缺氧缺血性脑损伤新生大鼠髓鞘损伤与修复的相关研究
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摘要
新生儿缺氧缺血性脑损伤(HIBD)是由于围产期各种因素引起的缺氧和脑供血减少所致的脑损伤,是围产期窒息的严重并发症,并成为脑瘫和其他神经系统慢性致残性疾病的主要原因。既往人们认为脑白质对缺氧高度耐受,因此对HIBD的研究一直都以神经元的病变及修复作为关注的重点。然而近年来研究发现,脑白质病变在HIBD中所起的作用非常关键,尤其是早产儿缺氧缺血(HI)导致的脑白质损伤发生率甚至高于皮质损伤,这些研究结果正在改变着人们对脑白质缺氧的传统认识。
缺血缺氧性脑白质损害的典型病变是脑白质中神经纤维的髓鞘化障碍。少突胶质细胞(OL)是CNS中的主要成髓鞘细胞,近年来研究表明OL系细胞对缺氧高度敏感,且成熟度越低、分化能力越强的OL系细胞对缺氧越敏感。新生儿脑内OL系细胞中以少突胶质细胞前体细胞(OPC)占主流,OPC对HI高度敏感,OPC受损后出现分化障碍,OL数量减少,功能异常,脑白质内髓鞘形成障碍,以至临床上出现脑瘫和智能发育落后等后遗症。然而,目前有关脑髓鞘的发育特点以及相关影响因素的研究甚少,尤其对HIBD条件下脑髓鞘的发育与形成的研究更少。可见,研究和探讨脑髓鞘的发育特点及HIBD对脑髓鞘形成的影响具有重要的理论与临床实用价值。
正常髓鞘化的神经纤维分为朗飞氏结区、结侧区、近结侧区及结间区等不同区域。郎飞氏结区钠通道高度聚集,这是神经冲动在有髓纤维跳跃式传导的分子基础。钾通道则主要分布于近结侧区,具有缓冲有髓神经纤维兴奋的作用。结侧区介于结区和近结侧区之间,起绝缘作用,对维持各区轴膜蛋白和离子通道的正常区域化,保证有髓纤维神经冲动快速传导十分重要。研究发现,结侧区主要由少突胶质细胞产生的神经束蛋白155(neurofascin 155,NF155)及其轴突源性配体caspr和contactin组成,它们之间互相作用,形成结侧区复合物。其中,NF155在结侧区复合物的形成中起募集、引导作用,是结侧区复合物形成的关键。结侧区复合物主要分布于结侧区脂筏中。在脂筏中的特征性分布使结侧区复合物更加稳定,利于有髓纤维各区离子通道的正常分布。但是,目前国内外对于髓鞘结侧区及NF155蛋白的研究尚在起步阶段,对NF155在实验动物脑中的发育规律还不清楚,研究其发育规律是揭示髓鞘结侧区在HIBD脑白质损伤中的改变及作用的基础。
基于上述理由,本研究首先研究大鼠不同发育时期的脑髓鞘发育规律,在此基础上深入探讨HIBD中发育期大鼠脑髓鞘的病变特点及神经行为学改变;并进一步从较为微观的角度观察HIBD模型鼠髓鞘关键区域----结侧区的NF155蛋白及脂筏的改变,并探讨补充外源性GM1对修复髓鞘结侧区脂筏的作用。研究方法及结果摘要叙述如下:
一、不同发育阶段大鼠脑髓鞘的发育
采用下述方法研究胚胎14d(E14),E18,出生后0d(P0)、P7、P15、P30、P90、P720大鼠脑髓鞘发育,结果如下:
1.应用劳克斯坚牢蓝(LFB)染色,髓鞘碱性蛋白( MBP)免疫组化染色的方法,我们发现E18、P0、P2均未见髓鞘阳性染色,P7大鼠胼胝体中部可见少量LFB染色,P15大鼠胼胝体,外囊,内囊及皮质两种染色均呈阳性,P30着色加深,与P90,P720相似。比较P15~P720各组与前一日龄组的MBP免疫组化染色积分光密度,差异均有显著意义(P<0.05)。
2.通过western-blot检测MBP蛋白在不同发育时期的表达,结果发现MBP蛋白在E14已有表达,出生后随日龄增长逐渐增加,至老年期含量最高,各日龄组与前一日龄组比较差异均有显著性(P<0.05)。
3.通过western-blot检测发现髓鞘结侧区蛋白NF155在胚胎期、P0、P2无明显表达,P7时仅有极少量表达,P30大量增加,P90达高峰,老年期又有所下降,各日龄组与前一日龄组比较差异均有显著性(P<0.05)。
4.通过Real-time PCR检测各发育期大鼠脑NF155 mRNA表达,结果与NF155蛋白表达趋势相似,各发育期鼠脑NF155mRNA的基因表达相对倍数随日龄增长逐渐增高,至3月龄时达到高峰,其后下降。
二、HIBD对发育期大鼠脑髓鞘及神经行为的影响
1.参照Levine法建立P7SD大鼠HIBD模型,建模后出现夹尾左旋行为者列为成功模型动物用于研究。P30时见HIBD模型大鼠脑大体左侧(缺血侧)脑可见明显萎缩。
2.应用LFB、MBP免疫组化髓鞘染色可见:P30HIBD模型患侧脑胼胝体,外囊,内囊,纹状体结构错乱,阳性染色分布异常,积分光密度降低,与假手术组比较有显著性差异(P<0.05)。
3.应用MBP,神经丝蛋白200(NF200)免疫荧光双标染色,激光共聚焦采图,见假手术组胼胝体、外囊髓鞘与神经丝伴行排列,结构清楚;P30HIBD大鼠脑髓鞘、神经丝染色变淡,排列错乱。
4.透射电镜观察P30各组大鼠脑室周围白质髓鞘的超微结构,见假手术组髓鞘结构致密,完整,神经丝排列有致。HIBD大鼠脑髓鞘疏松,髓鞘层间分层明显,结构紊乱,高倍镜下可见轴质萎缩。
5.应用western-blot方法检测P30HIBD大鼠脑MBP及NF155蛋白表达,见HIBD大鼠患侧脑组织MBP及NF155蛋白量均较假手术组显著下降(P<0.05)
6.通过Morris水迷宫行为学测试,P30假手术组大鼠游泳轨迹以直线式和趋向式为主, HIBD大鼠游泳轨迹以边缘式和随机式为主。HIBD大鼠在定位航行试验中逃避潜伏期较假手术组明显延长;在空间探索实验中穿台次数较假手术组明显减少,两组间差异有显著性(P<0.05)。
三、HIBD对大鼠髓鞘结侧区脂筏的影响及GM1的干预效应研究
1.通过去垢剂法提取大鼠脑脂筏,Optiprep梯度离心液超高速离心后用GM1鉴定,脂筏主要分布于梯度离心条段的第3~5层。
2.将各梯度离心条段经SDS-Page凝胶电泳和免疫印迹检测可见:P30HIBD大鼠患侧脑脂筏中GM1含量较假手术组显著降低,GM1组大鼠脑脂筏中的GM1含量较HIBD组显著提高(P<0.05)。
3.通过western-blot检测见HIBD大鼠患侧脑NF155蛋白表达及NF155在脂筏中的含量均较假手术组明显减少。GM1组大鼠脑NF155蛋白量及NF155在脂筏中的含量较HIBD组显著提高(P<0.05)。
4. MBP免疫组化染色及脑髓鞘透射电镜检查见HIBD组大鼠脑髓鞘结构破坏,GM1组髓鞘得到明显修复,MBP免疫组化染色积分光密度值高于HIBD组,略低于假手术组。
全文结论如下:
1.初生SD大鼠脑髓鞘化程度很低,出生后随日龄增长快速髓鞘化,P15全脑大部分区域髓鞘化已经开始,P30接近成熟,P90已发育成熟。提示观察大鼠脑髓鞘的时间不宜早于15d,最佳时间在30d以后。NF155蛋白表达晚于MBP,亦随日龄逐渐增加,从P7有少量表达,P30大量增加,P90达高峰,老年期呈逐渐下降趋势。提示在发育早期髓鞘结侧区结构的分化晚于髓鞘的形成,在老年期结侧区成分减少,有可能是老年期髓鞘功能减退的原因之一。
2. P30HIBD模型大鼠脑髓鞘量减少,脑组织MBP、NF155蛋白表达下调,学习记忆能力下降。髓鞘结构出现错乱,松散,分层等形态学改变,伴有轴突轴质萎缩。提示HIBD大鼠模型脑中髓鞘结构明显异常,导致神经功能异常。此外,还可能因髓鞘结侧区结构破坏使神经纤维轴膜离子通道区域化分布失败,导致有髓神经传导功能异常。
3. P30HIBD模型大鼠脑脂筏量减少,结侧区蛋白NF155在脂筏中的分布亦明显减少,提示结侧区脂筏受损。补充外源性GM1不仅能显著增加大鼠脑脂筏量,还能增加HIBD大鼠脑中NF155的表达量及其在脂筏中的分布。提示外源性GM1可能通过补充脂筏中重要脂质,修复髓鞘结侧区脂筏,保护结侧区重要髓鞘蛋白NF155及结侧区复合物,最终起到修复髓鞘的作用。
综上所述,脑白质损伤是HIBD中的重要部分,HIBD中脑髓鞘及其结侧区结构均有明显病变。通过改善脂筏的成分可能修复髓鞘结侧区结构和功能,最终达到修复髓鞘的效果。
Neonatal hypoxic-ischemic brain damage (HIBD) is a common brain injury due to hypoxia and decrease of cerebral blood supply caused by various perinatal factors. HIBD is a serious complication of perinatal asphyxia and the main reason for cerebral palsy and other neurological chronic disabling diseases. People used to think that white matter was of high tolerance to hypoxia, so most researches on HIBD had focused on neuron damage and repair. However, recent studies found that white matter damage play crucial role in HIBD, and especially in premature infants with hypoxic-ischemic (HI), the incidence of white matter damage was even higher than that of cortical damage. These findings are changing people’s traditional understanding of white matter anoxia.
Typical white matter damage due to hypoxic-ischemia is myelinization disturbance of nerve fibers. Oligodendrocytes (OL) are the major cells who forming myelin sheath in central nervous system (CNS). In recent years, studies have shown that OL are of high sensitivity to hypoxia, especially the cells at lower maturity level and with strong differentiation. In the neonatal brain, oligodendrocyte precursor cells (OPC) are dominant OL, and highly sensitive to HI. After damage, OPC can’t differentiate well, and thus, reduce the number of OL and make dysfunction. In this way, dysmyelination of white matter happens, and as a result, some sequelae occur, such as developmental disorders, cerebral palsy and mental retardation and so on. However, there are rare studies about the brain myelin development and related influencing factors, especially under HIBD. As it can be seen, studies on developmental characteristics of brain myelin and effects of HIBD on brain myelination have important theoretical and clinical value.
Myelinated axons are commonly divided into four functional regions: nodes of Ranvier, paranodes, juxtaparanodes, and internodes. The highly concentrated sodium channels in nodes of Ranvier are the molecular basis of nerve impulse’s saltatory conduction in myelinated nerve fibers. Potassium channels are mainly located in juxtaparanodes, which buffer the exciting of mylinated nerve fibers. The paranodes, areas between node and juxtaparannodes, have insulated effect and maintain various axilemma proteins and ion channels to be normal regionalization, and to ensure rapid transmission of nerve impulses of myelinated fiber is very important.
Study found that the paranodes composes mainly of the myelin protein neurofascin 155 (NF155) produced by oligodendrocytes and their axons endogenous ligand caspr and contactin, and they interact to each other to form the paranodal complex. In which, NF155 guide the formation of paranodal complexes, and plays a key for complex formation in paranode. The paranodal complexes are mainly distributed in lipid rafts of paranode. This distribution makes paranodal complex more stable, which will help the normal distribution of various ion channels. However, the study of paranode of the myelin sheath and NF155 proteins is still at an initial stage, and development profile of NF155 in the brain of the experimental animals is unclear. It will be an important basis to study and explore development law of medullary sheath in paranode and its effect in white matter damage due to HIBD.
In summary, this study will firstly study myelin development profile of rat brain at different developmental stages, and then, on this basis, to study the features of myelin sheath damage and neurological behavioral changes after HIBD; and further from a more microscopic point of view to study the changes of NF155 protein and lipid rafts of critical areas of myelin sheath—paranode in HIBD rat model, and explore the repair effect of exogenous GM1 on lipid rafts in myelin sheath of paranode. Research methods and results summarized as follows:
1. The development of rat brain myelin at different developmental stages
We use the following methods to study rat brain myelin development of embryo 14d (E14) and E18, and postnatal 0 d (P0), P7, P15, P30, P90 and P720. The results are as follows:
(1). With Luxol fast blue (LFB) staining and immunohistochemical staining against myelin basic protein (MBP), we found that E18, P0 and P2 rats showed no positive staining of myelin, P7 rats had mild LFB staining in the corpus callosum, P15 rats were positive to both staining in the corpus callosum, internal capsule and capsula externa, and cortex. The double positive staining was deepened in P30 rats, similar to those in the rats of P90 and P720. MBP was expressed significantly in a time-dependent manner from P15 to P720 (P<0.05).
(2). Western blot analysis indicated that MBP was expressed since E14, then increased with age after birth, and gradually increased to the highest level of old age. The difference between every age groups was significant (P<0.05).
(3) Western blot analysis found that myelin protein NF155 was not expressed in the brain during the embryonic stage, P0, and P2. It was slightly expressed at P7, strongly expressed at P30, reached a peak at P90, and declined in old age. The difference between every age groups was significant (P<0.05).
(4) Real-time PCR showed that the tendency of NF155 mRNA expression was similar to that of protein expression. The mRNA expression of NF155 was increased with age, and reached a peak at P90, and then decreased.
2 The influence of HIBD on myelin sheath and neurobehavioral function in developing rat brain.
(1). P7 SD rats were established according to Levine HIBD model. The rats with their head turn to the left when their tails were clamped were identified as successful. Obvious brain atrophy was observed in left sides (the ischemia sides) of P30 HIBD rat brain.
(2). With LFB staining and MBP immunohistochemical staining, the affected brain side showed structural disorder in the corpus callosum, external capsule, internal capsule and striatum in P30 HIBD model. Abnormal positive staining was observed with decreased integral optical density, which had significant difference with the sham group (P<0.05).
(3). Double immunofluorescence staining for MBP and neurofilament 200 (NF200) showed that myelin sheath displayed parallel arrangement with neurofilament in the corpus callosum and external capsule of sham group, but in P30HIBD rat brain, myelin sheath and neurofilament was slightly stained in a disturbed order.
(4). Transmission electron microscopy showed that the ultrastructure of rat periventricular myelin was compact, intact and in well arrangement in sham group. But in HIBD rat brain, myelin sheath was loosen, delaminated, and disarranged order, and axonal atrophy was found under high-power microscope.
(5). Application of western-blot method for detection of MBP and NF155 P30HIBD rat brain protein expression.The MBP and NF155 expressions both decreased significantly in HIBD rat brains compared with sham group (P<0.05).
(6). Morris water maze showed that P30 sham rats swam to track straight and tend to type-based, while HIBD rats swam to the edge of track- and stochastic-based. The escape latency in HIBD rats was significantly longer than the sham group (P<0.05). In spatial probe test, the number of shuttling was significantly reduced in sham group than in HIBD group (P<0.05).
3. The influence of HIBD on paranodal lipid raft of rat myelin sheath, and the intervention effect of GM1.
(1) Rat brain lipid rafts were extracted by detergent cracking and Optiprep gradient ultracentrifugation, and then identified by GM1. Lipid raft are mainly distributed in the 3rd to 5th layer of gradient centrifugation strip.
(2) Detecting each gradient centrifugation strip with SDS-Page gel electrophoresis and immunoblotting, the content of GM1 in lipid raft of P30 HIBD left rat brains decreased greatly compared to those in sham group. The content of GM1 in lipid raft of GM1 group was increased greatly compared to that in HIBD group (P<0.05).
(3) NF155 protein levels and NF155 content in lipid rafts in P30 left rat brains were detected by Western blotting. Both of them were decreased in HIBD group compared with sham operated group, significantly increased in GM1 group compared with HIBD group (P<0.05).
(4) MBP immunohistochemical staining and transmission electron microscopy showed the myelins of HIBD rat brains were structural damaged, however, the myelin of GM1 group were significantly repaired. MBP immunohistochemical staining integral OD of GM1 group was higher than that of HIBD group, slightly lower than the sham group (P<0.05).
Conclusions are as follows:
1. Neonatal rat brains are poor myelinited and rapidly development after birth. The myelination has started before P15 in rat brain and nearly be mature in P30. This suggest that the optimal time to observe myelin would be P30 and later. Expression of NF155 protein is later than MBP, increased with age too. NF155 is Micro expressed in P7, increases greatly in P30, and reaches the peak in P90, then declines in old age. The expression of NF155mRNA has a similar trend as NF155 protein. These suggest that differentiation of paranodal structure is later than myelinization.
2. Volume of myelin sheath in HIBD rat brain is reduced, and the protein expression of MBP and NF155 in brain tissue decreased, inducing descent of study and memory abilities. The myelin sheath emerge structural disorder, loose, layered with axonal atrophy. These suggest that HI not only reduces the myelin structural proteins, but also the paranodal proteins. These may induces abnormal regional distribution of membrane ion channels, thus leading to abnormal nerve conduction.
3. The Content of lipid raft and the distribution of NF155 in lipid raft are reduced in P30 HIBD model rats. Applying exogenous GM1 can increase the content of lipid raft, as well as NF155 expression and its lipid rafts association in HIBD rat brains. GM1 may repair the structure of lipid rafts, promote the association of important myelin proteins with lipid rafts, stabilize the structure of paranodes, and eventually prevent myelin sheath damage. The results suggest a novel and potential mechanistic explanation for the neuroprotective properties of GM1.
Above all, white matter injury is an important part in the process of HIBD. There are significant pathological changes of myelin sheath and paranodal structure. Myelin sheath could be repaired by improving the composition of lipid rafts in paranode.
缺血缺氧性脑白质损害的典型病变是脑白质中神经纤维的髓鞘化障碍。少突胶质细胞(OL)是CNS中的主要成髓鞘细胞,近年来研究表明OL系细胞对缺氧高度敏感,且成熟度越低、分化能力越强的OL系细胞对缺氧越敏感。新生儿脑内OL系细胞中以少突胶质细胞前体细胞(OPC)占主流,OPC对HI高度敏感,OPC受损后出现分化障碍,OL数量减少,功能异常,脑白质内髓鞘形成障碍,以至临床上出现脑瘫和智能发育落后等后遗症。然而,目前有关脑髓鞘的发育特点以及相关影响因素的研究甚少,尤其对HIBD条件下脑髓鞘的发育与形成的研究更少。可见,研究和探讨脑髓鞘的发育特点及HIBD对脑髓鞘形成的影响具有重要的理论与临床实用价值。
正常髓鞘化的神经纤维分为朗飞氏结区、结侧区、近结侧区及结间区等不同区域。郎飞氏结区钠通道高度聚集,这是神经冲动在有髓纤维跳跃式传导的分子基础。钾通道则主要分布于近结侧区,具有缓冲有髓神经纤维兴奋的作用。结侧区介于结区和近结侧区之间,起绝缘作用,对维持各区轴膜蛋白和离子通道的正常区域化,保证有髓纤维神经冲动快速传导十分重要。研究发现,结侧区主要由少突胶质细胞产生的神经束蛋白155(neurofascin 155,NF155)及其轴突源性配体caspr和contactin组成,它们之间互相作用,形成结侧区复合物。其中,NF155在结侧区复合物的形成中起募集、引导作用,是结侧区复合物形成的关键。结侧区复合物主要分布于结侧区脂筏中。在脂筏中的特征性分布使结侧区复合物更加稳定,利于有髓纤维各区离子通道的正常分布。但是,目前国内外对于髓鞘结侧区及NF155蛋白的研究尚在起步阶段,对NF155在实验动物脑中的发育规律还不清楚,研究其发育规律是揭示髓鞘结侧区在HIBD脑白质损伤中的改变及作用的基础。
基于上述理由,本研究首先研究大鼠不同发育时期的脑髓鞘发育规律,在此基础上深入探讨HIBD中发育期大鼠脑髓鞘的病变特点及神经行为学改变;并进一步从较为微观的角度观察HIBD模型鼠髓鞘关键区域----结侧区的NF155蛋白及脂筏的改变,并探讨补充外源性GM1对修复髓鞘结侧区脂筏的作用。研究方法及结果摘要叙述如下:
一、不同发育阶段大鼠脑髓鞘的发育
采用下述方法研究胚胎14d(E14),E18,出生后0d(P0)、P7、P15、P30、P90、P720大鼠脑髓鞘发育,结果如下:
1.应用劳克斯坚牢蓝(LFB)染色,髓鞘碱性蛋白( MBP)免疫组化染色的方法,我们发现E18、P0、P2均未见髓鞘阳性染色,P7大鼠胼胝体中部可见少量LFB染色,P15大鼠胼胝体,外囊,内囊及皮质两种染色均呈阳性,P30着色加深,与P90,P720相似。比较P15~P720各组与前一日龄组的MBP免疫组化染色积分光密度,差异均有显著意义(P<0.05)。
2.通过western-blot检测MBP蛋白在不同发育时期的表达,结果发现MBP蛋白在E14已有表达,出生后随日龄增长逐渐增加,至老年期含量最高,各日龄组与前一日龄组比较差异均有显著性(P<0.05)。
3.通过western-blot检测发现髓鞘结侧区蛋白NF155在胚胎期、P0、P2无明显表达,P7时仅有极少量表达,P30大量增加,P90达高峰,老年期又有所下降,各日龄组与前一日龄组比较差异均有显著性(P<0.05)。
4.通过Real-time PCR检测各发育期大鼠脑NF155 mRNA表达,结果与NF155蛋白表达趋势相似,各发育期鼠脑NF155mRNA的基因表达相对倍数随日龄增长逐渐增高,至3月龄时达到高峰,其后下降。
二、HIBD对发育期大鼠脑髓鞘及神经行为的影响
1.参照Levine法建立P7SD大鼠HIBD模型,建模后出现夹尾左旋行为者列为成功模型动物用于研究。P30时见HIBD模型大鼠脑大体左侧(缺血侧)脑可见明显萎缩。
2.应用LFB、MBP免疫组化髓鞘染色可见:P30HIBD模型患侧脑胼胝体,外囊,内囊,纹状体结构错乱,阳性染色分布异常,积分光密度降低,与假手术组比较有显著性差异(P<0.05)。
3.应用MBP,神经丝蛋白200(NF200)免疫荧光双标染色,激光共聚焦采图,见假手术组胼胝体、外囊髓鞘与神经丝伴行排列,结构清楚;P30HIBD大鼠脑髓鞘、神经丝染色变淡,排列错乱。
4.透射电镜观察P30各组大鼠脑室周围白质髓鞘的超微结构,见假手术组髓鞘结构致密,完整,神经丝排列有致。HIBD大鼠脑髓鞘疏松,髓鞘层间分层明显,结构紊乱,高倍镜下可见轴质萎缩。
5.应用western-blot方法检测P30HIBD大鼠脑MBP及NF155蛋白表达,见HIBD大鼠患侧脑组织MBP及NF155蛋白量均较假手术组显著下降(P<0.05)
6.通过Morris水迷宫行为学测试,P30假手术组大鼠游泳轨迹以直线式和趋向式为主, HIBD大鼠游泳轨迹以边缘式和随机式为主。HIBD大鼠在定位航行试验中逃避潜伏期较假手术组明显延长;在空间探索实验中穿台次数较假手术组明显减少,两组间差异有显著性(P<0.05)。
三、HIBD对大鼠髓鞘结侧区脂筏的影响及GM1的干预效应研究
1.通过去垢剂法提取大鼠脑脂筏,Optiprep梯度离心液超高速离心后用GM1鉴定,脂筏主要分布于梯度离心条段的第3~5层。
2.将各梯度离心条段经SDS-Page凝胶电泳和免疫印迹检测可见:P30HIBD大鼠患侧脑脂筏中GM1含量较假手术组显著降低,GM1组大鼠脑脂筏中的GM1含量较HIBD组显著提高(P<0.05)。
3.通过western-blot检测见HIBD大鼠患侧脑NF155蛋白表达及NF155在脂筏中的含量均较假手术组明显减少。GM1组大鼠脑NF155蛋白量及NF155在脂筏中的含量较HIBD组显著提高(P<0.05)。
4. MBP免疫组化染色及脑髓鞘透射电镜检查见HIBD组大鼠脑髓鞘结构破坏,GM1组髓鞘得到明显修复,MBP免疫组化染色积分光密度值高于HIBD组,略低于假手术组。
全文结论如下:
1.初生SD大鼠脑髓鞘化程度很低,出生后随日龄增长快速髓鞘化,P15全脑大部分区域髓鞘化已经开始,P30接近成熟,P90已发育成熟。提示观察大鼠脑髓鞘的时间不宜早于15d,最佳时间在30d以后。NF155蛋白表达晚于MBP,亦随日龄逐渐增加,从P7有少量表达,P30大量增加,P90达高峰,老年期呈逐渐下降趋势。提示在发育早期髓鞘结侧区结构的分化晚于髓鞘的形成,在老年期结侧区成分减少,有可能是老年期髓鞘功能减退的原因之一。
2. P30HIBD模型大鼠脑髓鞘量减少,脑组织MBP、NF155蛋白表达下调,学习记忆能力下降。髓鞘结构出现错乱,松散,分层等形态学改变,伴有轴突轴质萎缩。提示HIBD大鼠模型脑中髓鞘结构明显异常,导致神经功能异常。此外,还可能因髓鞘结侧区结构破坏使神经纤维轴膜离子通道区域化分布失败,导致有髓神经传导功能异常。
3. P30HIBD模型大鼠脑脂筏量减少,结侧区蛋白NF155在脂筏中的分布亦明显减少,提示结侧区脂筏受损。补充外源性GM1不仅能显著增加大鼠脑脂筏量,还能增加HIBD大鼠脑中NF155的表达量及其在脂筏中的分布。提示外源性GM1可能通过补充脂筏中重要脂质,修复髓鞘结侧区脂筏,保护结侧区重要髓鞘蛋白NF155及结侧区复合物,最终起到修复髓鞘的作用。
综上所述,脑白质损伤是HIBD中的重要部分,HIBD中脑髓鞘及其结侧区结构均有明显病变。通过改善脂筏的成分可能修复髓鞘结侧区结构和功能,最终达到修复髓鞘的效果。
Neonatal hypoxic-ischemic brain damage (HIBD) is a common brain injury due to hypoxia and decrease of cerebral blood supply caused by various perinatal factors. HIBD is a serious complication of perinatal asphyxia and the main reason for cerebral palsy and other neurological chronic disabling diseases. People used to think that white matter was of high tolerance to hypoxia, so most researches on HIBD had focused on neuron damage and repair. However, recent studies found that white matter damage play crucial role in HIBD, and especially in premature infants with hypoxic-ischemic (HI), the incidence of white matter damage was even higher than that of cortical damage. These findings are changing people’s traditional understanding of white matter anoxia.
Typical white matter damage due to hypoxic-ischemia is myelinization disturbance of nerve fibers. Oligodendrocytes (OL) are the major cells who forming myelin sheath in central nervous system (CNS). In recent years, studies have shown that OL are of high sensitivity to hypoxia, especially the cells at lower maturity level and with strong differentiation. In the neonatal brain, oligodendrocyte precursor cells (OPC) are dominant OL, and highly sensitive to HI. After damage, OPC can’t differentiate well, and thus, reduce the number of OL and make dysfunction. In this way, dysmyelination of white matter happens, and as a result, some sequelae occur, such as developmental disorders, cerebral palsy and mental retardation and so on. However, there are rare studies about the brain myelin development and related influencing factors, especially under HIBD. As it can be seen, studies on developmental characteristics of brain myelin and effects of HIBD on brain myelination have important theoretical and clinical value.
Myelinated axons are commonly divided into four functional regions: nodes of Ranvier, paranodes, juxtaparanodes, and internodes. The highly concentrated sodium channels in nodes of Ranvier are the molecular basis of nerve impulse’s saltatory conduction in myelinated nerve fibers. Potassium channels are mainly located in juxtaparanodes, which buffer the exciting of mylinated nerve fibers. The paranodes, areas between node and juxtaparannodes, have insulated effect and maintain various axilemma proteins and ion channels to be normal regionalization, and to ensure rapid transmission of nerve impulses of myelinated fiber is very important.
Study found that the paranodes composes mainly of the myelin protein neurofascin 155 (NF155) produced by oligodendrocytes and their axons endogenous ligand caspr and contactin, and they interact to each other to form the paranodal complex. In which, NF155 guide the formation of paranodal complexes, and plays a key for complex formation in paranode. The paranodal complexes are mainly distributed in lipid rafts of paranode. This distribution makes paranodal complex more stable, which will help the normal distribution of various ion channels. However, the study of paranode of the myelin sheath and NF155 proteins is still at an initial stage, and development profile of NF155 in the brain of the experimental animals is unclear. It will be an important basis to study and explore development law of medullary sheath in paranode and its effect in white matter damage due to HIBD.
In summary, this study will firstly study myelin development profile of rat brain at different developmental stages, and then, on this basis, to study the features of myelin sheath damage and neurological behavioral changes after HIBD; and further from a more microscopic point of view to study the changes of NF155 protein and lipid rafts of critical areas of myelin sheath—paranode in HIBD rat model, and explore the repair effect of exogenous GM1 on lipid rafts in myelin sheath of paranode. Research methods and results summarized as follows:
1. The development of rat brain myelin at different developmental stages
We use the following methods to study rat brain myelin development of embryo 14d (E14) and E18, and postnatal 0 d (P0), P7, P15, P30, P90 and P720. The results are as follows:
(1). With Luxol fast blue (LFB) staining and immunohistochemical staining against myelin basic protein (MBP), we found that E18, P0 and P2 rats showed no positive staining of myelin, P7 rats had mild LFB staining in the corpus callosum, P15 rats were positive to both staining in the corpus callosum, internal capsule and capsula externa, and cortex. The double positive staining was deepened in P30 rats, similar to those in the rats of P90 and P720. MBP was expressed significantly in a time-dependent manner from P15 to P720 (P<0.05).
(2). Western blot analysis indicated that MBP was expressed since E14, then increased with age after birth, and gradually increased to the highest level of old age. The difference between every age groups was significant (P<0.05).
(3) Western blot analysis found that myelin protein NF155 was not expressed in the brain during the embryonic stage, P0, and P2. It was slightly expressed at P7, strongly expressed at P30, reached a peak at P90, and declined in old age. The difference between every age groups was significant (P<0.05).
(4) Real-time PCR showed that the tendency of NF155 mRNA expression was similar to that of protein expression. The mRNA expression of NF155 was increased with age, and reached a peak at P90, and then decreased.
2 The influence of HIBD on myelin sheath and neurobehavioral function in developing rat brain.
(1). P7 SD rats were established according to Levine HIBD model. The rats with their head turn to the left when their tails were clamped were identified as successful. Obvious brain atrophy was observed in left sides (the ischemia sides) of P30 HIBD rat brain.
(2). With LFB staining and MBP immunohistochemical staining, the affected brain side showed structural disorder in the corpus callosum, external capsule, internal capsule and striatum in P30 HIBD model. Abnormal positive staining was observed with decreased integral optical density, which had significant difference with the sham group (P<0.05).
(3). Double immunofluorescence staining for MBP and neurofilament 200 (NF200) showed that myelin sheath displayed parallel arrangement with neurofilament in the corpus callosum and external capsule of sham group, but in P30HIBD rat brain, myelin sheath and neurofilament was slightly stained in a disturbed order.
(4). Transmission electron microscopy showed that the ultrastructure of rat periventricular myelin was compact, intact and in well arrangement in sham group. But in HIBD rat brain, myelin sheath was loosen, delaminated, and disarranged order, and axonal atrophy was found under high-power microscope.
(5). Application of western-blot method for detection of MBP and NF155 P30HIBD rat brain protein expression.The MBP and NF155 expressions both decreased significantly in HIBD rat brains compared with sham group (P<0.05).
(6). Morris water maze showed that P30 sham rats swam to track straight and tend to type-based, while HIBD rats swam to the edge of track- and stochastic-based. The escape latency in HIBD rats was significantly longer than the sham group (P<0.05). In spatial probe test, the number of shuttling was significantly reduced in sham group than in HIBD group (P<0.05).
3. The influence of HIBD on paranodal lipid raft of rat myelin sheath, and the intervention effect of GM1.
(1) Rat brain lipid rafts were extracted by detergent cracking and Optiprep gradient ultracentrifugation, and then identified by GM1. Lipid raft are mainly distributed in the 3rd to 5th layer of gradient centrifugation strip.
(2) Detecting each gradient centrifugation strip with SDS-Page gel electrophoresis and immunoblotting, the content of GM1 in lipid raft of P30 HIBD left rat brains decreased greatly compared to those in sham group. The content of GM1 in lipid raft of GM1 group was increased greatly compared to that in HIBD group (P<0.05).
(3) NF155 protein levels and NF155 content in lipid rafts in P30 left rat brains were detected by Western blotting. Both of them were decreased in HIBD group compared with sham operated group, significantly increased in GM1 group compared with HIBD group (P<0.05).
(4) MBP immunohistochemical staining and transmission electron microscopy showed the myelins of HIBD rat brains were structural damaged, however, the myelin of GM1 group were significantly repaired. MBP immunohistochemical staining integral OD of GM1 group was higher than that of HIBD group, slightly lower than the sham group (P<0.05).
Conclusions are as follows:
1. Neonatal rat brains are poor myelinited and rapidly development after birth. The myelination has started before P15 in rat brain and nearly be mature in P30. This suggest that the optimal time to observe myelin would be P30 and later. Expression of NF155 protein is later than MBP, increased with age too. NF155 is Micro expressed in P7, increases greatly in P30, and reaches the peak in P90, then declines in old age. The expression of NF155mRNA has a similar trend as NF155 protein. These suggest that differentiation of paranodal structure is later than myelinization.
2. Volume of myelin sheath in HIBD rat brain is reduced, and the protein expression of MBP and NF155 in brain tissue decreased, inducing descent of study and memory abilities. The myelin sheath emerge structural disorder, loose, layered with axonal atrophy. These suggest that HI not only reduces the myelin structural proteins, but also the paranodal proteins. These may induces abnormal regional distribution of membrane ion channels, thus leading to abnormal nerve conduction.
3. The Content of lipid raft and the distribution of NF155 in lipid raft are reduced in P30 HIBD model rats. Applying exogenous GM1 can increase the content of lipid raft, as well as NF155 expression and its lipid rafts association in HIBD rat brains. GM1 may repair the structure of lipid rafts, promote the association of important myelin proteins with lipid rafts, stabilize the structure of paranodes, and eventually prevent myelin sheath damage. The results suggest a novel and potential mechanistic explanation for the neuroprotective properties of GM1.
Above all, white matter injury is an important part in the process of HIBD. There are significant pathological changes of myelin sheath and paranodal structure. Myelin sheath could be repaired by improving the composition of lipid rafts in paranode.
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21. Hu BR,Liu CL,Yesa L, et al. Involvement of caspase-3 in cell death after hypoxia -ischemia declines during brain mat- uration[J]. Cereb Blood Flow Metab, 2000, 20(9):1294- 1300.
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24. Warrington AE, Barbarese E, Pfeiffer SE. Differential myelinogenic capacity of specific developmental stages of the oligodendrocyte lineage upon transplantation into hypomyelinating hosts[J].J Neurosci Res 1993;34 (1):1-13
25. Gard AL, Pfeiffer SE. Oligodendrocyte progenitors isolated directly from developing telencephalon at a specific phenotypic stage: myelinogenic potential in a defined environment[J].Development 1989;106(1):119-132
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34. Levison SW, Rothstein RP, Romanko MJ, et al. Hypoxia/ischemia depletes the rat perinatal subventricular zone of oligodendrocyte progenitors and neural stem cells[J]. Dev Neurosci, 2001, 23(3): 234-247.
35. Stephen A.Back, Byung Hee Han, Ning Ling Luo, et al. Selective vulnerability of late oligodendrocyte progenitors to hyposia-ischemia [J]. J Neurosci, 2002,22(2):455-463
36. Blomgren K, Hallin U, Andersson AL, et al. Calpastatin is up-regulated in response to hypoxia and is a suicide substrate to calpain after neonatal cerebral hypoxia-ischemia [J]. J Biol Chem 1999,274: 14046-14052
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24. Warrington AE, Barbarese E, Pfeiffer SE. Differential myelinogenic capacity of specific developmental stages of the oligodendrocyte lineage upon transplantation into hypomyelinating hosts[J].J Neurosci Res 1993;34 (1):1-13
25. Gard AL, Pfeiffer SE. Oligodendrocyte progenitors isolated directly from developing telencephalon at a specific phenotypic stage: myelinogenic potential in a defined environment[J].Development 1989;106(1):119-132
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