Dissertation
Dissertation > Medicine, health > Neurology and psychiatry > Neurology > Brain diseases > Epilepsy

Protective Effects and Mechanisms of EGCG on Pentylenetetrazole-induced Seizures in Rats

Author XieTao
Tutor WangWeiPing
School Hebei Medical University
Course Neurology
Keywords PTZ epilepsy oxidative stress EGCG learning andmemory neuroprotective effect
CLC R742.1
Type PhD thesis
Year 2012
Downloads 200
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Epilepsy, one of the most common neurological disorders, affects1%ofthe world population. Among the50million epilepsy patients, about30%suffering with intractable epilepsy, most of which were temple lobe epilepsy.Many patients with epilepsy have been diagnosed with affective andpersonality disorders. Clinical investigations have shown that over half of theepileptic patients suffer from cognitive impairment. A variety of factory canadversely affect the cognition in patients with epilepsy, including the etiologyof the seizures, seizure type, and psychosocial problems. Another factor thatmay affect cognition is antiepileptic drugs (AEDs). Thus there is an urgentneed to find new drugs that can suppress seizures effectively and preventcognitive decline in the meantime.Oxidative stress, which has has been recognized to play an importantrole in pathophysiology of neurological diseases such as Amyotrophic LateralSclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD),aswell as in epilepsy pathophysiolocy. Reactive oxygen species have beenshown in the initial phases of seizure. When the free radical generationexceeds the antioxidant defence, oxidative damage of cellular maromoleculesincluding DNA, cell lipids, protein will be happen. Lots studies havedemonstrated oxidative damage in epileptic rat model.Temporal lobe epilepsy is most commonly associated with hippocampusneuron loss. This is a result of epilepsy but also can reorganize the networkand induce seizures recrudescence. Seizures induce a mixed pattern of celldeath that includes features consistent with both apoptosis and necrosis.Mitochondrion linked apoptosis like signaling pathways has been describedafter seizures. The Bcl-2family comprises proapoptotic and antiapoptoticproteins. The disorder of the ratio of Bcl-2to Bax will increase the mitochondrial membrane permeability and release cytochrome c frommitochondria, and then activate caspase-3to induce apoptosis.Researches have shown that antioxidative agent such as VitC, VitE couldeffectively suppress epilepsy in animal models. As a result, medicinal plantsrecently have given particular attention as a protective agent against epilepsyand oxidative stress. Centella asiatica, curcumin and Emblica officinalis havebeen shown could suppress epilepsy effectively and mitigate oxidative damage.Epigallocatechin-3-gallate (EGCG), the main polyhenol of green tea, has beencharacterized as having anti-oxidant, anti-inflammatory and anti-apoptoticproterties. EGCG anti-oxidant property may be stronger than VitC and Vit E16times and18times. EGCG could demonstrate the antioxidative propertythrough act on the free radical, iron-chelating and increasing the body’sendogenous antioxidants to reduce oxidative damage. In vivo, EGCG couldcross the blood-brain barrier, direct exerting neuroprotective effects. Recentresearches demonstrated that EGCG could attenuate cognitive impairmentinduced by lead, alcohol and diabetes mellitus. Furthermore, EGCG couldenhance the normal rats’ leanring ability. EGCG also could showneuroprotection effect on cerebral ischemia and spinal cord injury. In our study,we used PTZ kindled rat to evaluate the effects of EGCG on seizures,seizure-induced neuron loss and cognitive impairment and furether explore theunderling mechanisms.Part Ⅰ Effects of EGCG on pentylenetetrazole-induced kindling and theunderlying mechanismsObjective: To establish a chronic epileptic rat model kindled by PTZ andobserve the effects of EGCG on pentylenetetrazole-induced kindling and thechanges of oxidative stress parameter (malondialdenhyde and glutathione) inkindled rats, evaluate the antiepilepsy property of EGCG and further explorethe underlying mechanisms.Methods: Adult male Spraque-Dawley (SD) rats weighting180-220g,obstained from Hebei Medical University, were housed in groups of four tofive per cage in a room that was maintained at a constant temperature and humidity. Prior to the experiments, EGCG and PTZ were dissolved inphysiological saline. Then, PTZ was injected intraperitoneally on alternate dayin a dose of35mg/kg (13injections total), while EGCG was injectedintraperitoneally daily. The administration work was conducted between08:00-09:00AM. The animals were randomly divided into five groups of tenanimals each group. GroupⅠ(control group) received0.9%saline i.p. everyother day (3.5ml/kg,13injections total), GroupⅡ (PTZ group) receivedsaline pretreatment along with PTZ every other day, Group Ⅲ Ⅳ(PTZ+EGCG group) received EGCG pretreatment in doses of25and50mg/kg, respectively in addition to alternate-day PTZ for13injections. In thesegroups, EGCG was given30min before PTZ; GroupⅤ,(EGCG group)EGCG50mg/kg was administered alone. Animals were observed for30minafter each PTZ administration. The latency to myoclonic jerks and thegeneralized tonic clonic seizures (GTCS) as well as duration of GTCS wererecorded. Seizure stage was evaluated using the following scale stage0: noresponse; Stage1: hyperactivity, vibrissae twitching; Stage2: head nodding,head clonus and myoclonic jerk; Stage3: unilateral forelimb clonus; Stage4:rearing with bilateral forelimb clonus; Stage5: generalized tonic-clonicseizure (GTCS) with loss of postural control.24h after the last administion,the animals were sacrificed and the brains were removed and stored at-80℃to evaluate MDA and GSH levels.Results: In the PTZ group and the EGCG25mg/kg+PTZ group,mortality rate was30%and20%respectively. There was no mortality in thegroups administered50mg/kg EGCG group and control animals. The repeatedadministration of subconvulsive PTZ (35mg/kg) induced severe seizuresduring the13kindling injection. Pretreatment with EGCG dose dependentlydecreased the mean seizure stage as compared to the PTZ group (F (2,22)=25.073, p<0.001). In the control and EGCG group, there was no seizureactivity. The latency to myoclonic jerk (F (2,24)=13.07, p<0.001) and GTCS(F (2,20)=13.056, p<0.001) were dose-dependent increased in PTZ+EGCGgroup as compared to the PTZ group. The latency to myoclonic jerk increased from114.38±12.99s in PTZ group to142.20±20.59and164.51±22.88s inthe groups administered EGCG25mg/kg and50mg/kg respectively,meanwhile the latency to GTCS increased from184.55±52.51in the PTZgroup to243.38±33.30and307.76±49.60in the groups administered EGCG25mg/kg and50mg/kg respectively. Our research also showed that EGCGmarkedly decreased the duration of GTCS from21.82±5.85in the PTZ groupto16.67±2.35and14.29±1.38in the PTZ+EGCG25mg/kg and50mg/kgrespectively. The content of GSH in the PTZ group was significantly lowercompared with that of control group(GSH: control:6.89±0.17mg/g prot,n=10; PTZ:3.42±0.20mg/g prot n=7, p<0.05), and the level of MDA in PTZgroup was much higher than that of control group (MDA: control:1.99±0.13nmol/mg prot, n=10; PTZ:4.66±0.17nmol/mg prot, n=7p<0.05), whilepretreatment with EGCG led to a significant increase in GSH level in adose-dependent manner.(PTZ:3.42±0.20mg/g prot, n=7; PTZ+25mg/kgEGCG:4.79±0.11mg/g prot, n=8; PTZ+50mg/kg EGCG:5.36±0.30mg/gprot n=10). A noticeable decrease in the concentration of MDA was also notedwith the application of EGCG as compared to the PTZ group.(PTZ:4.66±0.17nmol/mg prot, n=7; PTZ+25mg/kg EGCG:3.23±0.12nmol/mg prot,n=8; PTZ+50mg/kg EGCG:2.57±0.27nmol/mg prot n=10). EGCG per secaused no change as compared to the control group (p>0.05).Conclusion: PTZ induced kindling provides a useful model of postseizuredysfunction. The present study showed that EGCG could effectively decreasethe mean seizure stage and the duration of GTCS, EGCG also could increasethe latency to myoclonic jerk and latency to GTCS. We also found that EGCGcould ameliorate the oxidative damage induced by seizures. Thus, EGCGcould successfully suppress PTZ induced kindling and this may be through itsanti-oxidative property.PartⅡ Effects of EGCG on pentylenetetrazole-induced cognitive impairmentand the underling mechanismsObjective: To observe the effects of EGCG on pentylenetetrazole inducedcognitive impairment and synaptic ultrasturcture changes, evaluate the anti-dementia property of EGCG and further explore the underlyingmechanisms.Methods: Adult male Spraque-Dawley (SD) rats weighting180-220g,obstained from Hebei Medical University, were housed in groups of four tofive per cage in a room that was maintained at a constant temperature andhumidity. Prior to the experiments, EGCG and PTZ were dissolved inphysiological saline. Then, PTZ was injected intraperitoneally on alternate dayin a dose of35mg/kg (13injections total). While EGCG was injectedintraperitoneally daily. The administration work was conducted between08:00-09:00AM. The animals were randomly divided into five groups of tenanimals each group. GroupⅠ(control group) received0.9%saline i.p. everyother day (3.5ml/kg,13injections total), GroupⅡ (PTZ group) receivedsaline pretreatment along with PTZ every other day, Group Ⅲ Ⅳ(PTZ+EGCG group) received EGCG pretreatment in doses of25and50mg/kg, respectively in addition to alternate-day PTZ for13injections. In thesegroups, EGCG was given30min before PTZ; GroupⅤ,(EGCG group)EGCG50mg/kg was administered alone.24h after the last administration,Morris water maze test was performed. Place navigation test was to test therats’ learning ability. Before the traning started, rats were allowed to swimfreely in the pool for120s without platform. Rats were given two sessions perday for5days. Each session comprised four trials, with an intertribal intervalof60s, and the intersession interval was>2h. In each trail, the rat was gentlyplaced into the pool at the middle of the circular edge in a randomly selectedquadrant, with the nose pointing toward the wall. If rats could not find escapeto the platform within120s by themselves, they were placed on the platformby hand and allowed to remain there for30s and their escape latency wasaccepted as120s. After climbing onto the platform, the animal remained therefor30s before the commencement of the next trial. Spatial probe test: on thesixth day, a probe trial without the platform was assessed, and the time spentin the target quadrant where the platform had been located was recorded.Visible platform trial: to exclude the sensorimotor or motivational factors in rats on learning performance, we added the visible trial on the last day. Ratswere given four trials per day similar to those described above for the hiddenplatform trial, but the escape platform was elevated above water surface2cm,the escape latency and the swimming speed were recorded. After the behaviortest, three rats from each group were anesthetized by10%chloral hydrate andperfused with4%paraformaldehyde solution (containing2.5%glutaraldehyde). The synaptic ultrastructure of the hippocampal CA1are wereobserved through the electron microscopy.Results: In the Morris water maze, all animals showed a progressivedecline in the escape latency with training, and main effects for day (F (4,160)=301.015, p<0.001) and for group (F (4,40)=21.10, p<0.001) were significant.Rats in PTZ group exhibited a significant prolonged of escape latency duringall sessions compared with control group (p<0.05). However, the poorperformance was mitigated by the pretreated with EGCG. These two groupsperformed equivalently to the control group (25mg/kg EGCG+PTZ vs CON,p=0.177;50mg/kg EGCG+PTZ vs CON, p=0.192). On the probe trial, withthe platform removed, there was a significant difference among groups (F (4,44)=6.071, p<0.05). The PTZ group spent significantly less time in the targetquadrant than the control group (p<0.05). In25mg/kg and50mg/kgpre-treatment EGCG groups, the deficits were significantly improved andshowed no difference from control group (25mg/kg EGCG+PTZ vs CON,p=0.065, EGCG+PTZ vs CON, p=0.489). For swimming speed, no significantdifferences were found among the five groups (F (4,38)=1.066, p>0.05). Inaddition, EGCG per se had no effect on cognition. There was no significantdifference in the visible plat form among the groups (p>0.05). The results ofthe width of synaptic cleft: there was a significant difference among thegroups (F(4,99)=19.903, p<0.001), kindled rats significantly enlarged thewidth of synaptic cleft compared to the normal group (P<0.05)(PTZ:26.79±1.92nm; CON:21.6045±2.29nm), while EGCG significantly reduced thesynaptic cleft compared to the PTZ group(p<0.05)(PTZ+25mg/kg EGCG:23.25±1.90nm; PTZ+50mg/kg EGCG:25.52±2.80nm), there was no statistic difference among PTZ+25mg/kg EGCG, PTZ+50mg/kgEGCG andcontrol group; The results of the length of active zone: there was a significantdifference among the group (F(4,99)=56.591, p<0.001), kindled ratssignificantly reduced the active zone compared to the controlgroup(p<0.05)(PTZ:257.17.58±17.58nm; CON:319.00±10.66nm), whileEGCG significantly increased the active zone compared to the PTZgroup(p<0.05)(PTZ+25mg/kg EGCG:308.94±17.24nm; PTZ+50mg/kgEGCG:313.06±15.06nm), there was no statistic difference amongPTZ+25mg/kg EGCG, PTZ+50mg/kgEGCG and control group. Results of thethickness of PSD: there was a significant difference among the groups(F(4,99)=21.150, p<0.001), kindled rats significantly reduced the thickness ofPSD compared to the control group(p<0.05)(PTZ:24.28±2.63nm; CON:32.14±3.32nm), while EGCG significantly increase the PSD thicknesscompared to the PTZ group (p<0.05)(PTZ+25mg/kg EGCG:29.99±3.93nm;PTZ+50mg/kg EGCG:31.14±3.32nm), there was no statistic differenceamong PTZ+25mg/kg EGCG, PTZ+50mg/kgEGCG and control group.Theresults of the synaptic curvature: there was no significant difference among thegroup (p>0.05).(PTZ:1.07±0.41; CON:1.11±0.52; PTZ±25mg/kg EGCG:1.09±0.79; PTZ+50mg/kg EGCG:1.1±0.50). Furthermore, there was nostatistic difference between the EGCG group and control group among thesynaptic parameter (p>0.05).Conclusion: PTZ kindled rat impaired spatial learning and memory withthe abnormal changes in synaptic ultrastructure in hippocampal CA1area.EGCG could ameliorate the cognitive impairment and protect the synapticultrastructure. There might be a close relationship between protective effectsof synaptic ultrastructure and anti-dementia property of EGCG. Furthermore,EGCG per se had no significant effects on cognition.PartⅢ Effects of EGCG on PTZ induced neuron loss and the underlyingmechanisms.Objective: To observe the effects of EGCG on hippocampal neuron lossinduced by PTZ and the effects of EGCG on the PI3K/Akt signaling pathway and the mitochondrial apoptosis-related protein, then evaluate theneuroprotective effects of EGCG and further explore the underlyingmechanisms.Methods: Adult male Spraque-Dawley (SD) rats weighting180-220g,obstained from Hebei Medical University, were housed in groups of four tofive per cage in a room that was maintained at a constant temperature andhumidity. Prior to the experiments, EGCG and PTZ were dissolved inphysiological saline. Then, PTZ was injected intraperitoneally on alternate dayin a dose of35mg/kg (13injections total), while EGCG was injectedintraperitoneally daily. The administration work was conducted between08:00-09:00AM. The animals were randomly divided into four groups of tenanimals each group. GroupⅠ(control group) received0.9%saline i.p. everyother day (3.5ml/kg,13injections total), GroupⅡ (PTZ group) receivedsaline pretreatment along with PTZ every other day, Group Ⅲ Ⅳ(PTZ+EGCG group) received EGCG pretreatment in doses of25and50mg/kg, respectively in addition to alternate-day PTZ for13injections. In thesegroups, EGCG was given30min before PTZ.24h after the last adiminstration,Nissl staining was performed to examine the number of surving neurons inhippocampal CA1and CA3regions in rats. The changes of phosphor-PI3Kp85, phosphor-Akt, Bax, Bcl-2and caspase-3in hippocampus were test bywestern blot.Results: Nissl staining showed that: neurons in CA1and CA3of controlgroup were clear with normal nucleolus, well-distributed karyotin and richnissl bodies in kytoplasm, there was no significantly neuron loss. While in thekindled rats, neuron loss was obviously, with shrunken plasma body andpyknotic nuclei. In the EGCG group, most pyramid cells were normal andonly a few showed chromatin condensation. The number of surviving neurons:compared to the control group, the number of surving neurons in the PTZgroup was significantly decreased (p<0.05)(CA1: PTZ:137.7±25.36; CON:235.8±20.43)(CA3: PTZ:142.2±23.63; CON:252.6±17.02), comparedwith the PTZ gourp, EGCG significantly reduced the neuron loss(p<0.05) (CA1: PTZ+25mg/kg EGCG:217.9±11.14; PTZ+50mg/kg EGCG;223.5±20.82)(CA3: PTZ+25mg/kg EGCG:233.8±13.96; PTZ+50mg/kg EGCG:240.7±18.03). There was no statistic difference among PTZ+25mg/kg EGCG,PTZ+50mg/kgEGCG and control group. Bcl-2protein level: CON:0.72±0.03; PTZ:0.50±0.04; PTZ+25mg/kg EGCG:0.65±0.07; PTZ+50mg/kgEGCG=0.70±0.08. Bax protein level: CON:0.48±0.02; PTZ:0.60±0.03;PTZ+25mg/kg EGCG:0.52±0.01; PTZ+50mg/kg EGCG:0.50±0.03;caspase3protein level: CON:0.28±0.02; PTZ:0.54±0.03; PTZ+25mg/kgEGCG:0.35±0.03; PTZ+50mg/kg EGCG:0.30±0.01. Compared with thecontrol group, the protein level of Bcl-2was significantly lower, while theprotein levels of Bax and caspase-3were significantly higher in the PTZgroup(p<0.05). Compared with the PTZ group, the protein level of Bcl-2wasmarkedly increased, while the protein levels of Bax and caspase-3weremarkedly decreased in the EGCG group (p<0.05). There was no statisticdifference among the PTZ+25mg/kg EGCG, PTZ+50mg/kg EGCG andcontrol group. p-PI3K protein level: CON:0.74±0.05; PTZ:0.45±0.08;PTZ+25mg/kg EGCG:0.64±0.05; PTZ+50mg/kg EGCG:0.70±0.07. p-Aktprotein level: CON:0.78±0.02; PTZ:0.46±0.04; PTZ+25mg/kg EGCG:0.72±0.03; PTZ±50mg/kg EGCG:0.75±0.04. Compared to the controlgroup, the protein levels of p-PI3K and p-Akt were significantlty decreased inthe PTZ group(p<0.05), while the protein levels of p-PI3K and p-Akt weremarkedly increased after EGCG adiminstration (p<0.05), there was no statisticdifference among the group PTZ+25mg/kg EGCG, PTZ+50mg/kg EGCGand control group. There was no significant dfference of the total protein ofPI3K and Akt among the groups.Conclusion: There was a significant neuron loss in PTZ kindled rats.EGCG could attenuate the deficits, and the neuroprotective effects may bethrough promoting the PI3K/Akt signaling pathway and inhibiting themitochondrial apoptosis pathway.

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