Cholinesterases and peanut agglutinin binding related to cell proliferation and axonal growth in embryonic chick limbs

Embryonic cholinesterases are assigned important functions during morphogenesis. Here we describe the expression of butyrylcholinesterase and acetylcholinesterase, and the binding of peanut agglutinin, and relate the results to mitotic activity in chick wing and leg buds from embryonic day 4 to embryonic day 9. During early stages, butyrylcholinesterase is elevated in cells under the apical ectodermal ridge and around invading motoraxons, while acetylcholinesterase is found in the chondrogenic core, on motoraxons and along the ectoderm. Peanut agglutinin binds to the apical ectodermal ridge and most prominently to the chondrogenic core. Measurements of thymidine incorporation and enzyme activities were consistent with our histological findings. Butyrylcholinesterase is concentrated near proliferative zones and periods, while acetylcholinesterase is associated with low proliferative activity. At late stages of limb development, acetylcholinesterase is concentrated in muscles and nonexistent within bones, while butyrylcholinesterase shows an inverse pattern. Thus, as in other systems, in limb formation butyrylcholinesterase is a transmitotic marker preceding differentiation, acetylcholinesterase is found on navigating axons, while peanut agglutinin appears in non-invaded regions. These data suggest roles for cholinesterases as positive regulators and peanut-agglutinin-binding proteins as negative regulators of neural differentiation.     

A simple procedure for quantification of neurite outgrowth based on stereological principles

The molecular mechanisms controlling formation and remodelling of neuronal extensions are of considerable interest for the understanding of neuronal development and plasticity. Determination of neurite outgrowth in cell culture is a widely used approach to investigate these phenomena. This is generally done by a time consuming tracing of individual neurites and their branches. We have used stereological principles to determine the length of neurites. The total neuritic length per cell was estimated by counting the number of intersections between neurites and test lines of an unbiased counting frame superimposed on images of cell cultures obtained by conventional computer-assisted microscopy. The absolute length, L, of neurites per cell was subsequently estimated from the number of neurite intersections, I, per cell by means of the equation L=(πd/2)I describing the relationship between the number of neurite intersections and the vertical distance, d, between the test lines used. When measuring neurite outgrowth from PC12 cells and primary hippocampal neurons, data obtained by counting neuritic intersections correlated statistically significantly with data obtained using a conventional tracing technique. However, information was acquired more efficiently using the stereological approach. Thus, using the described set-up, the stereological procedure was approximately five times less time consuming than the conventional method based on neurite tracing. The study shows that stereological estimation of neuritic length provides a precise and efficient method for the study of neurite outgrowth in cultures of primary neurons and cell lines.     

Ultrasound can Modulate Neuronal Development: Impact on Neurite Growth and Cell Body Morphology

Neuronal development is known to be a dynamic process that can be modulated by presenting guidance cues to neuronal cells. We show that ultrasound, when applied at pulsed settings and with intensities slightly greater than clinical diagnosis levels, can potentially act as a repulsive cue for modulating neuronal growth dynamics. Using differentiated Neuro-2a cells as the model, we have examined in vitro how neuronal development can change during and after exposure to 1-MHz ultrasound for different acoustic settings. Neurite retraction and cell body shrinkage were found in neuronal cells over a 10-min exposure period with 1.168 W/cm² spatial-peak, time-averaged intensity (based on 0.84 MPa peak acoustic pressure, 100-cycle pulse duration, and 500-Hz pulse repetition frequency). These effects were found to result in instances of neuronal cell body displacement. The extent of the effects was dependent on acoustic intensity, with peak acoustic pressure being a more important contributing factor compared with pulse duration. The morphological changes were found to be non-destructive, in that post-exposure neurite outgrowth and neuritogenesis were respectively observed in neurite-bearing and neurite-less neuronal cells. Our results also showed that mechanotransduction might be involved in mediating ultrasound-neuron interactions, as the morphological changes were suppressed if stretch-activated ion channels were blocked or if calcium messenger ions were chelated. Overall, these findings suggest that ultrasound can potentially influence how neuronal cells develop through modifying their cytomechanical characteristics.     

编码轴突生长抑制因子的重组DNA疫苗的免疫原性研究

目的 探讨以前期实验所构建的腺病毒骨架质粒pAdEasy为载体,编码神经损伤后轴突生长抑制因子Nogo-A、少突胶质细胞髓磷脂糖蛋白(OMgp)、腱糖蛋白-R(TN-R)和髓磷脂相关糖蛋白(MAG)的重组DNA疫苗的免疫原性. 方法 16只5周龄Lewis大鼠按随机数字表法分为DNA疫苗注射组(Vaccine组)和空质粒注射组(pAdEasy组).Vaccine组大鼠以DNA疫苗经双侧胫骨肌注射免疫,1次/周,共持续8周.每次进行疫苗注射前采血和分离血清,Dot-blot和ELISA法对血清中抗体进行定性和定量检测. 结果 6周后Vaccine组大鼠血清能与GST-TN-R和GST-OMgp融合蛋白产生较强的免疫反应,点杂交反应较明显;pAdEasy组大鼠血清则不能与GST-TN-R和GST-OMgp融合蛋白产生免疫反应.6周后Vaccine组大鼠血清中抗体效价则可以达到1:100万,并保持稳定的水平. 结论 编码轴突生长抑制因子Nogo-A、OMgp、TN-R和MAG的重组DNA疫苗接种大鼠后能够产生特异性的抗体.说明该重组DNA疫苗具有良好的免疫原性.     

Induction of functional and morphological expression of neuropeptide Y (NPY) in cortical cultures by brain-derived neurotrophic factor (BDNF): evidence for a requirement for extracellular-regulated kinase (ERK)-dependent and ERK-independent mechanisms

Previous studies have demonstrated that brain-derived neurotrophic factor (BDNF) induces expression of neuropeptide Y (NPY) neurons in aggregate cultures derived from the fetal rat cortex. Using BDNF induction of NPY production and neurite extension of NPY neurons as functional and morphological criteria, respectively, we addressed the question: Does BDNF activate the extracellular-regulated kinase (ERK) pathway and if so, is activated (phosphorylated, P)-ERK required for the induction of both the functional and morphological expression of NPY? BDNF led to a rapid (30 min) and sustained (6 h) phosphorylation of ERK. PD98059 (PD, a specific inhibitor of the ERK kinase MEK), drastically inhibited, LY294002 (LY, a specific inhibitor of phosphatidylinositol-3-kinase, PI-3K) partially inhibited, and GF 109203X (GF, a specific inhibitor of protein kinase C) did not inhibit phosphorylation of ERK. A 24-h exposure to BDNF led to approximately 2-fold increase in the total culture content of NPY ( approximately 60% of which was secreted and approximately 40% remained in the aggregates) and to an abundance of neurite-bearing NPY neurons. BDNF-induced NPY produced and secreted into the medium was inhibited 73% by PD, 52% by LY and not at all by GF. In contrast, BDNF-induced NPY produced and sequestered in the aggregates was not inhibited by any of these inhibitors, suggesting a role for the ERK pathway in induced secretion of NPY. PD or LY did not inhibit BDNF-induced abundance of neurite-bearing NPY neurons. K252a (an inhibitor of TrkB-tyrosine kinase) abolished all the effects of BDNF assessed in our cultures. In summary, we demonstrate that TrkB-mediated activation of the ERK pathway is preferentially required for BDNF induction of NPY produced and secreted but not for the induction of the expression of neurite-bearing NPY neurons. Thus, BDNF induction of the functional and morphological expression of NPY is brought about by ERK-dependent and ERK-independent mechanisms.     

Directed nerve outgrowth is enhanced by engineered glial substrates

In the present study, the influence of astrocyte alignment on the direction and length of regenerating neurites was examined in vitro. Astrocytes were experimentally manipulated by different approaches to create longitudinally aligned monolayers. When cultured on the aligned monolayers, dorsal root ganglion neurites grew parallel to the long axis of the aligned astrocytes and were significantly longer than controls. Engineered monolayers expressed linear arrays of fibronectin, laminin, neural cell adhesion molecule, and chondroitin sulfate proteoglycan that were organized parallel to one another, suggesting that a particular spatial arrangement of these molecules on the astrocyte surface may be necessary to direct nerve regeneration in vivo. In contrast, no bias in directional outgrowth was observed for neurites growing on unorganized monolayers. The results suggest that altering the organization of astrocytes and their scar-associated matrix at the lesion site may be used to influence the direction and the length of adjacent regenerating axons in the damaged brain and spinal cord.     

White matter inhibitors in CNS axon regeneration failure

Multiple lines of evidence have indicated that the inability of adult mammalian central nervous system (CNS) axons to regenerate after injury is partly due to the growth inhibitory property of central myelin. Three prototypical myelin-associated inhibitors of neurite outgrowth have been identified, including Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp). These inhibitory ligands, their receptors and signaling pathways are being intensively investigated for their roles in CNS axon regeneration failure. In addition, several members of the axon guidance molecules have been implicated in restricting CNS axon regeneration, some of which are expressed by mature oligodendrocytes. Here we review in vitro and in vivo studies of these molecules in neurite growth and in axon regeneration failure and discuss the implications of these studies. While the increasing number of potential axon regeneration inhibitors highlights the complexity of the restrictive CNS environment, it provides new windows of opportunity as well as new challenges for therapeutic development for spinal cord injury and related neurological conditions.     

吗啡增强备用根大鼠脊髓后角组织提取液对培养鸡胚背根节的促神经突起生长的作用

用组织培养方法，探讨备用很大鼠手术侧和非手术侧脊髓后角组织提取液对鸡胚背根节（ＤＲＧ）神经突起的促生长作用，以及应用吗啡对此作用的影响。结果显示：备用根大鼠手术侧脊髓后角组织提取液作用的ＤＲＧ神经突起密度（３６．４２±４．６９），比非手术侧的（２３．９６±３．４７）明显增大。提示去初级传入纤维的脊髓后角组织具有促进ＤＲＧ神经突起生长的神经营养活性作用。应用吗啡的备用根大鼠手术侧脊髓后角组织提取液作用的ＤＲＧ神经突起密度（６４．１９±９．２４），又比备用根大鼠手术侧的大。这表明，吗啡具有进一步增强去初级传入纤维支配的脊髓后角组织提取液促进培养的ＤＲＧ神经突起生长的效应。     

Adhesive proteins linked with focal adhesion kinase regulate neurite outgrowth of PC12 cells

Adhesive proteins existing in the extracellular matrix (ECM) play important roles in the regulation of neuronal cell behavior, including cell adhesion, motility and neurite outgrowth. Herein we show the effects of a series of adhesive proteins on the neurite outgrowth of PC12 cells and elucidate that this is closely related to the activation of focal adhesion kinase (FAK). For this we prepared culture substrates by coating tissue culture plastic with either collagen (Col), fibronectin (FN) or laminin (LN) and investigated the neurite outgrowth behavior. The results demonstrated that neurite outgrowth was highly dependent on the particular type of adhesive protein. While neurite number was comparable on all the coated surfaces, the length of neurites was greater on the FN- and LN-coated ones (greatest on the LN-coated one). In particular, FAK expression was highly up-regulated in the FN- and LN-coated surfaces, as revealed by Western blot analysis. A knock-down experiment further supported the idea that neurite outgrowth was largely suppressed in cells transfected with a FAK knock-down gene. Taken together, the neurite outgrowth of PC12 cells was greatly affected by adhesive proteins of the ECM, particularly FN and LN, and this is considered to be closely related to FAK intracellular signaling. This study may be useful in the consideration and design of nerve guidance and three-dimensional scaffolds which are appropriate to promote neuronal growth and nerve tissue regeneration.     

Optimal Micropattern Dimensions Enhance Neurite Outgrowth Rates,Lengths, and Orientations

Micropattern dimensions can significantly influence neurite outgrowth orientation, rate, and length. Laminin micropatterns of various widths from 10 to 50 μm at 10 μm intervals separated by 40 μm spaces were generated on poly(methyl methacrylate) surfaces using microscale plasma-initiated patterning (μPIP). Dissociated dorsal root ganglion (DRG) neurons were seeded on the micropatterned surfaces and cultured for 24 h in serum-free media. Neurite outgrowth numbers, lengths, rates, and orientations were measured on all micropatterned substrates. The results indicated that the dimension of the laminin pattern influenced the neurite outgrowth length, rate, and orientation, but not the numbers of neurite outgrowth. Neurons on more than 30 μm wide laminin pattern showed faster neurite outgrowth compared to other dimensions, and relatively low orientation at 50 μm pattern dimensions. Neurites at 40 μm laminin pattern widths demonstrated the fastest outgrowth rates and were highly oriented. The 40 μm laminin dimension is wide enough to provide sufficient laminin amounts for neuron growth and narrow enough to efficiently guide neurites. Based on these results, adhesive protein micropatterns of 40 μm dimensions are recommended when investigating DRG neurons.