活体磁共振示踪磁标记大鼠神经干细胞**☆

背景：要将神经干细胞替代治疗神经系统疾病应用于临床,必须解决一个重要问题,就是植入人脑内神经干细胞的存活、迁移、识别和动态监测。 目的：拟建立菲立磁体外标记胎鼠神经干细胞的方法及检测手段,观察标记细胞移植后在活体上磁共振信号的改变。 设计、时间及地点：MRI动态评估,体内实验,于2005-01/08在武警医学院附属医院完成。 材料：孕13~18 d SD胚胎大鼠10只用于分离、培养大鼠胚胎源性神经干细胞,健康成年清洁级SD大鼠32只,用于制作局灶性脑缺血再灌注模型。 方法：分离培养大鼠胚胎源性神经干细胞,使用菲立磁-多聚赖氨酸复合物标记神经干细胞。制作局灶性脑缺血再灌注模型,将菲立磁-多聚赖氨酸复合物标记的神经干细胞分别移植入模型大鼠左侧脑内,右侧移植未标记的神经干细胞。 主要观察指标：对标记细胞进行普鲁士蓝染色、电镜观察。细胞活体移植后1,5,14 d体内磁共振示踪。 结果：菲立磁可以高效率地标记神经干细胞,普鲁士蓝染色显示菲立磁-多聚赖氨酸复合物标记神经干细胞胞质内出现细小的蓝色铁颗粒,电镜结果显示菲立磁-多聚赖氨酸复合物标记的神经干细胞胞质内含有许多包裹铁颗粒的囊泡。磁共振成像检查发现脑内移植的标记细胞在磁共振上呈明显的低信号改变,移植第5天低信号物质沿胼胝体腹侧迁移。在移植第14天,对称平行的两个针道已经基本看不见,病灶侧侧脑室部位低信号物质向对侧迁移,左侧侧脑室部位低信号物质基本看不见。 结论：菲立磁经多聚左旋赖氨酸转染后可体外标记神经干细胞,标记后体内移植的神经干细胞可以在磁共振上产生明显的低信号改变。     

人骨髓间质干细胞的体外菲立磁标记

背景：常规干细胞示踪的检测方法,需取实验动物组织进行检查,缺乏示踪的动态检测和无创性。 目的：通过体外菲立磁标记骨髓间质干细胞并进行磁共振成像扫描,明确不同浓度菲立磁对人骨髓间质干细胞细胞活性的影响和检测菲立磁标记的最佳浓度,并筛选出适合人骨髓间质干细胞磁共振成像扫描的成像序列。 方法：使用不同质量浓度菲立磁(100,50,25,12.5 mg/L)与生长状态良好第3代人骨髓间质干细胞孵育24~48 h,另设未进行标记的第3代细胞为空白对照组。两组细胞进行四甲基偶氮唑蓝法检测增殖生长情况并描绘生长曲线。细胞普鲁士蓝染色鉴定人骨髓间质干细胞的标记情况。进行磁共振成像检测,确定最佳磁共振成像序列。 结果与结论：质量浓度≤25 mg/L的菲立磁标记对人骨髓间质干细胞的增殖能力不会产生影响,并且以25 mg/L的标记效果最佳。而≥50 mg/L的菲立磁标记则明显抑制人骨髓间质干细胞的增殖。磁共振成像扫描显示菲立磁标记24,48 h或子代的人骨髓间质干细胞标本T1WI、T2WI以及T2*WI 3个序列上均可见信号降低,以T2*WI变化最为明显。证实菲立磁可用于体外标记人骨髓间质干细胞连续性研究。菲立磁标记对人骨髓间质干细胞增殖能力的影响存在浓度相关性,25 mg/L为最佳标记浓度。磁共振成像T2*WI序列更适合追踪菲立磁标记的人骨髓间质干细胞。     

菲立磁标记兔BMSCs自体移植脊髓后的核磁共振活体示踪及形态学研究

目的通过观察菲立磁标记兔骨髓源性神经干细胞(BMSCs)自体移植脊髓后的核磁共振活体示踪及形态,期待找到一种应用非侵袭性方法来识别、跟踪BMSCs的存活状态及与宿主组织整合情况的方法。方法无菌条件下股骨取骨髓,梯度密度离心法分离获取兔骨髓基质细胞;使用“Feridex-多聚赖氨酸复合物(FE-PLL)”标记骨髓基质细胞,采用普鲁士兰染色和台盼蓝排除实验等方法鉴定FE-PLL标记兔骨髓基质细胞的效率和细胞的活力;体外标记的细胞自体脊髓移植,磁共振、免疫组织化学染色和透射电镜检查。结果普鲁士蓝染色显示FE-PLL标记骨髓基质细胞胞质内出现细小的蓝色铁颗粒;与正常未标记的细胞相比较,FE-PLL标记对骨髓基质细胞的活力、增殖和分化等能力没有明显的影响;经菲立磁标记的兔BMSCs自体脊髓移植后,可在核磁共振上活体示踪。结论菲立磁与核磁共振联合可无创性活体标记检测移植的神经干细胞基本的存在部位、存在方式及其一些生物学特性,可以用来活体示踪移植的BMSCs。     

USPIO标记大鼠骨髓源性神经干细胞移植脑皮层影像及形态学初步研究

目的将超小超顺磁性氧化铁（USPIO）Sinerem标记的大鼠骨髓源性神经干细胞移植鼠脑后，初步观察其在大脑中的活性、迁移和整合情况，确定MRI成像示踪Sinerem标记神经干细胞的可行性。方法分离SD大鼠骨髓基质细胞，体外培养诱导成骨髓源性神经干细胞。将Sinerem氧化铁和神经干细胞共孵育培养过夜。采用普鲁士蓝染色和透射电镜确定细胞内铁的摄取、定位情况。将标记细胞立体定向移植微注射到大鼠脑皮层。在不同时间点以SE序列T2WI行4．7T磁共振干细胞成像示踪，然后用组织学方法观察标记细胞在脑内的转归情况。结果Sinerem标记神经干细胞效率为98％～100％，普鲁士蓝染色显示铁颗粒存在胞质中，电镜显示铁颗粒集中于内涵体／溶酶体中；移植细胞体内的T2WI信号强度明显降低．可在4周时检测到细胞沿胼胝体迁移；组织学检测结果表明标记后的细胞可在脑内存活，并能沿神经纤维迁移。结论USPIO能有效地标记骨髓源性神经干细胞，利用磁共振成像可进行大脑中活体示踪监测。     

菲立磁标记骨髓基质细胞动脉移植治疗帕金森病的实验研究

目的:探讨应用骨髓基质细胞(BMSCs)经颈动脉移植治疗帕金森病(PD)大鼠的机制及疗效,以及菲立磁(Feridex)标记的BMSCs移植入PD大鼠体内后,MRI示踪观察的可行性。方法:建立PD大鼠模型,体外分离培养扩增SD大鼠BMSCs,从右侧颈动脉移植治疗10只PD大鼠,移植后进行行为学观察和MRI示踪观察。结果:移植治疗的PD大鼠阿扑吗啡诱发旋转实验较对照组明显减少;组织学和MRI示踪发现移植的BMSCs在PD大鼠脑内存活、迁移,并向神经细胞方向分化。结论:BMSCs移植能显著改善PD大鼠的生物学行为,利用MRI技术可以对菲立磁标记的BMSCs进行活体追踪。     

菲立磁标记大鼠骨髓基质细胞及自体移植后磁共振示踪的研究

目的初步探索利用菲立磁(Feridex)和转染试剂体外磁性标记大鼠骨髓基质细胞这一方法的可行性，为将来临床上应用磁共振成像(MRI)追踪标记细胞奠定基础。方法无菌条件下行股骨取骨髓，梯度密度离心法分离获取大鼠骨髓基质细胞，使用“Feridex-多聚赖氨酸复合物(FE-PLL)”标记骨髓基质细胞，采用普鲁士蓝染色、电镜和台盼蓝排除实验等方法鉴定FE-PLL标记大鼠骨髓基质细胞的效率和细胞的活力，并对FE-PLL标记骨髓基质细胞的增殖和分化能力进行评估。将FE-PLL标记和未标记后的骨髓基质细胞分别移植人大鼠左有侧尾壳核脑内．细胞移植后1、4、7周应用MRI对脑内移植的细胞进行活体示踪，最后利用组织切片进行普鲁士蓝染色。结果菲立磁可以高效率地标记骨髓基质细胞，标记效率在99%左右。普鲁士蓝染色显示FE-PLL标记骨髓基质细胞胞质内出现细小的蓝色铁颗粒。电镜结果显示FE-PLL标记的骨髓基质细胞胞质内含有许多包裹铁颗粒的囊泡。与正常未标记的细胞相比较，FE-PLL标记对骨髓基质细胞的活力、增殖和分化等能力没有明显的影像。MRI检查发现脑内移植的标记细胞在磁共振上呈明显的低信号改变，未标记细胞侧脑组织无明显的低信号改变，与组织学切片结果基本相一致。结论以上结果提示菲立磁可以用来体外标记骨髓基质细胞，利用MRI技术可以对脑内移植后的标记细胞进行初步的活体追踪。     

活体示踪BMSCs在大鼠癫痫模型中的可行性研究

目的探索活体示踪骨髓间充质干细胞(BMSCs)在大鼠癫痫模型中的可行性。方法体外分离纯化SD大鼠BMSCs,超微超顺磁性氧化铁(USPIO)纳米颗粒标记,而后诱导大鼠癫痫模型并接受USPIO标记的BMSCs(U-BMSCs)移植,进而通过MRI检测U-BMSCs体内外影像,示踪UBMSCs在癫痫模型中的分布,最后通过免疫组化验证USPIO标记对BMSCs抑制癫痫海马神经元损失的影响。结果 USPIO体外标记BMSCs的效率为99.2%±1.24%,体内外MRI T2W2显示U-BMSCs为可与脑实质区别明显的低信号影像。细胞移植两周后发现U-BMSCs低信号影像可分布于癫痫模型脑实质包括海马,免疫组化结果示U-BMSCs移植组海马CA1和齿状回门区(DH)神经元数量明显多于PBS组(P0.05),且与BMSCs移植组无明显区别(P0.05)。结论 USPIO标记BMSCs可依赖MRI检测实现其在大鼠癫痫模型中的活体示踪且不影响其神经保护功能。     

超顺磁氧化铁标记骨髓基质干细胞移植治疗帕金森病鼠的实验研究

目的:研究应用超顺磁氧化铁(SPIO)标记骨髓基质干细胞(MSCs)移植治疗帕金森病(PD)大鼠后的在体MRI观察。方法:分离、获取大鼠骨髓基质细胞,脂质体转染法将SPIO标记MSCs;制作PD模型,SPIO标记的MSCs移植到PD大鼠右侧纹状体区,应用MRI在体观察脑内移植的骨髓基质细胞的存活和迁徙情况。结果:体外SPIO标记的骨髓基质细胞普鲁蓝染色阳性;脑内移植SPIO标记MSCs的PD大鼠磁共振T2和T2GRE扫描检查显示在移植区呈低信号改变。随时间的延长,移植区信号向周围扩大。脑纹状体区的铁染色也可见SPIO标记MSCs从移植部位向四周迁移。结论:SPIO可用于体外标记MSCs,通过MRI技术可以对标记细胞脑内移植后进行初步的活体示踪,有利于MSCs移植治疗PD后的疗效观察。     

超顺磁性纳米铁粒子标记骨髓基质干细胞及对其分化能力的影响

目的研究超顺磁性纳米铁粒子标记骨髓基质干细胞(BMSCs)对其存活、增殖及向肝细胞分化能力的影响,确定最佳标记浓度,为磁共振成像(MRI)技术追踪同种异体移植的磁标记细胞奠定基础。方法使用菲立磁(Ferumoxides)标记大鼠BMSCs,采用Prussianblue染色、电镜等鉴定Ferumoxides标记BMSCs的效率;检测标记后BMSCs的诱导分化率评估其向肝细胞分化能力。门静脉和肝内局部移植标记BMSCs后行MRI肝脏扫描。结果标记培养基Fe3 浓度在11.2￣16.8μg/mL时,Prussianblue染色可见90%以上细胞阳性,透射电镜可见铁颗粒位于胞浆中;标记后BMSCs向肝细胞分化的能力与正常未标记BMSCs差异无统计学意义(P>0.05);Fe3 浓度在5.6μg/mL时,着色率<60%;Fe3 浓度为22.4μg/mL、28.0μg/mL时培养基中死细胞增多,且诱导分化后AFP与白蛋白阳性率降低,与对照组比较差异有统计学意义(P<0.05)。标记BMSCs门静脉和肝内局部移植后大鼠肝脏在MRI的SE-T2WI序列扫描下与移植前比较局部呈明显低信号改变。结论标记培养基Fe3 浓度在11.2￣16.8μg/mL时,Ferumoxides对BMSCs标记效率较高,且不影响其存活、增殖及向肝细胞分化能力,标记后的BMSCs可用于进一步实验。活体MRI示踪肝内或门静脉移植Ferumoxides标记的BMSCs有一定的可行性。     

磁共振活体示踪经冠状动脉骨髓间充质干细胞移植：验证其与病理组织学结果的一致性*☆

背景：目前动物实验及临床研究证明经冠状动脉移植骨髓间充质干细胞可改善心肌梗死后的心脏功能,但骨髓间充质干细胞经冠状动脉移植后能否到达心肌梗死部位仍存在争议。 目的：磁共振活体示踪经冠状动脉注射的骨髓间充质干细胞能否到达心肌梗死部位。 方法：全骨髓法分离培养猪骨髓间充质干细胞,超顺磁性氧化铁标记后胰酶消化,调整细胞浓度为1010 L-1。10只猪均建立心肌梗死模型,造模后1周通过OTW球囊导管经冠状动脉将标记好的骨髓间充质干细胞悬液定向移植至梗死区。普鲁士蓝染色评价细胞标记效率,采用快速梯度回波序列完成长轴位四腔心和二腔心扫描,在以长轴位四腔心和二腔心为定位相,垂直于室间隔获得左心室短轴位图像。 结果与结论：超顺磁性氧化铁可安全有效标记骨髓间充质干细胞,经冠状动脉注射的骨髓间充质干细胞能到达心肌梗死区及交界区,磁共振能示踪到超顺磁性氧化铁标记的骨髓间充质干细胞,示踪结果与病理组织学检查一致,且磁共振示踪时间可长达5周。     

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.