Neurturin和NGF联合应用对AD模型鼠基底前脑NGFR阳性神经元的保护作用

本研究目的是探讨neurturin和神经生长因子(NGF)联合应用对穹窿-海马伞(FF)切断后基底前脑胆碱能神经元的保护作用。将这两种因子联合注射到FF切断的大鼠侧脑室,注射一个月后,取脑进行免疫组化结合图像分析方法对内侧隔核(MS)和斜角带核垂直支(VDB)的神经生长因子受体(NGFR)阳性神经元存活情况进行比较分析。结果表明:FF切断一个月后,损伤组损伤侧MS和VDB的NGFR阳性神经元大量减少(分别减少64.8％和51.4％),MS和VDB的NGFR阳性细胞的面积和周长显著降低(P<0.01),OD值显著增高(P<0.01);NGF组损伤侧NGFR阳性神经元受到保护,NGFR阳性经元数显著高于损伤组损伤侧(P<0.01);联合组损伤侧NGFR阳性神经元也受到保护(MS和VDB细胞数分别只下降7.3％和15.4％),与损伤组损伤侧比较NGFR阳性细胞数增加了57.4％和36.0％(P<0.01),也明显高于NGF组(P<0.05);细胞形态学参数明显改善(P<0.05),细胞膜受体含量显著提高(P<0.05)。结果提示,neurturin和NGF联合应用和NGF单独应用均能不同程度地保护基底前脑胆碱能神经元,联合应用效果更佳。     

Cholinergic medial septum neurons do not degenerate in aged 129/Sv control or p75(NGFR)-/-mice

Cholinergic medial septum neurons express TrkA and p75 nerve growth factor receptor (p75(NGFR)) and interactions between TrkA and p75(NGFR) are necessary for high-affinity binding and signaling of nerve growth factor (NGF) through TrkA. In adult p75(NGFR)-deficient (-/-) mice, retrograde transport of NGF and other neurotrophins by these neurons is greatly reduced, however, these neurons maintain their cholinergic phenotype and size. Reduced transport of NGF has been proposed to play a role in Alzheimer's disease. Here, we investigated whether chronic and long-term absence of p75(NGFR) (and possibly reduced NGF transport and TrkA binding) would affect the cholinergic septohippocampal system during aging in mice. In young (6-8 months), middle aged (12-18 months), and aged (19-23 months) 129/Sv control mice the total number of choline acetyltransferase-positive medial septum neurons and the mean diameter and cross sectional area of the cholinergic cell bodies were similar. The cholinergic hippocampal innervation, as measured by the density of acetylcholinesterase-positive fibers in the outer molecular layer of the dentate gyrus was also similar across all ages. These parameters also did not change during aging in p75(NGFR) -/- mice and the number and size of the choline acetyltransferase-positive neurons and the cholinergic innervation density were largely similar as in control mice at all ages. These results suggest that p75(NGFR) does not play a major role in the maintenance of the number or morphology of the cholinergic basal forebrain neurons during aging of these mice. Alternatively, p75(NGFR) -/- mice may have developed compensatory mechanisms in response to the absence of p75(NGFR).     

Bone mesenchymal stem cells for gene transfer of NGF to the adult rat brain: rescue the NGFR p75 positive neurons from fimbria-fornix lesion-induced degeneration

The study was to evaluate the therapeutic benefit of transplanted bone mesenchymal stem cells (BMSCs) transfected never growth factor (NGF) gene and GFP gene (as a reporter gene), in treating the rat with fimbria-fornix lesion. After transduction of NGF gene via recombinant retroviral vectors into the rat BMSCs, BMSCs were therefore transformed into the GFP-NGF positive BMSCs, nearly 100% of BMSCs expressed NGF, and then transplanted into basal forebrain of rat with fimbria-fornix lesion. After 2 weeks post-transplantation, the GFP-NGF positive BMSCs survive and fuse in vivo with astroglia or NGFR p75 positive neurons in the basal forebrain, no evidence of transdifferentiation was observed in this study. The number of NGFR p75 positive neurons in basal forebrain of NGF group was significantly higher than those of the void plasmid group (p < 0.05) or the PBS group (p < 0.01). These results indicate that the GFP-NGF positive BMSCs provide, by way of paracrine, NGF that effectively perform the functions of neuroprotection, which cell fusion may be also contribute to.     

Nerve growth factor receptor (NGFR)-like immunoreactivity in the perineurial cells of the adult rat peripheral nerves: normal appearance and response to nerve section]

The expression of the nerve growth factor receptor (NGFR)-like immunoreactivity in the perineurial cells of normal rat sensory and sympathetic nerves and after surgical severance was analyzed using a specific anti-rat NGFR monoclonal antibody (192-IgG). The perineurial cells as well as axonal endings showed NGFR-like immunoreactivity in the terminal region of normal nerves and the immunoreactivity in the terminal perineurial cells was enhanced after nerve severance. In the nerve trunk region, no cellular elements exhibited NGFR-like immunoreactivity in normal rats, but immunoreactivity was induced in the innermost perineurial cells and all Schwann cells in portions distal to the severance. These findings suggest that the perineurial cells function to provide NGF for intact and regenerating axons by binding NGF to NGFR on the perineurial cell surface.     

Effects of LNG-IUS on nerve growth factor and its receptors expression in patients with adenomyosis

The levonorgestrel-releasing intrauterine system (LNG-IUS) is effective in the treatment of dysmenorrhea associated with adenomyosis. However, the mechanism of pain relief of LNG-IUS in patients with adenomyosis is unclear. We aimed to investigate the effects of LNG-IUS on the expression of nerve growth factor (NGF) and its receptors, NGFR p75 and TrkA in patients with adenomyosis. Endometrial and myometrial tissues were prepared from 17 LNG-IUS-treated patients and 15 hormonally untreated patients who had undergone hysterectomies for adenomyosis. Immunohistochemistry with antibodies against NGF, NGFR p75, and TrkA, was performed. The expression of NGF, NGFR p75, and TrkA in endometrium and myometrium of LNG-IUS-treated patients was significantly decreased compared to those of hormonally untreated patients. Our findings may indicate that the suppression of NGF and its receptors by LNG-IUS is another possible mechanism of relieving pain in patients with adenomyosis.     

Protracted effects of chronic oral haloperidol and risperidone on nerve growth factor, cholinergic neurons, and spatial reference learning in rats

The primary therapeutic agents used for schizophrenia, antipsychotic drugs, ameliorate psychotic symptoms; however, their chronic effects on cognition (or the physiologic processes of the brain that support cognition) are largely unknown. The purpose of this rodent study was to extend our previous work on this subject by investigating persistent effects (i.e. during a 14 day drug-free washout period) of chronic treatment (i.e. ranging from 45 days to 6 months) with a representative first and second generation antipsychotic. Drug effects on learning and memory and important neurobiological substrates of memory, the neurotrophin, nerve growth factor (NGF) and its receptors, and certain components of the basal forebrain cholinergic system were investigated. Behavioral effects of oral haloperidol (2.0 mg/kg/day), or risperidone (2.5 mg/kg/day) were assessed in an open field, a water maze task, and a radial arm maze procedure and neurochemical effects in brain tissue were subsequently measured by enzyme-linked immunosorbent assays (ELISAs). The results indicated that both antipsychotics produced time-dependent and protracted deficits in the performance of a water maze procedure when compared with vehicle-treated controls, while neither drug was associated with significant alterations in radial arm maze performance. Interestingly, haloperidol, but not risperidone, was detectible in the rodent brain in appreciable levels for up to 2 weeks after drug discontinuation. Both antipsychotics were also associated with reduced levels of NGF protein in the basal forebrain and prefrontal cortex and significant (or nearly significant) decreases in phosphorylated tropomyosin-receptor kinase A (TrkA) protein and the vesicular acetylcholine transporter (depending on the brain region analyzed). Neither antipsychotic markedly affected TrkA or p75 neurotrophin receptor levels. These data in rats indicate that chronic treatment with either haloperidol or risperidone may be associated with protracted negative effects on cognitive function as well as important neurotrophin and neurotransmitter pathways that support cognition.     

Amelioration of cholinergic neurons dysfunction in aged rats depends on the continuous supply of NGF

The present study was designed to examine whether NGF-induced improvement in morphology of senile basal forebrain cholinergic neurons persist after discontinuation of NGF treatment. Trophic effect of continuous intraventricular infusion of NGF was tested in the 4- and 28 months old male Wistar rats immediately after cessation of NGF and 3 or 6 weeks after termination of treatment. Immunohistochemical procedure for ChAT, TrkA, and p75(NTR) receptor has been applied to identify cholinergic cells in the basal forebrain structures. Using the quantitative image analyzer, morphometric and densitometric parameters of cholinergic cells were measured. In untreated 28-month-old rats a reduction in the number, size and intensity of staining of cholinergic neurons was observed in all basal forebrain structures. NGF significantly improved morphological parameters of ChAT- and TrkA-positive cells in aged rats. In 28-month-old rats tested within 3 and 6 weeks after discontinuation of infusion a renewed progressive deterioration of cholinergic phenotype of basal forebrain neurons was observed when compared with the NGF-treated immediately tested group. The parallel staining for p75(NTR) revealed normal morphology of the basal forebrain neurons, despite of the age of rats or the NGF treatment. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neurons in the basal forebrain when compared with the young animals. These findings demonstrate that senile impairment of cholinergic neurons is induced by a loss of cholinergic phenotype rather than an acute degeneration of cell bodies. NGF may be beneficial in enhancing cholinergic neurochemical parameters, but the protective effects seem to be dependent on the continuous supply of NGF.     

基底前脑神经元移植到海马后ChAT、NGFR和NOS的表达变化

为了比较基底前脑神经元移植至海马后胆碱乙酰转移酶、神经生长因子受体和一氧化氮合酶神经元的变化规律。选用雄性 SD大鼠 35只 ,在单侧穹隆海马伞损伤后一周 ,将胚胎基底前脑神经元植入损伤侧海马。在移植术后存活 5 d,7d,14 d,30d,6 0 d,90 d,和 180 d共分为 7个时间组灌注取材 ,进行 Ch AT和 NGFR的免疫组织化学反应及 NOS反应 ,观察其神经元的变化。结果证明 ,Ch AT神经元在移植后 30 d方出现阳性反应 ;NGFR神经元在移植后 7d可见淡染的胞体 ,随后逐渐增强 ;NOS神经元在移植后 7d时呈明显的阳性反应 ,而且数量多。三种神经元直到 180 d时均尚呈阳性反应。提示移植物内 NOS神经元出现早且数量多 ,NGFR神经元出现的也较早 ,Ch AT神经元出现最晚。这三种物质在细胞内出现的时间和数量差异与它们在细胞内发挥的功能有关     

重组神经生长因子受体基因在受体缺乏的神经母细胞瘤细胞系中的表达

神经母细胞瘤是儿童期的常见恶性肿瘤之一。临床上,半数以上神经母细胞瘤有明显的N-myc癌基因扩增。神经生长因子(NGF)可使某些神经母细胞瘤细胞分化成神经元样的细胞。但对有N-myc扩增的神经母细胞瘤则可能因NGF受体缺乏而无明显促分化反应。本研究运用重组DNA技术将人NGF受体基因重组到有N-myc扩增,且NGF受体表达很低的神经母细胞瘤系(IMR-32)中,经克隆化培养、筛选,建立了稳定的细胞系—IMR-32/NGFR和对照细胞系IMR-32/NEO。经用抗NGFR的单克隆抗体检测用Flow cytometry技术证实IMR-32/NGFR系中有明显的NGF受体表达,而其母系IMR-32和空病毒对照系IMR-32/NEO则无表达迹象,说明NGF受体表达在IMR-32/NGFR系中是特异的,并能同抗NGFR单克隆抗体特异结合。用Northern B10ting技术亦测得IMR-32/NGFR中NGFR的mRN A明显表达。而其母系IMR-32和对照系IMR-32/NEO则无明显表达。这说明IMR-32/NGFR系中NGFR在mRNA水平上亦是特异的。N-myc和K-ras癌基因在IMR-32、IMR-32/NEO、IMR-32/NGFR三系中无明显变化。在NGF处理后,形态上三系均无明显的分化迹象。但IMR-32/NGFR在神经原纤维轻链的表达上轻度增高。c-fos癌基因的表达见于所有三个细胞系,IMR-32/NGFR略强些。这些说明,在恢复了NGFR基因和表达之后,对N-myc和K-ra     

The effects of denervation and chronic haloperidol treatment on neostriatal dopamine receptor density are not additive in the rat

The effects of combined denervation and chronic haloperidol treatment on neostriatal D-2 receptor density were examined, and a lack of additivity was found. In the first experiment, haloperidol treatment for 14 days initiated on day 4 after the unilateral intracerebral injection of 6-hydroxydopamine (6-OHDA) abolished the lesion-induced asymmetry of [3H]spiroperidol binding by elevating the density of sites in the intact striatum. These results were replicated in the second experiment, in which the drug treatment was begun 4-5 weeks after 6-OHDA. In both experiments the apomorphine-induced rotation did not differ significantly between lesioned rats treated with haloperidol or its vehicle.     

Nerve growth factor-induced loss of cell-associated nerve growth factor receptor in human melanoma A875 cells.

Human A875 melanoma cells are known to express the low-affinity nerve growth factor receptor p75NGFR in a monomeric and a covalently linked probably dimeric form. Kinetic analysis of the association of nerve growth factor (NGF) with its receptor revealed a rapid loss of binding sites at high ligand concentrations. Using cross-linking and immunoprecipitation with an anti-p75NGFR antibody, this was found to be due to a decrease of the high molecular weight form of the receptor. Mechanisms for such a ligand-induced receptor loss are discussed.     

Chronic ascorbate potentiates the effects of chronic haloperidol on behavioral supersensitivity but not D2 dopamine receptor binding.

Ample behavioral evidence suggests that ascorbate parallels the action of haloperidol, a widely used neuroleptic. To determine the extent to which this parallel extends to chronic treatment, 21 days of exposure to ascorbate (100 or 500 mg/kg) alone or combined with haloperidol (0.5 mg/kg) were assessed on stereotyped behavior and neostriatal D2 dopamine receptor binding in rats. Our results indicate that when challenged with the dopamine agonist, apomorphine (0.5 mg/kg), animals chronically treated with haloperidol or high-dose ascorbate alone display a supersensitive sniffing response relative to controls, while animals chronically treated with the combination of haloperidol and high-dose ascorbate display a further potentiation of sniffing relative to the haloperidol groups. In addition, [3H]spiperone saturation studies showed, as expected, an up-regulation of striatal D2 dopamine receptors in rats treated with haloperidol as reflected by a change in receptor density (Bmax) but not affinity (KD). Ascorbate treatment, however, had no effect on D2 receptor density or the distribution of [3H]apomorphine in whole brain. Even though chronic treatment with the haloperidol-high-dose-ascorbate combination produced an up-regulation of striatal D2 dopamine receptors, this treatment did not cause a further up-regulation relative to haloperidol alone nor did it have any effect on [3H]apomorphine distribution. Taken together, these findings indicate that although chronic ascorbate produces behavioral supersensitivity to apomorphine through central mechanisms, they appear to differ from those induced by chronic haloperidol.     

Expression of Nerve Growth Factor and its Receptor in Distracted Tibial Nerve after Limb Lengthening

Despite many experimental and clinical studies conducted on distraction osteogenesis (DO) in the past decade, changes in the surrounding tissues that occur after the procedure remains poorly understood. To study the biochemical changes of recovery in nerve tissues upon DO‐induced nerve injury, we prepared a rabbit model of tibia lengthening to observe the expression pattern of nerve growth factor (NGF) and low‐affinity NGF receptor (p75NGFR) in the distracted tibial nerve. The distracted tibial nerve was harvested at various time points during the consolidation period of new bone formation and immunohistochemical staining was performed to detect the expression of NGF and p75NGFR. The expression levels of NGF and p75NGFR were found to be different at various times after DO. The changes in expression of these two cellular factors show similar tendencies with significantly elevated expression in Schwann cells at 7 and 14 days after distraction, but low or undetectable levels of expression at 0, 28, and 56 days. These results suggest that NGF and p75NGFR may play important roles in the adaptive process of the distracted nerve. NGF and p75NGFR are autocrine growth factors present in the distracted nerve during the early consolidation period. NGF interacts with p75NGFR to promote damage repair and reconstruction of nerves. Together, this study furthers the understanding of the relative mechanisms of nerve repair, as well as provides a further basis for the clinical application of neurotrophins. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.     

The pro-fibrogenic effect of nerve growth factor on conjunctival fibroblasts is mediated by transforming growth factor-β

BACKGROUND: Nerve growth factor (NGF) and nerve growth factor receptor (NGFR) expressions have been found to be increased in sub-conjunctival scarring. OBJECTIVE: The aim of this study was to investigate the in vitro effects of NGF on some pro-fibrogenic properties of human conjunctival fibroblasts. METHODS: Expression of NGF, trkA(NGFR) and p75NTR on human fibroblasts grown from conjunctival biopsies and incubated for 2 or 6 days with NGF were evaluated by immunofluorescence, RT-PCR, flow cytometry and ELISA. The fibrogenic effect of NGF on conjunctival fibroblasts was investigated by evaluating their migration (wound model), proliferation ([3H]-thymidine incorporation), collagen production (3H]-proline incorporation), expression of alpha-smooth muscle actin (alpha-SMA) (cell surface ELISA) and contraction of 3D collagen gels. RESULTS: NGF induced the expression of p75NTR in the fibroblasts that constitutively expressed only trkA(NGF) and increased the migration of wounded fibroblasts, but not their proliferation and collagen production. NGF induced the conversion of fibroblasts into myofibroblasts expressing alpha-SMA, and enhanced their contraction of a collagen matrix. Interestingly, chronic NGF treatment induced transforming growth factor-beta1 (TGF-beta1) production by fibroblasts, and following specific TGF-beta neutralization, all the NGF-induced effects were completely abrogated. CONCLUSION: Our findings indicate that NGF, via TGF-beta induction, is likely to be involved in the healing or fibrotic processes occurring in conjunctiva during some pathological conditions.     

Nerve growth-factor and anti-CD40 provide opposite signals for the production of IgE in interleukin-4-treated lymphocytes

Nerve growth factor (NGF) is a well-known neurotrophic factor acting on both the peripheral and the central nervous systems. In addition, it has been shown to play a role in the function of the immune system through specific receptors. Both high-affinity and low-affinity NGF receptors (NGFR) are expressed on human B lymphocytes. The low-affinity NGFR has been shown to have structural homology with another specific B cell surface molecule, CD40, which plays an important role in IgE production. In view of the structural similarities of the p75 NGFR and CD40 we examined whether NGF may also be involved in the regulation of IgE production. We found that NGF and anti-CD40 exerted opposite effects on the induction of IgE by IL-4 in peripheral blood mononuclear cells. NGF inhibited the induction of IgE by IL-4 and this inhibition was not mediated through blocking of the induction of CD23 nor through inhibition of IL-4R expression. The inhibition of IL-4-dependent IgE production was observed on surface (s)IgE⁺ and sIgE^?/sIgM⁺ B lymphocytes. Anti-CD40 on the other hand, exerted an enhancing effect on IgE production and its addition to IL-4 provided a signal that was resistant to the inhibitory effect of NGF. Antagonistic effects of NGF and IL-4 were also observed for other Ig isotypes since IL-4 prevented the increase in IgA and IgM production induced by NGF. These data indicate that although NGFR and CD40 belong to the same receptor super-family and exert similar proliferative effects on B lymphocytes, they interact differently with IL-4 in the regulation of IgE production.     

The effects of haloperidol treatment on the distribution of NK1 receptor immunoreactive neurons in guinea-pig brain

Previous studies have observed increased tachykinin NK(1) receptor immunoreactivity (NK(1)-IR) in the prefrontal cortex in subjects with schizophrenia. Since the subjects were medicated the possibility of a treatment effect could not be excluded. Thus, the present study was undertaken to determine the effect of chronic treatment with the antipsychotic drug, haloperidol, on the distribution of NK(1)-IR neurons in the guinea-pig brain. Guinea pigs were treated each day for 21 days with either haloperidol (1mg/kg) or vehicle and the brains were then processed for immunohistochemistry using an NK(1) receptor-specific polyclonal antibody. NK(1)-IR neurons and fibres were abundant in the forebrain cortex and caudate putamen and more sparsely distributed in a number of other brain regions. The relative density of NK(1)-IR neurons was significantly increased in the forebrain cortex, but not in the caudate putamen in guinea pigs treated with haloperidol. This study has shown that haloperidol causes region-specific changes to the density of NK(1)-IR neurons. Whether these changes are related to the therapeutic effects or to the side effects of haloperidol in individuals with schizophrenia, remains to be determined.     

BDNF improves the effects of neural stem cells on the rat model of Alzheimer's disease with unilateral lesion of fimbria-fornix

In this study, neural stem cells (NSCs) were obtained from the hippocampus using the serum-free culturing. NSCs labeled with 5'-bromo-2'-deoxyuridine (BrdU) were transplanted into transected rat basal forebrain followed by the injection of brain-derived neurotrophic factor (BDNF) into the lateral ventricle. Nestin staining and double-labeling immunohistochemistry were used to detect cell survival and neuronal differentiation of the BrdU labeled cells in the basal forebrain and it was observed that labeled NSCs differentiated into neurons and astrocytes in the basal forebrain. Immunohistochemical detection of p75(NGFR) indicated that the number of cholinergic neurons of the combination groups treated by NSCs, BDNF, and NSCs groups had more significant improvement than that of the injured groups in medial septum (MS) and vertical diagonal branch (VDB). Learning and memory abilities were also measured by Y-maze test and the results support that BDNF can enhance the treatment effects of NSCs transplanted into brain lesion model.     

Role of nerve growth factor in experimental autoimmune encephalomyelitis

The expression of neural regulatory molecules by immune cells that infiltrate the nervous system upon injury may be a mechanism for cross-regulation between the nervous system and the immune system. Several lines of evidence implicate nerve growth factor (NGF) signaling through its receptors (TrkA and p75(NGFR)) as a potential source of communication between the two systems. We observed changes in NGF mRNA expression and protein secretion by T lymphocytes polarized toward the Th2 phenotype. The presence of NGF did not affect T cell proliferation or cytokine production in vitro. Mice treated with NGF by i. p. injection following induction of experimental autoimmune encephalomyelitis, an inflammatory, demyelinating disease of the central nervous system, showed a delayed onset of disease and lower clinical scores during the course of disease. These data suggest a role for NGF signaling in the regulation of the immune response, possibly by enhancing sympathetic innervation of lymphoid tissues.     

Nerve growth factor and its receptors TrkA and p75 are upregulated in the brain of mdx dystrophic mouse

Increased angiogenesis and an altered blood–brain barrier have been reported in the brain of dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy. To further elucidate the mechanisms underlying angiogenesis in Duchenne muscular dystrophy, in this study we evaluated whether nerve growth factor (NGF) and nerve growth factor receptors (NGFRs) are involved, then correlated NGF-NGFRs expression with vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) content and matrix metalloproteinases-2 and -9 (MMP-2 and -9) activity, by confocal laser microscopy and immunohistochemistry. Results showed that neurons, astrocytes and ependymal cells were strongly labeled by NGF in mdx brain, expressing NGFRs on glial and endothelial cells. In controls, NGF faintly labeled neurons and astrocytes, whereas endothelial cells were negative for NGFRs. Immunogold electron microscopy demonstrated NGFR gold particles on endothelial cells in mdx brain, while in controls few particles were recognizable only on glial end feet. Western blotting and real time polymerase chain reaction (RT-PCR) demonstrated a higher expression of NGF and NGFR mRNA and protein in mdx brain as compared to controls, and increase of VEGF-VEGFR-2 and active MMP-2 and -9 content. Overall, these data suggest that in the brain of mdx mice, an upregulation of the NGF-NGFRs system might be involved directly, or indirectly through the activation of VEGF-VEGFR-2 and MMP-2 and -9, in the angiogenic response taking place in this pathological condition.