亚低温通过抑制AIF核转位改善大鼠颅脑损伤

目的 探讨亚低温对颅脑损伤（TBI）大鼠脑组织凋亡诱导因子（AIF）核转位的影响。方法 将36只成年雄性SD大鼠随机分为假手术组、TBI组、亚低温组，每组12只。采用控制性皮质撞击建立TBI模型，亚低温组大鼠给予亚低温处理。TBI后24 h，HE染色观察大鼠脑组织病理学变化；免疫组织化学方法检测大鼠脑组AIF的表达部位；免疫印迹法检测损伤脑组织线粒体和细胞核AIF、caspase-3的表达情况。结果 HE染色结果显示，TBI后，损伤侧脑组织可见沿灰白质界面的挫伤和出血，亚低温组挫伤和出血灶明显减轻。免疫印迹法检测结果显示，TBI后，损伤脑组织caspase-3表达量明显增加（P<0.01），细胞核AIF表达量明显增加（P<0.01），而线粒体AIF表达量明显降低（P<0.05）；亚低温组损伤脑组织caspase-3表达量明显下降（P<0.01），细胞核AIF表达量明显下降（P<0.01），而线粒体AIF表达量明显升高（P<0.05）。免疫组织化学染色结果显示，假手术组AIF位于大脑皮质和海马神经元细胞核外；TBI组后，损伤侧皮质及海马区AIF从线粒体转移至细胞核内的阳性细胞数量明显增多（P<0.01）；亚低温组损伤侧皮质及海马区AIF发生核内转移的阳性细胞数量减少（P<0.01）。结论 亚低温可能通过抑制AIF的核转位减轻颅脑损伤后神经细胞凋亡，从而起到神经保护作用。     

人脐血间充质干细胞静脉移植治疗大鼠局灶性脑缺血的实验研究

目的 探讨来源于人脐血的间充质干细胞经静脉移植治疗大鼠局灶性脑缺血的可行性及其机制.方法 将人脐血间充质干细胞在体外纯化、扩增并经BrdU标记后,经尾静脉移植到局灶性脑缺血大鼠体内,通过神经缺损评分观察移植后大鼠神经行为学改善情况,通过组织学方法观察移植到脑内的人脐血间充质干细胞表达脑源性神经营养因子和缺血灶周围微血管密度变化的情况.结果 人脐血间充质干细胞移植组大鼠的神经缺损评分显著低于对照组(P＜0.05);移植到脑内的人脐血间充质干细胞主要选择性分布于缺血灶周围区域并表达脑源性神经营养因子,移植组大鼠梗死灶周围的微血管密度显著高于对照组(P＜0.01).结论 经静脉注射移植人脐血间充质干细胞能明显促进局灶性脑缺血大鼠的神经行为功能恢复,促进缺血灶周边区微血管增生可能是人脐血间充质干细胞移植治疗局灶性脑缺血的机制之一.     

骨髓基质细胞条件液联合神经干细胞移植对帕金森病大鼠行为认知功能的影响：效果优于单纯神经干细胞移植吗？

背景：将骨髓基质细胞和神经干细胞分别单独移植于帕金森病模型鼠脑内已取得良好效果,但尚未见联合移植的报道。 目的：观察骨髓基质细胞条件液与神经干细胞联合移植对帕金森病模型大鼠行为认知功能的影响,并与单纯神经干细胞移植效果进行比较。 方法：用Neurobasal+B27培养3~6代的骨髓基质细胞24 h后,收集并离心上清液,即为骨髓基质细胞条件液。体外分离培养大鼠神经干细胞后,行BrdU标记。55只大鼠随机取8只作为正常组,剩余47只采用立体定向仪单侧黑质致密部和腹侧被盖区注射6-羟基多巴胺建立帕金森病动物模型。32只造模成功,随机分为3组,选取右侧纹状体两个坐标点为移植点,单纯神经干细胞组每点注入神经干细胞悬液5 μL,联合组每点注入神经干细胞悬液+骨髓基质细胞条件液5 μL,模型组不注入任何液体。观察帕金森病大鼠旋转行为的改变,通过水迷宫试验对大鼠认知功能进行评价,免疫组织化学染色检测 黑质区酪氨酸羟化酶蛋白的表达及移植区BrdU的表达。 结果与结论：与模型组比较,移植后1~8周单纯神经干细胞组、联合组大鼠旋转次数均显著减少(P < 0.01),移植后第4,8周单纯神经干细胞组、联合组大鼠逃避潜伏期均明显缩短(P < 0.05),在平台象限的游泳时间百分比、游泳距离百分比、穿越平台次数均明显增加(P < 0.05);后2组间各指标比较均无明显差异(P > 0.05)。移植后第8周,各组大鼠6-羟基多巴胺注射侧黑质区酪氨酸羟化酶阳性神经元和神经纤维表达基本缺失;单纯神经干细胞组、联合组移植区可见一定数量的BrdU阳性细胞表达,多数位于针道附近,部分细胞沿胼胝体迁移。提示骨髓基质细胞条件液与神经干细胞联合移植能改善帕金森病模型大鼠的旋转行为和认知能力,与单纯神经干细胞纹状体移植的效果基本相似。     

亚低温与脑缺血大鼠骨髓间充质干细胞的迁移

背景：关于亚低温运用到神经损伤修复领域的研究已有一些报道,但亚低温对神经干细胞在脑内移植迁移的影响还不太清楚。 目的：观察亚低温对移植入脑缺血大鼠侧脑室的骨髓间质干细胞迁移的影响。 方法：采用Longa法永久性闭塞SD大鼠大脑中动脉制作局灶性脑缺血损伤模型,亚低温组于移植前应用亚低温处理大鼠急性脑缺血损伤,对照组于移植前应用常温处理大鼠急性脑缺血损伤;造模术后24 h,在脑立体定向下,经侧脑室注射移植用5-BrdU标记的骨髓间充质干细胞。经过5,14,21,28 d,7周后用免疫组织化学方法检测各组大鼠脑组织BrdU阳性细胞数。 结果与结论：移植第14天多数标记的骨髓基质细胞细胞已经迁移至梗死灶周围,移植后各时间点亚低温组梗死灶周边皮质的BrdU阳性细胞数明显多于对照组(P < 0.05)。提示移植前宿主亚低温处理可以促进骨髓间充质细胞的定向迁移,对局灶性脑缺血有神经保护作用。     

低氧预适应刺激脑海马区内源性神经干细胞的增殖**

背景：缺血/缺氧/低氧等刺激均能导致内源性神经干细胞的增殖和分化,起到脑组织修复作用,但低氧预适应能否影响内源性神经干细胞的增殖尚不清楚。 目的：探讨低氧预适应对小鼠脑海马区内源性神经干细胞增殖的影响。 方法：清洁级Balb/c近交系小鼠随机分为3组,低氧对照组小鼠放入广口瓶内,立即用橡皮塞封紧,以动物出现第1次喘呼吸为低氧耐受极限的标志,完成低氧暴露1次;低氧预适应组小鼠按此法重复操作4次;正常对照组小鼠不进行低氧暴露。通过免疫荧光和激光共聚焦显微镜等技术,测定海马BrdU阳性细胞数和荧光强度。 结果与结论：与低氧对照组比较,低氧预适应组小鼠耐受时间显著延长(P < 0.01)。正常对照组海马区BrdU标记的内源性神经干细胞荧光强度微弱,海马区可见少量BrdU阳性细胞;低氧对照组、低氧预适应组荧光强度及BrdU阳性细胞数均明显增加(P < 0.01),且低氧预适应组增加幅度大于低氧对照组(P < 0.01)。证实在低氧预适应过程中,海马区内源性神经干细胞明显增殖,可能参与低氧预适应脑保护机制。     

食蟹猴骨髓源性神经干细胞自体脑内移植的研究

目的探讨食蟹猴自体骨髓基质细胞诱导分化成神经干细胞后移植到脑内的生长情况.方法对6只食蟹猴进行骨髓基质干细胞体外培养、诱导分化成神经干细胞,经BrdU标记后进行自体脑移植.动物分为即时移植组和延迟移植组,采用立体定向多点注射的方法将神经干细胞悬液移植到猴皮层创伤灶.结果HE染色显示即时移植和延迟移植的创伤区细胞数量都明显多于假移植治疗对照;免疫组织化学染色显示两组动物在干细胞移植后1～6个月脑皮层创伤灶内均有BrdU阳性表达细胞,移植后半年的动物在移植区邻近的脑白质内也可观察到有BrdU阳性表达的细胞,而创伤对照动物、假移植治疗动物和正常脑组织中则未见BrdU阳性表达. 结论BMSCs体外培养得到的神经干细胞经过自体移植能在脑皮层内存活,并且能增殖、分化和迁移,可成为神经干细胞的替代细胞或来源细胞;干细胞移植到陈旧性的脑皮层损伤灶也具有成活、增殖和迁移能力.     

法舒地尔联合骨髓间充质干细胞移植治疗大鼠急性心肌梗死：有协同治疗作用吗？

背景：RhoA激酶抑制剂法舒地尔能有效抑制心肌梗死后的心肌肥大。 目的：观察法舒地尔联合骨髓间充质干细胞移植对急性心肌梗死模型鼠心功能的影响,探讨两者是否具有协同治疗作用。 方法：体外培养扩增SD大鼠骨髓间充质干细胞,另选取42只雌性SD大鼠结扎前降支,建立急性心肌梗死模型,随机分成3组。干细胞组, 干细胞+法舒地尔组梗死心肌中移植骨髓间充质干细胞,干细胞+法舒地尔组同时给予法舒地尔治疗,梗死组不干预。4周后,以二维超声心动图分析心功能变化,SRY-PCR检测大鼠Y染色体上特有的基因SRY,Western-Blot检测RhoA蛋白表达,并进行病理观察。 结果与结论：与梗死组比较,干细胞组和联合组左室舒张末内径及左室收缩末内径均显著减小(P < 0.01),射血分数均显著升高(P < 0.01),其中干细胞+法舒地尔组变化幅度优于细胞移植组(P < 0.05)。干细胞组和联合组基因有SRY表达,梗死组未检测到基因SRY。干细胞+法舒地尔组RhoA蛋白表达水平较梗死组、干细胞组显著降低 (P < 0.05)。病理观察干细胞组和联合组心肌修复优于梗死组,干细胞+法舒地尔组较干细胞组明显。结果表明单纯骨髓间充质干细胞移植以及法舒地尔联合骨髓间充质干细胞移植均可改善心肌梗死大鼠心功能,减少心室扩张程度,两者联合情况下效果最佳,对心肌梗死具有协同治疗作用。     

静脉移植骨髓间充质干细胞联合重组人生长激素对充血性心肌病大鼠心肌和血管组织的修复**☆

背景：骨髓间充质干细胞静脉移植后,能否归巢至心脏受损部位和分化为心肌样细胞尚无统一定论。 目的：探讨重组人生长激素联合骨髓间充质干细胞静脉移植对充血性心肌病大鼠心肌和血管新生的影响。 方法：密度梯度离心和贴壁筛选法获得大鼠骨髓间充质干细胞。模型组、细胞移植组、生长激素组、联合组大鼠均在阿霉素诱导下建立心脏衰竭模型。造模后,细胞移植组经静脉注入BrdU标记的骨髓间充质干细胞8×1013 L-1;生长激素组皮下注射重组人生长激素2 U/(kg•d),连续14 d;联合组行骨髓间充质干细胞移植与重组人生长激素注射。4周后取材,BrdU+MHC及BrdU+Actin免疫组化染色确定骨髓间充质干细胞的归巢情况,评价移植细胞向心肌样细胞和血管内皮细胞的分化,苏木精-伊红染色检测血管密度。 结果与结论：与细胞移植组比较,联合组BrdU免疫组化阳性率显著升高(P < 0.001);BrdU+MHC双染和BrdU+Actin双染后心肌样细胞、血管内皮细胞均显著增多(P < 0.001)。与模型组比较,生长激素组、细胞移植组、联合组的总血管密度、微血管密度、毛细血管密度均显著升高(P < 0.001),后3组间比较无明显差异(P > 0.05)。结果证实骨髓间充质干细胞静脉移植后可归巢到心脏,对充血性心肌病大鼠心肌和血管有明显修复作用,能在损伤处区存活、生长,并向心肌样细胞、血管内皮细胞方向分化,增加损伤处血管密度;生长激素可以改善微环境,加强骨髓间充质干细胞向心肌样细胞、血管内皮细胞的转化率。     

亚低温对颅脑创伤大鼠脑组织中β-分泌酶表达的影响

目的 研究亚低温对大鼠颅脑创伤(TBI)后β-分泌酶(BACE)的影响.方法 应用液压冲击装置制作大鼠颅脑创伤模型,实验分为假手术组、创伤常温组(37℃)和创伤亚低温组(32℃),每组12只大鼠.采用免疫组织化学和免疫荧光法检测BACE表达部位,采用RT - PCR和免疫印迹法研究BACE mRNA和蛋白表达水平.结果 与假手术组比较,创伤常温组大鼠脑组织BACE免疫阳性细胞数明显增多,BACE mRNA和蛋白表达上升(P＜0.05).但是,可以看出BACE在给予亚低温处理后的免疫阳性细胞数量以及蛋白水平显著低于常温处理组(P＜0.05),与假手术组比较差异无统计学意义.结论 亚低温可以通过降低颅脑创伤后BACE表达水平来减轻继发性脑损伤程度,起到神经保护作用.     

PTEN/mTOR信号通路对大鼠神经干细胞分化成熟作用的研究

目的 探讨PTEN/mTOR信号通路对体外分离培养的大鼠神经干细胞(neural stem cells,NSCs)分化成熟的作用.方法 体外分离培养大鼠海马神经干细胞,收集第三代神经干细胞分别诱导分化0d、1d、3d和7d,倒置显微镜下观察细胞形态变化,应用Western blot和细胞免疫荧光检测第10号染色体缺失的磷酸酶与张力蛋白同源物基因(PTEN)和反映雷帕霉素靶蛋白(mTOR)活性的磷酸化核糖体(P-S6R)蛋白的表达.结果 与0d组和1d组相比,3d组PTEN表达明显增加(均P＜0.01)；7d组PTEN表达亦明显增加(P＜0.01和P＜0.05),而3d组和7d组P-S6R表达均明显下降(均P＜0.01).神经干细胞诱导分化后PTEN和P-S6R细胞免疫荧光染色均为阳性.结论 PTEN/mTOR信号通路参与调控神经干细胞的增殖分化和轴突生长,可能是导致神经干细胞分化成熟至一定阶段后增殖分化和轴突生长能力下降、不能建立有效的突触联系的重要原因之一.     

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.     

Special Pharmacokinetic Considerations in Children

Summary: Pediatric patients have greater degrees of pharmacokinetic variability and unpredictability than adults. This variability results from the effects of pharmacogenetics, age and growth, prior and current comedication, and disease. Newborns with seizures have the least predictable dosage requirements, and their needs change as drug-eliminating mechanisms mature in the neonatal period. Infants have the highest relative capacities to eliminate antiepileptics of any age group and require the largest relative doses. In addition to age-related trends, children demonstrate the same drug-specific, pharmacokinetic phenomena that adults do, including nonlinear phenytoin elimination, nonlinear valproate binding, and autoinduction of carbamazepine. Intercurrent illness and drug interactions further modify the age-related pharmacokinetic patterns in children and make dosage requirements even more unpredictable. Recent studies have shown that febrile illness can affect drug elimination, sometimes decreasing drug levels by 50% or more. Intermittent treatment with benzodiazepines administered either orally or rectally can be an important adjunct and help minimize this type of problem for children with marginally controlled epilepsy. Intermittent benzodiazepines are also helpful for children who have febrile seizures and who need only occasional antiepileptic protection.