.     

NT-3基因修饰许旺细胞与神经干细胞联合移植促进大鼠全横断脊髓受损伤神经元的存活及其轴突再生

目的探讨神经营养素-3基因修饰许旺细胞（NT-3-SCs）与神经干细胞（NSCs）联合移植对大鼠因脊髓全横断而受损伤的神经元存活及其轴突再生的作用。方法将NT-3-SCs及LacZ报告基因修饰SCs（LacZ-SCs）与NSCs联合移植到全横断性脊髓损伤处，60d后在脊髓横断处尾侧注射荧光金（FG）进行逆行标记。第67天，取材进行组织学检测大脑皮质体感区、红核及脊髓横断处头侧FG标记神经元；大脑皮质体感区、红核和脊髓背核内存活的神经元以及脊髓损伤处再生的轴突。结果大脑皮质体感区、红核及脊髓L1背核内存活的神经元由多到少依次为NT-3-SCs与NSCs联合组、LacZ—SCs与NSCs联合组和实验对照组。NT-3-SCs与NSCs联合组和LacZ—SCs与NSCs联合组的大脑皮质体感区、红核和脊髓横断处头侧有FG标记神经元；脊髓横断处及其附近组织有5-HT、CGRP和SP阳性神经纤维。结论NT-3-SCs与NSCs联合移植能够促进脊髓全横断损伤后神经元的存活以及轴突再生。     

不同方法共移植的嗅鞘细胞和雪旺氏细胞在损伤脊髓内的迁移和对轴突再生的影响

目的:观察经不同方法共移植的嗅鞘细胞和雪旺氏细胞在脊髓内的迁移特点和对轴突再生的影响.方法:将8只75±1d雌性SD大鼠随机分为2组,每组4只,利用NYU打击器制作TIO脊髓损伤模型,打击高度25mm,打击杆重量10g.造模后2周,一组采用联合移植,雪旺氏细胞移植于损伤鄢位中心,嗅鞘细胞移植于距离损伤中心0.5mm处的头侧和尾侧的脊髓中线上;另一组采用混合移植,将嗅鞘细胞和雪旺氏细胞混合后移植于距离损伤中心0.5mm处的头侧和尾侧的脊髓中线上.每个部位注射4个点,深度为1.75mm,1.25mm、1mm、0.5mm.注射速度为0.1μl/min.联合移植组嗅鞘细胞晕每点0.5μl含5×10~4个,雩旺氏细胞晕为每点1μl含10~5个雪旺氏细胞;混合移植组为每点1μl含嗅鞘细胞和雪旺氏细胞各5×10~4个.细胞移植后1周和8周时各组分别取2只大鼠.以损伤部位为中心取包含细胞移植部位的脊髓,荧光和共聚焦显微镜下观察细胞迁移情况,利用神经丝(neurofilment,NF)和乍长相关蛋白-43(growth associated protein-43,GAP-43)免疫荧光染色观察移植细胞对轴突再生的影响.结果:两组中均可见嗅鞘细胞迁移,主要在灰质和白质内沿脊髓纵轴向损伤部位迁移,还分别有一小部分沿中央管和蛛网膜下腔迁移;但雪旺氏细胞仅存与嗅鞘细胞混合移植于距离损伤中心0.5mm处的头侧和尾侧脊髓时才可见有限距离的迁移.联合移植时,NF阳性(NF+)和GAP-43阳性(GAP-43+)纤维伴随嗅鞘细胞迁移而沿脊髓纵轴延伸,雪旺氏细胞移植处可处NF+纤维从各个方向长入损伤部位(移植部位)并互相缠绕;混合移植时,大量NF+纤维伴随移植细胞迁移而延伸,损伤部佗虽然NF+纤维较少,但没有互相缠绕现象.结论:雪旺氏细胞在损伤脊髓内迁移能力差;嗅鞘细胞不仅具有良好的迁移能力,而且可促进雪旺氏细胞迁移.无论是联合移植还是混合移植,移植细胞均能促进轴突再生,但联合移植时雪旺氏细胞移植处再生纤维互相缠绕、无法延伸.     

嗅鞘细胞移植对坐骨神经切断后神经元的作用

目的研究嗅鞘细胞(olfactory ensheathing cells,OECs)移植对周围神经切断后脊髓及神经节内神经元的保护作用。方法SD大鼠55只,随机分为3个组:空白组5只、实验组及对照组各25只。行右侧坐骨神经切断,近端断端行肌肉内包埋,实验组和对照组分别予OECs及细胞培养液,空白组暴露神经后不作任何处理。术后1、2、3、7和14d,分批处死,行组织学观察及TUNEL标记观察神经元的改变。结果空白组术后14d均无阳性变化。大鼠坐骨神经切断后,脊髓及神经节内均有神经元凋亡发生。术后1、2、3d实验组细胞存活率分别为98.4%±6.5%、97.6%±6.5%及95.2%±6.7%,对照组分别为97.8%±6.7%、97.4%±6.4%及94.3%±6.8%,比较差异无统计学意义(P>0.05);7、14d实验组细胞存活率分别为92.4%±8.9%、87.7%±9.4%,较对照组87.4%±8.6%、83.4%±8.5%高,差异有统计学意义(P<0.05)。术后1、2d实验组及对照组脊髓前角运动神经元无细胞凋亡;3、7、14d实验组脊髓前角运动神经元凋亡指数分别为1.2±0.8、1.4±0.6及4.1±1.3,较对照组2.1±1.1、3.1±1.1、6.1±1.8低,差异有统计学意义(P<0.05)。术后1、2、3d神经节内细胞无凋亡;7d实验组神经节内的凋亡指数2.10±0.32,较对照组4.40±0.56低,差异有统计学意义(P<0.05);14d实验组神经节内的凋亡指数4.3±1.80与对照组6.70±2.50比较,差异无统计学意义(P>0.05)。结论周围神经损伤后脊髓及神经节内神经元有神经元凋亡发生,OECs移植对神经元凋亡有保护作用。     

人胚胎神经干细胞移植治疗大鼠脊髓完全横断损伤

目的：探讨利用人胚胎神经干细胞（hNSC）移植治疗大鼠脊髓完全横断损伤的效果.方法：分离、培养和鉴定hNSC.将培养的hNSC植入脊髓完全横断的Wistar大鼠损伤局部,并设DMEM-F12培养液注射组（对照组）.移植术后第1、2、4、6、8、10周对两组大鼠进行BBB运动功能评分,观察脊髓功能恢复情况;并取损伤处脊髓组织行免疫组织化学染色,了解双苯亚甲胺（Hoechst）标记的移植细胞在体内存活和分化情况.结果：体外细胞培养可获得大量hNSC;细胞移植4周后,实验组的BBB运动功能评分较对照组明显提高（P＜0.01）;第4周、第10周免疫组化结果示移植的hNSC在损伤脊髓内存活、分化形成神经元和神经胶质细胞,并向损伤脊髓头尾两侧迁徙.结论：hNSC移植后仍保持其多向分化能力;hNSC移植可改善脊髓全横断损伤大鼠的运动功能.     

新生鼠和成年鼠脊髓损伤后胚胎脊髓移植对大鼠损伤脊髓功能恢复的影响

目的研究新生鼠和成年鼠脊髓损伤后胚胎脊髓移植对大鼠损伤脊髓功能恢复的影响。方法将新生鼠和成年鼠腰段脊髓半切洞损伤,取E14胚胎脊髓组织移植到损伤区,手术后4、8、12周,进行组织学检查,联合行为评分,感觉诱发电位,运动诱发电位检查。结果组织学检查发现移植的胚胎脊髓在宿主脊髓中存活。联合行为评分,感觉诱发电位,运动诱发电位潜峰时的恢复,新生鼠移植组均优于成鼠移植组(P<0.05)。结论通过各种功能检查(CBS,SEP,MEP)表明胚胎脊髓移植能够促进脊髓损伤后新生鼠和成鼠运动功能的恢复。     

嗅鞘细胞移植在大鼠截瘫模型中的应用

目的探讨嗅鞘细胞局部移植治疗脊髓损伤的可行性、安全性及细胞移植数量和时机与疗效的关系。方法应用改良Allen’s法制备大鼠T10脊髓损伤模型,第1部分：随机分4组,移植时间分别设定为手术即时0,1,2,3周,共分为上述4组（随机）,每组共10只,4周后进行CBS评分。第2部分：取40只大鼠,随机分为5组,A组：注细胞数量为5：〈10。个。B组：移植方法同A组。注入细胞数量为4×10^5个。C组：移植方法同A组,注入细胞数量为3×10^5个。D组：移植方法同A组,注入细胞数量为2×10^5个;E组：移植方法同A组,将20μlDMEM／F12培养液注入损伤部位。结果在移植细胞为固定的4×10^5个时,损伤后2周移植大鼠CBS评分（损伤后4周,20．12±5．23,P〈0．05）较其余时间好：而在移植时间均为损伤后两周的情况下。B、C两组大鼠CBS评分（损伤后4周,B：20．34±5．63,C：20．34±5．63,P〉0．05）无明显差别,但较其余各组好（P〈0．05）。结论嗅鞘细胞局部移植治疗脊髓损伤安全有效,移植细胞数量适中和移植时间在2周时脊髓神经功能恢复较好。     

OECs移植联合应用尼莫地平促进大鼠脊髓损伤后功能恢复

目的探讨嗅鞘细胞(olfactoryensheathingcells,OECs)移植联合应用尼莫地平,恢复大鼠脊髓损伤后的功能。方法将成年大鼠分为脊髓半切洞损伤组(A组),脊髓半切洞损伤 OECs移植组(B组)和脊髓半切洞 OECs 尼莫地平移植组(C组)。手术后应用联合行为评分(CBS),感觉诱发电位(SEP)和运动诱发电位(MEP)检查,测定脊髓功能恢复情况。结果3组CBS得分A组>B组>C组,SEP和MEP潜峰时,均A组>B组>C组。统计分析均差异显著性(P<0.05)。结论移植OECs 尼莫地平能促进损伤脊髓功能的恢复。     

骨髓间质干细胞与胚胎嗅鞘细胞联合移植治疗大鼠脊髓损伤的实验研究

目的：观察人骨髓间质干细胞（MSCs）与胚胎嗅鞘细胞（OECs）联合移植治疗大鼠脊髓损伤的效果，探讨共移植的OECs对MSCs分化的影响。方法：采用Allen’s法制作大鼠脊髓损伤模型．随机分为MSCs移植组（A组）、OECs移植组（B组）、MSCs与OECs联合移植组（C组）及PBS注射组（D组），术后1-5周采用BBB评分、经颅磁刺激运动诱发电位及组织学方法检查脊髓损伤修复情况。结果：术后2周起，A、B、C组BBB评分和诱发电位潜伏期与D组比较恢复明显，C组与A、B组相比亦有显著性差异（P〈0．05），术后5周时C组损伤区有较密的神经轴突分布，近端可见大量成束再生轴突；A、B组损伤区及近端再生轴突分布稀疏；D组损伤区及近端少见轴突。C组移植的MSCs中Nestin及NF表达阳性的比率较A组高（P〈0.05）。结论：联合移植OECs可促进MSCs向神经元方向转化，提高MSCs分化为神经元的比例；MSCs与OECs可以在脊髓损伤修复中发挥协同作用，联合细胞移植是提高脊髓损伤修复效果的可行方法。     

Increased expression of cyclo-oxygenase 2 and vascular endothelial growth factor in lesioned spinal cord by transplanted olfactory ensheathing cells

Olfactory ensheathing cells (OECs) were transplanted in adult rats after photochemical injury of the spinal cord. Rats received either 180,000 OECs suspended in DMEM or DMEM alone. Locomotor ability scored by the BBB-scale, pain sensibility, and motor and somatosensory evoked potentials were evaluated during the first 14 days post-surgery. At 3, 7, and 14 days, 5 rats per day of both groups were perfused and transverse sections from proximal, lesioned and distal spinal cord blocks were stained for COX-2, VEGF, GFAP and lectin. The BBB-score and the amplitude of motor and somatosensory evoked potentials were significantly higher in OEC- than in DMEM-injected animals throughout follow-up, whereas the withdrawal latency to heat noxious stimulus was lower in OEC- than in DMEM-injected rats. The area of preserved spinal cord and the levels of COX-2 and VEGF staining were significantly higher in OEC- than in DMEM-injected rats. GFAP- but no LEC-positive cells expressed COX-2 staining in OEC-transplanted rats. The density of blood vessels was also significantly increased in OEC- with respect to DMEM-injected rats. Our results show that OECs promote functional and morphological preservation of the spinal cord after photochemical injury, increasing neoangiogenesis and up-regulation of COX-2 and VEGF expression in astrocytes.     

Survival and number of olfactory ensheathing cells transplanted in contused spinal cord of rats

Objective： To observe the survival and the number of olfactory ensheathing cells （OECs） transplanted in the contused spinal cord, so as to provide a basis for further studying the biological action of OECs.Methods： The rat spinal cords were contused with NYU-impactor Ⅱ at T10 level by dropping a 10 g rod from a height of 25 mm. At the 1st week after injury, OECs isolated freshly from green fluorecense protein （GFP） of the rats were transplanted into the spinal cord at injured site and other two sites 1 mm apart from the caudal and rostral ends with the OECs number of 30000/μl×3 =90000. The survival and the number of OECs were qualitatively and semi-quantitatively observed under the fluorescense microscope from 1 week to 13 weeks after transplantation. The motor function of the cord was evaluated with BBB score.Results： GFP-OECs could survive at least for 13 weeks within the contused spinal cord. Their arrangement was from tight to loose and their number was decreased from 1 week to 13 weeks after injury. The average number of GFP-OECs was 536 at the 1st week, which was less than 1% of the number as compared with original transplantation. After then, the number of GFP-OECs was continually decreased,but the most obvious decrease was found during 1 week to 2 weeks. The extent of decrease at other time points was relatively mild. In contrast to the cell number, motor function of the cord was gradually recovered after transplantation.Conclusions： The survival and the number of GFPOECs are different between the animals and are affected by the pathological reaction of the host cord. Also it is related to the motor function recovery of the contused cord.     

Experimental study of neural repair of the transected spinal cord using peripheral nerve graft

It has been reported that transected spinal cord shows signs of axonal regeneration after peripheral nerve (PN) graft. We studied the membrane excitability and ion distribution in axons from transected rat spinal cord 3 weeks after PN graft using the spinal cord evoked potential, electron probe X-ray microanalysis, and the patch-clamp technique. Axonal structures were also observed using conventional electron microscopy. At the Th11 level, laminectomy was performed (=control) and the left thoracic segments of the spinal cord 2mm in length were excised (=nongrafted group). PN sections from 8-week-old male Wistar rats were grafted into the spinal cord gap (=PN-grafted group). The spinal cord evoked potential in the PN-grafted group partly recovered in contrast to that in the nongrafted group, which showed no recovery. Higher Na, Cl, and Ca peaks and lower K peaks in the PN-grafted group were demonstrated compared with those in the nongrafted group. In the PN-grafted group, a higher current signal appeared in the axonal membrane of the spinal cord, suggesting a greater membrane activity compared with that in the nongrafted group. Unlike the nongrafted group, in which no myelinated axons were found, demyelinated axons that were myelinated by Schwann cells from the grafted peripheral nerve were observed in the PN-grafted group. These findings suggested that Schwann cells from the transplanted PN contributed to the repair of the transected spinal cord.