| Objective Through exaction, separation and purification to get the low molecular weight Angelica sinensis polysaccharide (LMW-ASP). A classical middle cerebral artery occlusion (MCAO) reperfusion rat model was established to study the protective effects of LMW-ASP/tetramethylpyrazine (TMP) on cerebral ischemia reperfusion injury. To explore the effects of LMW-ASP/TMP on neural plasticity through measuring neurological function, the expression of some protein associated with plasticity, structure of dendrites and ultrastructure of synapses, which provide a theoretical basis of the treatment of LMW-ASP/TMP to ischemic cerebral injury.Methods (1) The root of Angelica sinensis was cut up and fully dried, then Angelica sinensis polysaccharide was exacted by water boiled-alcohol precipitation method after preconditioning and deproteinization was conducted using Sevage method. Finally, LMW-ASP (MW<5kDa) was separated through an ultrafiltration membrane.(2) Totally 170 adult healthy male rats were employed in this study.120 of them were randomly divided into five groups for immunohistochemical labeling:sham operation group (S), model group (M), LMW-ASP treated group (A), TMP treated group (T) and LMW-ASP+TMP treated group (AT). In each group, rats were randomly allocated into four subgroup of 3d,7d,14d and 28d with 6 rats per subgroup. The rest 50 rats were randomly divided into aforementioned five groups with 10 rats per group (6 for Golgi staining and 4 for detection of transmission electron microscope (TEM)). The classical MCAO reperfusion models were established in rats of M?A?T and AT groups, while the rats in S group were operated in the same way with M group but without inserting the intraluminal suture. LMW-ASP, TMP and LMW-ASP+TMP were injected to rats in A?T?AT groups intraperitoneally and 0.9% NaCl was injected to rats of S and M groups every day until the day before the rats were sacrificed. The neurological function test was evaluated using the modified neurological severity score (mNSS) at 3d,7d,14d and 28d after MCAO reperfusion. Animals were euthanized for immunohistochemical labeling to measure SYP, GAP-43 and MAP-2 levels in the cortex of peri-infarct area at 3d,7d,14d and 28d after MCAO reperfusion. Golgi staining was performed to observe changes of dendritic morphology in the forelimb region of the motor cortex in peri-infarct area at 14d after MCAO reperfusion. Finally, the synaptic ultrastructure in the cortex of peri-infarct area was investigated using TEM at 28d after MCAO reperfusion.Results (1) We get LMW-ASP successfully with a yield of 0.92%.(2) The rats in M group and treatment groups showed functional impairment after ischemic injury, which improved with time going on. Compared to M group, the mNSS score of rats in T and AT groups decreased significantly (P=0.041, P=0.012) at 14d after MCAO reperfusion without significant difference between the two groups. At 28 after MCAO reperfusion, the mNSS score of rats in A, T and AT groups decreased significantly compared to M group (P=0.034, P=0.004, P=0.002) without significant difference between the three groups.(3) The expression of SYP in the cortex of peri-infarct area of rats in M group was significantly higher than that of S group at 7d,14d and 28d (P<0.001, P<0.001, P<0.001) after MCAO reperfusion and gradually increased from 3d to 28d. At 3d after MCAO reperfusion, the expression of SYP in the cortex of peri-infarct area of rats in AT group was significantly higher than M group (P<0.001). At 7d,14d and 28d, compared with M group, the expression of SYP in the cortex of peri-infarct area of rats in A, T and AT groups was significantly higher than M group (7d:P<0.001, P<0.001, P<0.001; 14d:P=0.001, P=0.008, P<0.001; 28d:P=0.005, P=0.017, P<0001). Especially at 28d after MCAO reperfusion, the level of SYP in AT group was higher than A (P=0.009) group and T group (P=0.002).(4) Compared with S group, the expression of GAP-43 in the cortex of peri-infarct area of rats in M group increased at 3d after MCAO reperfusion (P<0.001), reached the peak at 7d, declined in the later days, but it was still significantly higher than S group at 28d after MCAO reperfusion (P<0.001). At 3d,7d, 14d and 28d after MCAO reperfusion, the expression of GAP-43 in the cortex of peri-infarct area of rats in A and AT groups remarkably increased compared to M group (3d:P<0.001, P<0.001; 7d:P=0.002, P<0.001; 14d:P=0.024, P<0.001; 28d: P=0.029, P<0.001). Especially at 7d after MCAO reperfusion, the expression of GAP-43 in AT group was higher than A group (P=0.03).(5) The expression of MAP-2 in the cortex of peri-infarct area of rats in M group decreased significantly at 3d (PO.001) after MCAO reperfusion and then increased gradually from 3d to 28d but still lower than S group at 28d after MCAO reperfusion (P<0.001). At 7d after MCAO reperfusion, the expression of MAP-2 in the cortex of peri-infarct area of rats in T and AT groups significantly increased compared with M group (P<0.001, P<0.001). At 14d and 28d after MCAO reperfusion, the expression of MAP-2 in the cortex of peri-infarct area of rats in A, T and AT groups significantly increased compared with M group (14d:P<0.001, P<0.001, P<0.001,28d:P<0.001, P<0.001, P<0.001). Especially at 28d after MCAO reperfusion, the expression of MAP-2 in AT group was higher than A group (P=0.013).(6) At 14d after MCAO reperfusion, there was no significant difference between the five groups about total dendritic length of layer V pyramidal cells in the forelimb region of the motor cortex in peri-infarct area (P=0.2). The basilar dendritic spine density in rats of M group was lower than that of S group (P=0.005), while the basilar dendritic spine density in rats of T and AT groups increased obviously compared to M group (P=0.049, P<0.001) without significant difference between the two groups. The apical dendritic spine density in rats of M group was lower than that of S group (P=0.032), while there was no significant difference among other groups.(7) At 28d after MCAO reperfusion, Compared with the S group, the synaptic ultrastructural parameters in the cortex of peri-infarct area in the rats of M group were partially changed with a less curved synaptic interface (P=0.013), a thinner PSD (P<0.001) and a wider synaptic cleft (P=0.011). Synaptic interface curvature in the rats of T and AT groups significantly increased compared with M group (P=0.009, P=0.001) without significant difference between the two groups. PSD thickness in the rats of AT group significantly increased compared with M group (P=0.002). Conclusions (1) Angelica sinensis polysaccharide could be successfully extracted by the method of water boiled-alcohol precipitation. Moreover, we could get the LMW-ASP (MW<5kDa) through Sevage method and ultrafiltration technology.(2) The neurological function of rats after cerebral ischemia reperfusion injury could recover spontaneously and LMW-ASP, TMP and LMW-ASP+TMP could enhance this recovery process.(3) The expression of SYP, GAP-43 and MAP-2 in the cortex of peri-infarct area in rats after cerebral ischemia reperfusion injury changed constantly with time going on, indicating that cerebral ischemia reperfusion injury induced modification of neural plasticity. LMW-ASP, TMP and LMW-ASP+TMP could improve plasticity of axons, dendrites and synapses by increasing the expression of SYP, GAP-43 and MAP-2 in the cortex of peri-infarct area and LMW-ASP+TMP may obtain better effects.(4) The basilar and apical dendritic spine density of layer V pyramidal cells in the forelimb region of the motor cortex in peri-infarct area decreased at 14d after cerebral ischemia reperfusion injury, indicating that ischemia could induce a down-regulation of dendritic spine density. TMP and LMW-ASP+TMP could modify dendritic plasticity through increasing the basilar dendritic spine density.(5) The synaptic ultrastructure in the cortex of peri-infarct area changed obviously at 28 after ischemia reperfusion injury, including a less curved synaptic interface, a thinner PSD and a wider synaptic cleft, indicating that ischemia could induce changes of synaptic ultrastructure. TMP and LMW-ASP+TMP could increase the curvature of synaptic interface and PSD thickness, indicating that TMP and LMW-ASP+TMP could enhance synaptic transmission efficacy by regulating the synaptic structural plasticity. |
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