| 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 stagesWe 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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