The Experimental Exploration Of The Mechanism Of Damaged Cognition Of Epileptic Rats And The Intervening Effects Of Nimodipine

Epilepsy, which is essentially an abnormality of cerebral function resulting from the discharges of some cerebral neuron in over high excitement state, has been a common chronic clinical syndrome. Besides the common symptoms of epilepsy, about 30%~40% of the epileptic patients have been found to have deleterious cognitive consequences, which may affect the patients'life greatly, such as the declination of memory, the scattering of attention, etc. Despite the fact that the relationship between epilepsy and the cognitive deficiency has drawn much attention and become one of the focuses of modern epilepsy research, the mechanics of damaged cognition after epilepsy is not known yet.Synapse has been well recognized as the key of transmitting neural information for its being touch points among neurons. It is therefore that whether synapse is intact or not determines the quality of information transmitting, processing and storage. The synaptic plasticity refers to the ability of synapse to change shape and adjust functions under some certain conditions, which plays a crucial role in the whole developing process of neural system, study ability and memory. The synaptic plasticity, which is closely related with long-term potentiation (LTP) of hippocampus, is measured by the changes of the number of synaptic vesicles density (SVD), the width of synaptic cleft (WSC) and the postsynaptic density (PSD).The density and distribution of synaptophysin (SYN, P38), a typical proteinous marker of synaptic vesicles, is not only an indirect reflection of the quantity and distribution of synapse but also closely related to the reconstruction of synapse and cognitive process. Postsynaptic density 95(PSD-95) is an essential factor of inducing LTP for its role in clustering N-methy1-D-aspartate (NMDA). As the co-functioning results of the presynaptic and postsynaptic mechanics, the formation of LTP requires the increase of both the release of presynaptic transmitter and the efficiency of postsynaptic receptor. P38 reflects the release of presynaptic transmitter and PSD-95 reflects the efficiency of postsynaptic receptor.Calcium, an important messager of neural information, plays an important part in learning ability and memory. Any change of the intracellular concentration of Calcium ([Ca2+]i) will lead to a series of biological and even pathological changes. Calcium/calmodulin-dependent protein kinase IIα(CaMK IIα) is the most protein inside hippocampus and a main element of PSD simultaneously. There is a close relationship between autophosphorylation of CaMK IIαand the maintenance of LTP within 3 hours. Moreover the maintenance of LTP more than 3 hours requires the expression of a series of genes and the synthesis of new proteins. And cAMP response element binding protein (CREB), the intracellular transcription factor, which mediates the genetic transcription and the proteinous synthesis, plays a key role in the transition from short term memory to long term memory.Epilepsy, one of the high frequency stimulations (HFS), will cause the release of glutamic acid (Glu) and the inward flow of Calcium ion, which will induce the poisonous effect of excitatory amino-acid (EAA) and the overloading of Ca2+. And we have not found the truths, which may help find some new breakthroughs in the research on the mechanics of damaged cognition after epilepsy, such as whether the HPS will possibly lead to the declination of learning ability and memory, how the Calcium message transmits along Ca2+-CaMK II-CREB, how P38 in charge of the Glu release and PSD-95 in charge of clustering the Glu receptor change, and what the change of synaptic ultrastructure is, etc.Nimodipine (NMD) is an inhibitor of L-type voltage-dependent Ca2+ channels (VDCCs). Recent researches have showed that NMD could help improve the impairments of learning ability and memory resulted from various diseases. Still not published is the experimental research on NMD's effect on damaged learning ability and memory after epilepsy. Based on the above mentioned facts, the dissertation intends to explore the possible mechanics of the damaged cognition after epilepsy through the research of synaptic plasticity and Calcium message transcription and observe the positive effects of NMD upon epilepsy and damaged cognition after epilepsy.Part I Establishment of chronic epileptic rat model, evaluation of behavioral features and the effect of NMDObjective: To establish a chronic epileptic rat model kindled by PTZ and observe the changes of learning ability and memory of epileptic rats and the effect of NMD.Methods: Adult male Sprague–Dawley (SD) rats were selected through Y-Maze tests and then divided into 4 groups randomly. The 35 rats in the normal control group (NC) were injected saline intraperitoneally (i.p) by 3.5 ml/kg for consecutive 44 days. The 40 rats in PTZ group received intraperitoneal injection of 1% pentylenetetrazol (PTZ) by 35 mg/kg for consecutive 44 days. From the 31st day on, each rat was injected saline intraperitoneally by 3.5 ml/kg 15 minutes before PTZ in the rest 14 days. Rats in NMD1 group and in NMD2 group (40 rats each group) were injected intraperitoneally 1% PTZ by 35mg/kg for consecutive 44 days. From the 31st day on, each rat was injected NMD intraperitoneally by 1.25 mg/kg and by 2.5 mg/kg respectively 15 minutes before PTZ in the rest 14 days. Then those rats were recorded with electroencephalogram (EEG) and tested with Morris Water Maze (MWM) and Y-Morris for the measurement of the rats'learning ability and memory.Results: Beginning from day 6 on, rats in epileptic and NMD groups had seizures induced by repetitive PTZ, which were characterized by head shaking, squealing and crawling. Moreover, wild running, loss of righting reflex and generalized tonic–clonic convulsions were seen on day 18~day 24, which indicated reaching the kindling standard. On day 30, the numbers of the rats being kindled in PTZ group, NMD1 group and NMD2 group were 35, 34, and 35 respectively. The Racine's stage greatly reduced after NMD injection in the NMD groups. No seizures were found in the normal group. EEG changes: The EEG records of the rats in NC group were dominant withαwaves andβwaves and the background was normal without any discharges. The rats in PTZ group were recorded in EEG as sharps, spike-slow waves and most polyspike discharge, with high potential on a relatively normal background. The EEG records of the rats in NMD1 group and NMD2 group were demonstrated the repressed discharges. Place navigation results in MWM test: Comparisons were made among the mean escape latencies on each day. On day 1, comparisons of the four groups were of no difference in significance (P>0.05). On day 2, the mean escape latency of rats in PTZ group (27.36±6.13) s was much longer than that in NC group (15.14±4.69) s (P<0.05). Moreover, the mean escape latencies of the rats in both NMD1 group (21.66±5.21) s and NMD2 group (19.76±5.81) s were shorter than that of the rats in PTZ group (P<0.05), but much longer than that in NC group (P<0.05). The comparison of mean escape latency in NMD1 group and NMD2 group had no distinct difference (P>0.05). On day 3, the mean escape latency of the rats in PTZ group (7.71±1.30) s was obvious different from that in NC group (4.46±1.11) s (P<0.01). The mean escape latencies of the rats in both NMD1 group (5.94±1.19) s and NMD2 group (5.69±1.03) s were shorter than that in PTZ group (P<0.05), but longer than that of those in NC group (P<0.05). The comparison of mean escape latencies of the rats in NMD1 group and NMD2 group had no statistic difference (P>0.05). Spatial probe test: The number of crossing times through the platform quadrant within 120 s in PTZ group (5.73±2.14) decreased obviously compared with that in NC group (9.25±2.18) ( P<0.01). And the numbers of crossing times through the platform quadrant within 120 s in both NMD1 group (8.58±2.12) and NMD2 group (7.51±2.05) increased significantly compared with that in PTZ group (P<0.01), but were of no statistic significance compared with that in NC group(P>0.05). And the comparison of the numbers in NMD1 group and NMD2 group was not statistically significant (P>0.05). There was no significant difference of the swimming time spent in the platform quadrant of the rats in four groups (P>0.05). Y-maze results: The error number (EN) of the rats in PTZ group (12.75±2.50) increased obviously compared with that in NC group (6.25±3.10) (P<0.05). The ENs in both NMD1 (8.75±2.22) and NMD2 group (8.00±3.16) significantly decreased compared with that in PTZ group (P<0.05), but was of no obvious difference from that in NC group (P>0.05). There was no obvious difference between NMD1 group and NMD2 group (P>0.05). Comparisons of the total reaction time (TRT) of the rats in four groups were of no significant difference (P>0.05).Conclusion: The epileptic model kindled by PTZ in the experiment did have impairments in learning ability and memory. This model is the perfect animal model which is feasible to study the damaged cognition after epilepsy. NMD could partly improve the damaged cognitive function of epileptic rats.Part II Changes of postsynaptic density 95(PSD-95) and synaptophysin (P38) expression levels in hippocampus of rats kindled by PTZ and the effect of NMDObjective: To observe the changes of P38 protein, PSD-95 protein and PSD-95 mRNA in hippocampus of rats kindled by PTZ and the effect of NMD and to explore the role of PSD-95 and P38 in damaged cognition after epilepsy.Methods: 8 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin. The changes of P38 and PSD-95 were tested with immunohistochemical SP methods and the absorbency of immunohistochemical products was also detected. Another 8 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total RNA was extracted strictly by Trizol. The expression of PSD-95 mRNA was measured by RT-PCR.Results: The absorbency of PSD-95 immunohistochemical products in the hippocampal CA1 area of the rats in PTZ group (0.6525±0.0648) decreased distinctly compared with that in NC group (0.7108±0.0584) (P<0.05). Compared with the absorbency of PSD-95 products in PTZ group, those in NMD1 group (0.6921±0.0620) and NMD2 group (0.7033±0.0659) increased obviously (P<0.05). There was no distinct difference among NMD1 group, NMD2 group and NC group (P>0.05). The absorbency of P38 immunohistochemical products in the hippocampal CA1 area of the rats in PTZ group (0.4475±0.0505) reduced distinctly compared with that in NC group (0.6033±0.0444) (P<0.05). Compared with the absorbency in PTZ group, those in NMD1 group (0.5819±0.0367) and NMD2 group (0.5723±0.0528) increased significantly (P<0.05). The difference of the absorbencies among NMD1 group, NMD2 group and NC group was not distinct (P>0.05). The expression levels of PSD-95 mRNA were determined by calculating the density ratio of PSD-95 mRNA/β-actin mRNA. The expression level in PTZ group (0.325±0.030) reduced obviously compared with that in NC group (0.607±0.051) (P<0.05). Compared with that in PTZ group, the PSD-95 mRNA expression levels in both NMD1 group (0.512±0.035) and NMD2 group (0.575±0.041) increased significantly (P<0.05). The expression levels in NMD1 group, NMD2 group and NC group were of no statistic significance (P>0.05).Conclusion: There were reductions in expression levels of P38, PSD-95 and PSD-95 mRNA in the hippocampus of the PTZ-kindled rats in accordance with the decline of the rats'learning ability and memory, which suggests that both P38 and PSD-95 might participate in the mechanism of damaged cognition after epilepsy. NMD could improve the expression levels of P38, PSD-95 and PSD-95 mRNA and the damaged cognitive function of epileptic rats.Part III Changes of neuronal and synaptic ultrastructure in hippocampus of rats kindled by PTZ and the effect of NMDObjective: To observe the changes of neuronal ultrastructure and synaptic ultrastructure (SVD, PSD and WSC) in hippocampal CA1 area of rats kindled by PTZ and the effect of NMD and to explore the relationship between synaptic plasticity and damaged cognition after epilepsy.Methods: 3 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution (containing 2.5% glutaraldehyde). The changes of neuronal ultrastructure and synaptic ultrastructure (SVD, PSD and WSC) in hippocampal CA1 area of rats in each group were observed through the electron microscopy.Results: In hippocampal CA1 area of the rats in NC group, there were intact neurons, distinct structure, uniform chromatin, abundant apparatus, and integrate myelin. In PTZ group, however, there were condensed nucleus, edematous neuron, swollen perikaryon with vacuole, reduced mitochondria and Golgi apparatus and polyribosome; there was also Myelin-splitting nerve fiber. In NMD1 and NMD2 group, the neuron and neuropile were less swollen, karyotheca were normally intact, chromatin were uniform, the apparatus were rich and basically normal, and the myelin was complete and integrate. In NC group, Gray I type synapses in hippocampal CA1 area were abundant with distinct pre-and-post synaptic membranes and rich synaptic vesicles. The synaptic cleft was clear and the PSD was thick and uniform. In PTZ group, there were reduced synapses with indistinct pre-and-post synaptic membranes, increased WSC, thinner PSD, and decreased SVD. In both NMD1 group and NMD2 group, there were more synapses than that in PTZ group. The synapses had relatively distinct pre-and-post synaptic membranes, and uniformed PSD, and more synaptic vesicles. The PSD in hippocampal CA1 area of the rats in PTZ group (31.37±1.94) nm reduced notably compared with that in NC group (49.86±3.38) nm (P<0.05). Compared with that in PTZ group, PSD in both NMD1 group (45.69±2.70) nm and NMD2 group (48.20±2.15) nm increased notably (P<0.05). Comparisons of PSD in NMD1, NMD2 group and NC group were of no significant difference (P>0.05). The WSC in PTZ group (22.34±1.84) nm also increased obviously compared with that in NC group (14.62±1.19) (P<0.05). Compared with that in PTZ group, the WSC in both NMD1 group (16.37±1.72) nm and NMD2 group (15.02±1.63) nm reduced significantly (P<0.05). Comparisons of WSC of NMD1 group, NMD2 group and NC group were of no distinct difference (P>0.05). Compared with that in NC group (3362.61±102.08)/um2, the SVD in PTZ group (1078.36±111.03)/um2 decreased significantly (P<0.05). Compared with that in PTZ group, the SVD in hippocampal CA1 area in both NMD1 group (2932.01±142.95)/um2 and NMD2 group (3155.50±137.49)/um2 increased significantly (P<0.05). Comparisons of SVD of NMD1 group, NMD2 group and NC group had no obvious difference (P>0.05).Conclusion: There were abnormal changes in neuronal and synaptic ultrastructure in hippocampal CA1 area of the epileptic rats. NMD could ameliorate the neuronal and synaptic abnormal ultrastructure, which was consistent with the changes of learning ability and memory. There might be a close relationship between synaptic plasticity and damaged cognition after epilepsy.Part IV Changes of [Ca2+]i and calcium/calmodulin-dependent protein kinase IIα(CaMK IIα) expression levels in hippocampus of rats kindled by PTZ and the effect of NMDObjective: To observe the changes of [Ca2+]i, CaMK IIα, P-CaMK IIαprotein and CaMK IIαmRNA in hippocampus of rats kindled by PTZ and the effect of NMD and to explore the relationship between calcium signal transduction and damaged cognition after epilepsy.Methods: 6 rats in each group were sacrificed and the hippocampi were quickly separated, homogenized and the suspension was collected and incubated with Fluo-3/AM. The changes of [Ca2+]i were tested with flow cytometry; The expressions of CaMK IIαmRNA of the hippocampus in each group were tested with RT-PCR; 8 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for CaMK IIαand membrane protein for P-CaMK IIαwas extracted. The expression levels of CaMK IIαand P-CaMK protein were detected by Western blot.Results: The [Ca2+]i in hippocampus of the rats in PTZ group (1.94±0.33) increased obviously compared with that in NC group (1.26±0.21) (P<0.05). Compared with that in PTZ group, the [Ca2+]i in hippocampus of the rats in both NMD1 group (1.35±0.28) and NMD2 group (1.32±0.26) reduced notably (P<0.05). The comparisons of [Ca2+]i in hippocampus among NMD1 group, NMD2 group and NC group were of no significant difference (P>0.05). The protein levels of CaMK IIαand P-CaMK IIαin hippocampus of rats in each group were determined by CaMK IIα/β-actin and P-CaMK IIα/β-actin respectively. The protein levels of CaMK IIαin hippocampus in PTZ group (0.700±0.061) reduced significantly compared with that in NC group (1.030±0.133) (P<0.05). Compared with that in PTZ group, the protein levels in hippocampus of the rats in both NMD1 group (1.000±0.130) and NMD2 group (1.000±0.130) increased significantly (P<0.05). The comparisons of the protein levels of CaMK IIαin hippocampus among NMD1 group, NMD2 group and NC group had no statistic difference (P>0.05). The protein levels of P-CaMK IIαin hippocampus in PTZ group (0.250±0.036) reduced significantly compared with that in NC group (0.693±0.035) (P<0.05). Compared with that in PTZ group, the protein levels in hippocampus of the rats in both NMD1 group (0.679±0.058) and NMD2 group (0.665±0.043) increased significantly (P<0.05). The comparisons of the protein levels of P-CaMK IIαin hippocampus among NMD1 group, NMD2 group and NC group were of no statistic difference (P>0.05). The expression levels of CaMK IIαmRNA in hippocampus of rats in each group were determined by calculating the density ratio of CaMK IIαmRNA/β-actin mRNA. The expression levels of CaMK IIαmRNA in hippocampus in PTZ group (0.758±0.050) reduced notably compared with that in NC group (1.217±0.074) (P<0.05). Compared with that in PTZ group, the CaMK IIαmRNA levels in hippocampus of the rats in both NMD1 group (1.149±0.083) and NMD2 group (1.300±0.071) increased notably (P<0.05). The comparisons of the CaMK IIαmRNA levels in hippocampus among NMD1 group, NMD2 group and NC group had no statistic difference (P>0.05).Conclusion: The [Ca2+]i in hippocampus of the epileptic rats increased and the expression levels of P-CaMK IIαprotein,CaMK IIαprotein and CaMK IIαmRNA in hippocampus reduced, which resulted in the imbalance of calcium signal transduction pathway. These might be one part of the molecule pathogenesis of damaged cognition after epilepsy. NMD could adjust [Ca2+]i and improve the expression levels of CaMK IIαprotein and mRNA in accordance with the improvement of learning ability and memory of the epileptic rats, which demonstrates NMD's pharmacological effects on improving cognitive functions.Part V Changes of expression levels of cAMP response element binding protein (CREB) in hippocampus of rats kindled by PTZ and the effect of NMDObjective: To observe the changes of P-CREB protein and CREB mRNA in hippocampus of rats kindled by PTZ and the effect of NMD and to explore the relationship between transcription factor CREB and damaged cognition after epilepsy.Methods: The changes of the expressions of P-CREB(Ser133) and CREB mRNA were tested with Western blot and RT-PCR methods, respectively.Results: The protein levels of P-CREB (Ser133) in hippocampus of rats in each group were determined by P-CREB/β-actin. The protein levels of P-CREB in hippocampus in PTZ group (0.275±0.041) decreased significantly compared with that in NC group (0.643±0.054) (P<0.05). Compared with that in PTZ group, the protein levels in hippocampus of the rats in both NMD1 group (0.602±0.045) and NMD2 group (0.621±0.071) increased obviously (P<0.05). The comparisons of the protein levels of P-CREB in hippocampus among NMD1 group, NMD2 group and NC group had no statistic difference (P>0.05). The expression levels of CREB mRNA in hippocampus of rats in each group were determined by calculating the density ratio of CREB mRNA/β-actin mRNA. The CREB mRNA levels in hippocampus in PTZ group (0.319±0.037) reduced notably compared with that in NC group (0.605±0.071) (P<0.05). Compared with that in PTZ group, the CREB mRNA levels in hippocampus of the rats in both NMD1 group (0.560±0.058) and NMD2 group (0.582±0.083) increased notably (P<0.05). The comparisons of the CREB mRNA levels in hippocampus among NMD1 group, NMD2 group and NC group had no statistic difference (P>0.05).Conclusion: The expression levels of both CREB protein and mRNA in hippocampus of the epileptic rats reduced and NMD could improve that, which was consistent with the changes of learning ability and memory. Transcription factor CREB might participate in the mechanism of damaged cognition after epilepsy.