Prothrombin and PIVKA-II Levels in Cord Blood from Newborn Exposed to Anticonvulsants During Pregnancy

PURPOSE: To determine whether anticonvulsant exposure during human pregnancy caused an increase of the abnormal form of prothrombin, known as PIVKA-II (prothrombin induced by vitamin K absence for factor II), and a decrease in total prothrombin, in the blood of the newborn. METHODS: Cord blood was collected from the placenta at the time of parturition from 12 women who had received anticonvulsant therapy during pregnancy and from 11 control women. RESULTS: PIVKA-II was present in cord blood from control mothers at low or nondetectable levels. In the same samples, total prothrombin concentrations were approximately 50% of adult levels, but there was wide variation between individuals. Exposure to carbamazepine (CBZ) alone during pregnancy was associated with markedly increased PIVKA-II levels in four of six samples and decreased total prothrombin levels for the whole group. High PIVKA-II levels also were recorded in one cord blood sample from a mother who received phenytoin (PHT) and vigabatrin (VGB). Two cases of PHT alone and one of valproic acid (VPA) alone were not associated with increased PIVKA-II levels. CONCLUSIONS: These results are consistent with the hypothesis that some anticonvulsants (particularly CBZ) interfere with vitamin K metabolism during pregnancy and may result in hematologic signs of vitamin K deficiency in the newborn.     

人胚神经干细胞及人脐血干细胞移植治疗大鼠脑缺血的实验研究

目的 研究人胚神经干细胞（hNSCs）、人脐血干细胞（hUCBCs）移植治疗脑缺血大鼠的效果及其在缺血大鼠脑内的增殖、分化状况,并对两种干细胞移植的效果进行比较。方法 从自然流产的孕10～13周的人胚脑组织中分离、培养hNSCs;采集足月新生儿脐带血60～100ml,分离出其中的单个核细胞;移植前hNSCs、hUCBCs均经5–溴脱氧嘧啶尿苷（Brdu）标记48h。采用线栓法制作大鼠脑缺血模型,1d后经尾静脉移植未分化的hNSCs、hUCBCs入脑缺血大鼠体内,对移植后大鼠进行神经损害严重程度评分（NSS）,用免疫组化方法观察移植后hNSCs、hUCBCs的存活、迁移、分化状况。结果 从人胚脑中成功培养出hNSCs,培养条件下呈悬浮状态生长,形成神经球;hUCBCs在体外具有增殖能力。两移植组大鼠均自移植后21d起其NSS显著低于对照组（P<0.05）,两移植组移植前及移植后各时间点NSS分别比较未发现显著性差异（P>0.05）;移植后14、21、28、35d脑组织切片中均可见Brdu染色阳性细胞,缺血侧明显多于对侧（P<0.05）,移植后21、28、35d明显多于移植后14d（P<0.05）;hNSCs组Brdu染色阳性细胞数多于hUCBCs组（P<0.05）;移植组各时间点脑组织切片中均可见nestin染色阳性细胞;在Brdu阳性细胞群中,hNSCs组73.8%为胶质纤维酸性蛋白（GFAP）染色阳性细胞,16.7%为2,3–环核苷酸磷酸二脂酶（CNPase）染色阳性 细胞,9.5%为神经元特异性烯醇化酶（NSE）染色阳性细胞;hUCBCs移植组74.5%为GFAP染色阳性细胞,15.4%为CNPase染色阳性细胞,10.1%为NSE染色阳性细胞;两组比较差异无显著性（P>0.05）。结论 hNSCs、hUCBCs均具有多分化潜能,受缺血部位微环境信号的影响分化成3种主要类型的神经细胞;静脉移植hNSCs、hUCBCs能有效改善脑梗死动物的神经功能;除了hNSCs外,hUCBCs移植也是治疗缺血性脑血管病的一种可能手段。     

A Comprehensive Study of the Relation Between Serum Concentrations, Concentration Ratios, and Level/Dose Ratios of Carbamazepine and Its Metabolites with Age, Weight, Dose, and Clearances in Epileptic Children

Summary: We made a comprehensive study of the relation between age, weight, carbamazepine (CBZ) dose, total clearance (TC), and intrinsic clearance (IC) and concentrations, concentration ratios, and level/dose ratios of CBZ, carbamazepine-10,11-epoxide (CBZ-E) and trans -10,11-dihydroxy-10,11-dihydro-carbamazepine (CBZ-H) in a group of epileptic children receiving CBZ monotherapy. Body weight and age showed negative correlations with TC, IC, CBZ dose, and CBZ-E/CBZ and CBZ-HI CBZ concentration ratios, and had positive relation with CBZ, CBZ-E, and CBZ-H level/dose ratios. These results indicate decreased CBZ metabolism with patient maturity. Correlations between CBZ dose with TC, IC, and the concentration ratios of CBZ-E/CBZ, CBZ-H/CBZ-E, and CBZ-H/CBZ were positive. CBZ dose also had negative associations with CBZ and CBZ-E level/dose ratios, indicating dose-dependent autoinduction of CBZ metabolism. Our data suggest that weight, age, and CBZ dose have less influence on epoxidehydrolase activities than on epoxidase activities. The CBZ-E/CBZ concentration ratio can be used as an indicator of the degree of autoinduction of CBZ metdbohn, even in patients receiving CBZ monotherapy.     

不同途径移植人脐血单核细胞对脑出血大鼠神经功能的影响

目的 观察经不同途径移植人脐血单核细胞14天后脑出血大鼠神经功能评分的变化,探讨干细胞移植的最佳途径。方法 采用自体血二次注血/退针法制作脑出血模型,经计算机断层扫描（computed tomography,CT）检查证实脑出血模型构建成功后,将从人脐血中分离出的新鲜人脐血单核细胞（human umbilical cord blood mononuclear cells,HUCBMC）分别经Wistar大鼠尾静脉、左心室及脑出血局部移植入实验动物体内,对照组造模成功后,不予治疗,自然转归。各组模型均于移植1、3、7、14天采用Longa评分法评价大鼠神经功能。结果 经尾静脉、左心室及脑出血局部移植HUCBMC的大鼠神经功能评分,经统计学处理后提示不同时间点大鼠的神经功能评分差异有显著性（F =131.87,P＜0.001）;移植时间（time）与移植方法（group）的交互效应显示不同的治疗方法时间趋势相同（F =35.54,P＞0.05）。大鼠神经功能评分组间比较显示,不同移植方法对大鼠神经功能评分变化的差异有显著性（F =6.434,P =0.001）;HUCBMC移植术后1天脑局部移植组大鼠神经功能评分（2.35±0.67）高于其他组,移植术后3、7、14天脑局部移植组大鼠神经功能评分（分别为0.40±0.60,0.25±0.37,0.03±0.22）低于其他组;HUCBMC移植后1天4组大鼠神经功能评分差异无显著性（F =2.14,P =0.10）;移植3、7、14天4组大鼠神经功能评分差异有显著性（F值分别为5.59,22.94,11.07,其对应P值均＜0.01）;相同移植途径不同时间点多个样本均数之间比较各组差异有显著性（F值分别为27.71,29.07,92.11,13.47,其对应P＜0.001）;尾静脉与左心室移植组各时间点神经功能评分差别无显著性（P分别为0.85,0.08,0.70,0.68）。结论 经尾静脉、左心室及脑出血局部途径移植人脐血单核细胞治疗脑出血大鼠神经功能均有改善;脑局部移植是脐血单核细胞移植的最佳途径。     

Neuronal Conditioning Medium and Nerve Growth Factor Induce Neuronal Differentiation of Collagen-Adherent Progenitors Derived from Human Umbilical Cord Blood

The aim of the study was to isolate and characterize a population of neuronal progenitors in the human umbilical cord blood (HUCB) mononuclear cell (MNC) fraction, for in vitro manipulation towards neuronal differentiation. Selection of the HUCB neuronal progenitors (HUCBNPs) was based on the neuronal prerequisite for adherence to collagen. Populations of collagen-adherent, nestin-positive (94.8 ± 2.9%) progenitors expressing α1/2 integrin receptors, as revealed by Western blot and adhesion assay using selective antagonists, were isolated and survived for more than 14 days. In vitro differentiation of the HUCBNPs was achieved by treatment with 10% human SH-SY5Y neuroblastoma cell-conditioning media (CM) supplemented with 10 ng/ml nerve growth factor (NGF). Some 83 ± 8.2% of the surviving progenitors acquired a neuronal-like morphology, expressed by cellular outgrowths of different lengths. About 35 ± 6% of the HUCBNPs had long outgrowths with a length/cell diameter ratio greater than 2, typical of developing neurons. The majority of these progenitors, analyzed by immunocytochemistry and/or RT-PCR, expressed common neuronal markers such as microtubule-associated protein 2 (MAP-2; 98.5 ± 2%), neurotrophin receptor (TrkA; 98.5 ± 0.06%), neurofillament-160 (NF-160; 94.2 ± 1%), beta-tubulin III (89.8 ± 4.2%) and neuron specific enolase (NSE). Combined CM and NGF treatment induced constitutive activation of the mitogen-activated protein kinases ERK2 (36-fold vs control), p38α (nine-fold vs control) and p38beta (23-fold vs control), most likely related to survival and/or differentiation. The results point to operationally defined conditions for activating neuronal differentiation of HUCBNPs ex vivo and emphasize the crucial role of neuronal CM and NGF in this process. This study is part of a PhD thesis to be submitted to the Hebrew University of Jerusalem by H.A.Z.     

Interactions of Phenobarbital and Phenytoin with Carbamazepine and Its Metabolites' Concentrations, Concentration Ratios, and Level/Dose Ratios in Epileptic Children

Summary: The effects of phenytoin (PHT) or phenobarbital (PB) comedication on the concentrations, concentration ratios, and level/dose ratios of carbamazepine (CBZ) and its metabolites were investigated. The hetero-induction effects of CBZ metabolism by PHT or PB were clearly demonstrated. Serum CBZ level/dose ratios in patients with CBZ polytherapy were decreased while CBZ- l0,11-epoxide (CBZ-E) and trans -l0,11-dihydroxy-10, 1-dihydro-CBZ (CBZ-H) concentrations were increased as compared with those of patients receiving CBZ alone. The concentration ratios of CBZ-H/CBZ and CBZ-E/ CBZ were also greater in patients receiving CBZ +PHT or CBZ+PB than in patients receiving CBZ alone. In addition, positive correlations between serum PHT concentration and CBZ-H/CBZ or CBZ-E/CBZ concentration ratios were observed. There were no significant differences in CBZ-H/CBZ-E concentration ratios, the free fractions of CBZ and its metabolites, and CBZ-E or CBZ-H 1eveYdose ratios among the three groups of patients. Because this approach investigates the in vivo relation between the substrates and products of the enzymes involved in CBZ biotransformation, more detailed information about the drug interactions was obtained. The results suggest that the PHT has a potent induction effect on CBZ epoxidase, whereas PB is a moderate inducer.     

Regional Spinal Cord and Brain Blood Flows in the Rat

Regional spinal cord blood flow and blood flow in different brain areas were measured in 18 rats by the microsphere technique. By the use of spheres labeled with two different isotopes injected 5 min apart, double determinations were made. The consistency of the results from the two injections was very good. Blood flow was highest in the lumbar part of the spinal cord, somewhat lower in the lower thoracic part, lowest in the upper thoracic part, and again higher in the cervical cord; the mean values of the two determinations were 0.98, 0.74, 0.42, and 0.48 ml · min^-1 · g^-1 tissue for these regions, respectively. The brain areas investigated were the brain stem, cerebellum, and right and left hemispheres; the respective mean values of the two blood flow determinations in these areas were 0.54, 0.58, 0.33, and 0.38 ml · min^-1 · g^-1 tissue.     

Simultaneous Measurement of Local Blood Flow and Tissue Oxygen in Rat Spinal Cord

Measurement of local blood flow by hydrogen clearance is a useful technique and is compatible with simultaneous measurement of oxygen tension over long periods. However, existing methods present serious limitations of spatial resolution due to high diffusion rates and other factors. Improved methods permit local measurements in both gray and white matter of the rat spinal cord that correlate well with data from autoradiographic techniques, and indeed distinguish between individual gray matter laminae. Applications of similar methods should be useful in other systems where high spatial resolution is required.     

Intradose and Circadian Variation in Circulating Carbamazepine and Its Epoxide in Epileptic Patients: A Consequence of Autoinduction of Metabolism

Total and free carbamazepine (CBZ), and CBZ 10,11 epoxide (CBZ-E) concentrations were measured over 24 h in 19 patients receiving CBZ 400 mg b.i.d. either as monotherapy (n = 13) or combined with another anticonvulsant (n = 6). Differences in CBZ and CBZ-E disposition between day and night dosing were minor. Mean plasma CBZ concentrations were higher and CBZ-E/CBZ ratios were lower in the monotherapy patients. Variations in total and free plasma CBZ levels were comparable in the monotherapy and polypharmacy groups. Peak free and total CBZ concentrations coincided at approximately 4 h postdose. Free CBZ levels correlated significantly with total in each patient. The extent of variation in total plasma CBZ concentration during 24 h correlated significantly with antipyrine clearance in the monotherapy group. Circadian rhythms are unlikely to influence CBZ disposition to a clinically relevant extent. Measurement of peak and trough CBZ concentrations should improve the value of therapeutic drug monitoring. The diurnal variation in CBZ concentration appears related to the degree of autoinduction of metabolism and is substantial enough to warrant the development of a slow-release preparation of the drug.     

Blood Flow Increase by Cervical Spinal Cord Stimulation in Middle Cerebral and Common Carotid Arteries

The effect of spinal cord stimulation (SCS) on cerebral blood flow (CBF) has, in the past, been evaluated by semiquantitative techniques, but has not been used to treat CBF diseases. The aim of this study was to assess the effect of cervical SCS on regional blood flow by both semiquantitative and quantitative methods. Thirty‐five patients with cervical SCS‐implanted devices were enrolled. The following parameters were measured before and after cervical SCS: systolic and diastolic velocity (cm/s) in the middle cerebral artery (MCA) by transcranial Doppler (TCD) and volume blood flow quantification (ml/min) in the common carotid artery (CCA) by color Doppler. During cervical SCS there was a significant and bilateral increase in systolic (21%) and diastolic (26%) velocity in the MCA and in CCA blood flow (50%). We conclude that cervical SCS increases blood flow in the middle cerebral artery and common carotid artery. The consistent increase supports the potential usefulness of cervical SCS as an adjuvant treatment for cerebral blood flow diseases.     

脑梗死急性期患者脑血流自动调节功能观察

目的 利用临界关闭压（critical closing pressure,CCP）探讨脑梗死急性期自动调节功能及与病情的相关性。      

Human Umbilical Cord Blood CD45<Superscript>+</Superscript> Pan-Hematopoietic Cells Induced a Neurotherapeutic Effect in Mice with Traumatic Brain Injury: Immunophenotyping,Comparison of Maternal and Neonatal Parameters,and Immunomodulation

Human umbilical cord blood (HUCB) transplantation has become an alternative cell therapy for hematological and oncological malignancies in the clinic and is considered for neurological disorders. The heterogeneity in the content of the different stem and progenitor cells composing HUCB mononuclear cells (MNC) may influence their engraftment and neurotherapeutic effect. We hypothesized that CD45 pan-hematopoietic marker expression is heterogeneous in MNC, and therefore, CD45⁺ subpopulation enrichment for neurotherapy may provide a tool to overcome cellular variance in different HUCB units. We employed an immunomagnetic separation method to isolate and characterize HUCB CD45⁺ pan-hematopoietic subpopulation and to investigate whether the vaginal or cesarean deliveries influence their neurotherapeutic effect in a traumatic brain injury (TBI) mouse model. Adult C57BL/6J male mice were subjected to moderate TBI and intravenously xenotransplanted with 1?×?10⁶ CD45⁺ cells derived from either vaginal or cesarean HUCB units. A large heterogeneity in the expression of CD45 marker in MNC, both in vaginal and cesarean HUCB units, was found, regardless of the number of live births. A higher expression of hematopoietic markers was found in the CD45⁺ subpopulation while low expressional levels of typical mesenchymal markers were detected. Neurotherapeutic effects, evaluated with an established neurological severity score and novel object recognition test, indicated improved functional motor and memory recovery and found independent of delivery type. Cytokine analysis in extracts of TBI brain cortices indicated an acute immunomodulatory effect by HUCB CD45⁺ subpopulation upon xenotransplantation. These results may provide insights to CD45 marker as a predictor of HUCB units’ quality for neurotherapy in TBI.     

多发性硬化患者外周血肿瘤坏死因子超家族成员LIGHT及其受体HVEM的表达

目的 为分析TNF家族成员LIGHT及其受体疱疹病毒侵入介体（herpesvirus entry mediator，HVEM）与多发性硬化（MS）的关系，尝试性地研究了MS患者外周血T细胞LIGHT和HVEM的表达。方法 用流式细胞仪检测了LIGHT和HVEM在未治疗MS、免疫抑制治疗MS、脑卒中患者以及正常对照组外周血CD4^＋、CD8^＋T细胞的表达。结果 与正常对照比较．免疫调节治疗MS患者表达HVEM的外周血单个核细胞（PBMC）、CD4^＋和CD8^＋T细胞明显增多（分别为P〈0．001、P〈0．01和P〈0．001）。LIGHT尽管在未治疗MS组CD8^＋T细胞的表达呈增高趋势．但各组间差异无显著性。结论 LIGHT上调可能与CD8^＋T细胞的表达相关，LIGHT和HVEM之间是否存在调节性反馈环路尚需探讨。