Lymphoid tissues induce NGF-dependent and NGF-independent neurite outgrowth from rat superior cervical ganglia explants in culture

Induction of neurite outgrowth from superior cervical ganglia (SCG) by rat lymphoid tissues was studied using a tissue culture model. Neonatal rat SCG were cultured with 6–12-week-old rat thymus, spleen, or mesenteric lymph node (MLN) explants in a Martrigel layer, in defined culture medium without exogenous nerve growth factor (NGF). SCG were also co-cultured with neonatal rat heart (as positive control) or spinal cord (SC; as negative control). To determine whether inflammation affects the ability of lymphoid tissues to induce neurite outgrowth, we also examined MLN at various times after infecting rats with Nippostrongylus brasiliensis (Nb-MLN). In one series of experiments, a single lymphoid tissue explant was surrounded by four SCG at a distance of 1 mm. The extent of neurite outgrowth was determinded by counting the number of neurites 0.5 mm away from each ganglion at several time points. Adult thymus and, to a lesser extent, spleen had strong stimulatory effects on neurite outgrowth from SCG after 12 hr or more in culture. For thymus tissue, this was similar to the positive control heart explants. MLN from normal rats had minimal effect on neurite outgrowth; however, Nb-MLN showed a time-dependent enhancement of the neurite outgrowth, maximal at 3 weeks after infection. The relative efficacy of neurite outgrowth induction (heart ≥ thymus ≥ Nb-MLN ≥ spleen ≥ MLN ≥ SC) was confirmed in a second series of experiments where one SCG was surrounded by three different tissue explants. We then examined the role of 2.5S NGF, a well-known trophic factor for sympathetic nerves, in the lymphoid tissue-induced neurite outgrowth. Anti-NGF treatment of co-cultures of SCG and heart almost completely blocked the neurite outgrowth. Anti-NGF also significantly inhibited thymus- and spleen-induced neurite outgrowth, but not as effectively as heart-induced neuritogenesis (93,80, and 77% inhibition at 24 hr; 86,70, and 68% inhibition at 48 hr for heart, thymus, and spleen, respectively). On the other hand, anti-NGF inhibited only 8% of neurite outgrowth induced by 3-week post-infection Nb-MLN at 24 hr, and 41% at 48 hr. These data show that several adult rat lymphoid tissues exert neurotrophic/tropic effects. The predominant growth factor in thymus and spleen is NGF, while Nb-MLN produces factor(s) which is (are) immunologically distinguishable from NGF. These neurotrophic/tropic factors are produced during the reactive lymphoid hyperplasia that forms part of the inflammatory response against the nematode, N. brasiliensis. This suggests the possibility that cytokines produced by lymphocytes or other inflammatory cells may stimulate sympathetic neurite outgrowth in vivo. © 1994 Wiley-Liss, Inc.     

S-100 beta and insulin-like growth factor-II differentially regulate growth of developing serotonin and dopamine neurons in vitro.

To study the phenotypic specificity of S-100 beta and insulin-like growth factor II (IGF-II) for developing monoamine neurons, serotonin (5-HT) neurons from the embryonic day 14 (E14) rostral raphe or dopamine (TH) neurons from the substantia nigra/ventral tegmental area were cultured for 3 days in vitro (3 DIV) in the presence of these factors. Neuronotrophic effects were analyzed by computer-assisted morphometry of 5-HT and TH-immunoreactive neurons. S-100 beta and IGF-II differentially regulated the growth of 5-HT and TH neurons but did not affect their survival. S-100 beta significantly increased several parameters of neurite outgrowth by 5-HT neurons but inhibited the spatial extent (field area) of TH neurites. IGF-II promoted growth of cell bodies of both phenotype, but only stimulated neurite outgrowth by TH neurons. S-100 beta and IGF-II differentially affected the number of GFAP immunoreactive cells from raphe and substantia nigra, but these effects did not correlate with the specificity of neuronotrophic effects. S-100 beta and IGF-II immunoreactivities were expressed in glial cultures derived from the same brain regions, raising the possibility that these factors have autocrine effects on glia as well as paracrine actions on neurons. The results of this study suggest that specificity of neurotrophic factors for particular embryonic neurons may be correlated with their neurotransmitter phenotype.     

NGF-dependent and -independent growth of neurites from sympathetic ganglion cells of the aged human in a serum-free culture

Small pieces of tissue isolated from abdominal sympathetic ganglia in aged male patients were cultured in a chemically defined, serum-free medium. The growth of neurites from pieces of ganglia in cultures with and without 50 ng/ml mouse 2.5S nerve growth factor (NGF) was compared. The NGF stimulated significantly greater regeneration of neurites, causing the growth of long fibers from the ganglion pieces. Many short neurites grew, even in the absence of the NGF, but these were generally short, except for long neurites generated in several nerve cells. A method was devised for the evaluation of NGF-dependent growth of neurites in the culture. The rate of the NGF-dependent growth of neurites, which was calculated by the difference in the total lengths of the NGF-dependent neurites between 2- and 4-day-old cultures, was approximately 160 micron/day. The results indicate that although the growth of neurites from some sympathetic nerve cells of the aged human become independent of the NGF, most of the nerve cells remain dependent on the NGF, even in the stage of senescence.     

可溶性PCP-2EC/Fc蛋白在神经粘附和轴突生长中的促进作用

为了探讨酪氨酸磷酸酶PCP- 2在神经粘附和轴突生长中的作用,本实验构建了PCP- 2胞外区与人免疫球蛋白IgGFc融合蛋白表达载体,在哺乳动物细胞中表达并纯化PCP- 2EC/Fc蛋白,进行荧光球聚集实验,然后以PCP 2胞外区融合蛋白作为基质,观察其对原代培养神经元的粘附和轴突生长的影响。结果显示,PCP- 2胞外区可同源粘附,对原代培养神经元的粘附和轴突生长有促进作用,提示PCP -2作为同源介导的神经粘附分子,可能在维持正常神经的功能和促进损伤神经的修复中发挥重要作用。     

X线照射对培养脊髓神经元突起生长的作用

目的 :观察 X线照射对脊髓神经元突起的影响。方法 :应用大鼠 E18脊髓细胞 ,在培养第 2天进行 4Gy定量照射。结果 :培养 1、2、3、4周时 ,神经元突起的长度分别为 (30 .16± 0 .71)、(37.0 3± 0 .2 9)、 (45 .2 2± 0 .36 )、(5 0 .83± 0 .72 )μm ,明显高于对照组 (P <0 .0 1)。结论 :X线照射有维持 NBN存活及促进神经突起生长的作用     

Electrospinning of Matrigel to Deposit a Basal Lamina-Like Nanofiber Surface

Schwann cell basal lamina is a nanometer-thin extracellular matrix layer that separates the axon-bound Schwann cells from the endoneurium of the peripheral nerve. It is implicated in the promotion of nerve regeneration after transection injury by allowing Schwann cell colonization and axonal guidance. Hence, it is desired to mimic the native basal lamina for neural tissue engineering applications. In this study, basal lamina proteins from BD Matrigel (growth factor-reduced) were extracted and electrospun to deposit nonwoven nanofiber mats. Adjustment of solute protein concentration, potential difference, air gap distance and flow rate produced a basal lamina-like construct with an average surface roughness of 23 nm and composed of 100-nm-thick irregular and relatively discontinuous fibers. Culture of embryonic chick dorsal root ganglion explants demonstrated that the fabricated nanofiber layer supported explant attachment, elongation of neurites, and migration of satellite Schwann cells in a similar fashion compared to electrospun collagen type-I fibers. Furthermore, the presence of nanorough surface featues significantly increased the neurite spreading and Schwann cell growth. Sciatic nerve segment incubation also showed that the construct is promigratory to nerve Schwann cells. Results, therefore, suggest that the synthetic basal lamina fibers can be utilized as a biomaterial for induction of peripheral nerve repair.     

PQBP1, a factor linked to intellectual disability,affects alternative splicing associated with neurite outgrowth

Polyglutamine-binding protein 1 (PQBP1) is a highly conserved protein associated with neurodegenerative disorders. Here, we identify PQBP1 as an alternative messenger RNA (mRNA) splicing (AS) effector capable of influencing splicing of multiple mRNA targets. PQBP1 is associated with many splicing factors, including the key U2 small nuclear ribonucleoprotein (snRNP) component SF3B1 (subunit 1 of the splicing factor 3B [SF3B] protein complex). Loss of functional PQBP1 reduced SF3B1 substrate mRNA association and led to significant changes in AS patterns. Depletion of PQBP1 in primary mouse neurons reduced dendritic outgrowth and altered AS of mRNAs enriched for functions in neuron projection development. Disease-linked PQBP1 mutants were deficient in splicing factor associations and could not complement neurite outgrowth defects. Our results indicate that PQBP1 can affect the AS of multiple mRNAs and indicate specific affected targets whose splice site determination may contribute to the disease phenotype in PQBP1-linked neurological disorders.     

uPA‐uPAR molecular complex is involved in cell signaling during neuronal migration and neuritogenesis

Background: In the development of the central nervous system (CNS), neuronal migration and neuritogenesis are crucial processes for establishing functional neural circuits. This relies on the regulation exerted by several signaling molecules, which play important roles in axonal growth and guidance. The urokinase‐type plasminogen activator (uPA)—in association with its receptor—triggers extracellular matrix proteolysis and other cellular processes through the activation of intracellular signaling pathways. Even though the uPA‐uPAR complex is well characterized in nonneuronal systems, little is known about its signaling role during CNS development. Results : In response to uPA, neuronal migration and neuritogenesis are promoted in a dose‐dependent manner. After stimulation, uPAR interacts with α₅‐ and β₁‐integrin subunits, which may constitute an αβ‐heterodimer that acts as a uPA‐uPAR coreceptor favoring the activation of multiple kinases. This interaction may be responsible for the uPA‐promoted phosphorylation of focal adhesion kinase (FAK) and its relocation toward growth cones, triggering cytoskeletal reorganization which, in turn, induces morphological changes related to neuronal migration and neuritogenesis. Conclusions : uPA has a key role during CNS development. In association with its receptor, it orchestrates both proteolytic and nonproteolytic events that govern the proper formation of neural networks. Developmental Dynamics 243:676–689, 2014. © 2014 Wiley Periodicals, Inc.     

Zic2 is required for enteric nervous system development and neurite outgrowth: a mouse model of enteric hyperplasia and dysplasia

From a forward genetic screen in mice we isolated a novel Zic2 allele with abnormal aggregation of meconium in the gastrointestinal tract. Zic2^m1Nisw mutant embryos show an increase in the number of enteric neurons in vivo and disorganization of the neurite network. Explant culture of Zic2^m1Nisw gastrointestinal tract show extensive neurite outgrowth, suggesting that Zic2 is a negative regulator of nerve fiber growth. These studies demonstrate a previously unknown function of Zic2 and provide a novel animal model of enteric nervous system dysplasia and hyperplasia.     

The sonic hedgehog pathway mediates brain plasticity and subsequent functional recovery after bone marrow stromal cell treatment of stroke in mice

Bone marrow stromal cells (MSCs) improve neurologic recovery after middle cerebral artery occlusion (MCAo). To examine whether in vivo blockage of the endogenous sonic hedgehog (Shh) pathway affects grafted MSC-induced neurologic benefits, MCAo mice were administered: vehicle (control); cyclopamine (CP)— a specific Shh pathway inhibitor; MSC; and MSC and cyclopamine (MSC-CP). Neurologic function was evaluated after MCAo. Electron microscopy and immunofluorescence staining were employed to measure synapse density, protein expression of tissue plasminogen activator (tPA), and Shh in parenchymal cells in the ischemic boundary zone (IBZ), respectively. Marrow stromal cell treatment significantly enhanced functional recovery after ischemia, concurrent with increases of synaptophysin, synapse density, and myelinated axons along the IBZ, and significantly increased tPA and Shh expression in astrocytes and neurons compared with control. After treatment with MSC-CP or CP, the above effects were reversed. Co-culture of MSCs with cortical neurons confirmed the effect of Shh on MSC-mediated neurite outgrowth. Our data support the hypothesis that the Shh pathway mediates brain plasticity via tPA and thereby functional recovery after treatment of stroke with MSCs.