| Hypoxic ischemic encephalopathyis a common neurological disease during neonatal period. Survivors often suffer from learning, memory and other developmental disorders of the nervous system, such as cerebral palsy, epilepsy, dysentery, etc. It's a hot point to interfere the HIBD early. But till today we haven't found a satisfactory way to cure it. It's important to explore a good way to maximum the activation of the brain against injury after HIE and remodel potential recovery methods, thus we can obtain a better functional recovery of patients and will improve their quality of life.Objective:To explore the learning and memory impairment in neonatal rats after hypoxic ischemic brain injury and the role and possible mechanism of traditional Chinese herb monomer Polydatin on it. We determined learning and memory ability, observed the expression of ICAM-1, analysed the cells'apoptosis, and studied the central synaptic plasticity and microstructure of nervous system.Methods:In this study,7-day SD rats with neonatal hypoxic-ischemic brain injury model was examined. The rats were divided into sham operation group, HIBD intervention group and PD group separately. After left main carotid artery was isolated and ligated for 2 hours, rats were put into a container filled with nitrogen-oxygen mixture air for 2 hours which oxygen concentration was 8% inside, thus the hypoxic-ischemic brain injury model was made. For sham group, we only separated the left common carotid artery, not ligated it. Morris Water Maze test was used to assess the change of learning and memory 21 days after HIBD. Expression of ICAM-1 in synaptophysin of brain tissue was observed by immunohistochemistry. Neuronal apoptosis was observed by TUNEL method, Electro microscope was used to observe the changes in synaptic structure of hippocampus CA1 area.Results:1. The study of the effects of Polydatin on long-term learning and memory in HIBD newborn rats.(1)Place navigation test (PNT):HIBD group (52.37±8.03S), Mean escape latency was significantly longer comparing with the sham group (39.55±8.08S) (P<0.001). While for PD treatment group (43.29±7.63S) was significantly shorter than the HIBD group (P<0.05). On the first day, the mean escape latency of these three groups'rats showed no significant difference (P> 0.05). After training, the mean escape latency in the three groups of rats was shortened gradually. During the third day, fourth day, the mean escape latency between these three groups were statistically significant (P<0.05 or P<0.01) after pair comparison by single-factor analysis of variance, For PD treatment group and sham operation group, the mean escape latency was significantly shorter than the HIBD group.(2) Spatial probe test (SPT):For HIBD group, the times of platform crossing (2.36±1.80) was significantly reduced comparing the sham group (5.29±2.62) and PD intervention group (4.25±1.66). For HIBD group, the swimming time (10.63±3.66S) in target quadrant was significantly shorter comparing the sham group (15.74±3.85) and PD treatment group (14.32±2.52) (P all<0.05). For PD treatment group, the times of platform crossing and swimming time (14.32±2.52S) in objectives quadrant were lower than the sham group. However, it showed no significant difference between groups (P all> 0.05). The results showed that the space exploration capabilities of rat were damaged after hypoxic-ischemic injury. Polydatin can reduce the damage degree of space exploration capabilities caused by acute hypoxic-ischemic brain injury in rats.(3)Immunohistochemistry result:In hypoxic-ischemic group, the left cerebral cortex and hippocampus CA1 area, the arrangement of pyramidal cells in layers was disordered. The number of cells in field were reduced significantly and arranged loosely, nuclear profile was not clear. Nissl granules were lost significantly. For PD treatment group, the number of neurons Nissl was more rich and was stained deeper than hypoxic-ischemic group. Nuclear profile was relatively clear, layers of cells were abundant. Cells were arranged more neatly comparing with the hypoxic ischemia group.2. The expression of Synaptophysin and microstructural changes in hippocampal CA1 area after HIBD and the role of Polydatin intervention(1) The expression of Synaptophysin:7 days after hypoxic ischemia injury, for HIBD group, the synaptophysin average density value of hippocampal CA1 ischemic (0.1628±0.0123) was reduced comparing with the sham operation group (0.2857±0.0709).21 day after the hypoxic ischemia injury, for HIBD group, synaptophysin expression density (0.3436±0.0569) was significantly higher in ischemic hippocampal CA1 area. For PD intervention group, it also showed the same tendency:7 days after hypoxic and ischemia injury, for the expression of synaptophysin in ischemic side hippocampal CAlarea, the average density is nearly close to or the same level with sham group.21 day after hypoxic ischemia injury, for PD group, the expression of synaptophysin was further enhanced in ischemic hippocampal CA1 area (0.4491±0.2230) comparing the other groups. And at the same time, differences were significant between sham group and model group (P<0.05, P<0.01).(2) Electron microscopy results:①HIBD group:hippocampal neural cell shrinked, nuclear membrane folded, chromosomes compacted, Nucleolus doubled, cytoplasm densed, mitochondria swelled, mitochondria disappeared, and endoplasmic reticulum expanded. Brain Punctate foci dissoluted, the surrounding myelin degeneration, Glial cells Proliferated, glial foot around the microvascular swelled, ruptured, microvascular cavity compressive deformation. Neurofilament decreased, array scattered.Neurons Synaptic declined, synaptic cleft widened, synaptic vesicles decreased, and postsynaptic density was thin, nuclear membrane integrity of hippocampal neurons was smooth, nuclear chromatin Uniform, cytoplasmic rough endoplasmic reticulum, ribosomes, Golgi bodies, mitochondria and other organelles were full and rich. Neurofilament orderly arranged. Presynaptic membrane, postsynaptic membrane and synaptic cleft were clear. Presynaptic terminals had more round clearest vesicles, and postsynaptic membrane has a high density electron.3. The study of ICAM-lexpression and the apoptosis on brain after hypoxic and ischemia injury(1) The expression of ICAM-1:For sham group, there were little brain microvessel immunostained positively. In HIBD group,6h after HI, the number of ICAM-1 immuno-positive staining blood vessels was increased significantly,12h reached peak point, after 24h, ICAM-1 immunoreactive staining of blood vessels levels continued in the peak. For treatment group, after 6h,12h,24h HI, ICAM-1 expression on brain microvascular endothelial was decreased significantly (P<0.05 or P<0.01) comparing with HIBD group; HE staining also showed that pathological changes in injured brain tissue infiltration of neutrophil reduced significantly. Neuron cell's degeneration, brain's edema and the organizational structure of disorder were reduced. After hypoxic and ischemia injury, once Polydatin was given early to the rat, it showed upregulation of ICAM-1 in cerebral microvascular endothelial, and brain inflammatory response was inhibited.(2) The apoptosis of hippocampal CA1 and the changes of apoptotic index:Sham group, it showed no or only a few apoptotic cells appeared. In HIBD group, there were a number of apoptotic cells. For PD intervention group, the number of apoptotic cells was reduced significantly comparing with HIBD group. Nuclei of apoptotic cells had brown granules.The characteristics of apoptotic morphological should be as following: the nucleus shrink, chromatin condensation, nuclear lysis and perinuclear crescent-like chromatin concentration. Dark apoptotic bodies appeared in cell. For hypoxic-ischemic group, neuronal apoptosis index were significantly higher in the left hippocampus CA1 (P<0.001) at different time points after hypoxia comparing with sham operation group.The apoptotic index increased after hypoxia with the increasing of time. Compared with hypoxic-ischemic group, at the same time point, apoptosis index of PD treatment group decreased significantly (P all<0.05). Conclusions:1. HIBD can cause a decline of neonatal rats in spatial learning and memory. Polydatin treatment can improve spatial learning and memory in HIBD rats.2. ICAM-1 took part in the inflammatory response of hypoxic-ischemic brain injury. Polydatin treatment on hypoxic-ischemic brain had a neuroprotective effect; the mechanism may be related to blocking ICAM-1 protein expression.3. After neonatal rat hypoxic-ischemic brain injury, apoptosis involved in the death of neurons. Polydatin intervention can inhibit neuronal apoptosis caused by ischemia and hypoxia.4. Synaptophysin immune reaction in hippocampus reduced signify-cantly and microstructure was damaged after HIBD. It showed that the structure and function of the central nervous system plasticity reduced after HIBD.Polydatin therapy can increase synaptophysin expression in hippocampus CA1 area, and improve the microstructure of synapses, and promote rebuilding of synapse.In summary, after cerebral ischemia, learning and memory injury is a process concerning many factors. Inflammatory injury which caused by cerebral ischemia-reperfusion, can be used as signals to stimulate the induction of apoptosis in apoptosis-related gene expression changes, inflammation and synaptic plasticity damage of the structure and function, which will lead to nerve damaging, eventually lead to learning and memory dysfunction. Polydatin possible play the role of improving learning and memory induced by cerebral ischemia and hypothyroidism through the way of anti-inflammatory and anti-apoptosis, thus increase central nervous system structure and function plasticity.
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