米诺环素对缺血再灌注脑损伤后轴突再生的影响及机制研究
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摘要
目的脑卒中是目前世界范围内严重危害人类健康的疾病之一，其发病率随着年龄的增加而上升，对家庭和社会造成严重的经济负担。脑卒中患者残留严重的神经功能缺损，是因为中枢神经系统损伤后轴突再生困难。传统的观点认为，中枢神经再生困难最主要的原因是因为受损神经元缺乏内源性再生的能力。但目前的观点认为，中枢神经系统病灶周围存在大量轴突再生抑制因子，这是阻碍受损神经元轴突再生的关键因素。排斥性导向分子（repulsive guidance molecule A,RGMa）是目前认为最有潜力的轴突再生抑制因子之一，可以激活细胞内信号传导分子RhoA及其效应器ROCK，使下游底物肌球蛋白轻链（myosin light chains, MLC）磷酸化水平增加，从而介导生长锥的塌陷和轴突再生抑制作用。米诺环素是一种能通过血脑屏障的四环素类抗生素，在多种急性脑缺血的动物模型中具有广谱的抗炎、抗凋亡、抗氧化及血管保护作用。在前期的临床研究中也发现，米诺环素可促进急性脑梗死患者神经功能的恢复。然而，其潜在的细胞和分子机制尚未完全阐明。本研究拟观察米诺环素是否可以通过下调缺血灶周围RGMa的表达，促进轴突再生，从而改善大鼠的神经功能，并且在体外进一步观察米诺环素是否通过MLCP/MLC信号通路促进氧糖剥夺/复氧损伤后PC12细胞神经突的生长作用。
方法
第一部分
1.将72只清洁级健康成年雄性SD大鼠，按随机数字表法分为以下四组：假手术组，缺血再灌注组，生理盐水组及米诺环素组，各组各18只。采用线栓法闭塞大鼠右侧大脑中动脉2h制备局灶性脑缺血再灌注损伤（I/R）模型，于再灌注的同时静脉注射3mg/kg的米诺环素，每天2次，持续14天。在缺血再灌注24h后，采用伊文思蓝（EB）法检测缺血脑组织血脑屏障通透性的改变。
2.再灌注2周时，采用免疫组织化学及Western Blot法检测缺血侧皮质及海马中RGMa蛋白的表达，免疫组化染色神经微丝蛋白200（NF-200）评估神经元轴突的生长。
3.分别在缺血再灌注第2，7，14及28天，采用改良的神经功能缺损评分（Modified neurological severity scores，mNSS)）及Montoya楼梯实验（staircase test）评估大鼠的神经功能。
第二部分
1.采用氧糖剥夺/复氧（oxygen glucose deprivation/reperfusion,OGD/R）模型模拟体内脑缺血再灌注损伤，体外培养的PC12细胞随机分为正常组，氧糖剥夺2，4，6，8h组和不同浓度的（0.1,1,10μM）米诺环素治疗组。在氧糖剥夺再复氧24h后采用CCK-8法测定PC12细胞的存活率。
2.在氧糖剥夺再复氧24h后采用MAP-2免疫细胞荧光染色标记PC12细胞的神经突，荧光显微镜下观察神经突长度，Western blot法检测各组GAP-43蛋白的表达水平。
3.体外培养的PC12细胞随机分为正常组，模型组，1μM米诺环素组和MLCP抑制剂组（1nM, Calyculin A）。在氧糖剥夺再复氧24h后采用Western blot法测定MLC及p-MLC的表达，荧光显微镜下观察神经突的长度及检测GAP-43蛋白的表达水平。
结果
第一部分
1.缺血再灌注24h后，大鼠缺血侧脑组织有明显的EB渗出（4.40±0.73μg/g），静脉给予3mg/kg米诺环素治疗后能降低缺血侧脑组织EB的渗出量（2.71±0.68μg/g，P<0.05）。
2.缺血再灌注2周后，缺血侧皮质及海马RGMa蛋白表达增加，轴突损伤严重，NF-200蛋白表达明显降低（P<0.05），而米诺环素组大鼠缺血脑组织内RGMa表达较模型组明显降低（P<0.05），NF-200蛋白表达增加（P<0.05）。
3.米诺环素使大鼠缺血再灌注损伤后改良的神经功能评分（mNSS）显著下降并改善大鼠的前肢运动功能（P<0.05）。
第二部分
1. PC12细胞的存活率随缺氧缺糖时间的延长而逐渐降低，OGD6h时细胞存活率为(46.1±2.9)。（0.1～10μM）米诺环素呈非线性浓度依赖性提高氧糖剥夺6h后PC12细胞的存活率，其中1μM浓度作用最强（P<0.05）。
2.氧糖剥夺损伤引起PC12细胞神经突回缩，米诺环素能显著促进PC12细胞氧糖剥夺损伤后神经突的生长，同时上调轴突再生蛋白GAP-43的表达（P<0.05），Calyculin A能阻断米诺环素的促进作用。此外，氧糖剥夺诱导PC12细胞MLC磷酸化水平增加，而米诺环素能显著降低PC12细胞MLC磷酸化水平（P<0.01）。
结论：米诺环素能促进大鼠局灶性缺血再灌注损伤后神经功能的恢复及轴突再生相关蛋白的表达，其机制可能与下调RGMa蛋白的表达有关。米诺环素可能是通过激活MLCP/MLC信号通路调控PC12细胞内MLC磷酸化水平，促进神经突的生长。
Objective Stroke is one of the most common diseases which is amajor public health problem threaten to human health worldwide andshows an increase in prevalence as population ages. Stroke has alreadyinduced severe economic burden on the family and society in developingcountries. Cerebral ischemia generally leads to persistent and severefunctional deficits. Accumulated evidence has indicated that the limitedcapacity of the adult central nervous system (CNS) to support therearrangement and re-extension of axonal connections is the majordeterminant of failed functional recovery. Axonal regeneration failure ismost likely attributable to the non-permissive adult CNS environment.There are many axonal regeration inhibitors near the site of injury. Therepulsive guidance molecule A (RGMa) is a membrane-bound proteinoriginally identified as an axon guidance molecule in chicken temporalretina. It recently has been identified as a powerful myelin-derived neuriteoutgrowth inhibitor in adult CNS. RGMa is mediated by the RhoA/ROCKpathway. The active ROCK regulates the phosphorylation level of MLC in an indirect manner by phosphorylating myosin light chains phosphatase(MLCP), these lead to up-regulation of phosphorylated MLC, which play akey role in neurite retraction and growth cone collapse. Minocycline is asecond-generation semi-synthetic tetracycline drug, which freely crossesthe blood-brain barrier and possesses a wide-spectrum anti-inflammatory,anti-apoptotic, antioxidative activities and vascular protective properties inmany animal models of cerebral ischemia. Accumulating clinical trialssuggest that minocycline could improve neurological function recovery inpatients with acute cerebral ischemia. However, the underlying cellular andmolecular bases for its neuroprotective effects have not been fullyelucidated. In this study, we investigated whether minocycline maydown-regulate the expression of RGMa protein and improve neurologicalfunction recovery after focal cerebral ischemia reperfusion. Furthermore,we investigated whether minocycline could enhance neurite outgrowth ofPC12cells exposed to oxygen glucose deprivation, which was associatedwith the activation of MCLP/MLC signaling pathway.
Methods
Part I
1. Seventy-two adult male healthy Sprague-Dawley (SD) rats wererandomly divided into the following four groups: sham-operated (n=18),cerebral ischemia and reperfusion (n=18), saline vehicle (n=18) andminocycline treated groups (n=18). The focal cerebral ischemia/reperfusion model (I/R) was induced by ligation the right middle cerebral artery of ratswith nylon monofilament for2h and3mg/kg minocycline was injectedintravenously immediately after reperfusion twice a day for14days in ratsof minocycline treated group. The permeability of blood-brain barrier wasassessed by Evan’s blue dye extravasation (EB) in the infarct brain24h afterMCAO/R.
2. The expression of RGMa protein in ischemic cortex andhippocampus was measured by immunohistochemical staining and Westernblot. Neurofilament protein200(NF-200) immunohistochemical stainingwas used to assess axonal damage2weeks after MCAO/R.
3. The staircase test and modified neurological severity score wereperformed to evaluate functional outcome at2,7,14and28days afterMCAO/R.
Part II
1. PC12cells were subjected to oxygen glucose deprivation andreoxygenation (OGD/R) to mimic cerebral ischemia reperfusion injury invivo. The cultured PC12cells were randomly divided into three groups:normal control group, OGD/R group (PC12cells were exposed to2,4,6,8h of OGD) and various doses of minocycline (0.1,1,10μM) treated group.24hours after reoxygenation, the cell viability of PC12cells was detectedwith CCK-8assay.
2. Twenty four hours after OGD/R, The expression of neurite outgrowth maker protein, growth associated protein (GAP-43) wasmeasured by western blot, and the mean length of neurite outgrowth wasalso measured with microtubule associated protein-2immunostainingunder fluorescence microscope.
3. PC12cells were randomly divided into normal control group,OGD/R group (6h), minocycline treated group (1μM) and MLCP inhibitorgroup (1nM, Calyculin A). The expression of myosin light chain (MLC),p-MLC and GAP-43protein were measured by Western blot and neuriteoutgrowth was visualized by immunofluorescence staining with MAP-2antibody24h after OGD/R.
Results
Part I
1. The Evans blue extravasation in ischemic brain tissue of rats wasobvious24h after I/R（4.40±0.73μg/g. Minocycline at a dose of3mg/kg(caudal vein) significantly reduced the extravasation of EB (2.71±0.68μg/g, P<0.05).
2. The expression of RGMa protein in ischemic cortex andhippocampus was significantly increased in the I/R group compared to thesham group assessed by both immunochemical staining and Western blottwo weeks after I/R, axonal regrowth was enhanced in the minocyclinetreated group compared to the I/R group (P<0.05). In addition, minocyclinesignificantly reduced the expression of RGMa protein2weeks after I/R. (P<0.05).
3. Minocycline could decrease mNSS and improve forelimb motorfunction as assessed by the staircase test of rats compared to the rats in theI/R group (P<0.05).
Part II
1. Oxygen glucose deprivation injury induced a time-dependentdecrease in cell viability of PC12cells, and the cell viability was46.1±2.9%at6h after OGD. Minocycline elicited a non-linear concentration-dependentneuroprotective effect on cell viability of PC12cells exposed to OGD for6h.The maximal neuroprotective effect of minocycline was achieved at1μM(77.0±2.5%, P<0.05).
2. OGD/R injury induced neurite retraction, minocycline improvedneurite outgrowth and increased the expression of GAP-43protein in PC12cells after OGD/R injury (P<0.05). Calyculin A (the inhibitor of MLCP)could abolish the promotive effects of minocycline. In addition, OGD/Rinduced an increase in the phosphorylation of MLC and minocyclinedecreased the phosphorylation of MLC in PC12cells after6h of OGD/Rinjury (P<0.01).
Conclusions Minocycline at a dose of3mg/kg promotes neurologicalfunctional recovery and axonal regeneration in rats after I/R injury, whichmight be mediated by down-regulating RGMa expression. Minocyclinecould activate MLCP/MLC signaling pathway, induce lower levels of p-MLC in PC12cells after OGD/R, which leads to the enhancement ofneurite outgrowth.
方法
第一部分
1.将72只清洁级健康成年雄性SD大鼠，按随机数字表法分为以下四组：假手术组，缺血再灌注组，生理盐水组及米诺环素组，各组各18只。采用线栓法闭塞大鼠右侧大脑中动脉2h制备局灶性脑缺血再灌注损伤（I/R）模型，于再灌注的同时静脉注射3mg/kg的米诺环素，每天2次，持续14天。在缺血再灌注24h后，采用伊文思蓝（EB）法检测缺血脑组织血脑屏障通透性的改变。
2.再灌注2周时，采用免疫组织化学及Western Blot法检测缺血侧皮质及海马中RGMa蛋白的表达，免疫组化染色神经微丝蛋白200（NF-200）评估神经元轴突的生长。
3.分别在缺血再灌注第2，7，14及28天，采用改良的神经功能缺损评分（Modified neurological severity scores，mNSS)）及Montoya楼梯实验（staircase test）评估大鼠的神经功能。
第二部分
1.采用氧糖剥夺/复氧（oxygen glucose deprivation/reperfusion,OGD/R）模型模拟体内脑缺血再灌注损伤，体外培养的PC12细胞随机分为正常组，氧糖剥夺2，4，6，8h组和不同浓度的（0.1,1,10μM）米诺环素治疗组。在氧糖剥夺再复氧24h后采用CCK-8法测定PC12细胞的存活率。
2.在氧糖剥夺再复氧24h后采用MAP-2免疫细胞荧光染色标记PC12细胞的神经突，荧光显微镜下观察神经突长度，Western blot法检测各组GAP-43蛋白的表达水平。
3.体外培养的PC12细胞随机分为正常组，模型组，1μM米诺环素组和MLCP抑制剂组（1nM, Calyculin A）。在氧糖剥夺再复氧24h后采用Western blot法测定MLC及p-MLC的表达，荧光显微镜下观察神经突的长度及检测GAP-43蛋白的表达水平。
结果
第一部分
1.缺血再灌注24h后，大鼠缺血侧脑组织有明显的EB渗出（4.40±0.73μg/g），静脉给予3mg/kg米诺环素治疗后能降低缺血侧脑组织EB的渗出量（2.71±0.68μg/g，P<0.05）。
2.缺血再灌注2周后，缺血侧皮质及海马RGMa蛋白表达增加，轴突损伤严重，NF-200蛋白表达明显降低（P<0.05），而米诺环素组大鼠缺血脑组织内RGMa表达较模型组明显降低（P<0.05），NF-200蛋白表达增加（P<0.05）。
3.米诺环素使大鼠缺血再灌注损伤后改良的神经功能评分（mNSS）显著下降并改善大鼠的前肢运动功能（P<0.05）。
第二部分
1. PC12细胞的存活率随缺氧缺糖时间的延长而逐渐降低，OGD6h时细胞存活率为(46.1±2.9)。（0.1～10μM）米诺环素呈非线性浓度依赖性提高氧糖剥夺6h后PC12细胞的存活率，其中1μM浓度作用最强（P<0.05）。
2.氧糖剥夺损伤引起PC12细胞神经突回缩，米诺环素能显著促进PC12细胞氧糖剥夺损伤后神经突的生长，同时上调轴突再生蛋白GAP-43的表达（P<0.05），Calyculin A能阻断米诺环素的促进作用。此外，氧糖剥夺诱导PC12细胞MLC磷酸化水平增加，而米诺环素能显著降低PC12细胞MLC磷酸化水平（P<0.01）。
结论：米诺环素能促进大鼠局灶性缺血再灌注损伤后神经功能的恢复及轴突再生相关蛋白的表达，其机制可能与下调RGMa蛋白的表达有关。米诺环素可能是通过激活MLCP/MLC信号通路调控PC12细胞内MLC磷酸化水平，促进神经突的生长。
Objective Stroke is one of the most common diseases which is amajor public health problem threaten to human health worldwide andshows an increase in prevalence as population ages. Stroke has alreadyinduced severe economic burden on the family and society in developingcountries. Cerebral ischemia generally leads to persistent and severefunctional deficits. Accumulated evidence has indicated that the limitedcapacity of the adult central nervous system (CNS) to support therearrangement and re-extension of axonal connections is the majordeterminant of failed functional recovery. Axonal regeneration failure ismost likely attributable to the non-permissive adult CNS environment.There are many axonal regeration inhibitors near the site of injury. Therepulsive guidance molecule A (RGMa) is a membrane-bound proteinoriginally identified as an axon guidance molecule in chicken temporalretina. It recently has been identified as a powerful myelin-derived neuriteoutgrowth inhibitor in adult CNS. RGMa is mediated by the RhoA/ROCKpathway. The active ROCK regulates the phosphorylation level of MLC in an indirect manner by phosphorylating myosin light chains phosphatase(MLCP), these lead to up-regulation of phosphorylated MLC, which play akey role in neurite retraction and growth cone collapse. Minocycline is asecond-generation semi-synthetic tetracycline drug, which freely crossesthe blood-brain barrier and possesses a wide-spectrum anti-inflammatory,anti-apoptotic, antioxidative activities and vascular protective properties inmany animal models of cerebral ischemia. Accumulating clinical trialssuggest that minocycline could improve neurological function recovery inpatients with acute cerebral ischemia. However, the underlying cellular andmolecular bases for its neuroprotective effects have not been fullyelucidated. In this study, we investigated whether minocycline maydown-regulate the expression of RGMa protein and improve neurologicalfunction recovery after focal cerebral ischemia reperfusion. Furthermore,we investigated whether minocycline could enhance neurite outgrowth ofPC12cells exposed to oxygen glucose deprivation, which was associatedwith the activation of MCLP/MLC signaling pathway.
Methods
Part I
1. Seventy-two adult male healthy Sprague-Dawley (SD) rats wererandomly divided into the following four groups: sham-operated (n=18),cerebral ischemia and reperfusion (n=18), saline vehicle (n=18) andminocycline treated groups (n=18). The focal cerebral ischemia/reperfusion model (I/R) was induced by ligation the right middle cerebral artery of ratswith nylon monofilament for2h and3mg/kg minocycline was injectedintravenously immediately after reperfusion twice a day for14days in ratsof minocycline treated group. The permeability of blood-brain barrier wasassessed by Evan’s blue dye extravasation (EB) in the infarct brain24h afterMCAO/R.
2. The expression of RGMa protein in ischemic cortex andhippocampus was measured by immunohistochemical staining and Westernblot. Neurofilament protein200(NF-200) immunohistochemical stainingwas used to assess axonal damage2weeks after MCAO/R.
3. The staircase test and modified neurological severity score wereperformed to evaluate functional outcome at2,7,14and28days afterMCAO/R.
Part II
1. PC12cells were subjected to oxygen glucose deprivation andreoxygenation (OGD/R) to mimic cerebral ischemia reperfusion injury invivo. The cultured PC12cells were randomly divided into three groups:normal control group, OGD/R group (PC12cells were exposed to2,4,6,8h of OGD) and various doses of minocycline (0.1,1,10μM) treated group.24hours after reoxygenation, the cell viability of PC12cells was detectedwith CCK-8assay.
2. Twenty four hours after OGD/R, The expression of neurite outgrowth maker protein, growth associated protein (GAP-43) wasmeasured by western blot, and the mean length of neurite outgrowth wasalso measured with microtubule associated protein-2immunostainingunder fluorescence microscope.
3. PC12cells were randomly divided into normal control group,OGD/R group (6h), minocycline treated group (1μM) and MLCP inhibitorgroup (1nM, Calyculin A). The expression of myosin light chain (MLC),p-MLC and GAP-43protein were measured by Western blot and neuriteoutgrowth was visualized by immunofluorescence staining with MAP-2antibody24h after OGD/R.
Results
Part I
1. The Evans blue extravasation in ischemic brain tissue of rats wasobvious24h after I/R（4.40±0.73μg/g. Minocycline at a dose of3mg/kg(caudal vein) significantly reduced the extravasation of EB (2.71±0.68μg/g, P<0.05).
2. The expression of RGMa protein in ischemic cortex andhippocampus was significantly increased in the I/R group compared to thesham group assessed by both immunochemical staining and Western blottwo weeks after I/R, axonal regrowth was enhanced in the minocyclinetreated group compared to the I/R group (P<0.05). In addition, minocyclinesignificantly reduced the expression of RGMa protein2weeks after I/R. (P<0.05).
3. Minocycline could decrease mNSS and improve forelimb motorfunction as assessed by the staircase test of rats compared to the rats in theI/R group (P<0.05).
Part II
1. Oxygen glucose deprivation injury induced a time-dependentdecrease in cell viability of PC12cells, and the cell viability was46.1±2.9%at6h after OGD. Minocycline elicited a non-linear concentration-dependentneuroprotective effect on cell viability of PC12cells exposed to OGD for6h.The maximal neuroprotective effect of minocycline was achieved at1μM(77.0±2.5%, P<0.05).
2. OGD/R injury induced neurite retraction, minocycline improvedneurite outgrowth and increased the expression of GAP-43protein in PC12cells after OGD/R injury (P<0.05). Calyculin A (the inhibitor of MLCP)could abolish the promotive effects of minocycline. In addition, OGD/Rinduced an increase in the phosphorylation of MLC and minocyclinedecreased the phosphorylation of MLC in PC12cells after6h of OGD/Rinjury (P<0.01).
Conclusions Minocycline at a dose of3mg/kg promotes neurologicalfunctional recovery and axonal regeneration in rats after I/R injury, whichmight be mediated by down-regulating RGMa expression. Minocyclinecould activate MLCP/MLC signaling pathway, induce lower levels of p-MLC in PC12cells after OGD/R, which leads to the enhancement ofneurite outgrowth.
引文
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[15] McKerracher L, David S, Jackson DL, et al. Identification of myelin-associatedglycoprotein as a major myelin-derived inhibitor of neurite growth[J]. Neuron,1994,13(4):805-11.
[16] Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is aNogo receptor ligand that inhibits neurite outgrowth[J]. Nature,2002,417(6892):941-4.
[17] GrandPré T, Nakamura F, Vartanian T, et al. Identification of the Nogo inhibitor ofaxon regeneration as a Reticulon protein[J]. Nature,2000,403(6768):439-44.
[18] Stahl B, Müller B, von Boxberg Y, et al. Biochemical characterization of a putativeaxonal guidance molecule of the chick visual system.[J]. Neuron,1990,5(5):735-43.
[19] Severyn C J, Shinde U, Rotwein P, et al. Molecular biology, genetics andbiochemistry of the repulsive guidance molecule family[J]. Biochem J,2009,422(3):393-403
[20] Schwab JM, Monnier PP, Schluesener HJ, et al. CNS injury induced RGM(repulsive guidance molecule) expression in the adult human brain [J]. ArchNeurol,2005,62(10):1561-8.
[21] Hata K, Fujitani M, Yasuda Y, et al. RGMa inhibition promotes axonal growth andrecovery after spinal cord injury[J]. J Cell Biol,2006;173(1):47-58.
[22] Franco EC, Cardoso MM, Gouvêia A, et al. Modulation of microglial activationenhances neuroprotection and functional recovery derived from bone marrowmononuclear cell transplantation after cortical ischemia[J]. Neurosci Res,2012,73(2):122-32.
[23] Hayakawa K, Mishima K, Nozako M, et al. Delayed treatment with minocyclineameliorates neurologic impairment through activated microglia expressing ahigh-mobility group box1-inhibiting mechanism[J]. Stroke,2008,39(3):951-8.
[24] Xu L, Fagan SC, Waller JL, et al. Low dose intravenous minocycline isneuroprotective after middle cerebral artery occlusion-reperfusion in rats[J]. BMCNeurol,2004,4:7.
[25] Fagan SC, Waller JL, Nichols FT, et al. Minocycline to improve neurologicoutcome in stroke (MINOS): a dose-finding study[J]. Stroke,2010,41(10):2283-7.
[26] Shaw G, Yang C, Ellis R, et al. Hyperphosphorylated neurofilament NF-H is aserum biomarker of axonal injury[J]. Biochem Biophys Res Commun,2005,336(4):1268-77.
[1] Papadopoulos CM, Tsai SY, Cheatwood JL, et al. Dendritic plasticity in the adultrat following middle cerebral artery occlusion and Nogo-a neutralization[J]. CerebCortex.2006,16(4):529-36.
[2] Fagan SC, Cronic LE, Hess DC. Minocycline Development for Acute IschemicStroke[J]. Transl Stroke Res,2011,1;2(2):202-208.
[3] Plane JM, Shen Y, Pleasure DE, et al. Prospects for minocycline neuroprotection[J].Arch Neurol,2010,67(12):1442-8.
[4] Lampl Y, Boaz M, Gilad R, et al. Minocycline treatment in acute stroke: anopen-label, evaluator-blinded study[J]. Neurology.2007,69(14):1404-10.
[5] Hashimoto K, Ishima T. A novel target of action of minocycline in NGF-inducedneurite outgrowth in PC12cells: translation initiation [corrected] factor eIF4AI[J].PLoS One.2010,5(11):e15430.
[6] Walsh MP, Cole WC. The role of actin filament dynamics in the myogenicresponse of cerebral resistance arteries[J]. J Cereb Blood Flow Metab.2013,33(1):1-12.
[7] Kubo T, Endo M, Hata K, et al. Myosin IIA is required for neurite outgrowthinhibition produced by repulsive guidance molecule[J]. J Neurochem,2008,105(1):113-26.
[8] Tabakman R, Lazarovici P, Kohen R. Neuroprotective effects of carnosine andhomocarnosine on pheochromocytoma PC12cells exposed to ischemia[J]. JNeurosci Res,2002,15;68(4):463-9.
[9] Singh G, Siddiqui MA, Khanna VK, et al. Oxygen glucose deprivation model ofcerebral stroke in PC-12cells: glucose as a limiting factor[J]. Toxicol MechMethods,2009,19(2):154-60.
[10] Fan X, Lo EH, Wang X. Effects of minocycline plus tissue plasminogen activatorcombination therapy after focal embolic stroke in type1diabetic rats[J]. Stroke,2013,44(3):745-52.
[11] Song Y, Wei EQ, Zhang WP, et al. Minocycline protects PC12cells fromischemic-like injury and inhibits5-lipoxygenase activation[J]. Neuroreport.2004Oct5;15(14):2181-4.
[12] Chen X, Chen S, Jiang Y, et al. Minocycline reduces oxygen-glucosedeprivation-induced PC12cell cytotoxicity via matrix metalloproteinase-9, integrinβ1and phosphorylatedAkt modulation. Neurol Sci.2012Dec7.
[13] Fujita A, Hattori Y, Takeuchi T, et al. NGF induces neurite outgrowth via adecrease in phosphorylation of myosin light chain in PC12cells. Neuroreport.2001,12(16):3599-602.
[1] Domeniconi M, Filbin MT. Overcoming inhibitors in myelin to promote axonalregeneration[J]. J Neurol Sci2005,233:43-47.
[2] Schimchowitsch S, Cassel JC. Polyamine and aminoguanidine treatments topromote structural and functional recovery in the adult mammalian brain after injury:a brief literature review and preliminary data about their combined administration[J].J Physiol Paris2006,99:221-223.
[3] Prinjha R, Moore SE, Vinson M, et al. Inhibitor of neurite outgrowth in humans[J].Nature.2000,403(6768):383-4.
[4] McKerracher L, David S, Jackson DL, et al. Identification of myelin-associatedglycoprotein as a major myelin-derived inhibitor of neurite growth[J]. Neuron.1994,13(4):805-11.
[5] Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is aNogo receptor ligand that inhibits neurite outgrowth. Nature.2002,417(6892):941-4.
[6] Domeniconi M, Cao Z, Spencer T, et al. Myelin-associated glycoprotein interactswith the Nogo66receptor to inhibit neurite outgrowth[J]. Neuron.2002,35(2):283-90.
[7] Mi S, Lee X, Shao Z, et al. LINGO-1is a component of the Nogo-66receptor/p75signaling complex[J]. Nat Neurosci.2004,7(3):221-8.
[8] Park JB, Yiu G, Kaneko S, et al. A TNF receptor family member, TROY, is acoreceptor with Nogo receptor in mediating the inhibitory activity of myelininhibitors[J]. Neuron.2005,45(3):345-51.
[9] Atwal JK, Pinkston-Gosse J, Syken J, et al. PirB is a functional receptor formyelin inhibitors of axonal regeneration. Science.2008,322(5903):967-70.
[10] Dubreuil CI, Winton KJ, Mekerraeher L. Rho activation patterns after spinalcord injury and the role of activated Rho in apoptosis in the central nervous system[J].J Cell Biol,2003,162(2)：233-243
[11] Ito M, Nakano T, Erdodi F, et al. Myosin phosphatase: structure, regulation andfunction[J]. Mol Cell Biochem,2004,259(1-2):197-209.
[12] Hartshorne DJ. Myosin phosphatase: subunits and interactions[J]. Acta PhysiolScand.1998,164(4):483-93.
[13] Feng J, Ito M, Ichikawa K, et al. Inhibitory phosphorylation site forRho-associated kinase on smooth muscle myosin phosphatase[J]. J Biol Chem.1999,274(52):37385-90.
[14] Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth musclemediated by a Rho-associated protein kinase in hypertension[J]. Nature.1997,389(6654):990-4.
[15] Kimura K, Ito M, Amano M, et al. Regulation of myosin phosphatase by Rhoand Rho-associated kinase (Rho-kinase)[J]. Science.1996,273(5272):245-8.
[16] Kim Y, Chang S. Modulation of actomyosin contractility by myosin light chainphosphorylation/dephosphorylation through Rho GTPases signaling specifies axonformation in neurons[J]. Biochem Biophys Res Commun,2004,318(2):579-87.
[17] Fujita A, Hattori Y, Takeuchi T, et al. NGF induces neurite outgrowth via adecrease in phosphorylation of myosin light chain in PC12cells[J]. Neuroreport,2001,12(16):3599-602.
[18] Amano M, Chihara K, Nakamura N, et al. Myosin II activation promotes neuriteretraction during the action of Rho and Rho-kinase[J]. Genes Cells,1998,3(3):177-88.
[19] Profyris C, Cheema SS, Zang D, et al. Degenerative and regenerativemechanisms governing spinal cord injury[J]. Neurobiol Dis,2004,15(3):415-36.
[20] Berg JS, Powell BC, Cheney RE. A millennial myosin census[J]. Mol Biol Cell,2001,12(4):780-94.
[21] Rochlin MW, Itoh K, Adelstein RS, et al. Localization of myosin II A and Bisoforms in cultured neurons. J Cell Sci,1995,108(Pt12):3661-70.
[22] Lewis AK, Bridgman PC. Mammalian myosin I alpha is concentrated near theplasma membrane in nerve growth cones[J]. Cell Motil Cytoskeleton,1996;33(2):130-50.
[23] Suter DM, Espindola FS, Lin CH, et al. Localization of unconventional myosinsV and VI in neuronal growth cones[J]. J Neurobiol,2000,42(3):370-82.
[24] Berg JS, Cheney RE. Myosin-X is an unconventional myosin that undergoesintrafilopodial motility[J]. Nat Cell Biol,2002,4(3):246-50.
[25] Wylie SR, Wu PJ, Patel H, et al. A conventional myosin motor drives neuriteoutgrowth[J]. Proc Natl Acad Sci U S A.1998,95(22):12967-72.
[26] Wylie SR, Chantler PD. Myosin IIA drives neurite retraction[J]. Mol Biol Cell,2003,14(11):4654-66.
[27] Ahmed A, Eickholt BJ. Intracellular kinases in semaphorin signaling[J]. AdvExp Med Biol,2007,600:24-37.
[28] Acevedo K, Moussi N, Li R, et al. LIM kinase2is widely expressed in alltissues. J Histochem Cytochem[J].2006,54(5):487-501.
[29] Li R, Soosairajah J, Harari D, et al. Hsp90increases LIM kinase activity bypromoting its homo-dimerization[J]. FASEB J,2006,20(8):1218-20.
[30] Soosairajah J, Maiti S, Wiggan O, et al. Interplay between components of anovel LIM kinase-slingshot phosphatase complex regulates cofilin[J]. EMBO J,2005,24(3):473-86.
[31] Tursun B, Schlüter A, Peters MA, et al. The ubiquitin ligase Rnf6regulates localLIM kinase1levels in axonal growth cones. Genes Dev[J].2005,19(19):2307-19.
[32] Schratt GM, Tuebing F, Nigh EA, et al. A brain-specific microRNA regulatesdendritic spine development[J]. Nature,2006,439(7074):283-9.
[33]Ohashi K, Nagata K, Maekawa M, et al. Rho-associated kinase ROCK activatesLIM-kinase1by phosphorylation at threonine508within the activation loop[J]. JBiol Chem,2000,275(5):3577-82.
[34] Ohta Y, Kousaka K, Nagata-Ohashi K, et al. Differential activities, subcellulardistribution and tissue expression patterns of three members of Slingshot familyphosphatases that dephosphorylate cofilin[J]. Genes Cells,2003,8(10):811-24.
[35] Endo M, Ohashi K, Mizuno K. LIM kinase and slingshot are critical for neuriteextension[J]. J Biol Chem,2007,282(18):13692-702.
[36] Meng Y, Zhang Y, Tregoubov V, et al. Abnormal spine morphology andenhanced LTP in LIMK-1knockout mice[J]. Neuron,2002,35(1):121-33.
[37] Meng Y, Takahashi H, Meng J, et al. Regulation of ADF/cofilin phosphorylationand synaptic function by LIM-kinase[J]. Neuropharmacology.2004,47(5):746-54.
[38] Rosso S, Bollati F, Bisbal M, et al. LIMK1regulates Golgi dynamics, traffic ofGolgi-derived vesicles, and process extension in primary cultured neurons[J]. MolBiol Cell,2004,15(7):3433-49.
[39] Tursun B, Schlüter A, Peters MA, et al. The ubiquitin ligase Rnf6regulates localLIM kinase1levels in axonal growth cones[J]. Genes Dev,2005,19(19):2307-19.
[40] Arimura N, Inagaki N, Chihara K, et al. Phosphorylation of collapsin responsemediator protein-2by Rho-kinase. Evidence for two separate signaling pathways forgrowth cone collapse[J]. J Biol Chem,2000,275(31):23973-80.
[41] Mimura F, Yamagishi S, Arimura N, et al. Myelin-associated glycoproteininhibits microtubule assembly by a Rho-kinase-dependent mechanism[J]. J BiolChem,2006,281(23):15970-9.
[42] Fukata Y, Itoh TJ, Kimura T, et al. CRMP-2binds to tubulin heterodimers topromote microtubule assembly[J]. Nat Cell Biol,2002,4(8):583-91.
[43] Arimura N, Inagaki N, Chihara K, et al. Phosphorylation of collapsin responsemediator protein-2by Rho-kinase. Evidence for two separate signaling pathways forgrowth cone collapse[J]. J Biol Chem,2000,275(31):23973-80.
[44] Inagaki N, Chihara K, Arimura N, et al. CRMP-2induces axons in culturedhippocampal neurons[J]. Nat Neurosci,2001,4(8):781-2.
[45] Yoshimura T, Kawano Y, Arimura N, et al. GSK-3beta regulates phosphorylationof CRMP-2and neuronal polarity[J]. Cell,2005,120(1):137-49.
[46] Wang T, Wu X, Yin C, et al. CRMP-2Is Involved in Axon Growth InhibitionInduced by RGMa In Vitro and In Vivo[J]. Mol Neurobiol.2012
[2] Sahota P, Savitz SI. Investigational therapies for ischemic stroke: neuroprotectionand neurorecovery. Neurotherapeutics.2011Jul;8(3):434-51.
[3] Akbik F, Cafferty WB, Strittmatter SM. Myelin associated inhibitors: a linkbetween injury-induced and experience-dependent plasticity[J]. Exp Neurol,2012,235(1):43-52.
[4] Kubo T, Hata K, Yamaguchi A, Yamashita T. Rho-ROCK inhibitors as emergingstrategies to promote nerve regeneration[J]. Curr Pharm Des,2007,13(24):2493-9.
[5] Jiang F, Yin H, Qin X. Fastigial nucleus electrostimulation reduces the expressionof repulsive guidance molecule, improves axonal growth following focal cerebralischemia[J]. Neurochem Res.2012,37(9):1906-14.
[6] Feng J, Wang T, Li Q, et al. RNA interference against repulsive guidance moleculeA improves axon sprout and neural function recovery of rats afterMCAO/reperfusion[J]. Exp Neurol,2012,238(2):235-42.
[7] Kitayama M, Ueno M, Itakura T, et al. Activated microglia inhibit axonal growththrough RGMa[J]. PLoS One,2011,6(9):e25234.
[8] Lampl Y, Boaz M, Gilad R, et al. Minocycline treatment in acute stroke: anopen-label, evaluator-blinded study[J]. Neurology,2007,69(14):1404-10.
[9] Zhang G, Zhang JH, Feng J, et al. Electrical stimulation of olfactory bulbdownregulates RGMa expression after ischemia/reperfusion injury in rats[J]. BrainRes Bull,2011,86(3-4):254-61.
[10] Belayev L, Busto R, Zhao W, et al. Quantitative evaluation of blood-brain barrierpermeability following middle cerebral artery occlusion in rats[J]. Brain Res,1996,739(1-2):88-96.
[11] Chen J, Sanberg PR, Li Y, et al. Intravenous administration of human umbilicalcord blood reduces behavioral deficits after stroke in rats[J]. Stroke J Cereb Circ,2001,32:2682–2688.
[12] Montoya CP, Campbell-Hope LJ, Pemberton KD, et al. The "staircase test": ameasure of independent forelimb reaching and grasping abilities in rats[J]. JNeurosci Methods,1991,36:219-28.
[13] Domeniconi M, Filbin MT. Overcoming inhibitors in myelin to promote axonalregeneration[J]. J Neurol Sci2005,233:43-47.
[14] Schimchowitsch S, Cassel JC. Polyamine and aminoguanidine treatments topromote structural and functional recovery in the adult mammalian brain afterinjury: a brief literature review and preliminary data about their combinedadministration[J]. J Physiol Paris,2006,99:221-223.
[15] McKerracher L, David S, Jackson DL, et al. Identification of myelin-associatedglycoprotein as a major myelin-derived inhibitor of neurite growth[J]. Neuron,1994,13(4):805-11.
[16] Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is aNogo receptor ligand that inhibits neurite outgrowth[J]. Nature,2002,417(6892):941-4.
[17] GrandPré T, Nakamura F, Vartanian T, et al. Identification of the Nogo inhibitor ofaxon regeneration as a Reticulon protein[J]. Nature,2000,403(6768):439-44.
[18] Stahl B, Müller B, von Boxberg Y, et al. Biochemical characterization of a putativeaxonal guidance molecule of the chick visual system.[J]. Neuron,1990,5(5):735-43.
[19] Severyn C J, Shinde U, Rotwein P, et al. Molecular biology, genetics andbiochemistry of the repulsive guidance molecule family[J]. Biochem J,2009,422(3):393-403
[20] Schwab JM, Monnier PP, Schluesener HJ, et al. CNS injury induced RGM(repulsive guidance molecule) expression in the adult human brain [J]. ArchNeurol,2005,62(10):1561-8.
[21] Hata K, Fujitani M, Yasuda Y, et al. RGMa inhibition promotes axonal growth andrecovery after spinal cord injury[J]. J Cell Biol,2006;173(1):47-58.
[22] Franco EC, Cardoso MM, Gouvêia A, et al. Modulation of microglial activationenhances neuroprotection and functional recovery derived from bone marrowmononuclear cell transplantation after cortical ischemia[J]. Neurosci Res,2012,73(2):122-32.
[23] Hayakawa K, Mishima K, Nozako M, et al. Delayed treatment with minocyclineameliorates neurologic impairment through activated microglia expressing ahigh-mobility group box1-inhibiting mechanism[J]. Stroke,2008,39(3):951-8.
[24] Xu L, Fagan SC, Waller JL, et al. Low dose intravenous minocycline isneuroprotective after middle cerebral artery occlusion-reperfusion in rats[J]. BMCNeurol,2004,4:7.
[25] Fagan SC, Waller JL, Nichols FT, et al. Minocycline to improve neurologicoutcome in stroke (MINOS): a dose-finding study[J]. Stroke,2010,41(10):2283-7.
[26] Shaw G, Yang C, Ellis R, et al. Hyperphosphorylated neurofilament NF-H is aserum biomarker of axonal injury[J]. Biochem Biophys Res Commun,2005,336(4):1268-77.
[1] Papadopoulos CM, Tsai SY, Cheatwood JL, et al. Dendritic plasticity in the adultrat following middle cerebral artery occlusion and Nogo-a neutralization[J]. CerebCortex.2006,16(4):529-36.
[2] Fagan SC, Cronic LE, Hess DC. Minocycline Development for Acute IschemicStroke[J]. Transl Stroke Res,2011,1;2(2):202-208.
[3] Plane JM, Shen Y, Pleasure DE, et al. Prospects for minocycline neuroprotection[J].Arch Neurol,2010,67(12):1442-8.
[4] Lampl Y, Boaz M, Gilad R, et al. Minocycline treatment in acute stroke: anopen-label, evaluator-blinded study[J]. Neurology.2007,69(14):1404-10.
[5] Hashimoto K, Ishima T. A novel target of action of minocycline in NGF-inducedneurite outgrowth in PC12cells: translation initiation [corrected] factor eIF4AI[J].PLoS One.2010,5(11):e15430.
[6] Walsh MP, Cole WC. The role of actin filament dynamics in the myogenicresponse of cerebral resistance arteries[J]. J Cereb Blood Flow Metab.2013,33(1):1-12.
[7] Kubo T, Endo M, Hata K, et al. Myosin IIA is required for neurite outgrowthinhibition produced by repulsive guidance molecule[J]. J Neurochem,2008,105(1):113-26.
[8] Tabakman R, Lazarovici P, Kohen R. Neuroprotective effects of carnosine andhomocarnosine on pheochromocytoma PC12cells exposed to ischemia[J]. JNeurosci Res,2002,15;68(4):463-9.
[9] Singh G, Siddiqui MA, Khanna VK, et al. Oxygen glucose deprivation model ofcerebral stroke in PC-12cells: glucose as a limiting factor[J]. Toxicol MechMethods,2009,19(2):154-60.
[10] Fan X, Lo EH, Wang X. Effects of minocycline plus tissue plasminogen activatorcombination therapy after focal embolic stroke in type1diabetic rats[J]. Stroke,2013,44(3):745-52.
[11] Song Y, Wei EQ, Zhang WP, et al. Minocycline protects PC12cells fromischemic-like injury and inhibits5-lipoxygenase activation[J]. Neuroreport.2004Oct5;15(14):2181-4.
[12] Chen X, Chen S, Jiang Y, et al. Minocycline reduces oxygen-glucosedeprivation-induced PC12cell cytotoxicity via matrix metalloproteinase-9, integrinβ1and phosphorylatedAkt modulation. Neurol Sci.2012Dec7.
[13] Fujita A, Hattori Y, Takeuchi T, et al. NGF induces neurite outgrowth via adecrease in phosphorylation of myosin light chain in PC12cells. Neuroreport.2001,12(16):3599-602.
[1] Domeniconi M, Filbin MT. Overcoming inhibitors in myelin to promote axonalregeneration[J]. J Neurol Sci2005,233:43-47.
[2] Schimchowitsch S, Cassel JC. Polyamine and aminoguanidine treatments topromote structural and functional recovery in the adult mammalian brain after injury:a brief literature review and preliminary data about their combined administration[J].J Physiol Paris2006,99:221-223.
[3] Prinjha R, Moore SE, Vinson M, et al. Inhibitor of neurite outgrowth in humans[J].Nature.2000,403(6768):383-4.
[4] McKerracher L, David S, Jackson DL, et al. Identification of myelin-associatedglycoprotein as a major myelin-derived inhibitor of neurite growth[J]. Neuron.1994,13(4):805-11.
[5] Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is aNogo receptor ligand that inhibits neurite outgrowth. Nature.2002,417(6892):941-4.
[6] Domeniconi M, Cao Z, Spencer T, et al. Myelin-associated glycoprotein interactswith the Nogo66receptor to inhibit neurite outgrowth[J]. Neuron.2002,35(2):283-90.
[7] Mi S, Lee X, Shao Z, et al. LINGO-1is a component of the Nogo-66receptor/p75signaling complex[J]. Nat Neurosci.2004,7(3):221-8.
[8] Park JB, Yiu G, Kaneko S, et al. A TNF receptor family member, TROY, is acoreceptor with Nogo receptor in mediating the inhibitory activity of myelininhibitors[J]. Neuron.2005,45(3):345-51.
[9] Atwal JK, Pinkston-Gosse J, Syken J, et al. PirB is a functional receptor formyelin inhibitors of axonal regeneration. Science.2008,322(5903):967-70.
[10] Dubreuil CI, Winton KJ, Mekerraeher L. Rho activation patterns after spinalcord injury and the role of activated Rho in apoptosis in the central nervous system[J].J Cell Biol,2003,162(2)：233-243
[11] Ito M, Nakano T, Erdodi F, et al. Myosin phosphatase: structure, regulation andfunction[J]. Mol Cell Biochem,2004,259(1-2):197-209.
[12] Hartshorne DJ. Myosin phosphatase: subunits and interactions[J]. Acta PhysiolScand.1998,164(4):483-93.
[13] Feng J, Ito M, Ichikawa K, et al. Inhibitory phosphorylation site forRho-associated kinase on smooth muscle myosin phosphatase[J]. J Biol Chem.1999,274(52):37385-90.
[14] Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth musclemediated by a Rho-associated protein kinase in hypertension[J]. Nature.1997,389(6654):990-4.
[15] Kimura K, Ito M, Amano M, et al. Regulation of myosin phosphatase by Rhoand Rho-associated kinase (Rho-kinase)[J]. Science.1996,273(5272):245-8.
[16] Kim Y, Chang S. Modulation of actomyosin contractility by myosin light chainphosphorylation/dephosphorylation through Rho GTPases signaling specifies axonformation in neurons[J]. Biochem Biophys Res Commun,2004,318(2):579-87.
[17] Fujita A, Hattori Y, Takeuchi T, et al. NGF induces neurite outgrowth via adecrease in phosphorylation of myosin light chain in PC12cells[J]. Neuroreport,2001,12(16):3599-602.
[18] Amano M, Chihara K, Nakamura N, et al. Myosin II activation promotes neuriteretraction during the action of Rho and Rho-kinase[J]. Genes Cells,1998,3(3):177-88.
[19] Profyris C, Cheema SS, Zang D, et al. Degenerative and regenerativemechanisms governing spinal cord injury[J]. Neurobiol Dis,2004,15(3):415-36.
[20] Berg JS, Powell BC, Cheney RE. A millennial myosin census[J]. Mol Biol Cell,2001,12(4):780-94.
[21] Rochlin MW, Itoh K, Adelstein RS, et al. Localization of myosin II A and Bisoforms in cultured neurons. J Cell Sci,1995,108(Pt12):3661-70.
[22] Lewis AK, Bridgman PC. Mammalian myosin I alpha is concentrated near theplasma membrane in nerve growth cones[J]. Cell Motil Cytoskeleton,1996;33(2):130-50.
[23] Suter DM, Espindola FS, Lin CH, et al. Localization of unconventional myosinsV and VI in neuronal growth cones[J]. J Neurobiol,2000,42(3):370-82.
[24] Berg JS, Cheney RE. Myosin-X is an unconventional myosin that undergoesintrafilopodial motility[J]. Nat Cell Biol,2002,4(3):246-50.
[25] Wylie SR, Wu PJ, Patel H, et al. A conventional myosin motor drives neuriteoutgrowth[J]. Proc Natl Acad Sci U S A.1998,95(22):12967-72.
[26] Wylie SR, Chantler PD. Myosin IIA drives neurite retraction[J]. Mol Biol Cell,2003,14(11):4654-66.
[27] Ahmed A, Eickholt BJ. Intracellular kinases in semaphorin signaling[J]. AdvExp Med Biol,2007,600:24-37.
[28] Acevedo K, Moussi N, Li R, et al. LIM kinase2is widely expressed in alltissues. J Histochem Cytochem[J].2006,54(5):487-501.
[29] Li R, Soosairajah J, Harari D, et al. Hsp90increases LIM kinase activity bypromoting its homo-dimerization[J]. FASEB J,2006,20(8):1218-20.
[30] Soosairajah J, Maiti S, Wiggan O, et al. Interplay between components of anovel LIM kinase-slingshot phosphatase complex regulates cofilin[J]. EMBO J,2005,24(3):473-86.
[31] Tursun B, Schlüter A, Peters MA, et al. The ubiquitin ligase Rnf6regulates localLIM kinase1levels in axonal growth cones. Genes Dev[J].2005,19(19):2307-19.
[32] Schratt GM, Tuebing F, Nigh EA, et al. A brain-specific microRNA regulatesdendritic spine development[J]. Nature,2006,439(7074):283-9.
[33]Ohashi K, Nagata K, Maekawa M, et al. Rho-associated kinase ROCK activatesLIM-kinase1by phosphorylation at threonine508within the activation loop[J]. JBiol Chem,2000,275(5):3577-82.
[34] Ohta Y, Kousaka K, Nagata-Ohashi K, et al. Differential activities, subcellulardistribution and tissue expression patterns of three members of Slingshot familyphosphatases that dephosphorylate cofilin[J]. Genes Cells,2003,8(10):811-24.
[35] Endo M, Ohashi K, Mizuno K. LIM kinase and slingshot are critical for neuriteextension[J]. J Biol Chem,2007,282(18):13692-702.
[36] Meng Y, Zhang Y, Tregoubov V, et al. Abnormal spine morphology andenhanced LTP in LIMK-1knockout mice[J]. Neuron,2002,35(1):121-33.
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