| Epidemiological data show that there are about 65 million patients with epilepsy in the world,and up to a quarter of epilepsy patients have cognitive dysfunction.And there are about more than 6 million patients with epilepsy in China,of which 30% to 40% of epilepsy patients have cognitive dysfunction.Cognitive dysfunction in epilepsy patients is the result of a combination of various factors,including epilepsy itself(such as age of onset,duration,frequency of seizures,seizure types,epileptic foci),antiepileptic drugs,psychosocial factors,etc.Because most epilepsy patients have to take long-term oral antiepileptic drugs,the impact of antiepileptic drugs on cognitive function has been a concerned problem of epilepsy patients and clinicians.It is reported that most of the traditional antiepileptic drugs have relation to the cognitive decline of epilepsy patients.However,there are studies show that compared with other traditional antiepileptic drugs,sodium valproate(VPA)has less side effects on cognitive function.Some studies show that VPA may improve epilepsy-induced learning and memory dysfunction in animal models.The effect of VPA on cognitive function is related to the dose,and high dose of VPA may have adverse effect on cognition.Animal studies have shown that daily 250-300mg/kg of VPA could control seizures well without memory impairment.Lamotrigine(LTG)is a relatively newly broad-spectrum antiepileptic drug.It is reported that LTG has similar anti-epilepsy effect to VPA and also has less side effects on cognitive function.However,few studies have been conducted to validate the dose dependency of the effect of LTG on cognitive impairment caused by chronic epilepsy.It is generally believed that the risk of cognitive impairment would increase as the dose of antiepileptic drugs increased.We observed the anti-epileptic effect of different doses of LTG in the treatment of pentylenetetrazole(PTZ)-kindled rats.In order to determine whether LTG has relation to cognitive impairment in the treatment of epilepsy,we took daily 300mg/kg VPA treatment group as a positive control and compared the effect of different doses of LTG on the performance of PTZ-kindled rats in the water maze test,and further compared the neuronal apoptosis of hippocampal CA1 area and CA3 area of different treatment groups by TUNEL staining.In our study,we observed the effect of high dose of LTG on learning and memory function and hippocampal neuronal apoptosis during the treatment of PTZ-kindled rats and discussed the effects of lamotrigine on cognitive function of during epilepsy treatment.In order to further study the the signal transduction pathways leading to apoptosis of hippocampal neurons induced by epilepsy,we observed the expression and activation of STAT3,which is one member of proteins involved in JAK-STAT pathway.In our study,We compared the changes of STAT3 protein in hippocampus after treatment with lamotrigine and sodium valproate and discussed the implication of STAT3 expression and activation in hippocampus after kindling.Part one Antiepileptic effect of different doses of LTG on PTZ-kindled ratsObjective: To establish the PTZ-kindled rat model and observe the effect of low,middle and high doses of LTG on the seizure level of PTZ-kindled rats,evaluate the antiepileptic effect of different doses of LTG.Methods: Female Sprague-Dawley(SD)rats(8-12 weeks old)were treated with 35 mg/kg of PTZ by intraperitoneal injection on alternate days,for a total of 13 injections for the kindling,to establish the PTZ-kindled rat model.And 0.9% sodium chloride was injected in the same way at a dose of 3.5 mL/kg in the sham group.After each PTZ injection,Seizure stage was evaluated using Racine's scale for half an hour after each PTZ injection.Racine's scale is including 5 stages: stage 0: no response;stage 1: wet dog-like jitter,facial twitching such as blink,vibrissae moving or rhythmic chewing;stage 2: head nodding;stage 3: unilateral forelimb clonus;stage 4: rearing with bilateral forelimb clonus;5.generalized tonic-clonic seizure(GTCS)with loss of posture control and falling.The kindling was considered to be successful when the animal's appearance reached stage 3 after two consecutive administrations.At 48 hours after completion of the induction of kindling,the successfully kindled animals were randomly divided into five groups,and were intraperitoneally treated with LTG or VPA for two weeks following the protocol: control group,treated with 3.5 mL/kg of 0.9% sodium chloride daily;low-dose LTG group,treated with 12.5 mg/kg of LTG daily;middle-dose LTG group,treated with 25 mg/kg of LTG daily;high-dose LTG group,treated with 50 mg/kg of LTG daily;VPA group,treated with 300 mg/kg of VPA daily.Meanwhile,PTZ injection(35 mg/kg,i.p.)was continued on alternate days to keep the kindled station.Animals in the sham group were treated with 3.5 ml/kg of 0.9% sodium chloride in the same way,as described above.Results: There were no significant differences in average seizure stage among CON group and LTG and VPA treated groups before treatment(F[4,541]=0.623,P=0.646),while there were significant differences in average seizure stage among these groups after treatment(F[4,289]=29.996,P=0.000).The average seizure stage of rats significantly decreased in the LTG and VPA treated groups,compared with rats treated with vehicle in the control group(P<0.05).Treatment with middle-dose and high-dose LTG significantly reduced the seizure stage of rats,compared with rats in the low-dose LTG treated group(P<0.05).Conclusion: LTG significantly reduced the seizure stage of PTZ-kindled rats.Middle-and high-dose LTG had better antiepileptic effect compared with low-dose LTG in the treatment of PTZ-kindled rats.The antiepileptic effect of high-dose LTG on PTZ-kindled rats was not significant different from that of middle-dose LTG.Part two Effects of different doses of LTG on cognitive function during the treatment of PTZ-kindled ratsObjective: To compare the differences in escape latency and spatial probe frequency of PTZ-kindled rats treated with different doses of LTG,and to analyze the effect of LTG on cognitive impairment during antiepileptic treatment.Methods: Female Sprague-Dawley(SD)rats(8-12 weeks old)were treated with 35 mg/kg of PTZ by intraperitoneal injection on alternate days,for a total of 13 injections for the kindling,to establish the PTZ-kindled rat model.And 0.9% sodium chloride was injected in the same way at a dose of 3.5 mL/kg in the sham group.After each PTZ injection,Seizure stage was evaluated using Racine's scale for half an hour after each PTZ injection.The kindling was considered to be successful when the animal's appearance reached stage 3 after two consecutive administrations.At 48 hours after completion of the induction of kindling,the successfully kindled animals were randomly divided into five groups,and were intraperitoneally treated with LTG or VPA for two weeks following the protocol: control group,treated with 3.5 mL/kg of 0.9% sodium chloride daily;low-dose LTG group,treated with 12.5 mg/kg of LTG daily;middle-dose LTG group,treated with 25 mg/kg of LTG daily;high-dose LTG group,treated with 50 mg/kg of LTG daily;VPA group,treated with 300 mg/kg of VPA daily.Meanwhile,PTZ injection(35 mg/kg,i.p.)was continued on alternate days to keep the kindled station.Animals in the sham group were treated with 3.5 ml/kg of 0.9% sodium chloride in the same way,as described above.The Morris Water Maze(MWM)test started from the 10 th day of treatment with LTG or VPA.Each rat completed five trials over five days,with four entries to the tank(randomized each day)per trial.For the first four days,rats were gently placed in the middle of the circulate edge in a randomly selected quadrant,with the nose toward the wall.After finding the platform,rats were allowed a thirty second rest period before starting the next entry.If rats failed to find the platform within 120 seconds,they were picked up and placed onto the platform to allow for a 30-second rest period;and their escape latency was recorded as 120 seconds.On the 5th day,a spatial probe test without a platform was assessed,and the number of times the rat went across the place where the platform had previously been located within 120 seconds were recorded.Results: No significant difference in escape latency was found on day one(F[5,46]=0.974,P=0.444)and day two(F[5,46]=1.021,P=0.417),while a significant difference in escape latency was observed on day three(F[5,46]=2.988,P=0.020)and day four(F[5,46]=2.942,P=0.022).Escape latency was significantly reduced in the sham group,middle-and high-dose LTG and VPA treated groups on the 3rd and 4th day of the MWM test,when compared with rats treated with vehicle in the control group(P<0.05);while no significant difference was observed between low-dose LTG treated group and control group(P>0.05).Significant changes in spatial probe was observed in different groups on day five of the MWM test(F[5,46]=4.721,P=0.001).Spatial probe frequency was significantly improved in the sham group,middle-and high-dose LTG and VPA treated groups,when compared with rats treated with vehicle in the control group(P<0.05).However,treatment with low-dose LTG did not significantly improve spatial probe frequency,when compared with that in the control group(P>0.05)Conclusion: The learning and memory functions of PTZ-kindled rats were impaired.Middle-and high dose LTG may improve the learning and memory functions during the treatment of PTZ-kindled rats.On the basis of the maximum antiepileptic effect,the increase of LTG dose from daily 25mg/kd to 50mg/kg did not impair the learning and memory functions.Part three Effect of different doses of LTG on neuronal apoptosis in hippocampal CA1 and CA3 regions of PTZ-kindled ratsObjective: To compare the differences of TUNEL staining of hippocampal CA1 and CA3 regions of PTZ-kindled rats treated with different doses of LTG,and to analyze the effect of LTG on neuronal apoptosis in hippocampal CA1 and CA3 regions of PTZ-kindled rats.Methods: Female Sprague-Dawley(SD)rats(8-12 weeks old)were treated with 35 mg/kg of PTZ by intraperitoneal injection on alternate days,for a total of 13 injections for the kindling,to establish the PTZ-kindled rat model.And 0.9% sodium chloride was injected in the same way at a dose of 3.5 mL/kg in the sham group.After each PTZ injection,Seizure stage was evaluated using Racine's scale for half an hour after each PTZ injection.The kindling was considered to be successful when the animal's appearance reached stage 3 after two consecutive administrations.At 48 hours after completion of the induction of kindling,the successfully kindled animals were randomly divided into six groups,and were intraperitoneally treated with LTG or VPA for two weeks following the protocol: control group,treated with 3.5 mL/kg of 0.9% sodium chloride daily;low-dose LTG group,treated with 12.5 mg/kg of LTG daily;middle-dose LTG group,treated with 25 mg/kg of LTG daily;high-dose LTG group,treated with 50 mg/kg of LTG daily;VPA group,treated with 300 mg/kg of VPA daily.Meanwhile,PTZ injection(35 mg/kg,i.p.)was continued on alternate days to keep the kindled station.Animals in the sham group were treated with 3.5 ml/kg of 0.9% sodium chloride in the same way,as described above.Brain tissues were harvested under deep anesthesia after two weeks of LTG or VPA treatment,and were kept in 4% paraformaldehyde overnight.The brain tissues were paraffin-embedded and sliced into 4-?m consecutive sections.The sections in the series throughout the entire hippocampus were used for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)staining.TUNEL staining was carried out using an In situ Cell Death Detection Kit,according to manufacturer's protocol.Briefly,after dewaxing and dehydration,the hippocampus tissue sections were incubated with proteinase K(20?g/ml),and washed with phosphate buffer saline.Then,the sections were incubated in the TUNEL reaction mixture,and visualized using a converter with a DAB substrate kit.The sections were counter stained with hematoxylin,mounted onto slides,and covered with coverslips.For TUNEL staining anaysis,5feilds under the light microscope were chosen and the percent of TUNEL-positive cells within the 5 fields was calculated as apoptosis rate.Results: A significant difference was observed in apoptosis rates in the hippocampal CA1 region in different treatments groups(F[5,19]=4.386,P=0.008),but not in the CA3 region(F[5,19]=2.326,P=0.083).The apoptosis rates were significantly high in the hippocampal CA1 and CA3 regions in the control group,when compared with the sham group(P<0.05).Treatment with middle-and high-dose LTG and VPA significantly decreased the apoptosis rate of the hippocampal CA1 region,when compared with rats treated with vehicle in the control group(P<0.05).However,treatment with LTG and VPA did not change the apoptosis rate of the hippocampal CA3 region,as compared to rats treated with vehicle in the control group(P>0.05).Conclusion: There were significant neuronal apoptosis in hippocampal CA1 and CA3 regions of PTZ-kindled rats.Middle-and high-dose LTG may reduce the neuronal apoptosis in the hippocampal CA1 region during the treatment of PTZ-kindled rats,but may not reduce the neuronal apoptosis in the CA3 region.On the basis of the maximal antiepileptic effect,the increase of LTG dose did not aggravate neuronal apoptosis in the hippocampal CA1 region.Part four Effects of Lamotrigine on the Expression of STAT3 in Hippocampus of PTZ-kindled ratsObjective: To compare the STAT3 and P-STAT3 protein levels in hippocampal tissues in each group,and to observe the effect of PTZ kindling and different treatment on the expression of STAT3 protein in hippocampus.Methods: Female Sprague-Dawley(SD)rats(8-12 weeks old)were treated with 35 mg/kg of PTZ by intraperitoneal injection on alternate days,for a total of 13 injections for the kindling,to establish the PTZ-kindled rat model.And 0.9% sodium chloride was injected in the same way at a dose of 3.5 mL/kg in the sham group.After each PTZ injection,Seizure stage was evaluated using Racine's scale for half an hour after each PTZ injection.The kindling was considered to be successful when the animal's appearance reached stage 3 after two consecutive administrations.At 48 hours after completion of the induction of kindling,the successfully kindled animals were randomly divided into six groups,and were intraperitoneally treated with LTG or VPA for two weeks following the protocol: control group,treated with 3.5 mL/kg of 0.9% sodium chloride daily;low-dose LTG group,treated with 12.5 mg/kg of LTG daily;middle-dose LTG group,treated with 25 mg/kg of LTG daily;high-dose LTG group,treated with 50 mg/kg of LTG daily;VPA group,treated with 300 mg/kg of VPA daily.Meanwhile,PTZ injection(35 mg/kg,i.p.)was continued on alternate days to keep the kindled station.Animals in the sham group were treated with 3.5 ml/kg of 0.9% sodium chloride in the same way,as described above.The rats were decapitated under deep anesthesia and the hippocampus was quickly isolated after two weeks of LTG or VPA treatment.The isolated hippocampus tissue was homogenized in a RIPA lysate containing protease inhibitors,and protein concentrations were measured using the BCA protein concentration assay kit.Protein samples(50 ?g)were boiled under 95? for 10 minutes in 5×loading buffer before loading onto a SDS-PAGE gel.Gels were transferred on to PVDF membranes.Membranes were blocked in 5% milk for 1 h.Membranes were incubated with the blocking solution containing the antibody at 1:1000 dilution overnight at 4°C.After washing,membranes were incubated for 1h at room temperature with secondary antibodies diluted 1:5000 in corresponding blocking buffer.Immunoreactive bands were visualized using ECL chemiluminescence.Band intensity was recorded and analyzed.Results: A significant difference was observed in STAT3 protein levels of the hippocampus in different treatment groups(F[5,18]=35.647,P=0.000).And a significant difference was observed in P-STAT3 protein levels of the hippocampus in different treatment groups(F[5,18]=50.737,P=0.000).STAT3 and P-STAT3 levels in the hippocampus of the control group(CON group)were significantly higher than those in the Sham group(P<0.05).STAT3 and P-STAT3 levels in the hippocampus of lamotrigine treated groups and valproate treated group were significantly higher than those in Sham group(P<0.05).STAT3 and P-STAT3 protein levels were significantly decreased in the hippocampus of middle-dose lamotrigine treatment group(M-LTG group),high-dose lamotrigine treatment group(H-LTG group)and sodium valproate treatment group(VPA group)compared with the control group(CON group)(P<0.05).The levels of STAT3 and P-STAT3 protein in hippocampus from low-dose lamotrigine group(L-LTG group)were not significantly different from those in control group(CON group)(P>0.05).Conclusion: STAT3 and P-STAT3 protein levels in the hippocampus of PTZ-kindled rats were significantly increased.Middle-dose lamotrigine(25 mg/kg daily)and high-dose lamotrigine(50 mg/kg daily)treatmens made hippocampal STAT3 and P-STAT3 protein levels significantly decreased.Low-dose lamotrigine(12.5mg/kg daily)had no significant effect on STAT3 and P-STAT3 protein levels in the hippocampus of PTZ-kindled rats. |
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