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.     

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。     

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.     

Participation of interleukin 17A in neuroimmune interactions

Inflammation involving the helper T cell 17 (Th17) subset of lymphocytes has been implicated in a number of diseases that affect the nervous system. As the canonical cytokine of Th17 cells, interleukin 17A (IL-17A) is thought to contribute to these neuroimmune interactions. The main receptor for IL-17A is expressed in many neural tissues. IL-17A has direct effects on neurons but can also impact neural function via signaling to satellite cells and immune cells. In the central nervous system, IL-17A has been associated with neuropathology in multiple sclerosis, epilepsy syndromes and ischemic brain injury. Effects of IL-17A at the level of dorsal root ganglia and the spinal cord may contribute to enhanced nociception during neuropathic and inflammatory pain. Finally, IL-17A plays a role in sympathetic axon growth and regeneration of damaged axons that innervate the cornea. Given the widespread effects of IL-17A on neural tissues, it will be important to determine whether selectively mitigating the damaging effects of this cytokine while augmenting its beneficial effects is a possible strategy to treat inflammatory damage to the nervous system.     

Long-term coordinated microstructural disruptions of the developing neocortex and subcortical white matter after early postnatal systemic inflammation

Severe postnatal systemic infection is highly associated with persistent disturbances in brain development and neurobehavioral outcomes in survivors of preterm birth. However, the contribution of less severe but prolonged postnatal infection and inflammation to such disturbances is unclear. Further, the ability of modern imaging techniques to detect the underlying changes in cellular microstructure of the brain in these infants remains to be validated. We used high-field ex-vivo MRI, neurohistopathology, and behavioral tests in newborn rats to demonstrate that prolonged postnatal systemic inflammation causes subtle, persisting disturbances in brain development, with neurodevelopmental delays and mild motor impairments. Diffusion-tensor MRI and neurite orientation dispersion and density imaging (NODDI) revealed delayed maturation of neocortical and subcortical white matter microstructure. Analysis of pyramidal neurons showed that the cortical deficits involved impaired dendritic arborization and spine formation. Analysis of oligodendrocytes showed that the white matter deficits involved impaired oligodendrocyte maturation and axonal myelination. These findings indicate that prolonged postnatal inflammation, without severe infection, may critically contribute to the diffuse spectrum of brain pathology and subtle long-term disability in preterm infants, with a cellular mechanism involving oligodendrocyte and neuronal dysmaturation. NODDI may be useful for clinical detection of these microstructural deficits.     

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.     

Collagen-Dependent Neurite Outgrowth and Response to Dynamic Deformation in Three-Dimensional Neuronal Cultures

In vitro models of brain injury that use thick 3-D cultures and control extracellular matrix constituents allow evaluation of cell–matrix interactions in a more physiologically relevant configuration than traditional 2-D cultures. We have developed a 3-D cell culture system consisting of primary rat cortical neurons distributed throughout thick (>500 μm) gels consisting of type IV collagen (Col) conjugated to agarose. Neuronal viability and neurite outgrowth were examined for a range of agarose (AG) percentages (1.0–3.0%) and initial collagen concentrations ([Col]_i; 0–600 μg/mL). In unmodified AG, 1.5% gels supported viable cultures with significant neurite outgrowth, which was not found at lower (≤1.0%) concentrations. Varying [Col]_i in 1.25% AG revealed the formation of dense, 3-D neurite networks at [Col]_i of 300 μg/mL, while neurons in unmodified AG and at higher [Col]_i (600 μg/mL) exhibited significantly less neurite outgrowth; although, neuronal survival did not vary with [Col]_i. The effect of [Col]_i on acute neuronal response following high magnitude, high rate shear deformation (0.50 strain, 30 s⁻¹ strain rate) was evaluated in 1.5% AG for [Col]_i of 30, 150, and 300 μg/mL, which supported cultures with similar baseline viability and neurite outgrowth. Conjugation of Col to AG also increased the complex modulus of the hydrogel. Following high rate deformation, neuronal viability significantly decreased with increasing [Col]_i, implicating cell–matrix adhesions in acute mechanotransduction events associated with traumatic loading. These results suggest interrelated roles for matrix mechanical properties and receptor-mediated cell–matrix interactions in neuronal viability, neurite outgrowth, and transduction of high rate deformation. This model system may be further exploited for the elucidation of mechanotransduction mechanisms and cellular pathology following mechanical insult. D. Kacy Cullen and M. Christian Lessing contributed equally to this work.     

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.     

In vitro neurotoxic hazard characterization of different tricresyl phosphate (TCP) isomers and mixtures

Exposure to tricresyl phosphates (TCPs), via for example contaminated cabin air, has been associated with health effects including the so-called aerotoxic syndrome. While TCP neurotoxicity is mainly attributed to ortho-isomers like tri-ortho-cresyl phosphate (ToCP), recent exposure and risk assessments indicate that ToCP levels in cabin air are very low. However, the neurotoxic potential of non-ortho TCP isomers and TCP mixtures is largely unknown. We therefore measured effects of exposure (up to 48 h) to different TCP isomers, mixtures and the metabolite of ToCP (CBDP: cresyl saligenin phosphate) on cell viability and mitochondrial activity, spontaneous neuronal electrical activity, and neurite outgrowth in primary rat cortical neurons.The results demonstrate that exposure to TCPs (24–48 h, up to 10 μM) increases mitochondrial activity, without affecting cell viability. Effects of acute TCP exposure (30 min) on neuronal electrical activity are limited. However, electrical activity is markedly decreased for the majority of TCPs (10 μM) following 48 h exposure. Additional preliminary data indicate that exposure to TCPs (48 h, 10 μM) did not affect the number of neurites per cell or average neurite length, except for TmCP and the analytical TCP mixture (Sigma) that induced a reduction of average neurite length.The combined neurotoxicity data demonstrate that the different TCPs, including ToCP, are roughly equipotent and a clear structure-activity relation is not apparent for the studied endpoints. The no-observed-effect-concentrations (1 μM) are well above current exposure levels indicating limited neurotoxic health risk, although exposures may have been higher in the past. Moreover, prolonged and/or repeated exposure to TCPs may exacerbate the observed neurotoxic effects, which argues for additional research.