Study On The Regulative Mechanism For Astrocyte On Synaptic Plasticity

Part 1Effect of Astrocyte on Synapse in The CA1 Region ofHippocampusObjective: To investigate the relation between astrocyte(AS) and the synaptic number, vivid and maturity in the hippocampal CA1 region of growth rat.Method: Experiments were performed on the groups of rats: the new born (<24h,20 rats), the juvenile (28-30d,20), the adult rats(90-100d,20), the ara-c(90-100d,20,ara-c injection into intraventriculus lateralis), the mevastatin (90-100d,20, mevastatin injection into intraventriculus lateralis) and the saline (90-100d,20, saline injection into intraventriculus lateralis). The cholesterol content in the CA1 region of rat hippocampus was individually determined by high performance liquid chromatography (HPLC). The comparison of the growth statement of both AS and synapse among the three groups had been carried out in the region immunohistochemically by means of specific antiserum of either glial fibrillary acidic protein (GFAP) or synaptophysin (SPY), and neurons being stained by HE. The marked optical intensity of anti-GFAP immunoreation was determined with Q-500 microphotography management system. The optical density was received of minus back value, which was the value of optical density of uninjured corpus callosum and visual tract of the same sector, in order to avoid the incorrectness produced by no specific staining. The optical density of the three sections from each sample were determined, and therefore, their average value was obtained. The synaptic ultrastructure was observed under electron microscope in the CA1 region of rat hippocampus. The XY-540 processing system for biological images was used to determine the number of synaptic vesicles, the length of active zones, the thickness of post-synaptic density (PSD), the width of synaptic cleft and the curvature of synaptic interface. The shape of the synapse and the AS was also observed, their number being counted under the 30 vision-field of 8000 times, by means of the system. Results: (1). The cholesterol concentration in each of the three samples was 2.92±0.03, 11.20±3.41 and 12.91±1.25(g/kg) respectively, their differences being obvious significantly (p<0.01). (2). The optical density of GFAP staining in hippocampal CA1 regions of the new-born, the juvenile and the adult rats in turn were 2.53±1.76, 50.81±1.56 and 96.62±4.12, and their optical density value of SPY staining being 0.095±0.003,0.189±0.004 and 0.328+0.023 respectively, their differences were obvious statistically (P<0.01). (3). In the juvenil rats, a few AS (4.8+1.0/vision- field of 8000 times) and some unmature synapses (10.3±1.1/ vision-field of 8000 times) were found, less statistically than these (AS 9.5±1.2, synapse 16.3±1.4/ vision-field of 8000 times) in adult rat (P<0.01). The numbers of AS in the new born rat were much rare, and its synapse structures not being found. The electron microscopy showed that in the hippocampal CA1 region of the adult rats, the synaptic vesicles were round and clear, and there being thick postsynaptic densities (PSD). The synaptic interfaces were asymmetric and most synapses were of curve type. Some immature synapses could be seen in the hippocampal CA1 regions of the juvenile rats. The vesicle quanta (VQ) in the pre-synaptic structure were minimal and transparent, and the PSD was thin. Moreover, the synaptic clefts (SC) were wide and the length of active zones (LAZ) shorter. The differences in all the parameters of synaptic structure (PSS) were statistically significant (P<0.01). (4). After the injection of ara-c, the numbers of astrocyte and synapse, The optical density of GFAP and SPY and the cholesterol content decreased statistically compared with the adult group from as early as 3 day after injection (P<0.01), while only the the number of synapse, The optical density of SPY and the cholesterol content decreased statistically after theinjection of mevastatin (P<0.01).Conclusion: Astrocyte is closely related to synaptic number, vivid and structural maturity, in which cholesterol play a key role, in the CA1 area of hippocampi of growth rats. Part 2 Effect of glutamate on intracellular calcium change of neuronObjective: To study the effect of glutamate on the change of intracellular free calcium concentration ([Ca²⁺]i) in the primarily cultured neurons of rat hippocampus.Methods: The neurons were got from the hippocampus of SD rat born shorter than 24h, and those being acutely separated and primarily cultured in accordance with the ways of Larsen, et al. By means of microfluorescent technique the change of [Ca²⁺]i was observed, and the effect of glutamate on it being detected. The fluorescence rate of F340/F380 was designed to reflect the change of [Ca²⁺]i, and Fura-2 being loaded in by way of Fura-2/AM incubation. The effects of glutamate on the [Ca²⁺]i were studied while either the concentration or the effective time of glutamate was changed, or the antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor being administered simultaneously and respectively. Results: (1) Glutamate could increase the [Ca²⁺]i obviously. The increase of the [Ca²⁺]i induced by 100μmol/L glutamate was associated with effective time; The longer the effective time was, the higher the fluorescence rate got and the longer the time the rate descent from its top could be gradually. The increase of the fluorescence rate induced in the same effective time was related to the concentration of glutamate used, the rate reached its top as the effective glutamate was about 1mmol/L. While the concentration being 30mmol/L, the [Ca²⁺]i rose rapidly and extremely and the fluorescence rate occupied the highest value, which suggested that the cell was dead.(2) The increase of the [Ca²⁺]i induced by glutamate was inhibited obviously by the antagonists of NMDA receptor [100μmol/L D-2-amino-5-phosphonopentanoic acid (D-AP-5)] or AMPA receptor[50μmol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)], and the inhibitive effect being much more noted by the use of them both simultaneously. All of the negative effect above were incomplete. (3). The change of the [Ca²⁺]i induced by glutamate was attenuated significantly by the absence of extracellular calcium concentration, which could be reduced with the administration of D-AP-5 and CNQX, but the role of glutamate was not disappeared.Conclusion: The increase of the [Ca²⁺]i induced by glutamate is associated with both the effective time and the concentration of glutamate used in the primarily cultured neurons of rat hippocampus. Both NMDA and AMPA receptor play an important part in the Ca²⁺ transportation through the biological membrane of neuron. Glutamate can affect release of intracellular calcium stores by some mechanisms else. Part 3Change of astrocytse, synapse and motor skill after mobility training in rats with acute cerebral InfarctionObjective: To investigate the changes of astrocyte and synapse s in the region around the infarction area and of motor skill in the experimental rats with acute cerebral infarction (CI) after mobility training. Methods: In the sixteenth week since a total of 130 rat models with kidney hypertension being made, 120 ones of them were done into experimental CI models of right middle cerebral artery occlusion (MCAO) using intraluminal thread. The rats were randomly divided into 4 groups: rehabilitation, inhibitation, saline and control, 30 animals in each one. Each group included 3 subgroup with equal number: 1 , 3 and 6 weekend subgroup. The 10 rats failed in making into CI model was normal group. The 40 rats of inhibition group were administered continuous ara-c (25mg/1ml; 2ml/kg, a time each week) infusion with minipump to the intraventriculus lateralis after the fourth day the CI model made. The saline animals only received 1ml normal saline injections in the same way, and the rats of the rehabilitation and the control group given no drug. The CI models of the rehabilitation, the inhibitation and the saline group were made mobility training, 30min a day, 5 times a week. The motor skill of the rats was examined via beam walking test, motor skill mark 1～7. With the means of part 1, The counts of astrocyte and synapse were got by way of electron microscope, and the glial fibrillary acidic protein (GFAP), synaptophysin and growth associated protein 43(GAP-43) observed with the help of immunohistochemistry staining in the region around the infarction at 7,21 and 42d after making the model. The staining intensity was determined with Q-500 microphotography management system. The optical intensity was received of the staining intensity minus back value, which was the staining intensity of uninjured corpus callosum and visual tract of the same sector, in order to avoid the incorrectness produced by no specific staining. The optical intensities of the three sections from each sample were determined, and therefore, their average value was obtained.Results: (1). The scores of motor skill in the rehabilitation group(3.2±0.3 at the third weekend and 5.8±0.9 at the sixth weekend) were more significantly than either in the control(1.8±0.5 at the third weekend and 2.6±0.8 at the sixth weekend) or in the inhibitation(1.6±0.9 at the third weekend and 2.1±0.7 at the sixth weekend) respectively(p<0.01). (2). The synapse number increased gradually with the growth of astrocyte by electron microscope. Both the astrocytes [5.8±0.3 at the third weekend and 8.5±0.8 (/8000 times visual field) at the sixth weekend] and the synapses [12.3±1.3 at the third weekend and 16.3±1.4 (/8000 times visual field) at the sixth weekend] grew in number also more in the rehabilitation group than either in the control or in the inhibitation one significantly (p<0.01). (3). By immunohistochemistry staining, it was found that the optical densities of GFAP and synaptophysin of the rehabilitation group enhanced simultaneously as time went on in the region around the infarction area, which were higher than the optical densities of either the control or the inhibitation group significantly (p<0.01). The optical density of GAP-43 staining rose at the first weekend and lowered to usual level at the third weekend. The differences of the optical density of GAP-43 staining were not significant among the rehabilitation, the saline, the control and the inhibitation group. (4). There were not statistical significance of the difference between the indexesabove of the saline and the rehabilitation group (p>0.05). Conclusions: Both the astrocyte and the synapse can be enhanced in number and vivid in the region around the CI area in the experimental rats, the moment the motor skill get improved notedly, after mobility training, in which the astrocyte may play an crucial part. The growth and regeneration of neurons do not occupy the leading role in the course of motor skill recovery. Part 4Mechanism for synapse change after mobility training in region around cerebral infarction area of rat.Objective: To investigate the effect of mobility training on the vivid and function of synapse in the region around the infarction area of rats with acute cerebral infarction.Methods: In the sixteenth week since a total of 150 rat models with kidney hypertension being made, they were done into experimental CI models of right middle cerebral artery occlusion (MCAO) using intraluminal thread. The rats were randomly divided into 5 groups equally: the mobility training, the saline , the inhibitation, the mevastatin and the control. Each group included 3 subgroup with equal number: 1 , 3 and 6 weekend subgroup. The 50 rats of inhibition group were administered continuous ara-c (25mg/1ml; 2ml/kg, a time each week) infusion with minipump to the intraventriculus lateralis after the fourth day the CI model made. The saline animals only received 1ml normal saline injections in the same way, and the rats of the rehabilitation and the control group given no drug. The CI models of the rehabilitation, the inhibitation and the saline group were made mobility training, 30min a day, 5 times a week. The motor skill of the rats was examined via beam walking test, motor skill mark 1～7. With the means of part 1, the optical intensity of the glial fibrillary acidic protein (GFAP), synaptophysin and the cholesterol contents observed with the help of immunohistochemistry staining and the high performance liquid chromatography (HPLC) respectively in the region around the infarction at 7,21 and 42d after making the model. The staining intensity was determined with Q-500 microphotography management system. The optical intensity was received of the staining intensity minus back value, which was the staining intensity of uninjured corpus callosum and visual tract of the same sector, in order to avoid the incorrectness produced by no specific staining. The optical intensities of the three sections from each sample were determined, and therefore, their average value was obtained. Results: (1). In the rehabilitation group, the optical density(%) of either GFAP (7d:11.02±2.01, 21d: 19.54±3.12 , 42d: 28.47±2.32) or synaptophysin (7d: 34.29±1.39, 21d: 43.22±3.14, 42d: 61.42±2.32)and the cholesterol contents(g·kg^-1) (7d:4.50±0.24, 21d: 7.45±1.31, 42d: 14.52±1.45) enhanced as the time went on in the course of the mobility training, and there were statistical significance of the difference between the three time point of the three indexes (p<0.01). (2). In the control group, the optical density (%) of either GFAP (7d: 9.37±2.12, 21d: 14.57±1.42, 42d: 22.43±1.35) or synaptophysin (7d: 32.1±0.16, 21d: 36.64+0.01, 42d: 42.89±1.05) and the cholesterol contents (g.kg^-1) (7d: 3.62±0.05, 21d: 5.48±0.21, 42d: 8.76+1.20) also increased significantly as the time went on after acute cerebral infarction (p<0.01), but the values of optical density (%) and cholesterol contents (g.kg^-1) were significantly lower than these in the rehabilitation group (p<0.01). (3). In the inhibitation group, the optical density (%) of either GFAP (7d:7.35±1.27, 21d:10.23±2.21, 42d:11.13±1.41), SPY(7d:30.06±1.24, 21d:31.34±1.22, 42d:29.94±2.31) and cholesterol content (7d 2.33±0.14, 21d 3.17±3.37,42d 4.21±3.25)(g.kg^-1) were significantly lower than these in the control and the rehabilitation group (p<0.01). (4). In the mevastatin group, the optical density (%) of synaptophysin (7d: 31.26+1.04, 21d: 31.21±1.12, 42: 30.22±1.96) and the cholesterol contents (g.kg^-1) (7d: 2.32±0.13, 21d: 3.20±3.40, 42: 4.18±2.85) were statistically lower than these in either the rehabilitation or the control group (p<0.01). But the differences of the GFAP optical density between the mevastatin and the rehabilitation group were not statistically significant in the three time points (p>0.05). (5). There were not statistical significances of the difference between the indexes above of the saline and the rehabilitation group (p>0.05).Conclusions: The vivid of synapse can be produced by mobility training in the region around the infarction area of the experimental rats with acute cerebral infarction, on which astrocyte may play an crucial role by means of the secretion of cholesterol.