左旋组氨酸对匹罗卡品致痫大鼠癫痫发作和海马区神经细胞凋亡的影响

目的:观察左旋组氨酸(L-His)对匹罗卡品(PLO)致痫大鼠皮质脑电图及大鼠海马各区神经细胞凋亡的影响。方法:雄性SD大鼠30只,随机平均分为对照组(匹罗卡品组)、干预组(匹罗卡品+左旋组氨酸组)。各组给予相关干预处理后腹腔注射匹罗卡品建立癫痫模型,行皮质脑电图观察及海马区细胞凋亡染色观察。结果:经皮质脑电图显示,干预组潜伏期延长、痫波频率及大发作次数较对照组明显降低,差异有统计学意义(P<0.05)。凋亡染色显示,对照组的海马各区细胞在各时间点的凋亡数明显高于干预组,差异具有统计学意义(P<0.05)。结论:左旋组氨酸可以延迟匹罗卡品致痫的形成并降低点燃后癫痫发作程度,降低海马各区细胞凋亡数值,提示左旋组氨酸具有抗痫作用。     

锂-匹罗卡品颞叶癫模型的建立及苔藓纤维出芽的动态变化

目的研究锂-匹罗卡品颞叶癫模型大鼠致后性发作的行为学特点及海马结构病理改变的动态变化。方法将所有Wistar大鼠随机分为对照组和实验组,实验组大鼠腹腔依次注射氯化锂、匹罗卡品诱发癫持续状态（SE）后,观察其自发性癫发作（SRS）,分别于SE后1周至10周5个不同时间点取材,Nissl染色和Timm染色分别观察海马神经元损伤及苔藓纤维出芽（MFS）的变化。结果注射匹罗卡品后84%的大鼠可诱发出SE,经过10～20d的缄默期后,可观察到Ⅰ～Ⅲ级的反复SRS,病理学检查可见海马神经元的损伤及齿状回内分子层MFS。结论锂-匹罗卡品颞叶癫模型与人类颞叶癫有类似发作特点及病理改变,是一种理想的颞叶癫动物模型。     

口服补液盐降低氯化锂-匹罗卡品惊厥持续状态大鼠死亡率的研究

目的探讨通过胃管灌胃改良口服补液盐(oral rehydration salts,ORS)降低氯化锂-匹罗卡品诱发惊厥持续状态(status epilepticus,SE)模型的死亡率的方法。方法选用成年组(60 d)SD大鼠30只和幼年组(20 d)SD大鼠45只,随机分为成年SE+ORS组、成年SE组、幼年SE+ORS组、幼年SE组、幼年血糖组各15只。经腹腔注射氯化锂和匹罗卡品,诱发大鼠惊厥发作,惊厥60 min后,给予安定止惊。各ORS组于SE后1、12、24 h根据不同年龄予以不同剂量改良ORS灌胃。其余各组不给予处理。记录实验大鼠惊厥发作情况,惊厥前后体重变化及死亡情况;血糖测定组于SE后8 h取血检测血糖,探索降低该模型死亡率的方法。结果 (1)各组惊厥后体重均降低,约占体重的5%~8%。(2)氯化锂-匹罗卡品惊厥持续状态模型死亡率较高,幼年组40.00%,成年组57.14%;死亡发生时间集中在惊厥后24 h内,72 h后死亡数趋于平稳。(3)SE后实验大鼠血糖明显降低,给予ORS液灌胃后,血糖趋于正常水平。(4)以改良ORS灌胃后,幼年组和成年组72 h内死亡率明显降低。结论 SE后改良ORS液灌胃有效改善实验动物死亡率,是有效提高其生存率的方法。     

MicroRNA-134/CREB/pCREB通路在癫痫中的表达及意义

目的探讨miR-134、CREB、pCREB在癫痫大鼠海马及难治性癫痫患者颞叶脑组织中的表达及意义。方法难治性癫痫患者及非癫痫对照组颞叶组织、氯化锂-匹罗卡品癫痫大鼠及空白对照组海马组织中,应用实时荧光定量PCR技术检测microRNA-134（miR-134）的表达,用Western blot方法检测CREB及p CREB的表达,用免疫组织化学方法检测人脑颞叶皮质及大鼠海马区CREB、p CREB的表达。结果与对照组相比miR-134表达在难治性癫痫患者中明显降低（P〈0.05）,在癫痫模型组中点燃后3、7、14、60 d明显降低（P〈0.05）,1 d与30 d表达降低较对照组差异无显著性（P〉0.05）;癫痫模型组CREB在3、7、14、60 d时间点明显升高（P〈0.05）、pCREB各时间点表达均高于空白对照组（P〈0.05）。结论难治性癫痫患者颞叶皮质及癫痫动物海马中miR-134表达下降,CREB、pCREB表达升高,提示其可能在癫痫发生发展机制中起重要作用。     

鞘氨醇激酶1和1-磷酸鞘氨醇受体2在癫痫大鼠海马组织中表达的变化*

目的:检测鞘氨醇激酶1 (SphK1)和1-磷酸鞘氨醇受体2 (S1PR2) 在癫痫大鼠海马中的表达,探讨SphK1和S1PR2在癫痫中的作用机制。方法:成年雄性SD大鼠108只,随机分为对照(Control)组(n=48)和癫痫(PILO)组(n=60)。癫痫组腹腔注射氯化锂(127 mg/kg),18～20 h后注射匹罗卡品,首剂量为30 mg/kg,发作＜IV级的大鼠重复注射匹罗卡品(10 mg/kg);对照组给予等剂量的生理盐水代替匹罗卡品。根据造模后观察时间和行为学改变,随机分为3个大组,6个亚组:急性期组(E6 h、E1 d、E3 d)、潜伏期组(E7 d)和慢性期组(E30 d、E56 d),每个亚组中对照大鼠和癫痫大鼠各8只。每组取4只大鼠麻醉取海马,另4只取大脑组织。运用Western blot检测SphK1、S1PR2在大鼠海马组织中的表达变化,免疫荧光检测星形胶质细胞活化增生情况及SphK1、S1PR2在星形胶质细胞中的定位表达。结果:与Control组比较,SphK1在造模后急性期(E3 d)、潜伏期(E7 d)和慢性期(E30 d、E56 d)海马中的表达均明显升高(P＜0.05或P＜0.01);S1PR2在急性期(E3 d)、潜伏期(E7 d)和慢性期(E30 d、E56 d)海马组织中的表达均明显下降(P＜0.05或P＜0.01);癫痫大鼠(E7 d)海马星形胶质细胞活化、增生明显(P＜0.05),SphK1和S1PR2在E7d的表达到位为海马星形胶质细胞中。结论:SphK1和S1PR2可能通过调控海马星形胶质细胞活化增生和影响神经元兴奋性参与了癫痫的发病。     

不同剂量瑞舒伐他汀对急性冠脉综合征患者炎症反应及预后的影响

目的:探讨不同剂量瑞舒伐他汀对急性冠脉综合征患者(ACS)炎症反应及预后的影响。方法:选择2014年5月至2015年10月间收治的120例ACS患者为研究对象,随机分为A、B、C三组,每组40例。所有患者先给予常规治疗,A组患者给予20mg/d普伐他汀治疗;B组患者给予10 mg/d瑞舒伐他汀;C组患者给予20 mg/d瑞舒伐他汀,3组均持续治疗6个月。比较3组患者治疗前后血脂、超敏C反应蛋白(hs-CRP)、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)的水平;随访6个月心血管事件发生情况。结果:3组患者治疗后hs-CRP、TNF-α、IL-6水平均明显下降,且C组下降最明显,其次为B组,比较差异均有统计学意义(P0.05);治疗后血脂水平均的改善,且C组改善最明显,其次是B组,比较差异均有统计学意义(P0.05);随访6个月,C组心血管事件发生率为2.5%,远低于B组(10.0%)及A组(20.0%),差异具有显著性(P0.05)。结论:大剂量(20 mg/d)瑞舒伐他汀治疗ACS降脂、抗炎效果明显,预后相对较好,值得临床推广使用。     

白芍总苷治疗慢性荨麻疹的疗效观察

目的:观察白芍总苷治疗慢性荨麻疹的疗效及不良反应.方法:采用随机对照临床试验将60例符合纳入标准的患者随机分为三组,分别为白芍总苷胶囊1.8 g/d加依匹斯汀10 mg/d组(A组),依匹斯汀加复方甘草酸苷150 mg/d组(B组)和单用依匹斯汀10 mg/d组(C组).疗程均为12周.治疗前后检查患者的血、尿、粪常规及肝、肾功,同时观察记录患者的不良反应.结果:A组有效率为90.0％,B组有效率为85.0％,C组有效率为55.0％.A组和B组有效率差异无统计学意义(P＞0.05);A、B组疗效高于对照组,差异有统计学意义(P＜0.05).三组的不良反应均较少.结论:白芍总苷联合依匹斯汀治疗慢性荨麻疹疗效显著且不良反应少,是慢性荨麻疹可供选择的有效治疗方法.     

不同剂量盐酸羟考酮复合右美托咪定对开腹手术患者镇痛效应及血流动力学的影响

目的:探讨不同剂量盐酸羟考酮复合右美托咪定对开腹手术患者镇痛效应及血流动力学的影响。方法:选择从2015年3月到2017年3月期间在我院接受开腹手术治疗的患者100例进行研究。根据随机数字表法对患者进行分组,A组(应用1.0 mg/kg的盐酸羟考酮及2.5μg/kg的右美托咪定麻醉)、B组(应用0.75 mg/kg的盐酸羟考酮及2.5μg/kg的右美托咪定麻醉)、C组(应用0.5 mg/kg的盐酸羟考酮及2.5μg/kg的右美托咪定麻醉)和D组(应用1.0 mg/kg的盐酸羟考酮麻醉),每组各25例。对比各组镇痛效应满意度以及麻醉诱导前(T0)、插管即刻(T1)、插管后5 min(T2)时的血流动力学指标,并统计各组不良反应情况。结果:A、B、C三组的镇痛效应满意度均分别明显高于D组,差异均有统计学意义(均P0.05),A、B、C三组的镇痛效应满意度对比差异无统计学意义(P0.05)。A、B组T1时的收缩压(SBP)、舒张压(DBP)、平均动脉压(MAP)及心率(HR)水平均分别高于C、D组,差异有统计学意义(P0.05);T0和T2时,各组的SBP、DBP、MAP及HR水平相比差异均无统计学意义(均P0.05)。各组T1时SBP、DBP、MAP及HR水平与T0时相比明显上升,而T2时又明显下降,差异均有统计学意义(P0.05)。B组不良反应总发生率为12.00%,明显低于A组的36.00%、C组的44.00%及D组的40.00%,差异均有统计学意义(均P0.05)。结论:不同剂量的盐酸羟考酮与右美托咪定复合麻醉对开腹手术患者的镇痛效应较好,并对患者的血流动力学水平均造成一定影响,但随着时间的延长而逐渐减弱,2.5μg/kg的右美托咪定以及0.75 mg/kg的盐酸羟考酮复合麻醉的安全性较高,值得在临床上推广应用。     

脑内5-羟色胺减少对致痫大鼠癫痫发作及学习记忆的影响

目的:于中脑正中中缝核局部微量注射5,7-二羟色胺(5,7-DHT),探讨5-羟色胺(5-HT)与癫痫的关系及匹罗卡品(PILO)致痫大鼠学习记忆改变的可能机制。方法:成年SD大鼠随机分为PILO组、PILO+5,7-DHT组、空白对照组三组,然后根据是否出现癫痫持续状态(SE)再将PILO组分成:PILO+SE组和PILO-SE组两亚组;利用视频脑电图观察大鼠癫痫发作及皮层脑电变化;运用Morris水迷宫测评大鼠空间学习记忆水平;最后运用免疫组化法观察大鼠中缝核5-HT能神经元。结果:大鼠予以5,7-DHT(PILO+5,7-DHT组)处理后造模成功率、死亡率及慢性期自发性发作频率均增高;与空白组比较PILO+SE组中缝核5-HT能神经元数目有所下降(P<0.05),而PILO+5,7-DHT组下降更明显(P<0.01);与空白组比较PILO+SE组平均逃避潜伏期延长、穿越平台次数减少、原平台象限停留时间缩短(P<0.05),而与PILO+SE组比较PILO+5,7-DHT组变化不明显。结论:脑内5-HT水平的降低容易诱发癫痫发作,尚不能认为癫痫大鼠合并认知功能障碍与脑内5-HT水平下降有关。     

匹罗卡品癫痫模型中海马区TREK-2钾离子通道表达变化及意义

目的:研究匹罗卡品癫痫模型中海马区TREK-2双孔钾离子通道的表达变化,初步探讨TREK-2在癫痫发病过程中的机制及意义。方法:选用成年雄性SD大鼠腹腔注射氯化锂-匹罗卡品(lithium-pilocarpine)构建癫痫模型,分别在癫痫持续状态(status epilepticus,SE)后不同时间点(6 h、1 d、3 d、1 w、2 w、4 w、8 w)提取海马组织,利用western-blot检测海马区TREK-2随时间表达变化。并用TREK-2 si RNA下调海马区TREK-2表达,进一步观察对大鼠癫痫状态的影响。结果:与对照组相比,TREK-2在诱导癫痫持续状态发作后的3d开始降低(P0.05),1 w,2 w,4 w明显降低(P0.01),8 w时仍维持在很低水平(P0.001)。在TREK-2表达下调后,大鼠癫痫潜伏时间(latent period)明显缩短,癫痫持续状态1 h 5级以上发作频率(seizure frequency)明显增加。结论:TREK-2在氯化锂-匹罗卡品致痫大鼠海马组织中表达的降低,且其下调加重癫痫状态的事实提示TREK-2参与了癫痫的发生发展过程。     

Hippocampal desynchronization of functional connectivity prior to the onset of status epilepticus in pilocarpine-treated rats

Status epilepticus (SE), a pro-epileptogenic brain insult in rodent models of temporal lobe epilepsy, is successfully induced by pilocarpine in some, but not all, rats. This study aimed to identify characteristic alterations within the hippocampal neural network prior to the onset of SE. Sixteen microwire electrodes were implanted into the left hippocampus of male Sprague-Dawley rats. After a 7-day recovery period, animal behavior, hippocampal neuronal ensemble activities, and local field potentials (LFP) were recorded before and after an intra-peritoneal injection of pilocarpine (350 mg/kg). The single-neuron firing, population neuronal correlation, and coincident firing between neurons were compared between SE (n?=?9) and nonSE rats (n?=?12). A significant decrease in the strength of functional connectivity prior to the onset of SE, as measured by changes in coincident spike timing between pairs of hippocampal neurons, was exclusively found in SE rats. However, single-neuron firing and LFP profiles did not show a significant difference between SE and nonSE rats. These results suggest that desynchronization in the functional circuitry of the hippocampus, likely associated with a change in synaptic strength, may serve as an electrophysiological marker prior to SE in pilocarpine-treated rats.     

Effects of L-arginine on prevention and treatment of lithium-pilocarpine-induced status epilepticus

The effects of various doses of L-arginine, a nitric oxide substrate, on lithium-pilocarpine-induced seizures were studied in rats. Rats were implanted with chronic, stainless steel screw electrodes epidurally for electrocortical recordings. A control group received 3 mEq/kg LiCl (i.p.) and 24 h later 45 mg/kg pilocarpine HCl (i.p.). Two different experimental procedures were followed: (1) L-arginine was applied in doses of 100 mg/kg, 300 mg/kg or 500 mg/kg (i.p.), 30 min before pilocarpine injection; (2) 300 mg/kg, 500 mg/kg or 1000 mg/kg (i.p.) L-arginine was injected either 5 min or 30 min after the onset of status epilepticus (SE). L-arginine (300 mg/kg) injected 30 min before pilocarpine significantly reduced the percentage of SE, but did not change the latency to SE or 24-hour survival. These parameters were not significantly affected by the 100 mg/kg or 500 mg/kg dose of L-arginine. On the other hand, no dose of L-arginine that was applied after SE had begun, had any significant influence on the seizures. We concluded that L-arginine may prevent seizure activity in some but not all doses, and does not have any effect on the ongoing seizure activity.     

Does Pilocarpine-Induced Epilepsy in Adult Rats Require Status epilepticus?

Pilocarpine-induced seizures in rats provide a widely animal model of temporal lobe epilepsy. Some evidences reported in the literature suggest that at least 1 h of status epilepticus (SE) is required to produce subsequent chronic phase, due to the SE-related acute neuronal damage. However, recent data seems to indicate that neuro-inflammation plays a crucial role in epileptogenesis, modulating secondarily a neuronal insult. For this reason, we decided to test the following hypotheses: a) whether pilocarpine-injected rats that did not develop SE can exhibit long-term chronic spontaneous recurrent seizures (SRS) and b) whether acute neurodegeneration is mandatory to obtain chronic epilepsy. Therefore, we compared animals injected with the same dose of pilocarpine that developed or did not SE, and saline treated rats. We used telemetric acquisition of EEG as long-term monitoring system to evaluate the occurrence of seizures in non-SE pilocarpineinjected animals. Furthermore, histology and MRI analysis were applied in order to detect neuronal injury and neuropathological signs. Our observations indicate that non-SE rats exhibit SRS almost 8 (+/22) months after pilocarpine-injection, independently to the absence of initial acute neuronal injury. This is the first time reported that pilocarpine injected rats without developing SE, can experience SRS after a long latency period resembling human pathology. Thus, we strongly emphasize the important meaning of including these animals to model human epileptogenesis in pilocarpine induced epilepsy.     

Brain-derived neurotrophic factor superinduction parallels anti-epileptic--neuroprotective treatment in the pilocarpine epilepsy model

Antiepileptic drugs provide neuroprotection in several animal models of brain damage, including those induced by status epilepticus (SE). The mechanisms involved in this action are unknown, but neurotrophic factors such as brain-derived neurotrophic factor (BDNF) may play a role. In this study we investigated the changes in BDNF levels in rats in which SE had been induced by pilocarpine injection (400 mg/kg i.p.) and continued for several hours (unprotected group). In other animals (protected groups), SE was suppressed after 30 min by intraperitoneal injection of either diazepam (10 mg/kg) + pentobarbital (30 mg/kg) or paraldehyde (0.3 mg/kg). In diazepam + pentobarbital-treated rats the hippocampal damage caused by SE was significantly lower (p < 0.05) than in unprotected animals. In addition, 2 and 24 h after pilocarpine injection, the levels of BDNF mRNA were moderately increased in the unprotected group, but 'superinduced' in protected animals, especially in the neocortex and hippocampus. A time-dependent increase in BDNF immunoreactivity was also found by western blot analysis in rats treated with diazepam + pentobarbital. In contrast, a decrease of BDNF immunoreactivity occurred in the unprotected group. In conclusion, these results show that neuroprotection induced by anti-epileptic drugs in pilocarpine-treated rats is accompanied by strong potentiation of BDNF synthesis in brain regions involved in SE.     

Differences in sensitivity to the convulsant pilocarpine in substrains and sublines of C57BL/6 mice

In rodents, the cholinomimetic convulsant pilocarpine is widely used to induce status epilepticus (SE), followed by hippocampal damage and spontaneous recurrent seizures, resembling temporal lobe epilepsy. This model has initially been described in rats, but is increasingly used in mice, including the C57BL/6 (B6) inbred strain. In the present study, we compared the effects of pilocarpine in three B6 substrains (B6JOla, B6NHsd and B6NCrl) that were previously reported to differ in several behavioral and genetic aspects. In B6JOla and B6NHsd, only a small percentage of mice developed SE independently of whether pilocarpine was administered at high bolus doses or with a ramping up dosing protocol, but mortality was high. The reverse was true in B6NCrl, in which a high percentage of mice developed SE, but mortality was much lower compared to the other substrains. However, in subsequent experiments with B6NCrl mice, striking differences in SE induction and mortality were found in sublines of this substrain coming from different barrier rooms of the same vendor. In B6NCrl from Barrier #8, administration of pilocarpine resulted in a high percentage of mice developing SE, but mortality was low, whereas the opposite was found in B6NCrl mice from four other barriers of the same vendor. The analysis of F1 mice from a cross of female Barrier 8 pilocarpine‐susceptible mice with resistant male mice from another barrier (#9) revealed that F1 male mice were significantly more sensitive to pilocarpine than the resistant parental male mice whereas female F1 mice were not significantly different from resistant Barrier 9 females. These observations strongly indicate X‐chromosome linked genetic variation as the cause of the observed phenotypic alterations. To our knowledge, this is the first report which demonstrates that not only the specific B6 substrain but also sublines derived from the same substrain may markedly differ in their response to convulsants such as pilocarpine. As the described differences have a genetic basis, they offer a unique opportunity to identify the genes and pathways involved and contribute to a better understanding of the underlying molecular mechanisms of seizure susceptibility.     

Effects of a free radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN) on short-term recovery of immature rats after status epilepticus

The present study examined the effects of a free radical scavenger, N-tert-butyl-alfa-phenylnitrone (PBN) on lithium-pilocarpine-induced status epilepticus (SE) and its short-term consequences in rats 12 (P12) or 25 (P25) days old. PBN (2 x 100 mg/kg i.p.) was injected according to the following schedules: 1) PBN-pretreated animals received the first dose 30 min prior to pilocarpine, the second dose was given 1 min after SE onset, and 2) PBN-treated animals received the first dose of PBN 1 min after SE onset and the second one 60 min later. Paraldehyde was administered to decrease mortality. Effects of PBN were highly age-dependent. In P25 group, PBN-pretreatment increased latency to SE onset and significantly suppressed the severity of motor manifestation of SE. Both PBN pretreatment and treatment improved recovery after SE. In contrast, administration of PBN in P12 animals did not affect SE pattern or recovery after SE. Administration of PBN had no effects on the motor performance of animals 3 and 6 days after SE. Neuronal damage was examined 24 h and 7 days after SE using Fluoro-Jade B staining. Mild neuroprotective effects of PBN in hippocampal fields CA1 and CA3 occurred in P25 rats in both experimental schedules. In contrast, administration of PBN aggravated neuronal injury in the hippocampus in P12 rats. Administration of PBN to intact rats did not induce neurodegeneration in either age group.     

Calpain I Activity and Its Relationship with Hippocampal Neuronal Death in Pilocarpine-Induced Status Epilepticus Rat Model

This study aims to establish pilocarpine-induced rat model of status epilepticus (SE), observe the activity of calpain I in the rat hippocampus and the subsequent neuronal death, and explore the relationship between calpain I activity and neuronal death in the hippocampus. Fifty-eight adult male Wistar rats were assigned randomly into either control group (n = 8) or epilepsy group (n = 50). SE was induced in the epilepsy group using pilocarpine. Before the injection, the rats were given atropine sulfate to reduce the side effect of pilocarpine. All rats in the seizure group were grouped into either SE or non-SE, depending on whether they developed convulsive seizures. The rats in SE group were treated with chloral hydrate to stop seizures after 60 min. Control animals were treated with the same dose of 0.9 % saline. All rats were monitored for seizures. At 24 h after SE, the rats’ left brain tissues were stained by HE and TUNEL. Neuronal necrosis and apoptosis in the hippocampal CA3 area were observed. Calpain I activity in the right hippocampus was also observed using western blotting. Eighty percent of the rats in the seizure group developed SE, of which 35 % died. No rat died in both the control and non-SE groups. At 24 h after SE, the number of HE-stained neurons decreased (SE group: 55.19 ± 8.23; control group: 102.13 ± 3.73; non-SE group: 101.2 ± 2.86) and the number of TUNEL-positive neurons increased (SE group: 4.91 ± 1.35; non-SE and control group: 0). No obvious changes were observed in the neurons of the control and non-SE group animals. The 76 kDa cleavage of calpain I (the average optical density ratio is 0.096 ± 0.015) emerged in the SE group. Neuronal death has a direct relationship with calpain I activity. There is high success rate and lower death rate for pilocarpine to induce SE. At 24 h after SE, activity of calpain I, neuronal necrosis and apoptosis increased in the hippocampus. Neuronal death has a direct relationship with calpain I activity, which suggests that calpain I plays an important role in neuronal damage during SE.