Objective:To evaluate the application value of electroencephalogram (EEG) in diagnosing brain death.Methods:From December 2015 to July 2019, basic profiles, clinical data and ancillary examinations were collected retrospectively for patients applying for brain death determination. Clinical test of brain death had to be performed by at least two qualified physicians. Ancillary examinations included electroencephalogram (EEG), transcranial Doppler ultrasonography (TCD) and short-latency somatosensory evoked potential (SLSEP). The diagnostic criteria were based upon the Chinese Guidelines for Determining Brain Death in Adults and Children.Results:Among a total of 77 patients, 72 of them fulfilled the diagnostic criteria for brain death. The average age was (42±5)(8-67) years and the completion rate of EEG 96.4%. And 13.9% of brain-dead patients demonstrated electromyographic artifact and EEG hinted at electrocerebral inactivity after using muscle relaxants. The diagnostic sensitivity of EEG was 95.8%in brain death. For one false positive case, EEG still predicted the patient's eventual mortality possibly due to the inability of EEG to show electrical activity in brain stem.Conclusions:EEG has an excellent sensitivity in brain death determination. Scalp electromyography artifact is not uncommon on EEG of brain-dead patients. And muscle relaxants may aid the determination process if necessary.

INTRODUCTION:Right-to-left shunt (RLS) induced by a patent foramen ovale (PFO) is associated with an increased risk of cryptogenic stroke (CS). However, little is known about the relationship between the amount of RLS and the stroke pattern. In this study, we aimed to evaluate the distinct clinical features of PFO-related CS in different RLS degrees resulting from PFO. METHODS:This is a cohort study of 222 CS patients admitted to the Tongji Hospital from 1st May 2014 to 31st April 2017. All patients underwent contrast transcranial Doppler examination. And then, 121 (54.5%) were classified as non-RLS group, while 76 (34.2%) were classified as mild RLS group and 25 (11.3%) were large RLS group according to the number of micro-emboli signals. The groups were compared with respect to their clinical and neuroimaging characteristics. RESULTS:In terms of risk factors of stroke, the prevalence of hypertension was lower in mild group (p = 0.002). Regarding the infarct patterns in different CS patients, we found that the multiple cortical lesions were more frequently observed (p<0.001) with increasing RLS in DWI. Moreover, there was a rising trend in the proportion of small lesions (≤1 cm) with an increasing RLS (p < 0.01). And as RLS increased, the posterior circulation was more likely to be influenced (p < 0.05). In addition, the serum cholesterol concentration was lower in the large RLS group, compared to the non-RLS group (p = 0.003) and mild RLS group (p = 0.008). While the mean platelet volume (MPV) in mild group was significantly higher than that of non-RLS group (p = 0.013). CONCLUSION:Patients with larger RLS show more infarction in posterior circulation, higher frequency of small lesions or multiple cortical lesions. The results of our study indicate that the infarct patterns might be a clue of PFO-related stroke.

Whole-cell patch clamp recording has been successfully used in identifying the voltage-dependent gating and conductance properties of ion channels in a variety of cells. However, this powerful technique is of limited value in studying low membrane resistance cells, such as astrocytes in situ, because of the inability to control or accurately measure the real amplitude of command voltages. To facilitate the study of ionic conductances of astrocytes, we have developed a dual patch recording method which permits membrane current and membrane potential to be simultaneously recorded from astrocytes in spite of their extraordinarily low membrane resistance. The utility of this technique is demonstrated by measuring the voltage-dependent activation of the inwardly rectifying K+ current abundantly expressed in astrocytes and multiple ionic events associated with astrocytic GABAA receptor activation. This protocol can be performed routinely in the study of astrocytes. This method will be valuable for identifying and characterizing the individual ion channels that orchestrate the electrical activity of low membrane resistance cells.

Similar to astrocytes, NG2 glial cells are uniformly distributed in the central nervous system (CNS). However, little is known about the interspatial relationship, nor the functional interactions between these two star-shaped glial subtypes. Confocal morphometric analysis showed that NG2 immunostained cells are spatially organized as domains in rat hippocampal CA1 region and that each NG2 glial domain occupies a spatial volume of approximate to 178, 364 m(3). The processes of NG2 glia and astrocytes overlap extensively; each NG2 glial domain interlaces with the processes deriving from 5.8 +/- 0.4 neighboring astrocytes, while each astrocytic domain accommodates processes stemming from 4.5 +/- 0.3 abutting NG2 glia. In CA1 stratum radiatum, the cell bodies of morphologically identified glial cells often appear to make direct somatic-somata contact, termed as doublets. We used dual patch recording and postrecording NG2/GFAP double staining to determine the glial identities of these doublets. We show that among 44 doublets, 50% were NG2 glia-astrocyte pairs, while another 38.6% and 11.4% were astrocyte-astrocyte and NG2 glia-NG2 glia pairs, respectively. In dual patch recording, neither electrical coupling nor intercellular biocytin transfer was detected in astrocyte-NG2 glia or NG2 glia-NG2 glia doublets. Altogether, although NG2 glia and astrocytes are not gap junction coupled, their cell bodies and processes are interwoven extensively. The anatomical and physiological relationships revealed in this study should facilitate future studies to understand the metabolic coupling and functional communication between NG2 glia and astrocytes. (c) 2013 Wiley Periodicals, Inc.

Mammalian protoplasmic astrocytes are extensively coupled through gap junction channels but the biophysical properties of these channels under physiological and ischemic conditions in situ are not well defined. Using confocal morphometric analysis of biocytin-filled astrocytic syncytia in rat hippocampal CA1 stratum radiatum we found that each astrocyte directly couples, on average, to 11 other astrocytes with a mean interastrocytic distance of 45 microm. Voltage-independent and bidirectional transjunctional currents were always measured between directly coupled astrocyte pairs in dual voltage-clamp recordings, but never from astrocyte-NG2 glia or astrocyte-interneuron pairs. The electrical coupling ratio varied considerably among astrocytes in developing postnatal day 14 rats (P14, 0.5-12.4%, mean = 3.6%), but became more constant in young adult P21 rats (0.18-3.9%, mean = 1.6%), and the coupling ratio declined exponentially with increasing pair distance. Electrical coupling was not affected by short-term oxygen-glucose deprivation (OGD) treatment, but showed delayed inhibition in an acidic extracellular pH of 6.4. Combination of acidic pH (6.4) and OGD, a condition that better represents cerebral ischemia in vivo, accelerated the inhibition of electrical coupling. Our results show that, under physiological conditions, 20.7-24.2% of K(+) induced currents can travel from any astrocytic soma in CA1 stratum radiatum to the gap junctions of the nearest neighbor astrocytes, but this should be severely inhibited as a consequence of the OGD and acidosis seen in the ischemic brain.

Expression of a linear current–voltage (I–V) relationship (passive) K+ membrane conductance is a hallmark of mature hippocampal astrocytes. However, the molecular identifications of the K+ channels underlying this passive conductance remain unknown. We provide the following evidence supporting significant contribution of the two-pore domain K+ channel (K2P) isoforms, TWIK-1 and TREK-1, to this conductance. First, both passive astrocytes and the cloned rat TWIK-1 and TREK-1 channels expressed in CHO cells conduct significant amounts of Cs+ currents, but vary in their relative PCs/PK permeability, 0.43, 0.10, and 0.05, respectively. Second, quinine, which potently inhibited TWIK-1 (IC50 = 85 μm) and TREK-1 (IC50 = 41 μm) currents, also inhibited astrocytic passive conductance by 58% at a concentration of 200 μm. Third, a moderate sensitivity of passive conductance to low extracellular pH (6.0) supports a combined expression of acid-insensitive TREK-1, and to a lesser extent, acid-sensitive TWIK-1. Fourth, the astrocyte passive conductance showed low sensitivity to extracellular Ba2+, and extracellular Ba2+ blocked TWIK-1 channels at an IC50 of 960 μm and had no effect on TREK-1 channels. Finally, an immunocytochemical study showed colocalization of TWIK-1 and TREK-1 proteins with the astrocytic markers GLAST and GFAP in rat hippocampal stratum radiatum. In contrast, another K2P isoform TASK-1 was mainly colocalized with the neuronal marker NeuN in hippocampal pyramidal neurons and was expressed at a much lower level in astrocytes. These results support TWIK-1 and TREK-1 as being the major components of the long-sought K+ channels underlying the passive conductance of mature hippocampal astrocytes.

Objective:The morphological and electrophysiological characteristics of astrocytes and NG2 glia in rat hippocampal slices have been investigated in order to distinguish these two types of glial cells.Methods:All the recorded cells came from CA1 stratum radiatum(SR) regions in P21～P25 rat hippocampal slices.By employing patch clamp whole-cell recording technique,post-recording labeling method and confocal imaging system,we have studied the differences between astrocytes and NG2 glial cells in the morphological and basic electrophysiological properties.Results:Morphometrically,astrocytes harbored a large number of fine processes which were extremely dense and branched.Abundant processes formed the typical spongiform morphology of astrocytes.In comparison,NG2 glia had much less branched processes that were long and threadlike.The somata of NG2 cells were small and round and,unlike astorcytes,lack of classic primary processes.Electrophysiologically,the resting membrane potential(RMP) of these two types of glial cells were very close.However,the differences in the membrane capacitance(Cm) and membrane resistance(Rm) were significant.I-V relationship from astrocytes was linear which signified the typical passive electrical character of astrocytes,but I-V plot from NG2 glia showed apparent rectification.Fast-inactiving K+ currents(Ka),delayed rectifying K+ currents(Kdr) and a fraction of inwardly rectifying K+ currents(Kir) had contributed to the whole-cell currents of NG2 glia.In addition,some of NG2 glia expressed voltage-gated Na+ currents in a low density.Conclusion:Astrocytes and NG2 glial cells have different morphological and electrophysiological properties and based on the above-mentioned differences we can distinguish them very clearly.

目的 探讨骨髓间充质干细胞(MSCs)移植对脊髓损伤大鼠神经功能恢复的影响极其可能机制.方法 采集SD大鼠骨髓,分离出MSCs,并培养、纯化,取传至第6代的MSCs,移植前1 d以5溴2脱氧尿嘧啶核苷(Brdu)标记,然后用显微注射器将其缓慢注射到脊髓损伤大鼠模型(成年SD大鼠)的受损脊髓中心,以不进行移植的脊髓损伤大鼠模型(损伤组)和假手术组为对照.术后进行运动功能评分以判断神经功能恢复情况,并切取移植区域脊髓组织,应用荧光免疫化学染色检测脊髓损伤灶边周的神经元凋亡情况(TUNEI.与NeuN双标记)以及移植的MSCs的分布和向神经元分化情况(Brdu与NeuN双标记),应用逆转录聚合酶链反应检测损伤灶区域脑源性神经营养因子(BDNF)mRNA的表达.结果 移植后第3天开始,损伤组和移植组大鼠的BBB运动功能评分明显上升,第14天后进入平台期,但移植组各时间点的.BBB运动功能评分均明显高于损伤组(P<0.05).移植后3 d,在移植组的脊髓组织中可见Brdu标记阳性细胞,少部分Brdu标记阳性细胞同时表达神经元特异性标志物NeuN;移植后第3、7天,损伤组和移植组脊髓组织中的凋亡神经元均显著多于假手术组(P<0.01),凋亡的神经元多存在于损伤周边区,但移植组的凋亡神经元显著少于损伤组(P<0.01);移植后第3、7天,假手术组未检测到BDNF mRNA的表达,损伤组和移植组均可检测到BDNF mRNA的表达,移植组的表达量分别较损伤组高28.6%和39.2%(P<0.05).结论 移植的骨髓MSCs可促进脊髓损伤大鼠神经功能的恢复,其机制除MSCs直接分化为神经元进行修复外,还可能与其改善脊髓损伤区的微环境、上调BDNF、mRNA表达、减少神经细胞凋亡有关.

取胎鼠海马进行神经干细胞(NSC)的培养、鉴定与标记,以氯化锂-匹罗卡品诱导制成大鼠急性癫痫模型,于发作后第4天将以5-溴-2'-脱氧尿苷(Brdu)标记的NSC移植入大鼠海马,检测移植后NSC在宿主体内的存活情况,移植细胞对癫痫大鼠齿状回苔藓纤维出芽(MFS)的影响.发现移植后2个月NSC在宿主体内能够存活,并向移植区周围迁移;NSC移植组大鼠海马齿状回内分子层Timm染色颗粒明显少于未移植组.认为NSC移植入癫痫大鼠海马后能够存活,并能显著抑制海马齿状回MFS.

Objective: To explore the isolation, expansion and differentiation of murine bone marrow-derived mesenchymal stem cells (mMSCs) into neuron-like cells in vitro. Methods:Bone marrow was collected from 8- to 9-week-old KM mouse, suspended in the complete culture medium and planted into FN-coated plates. MMSCs were purified and expanded in vitro. After 9-10 passages, the growth status, cell cycle, phenotype and morphology of mMSCs were analyzed and observed by a microscope. The specific marking proteins of neurons, glia and neural stem cells were detected by immunofluorescence before and after induction. Results: MMSCs were adherent cells with a morphology of fibroblastoid presenting spindle-shaped and polygonal-shaped and the morphology was similar in culture of 15 passages. Cell cycle assay demonstrated 83 % of mMSCs were in G0/G1 phase. Phenotype showed that mMSCs were positive for the surface markers CD44, CD13 and negative for CD45, which was identical to human bone marrow-derived MSCs. After the inducement, most mMSCs turned into bipolar, multipolar and taper, then intersected as network structure. Moreover, they had the phenotypes of neurons, glia and neural stem cells. Conclusion: MMSCs sharing the same morphology, phenotype, growth characteristics, and potentiality of differentiation into neuron-like cells as human MSCs can be successfully isolated from bone marrow and expanded in vitro. Therefore, mMSCs will be an perfect tool to study the cellular and genetic therapy in vivo in mouse pathology models.

Objective: To compare the effect of growth factor (bFGF, EGF), B27 and serum on the differentiation of embryonic stem cells in culture. Methods: Embryonic E16-18 rats were obtained from pregnant Wistar rats. Neural stem cells were dissociated from ventricular zone and divided into 4 groups: B27 group, bFGF+EGF group, serum group and the control group. The growth of the cells was observed everyday. Results: Most of the cultured cells were nestin-positive. The growth and differentiation of the stem cells were promoted in all 4 groups. According to double-labeling immunofluorerscence and picture, more neural stem cells differentiate into neurons in the B27 and bFGF+EGF group, and the latter was more significant(P0.01);In the serum and control group, more neural stem cells differentiate into astrocytes, which is more significant in the serun group.Conclusion: Interior gene and exterior environment have effects on differentiation of neural stem cells.bFGF and EGF not only promote the growth of cells but also the differentiation.

目的探讨安全高效移植骨髓间充质干细胞(mensenchymal stem cells,MSCs)治疗脑缺血的方法.方法采用连续传代培养的方法纯化MSCs,Brdu标记后分别经颈动脉(1×104/μl,200 μl)和立体定位(1×105/μl,10 μl)注射至大鼠脑缺血2 h再灌注1 d模型,各自设立对照组,观察术后神经功能评分以及脑部Brdu阳性细胞的分布.结果经颈动脉注射组较立体定位注射组爬杆实验评分低(P＜0.05),Brdu阳性细胞存活多,迁移范围更广.结论 MSCs具有卒中后脑保护作用,而经颈动脉的给药方式优于立体定位注射.