摘要
癫痫是最常见的神经系统疾病之一，其定义为反复的、自发的大脑皮层神经元异常同步放电导致脑功能障碍。癫痫发作具有短暂性、发作性、重复性、刻板性的特点。据统计，发达国家癫痫的发病率为24~53/10万/年，我国目前大约有900万癫痫患者，发病率为35/10万/年左右，且呈逐年增高趋势，由此可以推段我国每年新增癫痫病人至少40万例。癫痫可导致神经元的丢失、胶质细胞的增生，从而导致致痫灶及远隔区域的脑功能障碍，影响患者的生存质量，甚至导致患者死亡。近年来，癫痫所致脑损伤的病理生理机制研究已取得了重大进步，并且涌现了一些新的抗癫痫药物。然而其临床治疗效果却仍未达到满意。已知的抗癫痫药物其治疗有效率约为70%，即有大约30%的癫痫患者应用某一药物治疗时无效，需更换或联用其他的治疗方法，其中有10%的患者需要手术治疗。目前癫痫治疗主要存在的问题为：1、抗癫痫药物的毒副作用；2、对癫痫并发症的治疗（尤其是认知、情感障碍的治疗）；3、耐药性癫痫的治疗；此外，对癫痫持续状态的治疗药物仍然有限。
癫痫的继发性脑损伤常导致病情加重，有些也是促进癫痫的因素，其机制主要涉及氧化应激、炎症反应、凋亡、细胞内钙超载、兴奋性氨基酸毒性学说等，这些发病机制之间相互作用、相互影响，形成了复杂的病理级联反应及调控网络，导致了癫痫的发生发展及继发性脑功能障碍。其中兴奋性氨基酸毒性、氧化应激和凋亡存在于癫痫起始病灶及非起始灶的受累脑组织中，是导致神经元功能障碍甚至死亡的主要原因。因此，抗氧化应激、抑制兴奋性氨基酸的过度兴奋、抑制凋亡成为保护癫痫及相关脑组织损伤的几种重要途径。
右美托嘧啶是一种高选择性的α2-受体激动剂，目前已广泛应用于镇静及辅助全身或局部麻醉作用，此外有研究表明该药还具有抑制兴奋性氨基酸释放、抗氧化应激、抗炎、抗凋亡作用。曾有学者发现，α2-受体激动剂可乐定具有抗癫痫效果，其抗癫痫作用机制可能为：1、由于钙离子流的阻滞，钾离子流的促进和囊泡泡吐自身的直接调节，最终是通过兴奋性递质释放的减少来实现了突触前抑制。2、突触后抑制：可乐定可与突触后α2-受体结合，抑制钙超载，产生长时程增强与受体变化。但由于可乐定激动α2-受体同时还激动α1-受体(220:1)，即对α2-受体选择性较差，因此未能应用于临床抗癫痫治疗。而右美托嘧啶的α2-受体选择性是可乐定的约8倍(1600:1)。因此其可能成为更理想的抗癫痫药物。
本研究选用雄性Wistar大鼠，应用杏仁基底外侧核电点燃法建立大鼠急慢性癫痫及自发性癫痫持续状态（self-sustaining status epilepticus,SSSE）模型。在这些模型上依据Racine分级及脑电图（electro-encephalogram，EEG）记录观察癫痫发作的严重程度及持续时间；并检测海马组织中兴奋性氨基酸-谷氨酸（glutamate，Glu）及抑制性氨基酸-γ-氨基丁酸（γ-AminobutyricAcid, GABA）、抗氧化物谷胱甘肽（glutathione,GSH）和氧化应激终产物丙二醛（Malondiadehyde，MDA）、凋亡抑制/促进蛋白Bcl-2/Bax、及基因转录因子核因子-κB(neuclear factor-κB, NF-κB)的表达。确定杏仁基底外侧核电刺激诱导大鼠癫痫发作的作用及癫痫发作后引起的大鼠脑海马组织中兴奋/抑制性氨基酸递质的失衡、氧化应激损伤、凋亡等病理生理过程，并通过腹腔注射右美托咪啶进行干预，对干预前后大鼠癫痫发作的严重程度及持续时间、脑海马组织中Glu/GABA递质含量、GSH/MDA水平、Bcl-2/Bax及NF-κB表达水平的变化进行评价，以研究右美托咪啶对大鼠杏仁核电点燃癫痫模型的抗癫痫及脑保护作用，并对其可能的机制进行探讨。本实验研究共分为三部分，各部分内容分述如下。
第一部分大鼠杏仁基底外侧核急慢性电点燃癫痫模型的建立
目的：观察不同参数刺激条件对大鼠杏仁基底外侧核电点燃癫痫模型的诱导效果。通过点燃过程中大鼠行为学、可操作性、点燃成功率、Racine分级、EEG等的观察，研究不同刺激参数对杏仁基底外侧核电点燃癫痫模型建立的影响。探讨其模拟人类不同癫痫类型的价值。
方法：取成年健康雄性Wistar大鼠（250-350g）60只为实验对象，随机分为3组，1、3组各15只，第2组再随机分为2个亚组，每组15只，经水合氯醛全麻后，将双股漆包镍镉电极经大鼠脑立体定向仪置入大鼠左侧杏仁基底外侧核，坐标为前囟后2.2mm，外4.8mm，自颅骨表面下8.5mm。术毕大鼠恢复1周。一周后测定后放电阈值，后放电阈值定义为出现≥3s的棘波后放电时的电流强度，且不超过400μA。测定后放电阈值后24小时，采用不同刺激参数对大鼠进行点燃程序刺激。实验1：慢性电点燃癫痫模型的建立：15只Wistar大鼠测定后放电阈值后，以高于后放电阈值的恒定电流(500μA)刺激杏仁基底外侧核，单向方波，频率60Hz，波宽1.0ms，串长1s，每日1次，观察大鼠行为学及EEG变化。癫痫严重程度依据Racine分级法评估；至大鼠连续5次出现5级发作，即认为大鼠点燃成功。点燃过程中观察大鼠行为学并记录EEG，点燃完毕后记录点燃成功率。实验2：快速电点燃癫痫模型的建立：取实验大鼠30只，再次随机分为A、B两个亚组，每组15只，测定后放电阈值24小时后以高于后放电阈值的恒定电流刺激杏仁基底外侧核，具体参数如下：A组：刺激频率16Hz，波宽1.0ms，串长10s，500μA恒流，单向方波，刺激间隔时间7分钟，刺激次数30次/天，以动物连续出现3次Racine5级发作为点燃成功标志。B组：刺激频率60Hz，波宽1.0ms，串长10s，强度500μA，刺激间隔时间30min，每天刺激10次，连续刺激2d，刺激电流为恒流，单向方波。以动物连续出现3次Racine5级发作为点燃成功标志。两组点燃过程中均观察大鼠行为学并监测EEG，计算点燃成功率。实验3：杏仁基底外侧核电点燃后诱导SSSE的建立：15只Wistar大鼠测定后放电阈值后，按下列参数进行电刺激以诱导SSSE的发生：首先以500μA恒定电流，单向方波,波宽1.0ms，频率60Hz，刺激杏仁基底外侧核，每日1次，每次1s。至连续出现5次Racine分级5级发作视为点燃成功。点燃成功后2周，以如下参数再次刺激杏仁基底外侧核诱导SSSE：刺激持续时间25分钟，刺激程序包括波宽1.0ms的双向方波连续100ms，以每秒2次的频率施加，2次刺激间给予脉冲频率为每秒50次，波宽1.0ms，单向方波的持续刺激，2种刺激参数的电流均为恒流700μA。刺激过程中及刺激后12小时采用EEG观测自发癫痫持续状态发生情况。
结果：
1慢性电点燃癫痫模型：15只大鼠经后放电阈值测定，有14只符合实验标准（后放电阈值≤400μA），继续进行后续实验；1只大鼠后放电阈值测定＞400μA时仍未引出，考虑电极位置放置欠佳，排除出实验组。14只实验大鼠经点燃程序刺激时，1只出现电极脱落，未完成点燃程序；13只大鼠中有12只大鼠表现出连续5次5级发作，成功点燃，1只大鼠虽曾表现出5级发作，但此后发作等级下降，未点燃成功。点燃成功时间10-19天，总点燃成功率（点燃成功例数/总纳入实验例数）：85.7%；实际点燃成功率（点燃成功例数/完成点燃程序例数）：92.3%。点燃成功后大鼠均存活。再次刺激引起的癫痫发作稳定可靠。
2快速电点燃癫痫模型：本实验组分为2个亚组，每组各15只大鼠。A组大鼠中，共13只大鼠后放电阈值≤400μA，纳入后续试验；2只大鼠后放电阈值测定＞400μA时仍未引出，考虑电极位置放置欠佳，排除出实验组。在程序刺激过程中，2只大鼠出现电极脱落。剩余11只大鼠完成点燃程序，其中8只大鼠表现出连续3次Racine分级5级发作，3只虽曾表现出5级发作，但未表现出连续3次该级别发作，或发作级别达到5级后出现下降趋势，未能点燃。点燃成功时间：1-2d，总点燃成功率：61.5%；实际点燃成功率：72.7%。B组大鼠中，共14只大鼠后放电阈值达标，纳入实验；1只大鼠后放电阈值未达标，排除出实验。进行程序刺激时，4只大鼠出现电极脱落，10只大鼠完成点燃程序，其中2只大鼠因未出现连续3次5级发作而未点燃成功，8只大鼠出现连续3次5级发作，视为点燃成功。点燃成功时间2天；总点燃成功率：57.1%；实际点燃成功率：80.0%。
3电点燃后诱导SSSE模型：本实验组共15只大鼠，15只大鼠后放电阈值测定均达标，纳入实验。其中1只点燃过程中电极脱落，13只经慢性杏仁基底外侧核点燃程序刺激后均达到点燃成功标准，1只未达到点燃标准。点燃成功时间：10-19天；总点燃成功率：86.7%；实际点燃成功率：92.9%。13只点燃大鼠进入25min的SSSE诱导电刺激程序，刺激结束后均成功诱导出SSSE，表现为自发的间断Racine3-5级发作，持续时间5-12小时，但其中2只大鼠死于SSSE观察过程中，死亡率：15.4%。
结论：大鼠杏仁核电点燃癫痫模型可很好地模拟人类颞叶癫痫的病理生理过程。实验数据表明，电极植入方法安全可靠，大鼠术后全部存活。在点燃过程中，不同的刺激方法所需点燃时间、总点燃成功率及实际点燃成功率不同。慢性杏仁基底外侧核电刺激时大鼠经10-19天可达点燃成功标准，需时长，但总点燃成功率及实际点燃成功率高。快速点燃模型中，A组点燃所需时间短，但总点燃成功率及实际点燃成功率较慢性点燃低。B组点燃时间同样较短，但总点燃成功率较A组低，实际点燃成功率较A组高；此外，点燃过程中，慢点燃组大鼠电极脱落可能性较小，快点燃组大鼠电极脱落可能性较大，尤其是B组。慢性点燃后，大鼠经SSSE诱导刺激程序，均能诱导出SSSE发作，可很好地模拟人类癫痫持续状态，其癫痫持续状态的死亡率与人类相近。
第二部分右美托咪啶对杏仁基底外侧核电刺激诱导SSSE模型抗癫痫作用及机制研究
目的：观察右美托咪啶对电刺激杏仁基底外侧核诱导的SSSE模型痫性发作次数及发作时间的影响，并测定SSSE后大鼠海马组织中Glu/GABA含量及GSH/MDA含量的变化，研究右美托咪啶在SSSE中的抗癫痫及脑保护作用，并探讨其涉及的兴奋性氨基酸毒性学说、氧化应激等机制。
方法：取健康成年雄性Wistar大鼠为研究对象，应用杏仁基底外侧核程序电刺激诱导法制备SSSE模型。Wistar大鼠共65只，随机分为5组，低剂量组（L组），SSSE+低剂量（50μg/kg）右美托咪啶腹腔注射；高剂量组（H组），SSSE+高剂量（100μg/kg）右美托咪啶腹腔注射；自发性癫痫持续状态组（SE组），仅诱导SSSE发作；假手术组（S组），仅给与电极植入，未行程序电刺激；空白对照组（C组）：未予任何处理。点燃及诱导SSSE程序如第一部分。L/H组分别于诱导成功后5分钟经大鼠腹腔注射50/100μg/kg的右美托咪啶。以脑电图观测痫性发作的次数及累计时间；留取大鼠海马标本后，以液质联用法测定海马中Glu/GABA含量，酶联免疫法测定海马中GSH/MDA含量。
结果：
1L组中有13只大鼠，H组中有11只大鼠，SE组中有15只大鼠点燃成功。经电刺激诱导后，点燃大鼠均可表现出SSSE，持续时间5-12小时。最终L组13只大鼠，H组11只大鼠，SE组中13只大鼠经SSSE后存活；SE组2只大鼠在发作过程中死亡，死亡率13.33%，排除出实验。与SE组相比，L组痫性发作次数和累计时间无明显减少，差异无统计学意义（p>0.05）；但H组痫性发作次数和累计发作时间明显下降，差异有统计学意义（p<0.05）。S组和C组未见痫性发作。
2液质联用中，SSSE发作12小时后，L组、H组、SE组海马组织中Glu含量和C组相比均升高，差异有统计学意义（p<0.05）；S组和C组无差别（p>0.05）；L组和SE组含量无差别（p>0.05）。但H组较SE组和L组含量下降，差异有统计学意义（p<0.05）。L组、H组、SE组海马中GABA水平与C组相比均增高（p<0.05）。S组和C组之间无差异（p>0.05）；L组和SE组之间无差异（p>0.05）。但H组含量较L组和SE组均降低，差异有统计学意义（p<0.05）。GABA含量未随着右美托咪啶的治疗有效性而升高，但随着Glu含量的变化而变化。
3酶联免疫法测定GSH/MDA，诱导SSSE后12小时，L组、H组、SE组海马组织匀浆中GSH含量较C组明显下降（p<0.05）。S组和C组之间GSH含量无明显差异（p>0.05）；L组和SE组之间含量亦无明显差异（p>0.05）。在H组中，虽然仍低于S组和C组，但高剂量的右美托咪啶使GSH含量较L组和SE组明显升高（p<0.05）。MDA含量在L组、H组、SE组中较在C组中明显升高（p<0.05）。S组和C组之间、L组和SE组之间MDA含量均无明显差异（p>0.05）。H组MDA含量明显低于L组及SE组（p<0.05）。上述数据表明高剂量右美托咪啶可以有效地治疗癫痫相关的氧化应激。
结论：大鼠经杏仁核电点燃后，再给与25min的SSSE诱导刺激程序，可成功诱导SSSE发作。SSSE发作12小时后，大鼠海马组织中可检测到Glu升高及GABA的代偿性升高，GSH含量下降及MDA含量升高，表明兴奋性氨基酸毒性及氧化应激与癫痫持续状态密切相关。应用高剂量右美托咪啶可以减少SSSE时痫性发作的次数及累计时间，并可缓解海马组织中Glu的升高、GSH的减少及MDA的增加。从而表现出抗癫痫及脑保护作用。
第三部分右美托咪啶对实验性慢性癫痫大鼠的抗癫痫作用及机制研究
目的：观察右美托咪啶对杏仁基底外侧核慢性电点燃大鼠癫痫模型后放电持续时间及Racine发作分级的影响，并经western blot法测定继续电刺激1周后海马组织中Bcl-2/Bax的表达变化，及NF-κB表达变化，研究右美托咪啶对慢点燃癫痫模型大鼠继发海马组织炎症及凋亡的作用，探讨右美托咪啶脑保护作用的相关机制。
方法：健康成年雄性Wistar大鼠65只，随机分为5组，低剂量组（L组）15只，慢性电点燃程序刺激+点燃后刺激+低剂量（50μg/kg）右美托咪啶腹腔注射；高剂量组（H组）15只，慢性电点燃程序刺激+点燃后刺激+高剂量（100μg/kg）右美托咪啶腹腔注射；慢性电点燃癫痫组（E组）15只，慢性电点燃程序刺激+点燃后刺激；假手术组（S组），仅给与电极植入，未行杏仁基底外侧核电刺激；空白对照组（C组）：未予任何处理。L、H、E组点燃成功后再次给予杏仁基底外侧核电刺激，每日1次，其中L/H组于电刺激前5分钟给予右美托咪啶50/100μg/kg腹腔内注射，1周后记录再刺激时每组癫痫发作的Racine分级5级的百分比及后放电持续时间，将5组大鼠共同处死，取海马组织，采用Western-blot法分别检测各样本脑组织中Bcl-2、Bax和NF-κB水平的变化。
结果：
1经慢性电点燃程序刺激后，L组中有13只大鼠点燃成功，H组中有13只，E组中有12只，各组均无大鼠死亡。点燃后继续给予同样刺激，E组大鼠表现同样5级发作，后放电持续时间较前无明显变化；L组大鼠5级发作百分比与后放电持续时间较E组无统计学差异（p>0.05）；H组大鼠5级发作百分比及后放电持续时间较E组明显下降，差异有统计学意义（p<0.05）；S组和C组均无癫痫发作。
2Western blot中，经每日1次点燃后杏仁基底外侧核再刺激，至1周后，L组、E组、H组海马组织Bcl-2水平和C组相比明显下降（p<0.05）。L组与E组之间无明显差异（p>0.05）；S组和C组之间亦无明显差异（p>0.05）；但H组较E组及L组Bcl-2含量明显升高，且具有统计学意义（p<0.05）。L组、E组、H组海马组织中Bax水平较C组明显升高（p<0.05）；L组与E组之间无明显差异（p>0.05）；S组和C组之间亦无明显差异（p>0.05）；但H组较E组和L组Bax含量明显下降，差异有统计学意义（p<0.05）。
3Western blot中，经每日1次点燃后杏仁基底外侧核再刺激，至1周后，L组、E组、H组海马组织NF-κB水平和C组相比明显下降（p<0.05）。L组与E组之间无明显差异（p>0.05）；S组和C组之间亦无明显差异（p>0.05）；但H组较E组和L组NF-κB含量明显升高，且具有统计学意义（p<0.05）。
结论：慢性杏仁基底外侧核电点燃癫痫模型中，右美托咪啶可减轻癫痫发作严重程度和后放电持续时间。大鼠海马组织中，高剂量右美托咪啶可增加Bcl-2蛋白的表达，减少Bax蛋白的表达，减少NF-κB的表达，提示右美托咪啶可缓解慢性癫痫模型的发作，并通过抗癫痫、抗炎、抗凋亡途径保护癫痫大鼠脑组织。
Epilepsy is one of the most common chronic neurologic diseases definedas spontaneously and repetitively synchronous abnormal discharge of cerebralcortical neuron resulting in cerebral dysfunction. As the characteristic ofepilepsy, transience, paroxysmal, repetition, stereotype can be observed. It hasbeen reported that the morbidity is24-53%per1hundred thousand people indeveloped countries. According as the current data, there are about ninemillion patients in China where the mobidity is about35per1hundredthousand people with ascending trend year by year. Epileptic seizurescontribute to the loss of neurons, gliosis and subsequently the dysfunction oflocal and remote cerebral areas leading to the declined quality of life and eventhe death of patients. Recently, the pathophysiological mechanisms underlyingthe brain injury attributed to epilepsy have been further clarified resulting inthe emergence of some novel antiepileptic compounds; however, theoptimized therapeutic effect has not been achieved. The focus problems ofcurrent antiepileptic treatments are that the toxic and side effects of drugs, theconsiderable complications, the remedy when refractory and the relativelylack of methods for status epilepticus (SE).
The second injury of brain, which involves the oxdative stress, theinflammatory reaction, apoptosis, intracellular calcium overload andexcitatory amino acids toxicity, is attributed to epileptic seizures andcontributes to not only the deteriorated condition but also the enhancement ofepileptogenesis. All of the pathophysiological changes interact resulting inpathological cascade reaction and regulatory network. Especially theexcitatory amino acids toxicity, oxidative stress and apoptosis acting in theprimary and non-primary epileptic focus play an important role in neuronaldysfunction. Accordingly, inhibition of the excitatory amino acids toxicity,anti-oxidative stress and discontinuation of apoptosis process are important ways to protect the brain from the injury associated with epilepsy.
Dexmedetomidine (DEX), a high selective alpha2-adrenoceptor agonist,has been widely used in sadation and general/local anaesthesia. Reports haveasserted that DEX can inhibit the excessive release of excitatory amino acids,attenuate the oxidative stress and inflammatory reaction and discontinue theprocess of apoptosis. Meanwhile, researchers have concluded theanti-epiepsia effect of clonidine, another alpha2-adrenoceptor agonist. Thepossible mechanisms involve the pre and postsynaptic inhibition associatedwith Ca2+influx and excessive release of excitatory amino acids; however,clonidine is still unacceptable because of the simultaneous selection ofalpha1-adrenoceptor. About8times selectivity for alpha2-adrenoceptorcompared to clonidine, DEX is an appropriate choice for treatment ofepilepsy.
In the current experiment, male healthy Wistar rats were chosen for theacute/chronic basal lateral amygadala (BLA) electrical kindling epilepsiamodels. According as Racine behavioral classification and electro-encephalogram (EEG), the severity and duration of epileptic seizure wererecorded to examine if it can be induced by BLA electrical stimulation. Theexcitatory amino acid-glutamate (Glu)/inhibitory amino acid-γ-aminobutyricacid (GABA), the glutathione (GSH)/malondiadehyde (MDA), the Bax/Bcl-2and the neuclear transcription factor-κB (NF-κB) in hippocampus tissuewere tested to observe the unbalance between excitatory and inhibitory aminoacids, the injury associated with the oxidative stress and the process ofapoptosis, as well as their changes after intraperitoneal injection of DEX. Allthe data would be analysised to assess the anti-epilepsy and brain protectiveefficacy of DEX and to explore the possible mechanisms. There were threeparts of the current study; each one of them was stated as below.
PartⅠEstablishment of the acute and chronic epilepsy in rat BLAelectrical kindling model
Objective: To test the inductive effect of epilepsy under the differentBLA electrical stimulus parameter. The success rate of kindling, theafterdischarge threshold, the Racine’s classification and the EEG wereobserved and recorded to explore the differentiation of the influences underthe diffent parameters and to assess the values of them.
Methods: Sixty male healthy adult Wistar rats weight250-350gramswere randomly devided into three groups. There were15rats in group1and3respectively, while30rats in group2were devided randomly and averagelyinto two subgroups again. Following the intraperitoneally injected chloralhydrate, the twisted enamel-covered nickel-cadmium electrode was implantedinto the left BLA (Bregma: anterior:2.8mm; lateral:4.8mm; ventral:8.5mm from skull surface) of the rat via brain stereotactic instrument. After oneweek recovery, the afterdischarge threshold (ADT), which was the minimumcurrent intensity (≤400μA) as the emergence of spike wave (duration≥3s),of each rat was detected. Subsequently, the rats were electrically stimulatedwith different kindling parameters. Experiment (1): the estabilishment ofchonic BLA kindling model. After the detection of ADT, the fifteen rats ingroup1were subject to BLA kindling program with a constent current (500μA), waviness width1ms, monophasic square-wave pulses, and60Hz for1s,once every day, during which the behavioral and EEG changes were recorded.The severity of seizure was assessed according to the Racine’s classification,and the rats were thought kindled successfully after consecutive5times of5stage seizures. The durations of afterdischarge were measured on the basis ofEEG. Experiment (2): the estabilishment of acute BLA kindling model. Thirtyrats were devided randomly and averagely into two subgroups after thedetection of ADT, group A and group B. Rats were subject to the BLAkindling program with the different parameters: group A, rats were kindledwith the constent current, intensity500μA, frequency16Hz, waviness width1.0ms, train length10.0s, biphasic square-wave pulse stimulation. Threr was7min interval among each stimulation, and rats were stimulated no more than2days with30times every day untile the consecutive3Racine5stage seizures were observed, which was thought the mark of kindled successfully.Group B, rats were kindled with the constent current, intensity500μA,frequency60Hz, waviness width1.0ms, train length10.0s, biphasicsquare-wave pulse stimulation. There was30min interval among eachstimulation, and rats were stimulated2days with10times every day untile theconsecutive3Racine5stage seizures were observed, which was thought themark of kindled successfully. Experiment (3): the establishment of a rat modelof self-sustaining SE (SSSE) with prolonged amygdala stimulating: after thedetectiong of ADT,15rats were kindled with the following paradigm: theconstent current, intensity500μA, frequency16Hz, waviness width1.0ms,train length1.0s, monophasic square-wave pulse stimulation. The rats werethought kindled successfully until consecutive5Racine5stage seizures. Aftera2weeks recovery period, the induction process of SSSE was performed asdescribed below: stimulus duration of25min and stimulus program consistingof100ms trains of1.0ms square-wave pulses. The trains were given at afrequency of2/s and the intra-train pulse frequency was50/s. Peak pulseintensity was700μA. Development and duration of SSSE were monitored for12h by EEG-recording.
Results:
1Chonic BLA kindling models: of15rats,14conformed to the standardof ADT (≤400μA) and were subject to the subsequent test. The rest one wasexcluded from experiment considering of the unmeasurable ADT associatedwith the incorrect placement of electrode. During the kindling process,1of14rats was excluded from test because of the deciduous electrode. Thirteen ratsfinished the process; however,12of them appeared the consecutive5Racine5stage seizure and were thought kindled successfully, the rest one had evershown Racine5stage seizure followed by descending seizure grades and wasthought failing to kindling. The time taken by kindling process was10-19days,the total successful kindling rate (successful kindling number vs total kindlingnumber) was85.7%, the actual successful kindling rate (successful kindlingnumber vs finished kindling number) was92.3%, and all of the rats were survival of the kindling process. Anytime after kindled, the same stimulationcan induce the Racine4-5stage seizure.
2Acute BLA kindling models:30rats were devided randomly andaveragely into two groups, group A and group B. In group A,13of the15ratsconformed to the standard of ADT (≤400μA) and were subject to thesubsequent test, while2rats were excluded. The electrodes of2within the13rats were come off; however the rest11rats finished the kindling process, inwhich8rats showed consecutive3Racine5stage seizures, while3ratsshowed nonconsecutive5stage seizres or5stage followed by descendingseizure grades. The time taken by kindling process were1-2days, the totalsuccessful kindling rate was61.5%, the actual successful kindling ratewas72.7%. In group B,14of the15rats conformed to the standard of ADT(≤400μA) and were subject to the subsequent test, while1rat was excluded. Theelectrodes of4rats within the14rats were come off; however the rest10ratsfinished the kindling process, in which8rats showed consecutive3Racine5stage seizures, while2rats showed nonconsecutive5stage seizres. The timetaken by kindling process was2days, the total successful kindling rate was57.1%, and the actual successful kindling rate was80.0%.
3The BLA electrical stimulated self-sustaining SE (SSSE) models:15rats conformed to the standard of ADT (≤400μA) and were subject to thesubsequent test. The electrodes of1rat within the14rats were come off;however the rest13rats finished the kindling process, in which13rats weresuccessfully kindled and subject to the subsequent induction process, while1rat failed to be kindled. The time taken by kindling process was10-19days,the total successful kindling rate was86.7%, and the actual successful kindlingrate was92.9%. After the inductiong of SSSE with prolonged25min BLAelectrical stimulation, all of the13rats showed self-sustaining intermittentRacine5seizures defined as SSSE, duration time5-12h. There were2ratsdied during SSSE, and the death rate was15.4%.
Conclusion:
The rat BLA electrical kindling epilepsia models can well imitate the pathophysiological progress of humen-bing temporal lobe epilepsy. Thecurrent data has demonstrated that the implanted electrode was safe andreliable without the death of the operated rats. During kindling process, thetime required for successfully kindled, the total successful kindling rate andthe actual successful kindling rate are significantly different among threegroups. In the chornic BLA kindling models, the time is about10-19days;however, the total successful kindling rate and the actual successful kindlingrate are significantly higher than the other two groups. In group A of the acuteBLA kindling models, the time is shorter than that of chronic models; however,the the total successful kindling rate and the actual successful kindling rate arenot so optimized. The group B has shown the same short time as group A, butmore lower the the total successful kindling rate. The actual successfulkindling rate of group B is statistically higher than group A, but still lowerthan the chronic models. In the BLA electrical stimulated SSSE models, all ofthe rats undergone the prolonged inductiong progress can show SSSE after thesuccessfully chronic BLA-kindling process, and the death rate during SSSE issimilar to human bing.
PartⅡ Anticonvulsant effect of dexmedetomidine in a rat model ofself-sustaining status epilepticus with prolonged amygdalastimulation
Objective: To explore the effect of DEX on the number and accumulatedtime of seizure in the rat model of SSSE with prolonged amygdale stimulation,and detect the level of Glu/GABA and GSH/MDA in hippocampus of rat afterSSSE to assess its anticonvulsant and cerebral protective action and discussthe involved mechanisms.
Methods: Sixty-five adult male healthy Wistar rats were devidedrandomly and averagely into5groups, Group L (SSSE plus low dosage DEX):intraperitoneally DEX (50μg/kg)-injected and electrically stimulated animals(n=15); Group H (SSSE plus high dosage DEX): intraperitoneally DEX(100μg/kg)-injected and electrically stimulated animals (n=15); GroupSE (SSSE): electrically stimulated and non-DEX-injected animals (n=15);Group S (sham): electrode-implanted but non-DEX-injected and non-electrically-stimulated animals (n=10); and Group C (control): without anytreatment (n=10). The kindling and induction programs were performd asdescribed in part I. The rats in group L/H were intraperitoneally injected DEX50/100μg/kg5min after the successful inductiong of SSSE. The number andaccumulative time were recorded according to the EEG; the levels ofGlu/GABA and GSH/MDA in hippocampus tissure of rat collected12h afterthe emergence of SSSE were detected by the liquid chromatography-tandemmass spectrometry (LC-MS) and the enzyme-linked immunosorbent assay(ELISA).
Results:
1Threr are13rats in group L,11rats in group H, and15rats in SE werekindled successfully, and showed5-12h induced SSSE. Eventually,13rats inL,11rats in H and13rats in SE were survived after SSSE,2rats died ofSSSE in SE were excluded from the experiment (death rate=13.33%).Compared with group SE, the number and accumulative time in group L werenot statistically decreased (p>0.05); however, the ones in group H weresignifically decreased (p<0.05). There were not epileptic seizures in the rats ofgroup S and C.
2In LC-MS. In the hippocampus tissures,12h after the SSSE, thecontents of Glu were increased in group L, H, and SE compared with group C(p<0.05). There was no difference between group S and group C (p>0.05), orbetween group L and SE (p>0.05). But significant decrease was observed ingroup H compared to that in group SE (p<0.05). The levels of GABA wereobviously increased in group L, H, SE compared with group C (p<0.05).There was no different between group S and C (p>0.05), or group L and SE(p>0.05). However, the level of GABA in group H was significantly decreasedthan that in group L and SE (p<0.05). The present data suggested that the levelof GABA was not decreased or increased corresponding to the dosage of DEX but to the level of Glu.
3In ELSIA. In the hippocampus tissures,12after the SSSE, the contentsof GSH were decreased in group L, H, SE compared with group C (p<0.05).There was no difference between group S and C (p>0.05), or between group Land SE (p>0.05). The level of GSH in group H was still lower than that ingroup S and C; however, it was higher than that in group L and SE (p<0.05).Simultaneously, the levels of MDA were significantly increased in group L, H,and SE compared to the group C (p<0.05). There was no difference betweengroup S and C (p>0.05), or between group L and SE (p>0.05). But the levelwas significantly decreased in group H than that in group L and SE (p<0.05).The current data suggested that high dosage DEX (100μg/kg) effectivelyattenuated the oxidative stress associated with SSSE.
Conclusions：After kindling process, SSSE can be successfully inducedin all rats with a25min prolonged stimulus program. Twelve hours afterSSSE, the levels of Glu/GABA and GSH/MDA in hippocampus tissure canbe increased/compensatively increased and decreased/increased, suggestingthe close relation between SSSE and the excitatory amino acid toxicity or theoxidative stress. The high dosage DEX can show anticonvulsant andbrain-protective effect by decreasing the number and accumulative time ofthe seizures during SSSE, the level of Glu and MDA, and increasing the levelof GSH.
PartⅢ Anti-epilepsia effect of dexmedetomidine and the underlyingmechanism in rat chronic BLA electric kindling model
Objective: to observe the action of DEX on the duration and Racineclassfication in the chronic BLA kindling epileptic rat models, detect the levelof Bax/Bcl-2and NF-κB in the hippocampus tissue, assess the effect of DEXon the inflammatory and apoptosis responses associated with epilepsy, andexplore the involved mechanisms underlying the brain-protective effect ofDEX.
Methods: Sixty-five male adult healthy Wistar rats were devided into5groups randomly and averagely. Group L (chronic BLA electric kindlingprocess plus stimulation after kindling plus low dosage DEX): chronickindling process followed by intraperitoneally DEX (50μg/kg)-injected andelectrically stimulated animals (n=15); Group H (chronic BLA electrickindling process plus stimulation after kindling plus high dosage DEX):chronic kindling process followed by intraperitoneally DEX(100μg/kg)-injected and electrically stimulated animals (n=15); Group E (chronic BLAelectric kindling process only): chronic kindling process followed byelectrically stimulated and non-DEX-injected animals (n=15); Group S (sham):electrode-implanted but non-DEX-injected and non-electrically-stimulatedanimals (n=10); and Group C (control): without any treatment (n=10). The ratsin group L, H, E were subject to continuing electrical stimulation once a dayafter successful BLA electrical kindling and the rats in group L/H wereintraperitoneally injected DEX50/100μg/kg every5min before stimulation.The Racine classification and ADD were recorded when the last stimulationfor the assessment of severity. At the1week after kindling, after the laststimulation, all rats in5groups were executed and the hippocampus tissueswere reserved for detection of Bax/Bcl-2and NF-κB.
Results:
1There were13rats in group L,13rats in group H, and12rats in groupE were kindled successfully, and no rats were dead during the kindling process.After kindled, all rats in group L, H, E were subject to electrical stimulationagain, once a day. The rats in group E showed stably5stage seizure and theADD was not significantly varied. After1week, the seizure degree and ADDof the rat in group L were not statistically different from that in group E(p>0.05). However, the seizure degree and ADD in group H were significantlydifferent from that in group H (p<0.05). There were no epileptic seizures inrats of group S and C.
2In Western blot. One week after the electrical stimulation once a day,the levels of Bcl-2in group L, H, E were significantly decreased compared with group C (p<0.05) in the hippocampus tissure. There was no differencebetween group L and group E (p>0.05), or between group S and group C(p>0.05); however, the level in group H was significantly increased comparedto that in group E (p<0.05). The level of Bax in group L, H, E weresignificantly increased compared to group C (p<0.05). there were nodifference in the level of Bax between group L and E (p>0.05), or betweengroup S and C (p>0.05); however, the level in group H was significantlydecreased compared to that in group E (p<0.05).
3In Western blot, after1week electrical stimulation once a day, thelevels of NF-κB in rat hippocampus tissues in group L, H, E weresignificantly decreased compare to group C (p<0.05). There was nodifference between group L and E (p>0.05), or group S and C (p>0.05);however, the level in group H was higher than that in group E (p<0.05).
Conclusions：DEX can decreased the seizure degree and the ADD in thechronic BLA electrical kindling models, increased the expressed levels ofBcl-2and NF-κB and decreased the expressed level of Bax in rat hippocampustissure, suggesting that the DEX can attenuated the severity of chronicepileptic seizure model, and show the protective effect by anti-epileptic,anti-inflammatory and anti-apoptosis action.
引文
1Nagaraja TS, Mohamood R, Krishna V, et al. Anticonvulsant activity ofErythrina mysorensis bark extract in an animal model of epilepsy. JPharmacol Pharmacother,2012,3:62-64
2周永,刘民,梁万年.癫痫流行病学研究进展.中华流行病学杂志,2007,28:92-94
3Aguiar CC, Almeida AB, Araujo PV., et al. Oxidative stress and epilepsy:literature review. Oxid Med Cell Longev,2012,2012:7952-7959
4Eid T, Lee TS, Wang Y, et al. Gene expression of glutamatemetabolizing enzymes in the hippocampal formation in human temporallobe epilepsy. Epilepsia,2013,54:871-890
5Kobow K, Auvin S, Jensen F, et al. Finding a better drug for epilepsy:antiepileptogenesis targets. Epilepsia,2012,53:1868-1876.
6Prommer, E. Review article: dexmedetomidine: does it have potential inpalliative medicine? Am J Hosp Palliat Care,2011,28:276-283
7Shouse MN, Scordato JC, Farber PR, et al. The alpha2adrenoreceptoragonist clonidine suppresses evoked and spontaneous seizures, whereasthe alpha2adrenoreceptor antagonist idazoxan promotes seizures inamygdala-kindled kittens. Brain Res,2007,1137:58-68
8Coulter DA, McIntyre DC, Loscher W. Animal models of limbicepilepsies: what can they tell us? Brain Pathol,2002,12:240-256
9Barness SJ, Pinel JP. Conditioned effects of kindling. Neurosci BilbehavRev,2001,25:745-751
10van de Bovenkamp-Janssen MC, Akhmadeev A, Kalimullina L, et al.Synaptology of the rostral reticular thalamic nucleus of absence epilepticWAG/Rij rats. Neurosci Res,2004,48:21-31
11Sah P, Faber ES, Lopez De Armentia M, et al. The amygdaloid complex:anatomy and physiology. Physiol. Rev,2003,83:803-834
12Morimoto K, Fahnestock M, Racine RJ. Kindling and status epilepticusmodels of epilepsy: rewiring the brain. Prog. Neurobiol,2004,73:1-60
13Richardson EJ, Griffith HR, Martin RC, Paige AL, et al. Structural andfunctional neuroimaging correlates of depression in temporal lobeepilepsy. Epilepsy Behav,2007,10:242-249
14White LE, Price JL. The functional anatomy of limbic status epilepticusin the rat. I-II. J. Neurosci,1993a-b,13:4787–4830
15Rogawski MA, Kurzman PS, Yamaguchi SI, et al. Role of AMPA andGluR5kainate receptors in the development and expression of amygdalakindling in the mouse. Neuropharmacology,2001,40:28-35
16Morimoto K, Katayama K, Inoue K, Sato K. Effects of competitive andnoncompetitive NMDA receptor antagonists on kindling and LTP.Pharmacol. Biochem. Behav,1991,40:893-899
17Von Bohlen und Halbach O, Schulze K, Albrecht D. Amygdala-kindlinginduces alterations in neuronal density and in density of degenerated fibers.Hippocampus,2004,14:311-318
18王维平,王晓鹏.癫痫电点燃的研究进展.脑与神经疾病杂志,2008,16:644-646
19李昱,刘川,杨素芬,等.快速海马电点燃癫痫模型的改良方法.遵义医学院学报,2002,25:106-108
20Osawa M, Uemura S, Kimura H, et al. Amygdala kindling developswithout mossy fiber sprouting and hippocampal neuronal degeneration inrats. Psychiatry Clin Neurosci,2001,55:549-557
21Armitage LL, Mohapel P, Jenkins EM, et al. Dissociation between mossyfiber sprouting and rapid kindling with low-frequency stimulation of theamygdala. Brain Res,1998,781:37-44
22L scher W. Animal models of epilepsy for the development of anti-epileptogenic and disease-modifying drugs. A comparison of thepharmacology of kindling and post-status epilepticus models of temporallobe epilepsy. Epilepsy Res,2002,50,105-123
23Claudia B, Maike G, Heidrun P, et al. Epileptogenesis and neuropathologyafter different types of status epilepticus induced by prolonged electricalstimulation of the basolateral amygdala in rats. Epilepsy Research,2003,55,83-103
[1] Strauss DJ, Day SM, Shavelle RM, et al. Remote symptomatic epilepsy:does seizure severity increase mortality? Neurology,2003,60:395-399
[2] Treiman DM, Meyers PD, Walton NY, et al. A comparison of fourtreatments for generalized convulsive status epilepticus. Veterans affairsstatus epilepticus cooperative study group. N. Engl. J. Med,1998,339:792-798
[3] Trinka E. The use of valproate and new antiepileptic drugs in statusepilepticus. Epilepsia,2007,48:49-51
[4] Mazarati AM, Baldwin RA, Sankar R, et al. Time-dependent decrease inthe effectiveness of antiepileptic drugs during the course of self-sustainingstatus epilepticus. Brain Res,1998a,814:179-185
[5] Bengzon J, Kokaia M, Brundin P, et al. Seizure suppression in kindlingepilepsy by intrahippocampal locus coeruleus grafts: evidence for an alpha2-adrenoreceptor mediated mechanism. Exp Brain Res,1990,81:433-437
[6] Scotti de Carolis A, Passarelli F, Pezzola A. Study on the anticonvulsantactivity of clonidine against pentylenetetrazol-induced seizures in rats:pharmacological evidence of alpha2-adrenoceptors mediation. Arch IntPharmacodyn Ther,1986,282:209-218
[7] Hoffman WE, Kochs E, Werner C, et al. Dexmedetomidine improvesneurologic outcome from incomplete ischemia in the rat. Reversal by thealpha2-adrenergic antagonist atipamezole. Anesthesiology,1991,75:328-332
[8] Whittington RA, Virag L, Vulliemoz Y, et al. Dexmedetomidine increasesthe cocaine seizure threshold in rats. Anesthesiology,2002,97:693-700
[9] Racine RJ. Modification of seizure activity by electrical stimulation: II.Motor seizure. Electroencephal Clin Neurophys,1972,32:281-294
[10]Brandt C, Glien M, Potschka H, et al. Epileptogenesis and neuropathologyafter different types of status epilepticus induced by prolonged electricalstimulation of the basolateral amygdala in rats. Epilepsy Res,2003,55:83-103
[11]Nissinen J, Halonen T, Koivisto E, et al. A new model of chronic temporallobe epilepsy induced by electrical stimulation of the amygdala in rat.Epilepsy Res,2000,38:177-205
[12]Wasterlain CG, Stohr T, Matagne A. The acute and chronic effects of thenovel anticonvulsant lacosamide in an experimental model of statusepilepticus. Epilepsy Res,2011,94:10-17
[13]Okhawa H, Ohishi N, Yagi K. Assay of lipid peroxides in animal tissue bythiobarbituric acid reaction. Ann. Biochem,1979,95:351-358
[14]Ellman GL. Tissue sulphydryl groups. Arch. Biochem. Biophys,1959,82:70-77
[15]Wasterlain CG. Mortality and morbidity from serial seizures. Anexperimental study. Epilepsia,1974,15:155-176
[16]Szot P, Lester M, Laughlin ML, et al. The anticonvulsant and pro-convulsant effects of alpha2-adrenoreceptor agonists are mediated bydistinct populations of alpha2A-adrenoreceptors. Neuroscience,2004,126:795-803
[17]Wasterlain CG, Chen JW. Mechanistic and pharmacologic aspects of statusepilepticus and its treatment with new antiepileptic drugs. Epilepsia,2008,49:63-73
[18]Rowley NM, Madsen KK, Schousboe A, et al. Glutamate and GABAsynthesis, release, transport and metabolism as targets for seizure control.Neurochem Int,2012,61:546-558
[19]Ueda Y, Tsuru N. Bilateral seizure-related changes of extracellularglutamate concentration in hippocampi during development of amygdaloidkindling. Epilepsy Res,1994,18:85-88
[20]Ueda M, Oyama T, Kuraishi Y, et al. Alpha2-adrenoceptor-mediatedinhibition of capsaicin-evoked release of glutamate from rat spinal dorsalhorn slices. Neurosci Lett,1995,188:137-139
[21]Chiu KM, Lin TY, Lu CW, et al. Inhibitory effect of glutamate releasefrom rat cerebrocortical nerve terminals by alpha2-adrenoceptor agonistdexmedetomidine. Eur J Pharmacol,2011,670,137-147
[22]Liu CS, Wu HM, Kao SH, et al. Phenytoin-mediated oxidative stress inserum of female epileptics: a possible pathogenesis in the fetal hydantoinsyndrome. Human Exp Toxicol,1997,16:177-181
[23]One H, Sakamoto A, Sakura N. Plasma total glutathione concentrations inepileptic patients taking anti-convulsants. Clinica Chimica Acta,2000,298:351-358
[24]Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neuro-degenerative disorders. Science,1993,262:689-695
[25]Waldbaum S, Patel M. Mitochondrial dysfunction and oxidative stress: acontributing link to acquired epilepsy? J Bioenerg Biomembr,2010,42:449-455
[26]Frantseva MV, Perez Velazquez JL, Tsoraklidis G, et al. Oxidative stress isinvolved in seizure-induced neurodegeneration in the kindling model ofepilepsy. Neuroscience,2000,97:431-435
[27]Ayoglu H, Gul S, Hanci V, et al. The effects of dexmedetomidine dosageon cerebral vasospasm in a rat subarachnoid haemorrhage model. J ClinNeurosci,2010,17:770-773
1DeFelipe Oroquieta J, Arellano JI, Alonso L, et al. The neuropathologyof temporal lobe epilepsy: primary and secondary changes in the corticalcircuits and epileptogenicity. Rev Neurol,2002,34:401-408
2Mann MW, Pons G. Various pharmacogenetic aspects of antiepilepticdrug therapy: a review. CNS Drugs,2007,21:143-164
3Kelly KM, Chung SS. Surgical treatment for refractory epilepsy: reviewof patient evaluation and surgical options. Epilepsy Res Treat,2011,2011:303624
4Krahl SE, Clark KB. Vagus nerve stimulation for epilepsy: A review ofcentral mechanisms. Surg Neurol Int,2012,3(Suppl4): S255-259
5Chabardes S, Minotti L, Chassagnon S, et al. Basal ganglia deep-brainstimulation for treatment of drug-resistant epilepsy: review and currentdata. Neurochirurgie,2008,54:436-440
6Keene DL. A systematic review of the use of the ketogenic diet inchildhood epilepsy. Pediatr Neurol,2006,35:1-5
7Fegade MV, Bhounsule SA, Pereira I. Can vitamin B-complex aggravatethe carbamazepine induced toxic epidermal necrolysis? J Clin Diagn Res,2012,6:1571-1573
8Faught E. Topiramate in the treatment of partial and generalized epilepsy.Neuropsychiatr Dis Treat,2007,3:811-821
9Voutsinos-Porche B, Koning E, Kaplan H, et al. Temporal patterns of thecerebral inflammatory response in the rat lithium-pilocarpine model oftemporal lobe epilepsy. Neurobiol Dis,2004,17:385-402
10Dericioglu N, Soylemezoglu F, Gursoy-Ozdemir Y, et al. Cell death andsurvival mechanisms are concomitantly active in the hippocampus ofpatients with mesial temporal sclerosis. Neuroscience,2013,237:56-65
11Engelhard K, Werner C, Eberspacher E, et al. The effect of the alpha2-agonist dexmedetomidine and the N-methyl-D-aspartate antagonist S(+)-ketamine on the expression of apoptosis-regulating proteins afterincomplete cerebral ischemia and reperfusion in rats. Anesth Analg,2003,96:524-531
12Hofer S, Steppan J, Wagner T, et al. Central sympatholytics prolongsurvival in experimental sepsis. Crit Care,2009,13: R11
13Tellez-Zenteno JF, Hernandez-Ronquillo L. A review of the epidemiologyof temporal lobe epilepsy. Epilepsy Res Treat,2012,2012:630853
14Bregman B, Le Saux F, Trottier S, et al. Chronic cobalt-induced epilepsy:noradrenaline ionophoresis and adrenoceptor binding studies in the ratcerebral cortex. J Neural Transm,1985,63:109-118
15Pitkanen A, Narkilahti S, Bezvenyuk Z, et al. Atipamezole, analpha(2)-adrenoceptor antagonist, has disease modifying effects onepileptogenesis in rats. Epilepsy Res,2004,61:119-140
16Gellman RL, Kallanos JA, McNamara JO. Alpha2-receptors mediate anendogenous noradrenergic suppression of kindling development. JPharmacol ExpTher,1987,241:891
17岳旺,张芳,王蕾.中枢α2去甲肾上腺素受体在点燃模型的调控作用[J].中国药学杂志,2001,36:164-167
18Hu K, Xie YY, Zhang C, et al. MicroRNA expression profile of thehippocampus in a rat model of temporal lobe epilepsy andmiR-34a-targeted neuroprotection against hippocampal neurone cellapoptosis post-status epilepticus. BMC Neurosci,2012,13:115
19Cory S, Adams JM. The Bcl-2family: regulators of the cellularlife-or-death switch. Nat Rev Cancer,2002,2:647-656
20Brunelle J K, Letai A. Control of mitochondrial apoptosis by the Bcl-2family. J Cell Sci,2009,122:437-441
21Billen L P, Kokoski C L, Lovell J F, et al. Bcl-XL inhibits membranepermeabilization by competing with Bax. PloS Biol,2008,6: e147
22Das A, Wallace GC, McDowell ML, et al. Hippocampal tissue of patientswith refractory temporal lobe epilepsy is associated with astrocyteactivation, inflammation, and altered expression of channels and receptors.Neuroscience,2012,220:237-246
23Yu N, Di Q, Liu H, et al. Nuclear factor-kappa B activity regulates brainexpression of P-glycoprotein in the kainic acid-induced seizure rats.Mediators Inflamm,2011,2011:670613
1. de Lanerolle NC, Kim JH, Williamson A, Spencer SS, Zaveri HP, EidT, Spencer DD.2003. A retrospective analysis of hippocampal pathol-ogy in human temporal lobe epilepsy: evidence for distinctive patientsubcategories. Epilepsia44:677-687.
2. Pitkanen A, Sutula TP.2002. Is epilepsy a progressive disorder? Prospectsfor new therapeutic approaches in temporal-lobe epilepsy. Lancet Neurol1:173-181.
3. Sharma AK, Reams RY, Jordan WH, Miller MA, Thacker HL, Snyder PW.2007. Mesial temporal lobe epilepsy: Pathogenesis induced rodent modelsand lesions. Toxicol Pathol35:984-999.
4. Bae EK, Jung KH, Chu K, Lee ST, Kim JH, Park KI, Kim M, Chung CK,Lee SK, Roh JK.2010. Neuropathologic and clinical features of humanmedial temporal lobe epilepsy. J Clin Neurol6:73-80.
5. Yang T, Zhou D, Stefan H.2010. Why mesial temporal lobe epilepsy withhippocampal sclerosis is progressive: uncontrolled inflammation drivesdisease progression? J Neurol Sci296:1-6.
6. Sloviter RS.1996. Hippocampal pathology and pathophysiology intemporal lobe epilepsy. Neurologia11Suppl4:29-32.
7. Wieser HG.2004. ILAE Commission Report. Mesial temporal lobeepilepsy with hippocampal sclerosis. Epilepsia45:695-714.
8. Linard B, Ferrandon A, Koning E, Nehlig A, Raffo E.2010. Ketogenic dietexhibits neuroprotective effects in hippocampus but fails to preventepileptogenesis in the lithium-pilocarpine model of mesial temporal lobeepilepsy in adult rats. Epilepsia51:1829-1836.
9. Kharatishvili I, Pitkanen A.2010. Posttraumatic epilepsy. Curr OpinNeurol23:183-188.
10.Boison D.2008. The adenosine kinase hypothesis of epileptogenesis. ProgNeurobiol84:249–262.
11.Crespel A, Coubes P, Rousset MC, Brana C, Rougier A, Rondouin G,Bockaert J, Baldy-Moulinier M, Lerner-Natoli M.2002. Inflammatoryreactions in human medial temporal lobe epilepsy with hippocampalsclerosis. Brain Res952:159-169.
12.Kharatishvili I, Nissinen JP, McIntosh TK, Pitkanen A.2006. A model ofposttraumatic epilepsy induced by lateral fluid-percussion brain injuryinrats. Neuroscience140:685-697.
13.Pitkanen A, Immonen RJ, Grohn OH, Kharatishvili I.2009. Fromtraumatic brain injury to posttraumatic epilepsy: what animal models tell usabout the process and treatment options. Epilepsia50(Suppl2):21-29.
14.Hayashi K, Ueshima S, Ouchida M, Mashimo T, Nishiki T, Sendo T,Serikawa T, Matsui H, Ohmori I.2011. Therapy for hyperthermia-inducedseizures in Scn1a mutant rats. Epilepsia52:1010–1017.
15.Zheng XY, Zhang HL, Luo Q, Zhu J.2011. Kainic acid-induced neuro-degenerative model: potentials and limitations. J Biomed Biotechnol2011:457079.
16.Aronica E, Zurolo E, Iyer A, de Groot M, Anink J, Carbonell C, van VlietEA, Baayen JC, Boison D, Gorter JA.2011. Upregulation of adenosinekinase in astrocytes in experimental and human temporal lobe epilepsy.Epilepsia52:1645-1655.
17.Gouder N, Scheurer L, Fritschy JM, Boison D.2004. Overexpression ofadenosine kinase in epileptic hippocampus contributes to epileptogenesis. JNeurosci24:692-701.
18.Ravizza T, Balosso S, Vezzani A.2011. Inflammation and prevention ofepileptogenesis. Neurosci Lett497:223–230.
19.Feng ZH, Hao J, Ye L, Dayao C, Yan N, Yan Y, Chu L, Shi FD.2011.Overexpression of mu-calpain in the anterior temporal neocortex ofpatients with intractable epilepsy correlates with clinicopathologicalcharacteristics. Seizure20:395–401.
20.Jung KH, Chu K, Lee ST, Kim J, Sinn DI, Kim JM, Park DK, Lee JJ, KimSU, Kim M, Lee SK, Roh JK.2006. Cyclooxygenase-2inhibitor, celecoxib,inhibits the altered hippocampal neurogenesis with attenuation ofspontaneous recurrent seizures following pilocarpine-induced statusepilepticus. Neurobiol Dis23:237–246.
21.Polascheck N, Bankstahl M, Loscher W.2010. The COX-2inhibitorparecoxib is neuroprotective but not antiepileptogenic in the pilocarpinemodel of temporal lobe epilepsy. Exp Neurol224:219–233.
22.Holtman L, van Vliet EA, van Schaik R, Queiroz CM, Aronica E, GorterJA.2009. Effects of SC58236, a selective COX-2inhibitor, onepileptogenesis and spontaneous seizures in a rat model for temporal lobeepilepsy. Epilepsy Res84:56–66.
23.Jaworska-Adamu J, Dmowska M, Cybulska R, Krawczyk A,Pawlikowska-Pawlega B.2011. Investigations of hippocampal astrocytesin lipopolysaccharide-preconditioned rats in the pilocarpine model ofepilepsy. Fol Histochem Cytobiol49:219-224.
24.Paradiso B, Zucchini S, Su T, Bovolenta R, Berto E, Marconi P, Marzola A,Navarro Mora G, Fabene PF, Simonato M.2011. Localized overexpressionof FGF-2and BDNF in hippocampus reduces mossy fiber prouting andspontaneous seizures up to4weeks after pilocar-pine-induced statusepilepticus. Epilepsia52:572–578.
25.Jankowsky JL, Patterson PH.2001. The role of cytokines and growthfactors in seizures and their sequelae. Prog Neurobiol63:125–149.
26.Matute C.2010. Therapeutic potential of kainate receptors. CNS NeurosciTher17:661-669.
27.Frasca A, Aalbers M, Frigerio F, Fiordaliso F, Salio M, Gobbi M, Cag-notto A, Gardoni F, Battaglia GS, Hoogland G, Di Luca M, Vezzani A.
2011. Misplaced NMDA receptors in epileptogenesis contribute toexcitotoxicity. Neurobiol Dis43:507-515.
28.Galanopoulou AS.2007. Developmental patterns in the regulation ofchloride homeostasis and GABAA receptor signaling by seizures. Epilepsia48(Suppl5):14-18.
29.Epsztein J, Represa A, Jorquera I, Ben-Ari Y, Crepel V.2005. Recurrentmossy fibers establish aberrant kainate receptor-operated synapses ongranule cells from epileptic rats. J Neurosci25:8229-8239.
30.Shinnar S, Hesdorffer DC, Nordli DR Jr, Pellock JM, O’Dell C, Lewis DV,Frank LM, Moshe SL, Epstein LG, Marmarou A, Bagiella E.2008.Phenomenology of prolonged febrile seizures: results of the FEBSTATstudy. Neurology71:170-176.
31.Ben-Ari Y, Dudek FE.2010. Primary and secondary mechanisms of epi-leptogenesis in the temporal lobe: there is a before and an after. EpilepsyCurr10:118-125.
32.Gorter JA, Goncalves Pereira PM, van Vliet EA, Aronica E, Lopes da SilvaFH, Lucassen PJ.2003. Neuronal cell death in a rat model for mesialtemporal lobe epilepsy is induced by the initial status epilepticus and notby later repeated spontaneous seizures. Epilepsia44:647-658.
33.Baram TZ, Jensen FE, Brooks-Kayal A.2011. Does acquiredepileptogenesis in the immature brain require neuronal death. EpilepsyCurr11:21-26.
34.Gorter JA, van Vliet EA, Aronica E, Lopes da Silva FH.2001. Progressionof spontaneous seizures after status epilepticus is associated with mossyfibre sprouting and extensive bilateral loss of hilar parvalbumin andsomatostatin-immunoreactive neurons. Eur J Neurosci13:657-669.
35.Lee TS, Eid T, Mane S, Kim JH, Spencer DD, Ottersen OP, de LanerolleNC.2004. Aquaporin-4is increased in the sclerotic hippocampus in humantemporal lobe epilepsy. Acta Neuropathol108:493-502.
36.Eid T, Lee TS, Thomas MJ, Amiry-Moghaddam M, Bjornsen LP, SpencerDD, Agre P, Ottersen OP, de Lanerolle NC.2005. Loss of perivascularaquaporin4may underlie deficient water and K homeostasis in the humanepileptogenic hippocampus. Proc Natl Acad Sci USA102:1193-1198.
37.Strohschein S, Huttmann K, Gabriel S, Binder DK, Heinemann U,Steinhauser C.2011. Impact of aquaporin-4channels on K buffering andgap junction coupling in the hippocampus. Glia59:973-980.
38.Tian GF, Azmi H, Takano T, Xu Q, Peng W, Lin J, Oberheim N, Lou N,Wang X, Zielke HR, Kang J, Nedergaard M.2005. An astrocytic basis ofepilepsy. Nat Med11:973-981.
39.Takahashi DK, Vargas JR, Wilcox KS.2010. Increased coupling andaltered glutamate transport currents in astrocytes following kainic-acid-induced status epilepticus. Neurobiol Dis40:573-585.
40.Vezzani A, Balosso S, Ravizza T.2008. The role of cytokines in thepathophysiology of epilepsy. Brain Behav Immun22:797-803.
41.Vezzani A, Granata T.2005. Brain inflammation in epilepsy: experimen-taland clinical evidence. Epilepsia46:1724-1743.
42.Marchi N, Fan Q, Ghosh C, Fazio V, Bertolini F, Betto G, Batra A, CarltonE, Najm I, Granata T, Janigro D.2009. Antagonism of peripheralinflammation reduces the severity of status epilepticus. Neurobiol Dis33:171-181.
43.Marchi N, Granata T, Freri E, Ciusani E, Ragona F, Puvenna V, Teng Q,Alexopolous A, Janigro D.2011. Efficacy of anti-inflammatory therapy in amodel of acute seizures and in a population of pediatric drug resistantepileptics. PLoS One6:e18200.
44.Sayyah M, Beheshti S, Shokrgozar MA, Eslami-far A, Deljoo Z, KhabiriAR, Haeri Rohani A.2005. Antiepileptogenic and anticonvulsant activityof interleukin-1beta in amygdala-kindled rats. Exp Neurol191:145-153.
45.Arganaraz GA, Konno AC, Perosa SR, Santiago JF, Boim MA, Vidotti DB,Varella PP, Costa LG, Canzian M, Porcionatto MA, Yacubian EM,Sakamoto AC, Carrete H Jr, Centeno RS, Amado D, Cavalheiro EA, JuniorJA, Mazzacoratti Mda G.2008. The renin-angiotensin system isupregulated in the cortex and hippocampus of patients with temporal lobeepilepsy related to mesial temporal sclerosis. Epilepsia49:1348-1357.
46.de Moura JC, Tirapelli DP, Neder L, Saggioro FP, Sakamoto AC, VelascoTR, Panepucci RA, Leite JP, Assirati JAJ, Colli BO, CarlottiCGJ.2012.Amygdala gene expression of NMDA and GABAA receptors in patientswith mesial temporal lobe epilepsy. Hippocampus22:92-97.
47.Marchetti C, Gavazzo P, Stafford GI, Van Staden J.2011. South Africanplants used in traditional medicine to treat epilepsy have an antagonisticeffect on NMDA receptor currents. J Ethnopharmacol137:382-388.
48.Sandoval R, Gonzalez A, Caviedes A, Pancetti F, Smalla KH, Kaehne T,Michea L, Gundelfinger ED, Wyneken U.2011. Homeostatic NMDAreceptor down-regulation via brain derived neurotrophic factor and nitricoxide-dependent signalling in cortical but not in hippocampal neurons. JNeurochem118:760-772.
49.Chamberlain SE, Jane DE, Jones RS.2012. Pre-and post-synapticfunctions of kainate receptors at glutamate and GABA synapses in the ratentorhinal cortex. Hippocampus22:555-576.
50.Li JM, Zeng YJ, Peng F, Li L, Yang TH, Hong Z, Lei D, Chen Z, Zhou D.
2010. Aberrant glutamate receptor5expression in temporal lobe epilepsylesions. Brain Res1311:166–174.
51.Zhang J, Yan H, Wu YP, Li C, Zhang GY.2011. Activation of GluR6-containing kainate receptors induces ubiquitin-dependent Bcl-2degradation via denitrosylation in the rat hippocampus after kainatetreatment. J Biol Chem286:7669–7680.
52.Fritschy JM, Kiener T, Bouilleret V, Loup F.1999. GABAergic neuronsand GABAA-receptors in temporal lobe epilepsy. Neurochem Int34:435–445.
53.Oliveira MS, Skinner F, Arshadmansab MF, Garcia I, Mello CF, Knaus HG,Ermolinsky BS, Otalora LF, Garrido-Sanabria ER.2010. Alteredexpression and function of small-conductance (SK) Ca2+-activated K+channels in pilocarpine-treated epileptic rats. Brain Res1348:187–199.
54.Riazanski V, Deriy LV, Shevchenko PD, Le B, Gomez EA, Nelson DJ.
2011. Presynaptic CLC-3determines quantal size of inhibitory trans-mission in the hippocampus. Nat Neurosci14:487–494.
55.Miceli F, Soldovieri MV, Iannotti FA, Barrese V, Ambrosino P, Martire M,Cilio MR, Taglialatela M.2011. The voltage-sensing domain of Kv7.2channels as a molecular target for epilepsy-causing mutations andanticonvulsants. Front Pharmacol2:2.
56.Das A, McDowell M, O’Dell CM, Busch ME, Smith JA, Ray SK, BanikNL.2010. Post-treatment with voltage-gated Na channel blocker attenuateskainic acid-induced apoptosis in rat primary hippocampal neurons.Neurochem Res35:2175–2183.
57.Stucki BL MD, Bolognani F, Perrone-Bizzozero NI, Banik NL, Grie-semer DA, Ray SK.2004. Upregulation of calpain in neuronal apoptosis inkainic acid induced seizures in rats. J Neurochem90(Suppl1):8.
58.Araujo IM, Carreira BP, Carvalho CM, Carvalho AP.2010. Calpains anddelayed calcium deregulation in excitotoxicity. Neurochem Res35:1966–1969.
59.Jantas D, Lorenc-Koci E, Kubera M, Lason W.2011. Neuroprotectiveeffects of MAPK/ERK1/2and calpain inhibitors on lactacystin-inducedcell damage in primary cortical neurons. Neurotoxicology32:845-856.
60.Kaur J, Mencl S, Sahaboglu A, Farinelli P, van Veen T, Zrenner E, EkstromP, Paquet-Durand F, Arango-Gonzalez B.2011. Calpain and PARPactivation during photoreceptor cell death in P23H and S334ter rhodopsinmutant rats. PLoS One6:e22181.
61.McDowell ML, Das A, Smith JA, Varma AK, Ray SK, Banik NL.2011.Neuroprotective effects of genistein in VSC4.1motoneurons exposed toactivated microglial cytokines. Neurochem Int59:175–184.
62.Mustafa AG, Wang JA, Carrico KM, Hall ED.2011. Pharmacologicalinhibition of lipid peroxidation attenuates calpain-mediated cytoskeletaldegradation after traumatic brain injury. J Neurochem117:579–588.
63.Ray SK, Samantaray S, Smith JA, Matzelle DD, Das A, Banik NL.2011.Inhibition of cysteine proteases in acute and chronic spinal cord injury.Neurotherapeutics8:180–186.
64.Yellanki S, Jagtap S, Masareddy R.2011. Dissofilm: a novel approach fordelivery of phenobarbital; design and characterization. J Young Pharm3:181-188.
65.Mantegazza M, Curia G, Biagini G, Ragsdale DS, Avoli M.2010.Voltage-gated sodium channels as therapeutic targets in epilepsy and otherneurological disorders. Lancet Neurol9:413–424.
66.Lǒscher W, Schmidt D.2011. Modern antiepileptic drug development hasfailed to deliver: ways out of the current dilemma. Epilepsia52:657–678.
67.Shorvon SD.2009. Drug treatment of epilepsy in the century of the ILAE:the second50years,1959–2009. Epilepsia50(Suppl3):93–130.
68.Olkkola KT, Ahonen J.2008. Midazolam and other benzodiazepines. HdbkExp Pharmacol182:335–360.
69.Johannessen Landmark C, Patsalos PN.2010. Drug interactions involvingthe new second-and third-generation antiepileptic drugs. Expert RevNeurother10:119-140.
70.Chong ZZ, Shang YC, Zhang L, Wang S, Maiese K.2010. Mammaliantarget of rapamycin: hitting the bull’s-eye for neurological disorders. OxidMed Cell Longev3:374–391.
71.Carson RP, Van Nielen DL, Winzenburger PA, Ess KC.2012. Neuronal andglia abnormalities in Tsc1-deficient forebrain and partial rescue byrapamycin. Neurobiol Dis45:369-380.
72.Buckmaster PS, Wen X.2011. Rapamycin suppresses axon sprouting bysomatostatin interneurons in a mouse model of temporal lobe epilepsy.Epilepsia52:2057-2064.
73.Wong M.2011. Rapamycin for treatment of epilepsy: antiseizure, anti-epileptogenic, both, or neither? Epilepsy Curr11:66–68.