Nerve Growth Factor Receptor-immunoreactive Fibres Innervate the Reticular Thalamic Nucleus: Modulation by Nerve Growth Factor Treatment in Neonate,Adult and Aged Rats

Terminal arborizations expressing nerve growth factor receptor (NGF-R) have been detected with immunohistochemistry in the reticular thalamic nucleus of neonate, adult and aged rats. Intracerebroventricular administration of nerve growth factor (NGF) resulted in a dramatic increase in NGF-R immunoreactivity throughout the lifespan. This effect was paralleled by a concomitant increase in NGF-R immunopositivity in the neurons of the basal forebrain, which was here demonstrated also in aged animals, thus indicating that the NGF-R immunoreactivity within the reticular thalamic nucleus derives in all likelihood from cholinergic neuronal cell bodies of the basal forebrain. Our results demonstrate a prominent ability of NGF to up-regulate its receptors within fibres innervating the reticular thalamic nucleus, and show that this up-regulation of NGF-R is maintained throughout the lifetime. Altogether this indicates that the reticular thalamic nucleus may represent a new, important site of action of endogenous NGF or NGF-like molecules within the brain. In view of the crucial role played by the reticular thalamic nucleus in gating thalamocortical information, the autoregulation of NGF-R in this structure may have important concomitants in both physiological and pathological conditions.     

Regional Increases in Brain-Derived Neurotrophic Factor and Nerve Growth Factor mRNAs During Amygdaloid Kindling, But Not in Acidic and Basic Fibroblast Growth Factor mRNAs

Summary: We studied mRNA levels for neurotrophic factors using the amygdaloid kindling model of epilepsy. One hour after stage 5 kindled seizures, there were four-to fivefold increases in brain-derived neurotrophic factor (BDNF) mRNA in rat dentate gyrus and perirhinal cortex. Nerve growth factor (NGF) mRNA levels were increased bilaterally in some (but not all) limbic areas. There were no detectable changes in acidic fibroblast GF (aFGF) mRNA or basic fibroblast GF (bFGF) mRNA for 24 h after the kindled seizures. During kindling, levels of BDNF mRNA in the dentate gyrus correlated with projection to generalized seizures, whereas NGF mRNA in the limbic regions continued to increase during seizure development. These results indicate that the induction of mRNAs for neurotrophic factors, especially for BDNF mRNA in the dentate gyrus, corresponds to the increases in metabolic and electrical ictal discharge associated with kindled seizures. The persistent increase observed in NGF mRNA may be related to enhanced synaptic efficacy during kindling, but aFGF and bFGF are presumed to have little relation to the kindling process.     

人神经生长因子治疗糖尿病性多发性神经病的随机双盲研究

目的:观察人神经生长因子(hNGF)治疗糖尿病性多发性神经病(DPN)的有效性及安全性。方法:采用双盲随机对照研究,48例DPN病人随机分为2组:hNGF组24例,每日给予hNGF 4 mL(1000 u),肌注:对照组 24例,每日给予安慰剂 4 mL,肌注,疗程均为12周。结果:hNGF组治疗12周后,患者MDNS和自觉感觉障碍有效率分别为73.83％和83.33％,明显高于安慰剂组。治疗结束后4及12周,其有效率仍显著高于对照组。治疗期间未发现严重不良反应。结论:hNGF治疗DPN有效且安全。     

Targeted Delivery of Nerve Growth Factor via Fibronectin Conduits Assists Nerve Regeneration in Control and Diabetic Rats

This study examined the influence of fibronectin conduits joining two halves of a sectioned sciatic nerve, with and without preimpregnation with nerve growth factor, on regeneration in rats with streptozotocin-induced diabetes mellitus. Regeneration, measured morphometrically in fibres containing immunoreactivity to calcitonin gene-related peptide (CGRP) and GAP-43, was significantly less ( P < 0.0001 for all comparisons) in diabetic rats with fibronectin mat grafts without nerve growth factor compared to similarly treated controls. Regeneration distances in diabetic rats were reduced to 43% (CGRP-reactive fibres) and 44% (GAP-43-reactive fibres) of controls, and the total amounts of immunoreactivities in the conduits were also reduced, though by lesser amounts (55 and 61% of controls respectively). Impregnation of the conduits with nerve growth factor before implantation increased the distance and amounts of regenerating immunoreactivity in both control and diabetic rats for both CGRP and GAP-43, such that these regeneration parameters were similar in nerve growth factor-treated diabetic rats to those in control rats implanted with untreated fibronectin mat conduits. These findings implicate impaired neurotrophic support in the defective regeneration characteristic of diabetic neuropathy.     

Comparison between Low Frequency Magnetic Field Stimulation and Nerve Growth Factor Treatment of Cultured Chromaffin Cells, on Neurite Growth, Noradrenaline Release, Excitable Properties, and Grafting in Nigrostriatal Lesioned Rats

Adrenal chromaffin cells in vitro respond to nerve growth factor (NGF) by expressing neuronal traits. Low frequency magnetic (LFM) field stimulation, while inducing a variety of effects on several cell types, has never been studied as to its effects on chromaffin cell cultures. The purpose of this study was to compare the effects of LFM field stimulation with that of NGF on the morphological phenotype, on noradrenaline (NA) release, and on membrane excitability of cultured chromaffin cells. We also tested the effects of grafting LFM and NGF-treated chromaffin cells into the caudate nucleus of rats with 6-hydroxydopamine lesions of the nigrostriatal pathway. The results of this study showed that LFM field stimulation produced neurite growth of cultured chromaffin cells in a manner similar to that of NGF exposure. The combination of the two procedures did not induce changes above those observed by NGF alone. Both NGF- and LFM-treated chromaffin cells released [³H]NA equally in response to a depolarizing concentration of KCl. On the other, Na⁺ current density of LFM field stimulation increased, but to a lesser extent than that seen in NGF-treated cells. In addition both types of cells when transplanted into nigrostriatal-lesioned animals induced a similar decrease in the motor asymmetries produced by the lesion. When NGF- or LFM-treated chromaffin cells where compared to untreated control cells, no significant differences were observed in [³H]NA release, on Na⁺ current densities, or on postgraft motor asymmetries. The results are discussed in terms of the fact that LFM-stimulated cells can be differentiated in a manner similar to NGF-treated cells, by acquiring sympathetic like traits which in turn can diminish motor asymmetries when grafted into nigrostriatal-lesioned rats.     

血管内皮生长因子和基质金属蛋白酶9及其抑制剂在脑膜瘤中的表达

目的探讨血管内皮生长因子(VEGF)和基质金属蛋白酶-9(MMP-9)及其抑制剂TIMP-1与脑膜瘤的生物学特性和侵袭性的关系。方法采用免疫组化S-P法测定手术切除的45例不同组织学类型脑膜瘤标本中VEGF、MMP-9及TIMP-1的蛋白表达。结果VEGF、MMP-9和TIMP-1在良性脑膜瘤中的阳性表达率分别为40%、36%、52%;在恶性脑膜瘤中的阳性表达率分别为70%、75%、55%。VEGF和MMP-9在恶性和良性脑膜瘤中的表达存在显著性差异(P<0.01),且二者在脑膜瘤中的表达呈正相关。结论VEGF、MMP-9与脑膜瘤的生物学特性相关,二者可能相互协同促进脑膜瘤的侵袭;MMP-9/TIMP-1的失衡与脑膜瘤的恶性程度和侵袭能力相关。     

包涵体中重组人神经生长因子的纯化,复性及鉴定

目的利用简单的纯化工艺，获得具有生物活性的重组人神经生长因子（rhNGF）。方法利用PET－11d－NGF／BL21工程菌表达rhNGF，通过改变培养基成份优化发酵条件。建立了一种在提取包涵体后经一步强阳离子柱层析的简单、快速、重复性好的rhNGF纯化方法。纯化产物用鸡胚背根神经节伸长法测定其生物学活性。结果优化发酵条件后，表达量提高至约占总菌体的10．9％。经一步层析，重组蛋白的纯度可达到80％以上。1BU（生物活性单位）约为0．5μg／mL。结论包涵体复性表明，包涵体的复性与复性液中氧化型与还原型含疏基化合物的比例密切相关。抑制杂菌生长，可明显提高rhNGF的表达量。纯化的rhNGF为分析NGF结构与功能及探讨其临床应用提供了材料。     

Increased Concentrations of Nerve Growth Factor and Brain-Derived Neurotrophic Factor in the Rat Cerebellum After Exposure to Environmental Enrichment

The molecular mechanism of environmental enrichment (EE) on brain function and anatomy has been partially attributed to the up-regulation of proteins involved in neuronal survival and activity-dependent plasticity, such as the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), in the cerebral cortex and hippocampus of animal models. Nevertheless, at present, little indication is available on the influence of EE on neurotrophin levels in the cerebellum. Thus, in this study, we exposed male Wistar rats to EE from weaning to 5 months of age and evaluated the production of NGF and BDNF in the cerebellum and compared the neurotrophin changes in this region with those obtained in other brain structures where neurotrophins are produced or transported. We found that in rats exposed to EE from 21st until 140th postnatal day, a significant increase of both BDNF and NGF concentrations was observed in the cerebellum, as compared to rats reared in standard conditions. In addition, cerebellum was the brain region where NGF and BDNF levels were more influenced by EE as compared to the changes observed in other regions. EE also caused a concomitant increase in NGF levels in the striatum while in the same brain region, BDNF levels were reduced. In summary, this study shows that a prolonged exposure to EE is associated with an increase in cerebellar NGF and BDNF production, thus suggesting that the beneficial effects of EE on the cerebellum of adult animals could be mediated, at least in part, by neurotrophins.     

Regulation of Nerve Growth Factor (NGF) Synthesis in the Rat Central Nervous System: Comparison between the Effects of Interleukin-1 and Various Growth Factors in Astrocyte Cultures and in vivo

In order to obtain information on the physiological regulation of NGF-synthesis in the central nervous system (CNS) we investigated the effects of a series of growth factors (known to be present in the CNS) in cultures of purified rat astrocytes and compared these effects with those observed after intraventricular injection of the same molecules. After preliminary experiments had shown that 10% fetal calf serum (FCS) produced a marked increase in NGF-mRNA levels in astrocytes (but neither in microglia nor oligodendrocytes) as demonstrated by Northern blot analysis and in situ hybridization the experiments were performed at low (0.5%) FCS concentrations. Supramaximal concentrations of IL-1 and various growth factors caused a 5- to 7-fold increase in NGF-mRNA after 6 h. By 24 h the NGF-mRNA levels approached control values again, most probably due to inactivation of the added factors since after readdition after 24 h the response was about the same as the initial one. Norepinephrine and 8-bromo-cAMP did not change NGF-mRNA levels. The growth factor-mediated changes in NGF-mRNA levels in astrocyte cultures were not consistently reflected by the changes observed after intraventricular injection. IL-1 produced by far the largest increase in hippocampal NGF-mRNA after intraventricular injection. This large response to IL-1 could result from a positive feedback mechanism, since IL-1beta injection not only increases NGF-mRNA but also IL-1beta-mRNA in the hippocampus. The understanding of the physiological regulation of NGF synthesis in the CNS is the basis for a rational approach to its pharmacological modification. This, in turn, is an attractive alternative to the (long-term) infusion of NGF or the transplantation of NGF-secreting cells with the goal of providing trophic support to the cholinergic neurons of the basal forebrain nuclei. These neurons are consistently affected in the early stages of Alzheimer's disease, their impaired function being essentially responsible for the cognitive deficits.     

Matrix Metalloproteinase-2 and Epidermal Growth Factor are Decreased in Platelets of Alzheimer Patients

Alzheimer's disease (AD) is the most prevalent type of dementia. Despite considerable advances in diagnostic accuracy, diagnostic procedures that are easily accessible are still sorely needed. Blood biomarkers are therefore in the focus of research. Platelets contain a high concentration of the amyloid precursor protein (APP), which has been mentioned as a potentially useful diagnostic marker. The aim of the present study was to analyze various cell adhesion molecules (CAMs), cytokines, growth factors, and matrix metalloproteinases (MMPs) in platelets of AD and mild cognitively impaired (MCI) patients as compared to healthy controls. Our data show a significant decrease in the levels of epidermal growth factor (EGF) and of MMP-2 in platelets of AD patients and decreased levels of MMP-2 in MCI. The APP ratio was slightly but not significantly decreased in AD patients, whereas CD40L and serotonin were unchanged. Our findings demonstrate specific changes in AD platelets. Whether these biomarkers can be established as potential early diagnostic biomarkers for AD remains to be established in longitudinal studies.     

神经生长因子对红藻氨酸诱导癫痫发作大鼠海马神经细胞凋亡的影响

目的　探讨外源性神经生长因子 (NGF)对癫痫发作所致海马神经细胞凋亡有无抑制作用。方法　采用红藻氨酸 (KA)诱导癫痫大鼠模型 ,以原位末端标记法 (TUNEL)标记 DNA片段 ,检测凋亡细胞在大鼠海马CA1区的动态变化及 NGF对其的影响。结果　海马 CA1区凋亡细胞在实验 2 4 h出现 ,4 8h明显增多 ,于 72h达高峰 ,7d时最少。给予 NGF脑室内注射后 ,在各相应时间点凋亡细胞数均明显减少 (P<0 .0 1)。结论　外源性 NGF能抑制癫痫发作所致的海马神经细胞凋亡 ,NGF对癫痫脑损伤有保护作用。     

Overexpression of Nerve Growth Factor in Skin Increases Sensory Neuron Size and Modulates Trk Receptor Expression

Sensory neuron development and differentiation is dependent on a family of growth factors known as neurotrophins. Neurotrophins modulate neuron development via trk tyrosine kinase receptor proteins trkA, trkB and trkC. To determine how elevated levels of a target-derived neurotrophin might affect neuronal differentiation, we analysed trk expression in the trigeminal ganglion of transgenic mice that overexpressed nerve growth factor (NGF) in the skin. increased levels of NGF caused a five-fold increase in neurons expressing trkA mRNA and a two-fold increase in neurons expressing trkC. In control mice, cell size distributions of neuronal subpopulations expressing each trk mRNA showed the three subpopulations distributed over a narrow, overlapping range. In contrast, cell size distribution in NGF-transgenic mice was significantly divergent due in large part to hypertrophy of trkA neurons and, to a lesser extent, trkC neurons. In addition, we examined neurons that bound the isolectin B4 from Bandeiraea simplicifolia (BS-IB4) because most of these neurons do not express any trk receptor in the adult. There was a significant increase in the size of BS-IB4–positive neurons in transgenic mice; however, there was no increase in their number. These studies indicate that an increased level of target-derived NGF affects the development of sensory neurons that in the adult express trkA or trkC, as well as neurons that do not express trk receptors.     

Potential Roles of Gene Expression Change in Adult Rat Spinal Motoneurons Following Axonal Injury: A Comparison among c-jun, Low-Affinity Nerve Growth Factor Receptor (LNGFR), and Nitric Oxide Synthase (NOS)

The present study investigates expression of nitric oxide synthase (NOS), immediate early genes (IEGs, c-jun, and c-fos), and low-affinity nerve growth factor receptor (LNGFR) in adult rat spinal motoneurons in response to three conditions of axonal injury: distal axotomy, root avulsion, and root avulsion followed by a peripheral nerve (PN) graft implantation. Expression of c-jun and LNGFR were predominately observed in motoneurons of the distal axotomized segment where most motoneurons survived. In contrast, expression of NOS was exclusively found in motoneurons of the root avulsed segment where most motoneurons died. c-fos was not expressed in motoneurons following either distal axotomy or root avulsion. In animals with PN graft implantation, a double fluorescent labeling technique was used to evaluate motoneuron regeneration. Expression of NOS was completely inhibited in all motoneurons that regenerated into the PN graft, but was not inhibited in those that did not regenerate. Moreover, regenerated motoneurons expressed LNGFR and c-jun while the nonregenerated motoneurons expressed NOS. Results of the present study have shown that motoneurons undergo changes in expression of cellular molecules in response to the axonal injury. The expression of c-jun and LNGFR may be related to the regenerative process while expression of NOS is more likely involved in the degenerative process. The results also show that PN graft implantation can alter the expression of cellular molecules and reduce motoneuron death due to root avulsion. The survival-promoting effects of PN graft implantation (presumably the effects of neurotrophic factors) may be achieved by modifying certain cellular molecules such as NOS.     

Cerebellar Brain-Derived Neurotrophic Factor, Nerve Growth Factor, and Neurotrophin-3 Expression in Male and Female Rats Is Differentially Affected by Hypergravity Exposure During Discrete Developmental Periods

We previously reported that the effects of perinatal exposure to hypergravity on cerebellum and motor functions in rat neonates are strongly dependent on the specific developmental period of exposure. In the present study, we explored the hypothesis that neurodevelopmental changes are associated with altered expression of brain neurotrophins critical for normal brain growth and differentiation. We compared the effects of hypergravity exposure during four developmental periods: period I extended from gestational day (G) 8 through G15; period II from G15 to birth, period III from birth to postnatal day (P) 6; and period IV extended from G8–P12. For comparison we used stationary control (SC) neonates not exposed to hypergravity. Neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin 3 (NT-3) levels were measured in cerebellar homogenates prepared from postnatal day 12 male and female rat neonates using specific ELISAs. Hypergravity exposure affected individual neurotrophins differently and the effect was further determined by the period of hypergravity exposure. ANOVA showed: (1) a significant effect of the period of exposure to hypergravity on cerebellar BDNF (p = 0.009), with maximal decrease of 28.7% in males and 32.1% in females following exposure during period III; (2) a significant effect on NGF (p < 0.0001), with maximal decrease of 35.6% in male and 48.8% in female neonates following exposure during period III; (3) no statistically significant effect on NT-3 expression with a trend towards decreased expression in female rats following exposure during period IV. Although the molecular mechanisms underlying the differential neurotrophins’ response to hypergravity are not clear, an altered pattern of their expression is likely to contribute to neurodevelopmental changes and impaired sensorimotor behavior in exposed neonates.     

Nerve Growth Factor is Required for Induction of c-Fos Immunoreactivity by Serum,Depolarization, Cyclic AMP or Trauma in Cultured Rat Sympathetic Neurons

Nerve growth factor (NGF) induces transient Fos-immunoreactivity (Fos-IR) independently of any other factor, both in newly isolated rat sympathetic neurons and in established cultures after NGF deprivation. The same proportion of neurons that express Fos-IR in response to NGF also survive. In addition to direct stimulation of Fos-IR expression, the presence or recent exposure to NGF is required to obtain Fos-IR expression by other stimuli. In newly isolated neurons no Fos-IR is detected in response to stimulation by serum alone and a response to depolarization or cyclic AMP is obtained only if neurons are stimulated within a short period after ganglion excision. In established cultures none of these stimuli, nor the trauma of cutting neurites or spiking cell bodies with a microinjection needle induce Fos-IR unless NGF is present or had been removed for <8 - 16 h. The lack of response is not due to a general decrease in the rate of protein or RNA synthesis. These findings show that in regenerating sympathetic neurons NGF induces c-Fos and suggest that NGF may activate a master trigger that is required for c-Fos expression to be induced by other stimuli.     

Effects of Nerve Growth Factor Treatment on Rats with Lesions of the Nucleus Basalis Magnocellularis Produced by Ibotenic Acid, Quisqualic Acid, and AMPA

Rats with bilateral lesions of the nucleus basalis magnocellularis (NBM) produced by ibotenic acid (IBO), quisqualic acid (QUIS), and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) received either human recombinant nerve growth factor (NGF) (5.0 μg/day) or cytochrome c (cc) (0.3 μg/day) treatment. Spatial memory impairments in the Morris water maze differed for the three toxins in the following order: IBO-cc > AMPA-cc > QUIS-cc. Treatment with NGF resulted in behavioral improvement for only the IBO-lesioned rats. Body weight was not affected by the different lesions before treatment; however, administration of NGF resulted in a decreased rate of body weight gain independent of the excitotoxin used. Reduction in choline acetyltransferase (ChAT) activity differed in the neocortex for the three toxins, whereas the hippocampus was unaffected. The cortical ChAT depletion was greatest for AMPA, intermediate for QUIS, and least for IBO. Restoration of ChAT activity by NGF differed for the three toxins (QUIS-NGF > IBO-NGF > AMPA-NGF), whereas ChAT activity in the hippocampus was increased equally. The loss of low-affinity NGF-receptor (p75NGFr)-immunoreactive neurons differed for the toxins (AMPA-cc > QUIS-cc > IBO-cc), whereas treatment with NGF increased the size of the remaining neurons independently of the used toxin. NGF induced a p75NGFr-immunoreactive thickening around intracerebral arteries for all three toxins. Lesions of the NBM produced by IBO, QUIS, and AMPA resulted in a quantitatively different pattern of behavioral, biochemical, and histological deficits. The quantitatively different pattern of recovery after administration of NGF may be determined by the responsivity of the remaining neurons. Intracerebroventricular administration of NGF affected noncholinergic systems as well, resulting in decreased food intake and cerebrovascular hyperinnervation.     

Altered Plasma Levels of Nerve Growth Factor and Transforming Growth Factor-β2 in Type-1 Diabetes Mellitus

Nerve growth factor (NGF) and transforming growth factor-β2 (TGF-β2) are cytokines which have known immunological effects. An elevated level of NGF has been reported in certain autoimmune diseases, whereas TGF-β2 is an immunosuppressor which is known to play a role in regulating cell proliferation. A role of this cytokine has been proposed in the pathogenesis of type-1 diabetes mellitus (IDDM), but no clinical studies have yet measured its serum level in this disease. In this study we measured the levels of NGF and TGF-β2 in the sera of patients with IDDM (n = 26) and values were compared to those of age-matched normal subjects (n = 27) and also to patients with type-2 diabetes mellitus (NIDDM) (n = 26) with similar HbA1c levels and an equal duration of diabetes. Serum NGF levels were significantly elevated in IDDM patients compared to those of age-matched controls (p < .001) and NIDDM controls (p < .01). TGF-β2 levels were lower in IDDM patients when compared with the healthy control (p < .001) and the NIDDM control (p < .05). There was no correlation between the levels of NGF and TGF-β2. The duration of diabetes and the level of HbA1c did not affect the NGF and TGF-β2 levels in the IDDM patients. We conclude that an increase in NGF and a suppression in TGF-β2 levels are present in patients with type-1 diabetes mellitus and that both cytokines may play independent roles in the pathogenesis of this disease.