高浓度七氟醚通过抑制VEGF/PI3K/AKT信号通路诱导神经干细胞凋亡的研究

目的探讨妊娠中期的七氟醚暴露对神经干细胞凋亡过程的影响和作用机制。 方法将孕中期大鼠随机分为3组,每组48只孕鼠：对照组,低浓度七氟醚组,高浓度七氟醚组。在妊娠第14天,以2﹪或3.5﹪七氟醚麻醉怀孕大鼠2?h。通过免疫荧光检查神经干细胞凋亡,收集麻醉后6、24和48?h以及出生后第0天（P?0）,第14天（P?14）和第28天（P?28）的脑组织进行Nestin-TUNEL免疫荧光双标染色以及Nestin、血管内皮生长因子（VEGF）和磷酸肌醇3-激酶（PI3K）AKT通路相关蛋白的免疫印迹检测。采用单因素方差分析和Bonferroni事后检验进行统计学分析。 结果麻醉后6、24和48?h以及P?0,P?14和P?28,脑组织中Nestin和TUNEL阳性细胞的百分比增加[6?h：对照组0.91±0.07,低浓度组1.01±0.08,高浓度组2.62±0.21（F?=?399,P?相似文献   

Novel bioreactors for the culture and expansion of aggregative neural stem cells

Neural stem cells have been cultured as three-dimensional aggregates in a number of different types of bioreactors. The design and configuration of the bioreactor are shown to be crucial factors for the successful propagation of the cells. A novel bioreactor with liquid re-circulation and a working volume of 200 ml has been designed, tested and shown to be able to produce a higher cell vitality compared to those produced in multi-well plates, shake flasks and stirred flasks. The novel reactor was able to produce a total density of cells of 3.5 x 10(6) cells/ml consisting of a larger number of smaller and proliferative aggregates, compared to only 1.8 x 10(6) cells/ml produced in a multi-well plate. Shake flasks and stirred flasks commonly used for facilitating mass transfer in the culture of micro-organisms are shown to be unsuitable for the propagation of neural stem cells.     

Population based models of cortical drug response: insights from anaesthesia

A great explanatory gap lies between the molecular pharmacology of psychoactive agents and the neurophysiological changes they induce, as recorded by neuroimaging modalities. Causally relating the cellular actions of psychoactive compounds to their influence on population activity is experimentally challenging. Recent developments in the dynamical modelling of neural tissue have attempted to span this explanatory gap between microscopic targets and their macroscopic neurophysiological effects via a range of biologically plausible dynamical models of cortical tissue. Such theoretical models allow exploration of neural dynamics, in particular their modification by drug action. The ability to theoretically bridge scales is due to a biologically plausible averaging of cortical tissue properties. In the resulting macroscopic neural field, individual neurons need not be explicitly represented (as in neural networks). The following paper aims to provide a non-technical introduction to the mean field population modelling of drug action and its recent successes in modelling anaesthesia.     

Baculovirus expression and bioactivity of a soluble 140 kDa extracellular cleavage fragment of L1 neural cell adhesion molecule

L1 neural cell adhesion molecule is the founding member of the L1 subfamily of the immunoglobulin superfamily and plays an important role in the overall development of both the central and peripheral nervous systems, making it an attractive candidate for promoting neural regeneration following injury. Currently, L1 used for experimental studies is primarily mammalian-derived; however, the insect cell expression system described here provides an alternative source of recombinant L1 with equivalent bioactivity. A 140 kDa L1 fragment based on a physiological plasmin cleavage site in the extracellular domain was cloned and expressed with a C-terminal 6x histidine tag. Recombinant insect cell-derived L1 was analyzed by Western blot using an antibody to human L1 to confirm immunogenicity and to optimize infection conditions for recombinant L1 production. The recombinant protein was secreted by insect cells, efficiently purified under non-denaturing conditions using dialysis followed by metal affinity chromatography, and analyzed by SDS-PAGE to produce a single band of the expected approximate 140 kDa size. The bioactivity of insect cell-derived L1 was compared to mammalian-derived L1-Fc and poly-L-lysine (PLL) using chick embryonic forebrain neurons. The results show comparable, robust neurite outgrowth at 24h on insect cell-derived L1 and mammalian-derived L1-Fc, with significantly longer neurites than those observed on PLL. Future studies will examine the immobilization of L1 to biomaterial surfaces in physiologically appropriate orientation via the C-terminal 6x histidine tag and will investigate their application in promoting axonal regeneration in the injured nervous system.     

Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1 × 10⁵ ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1 × 10⁶ ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm²) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm²). These findings provided fresh insight into the neural induction of mouse ES cells.     

System identification of muscle–joint interactions of the cat hind limb during locomotion

Neurophysiological experiments in walking cats have shown that a number of neural control mechanisms are involved in regulating the movements of the hind legs during locomotion. It is experimentally hard to isolate individual mechanisms without disrupting the natural walking pattern and we therefore introduce a different approach where we use a model to identify what control is necessary to maintain stability in the musculo-skeletal system. We developed a computer simulation model of the cat hind legs in which the movements of each leg are produced by eight limb muscles whose activations follow a centrally generated pattern with no proprioceptive feedback. All linear transfer functions, from each muscle activation to each joint angle, were identified using the response of the joint angle to an impulse in the muscle activation at 65 postures of the leg covering the entire step cycle. We analyzed the sensitivity and stability of each muscle action on the joint angles by studying the gain and pole plots of these transfer functions. We found that the actions of most of the hindlimb muscles display inherent stability during stepping, even without the involvement of any proprioceptive feedback mechanisms, and that those musculo-skeletal systems are acting in a critically damped manner, enabling them to react quickly without unnecessary oscillations. We also found that during the late swing, the activity of the posterior biceps/semitendinosus (PB/ST) muscles causes the joints to be unstable. In addition, vastus lateralis (VL), tibialis anterior (TA) and sartorius (SAT) muscle-joint systems were found to be unstable during the late stance phase, and we conclude that those muscles require neuronal feedback to maintain stable stepping, especially during late swing and late stance phases. Moreover, we could see a clear distinction in the pole distribution (along the step cycle) for the systems related to the ankle joint from that of the other two joints, hip or knee. A similar pattern, i.e., a pattern in which the poles were scattered over the s-plane with no clear clustering according to the phase of the leg position, could be seen in the systems related to soleus (SOL) and TA muscles which would indicate that these muscles depend on neural control mechanisms, which may involve supraspinal structures, over the whole step cycle.     

Unexpected activities of Smad7 in Xenopus mesodermal and neural induction

Neural induction is widely believed to be a direct consequence of inhibition of BMP pathways. Because of conflicting results and interpretations, we have re-examined this issue in Xenopus and chick embryos using the powerful and general TGFβ inhibitor, Smad7, which inhibits both Smad1- (BMP) and Smad2- (Nodal/Activin) mediated pathways. We confirm that Smad7 efficiently inhibits phosphorylation of Smad1 and Smad2. Surprisingly, however, over-expression of Smad7 in Xenopus ventral epidermis induces expression of the dorsal mesodermal markers Chordin and Brachyury. Neural markers are induced, but in a non-cell-autonomous manner and only when Chordin and Brachyury are also induced. Simultaneous inhibition of Smad1 and Smad2 by different approaches does not account for all Smad7 effects, indicating that Smad7 has activities other than inhibition of the TGFβ pathway. We provide evidence that these effects are independent of Wnt, FGF, Hedgehog and retinoid signalling. We also show that these effects are due to elements outside of the MH2 domain of Smad7. Together, these results indicate that BMP inhibition is not sufficient for neural induction even when Nodal/Activin is also blocked, and that Smad7 activity is considerably more complex than had previously been assumed. We suggest that experiments relying on Smad7 as an inhibitor of TGFβ-pathways should be interpreted with considerable caution.     

Saccade-related remapping of target representations between topographic maps: a neural network study

The goal of this study was to explore how a neural network could solve the updating task associated with the double-saccade paradigm, where two targets are flashed in succession and the subject must make saccades to the remembered locations of both targets. Because of the eye rotation of the saccade to the first target, the remembered retinal position of the second target must be updated if an accurate saccade to that target is to be made. We trained a three-layer, feed-forward neural network to solve this updating task using back-propagation. The network's inputs were the initial retinal position of the second target represented by a hill of activation in a 2D topographic array of units, as well as the initial eye orientation and the motor error of the saccade to the first target, each represented as 3D vectors in brainstem coordinates. The output of the network was the updated retinal position of the second target, also represented in a 2D topographic array of units. The network was trained to perform this updating using the full 3D geometry of eye rotations, and was able to produce the updated second-target position to within a 1 degrees RMS accuracy for a set of test points that included saccades of up to 70 degrees . Emergent properties in the network's hidden layer included sigmoidal receptive fields whose orientations formed distinct clusters, and predictive remapping similar to that seen in brain areas associated with saccade generation. Networks with the larger numbers of hidden-layer units developed two distinct types of units with different transformation properties: units that preferentially performed the linear remapping of vector subtraction, and units that performed the nonlinear elements of remapping that arise from initial eye orientation.     

On the stationary state of a network of inhibitory spiking neurons

The background activity of a cortical neural network is modeled by a homogeneous integrate-and-fire network with unreliable inhibitory synapses. For the case of fast synapses, numerical and analytical calculations show that the network relaxes into a stationary state of high attention. The majority of the neurons has a membrane potential just below the threshold; as a consequence the network can react immediately – on the time scale of synaptic transmission- on external pulses. The neurons fire with a low rate and with a broad distribution of interspike intervals. Firing events of the total network are correlated over short time periods. The firing rate increases linearly with external stimuli. In the limit of infinitely large networks, the synaptic noise decreases to zero. Nevertheless, the distribution of interspike intervals remains broad. Action Editor: Misha Tsodyks     

Experimental Study on Trace Marking and Oncogenicity of Neural Stem Cells Derived from Bone Marrow

It has been well accredited that the neural stem cells (NSCs) derived from bone marrow stroma cells (BMSCs) can be used as the therapeutic application. However, their efficacy and safety in therapeutic application are uncertain. In this experiment, the trace marking and oncogenicity of NSCs derived from BMSCs (BMSCs-D-NSCs) were studied. The BMSCs were harvested by gradient centrifugation and cultured in "NSCs medium" in vitro. The verified CD133/Nestin-positive BMSCs-D-NSCs were then transplanted into nude mice to detect the oncogenicity, into the right lateral cerebral ventricle or right caudae putamen and substantia nigra to examine, whether the symptoms were improved in Parkinson's Disease (PD) models after transplantation, by both SPECT image assay of dopamine transporter (DAT) in corpus striatum and its average standard uptake value (SUVave) in corpus striatum and thalamus. Tissue samples and surviving model animals were studied at 1, 3, and 6 months post-transplantation. Before transplantation, the cells were labeled with BrdU or rAAV-GFP for the pathological sections, and with Feridex for the in vivo trace by MRI assay. The concanavalin A (ConA) agglutination test, stop-dependence test with soft agar, karyotype analysis of chromosome G zone in BMSCs-D-NSCs, and the nude mouse neoplasia test were also performed. The BrdU, rAAV-GFP or Feridex can be used as trace markers of BMSCs-D-NSCs during transplantation. The transplanted BMSCs-D-NSCs displayed neither toxicity nor neoplasia up to 6 months in vivo, but could play an important role in improving the symptoms of the animals with degenerative diseases like PD.