| BackgroundHypoxic-ischemic brain damage (HIBD) refers to a fetal or neonatal brain damage, due to partial or complete lack of oxygen or cerebral blood flow reduction or suspension caused by perinatal asphyxia. The clinical feature is a series of abnormalities of central nervous system, which will be leave over a lot of sequels such as cerebral palsy, mental retardation, epilepsy and development delay, and it is one of the main reason of neonate acute death and chronic nervous system damage. Many moderate and severe hypoxic-ischemic brain damage children survive with the establishment of neonatal intensive care unit and the improvement level of medical technology, but the incidence of permanent brain dysfunction of them remains high. HIBD is a diffuse injury, the main lesion locations are brain hippocampus,cortex, corpus striatum,basal ganglia,thalamus and so on, its pathogenesis is complex, incited by hypoxic-ischemic falls, which is a series of biochemical chain reaction caused by the combined effects of a variety of mechanisms. Currently, clinical treatment for HIBD use comprehensive treatment such as symptomatic treatment, nutritional support and early intervention regularly, which still lack of a special effect and economical way. For the reason of that, the further study of the HIBD damage and repair mechanisms is of great significance to find a way of effective prevention and treatment, to improve the survival of neonate with HIBD, to reduce the long-lasting neurological sequel of survival children and to improve the population quality. Neonate is in the critical period of brain development, and HIBD has the characteristics of non-progressive and non-persistent ischemia hypoxia factors are existing. The brain development of seven-day-old SD rat is similar to the neonate, and the condition which caused transient brain hypoxic-ischemic is the same with HIBD, too. Therefore, at home and abroad, seven-day-old SD rat is used as the research object and reference to the Rice method to establish HIBD model in neonatal rat, which is widely used in research topics related to HIBD. Then, use animal model to explore the mechanism of damage after HIBD in depth, understand the effect of hypoxic-ischemic to long-lasting neurological function and ultra structure in neonatal rats, find method of reasonable and comprehensive prognosis and assessmeng of efficacy, and further to seek effective and economic intervention measures and to explore the repair mechanisms of these measures.HIBD is a diffuse injury, clinical accurate assessment of the long-lasting prognosis and intervention efficacy of HIBD is very difficult, for the complexity of the HIBD pathogenic factors and the specificity and plasticity of the brain development of infants. There is no consistent linear correlation between the prognosis and the severity of injury in the acute phase for many infants. In Current research on HIBD animal experiments, the evaluation for the HIBD long-lasting prognosis and interventions efficacy stays on the observation of changes in the two injury brain regions with the behavioral and routine pathological, but few ultra structure studies. Synapse, the core of brain plasticity, are the functional parts of the information transmission between neurons and effector cells, and have close relationship with behavioral performance. Therefore, observing changes in synaptic ultra structure can help to explore not only the HIBD mechanism and repair mechanism but also the correlation with behavioral changes, which is expected to make an early and objective evaluation about the long-lasting prognosis and intervention efficacy of HIBD.At present, hyperbaric oxygen, hypothermia, neurotrophic drugs and general support for symptomatic treatment are used for the treatment of HIBD. The application of hyperbaric oxygen treatment is not only limited by the hospital equipment conditions, but also affected by early severity, the treatment window and efficacy still have no final conclusion. While the efficacy of hypothermia treatment is uncertain with more limited application. The current study have reported that neurotrophic drug only have a certain effect in the acute phase of HIBD, and the high cost also affect its clinical application. Physical training is a treatment for active or passive movement through the patient’s own forces or therapist-aided or by means of equipment for the purpose to improve the function of local or systemic. It has been applied in the clinical treatment of HIBD for its easy implementation, low cost, treatment time can be long and implemented at home. However, its efficacy and mechanism are not yet entirely clear. On the one hand, the neonatal brain, which is in a critical period of development, has a strong plasticity and own repair capacity to damage, so it’s difficult to judge the progress of children associated with this intervention treatment or not clinically. On the other hand, studies have shown that appropriate time and amount of physical training can improve not only motor function but also the learning and memory abilities of human and animal. A lot of HIBD children are associated with mental retardation and sensory integrative dysfunction, in addition to motor dysfunction. Can early physical training improve these symptoms and how it improves?According to research reports, appropriate physical training can cause second messenger, such as cAMP and Ca2+, increasing appropriately in the central nervous system, to induce c-fos gene expression, control the transcription of downstream target genes, and then compose new proteins. Under normal circumstances, c-fos proteins involve in cell growth, differentiation, information transfer, learning and memory and other physiological processes. It has a very important role in memory encoded and the formation of long-term potentiation which reflect neuronal plasticity. Therefore, physical training is beneficial to the memory function in the brain, which provides evidence at the molecular level that movement can promote learning and memory.It has been pointed out that synaptophysin (p38) is an important symbol of synaptic remodeling, because p38involved in neuronal development and differentiation processes. p38expression in developing neurons take precedence over the composition of synapse, that is, before the synaptic structure formation, p38exist and express already, as neurons mature, synapses mature and the expression of p38is also a corresponding increase. p38also directly involved in synapse formation, and regulating the expression of other synaptic related proteins. So, this protein is widely used to mark the synaptic terminal.Based on the above theory, this study use the Rice method to establish a HIBD model in neonatal rats to observe the changes of long-lasting behavioral, pathological, and ultra structural, in order to explore HIBD mechanism, to focus on observing the ultra structural changes and its significance, guide long-lasting prognosis and efficacy assessment of HIBD. Then give early physical training intervention, observe its effects to the HIBD rats for forward movement, learning and memory ability and sensory function, and use immunohistochemical method to detect the expression of c-fos and synaptophysin, on the purpose to study its efficacy mechanism.Therefore, a series of experiment studies were performed as follows:Part I Construction of animal model of neonatal rat HIBD, examine long-lasting behavioral and ultrastructure ChangesObjectiveTo establish a hypoxic-ischemic brain damage (HIBD) model in neonatal rat, examine long-lasting ultrastructure Changes, provide functional assessment methods and guide clinical treatment.MethodsSixty seven-day-old SD rats were randomly assigned into a HIBD group and a sham operation group,each group was30cases. A battery of behavioral tests,including the Morris water maze test and sensorimotor function test, were performed at postnatal age5weeks. Nissl staining was used to counting the neurons number.The transmission electron microscopy was used to observing the structure of the synapse、 measuring the thickness of postsynaptic density and the length of active area,and their correlations with the results of behavioral tests were evaluated at last. ResultsIn the Morris water maze test,in all test points the incubation time to find the platform of the HIBD group was significantly longer than the sham operation group (P<0.05),and the frequency across the platform of the HIBD group rats was reduced,too(P<0.05). The sensorimotor function test showed the sensorimotor function was worse in the HIBD group than the sham operation group.Nissl staining showed that the neurons number in HIBD group was significantly reduced (P<0.01).The transmission electron microscopy test showed the synapses were reduced,the thickness of postsynaptic density became thinner and the length of active area became shorter in HIBD rats. The correlation of the thickness of postsynaptic density in the hippocampus and the incubation time to find the platform in the Morris water maze test was negative(r=-0.861, P=0.006),as also as the thickness of postsynaptic density in the cortex and the total scores in three sensorimotor function test in the HIBD group(r=-0.758,P=0.029).ConclusionsHypoxia ischemia can lead to the neurons loss and ultrastructure damage,caused long-lasting deficits of behavioral function,the ultrastructure changes is better than pathological observation to a certain degree,contribute to assessing prognosis and evaluating the efficacy of treatment,and guide clinical treatment in critical period of brain development based on synaptic plasticity of the immature brain.Part Ⅱ Effects of early physical training on long-lasting neurological function and ultrastructure changes following hypoxic-ischemic brain damage in neonatal ratsObjectiveTo investigate the effect of early physical training on long-lasting pathology、 ultrastructure and neurological in neonatal rats submitted to HIBD.Methodsninety7-day-old Sprague-Dawley rats were randomly divided into three groups:a group that was subjected to left carotid ligation followed by2hours hypoxic stress(the control group);a group that received physical training(Grab. Rotation. Walk, balance)1weeks after the HIBD event;a sham-operation group that was subjected to a sham-operation without ligation and hypoxic stress.Following four weeks physical training, Neurological score. Morris water maze tests and cortex sensorimotor tests were performed. Left hippocampal CA1and cortex neurons and ultrastructure were examined.The expression levels of Synaptophysin and c-fos were determined by immunohistochemistry.ResultsCompared with HIBD group, the Neurological score. Spatial learning and memory ability and sensorimotor tests of trained group were significantly increased, whereas there was no significant difference between the trained-HIBD and the sham-operation group.The ultrastructure of the left hippocampus and cortex was remarkably abnormal in the non-trained HIBD group by the transmission electron microscopy, while no obvious abnormality was observed in the trained group and the sham-operation group. The neurons in the left hippocampal CA1zone and cortex were decreased of non-trained group.The significant difference compared with trained group was obvious (P<0.05). The expression of Synaptophysin and c-fos in the trained group increased significantly compared with that in the non-trained HIBD group (P<0.05).ConclusionsEarly physical training can restrain brain damage and ameliorate spatial learning and memory impairments in rats with HIBD.Early exercise rehabilitation can reduce hippocampal and cortical neuronal death, enhanced synaptic plasticity,and ameliorate the long-term neurological function after HIBD.The strong expression of synaptophysin and c-fos in the hippocampus and cortex caused by early physical training may be one of the improvement mechanisms. |
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