The Study About Expression Changes Of MAPK And NMDAR In The Hippocampus Of Alzheimer's Rat

ObjectiveAlzheimer's disease (AD) is a kind of neural degenerating disease, quite common among the older people. The early pathological change is memory deficit, and then the neuronal loss in cortex, finally memory deterioration and the decline of language, spatial recognition etc. There are two obvious pathological characters: extracellular senile plagues (SP) in neocortex and hippocampus; intra - neuronal neurofibrillary tangles (NFT). The biochemical basis of those pathological changes is the extracellular Amyloid - beta peptide ( Aβ) deposition and intracellular excess phosphorylation of TAU. At present, the pathogenic factors and pathogenesis of Alzheimer' s disease are still unclear.Aβ composes of Alzheimer' s disease and is the core component of the senile plague; it' s also the biochemistry bases of AD neurofibrillary tangles and vascular amyloidosis. Acute injection of Aβ into brain can trigger behavior disturbance and memorial deficits, which are similar to AD, and Aβ deposition found in animal brain. It has been reported that in vitro and in vivo, Aβ neuro-toxicity will occur at the concentration of jxmol/L. The increase or change of Aβ deposit is the promoter for pathogenesis of AD, the neurotoxicity caused by Aβ maybe the final way to Alzheimer' s disease. However, the mechanism of Aβ neurotoxicity is still unknown at present.Hippocampus is the key part of the brain associated with learning and memory, long term potentiation (LTP) is considered as the essential component of learning and memory. Therefore, the mechanism of LTP induced by Aβ deposit in hippocampus of AD model rats is extremely important to the investigation oflearning and memory disturbance in Alzheimer' s disease. Hippocampus has highly ordered ply structure and all kinds of neurons are distributed independently, so to providing visual and concision morphological structures for researches. So as to we take hippocampus to produce AD model by dimensional injection of drugs. LTP was considered as the processing of long term enhancement of EPSP after high - frequency stimulation, was one important electrophysiological index of learning and memory; was facilitation of synaptic transmission and the function unit of memory store of learning. Whereas, CA₁ - CA₂ and CA₃ area of hippocampus were important areas closely related to learning and memory.Mitogen - activated protein- kinase (MAPK) signal pathway can mediate many kinds of physiological functions of the cell, i. e. cell growth, development, division, cell death and intercellular synchronization. MAPK is a family of protein kinases with serine - and threonine - residues, which distributed within the cytosols. MAPK families of mammal include Extracellular - signel regukated kinase (ERK),C - Jun - N - terminal kinase (JNK) and P³⁸ protein kinase, which consist of some basic MAPK signal pathways. Among them, MAPK pathway is also called ERK, which is the core part of MAPK signal pathway and constitutes an accuracy and high efficiency signal transduction system, combining with upward activated molecules and downward effective molecules. At present, MAPK pathway is the best - understood signal transduction pathway, which quite common among animal's cells; It can modulate the processing of cell cycle and the gene expression either.Concerning the research of relation between AD and MAPK, some scientists reported that the characteristic pathological changes of AD were closely associated with series of cellular signal transduction systems. The phosphorylation of microtubule - related - Tau protein modulated by MAPK and the deposit of Aβ were all related to the pathology of AD. Due to the discovery of MAPK in the AD neurofibrillary tangles and senile plagues of hippocampus, the researchers draw the conclusion that MAPK make Tau - protein phosphorylated normally , and play a very important role in the formation of AD neurofibrillary tangles. Somebody investigated the function of the MAPK family in the mechanism of Aβ' s neurotoxicity, observed the downward regulation of MAPK of cells afterexposure to Aβ 24 hr. Some others reported that MAPK pathway was quite essential to the formation of LTP in hippocampus. The activated MAPK expression of CA₁/CA₂ areas is increase after high - frequency stimulation (HFS) , If PD098059 and u0126 inhibite the MAPK's activation in advance, rats would suffer the damage of spatial memory. However most of those results drawn by researchers abroad were under the condition of cell - culture in vitro.N - methyl - D - aspartate receptor (NMDAR) is quite important to physiology and pathology of central nervous system, and closely associated with short - term memory. It also takes part in the formation of synaptic plasticity. Facilitation of NMDAR's current inputs the current into the pyramid cells of CA₁ areas , in order to regulate the learning and memory ability. we suppose that Aβ influence the induction and maintenance of LTP through ions current passing through NMDAR. It' s still unknown how to exactly regulate the current of NMDAR. There are two hypotheses: One supposes that hippocampal neurons regulate the current of NMDAR dependent on protein kinase C (PKC). Another one considers that may be mediated by activation of C - terminal tyrosine phos-phorylation of subunit of NMDAR. Therefore, the further researches will be needed to give proof of the two hypotheses.AD rat model was established by injection of Aβ 1⁴0, 1μL (10μg/μL) into rat's hippocampus. Behavior changes of learning and memory ability and LTP were determined by Morris water maze and electrophysiological methods respectively. HE staining, Nissl staining, immunohistochemistry and hybridization in situ were applied to determine the expression of MAPK protein and NMDAR in AD rat model, In order to investigate the pathogenesis of Alzheimer's disease.MethodsA total of 30 SD rats were divided into model group and control group randomly and equally. Aβ_1⁴0 had been incubated for a week at 37℃ to accumulate. Rats were fixed on stereotaxic instrument, and then Aβ was injected into the brain to produce AD animal model. Two weeks later, Morris water maze and electrophysiological method were applied to determine the learning and memoryability and LTP respectively. Then .brain sections were prepared. HE staining and Nissl staining were used. Immunohistochemistry detected the expression of phosphorylated MAPK protein and Aβ deposit. Immunohistochemistry and in situ hybridization determined the expression of NMDAR₁ protein and NMDAR₁ -mRNA of hippocampus. The results observed with optical microscope. The degree of average grey values in CA₁, CA₂, CA₃ and DG were measured with picture analysis system. Statistical data were expressed as Mean SD. T test was used.Results1. Behiviour observationMorris water maze:Two weeks after injection of Aβ, the escape latent was prolongated in Alzheimer rats compared with control rats (P <0. 05). It indicated that the ability of learning and memory in model animal was declined.2. Electrophysiological observationThe alteration of LTP amplitude indicate that the augmented amplitude of field — excitory postsynaptic potentiation (fEPSP) was decreased dramatically in Alzheimer rats than in control rats after high - frequency stimulation (HFS) 30 min(P<0.05)。The slope alteration of fEPSP between pre - and post - high - frequency stimulation (HFS) was extremely decreased in Alzheimer rats than in control rats(P<0.05)。3. Immunolocalization of Aβ deposit in hippocampusThe positive expression of Aβ deposit in CA₃ area of Alzheimer rats was significantly increased compared with control rats. The positive - immunoreactive substance was located on cell membrane and spikes, which was small brown granules.4. Nissl staining and HE stainingIn control group, Nissl staining showed that there were no obvious neuron damages in dorsal side of dentate gyrus of hippocampus; neurons densely arranged, all in order. Around the injection site, no obviously neurons loss wasfound in CA₁, CA₃ area. Away from the injection site, there was no obvious loss of neurons in parietal cortex. HE staining showed the existence of all layers cell in parietal cortex and hippocampus, no obvious hyperplasia of small glia. In AD group, Nissl staining showed that the cells belt in dorsal side of dentate gyrus of hippocampus was damaged dramatically, the numbers of cell were reduced, cells arranged out of order. The cell numbers around the injection site of CA₁, CA₃ area were decreased. Away from the injection site in parietal cortex, the neurons were lost, however, no obvious glia hyperplasia. HE staining indicated: the hyperplasia of a kind of cell with small and dark stained nucleus in the dorsal side of dentate gyrus of hippocampus, whose cell belt was absented. The cell was round and flat or pyramidal, less cytosol, should be small glia.5. Alteration of MAPK expression in rat's hippocampusMAPK expression was demonstrated in the cell nucleus. There were no positive - immunoreactive neurons in cortex both among model group and control group. The MAPK expression of model group was remarkably lower than control group in CA₁ area and CA₃ area and in dentate gyrus (p <0. 05, p <0. 01, p < 0.05).6. Expression and distribution of NMDAR₁ protein and NMDAR₁ - mRNA in hippocampus. Observed with optical microscope, the positive stained cells were found both in model group and control group; NMDAR₁ protein and NMDAR₁ - mRNA positive cells'cytosol was dark stained and mainly distributed in pyramidal layers and granular layers of dentate gyrus of hippocampus. There was no any specific staining on the plates pre - prepared with RNAase A or pre- hybridizations without probe. The degree of average grey value of NMDAR -mRNA positive cells in CA₁ and CA₃ area of model group was remarkably higher than control group (p<0.05, p<0.01); but the change in dentate gyrus was unobvious ( p > 0.05 ) .Conclusion1. Approved that injection of Aβ into hippocampus induced the behavior and electrophysiological changes of memory, and cause Aβ deposition. This in-...