骨髓间充质干细胞移植联合吡拉西坦修复大鼠脊髓损伤

背景：单纯的干细胞移植对脊髓损伤的修复作用并不理想,主要是因为脊髓损伤后损伤区域神经组织的水肿、缺血、缺氧等引起继发性损伤造成的。 目的：在骨髓间充质干细胞移植治疗大鼠脊髓损伤的同时应用吡拉西坦,观察两者对大鼠脊髓损伤恢复的影响。 方法：雌性Wistar大鼠参照改良Allen打击法制备大鼠脊髓损伤模型。随机分成3组,即单纯损伤组、骨髓间充质干细胞移植组及骨髓间充质干细胞移植联合吡拉西坦组。于伤后1,2,4,6,8周进行BBB评分和斜板实验等运动功能检测。第4周取材行病理切片苏木精-伊红染色,通过SRY-PCR检测雄性大鼠Y染色体上特有的基因SRY,从而得知移植骨髓间充质干细胞是否存活。8周后取材,行辣根过氧化物酶示踪观察,并通过透射电镜观察轴突的再生情况。 结果与结论：伤后4周,骨髓间充质干细胞移植组、联合治疗组大鼠后肢运动功能均有较明显恢复,联合治疗组较骨髓间充质干细胞移植组恢复快(P < 0.05)。单纯损伤组亦有所恢复,但程度较轻。病理切片单纯损伤组未见神经轴索通过;骨髓间充质干细胞移植组可见少量神经轴索样结构;联合治疗组可见较多神经轴索样结构。骨髓间充质干细胞移植组、联合治疗组有SRY基因表达,单纯损伤组未检测到SRY基因。辣根过氧化物酶阳性神经纤维数联合治疗组﹥骨髓间充质干细胞移植组>单纯损伤组,差异具有显著性意义(P < 0.05)。透射电镜下,骨髓间充质干细胞移植组、联合治疗组正中横断面可见新生的无髓及有髓神经纤维。提示骨髓间充质干细胞移植联合吡拉西坦促进大鼠损伤脊髓结构和功能恢复的效果明显优于单纯细胞移植组,两者联用具有协同效应。     

高压氧联合神经干细胞移植治疗大鼠脊髓损伤

背景：单纯神经干细胞移植已应用于对受损脊髓组织的修复。 目的：以神经干细胞移植同时应用高压氧治疗大鼠脊髓损伤,观察联合作用对脊髓损伤大鼠运动功能恢复的影响。 方法：雌性SD大鼠60只,以半切法制成胸段脊髓半横断大鼠模型。随机分成单纯损伤组、神经干细胞移植组及高压氧治疗组,每组20只。伤后第4周取材行病理切片苏木精-伊红染色及BrdU免疫组织化学染色,第8周取材行辣根过氧化物酶示踪,透射电镜观察轴突的再生情况,通过体感诱发电位观察神经电生理恢复情况。造模后1,2,4,6,8周进行BBB评分和斜板实验等运动功能检测。 结果与结论：观察伤后4周病理切片,单纯损伤组未见神经轴索通过,神经干细胞移植组可见少量神经轴索样结构,高压氧治疗组可见较多神经轴索样结构。BrdU的阳性细胞数及辣根过氧化物酶阳性神经纤维数,高压氧治疗组最多,神经干细胞移植组次之,单纯损伤组最少,且各组之间差异有显著性意义(P < 0.05)。透射电镜下神经干细胞移植组、高压氧治疗组正中横断面可见新生的无髓及有髓神经纤维。高压氧治疗组大鼠体感诱发电位的潜伏期短于神经干细胞移植组,波幅高于神经干细胞移植组(P < 0.05),明显优于单纯损伤组(P < 0.01)。伤后4周神经干细胞移植组、高压氧治疗组大鼠后肢运动功能均有较明显恢复,高压氧治疗组较神经干细胞移植组恢复快(P < 0.05);单纯损伤组亦有所恢复,但程度较轻。提示神经干细胞移植对于脊髓损伤大鼠后肢功能的恢复有促进作用,联合应用高压氧有协同效果。     

骨髓间充质干细胞立体定向移植治疗大鼠脊髓损伤*

摘要 背景：传统观念认为,神经组织损伤后几乎不能再生,以往对SCI的治疗缺乏有效手段,致使本病致残率高,疗效差。干细胞治疗关键在于移植具有再生能力的干细胞,通过多种作用机制,可以重建中枢神经系统的结构和功能,近年来引起了广泛的关注。 目的：探讨立体定向移植骨髓间充质干细胞（MSCs）对大鼠脊髓损伤修复的影响并探讨其机制 设计、时间及地点：随机对照动物实验,于2007-10/2008-6在天津市环湖医院完成。 材料：1月龄SD大鼠20只,用于制备骨髓间充质干细胞;健康成年Wistar大鼠45只,雌性、同系,体质量280±20 g。将动物随机分为对照组、假手术组与移植组,每组各15只。 方法：密度梯度离心法结合贴壁筛选法分离骨髓间充质干细胞,经流式细胞仪鉴定为MSCs。以动脉瘤夹夹闭法制备大鼠脊髓损伤（SCI）模型,在SCI大鼠致伤后第7天,通过立体定向途径移植MSCs到移植组大鼠脊髓损伤中心,移植等量生理盐水至假手术组大鼠脊髓损伤中心,对照组大鼠不做处理。 主要观察指标：SCI大鼠损伤前及损伤后第7天、14天、30天、60天、90天的BBB评分;损伤后第90天处死大鼠,观察其脊髓组织中有无BrdU阳性细胞、Brdu+NSE、Brdu+GFAP、Brdu+bFGF、Brdu+BDNF免疫组化双染阳性细胞并观察NSE、GFAP、bFGF、BDNF单染阳性细胞。 结果： ①BBB评分发现,MSCs移植组大鼠BBB后肢功能评分恢复优于对照组（p<0.05）;假手术组BBB评分在损伤后30天内恢复速度慢于对照组（p<0.05）,至第90天与对照组比较无显著差异（P>0.05）;②免疫组织化学染色发现,移植组大鼠脊髓内在损伤中心及头、尾端距离脊髓损伤中心1cm处均可见BrdU染色阳性细胞及Brdu+NSE、Brdu+GFAP、Brdu+bFGF、Brdu+BDNF免疫组化双染阳性细胞。移植组NSE、GFAP、bFGF、BDNF单染阳性细胞数明显高于对照组和假手术组（p<0.05）。 结论： MSCs移植可以促进SCI大鼠的神经功能的恢复,其机制可能与移植细胞分化为神经元样和神经胶质细胞样细胞,并分泌或促进宿主分泌神经营养因子有关。 关键词 脊髓损伤 骨髓间充质干细胞 立体定向 细胞移植     

人羊膜间充质干细胞移植对大鼠脊髓损伤神经功能恢复的影响

目的 观察人羊膜间充质干细胞( hAD - MSCs)移植对大鼠脊髓损伤神经功能恢复的影响.方法 建立大鼠脊髓全横断损伤模型,脊髓横断后立即以明胶海绵吸附10μl hAD- MSCs(约2×105个)或等量PBS液植入脊髓两断端之间.术后每周应用BBB评分评价大鼠后肢运动功能;采用免疫荧光染色观察hAD - MSCs在脊髓内的存活、分化情况;免疫组织化学染色观察受损脊髓远端组织NF - 200表达.结果 hAD - MSCs移植组神经功能明显恢复,BBB评分逐周增加,与对照组比较差异有统计学意义(P＜0.05).hAD - MSCs植入后2周在宿主脊髓中存在MAB1281染色阳性细胞,但不表达MAP -2和GFAP.hAD - MSCs移植后大鼠受损脊髓远端神经组织NF - 200表达明显强于对照组.结论 hAD - MSCs移植可促进大鼠脊髓损伤后的神经功能恢复,其机制可能与hAD - MSCs促进受损脊髓远端组织表达NF - 200有关.     

骨髓间质细胞源性神经球移植对大鼠脊髓损伤的修复作用

目的探讨骨髓间质细胞源性神经球(BMSCs-NS)移植在大鼠脊髓损伤修复中的作用。方法体外原代培养大鼠BMSCs-NS。Wistar大鼠45只,玻璃器压迫法制备胸髓(T9-10)完全损伤模型,随机分成三组:BMSCs-NS移植组(A组,n=15)、BMSCs移植组(B组,n=15)和培养液组(C组,n=15)。BBB评分评估三组大鼠后肢运动功能在不同时间段的恢复及双下肢运动诱发电位(MEPs)情况。结果 BBB平均分值为:A组,7.8±1.42;B组,5.7±0.90;C组,1.4±0.29。移植修复21d后BBB绝对分值,A组较B组和C组明显改善(B组:p<0.001;C组:p<0.0001)。移植5w后B组少数大鼠可以诱发出MEPs,但潜伏期较长(20ms)且波幅较低。2w时A组未能诱发出MEPs,但5w时的MEPs接近正常。结论细胞移植治疗能够恢复大鼠后肢的承重运动功能并能成功诱导出理想的MEPs。     

神经生长因子联合神经干细胞移植治疗大鼠脊髓损伤

背景：单纯的神经干细胞移植对受损脊髓组织的修复作用并不理想,研究证实神经生长因子兼有神经元营养和促突起生长双重作用,可以有效的促进脊髓损伤后神经功能的恢复。 目的：观察神经干细胞移植联合应用神经生长因子对脊髓损伤后大鼠运动功能恢复的影响。 方法：SD大鼠42只,建立急性脊髓损伤模型后随机分成3组,伤后1周于损伤处分别注入培养液、单纯神经干细胞或神经干细胞联合神经生长因子。于伤后1,2,4,6,8周进行BBB评分和斜板实验等运动功能检测。伤后4周取材行病理切片苏木精-伊红染色及BrdU免疫组化染色,伤后8周取材行辣根过氧化物酶示踪观察及体感诱发电位观察神经电生理恢复情况。 结果与结论：伤后4周单纯神经干细胞组、神经干细胞联合神经生长因子组大鼠后肢运动功能均有较明显恢复,神经干细胞联合神经生长因子组较单纯神经干细胞组快,差异有显著性意义(P < 0.05)。培养液组亦有所恢复,但程度较轻。病理切片显示培养液组未见神经轴索通过。单纯神经干细胞组可见少量神经轴索样结构,神经干细胞联合神经生长因子组可见较多神经轴索样结构。BrdU的阳性细胞数及HRP阳性神经纤维数：神经干细胞联合神经生长因子组>单纯神经干细胞组>培养液组且各组之间差异有显著性意义(P < 0.01)。神经干细胞联合神经生长因子组大鼠体感诱发电位的潜伏期、波幅优于单纯神经干细胞组(P < 0.05),明显优于培养液组(P < 0.01)。结果提示神经干细胞移植对于后肢功能的恢复有促进作用,联合应用神经生长因子有协同效果。     

转染绿色荧光蛋白的神经干细胞移植于半横断脊髓损伤处的体内外分化研究

目的 观察转染绿色荧光蛋白(GFP)的大鼠脊髓神经干细胞移植于半横断脊髓损伤处的体内外分化情况.方法 将表达GFP的慢病毒载体转染胎鼠脊髓神经干细胞,体外用10%胎牛血清诱导分化.转染后的神经干细胞与PLGA支架移植于大鼠半横断脊髓损伤处,术后1个月和3个月取材,行GFAP、NF和CNP免疫荧光染色.结果 转染GFP的神经干细胞球表达强烈的绿色荧光,体外分化可见GFAP/GFP、NF/GFP和CNP/GFP双阳性细胞,GFAP/GFP双阳性细胞明显多于其他两种.移植后3个月,GFP阳性细胞在脊髓内明显减少,可见少数GFAP/GFP和CNP/GFP舣阳性细胞,未见NF/GFP双阳性细胞.结论 转染GFP的神经干细胞可在体外增殖和分化,但大部分分化成胶质细胞.移植于急性期脊髓损伤处的神经干细胞不被诱导分化成神经元样细胞,可被诱导分化成神经胶质细胞.     

骨髓间充质干细胞移植对大鼠脑缺血再灌注损伤的神经保护作用

目的探讨骨髓间充质干细胞(BMSCs)移植对大鼠脑缺血再灌注损伤的神经保护作用,并探讨其可能的机制。方法经体外培养并纯化BMSCs,移植前以10 mg/L的BrdU进行标记。线栓法制作大鼠MCAO模型,随机分为移植A组、B组及对照C组。在模型建立后7 d,通过尾静脉分别将3×10~6个BMSCs、1×10~6个BMSCs移植入A、B组大鼠体内,C组不作处理。于移植前、后分别进行改良神经损伤严重程度评分(mNSS)及Morris水迷宫测试,并取大鼠脑组织行免疫组织化学染色。结果移植A组与移植B组、C组比较,行为学恢复更为明显,(P0.01);B、C两组相比无显著性差异(P0.05);移植A组大鼠学习和记忆能力较C组明显改善(P0.05)。A、B组大鼠在脑损伤中心及周围区,可见Brdu单染阳性细胞及Brdu+MAP-2、Brdu+GFAP、Brdu+vWF、Brdu+VEGF双染阳性细胞。结论 BMSCs经尾静脉移植至MCAO大鼠体内后,可在大鼠体内存活、分化,并促进大鼠伸进功能恢复。疗效与移植细胞数量相关。     

骨髓间充质干细胞立体定向移植治疗大鼠脑缺血损伤的实验研究

目的观察立体定向移植骨髓间充质干细胞(BMSC)治疗MCAO大鼠的效果并探讨其可能机制。方法用改良线拴法制作大脑中动脉闭塞(MCAO)模型。60只模型大鼠随机分为移植组(A组)、磷酸盐缓冲液溶液组(B组)与假手术组(C组)。在模型建立后7 d,通过立体定向方式将1×106个BMSCs移植入A组大鼠损伤侧纹状体,B组大鼠以同样方式在同样部位移植等体积的磷酸盐缓冲液,C组完成立体定向过程,但无液体注入。应用改良大鼠神经功能缺损评分(m NSS评分)、水迷宫测试观察大鼠神经功能恢复状况,并取大鼠脑组织行免疫组织化学染色。结果 A组m NSS评分优于B组、C组(P0.05);A组逃避潜伏期明显缩短(P0.05);跨越平台次数明显增多(P0.05)。A组大鼠在脑损伤中心及周围区,可见Brdu单染阳性细胞及Brdu+BDNF、Brdu+GFAP、Brdu+v WF、Brdu+VEGF双染阳性细胞。结论立体定向移植BMSCs可以显著改善MCAO大鼠神经功能恢复。     

神经干细胞移植治疗脊髓损伤

目的:探讨神经干细胞移植对脊髓损伤大鼠后肢运动功能修复的影响。方法:SD大鼠36只,制成T10脊髓全横断损伤模型。于造模成功后1周采用局部微量注射法移植。随机分三组：A损伤对照组（n=12）仅打开椎管暴露脊髓;B移植对照组（n=12）：注射10μl DMEM/F12培养液;C细胞移植组（n=12）：移植1.0?06/ml的神经干细胞悬液10μl。移植后通过不同时间点BBB行为评分、病理组织学、免疫荧光技术评价大鼠大鼠脊髓功能修复情况及移植细胞在体内的存活、迁移、分化。 结果:在体外成功建立SD大鼠海马源性神经干细胞培养体系;B、C两组大鼠随着时间延长BBB评分均不同程度提高,从移植后2W起C组大鼠评分明显高于B组,两组比较差异有统计学意义（P<0.05）;神经干细胞移植后能够在体内继续存活、迁移并且分化为NF-200、GFAP表达阳性的神经元及星形胶质细胞。 结论:神经干细胞移植治疗脊髓损伤是一种有效的方法。     

Use of FK506 and bone marrow mesenchymal stem cells for rat hind limb allografts

Dark Agouti rat donor hind limbs were orthotopically transplanted into Lewis rat recipients to verify the effects of bone marrow mesenchymal stem cells on neural regeneration and functional recovery of allotransplanted limbs in the microenvironment of immunotolerance. bone marrow mesenchymal stem cells were intramuscularly (gluteus maximus) injected with FK506 (tacrolimus) daily, and were transplanted to the injured nerves. Results indicated that the allograft group not receiving therapy showed severe rejection, with transplanted limbs detaching at 10 days after transplantation with complete necrosis. The number of myelinated axons and Schwann cells in the FK506 and FK506 + bone marrow mesenchymal stem cells groups were significantly increased. We observed a lesser degree of gastrocnemius muscle degeneration, and increased polymorphic fibers along with other pathological changes in the FK506 + bone marrow mesenchymal stem cells group. The FK506 + bone marrow mesenchymal stem cells group showed significantly better recovery than the autograft and FK506 groups. The results demonstrated that FK506 improved the immune microenvironment. FK506 combined with bone marrow mesenchymal stem cells significantly promoted sciatic nerve regeneration, and improved sensory recovery and motor function in hind limb allotransplant.     

Very efficient myoblast allotransplantation in mice under FK506 immunosuppression

Transgenic CD1 mice expressing β-galactosidase were used as myoblast donors. The myoblasts were injected in normal or mdx muscles previously irradiated and injected with notexin. Twenty-eight days after myoblast transplantation, the percentage of muscle fibers β-galactosidase-positive was low in mice not immunosuppressed but was high (80%) in those treated with FK506. In mdx mice, muscle fibers expressing β-galactosidase were also dystrophin positive. Most of the mice not treated with FK506 produced antibodies against the donor myoblasts. These results indicate that FK506 is a very useful immunosuppressive drug for myoblast transplantation in mice. Irradiation and notexin injection used in our experiments are, however, not feasible in humans. Other manipulations capable of increasing the participation of donor myoblasts to regeneration will therefore have to be identified before new clinical trials are attempted. © 1994 John Wiley & Sons, Inc.     

A small dose of the immunosuppressive agent FK506 (tacrolimus) protects peripheral nerve from ischemic fiber degeneration.

The immunosuppressant agent FK506 (tacrolimus) has proven to be neuroprotective against brain ischemia, but there are no data on potential neuroprotective effects of FK506 in peripheral nerve ischemia. We examined the potential effects of two doses of FK506 in protecting peripheral nerve from ischemic fiber degeneration. Ischemia in the left sciatic nerve of the rat was produced by injecting 2 x 10(6) microspheres (14 microm) into the left femoral, hypogastric, and superior gluteal arteries in proportions of 47%, 37%, and 17%, respectively. After embolization, FK506 was injected into the left femoral, hypogastric, and superior gluteal arteries in doses of 9.4, 7.4, and 3.4 microg, respectively, for the high-dose group and 4.7, 3.7, and 1.7 microg, respectively, for the low-dose group. The control rats were injected with saline. FK506 treatment resulted in dramatic behavioral improvement in nerve function, in the number of functioning nerve fibers, and in the salvage of a majority of nerve fibers from ischemic fiber degeneration in a dose-dependent fashion. These results suggest that a small dose of FK506 protects peripheral nerve from ischemic fiber degeneration and that it may have potential in the treatment of ischemic neuropathy.     

FK506 suppressed the inflammatory change of EAM in SJL/J mice.

Experimental autoimmune myositis (EAM) is a good model of human inflammatory myopathy. We induced EAM in SJL/J mice by injection with myosin and treated inflammatory changes with FK506. The mice developed inflammatory changes after the fifth myosin injection. After treatment with FK506, inflammation was suppressed and central nuclei of the muscle fibers increased. These findings indicate that FK506 is effective in the treatment of EAM. The data suggests that FK506 inhibits interaction with calcineurin. Intercellular adhesion molecule-1 (ICAM-1) positive cells were present in the inflammatory and non-inflammatory areas of EAM. The FK506-treated group stained more weakly for ICAM-1 than the untreated EAM group.     

同种异体肢体移植中他克莫司的应用剂量*☆

背景：他克莫司应用于同种异体肝移植的免疫耐受已多见报道。 目的：探索他克莫司在异体肢体移植时的最佳应用剂量。 方法：建立同种异体肢体移植大鼠模型,移植造模后设立给予不同他克莫司剂量的0.5,1,2 mg/(kg•d)组及对照组,对大鼠进行大体观察、组织学、淋巴细胞亚群测定。 结果与结论：移植后出现排斥反应的时间分别是对照组(3.43±0.79) d、0.5 mg/(kg•d)组(5.68±0.97) d、1 mg/(kg•d)组(9.13±1.17) d、2 mg/(kg•d) 组(9.61±2.38) d,排斥反应的病理分级4组分别为(2.61±0.38),(1.57±0.43),(0.85±0.24),(0.71±0.19)级。移植后各给药组CD4+、CD8+检测值均明显下降,CD4/CD8比值轻度增加或在正常值范围内;对照组CD4+、CD8+无明显变化,CD4/CD8比值明显增加。提示,他克莫司的应用剂量在1 mg/(kg•d)时就能达到理想的抑制免疫排斥反应,提高用量后意义不大。     

Synergistic effects of bone marrow stromal cells and a Rho kinase (ROCK) inhibitor,Fasudil on axon regeneration in rat spinal cord injury

Transplanted bone marrow stromal cells (BMSC) promote functional recovery after spinal cord injury (SCI) through multiple mechanisms. A Rho kinase inhibitor, Fasudil also enhances axonal regeneration. This study was aimed to evaluate whether combination therapy of BMSC transplantation and Fasudil further enhances axonal regeneration and functional recovery in rats subjected to SCI. Fasudil or vehicle was injected for 2 weeks. BMSC or vehicle transplantation into the rostral site of SCI was performed at 7 days after injury. Neurological symptoms were assessed throughout the experiments. Fluoro‐Ruby was injected into the dorsal funiculus of the rostral site of SCI at 63 days after injury. The fate of the transplanted BMSC was examined using immunohistochemistry. BMSC transplantation significantly increased the number of Fluoro‐Ruby ‐labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI, enhancing functional recovery of the hind limbs. Some of the engrafted BMSC were positive for Fluoro‐Ruby, neuronal specific nuclear protein and microtubule‐associated protein‐2, suggesting that they acquired neuronal phenotypes and built synaptic connection with the host's neural circuits. Fasudil treatment also improved axonal continuity, but did not promote functional recovery. Combination therapy dramatically increased the number of Fluoro‐Ruby‐labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI, but did not further boost the therapeutic effects on locomotor function by BMSC transplantation. The findings suggest that BMSC transplantation and Fasudil provide synergistic effects on axon regeneration after SCI, although further studies would be necessary to further enhance functional recovery.     

FK 506 reduces tissue damage and prevents functional deficit after spinal cord injury in the rat

We examined the efficacy of FK 506 in reducing tissue damage after spinal cord injury in comparison to methylprednisolone (MP) treatment. Rats were subjected to a photochemical injury (T8) and were given a bolus of MP (30 mg/kg), FK 506 (2 mg/kg), or saline. An additional group received an initial bolus of FK 506 (2 mg/kg) followed by daily injections (0.2 mg/kg intraperitoneally). Functional recovery was evaluated using open-field walking, inclined plane tests, motor evoked potentials (MEPs), and the H-reflex response during 14 days postoperation (dpo). Tissue sparing and glial fibrillary acidic protein (GFAP), biotinylated tomato lectin LEC, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and interleukin 1 beta (IL-1 beta) immunoreactivity were quantified in the injured spinal cord. FK 506-treated animals demonstrated significantly better neurologic outcome, higher MEP amplitudes, and lower H-wave amplitude compared to that of saline-treated rats. In contrast, administration of MP did not result in significant differences with respect to the saline-treated group. Histologic examination revealed that tissue sparing was largest in FK 506-treated compared to saline and MP-treated animals. GFAP and COX-2 reactivity was decreased in animals treated with FK 506 compared to that in animals given MP or saline, whereas IL-1 beta expression was similarly reduced in both FK 506- and MP-treated groups. Microglia/macrophage response was reduced in FK 506 and MP-injected animals at 3 dpo, but only in MP-treated animals at 7 dpo with respect to saline-injected rats. Repeated administrations of FK 506 improved functional and histologic results to a greater degree than did a single bolus of FK 506. The results indicate that FK 506 administration protects the damaged spinal cord and should be considered as potential therapy for treating spinal cord injuries.     

Effect of the calcineurin inhibitor FK506 on K+–Cl? cotransporter 2 expression in the mouse hippocampus after kainic acid-induced status epilepticus

Calcineurin (CaN)-mediated excitotoxicity impairs γ-aminobutyric acid (GABA) transmission and induces neuronal apoptosis. Ca(2+)-dependent K(+)-Cl(-) cotransporter 2 (KCC2) participates in GABAergic inhibitory transmission. However, the mechanism by which CaN mediates GABA receptor-mediated KCC2 in seizures is not fully understood. In the present study, we investigated the altered expression of KCC2 and the effects of the CaN inhibitor FK506 on KCC2 expression in the mouse hippocampus following kainic acid (KA) treatment. FK506 was injected twice 24 h and 30 min before KA treatment and then mice were treated with KA and killed 2 days later. FK506 had anticonvulsant effect on KA-induced seizure activities. CaN cleavage was evident in the hippocampus 24 h after KA treatment. FK506 pretreatment blocked the truncation of CaN in the KA-treated hippocampus. Cresyl violet and TUNEL staining showed that FK506 prevented KA-induced hippocampal cell death. In particular, Western blot analysis showed that KCC2 expression was time dependent, with a peak at 6 h and a return to decreased levels at 48 h, whereas FK506 pretreatment inhibited the KA-induced decrease in KCC2 expression in the hippocampus. Immunofluorescence showed that FK506 pretreatment protected the loss of inhibitory GABAergic KCC2-expressing neurons following KA treatment. Taken together, these results provide evidence that altered KCC2 expression may be associated with Ca(2+)-mediated seizure activity and indicate that neuron-specific KCC2 may be involved in neuroprotection after seizures.     

FK506 treatment inhibits caspase-3 activation and promotes oligodendroglial survival following traumatic spinal cord injury

The focus of this study is to examine the ability of FK506, an immunosuppressant that inhibits calcineurin activation, to limit caspase-3 activation in oligodendroglia following spinal cord injury (SCI). To better establish a role for calcineurin and caspase-3 activation in oligodendroglia following SCI, rats received a contusion injury to the spinal cord followed by treatment with FK506 or rapamycin (another immunosuppressant with no detectable inhibitory action on calcineurin activation). Animals were then sacrificed at 8 days postinjury and spinal cord tissue was processed using immunofluorescence histochemistry to examine cellular caspase-3 activation in ventral and dorsal white matter. In all treatment groups, numerous oligodendroglia were found to express the activated form of caspase-3 in regions proximal and distal to the injury epicenter. However, our findings suggest that treatment with FK506, but not rapamycin reduces the number of oligodendroglia expressing activated caspase-3 and increases the number of surviving oligodendroglia in dorsal white matter. These results provide initial evidence that agents that reduce the actions of calcineurin and subsequent caspase-3 activation may prove beneficial in the treatment of traumatic SCI.     

Comparison of continuous and discontinuous FK506 administration on autograft or allograft repair of sciatic nerve resection

An immunosuppressant drug that also possesses neuroregenerative properties, FK506 enhances the rate of axonal regeneration and improves recovery after nerve lesions. Nevertheless, prolonged immunosuppression may not be justified to assure the success of nerve regeneration. In this study, we compare the effects of continuous and discontinuous FK506 treatment on regeneration and reinnervation after sciatic nerve resection repaired with autologous or allogenic grafts in the mouse. For each type of repair, one group received FK506 (5 mg/kg) for 4 months, whereas a second group was treated with FK506 at 5 mg/kg for 5 weeks followed by 3 mg/kg for 4 weeks; a control group received saline only. Functional reinnervation was assessed by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hind paw over 4 months after operation. Morphological analysis of the regenerated nerves was performed at the termination of the study. Autografts and allografts treated with sustained FK506 (5 mg/kg) reached high levels of reinnervation and followed a course of recovery faster than controls. The numbers of myelinated fibers also were similar. Allografts without immunosuppression demonstrated a slower rate of regeneration, exhibiting lower final levels of recovery compared with other groups and containing fewer numbers of regenerating myelinated fibers. Withdrawal of immunosuppressant therapy resulted in a decline in the degree of reinnervation in all functions tested during the third month, with stabilization between the third and fourth months. The number of regenerated myelinated fibers in the group was significantly lower than in autografts. Thus, continuous or discontinuous FK506 administration slightly accelerated the rate of reinnervation in autografts. In allograft repair, FK506 significantly enhanced both the rate and degree of regeneration and recovery, but its withdrawal resulted in graft rejection, a marked deterioration in function, and loss of regenerating fibers.