脊髓全横断大鼠模型的构建☆

背景：脊髓全横断模型在造模时常难以保证神经纤维的完全离断。 目的：构建大鼠脊髓全横断损伤模型。 方法：将大鼠随机分为模型组和假手术组。模型组构建脊髓T10节段全横断模型;假手术组动物仅打开椎管与硬脊膜而后缝合,但不损伤脊髓。建模后1,3,5,7 d分别进行BBB评分以评估后肢运动功能,检测其体感诱发电位和运动诱发电位来评估神经传导通路的完整性,并行形态学观察来评估脊髓肉眼观病理形态。 结果与结论：与假手术组相比,模型组大鼠在建模后1,3,5,7 d时,其BBB评分降低(P < 0.01),未检测出体感和运动诱发电位。形态学观察结果显示模型组大鼠脊髓完全横断,而假手术组脊髓形态完整。结果提示实验成功构建了大鼠脊髓全横断模型。     

大鼠脊髓半横断损伤后神经营养因子bFGF、GDNF的表达变化

目的探讨脊髓半横断损伤后早期不同时间碱性纤维母细胞生长因子(bFGF)、胶质细胞源性神经营养因子(GDNF)的表达变化. 方法在成年SD大鼠脊髓T9～T10间半横断,取损伤位点尾侧段T10节段制作冰冻切片,运用bFGF、GDNF兔抗血清以免疫组化亲合素-生物素-过氧化物酶复合物法(ABC法)染色.观察并计数腹角bFGF、GDNF的阳性神经元数.结果 bFGF、GDNF主要分布于正常大鼠脊髓腹角神经元细胞浆,损伤后腹角bFGF、GDNF阳性神经元数在3 d组(n=6)、7 d组(n=6)、21 d组(n=6)均较假手术组(n=6)明显增加(P<0.01),bFGF阳性神经元数在术后3 d时达高峰,随伤后时间的延长进行性减少(P<0.01).而GDNF阳性神经元数在术后7 d时达高峰,3 d组与21 d组比较没有显著差异.结论脊髓半横断损伤(hSCI)后bFGF、GDNF表达明显增加,提示它们可能在hSCI早期修复中发挥作用.     

大鼠脊髓半横断损伤后凋亡相关基因的表达变化

目的探讨脊髓半横断损伤后凋亡相关基因Bc l-2、Bax、Fas在蛋白水平的表达变化规律及神经细胞凋亡的分子生物学机制。方法在成年SD大鼠脊髓T9~T10间半横断,取损伤位点尾侧段T10节段制作冰冻切片,运用Bc l-2、Bax、Fas兔抗血清以免疫组化亲合素-生物素-过氧化物酶复合物法(ABC)法染色。观察并计数腹角Bc l-2、Bax、Fas的阳性神经元数。结果Bc l-2、Bax、Fas主要分布于正常大鼠脊髓腹角神经元细胞浆,损伤后腹角Bc l-2、Bax、Fas阳性神经元数在3 d组(n=6)、7 d组(n=6)、21 d组(n=6)均较假手术组(n=6)明显增加(P<0.01),Bax、Fas阳性神经元数在术后3d时达高峰,随伤后时间的延长进行性减少(P<0.05)。Bc l-2阳性神经元数在术后7 d时达高峰,3 d组与21 d组比较没有显著差异。结论脊髓半横断损伤(hSC I)后,由Fas抗原参与的死亡受体途径及Bc l-2、Bax参与的线粒体途径均参与了hSC I后细胞的凋亡过程。     

动脉移植BMSCs对大鼠缺血再灌注损伤脊髓CNTF和STAT3的影响

目的探讨肾下腹主动脉移植骨髓间充质干细胞(BMSCs)对大鼠缺血再灌注损伤脊髓CNTF和STAT3的影响及对损伤脊髓功能恢复的作用。方法 24只成年SD雌性大鼠随机分为假手术组、对照组及移植组3组,每组8只。术后对大鼠进行后肢神经行为学评分和运动诱发电位检测,采用RT-PCR、Western blot检测大鼠缺血节段脊髓内CNTF和STAT3的表达变化。结果与假手术组比较,对照组和移植组BBB评分于术后0 d、5 d、10 d、15 d显著降低(P<0.01),MEP潜伏期延长(P<0.01)、波幅减小(P<0.01),脊髓CNTF和STAT3 mRNA和蛋白表达增加(P<0.01);与对照组比较,移植组BBB评分于术后5¹5 d增高(P<0.01),MEP潜伏期缩短(P<0.01)、波幅增加(P<0.01),CNTF和STAT3 mRNA和蛋白表达增加(P<0.01)。结论肾下腹主动脉移植BMSC可能通过增加损伤脊髓局部CNTF和STAT-3表达促进脊髓缺血再灌注损伤大鼠后肢功能恢复。     

驽药针刺对大鼠脊髓损伤后运动功能及BDNF表达的影响

目的研究驽药针刺在大鼠脊髓损伤后运动功能变化以及BDNF表达的变化。方法采用脊髓半横断损伤模型。100只SD大鼠随机分为对照组、假手术组、脊髓损伤组、单纯针刺组、驽药针刺组,每组分为3天、7天、14天、21天共4个亚组,每组5只。BBB法评定大鼠后肢运动功能变化,免疫组化法检测大鼠脊髓中BDNF的表达变化。结果 BBB评分显示驽药针刺组的各时间点评分均高于脊髓损伤组(P0.05),驽药针刺组7、14、21d的BDNF表达均高于脊髓损伤组(P0.05),且与BBB评分呈正相关(r=0.717,P0.05)。结论驽药针刺可明显改善脊髓损伤大鼠的运动功能,并可明显促进大鼠脊髓损伤后BDNF的表达。     

miR-138对脊髓全横断大鼠行为学的影响

目的探讨miR-138在脊髓全横断大鼠运动功能恢复过程中是否发挥调控作用。方法打开SD大鼠T9-T11椎板,脊髓内分别注射miR-138、DEPC处理水后制备脊髓全横断模型实验组与对照组。术后3d、65d取材,观察脊髓红肿范围、瘢痕长度;期间进行BBB运动功能评分;术后30d应用体感诱发电位(SEP)检测神经功能恢复情况。结果第5～9周miR-138组大鼠BBB评分明显低于对照组(P<0.05)。对照组后肢有运动的大鼠所占比例逐渐增多,而miR-138组该比例未见增加(27.03±16.2,6.5±11.72)(P<0.05)。SEP显示miR-138组大鼠左后肢均未测出潜伏期和波幅,右后肢潜伏期和波幅检出率均为16.7%,明显低于对照组(P<0.05)。术后3dmiR-138组脊髓横断处头端红肿长度较对照组增加(P<0.05)。术后65d脊髓横断处瘢痕明显,与周围软组织及椎骨粘连。miR-138组与对照组比较,瘢痕长度(1.07±0.21,1.03±0.12)cm及周长(1.00±0.17,1.05±0.09)cm均无显著性差异(P>0.05)。结论脊髓全横断大鼠局部应用miR-138后运动功能恢复较对照组...     

孕酮对兔脊髓急性缺血再灌注损伤后细胞凋亡的影响

目的探讨孕酮对兔脊髓急性缺血再灌注损伤后细胞凋亡的影响。方法将90只日本大耳白兔随机分为假手术组(n=30)、脊髓损伤组(n=30)与孕酮治疗组(n=30),各组又进一步分为12 h、24 h、36 h、48 h、72 h、14 d等6个亚组,每亚组5只。假手术组只行腹部切开术但不阻断腹主动脉。脊髓损伤组和孕酮治疗组均制作缺血再灌注模型,制模成功后孕酮治疗组每24h注射孕酮1次(将孕酮溶于玉米油,浓度10 mg/ml,注射剂量为8 ml/kg),脊髓损伤组在相同时间点注射等量生理盐水。采用TUNEl染色评估细胞凋亡,采用BBB评分评估运动功能。结果 HE染色结果显示,假手术组脊髓形态正常;脊髓损伤组可见脊髓结构被破坏,正常神经元数目减少;孕酮治疗组较脊髓损伤组正常神经元数目多。TUNEL染色结果显示,脊髓损伤组和孕酮治疗组各时间点凋亡细胞数均明显高于假手术组(P0.05);孕酮治疗组各时间点凋亡细胞的数目均明显少于脊髓损伤组(P0.05)。假手术组术后12 h BBB评分为17.3分,术后14 d时升高到21.0分;脊髓损伤组术后12 h BBB评分为0.6分,随后缓慢升高,术后14 d时评分为9.2分。孕酮治疗组BBB评分变化与脊髓损伤组相似,但术后36 h开始,各时间点评分均明显高于相应脊髓损伤组(P0.05)。结论孕酮治疗可改善脊髓急性缺血再灌注损伤兔运动功能,可能与抑制细胞凋亡有关。     

脊髓全横断大鼠椎管内睫状神经营养因子抗体封闭模型的建立

【摘要】　目的 建立一种特异性中和内源性CNTF表达的模型,用于研究CNTF在脊髓全横断损伤后的作用机制。方法 健康成年SD雌性大鼠45只,随机分为3组：假手术组15只、对照组15只、抗体封闭组15只。对照组和抗体封闭组大鼠行T11 脊髓全横断术和椎管内置管术,对照组大鼠经导管推注人工脑脊液20μl/只,抗体封闭组大鼠推注CNTF抗体（0.1g/ml）20μl/只。假手术组仅行T9椎板切除术。观察动物术后存活情况及24h时BBB评分,应用免疫组织化学和Western-blot技术检测术后24h、48h、72h 脊髓颈、胸、腰段CNTF的分布及表达变化。 结果　术后动物存活率100％,对照组和抗体封闭组BBB评分显著低于假手术组（Ρ<0.01）;对照组术后各时间点大量CNTF阳性细胞分布于灰质前角及白质前索、后索和外侧索;抗体封闭组T9、L2节段术后24h时未见CNTF阳性细胞,术后48h、72h出现阳性细胞,C5节段术后3个时间点均可见阳性细胞分布;Western-blot显示对照组脊髓各节段术后3个时间点CNTF表达量均较假手术组增加（Ρ<0.05）,抗体封闭组T10、L1脊髓术后24h时CNTF表达较对照组减少（Ρ<0.05）,而于48h、72h回升（Ρ<0.01）,C6节段术后各时点CNTF的变化无差异（Ρ>0.05）。 结论 该模型在24h内能够有效封闭内源性CNTF表达,为研究脊髓损伤后CNTF的作用和机制奠定了基础,并能推广应用于其它细胞因子的相关研究。 【关键词】　睫状神经营养因子(CNTF),抗体封闭,脊髓全横断,模型     

神经干细胞动脉移植治疗脊髓损伤后Bcl-2与Bax的变化

目的建立肾下腹主动脉输注神经干细胞(neural stem cell,NSC)治疗脊髓缺血损伤(spinal cord ischemia injury,SCII)大鼠模型,探讨神经干细胞移植治疗脊髓缺血损伤的机制。方法 54只成年SD雌性大鼠随机分为假手术组、对照组和NSC组,每组18只。假手术组进行手术操作,不阻断动脉;对照组大鼠行脊髓缺血再灌注损伤模型,阻断肾下腹主动脉120 min后开放;NSC组完成SCII操作后向留置针缓慢推注体外培养同源NSC100万。每组取5只动物分别于术后5 d、10 d、15 d行BBB评分和运动诱发电位检测,余下每组13只大鼠于术后7 d取材,其中8只动物用于RT-PCR和Western blot检测,5只动物用于Tunnel染色。结果术后7 d,TUNEL染色发现对照组和NSC组L2节段均存在大量凋亡细胞;术后7 d对照组和NSC组Bax和Bcl-2蛋白表达水平较假手术组明显增加(P<0.05),其中NSC组Bax蛋白表达水平低于对照组(P<0.05),Bcl-2蛋白表达水平高于对照组(P<0.05);NSC组Bcl-2/Bax蛋白质表达比值较对照组增加(P<0.05)。BBB评分和MEP显示对照组和NSC组大鼠术后均存在显著功能障碍,其中NSC组大鼠BBB评分于术后10¹5 d高于对照组(P<0.05),且MEP(10 d)的波幅增大(P<0.05)而潜伏期缩短(P<0.05)。结论经股动脉移植NSC,可通过上调Bcl-2表达和下调Bax表达而抑制缺血再灌注损伤脊髓细胞凋亡的发生,从而促进动物后肢运动功能的恢复。     

脊髓缺血再灌注损伤模型的改进及对大鼠神经行为学的影响

目的建立并改进大鼠脊髓缺血再灌注损伤模型,为研究脊髓缺血病理机制和保护策略提供方法学基础。方法 56只成年SD雌性大鼠随机分为7组,每组8只。A组仅行手术操作,不阻断动脉;B、C、D组分别于结扎肾下腹主动脉60min、90min、120min后开放动脉实现脊髓再灌注;E、F、G组电凝肾上腹主动脉发出的椎动脉,再结扎肾下腹主动脉60min、90min、120min后再灌注。分别于术后12h、24h、48h对大鼠进行神经行为学评分,观察术后48h大鼠L2节段病理形态变化并计数前角运动神经元。结果各组行腹主动脉阻断术大鼠BBB评分于术后12h、24h、48h均显著低于A组(P<0.01),F和G组后肢功能障碍最为明显,BBB评分显著低于其它组(P<0.01);术后48h,F组和G组大量神经元坏死,Ⅷ、Ⅸ板层内正常神经元数明显少于其它各组(Ρ<0.01)。结论改良大鼠脊髓缺血再灌注损伤模型能够有效阻断腰段脊髓血液供应,改良模型脊髓常温下耐受缺血时限为90min。     

Epilepsy and anomalies of neuronal migration: MRI and clinical aspects

Neuronal migration disorders are the result of disturbed brain development. In such disorders, neurons are abnormally located. In diagnosing these conditions, magnetic resonance imaging is superior to any other imaging technique. This enables us to improve our knowledge of the clinical correlates of neuronal migration. With reference to migrational disorder, a retrospective study of all 303 patients with epileptic seizures referred for magnetic resonance imaging during a 3-year period was performed, 13 patients (aged 12-41, mean age 27) were identified. They represent 4.3% of the entire study group. Of the patients with known epilepsy, 6.7% and of the mentally retarded, 13.7% had migrational disorders. Four patients had schizencephaly as the dominant finding, one was classified as hemimegalencephaly, 2 had isolated heterotopias, and 6 had localized pachy- and/or poly-microgyria. The clinical pictures are complex. Ectopias of grey matter are recognised foci of epilepsy, but from an epileptological and a clinical viewpoint little attention has been given to these disorders. The present study shows that malmigration is not rare in epilepsy patients, especially not in the mentally retarded.     

Hepatic Considerations in the Use of Antiepileptic Drugs

Summary: Virtually all of the major antiepileptic drugs (AEDs) can cause hepatotoxicity, although fatal hepatic reactions are rare. The mechanisms, incidences, and risk profiles for such reactions differ from drug to drug. With carbamazepine and phenytoin, hepatotoxicity may be due to drug hypersensitivity. Although the profiles of patients at risk have not been well-defined for these two antiepileptic drugs, it would appear from reports in the literature that older adolescents and adults are at higher risk than children of developing serious or fatal hepatotoxicity. Once hepatotoxicity develops, mortality rates are 10–38% with phenytoin and 25% for carbamazepine. The risk profile for valproate fatal hepatotoxicity has been more clearly defined. Those at primary risk of fatal hepatic dysfunction are children under the age of 2 years who are receiving multiple anticonvulsants and also have significant medical problems in addition to severe epilepsy. The risk is considerably lower for patients over the age of 2 years on valproate monotherapy. In contrast to the risk profile with other AEDs, adults receiving valproate as monotherapy have the lowest risk of hepatotoxicity. Fatal hepatic dysfunction coincident with valproate may be the result of aberrant drug metabolism. Concomitant use of AEDs that induce microsomal P450 enzymes (e.g., phenytoin and phenobarbital) may enhance the production of a toxic metabolite, and hence the greater risk of hepatotoxicity with polypharmacy.     

Vascular Malformations and Epilepsy: Clinical Considerations and Basic Mechanisms

Summary: Vascular malformations (VMs) are associated with epilepsy. The natural history of the various VMs, clinical presentation, and tendency to provoke epilepsy determine treatment strategies. Investigations have probed the mechanisms of epileptogenesis associated with these lesions. Electrophysiologic changes are associated with epileptogenic cortex adjacent to VMs. Putative pathophysiologic mechanisms of epileptogenesis include neuronal cell loss, glial proliferation and abnormal glial physiology, altered neurotransmitter levels, free radical formation, and aberrant second messenger physiology.     

Classification of methods in transcranial Electrical Stimulation (tES) and evolving strategy from historical approaches to contemporary innovations

Transcranial Electrical Stimulation (tES) encompasses all methods of non-invasive current application to the brain used in research and clinical practice. We present the first comprehensive and technical review, explaining the evolution of tES in both terminology and dosage over the past 100 years of research to present day. Current transcranial Pulsed Current Stimulation (tPCS) approaches such as Cranial Electrotherapy Stimulation (CES) descended from Electrosleep (ES) through Cranial Electro-stimulation Therapy (CET), Transcerebral Electrotherapy (TCET), and NeuroElectric Therapy (NET) while others like Transcutaneous Cranial Electrical Stimulation (TCES) descended from Electroanesthesia (EA) through Limoge, and Interferential Stimulation. Prior to a contemporary resurgence in interest, variations of transcranial Direct Current Stimulation were explored intermittently, including Polarizing current, Galvanic Vestibular Stimulation (GVS), and Transcranial Micropolarization. The development of these approaches alongside Electroconvulsive Therapy (ECT) and pharmacological developments are considered. Both the roots and unique features of contemporary approaches such as transcranial Alternating Current Stimulation (tACS) and transcranial Random Noise Stimulation (tRNS) are discussed. Trends and incremental developments in electrode montage and waveform spanning decades are presented leading to the present day. Commercial devices, seminal conferences, and regulatory decisions are noted. We conclude with six rules on how increasing medical and technological sophistication may now be leveraged for broader success and adoption of tES.     

Carbamazepine Efficacy and Utilization in Children

Summary: Carbamazepine is effective for preventing partial and generalized tonic-clonic seizures in children. Although absence epilepsies are more common in children than adults, an estimated 80% of children with epilepsy have seizure types or epilepsies that are potentially responsive to carbamazepine. The differential diagnosis of ictal staring is an especially important issue in children because absence and atypical absence seizures are more prevalent in children than adults. Age-related pharmacokinetic differences and drug interactions are major considerations in children. On average, children have higher clearance rates of carbamazepine, shorter half-lives, and higher ratios of carbamazepine-10, 11-epoxide to carbamazepine than adults. In addition, children with severe epilepsy are more likely to require multiple-drug therapy, which can lead to complex drug interactions. When carbamazepine is administered along with valproate, drug protein binding interactions can cause intermittent side effects.     

Neonatal Seizures: Problems in Diagnosis and Classification

Summary: The clinical identification of neonatal seizures is critical for the recognition of brain dysfunction; however, diagnosis is often difficult because of the poorly organized and varied nature of these behaviors. Current classification systems are limited in their ability to communicate motor, autonomic, and electroencephalo-graphic features of seizures precisely and to provide a basis for uniform effective diagnosis, therapy, and determination of prognosis. Recent investigations of neonates, utilizing bedside electroencephalographic/polygraphic/ video monitoring techniques, have provided the basis for improved diagnosis and classification of seizures in the newborn. These studies have demonstrated that not all clinical phenomena currently considered to be seizures require electrocortical epileptiform activity for their initiation or elaboration. In addition, the specific clinical character of the phenomena considered to be seizures, the clinical state of the infant, and the character of the EEG indicate the probable pathophysiological mechanisms involved and suggest probable etiologies, prognosis, and therapy. Similarities between animal models that demonstrate reflex physiology and neonates with motor automatisms and tonic posturing suggest that these clinical behaviors may not be epileptic in origin but, rather, primitive movements of progression and posture mediated by brainstem mechanisms. Although not all clinical behaviors currently considered to be neonatal seizures may have similar pathophysiological mechanisms, they are clinically significant because they all indicate brain dysfunction.     

Valproate Monotherapy in the Management of Generalized and Partial Seizures

Summary: For decades, therapeutic tradition has promoted the concept of polypharmacy in the management of epilepsy. In recent years, however, studies have shown that, for most patients, monotherapy can provide comparable or better seizure control than administration of multiple anticonvulsants, while diminishing the potential for adverse reactions, drug interactions, and poor compliance. Valproate is an important monotherapeutic agent that is highly effective in the control of idiopathic primary and secondarily generalized epilepsies, and partial seizures that do not generalize. Comparative studies have found that valproate is at least as effective as phenytoin and carbamazepine in the treatment of generalized and partial seizures. Given the similar efficacy, other factors such as pharmacokinetics and side effects may therefore determine anticonvulsant selection for monotherapy.     

Un nouveau cadre conceptuel de travail pour la psychiatrie

In an attempt to place psychiatric thinking and the training of future psychiatrists more centrally into the context of modern biology, the author outlines the beginnings of a new intellectual framework for psychiatry that derives from current biological thinking about the relationship of mind to brain. The purpose of this framework is twofold. First, it is designed to emphasize that the professional requirements for future psychiatrists will demand a greater knowledge of the structure and functioning of the brain than is currently available in most training programs. Second, it is designed to illustrate that the unique domain which psychiatry occupies within academic medicine, the analysis of the interaction between social and biological determinants of behavior, can best be studied by also having a full understanding of the biological components of behavior.