神经营养素-3能够促进体外受损伤大鼠脊髓神经元的存活及其神经突起生长

目的探讨神经营养素-3(NT-3)对体外机械性损伤的大鼠脊髓神经元存活及其神经突起生长影响。方法将体外培养的大鼠脊髓神经元分为4组:正常组、对照组、20 ng/ml NT-3组和40 ng/ml NT-3组。培养4 d后,除正常组外,其余3组建立划痕损伤模型。在划痕损伤后,对照组不做处理,另外2组分别在培养液中加入20 ng/mlNT-3和40 ng/ml NT-3继续培养。直至培养第6 d,应用4%多聚甲醛固定4组细胞。固定后的细胞分别做转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记(TUNEL)、微管相关蛋白2(MAP2)和生长相关蛋白-43(GAP43)免疫荧光染色,检测划痕损伤的神经元凋亡及其神经突起生长情况。结果免疫荧光化学染色显示,与对照组相比,应用NT-3处理的2组可以显著降低划痕损伤后的脊髓神经元凋亡率,并促进其神经突起生长。尤其是40 ng/mlNT-3组的神经元凋亡率最低,其神经突起可穿过划痕损伤边界。结论 NT-3能够促进体外机械性损伤的大鼠脊髓神经元存活及其神经突起生长。     

SOCS3通过抑制STAT3抑制受损伤大鼠初级感觉神经元生长

目的通过慢病毒转导SOCS3和作用相反的突变型SOCS3(mSOCS3),体外研究SOCS3在成年鼠的初级感觉神经元再生中的作用。方法将慢病毒载体质粒pRRL-SOCS3-IRES-GFP,pRRL-mSOCS3-IRES-GFP和pRRL-STAT3ER-IRES-GFP分别转染293T细胞,包装慢病毒载体并测定滴度。完全切断大鼠左侧坐骨神经,术后饲养8-128h,在不同时间点摘取双侧L5背根神经节。通过实时PCR和原位杂交检测DRGs中SOCS3 mRNA的存在;通过背根神经节分离神经元培养,分别将三种慢病毒载体感染神经元细胞,采用免疫荧光染色法观察神经元细胞核质反应和突起的长度。结果大鼠左侧坐骨神经损伤后,SOCS3 mRNA的表达在背根神经节神经元中明显增加,外源性SOCS3能阻止神经元中STAT3的磷酸化及核移位,mSOCS3增强了突起生长。结论 SOCS3可通过抑制STAT3抑制轴突生长。     

Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro

Olfactory ensheathing cells (OECs) together with olfactory nerve fibroblasts (ONFs) and neonatal astrocytes are potent stimulators of neurite growth in adulthood and during development, respectively. Since it is known that alignment of glial cells is important for the correct outgrowth of axon tracts, it was hypothesized that the alignment of glial cells stimulates directional and enhanced neurite outgrowth. Adult OEC/ONF and neonatal astrocytes were cultured either on biodegradable poly(d,l)-lactide matrices or in Petri dishes for 4 days. Thereafter neonatal cerebral cortical neurons were added. After a 2-days coculture period the cultures were fixed and processed for a combined MAP-2 and phosphorylated neurofilament (RT97) staining. The neurite growth (neurite elongation and neurite formation) and the neurite direction were assessed. We show that (1). OEC/ONF cultures are more potent in stimulating the length of the longest neurite of cocultured neurons, (2). alignment of glial is achieved in vitro on our biomatrices, (3). aligned glial/biomatrix complexes do not enhance neurite growth, and (4). aligned glial/biomatrix complexes direct neurite outgrowth. These data have significant implications for in vivo experiments focusing on glial transplantation. Transplanting glial/biomatrix complexes may stimulate the directional regrowth of severed axons across a lesion site.     

Molecular specificity of ganglioside effects on neurite regeneration of sensory neurons in vitro

Highly purified preparations of individual gangliosides have been tested for their ability to modulate the survival and morphological differentiation of embryonic chick dorsal root ganglion neurons. When added at 48 h to established cultures of nerve growth factor (NGF)-dependent neurons, all ganglioside species tested increased the expression of neurofilament protein. Poly- and trisialogangliosides were more effective than di- or monosialogangliosides. In contrast, neither NGF nor an antiserum against NGF influenced neurofilament protein expression over this period of culture. In addition, ganglioside-induced expression of neurofilament protein was not inhibited by the anti-NGF serum.     

Choroid plexus ependymal cells enhance neurite outgrowth from dorsal root ganglion neurons in vitro

The epithelial cells of the choroid plexus are a continuation of the ventricular ependymal cells and are regarded as modified ependymal cells. The present study was carried out to determine the influence of choroid plexus ependymal cells (CPECs) on axonal growth in vitro. Choroid plexuses were dissected from the fourth ventricle of postnatal day-1–10 mice, mechanically dissociated, and plated in fibronectin-coated culture dishes. CPECs had spread into monolayers with few endothelial cells in 3-week cultures. Some macrophages were scattered on the monolayer of CPECs. Dorsal root ganglia (DRG) were excised from mouse fetuses of 14-day gestation, dissociated with trypsin and cocultured on the CPEC monolayers. For comparison, dissociated DRG neurons were cocultured on astrocyte monolayers or cultured on laminin-coated plates. After 4.5 h culturing, the cultures were fixed and immunohistochemically double-stained for neurites and CPECs using antibodies against -tubulin III and S-100 , respectively. It was demonstrated that neurons extended many long neurites with elaborate branching on the surface of S-100-stained CPECs. In contrast, DRG neurons cultured on the astrocytes and on the laminin-coated plates had much shorter primary neurites with fewer branches than those cultured on the CPECs. The total length of neurites including primary neurites and their branches, of a single DRG neuron was 285 ± 14, 395 ± 15 and 565 ± 12 m on the laminin-coated plates, on astrocytes and on CPECs, respectively. Scanning electron microscopy revealed extension of neurites with well-developed growth cones on the ependymal cells. These results suggest that CPECs have a great capacity to promote neurite outgrowth from DRG neurons in vitro.     

Characterization of human mast cells developed in vitro from fetal liver cells cocultured with murine 3T3 fibroblasts.

Cocultures of dispersed human fetal liver cells with murine Swiss 3T3 fibroblasts resulted in the development of human mast cells after 1 to 4 weeks of culture. Mast cells were detected by immunohistochemistry using a murine monoclonal anti-tryptase antibody, before metachromasia appeared with toluidine blue. When subjected to double immunohistochemistry using murine monoclonal anti-chymase and anti-tryptase antibodies, 94% +/- 10% (SD) of the mast cells seen at day 30 of culture were of the MCT type. These results contrast with those obtained with human mast cells derived from cord blood mononuclear cells cocultured with murine 3T3 fibroblasts which are comprised of substantially greater numbers of MCTC cells, averaging 48% +/- 31% (SD) at day 30 of culture. Mast cells developed in vitro from fetal liver cells or cord blood mononuclear cells contained similar amounts (+/- SD) of histamine (0.9 +/- 0.5 pg/cell and 1.1 +/- 1 pg/cell, respectively) and tryptase (1.7 +/- 0.4 pg/cell and 1.9 +/- 1.2 pg/cell, respectively) on day 30 of culture. Fetal-liver-derived mast cells from a 30-day-old culture were identified by immunoelectron microscopy using gold-labelled antitryptase antibody. Typically, these mast cells appeared immature as they had large nuclear to cytoplasmic ratio and a small number of ill-formed cytoplasmic granules. For both fetal-liver- and cord-blood-derived mast cells, there was no evidence of conversion of the MCT type into the MCTC type provided by this study. These results suggest that commitment to develop as an MCT or MCTC type of mast cell may have occurred in mast cell precursors present in fetal liver and cord blood mononuclear cells, prior to granulation.     

Effects of serotonin on neurite outgrowth from thalamic neurons in vitro

Altering levels of serotonin in the primary somatosensory cortex during early postnatal life influences thalamocortical development. Recent in vivo experiments suggest that serotonin may have direct effects on the growth of thalamocortical axons, and the present study was undertaken to determine whether this amine influences process outgrowth from thalamic cells maintained in culture. Ventrobasal thalamic neurons were harvested from newborn rats and maintained in culture for eight days. At the end of this period, 0, 10, 25, 50 or 100 microM serotonin was added to the culture medium. After an additional six days, cultures were fixed and stained with neuron-specific enolase. Quantitative analysis of >500 cells from each condition indicated that 25 microM serotonin, but not the other concentrations of this amine, significantly increased the length of the primary (longest) process growing out from the cell body (P < 0.001), the total (summed) length of all processes (P < 0.0001), total neurites per cell (P < 0.05), number of branch points per cell (P < 0.01) and branch points on the primary neurite (P < 0.0005). These results demonstrate that exposing thalamic cells to serotonin increases process outgrowth from them in the absence of their cortical targets.     

Blockade of hexokinase activity and binding to mitochondria inhibits neurite outgrowth in cultured adult rat sensory neurons

Hexokinase is known as the first enzyme and rate-limiting step in glycolysis. The role of hexokinase activity and localization in regulating the rate of axonal regeneration was studied in cultured adult sensory neurons of dorsal root ganglia (DRG). Immunofluorescent staining of DRG demonstrated that small-medium neurons and satellite cells exhibited high levels of expression of hexokinase I. Large neurons had negative staining for hexokinase I. Intracellular localization and biochemical studies in cultured adult rat sensory neurons revealed that hexokinase I was almost exclusively found in the mitochondrial compartment. The hypothesis that neurotrophic factor dependent activation of Akt would regulate hexokinase association with the mitochondria was tested and quantitative Western blotting showed no effect of blockade of the phosphoinositide 3-kinase (PI 3-kinase)/Akt pathway using the inhibitor LY294002, indicating this interaction of hexokinase with mitochondria was not neurotrophic factor or Akt-dependent. Finally, pharmacological blockade of hexokinase activity and inhibition of localization to the mitochondrial compartment with hexokinase II VDAC binding domain (Hxk2VBD) peptide caused a significant inhibition of neurotrophic factor-directed axon outgrowth. The results support a key role for hexokinase activity and/or localization to the mitochondria in the regulation of neurite outgrowth in cultured adult sensory neurons.     

A lipoxygenase product,hepoxilin A(3), enhances nerve growth factor-dependent neurite regeneration post-axotomy in rat superior cervical ganglion neurons in vitro

Hepoxilins are 12-lipoxygenase metabolites of arachidonic acid found in the CNS. They can modulate neuronal signaling but their functions are not known. We examined the effects of hepoxilin A(3) on neurite outgrowth post-axotomy in an in vitro model of spinal cord transection using superior cervical ganglion neurons. In the absence of nerve growth factor, hepoxilin A(3) did not support neuronal survival, or regeneration post-axotomy but did significantly enhance neurite regeneration in the presence of nerve growth factor. As early as 1 h post-injury hepoxilin A(3)-treated cultures (+nerve growth factor) had significantly more neurites than controls (nerve growth factor alone). Average hourly rates of outgrowth in hepoxilin A(3)-treated cultures were significantly higher than in controls for at least 12 h post-injury, suggesting that the effect of hepoxilin A(3) is maintained in vitro for several hours post-injury. In uninjured neurons hepoxilin A(3) caused a rapid but transient increase in intracellular calcium in the somata; by 2 min post-addition, calcium levels decreased to a new stable plateau significantly higher than pre treatment levels. In injured neurons, hepoxilin A(3) addition immediately post-transection caused a rapid transient increase in intracellular calcium in cell bodies; however, peak calcium levels were significantly lower than in uninjured neurons and the new baseline lower than in uninjured cells. In uninjured cells hepoxilin A(3) addition in zero calcium produced the same pattern, a transient elevation and subsequent decline to a new stable baseline significantly above rest but in injured cells levels fell rapidly to pretreatment values. Taken overall, these findings demonstrate a novel role for hepoxilins as a potentiator of neurite regeneration. They also provide the first evidence that this lipoxygenase metabolite can alter intracellular calcium in neurons by causing release of calcium from intracellular stores and modulating calcium influx mechanisms.     

Enhanced neurite growth from mammalian neurons in three-dimensional salmon fibrin gels

Three-dimensional fibrin matrices have been used as cellular substrates in vitro and as bridging materials for central nervous system repair. Cells can be embedded within fibrin gels since the polymerization process is non-toxic, making fibrin an attractive scaffold for transplanted cells. Most studies have utilized fibrin prepared from human or bovine blood proteins. However, fish fibrin may be well suited for neuronal growth since fish undergo remarkable central nervous system regeneration and molecules implicated in this process are present in fibrin. We assessed the growth of mammalian central nervous system neurons in bovine, human, and salmon fibrin and found that salmon fibrin gels encouraged the greatest degree of neurite (dendrite and axon) growth and were the most resistant to degradation by cellular proteases. The neurite growth-promoting effect was not due to the thrombin used to polymerize the gels nor to any copurifying plasminogen. Copurified fibronectin partially accounted for the effect on neurites, and blockade of fibrinogen/fibrin-binding integrins markedly decreased neurite growth. Anion exchange chromatography revealed different elution profiles for salmon and mammalian fibrinogens. These data demonstrate that salmon fibrin encourages the growth of neurites from mammalian neurons and suggest that salmon fibrin may be a beneficial scaffold for neuronal regrowth after CNS injury.     

Conditioned medium promotes neurite growth from both central and peripheral neurons

Explants or dissociated neurons of spinal cord and other parts from chick embryos and rat fetuses were cultured for 1–4 days. Extensive outgrowth of neurites from the spinal cord was observed in the medium which had been conditioned by skeletal and heart muscle cells. In the fresh medium, the neurites were short and accompanied by the migrating non-neuronal cells. The conditioned medium promoted also the neurite outgrowth from retina, optic tectum, ciliary ganglion, superior cervical ganglion and dorsal root ganglion.     

Soluble and bound forms of GFRalpha1 elicit different GDNF-independent neurite growth responses in primary sensory neurons.

We have investigated the role of glial cell-line derived neurotrophic factor (GDNF) and the effect of soluble or immobilized localization of its GDNF family receptor alpha1 (GFRalpha1) on neurite growth in cultured embryonic Bax(-/-) dorsal root ganglion neurons, which survive in the absence of trophic support. Whereas GDNF alone has a moderate effect on neurite growth, soluble and immobilized GFRalpha1 elicit opposing and GDNF-independent effects on neurite growth by a phospholipase C (PLC) gamma-dependent mechanism. Thus, GFRalpha1 elicits nerve growth responses independent of GDNF. However, GDNF in the presence of soluble or immobilized GFRalpha1 reverse the GDNF-independent GFRalpha1 modulation of neurite growth. The different outcome of soluble and bound GFRalpha1 combined with our previous immunohistochemical data showing GFRalpha1-protein in Schwann cells but not axons suggest terminal Schwann cells as a source of locally administered target-derived GFRalpha1 and place this receptor in the path of axonal growth and guidance. Thus, target-derived GFRalpha1 play opposing roles when presented alone and with GDNF and, therefore, can function as a nerve growth cue that both can promote and prevent growth in the developing peripheral nervous system.     

Peripheral nerve contains heterogeneous growth factors that support sensory neurons in vitro

The availability of dissociated cultures of embryonic neurons has given impetus to a number of studies which have detected trophic factors in many mammalian and avian tissues maintained in vivo and in vitro [1, 2, 4–8, 10, 11, 13, 16, 18–21]. Using a single sensory neuron biological assay for mouse nerve growth factor (NGF) [17] as a reference standard, we report here that neurite promoting activity is highly enriched in segments of adult mouse peripheral nerve. Neurite outgrowth at 30 h, kinetics of neurite appearance and inhibition with rabbit antiserum to mouse NGF indicate that the trophic activities are heterogeneous and support both NGF-dependent and NGF-independent neuron populations of chick embryo sensory ganglia.     

Promotion of neurite outgrowth by fibroblast growth factor receptor 1 overexpression and lysosomal inhibition of receptor degradation in pheochromocytoma cells and adult sensory neurons

Basic fibroblast growth factor (FGF-2) is up-regulated in response to a nerve lesion and promotes axonal regeneration by activation of the tyrosine kinase receptor fibroblast growth factor receptor 1 (FGFR1). To determine the effects of elevated FGFR1 levels on neurite outgrowth, overexpression was combined with lysosomal inhibition of receptor degradation. In pheochromocytoma (PC12) cells, FGFR1 overexpression resulted in flattened morphology, increased neurite outgrowth and activation of extracellular signal-regulated kinase (ERK) and AKT. Degradation of FGFR1 was inhibited by the lysosomal inhibitor leupeptin and by the proteasomal inhibitor lactacystin. In rat primary adult neurons, FGFR1 overexpression enhanced FGF-2-induced axon growth which was further increased by co-treatment with leupeptin. Lysosomal inhibition of receptor degradation concomitant with ligand stimulation of neurons overexpressing FGFR1 provides new insight in tyrosine kinase receptor-mediated promotion of axon regeneration and demonstrates that adult sensory neurons express sub-optimal levels of tyrosine kinase receptors for neurotrophic factors.     

1,25-dihydroxyvitamin D3 induces nerve growth factor,promotes neurite outgrowth and inhibits mitosis in embryonic rat hippocampal neurons

There is an accumulation of evidence implicating a role for vitamin D(3) in the developing brain. The receptor for this seco-steroid is expressed in both neurons and glial cells, it induces nerve growth factor (NGF) and it is a potent inhibitor of mitosis and promoter of differentiation in numerous cells. We have therefore assessed the direct effect of vitamin D(3) on mitosis, neurite outgrowth, as well as NGF production as a possible mediator of those effects, in developing neurons. Using cultured embryonic hippocampal cells and explants we found the addition of vitamin D(3) significantly decreases the percentage of cultured hippocampal cells undergoing mitosis in conjunction with increases in both neurite outgrowth and NGF production. The role of vitamin D(3) during brain development warrants closer scrutiny.     

Primary sensory neurons and satellite cells after peripheral axotomy in the adult rat

The timecourse of cell death in adult dorsal root ganglia after peripheral axotomy has not been fully characterised. It is not clear whether neuronal death begins within 1 week of axotomy or continues beyond 2 months after axotomy. Similarly, neither the timecourse of satellite cell death in the adult, nor the effect of nerve repair has been described. L4 and L5 dorsal root ganglia were harvested at 1-14 days, 1-6 months after sciatic nerve division in the adult rat, in accordance with the Animals (Scientific Procedures) Act 1986. In separate groups the nerve was repaired either immediately or following a 1-week delay, and the ganglia were harvested 2 weeks after the initial transection. Microwave permeabilisation and triple staining enabled combined TUNEL staining, morphological examination and neuron counting by the stereological optical dissector technique. TUNEL-positive neurons, exhibiting a range of morphologies, were seen at all timepoints (peak 25 cells/group 2 weeks after axotomy) in axotomised ganglia only. TUNEL-positive satellite cell numbers peaked 2 months after axotomy and were more numerous in axotomised than control ganglia. L4 control ganglia contained 13,983 (SD 568) neurons and L5, 16,285 (SD 1,313). Neuron loss was greater in L5 than L4 axotomised ganglia, began at 1 week (15%, P=0.045) post-axotomy, reached 35% at 2 months ( P<0.001) and was not significantly greater at 4 months or 6 months. Volume of axotomised ganglia fell to 19% of control by 6 months ( P<0.001). In animals that underwent nerve repair, both the number of TUNEL-positive neurons and neuron loss were reduced. Immediate repair was more protective than repair after a 1-week delay. Thus TUNEL positivity precedes actual neuron loss, reflecting the time taken to complete cell death and elimination. Neuronal death begins within 1 day of peripheral axotomy, the majority occurs within the first 2 months, and limited death is still occurring at 6 months. Neuronal death is modulated by peripheral nerve repair and by its timing after axotomy. Secondary satellite cell death also occurs, peaking 2 months after axotomy. These results provide a logical framework for future research into neuronal and satellite cell death within the dorsal root ganglia and provide further insight into the process of axotomy induced neuronal death.     

Rat tooth pulp cells elicit neurite growth from trigeminal neurones and express mRNAs for neurotrophic factors in vitro

Extracellular concentration of nitrite (NO2-), an oxidized product of nitric oxide (NO), was measured consecutively in the dorsal region of the rat suprachiasmatic nucleus (SCN) by means of in vivo microdialysis. The NO2- concentrations in the dialysates showed robust circadian rhythm under a 12:12 h light/dark cycle and were higher during the dark phase than during the light phase. When the rats were transferred to constant darkness, the 24 h rhythm of NO2- persisted without damping the amplitude. The NO2- level was significantly lowered by an injection of NO synthase inhibitor (NG-monomethyl-L-arginine, 10 mg/kg i.p.). These findings indicate that the daily fluctuation of NO2- in the dorsal region of the SCN, which represents endogenous rhythm of NO, is regulated independently of photic inputs into the SCN and may be related to the circadian clock functions.     

重组白细胞介素13促进成纤维细胞增殖及胶原合成

目的：观察重组白细胞介素13（rIL-13）对3T3细胞的作用，探讨肺纤维化的发生机制。 方法： 3T3细胞分为实验组和对照组，分别加入rIL-13 (80 μg/L)及DMEM培养液, 作用24 h、48 h，利用透射电镜观察成纤维细胞的超微结构，用Hoechst 试剂盒观察细胞DNA形态；用MTT法测细胞增殖率，用Western blotting检测成纤维细胞分泌Ⅰ型胶原以及用放免法检测细胞上清中IL-6、IL-8水平。 结果： 实验组细胞DNA合成增加，细胞核增大，细胞质中可见较多核糖体及线粒体；rIL-13呈剂量依赖方式刺激成纤维细胞增殖，当rIL-13浓度在40-160 μg/L时，细胞增殖率几乎呈直线式增长。对照组及实验组均可检测到Ⅰ型胶原（CoⅠ），对照组分泌胶原的量显著高于对照组（P<0.01）。细胞培养上清中，实验组IL-6、IL-8含量显著高于对照组（P<0.01）。 结论： rIL-13通过促进成纤维细胞增殖及分泌炎性介质和Ⅰ型胶原，可能在肺纤维化的发病机制中发挥关键作用。     

Physiological properties of primary sensory neurons appropriately and inappropriately innervating skin in the adult rat.

1. We have studied the physiology of sensory neurons innervating skin of the rat hindlimb, in three groups of animals: 1) normal animals; 2) animals in which the sural nerve (Sn) had regenerated to its original cutaneous target; and 3) animals in which the gastrocnemius muscle nerve (Gn) had previously been cut and cross anastomosed with the distal stump of the cut Sn so that its axons regenerated to a foreign target, skin. 2. Single-unit recordings were made from 222 afferents in normal, intact animals. They had conduction velocities of 0.5-53.1 m/s. The conduction velocity distribution had distinct peaks at approximately 37.5, 2.5, and 1.25 m/s, presumably corresponding to A alpha beta-, A delta-, and C-fiber populations. Eighty-two percent of the characterized myelinated fibers had low-threshold mechanosensitive receptive fields, whereas 16% were high threshold, and only 2% appeared to have no receptive field. The very large majority of low-threshold mechanosensitive receptive fields (87%) were rapidly adapting hair follicle afferents. 3. In animals with regenerated Sn, 308 afferents were recorded with conduction velocities of 0.4-58.8 m/s. However, the mean conduction velocity was lower than in control animals (P less than 0.05), and only one peak, at 27.5 m/s, was apparent for myelinated fibers. Eighty-six percent of myelinated fibers were low-threshold mechanosensitive afferents, 8.5% were high-threshold mechanoreceptors (HTMRs), and 5.5% appeared to have no receptive fields. Fewer low-threshold mechanoreceptors (LTMRs; compared with controls) were activated by hair movement (63 vs. 87%). Most of the remainder appeared to be field receptors (which were therefore more commonly observed here than in normal animals). 4. In animals in which the Gn had regenerated to skin, 430 afferents were recorded. These had conduction velocities ranging from 0.6 to 71.4 m/s, and again only one peak was apparent in the myelinated conduction velocity histogram, at approximately 17.5 m/s. Of the myelinated fibers, 79% had low-threshold mechanosensitive receptive fields in skin and 10% high-threshold mechanosensitive receptive fields. The remaining 11% apparently had no receptive field (cf. 5.5% in regenerated Sn). In contrast to normal or regrown sural afferents, only 58% of low-threshold gastrocnemius afferents in skin were rapidly adapting. Of the 42% slowly adapting afferents, many surprisingly responded to hair movement. Thus some gastrocnemius afferents seemed to have retained the adaptation properties characteristic of muscle afferents. Also surprisingly, given that the Gn contains fewer fibers than the Sn, receptive-field areas were not significantly different from regrown or normal sural fibers.(ABSTRACT TRUNCATED AT 400 WORDS)