Immobilized concentration gradients of neurotrophic factors guide neurite outgrowth of primary neurons in macroporous scaffolds

Neurotrophic factors present as concentration gradients are neurotropic cues that direct axonal growth toward their targets. Multiple factors work together in vivo to ensure axons reach the proper targets, likely interacting with one another via intracellular signalling pathways. Nerve growth factor (NGF) and neurotrophin-3 (NT-3) are neurotrophins known to guide axons as well as promote axonal growth following injury to both the spinal cord and peripheral nerve. These molecules interact with neurons through different tyrosine kinase receptors. In this study, the receptors for these growth factors were shown to be co-localized on E10 chick dorsal root ganglion (DRG) cells, providing an opportunity for synergism. Well-defined concentration gradients of NGF and NT-3 were immobilized for the first time in a cell-penetrable, cell-adhesive scaffold of poly(2-hydroxyethylmethacrylate) and poly(L-lysine). An NGF concentration gradient of 310 ng/mL/mm was required to guide chick DRG neurites. A lower concentration gradient of 200 ng/mL/mm of NGF was shown to elicit guidance when an NT-3 concentration gradient of 200 ng/mL/mm was also present, indicating a synergistic response in the DRG neurons. These gradient scaffolds may be useful for guided regeneration following injury to the spinal cord or peripheral nerve and may also elucidate the mechanism for intracellular signaling of neurotrophic factors.     

Immobilized concentration gradients of nerve growth factor guide neurite outgrowth

Axons are guided to their targets by a combination of haptotactic and chemotactic cues. We previously demonstrated that soluble neurotrophic factor concentration gradients guide axons in a model system. In an attempt to translate this model system to a device for implantation, our goal was to immobilize a stable neurotrophic concentration gradient for axonal (or neurite) guidance. Nerve growth factor (NGF) was immobilized within poly(2-hydroxyethylmethacrylate) [p(HEMA)] microporous gels using a gradient maker. The NGF was stably immobilized, with only approximately 0.05% of the amount originally incorporated into the gel released over an 8-day period. Immobilized NGF was bioactive: the percent of PC12 cells extending neurites on NGF-immobilized p(HEMA) gels was 16 +/- 2%, which was statistically the same as those exposed to soluble NGF (22 +/- 6%). We were able to predict and reproducibly create stable NGF concentration gradients in the gel. At an NGF concentration gradient of 357 ng/mL/mm, PC12 cell neurites were guided up the gradient. The facile, flexible, and reproducible nature of this method allowed us to translate soluble growth factor gradient models to stable growth factor gradient devices that may ultimately enhance axonal guidance and regeneration in vivo.     

Environmental factors involved in axonal regeneration following spinal cord transection in rats

A recent study of a rat model treated with grafted collagen filament (CF) after spinal cord transection showed dramatic recovery of motor function but did not report on the acute-stage phenomenon. In the present study, we describe molecular and histological aspects of the axonal regeneration process during the acute stage following spinal cord transection. The spinal cord of 8-week-old rats was completely transected, and a scaffold of almost the same size as the resected portion was implanted in the gap. Changes in the mRNA expression of four neurotrophic factors [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, and glial cell-derived neurotrophic factor (GDNF)] were analyzed after 72 h. The expression of BDNF and NT-3 mRNA increased significantly in the CF-grafted group compared to the nongrafted group. Immunostaining for BDNF and NT-3 revealed that cells positive for these neurotrophic factors extended along the collagen filaments in the CF-grafted group. Similarly, astrocytes extended into the collagen filament scaffold together with the neurotrophic factors and partly across a border line. These findings indicate that collagen filament helps to reduce scar tissue, supports the expression of neurotrophic factors, and serves as a scaffold for the outgrowth of regenerating axons.     

Defining the concentration gradient of nerve growth factor for guided neurite outgrowth

The developing axon is believed to navigate towards its target tissue in response to a concentration gradient of neurotrophic factors, among other diffusible and surface-bound stimuli. However, the minimum concentration gradient required for guidance over the maximum distance is still unknown, largely because well-defined systems have not been utilized to address this question. In this study, a linear concentration gradient of nerve growth factor was achieved across a 5-mm agarose membrane that separated a nerve growth factor source compartment from a sink compartment. The concentrations in both compartments were maintained constant (and different). Both concentration and concentration gradient were well defined across the membrane, allowing us to study the relative importance of concentration gradient vs concentration for neurite guidance. The orientation of PC12 cell neurites was studied in response to a series of nerve growth factor concentration gradients in vitro. For effective guidance of PC12 cell neurite outgrowth, a minimum concentration gradient of 133ng/ml per mm was required, below which guidance was ineffective. Higher gradients were effective for guidance yet were limited by the concentration of nerve growth factor in the source compartment. At a nerve growth factor concentration of 995ng/ml, the PC12 cells' receptors were saturated, thereby limiting the maximum effective distance for guidance to less than 7.5mm in response to a diffusible nerve growth factor cue. This distance exceeds the 0.5-2mm distance observed by others for effective neurite guidance.Using this model system, we propose that the minimum concentration gradient can be defined for other cells and growth factors. Ultimately, it is anticipated that such concentration gradients could be included in a device to promote regeneration.     

Neurotrophin-3 is a target-derived neurotrophic factor for penile erection-inducing neurons

The aim of this study was to determine whether the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 could act as endogenous target-derived trophic factors for erection-inducing, i.e. penis-projecting major pelvic ganglion (MPG) neurons, and/or penile sensory neurons in adult rat. This was accomplished by studying the expression of NT mRNAs in the penis and their cognate receptors in the MPG and dorsal root ganglia (DRGs), and the retrograde axonal transport of radioiodinated NTs injected into the corpora cavernosa. Northern hybridization showed that NGF, BDNF, and NT-3 mRNAs are expressed in the shaft of the penis. In situ hybridization combined with usage of the retrograde tracer Fluoro-Gold showed that TrkC and p75 receptors are expressed in penis-projecting neurons of the MPG whereas the mRNAs for TrkA and TrkB receptors were undetectable. However, all the NT receptor mRNAs were expressed in penile sensory neurons of sacral level 1 (S1) DRG. (125)I-NT-3 injected into the shaft of the penis was retrogradely transported into the MPG and S1 DRG, whereas radioiodinated NGF and BDNF were transported specifically into the S1 DRG, thus confirming the existence of functional NT receptors in these penile neurons. In conclusion, these data suggest that NT-3 may act as a target-derived neurotrophic factor for both erection-inducing and penile sensory neurons, whereas NGF and BDNF may be more important for the sensory innervation of the penis.     

Brain-derived neurotrophic factor, nerve growth and neurotrophin-3 selected regions of the rat brain following kainic acid-induced seizure activity.

Changes in levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) in various regions of the rat brain following kainic acid-induced seizure activity were investigated. BDNF protein, as measured by a two-site enzyme immunoassay, increased transiently 12-24 h after the intraperitoneal administration of kainic acid to 61.6 ng/g wet weight in the hippocampus (approximately 10-fold increase), 19.5 ng/g in the piriform plus entorhinal cortex (approximately 10-fold) and 8.2 ng/g in the olfactory bulb (approximately 16-fold), and then rapidly decreased. Increases of 2- to 4-fold in levels of BDNF were also detected in the septum, cerebral cortex, striatum and hypothalamus, but not in the cerebellum. In contrast, levels of NGF and NT-3 decreased 24 h after the administration of kainic acid. Western and Northern blotting analyses of hippocampal tissues, respectively, revealed increase in levels of a 14-kDa protein corresponding to BDNF and its mRNA at both 4.2 and 1.4 kb. Hippocampal mRNAs for NGF and NT-3 increased and decreased, respectively, in kainic acid-treated rats. Immunohistological investigations showed that, in the hippocampus, the administration of kainic acid enhanced a homogeneous immunoreactivity of BDNF in the polymorph inner layer (the stratum radiatum of the CA3/CA4 regions and the hilar region) and in granule cells of the dentate gyrus. BDNF protein was found in neurons, but not at all in glial cells or in blood vessels, and was localized in the cytoplasm, the nucleoplasm and the primary dendrites of neurons as well as in perisynaptic extracellular spaces, but hardly in their axons. Our results show that kainic acid treatment increases levels of BDNF, but not NGF or NT-3, in various regions of the rat brain, other than the cerebellum. Also, the majority of BDNF newly synthesized by hippocampal granule neurons is secreted into the perisynaptic extracellular space in the polymorph inner layer of the dentate gyrus, supporting an autocrine-like role for the factor in synaptic functions.     

脊髓半横断损伤后腹角神经元神经生长因子、脑源性神经营养因子、神经营养因子-3和神经营养因子-4表达的变化

目的:探讨大鼠脊髓半横断损伤(htSCI)后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子(NT-3、NT-4)在脊髓腹角神经元表达的早期变化。方法:免疫组织化学ABC法分别染4种神经因子并作阳性细胞计数。结果:NGF主要分布于脊髓腹角神经元的胞核,BDNF、NT-4与NT-3主要分布于胞浆。htSCI前后它们在细胞内的分布范围没有变化。BDNF、NGF与NT-3的3 d在损伤尾侧段脊髓双侧腹角阳性神经元数与对照组相比显著减少。BDNF与NGF的14 d的双侧腹角阳性神经元数量均较正常组明显增多,NT-3与NT-4的14 d~21 d的双侧腹角阳性神经元数量均较正常组明显增多,BDNF7~21 d以及NGF14 d的健侧的阳性神经元数量均分别多于相应的伤侧。结论:内源性BDNF、NGF、NT-3、NT-4增加对脊髓损伤修复具有重要作用,BDNF和NGF在健侧表达的增加说明健侧代偿功能的活跃。     

Differential neurotrophin levels in cerebrospinal fluid and their changes during development in newborn rat

Cerebrospinal fluid concentration of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) was measured in normal developing rat from birth to postnatal day (PND) 21 by enzyme-linked immunosorbent assay. NGF levels were significantly higher than those of BDNF and NT-3 from PND 1–21. NGF levels decreased from PND 1–3 to PND 9. At PND 15 and 17, NGF levels peaked a second time and rapidly decreased to PND 21. BDNF peaked at PND 13–15, while NT-3 levels peaked at PND 7–9. Each of the three neurotrophins has its own characteristic pattern in changes in cerebrospinal fluid levels.     

Expression of mRNA of neurotrophic factors and their receptors are significantly altered after subchronic ketamine treatment

The neurotrophic factors play an important role in the maintenance of neurone viability and neuronal communication which are considered to be altered in schizophrenia. Subchronic application of ketamine (Ket) was found to be a useful model in schizophrenia research. To further validate this model the mRNA levels of neurotrophic factors NGF, NT-3, and BDNF and their receptors TrkA, TrkB, and TrkC, respectively, were measured in different brain areas in Ket-pretreated rats subchronically dosed with the atypical antipsychotic drug risperidone (Ris). With the exception of NGF in the frontal cortex, Ket pretreatment did change NGF, NT-3, and BDNF mRNA levels in the frontal cortex, the hippocampus, the striatum, the thalamus/hypothalamus region, and in the cerebellum. These changes correspond with changes at their tyrosine kinase receptors. Ris treatment normalised altered NT-3 levels in the hippocampus and balanced BDNF levels in the same structure. It was concluded that the Ket model might reflect distinct alterations in neurotrophic factor activity as found in schizophrenic patients and, moreover, that Ris treatment rebalances disturbed neurotrophic factor activity.     

Immunohistochemical study on the distribution of voltage-gated K(+) channels in rat brain following transient focal ischemia

Microchemotaxis chambers were used to explore macrophage chemotaxis in response to the neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). Both NGF and NT-3, but not BDNF, promoted macrophage chemotaxis that was receptor mediated and dependent on protein phosphorylation. These in vitro observations point to novel roles for neurotrophins that are present in nerve prior to and immediately after injury.     

BDNF,NGF和NT-3的mRNA和蛋白在猫左侧腰第六背根节共表达的不同方式(英文)

本实验研究了脑源性神经营养因子(brain derived neurotrophic factor,BDNF),神经生长因子(nerve growth factor,NGF)和神经营养因子-3(neurotrophin-3,NT-3)的mRNA和蛋白在猫左侧腰第六背根节(dorsal root ganglion,DRG)共表达的不同方式,并探讨了共表达的机制,为阐明神经营养因子的表达与脊髓可塑性的关系提供依据。本实验所使用的为未接受任何处理的健康猫。猫的左侧腰第六DRG被取出,行免疫组织化学染色与原位杂交方法结合的双重标记,确定是否有BDNF,NGF和NT-3的蛋白与mRNA共表达。实验结果显示BDNF,NGF和NT-3的蛋白与mRNA在猫DRG均有表达,但这三种神经营养因子mRNA和蛋白共表达的方式是不同的。免疫组化结果显示:BDNF阳性产物主要分布于细胞质和细胞核,细胞核的染色颜色较细胞质浅;NGF阳性产物主要分布于细胞核;NT-3阳性产物主要分布于细胞质。原位杂交结果显示:BDNF和NGF阳性信号主要分布于细胞质;NT-3阳性信号在细胞质和细胞核都有分布。由此可见,BDNF,NGF和NT-3的mRNA和蛋白在猫左侧腰第六DRG有不同的共表达方式,提示它们可能存在与脊髓可塑性有关的自分泌和/或旁分泌机制。     

Immunocytochemical colocalization of fibroblast growth factor-1 with neurotrophin-3 in mouse alveolar macrophages

Alveolar macrophages are known to express a variety of growth factors and neurotrophins. Fibroblast growth factor-1 (FGF-1) is abundantly present in the lung and has mitogenic and neurotrophic activities similarly to neurotrophins. In order to determine whether FGF-1 associates with neurotrophins in alveolar macrophages, we investigated the immunocytochemical colocalization of FGF-1 with neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), in mouse alveolar macrophages. The results showed that 34% of macrophages were immunoreactive for FGF-1, 10% for NGF, 9% for BDNF, and 17% for NT-3. Of FGF-1-immunoreactive (IR) macrophages, 16% were immunoreactive for NT-3, but only small percentages were immunoreactive for NGF (0.8%) and for BDNF (0.3%). FGF-1 and neurotrophins were all localized in the intracellular vesicles. In the vesicles, FGF-1 and NT-3 were frequently colocalized. All macrophages expressed lysosome-associated protein-2 (LAMP-2), a late endosomal and lysosomal marker, and early endosomes antigen 1 (EEA1), an early endosomal marker. FGF-1 and NT-3 were predominantly colocalized with LAMP-2 rather than with EEA1, whereas NGF and BDNF were colocalized with EEA1 rather than with LAMP-2. These results indicate that FGF-1 and NT-3 are substantially expressed in mouse alveolar macrophages and colocalized in vesicles, predominantly in late endosomes and lysosomes.     

Effects of neurotrophins on human bronchial smooth muscle cell migration and matrix metalloproteinase-9 secretion

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) have been found to be upregulated in inflammatory pulmonary diseases, including asthma. The functional role for the neurotrophins in the airways is still not known, but it has been proposed that neurotrophins induce airway hyperreactivity and tissue remodeling. Bronchial smooth muscle cells have been suggested to be involved in the remodeling process through their capacity to proliferate, migrate, and secrete inflammatory mediators and matrix metalloproteinases (MMPs). Therefore, we studied the effect of NGF, BDNF, and NT-3 on human bronchial smooth muscle cell (HBSMC) migration and MMP-2 and MMP-9 secretion. Immunocytochemistry studies showed that HBSMCs expressed the neurotrophin receptors TrkA, TrkB, and TrkC. BDNF, NT-3, and NGF increased MMP-9, but not MMP-2, secretion as shown by zymography. BDNF and NT-3, but not NGF, stimulated HBSMC migration as evaluated by Boyden chamber. Taken together, our data indicate that the neurotrophins may stimulate events important for airway remodeling.     

Expression of insulin-like growth factor-1 receptor (IGF-1R) and p27Kip1 in melanocytic tumors: a potential regulatory role of IGF-1 pathway in distribution of p27Kip1 between different cyclins

Alveolar macrophages play a crucial role in regulating lung immune responses and in maintaining the integrity of the respiratory tract. Neurotrophins (NTs), besides to their neurotrophic activities, exhibit physiological effects in the immune system. In this study, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), NT-3 and low- (p75) and high affinity (Trks) NT receptors were investigated by immunocytochemistry in cytospin centrifuged preparations of human alveolar macrophages. Approximately 2.5% alveolar macrophages were immunoreactive for NGF, whereas no macrophages displaying immunoreactivity for BDNF or NT-3 were observed. A 3.5% macrophages displayed immunoreactivity for TrkA-receptor protein, 10% for TrkB-receptor protein (full length isoform), and 2% for TrkC-receptor protein. No low-affinity p75 NT and TrkB[-] truncated isoform receptor immunoreactive macrophages were found. These findings support the hypothesis that NTs and the corresponding receptors may play a role in regulating immunological and functional activity of alveolar macrophages via paracrine/autocrine mechanisms.     

Neurotrophic factors promote and enhance locomotor recovery in untrained spinalized cats

In spinal cats, locomotor recovery without rehabilitation is limited, but weight-bearing stepping returns with treadmill training. We studied whether neurotrophins administered to the injury site also restores locomotion in untrained spinal cats and whether combining both neurotrophins and training further improves recovery. Ordinary rat fibroblasts or a mixture of fibroblasts secreting brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (Fb-NTF) were grafted into T12 spinal transection sites. Cats with each type of transplant were divided into two groups: one receiving daily training and the other receiving no training. As expected, trained cats with/without neurotrophin-producing transplants could step on the treadmill. Untrained cats without neurotrophin-producing transplants could not locomote. However, untrained cats with neurotrophin-secreting transplants performed plantar weight-bearing stepping at speeds up to 0.8 m/s as early as 2 wk after transection. Locomotor capability and stance lengths in these animals were similar to those in animals receiving training alone, suggesting that administration of BDNF/NT-3 was equivalent to treadmill training in restoring locomotion in chronically spinalized cats. Cats receiving both interventions showed the greatest improvement in step length. Anatomical evaluation indicated that all transections were complete and that axons did not enter the cord caudal to the graft. Thus BDNF/NT-3 secreting fibroblasts were equivalent to training in their ability to engage the locomotor circuitry in chronic spinal cats. Furthermore, the rapid time-course of recovery and the absence of axonal growth through the transplants indicate that the restorative mechanisms were not related to supraspinal axonal growth. Finally, the results show that transplants beneficial in rodents are applicable to larger mammals.     

脂肪源性干细胞修复周围神经损伤潜能的研究

目的:体外原代培养脂肪源性干细胞(adipose-derived stem cell,ADSC),并在细胞水平检测各种神经营养因子(neurotrophic factor,NF)的表达情况,探讨ADSC是否可以作为神经损伤修复的种子细胞。方法:体外原代培养和鉴定ADSC,应用RT-PCR方法检测ADSC细胞神经营养因子,包括神经生长因子(nerve growth factor,NGF)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、神经营养素-3(neurotrophin 3,NT-3);免疫组织化学法检测粘附因子(nerve cell adhesion molecule,NCAM)表达情况。结果:ADSC可以向脂肪和骨两方面分化;RT-PCR结果显示ADSC细胞NGF、BDNF、NT-3 mRNA水平明显升高;同时免疫荧光法检测到了高水平的NCAM。结论:ADSC具有干细胞分化潜能,并且能够表达NF和NCAM。     

Multifunctional, multichannel bridges that deliver neurotrophin encoding lentivirus for regeneration following spinal cord injury

Therapeutic strategies following spinal cord injury must address the multiple barriers that limit regeneration. Multiple channel bridges have been developed that stabilize the injury following implantation and provide physical guidance for regenerating axons. These bridges have now been employed as a vehicle for localized delivery of lentivirus. Implantation of lentivirus loaded multiple channel bridges produced transgene expression that persisted for at least 4 weeks. Expression was maximal at the implant at the earliest time point, and decreased with increasing time of implantation, as well as rostral and caudal to the bridge. Immunohistochemical staining indicated transduction of macrophages, Schwann cells, fibroblasts, and astrocytes within the bridge and adjacent tissue. Subsequently, the delivery of lentivirus encoding the neurotrophic factors NT-3 or BDNF significantly increased the extent of axonal growth into the bridge relative to empty scaffolds. In addition to promoting axon growth, the induced expression of neurotrophic factors led to myelination of axons within the channels of the bridge, where the number of myelinated axons was significantly enhanced relative to control. Combining gene delivery with biomaterials to provide physical guidance and create a permissive environment can provide a platform to enhance axonal growth and promote regeneration.     

Neurotrophic factor protection against ethanol toxicity in rat cerebellar granule cell cultures requires phosphatidylinositol 3-kinase activation

Neonatal rat cerebellar granule cells were used to assess the possible role of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway in the neuroprotective effects of neurotrophic factors against ethanol toxicity. Culture conditions included medium with ethanol (400 and 600 mg/dl), nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), ethanol+NGF or BDNF, the PI3-K inhibitor wortmannin (10 or 100 microM), and wortmannin+ethanol+NGF or BDNF. Neuronal survival was determined via the MTT assay. The results indicated that both NGF and BDNF ameliorate ethanol neurotoxicity, and wortmannin abolished this effect, except at the higher ethanol concentration combined with the lower wortmannin level. These data strongly implicate the PI3-K pathway in growth factor protection against ethanol neurotoxicity.