Temporal changes in the level of neurotrophins in the spinal cord and associated precentral gyrus following spinal hemisection in adult Rhesus monkeys

Neurotrophins (NTs) appear to be crucial for the survival and potential regeneration of injured neurons. However, their temporal changes and remote regulations following spinal cord injury (SCI) have been only partially determined, especially in primates. In this study, ELISA was performed on the extracts of injured spinal cord and the associated precentral gyrus contralateral to the site of spinal cord hemisection to investigate the temporal changes in the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in adult rhesus monkeys subjected to T8 spinal hemisection. Animals were allowed to survive 3, 7, 14, 30 and 90 days post-operation (dpo). In the spinal cord, the levels of NGF, BDNF and NT-3 sharply decreased between 3 and 7dpo. Thereafter, the levels of NGF and BDNF were transiently elevated while NT-3 level continuously increased and recovered to normal level at 30dpo. In the contralateral precentral gyrus (cPG), only the NT-3 level was altered and in fact elevated above the normal value. No obvious changes were observed in NT-4 level in any of the regions studied. Taken together, the present findings indicated that intrinsic NGF, BDNF and NT-3 may play a local role in the responses to the SCI in primates. Especially, the increase of NT-3 level occurred continuously in both the cPG and the spinal cord pointed to a possible transportation of NT-3 to the cord following SCI.     

Temporal changes in the level of neurotrophins in the spinal cord and associated precentral gyrus following spinal hemisection in adult Rhesus monkeys

Neurotrophins (NTs) appear to be crucial for the survival and potential regeneration of injured neurons. However, their temporal changes and remote regulations following spinal cord injury (SCI) have been only partially determined, especially in primates. In this study, ELISA was performed on the extracts of injured spinal cord and the associated precentral gyrus contralateral to the site of spinal cord hemisection to investigate the temporal changes in the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in adult rhesus monkeys subjected to T8 spinal hemisection. Animals were allowed to survive 3, 7, 14, 30 and 90 days post-operation (dpo). In the spinal cord, the levels of NGF, BDNF and NT-3 sharply decreased between 3 and 7 dpo. Thereafter, the levels of NGF and BDNF were transiently elevated while NT-3 level continuously increased and recovered to normal level at 30 dpo. In the contralateral precentral gyrus (cPG), only the NT-3 level was altered and in fact elevated above the normal value. No obvious changes were observed in NT-4 level in any of the regions studied. Taken together, the present findings indicated that intrinsic NGF, BDNF and NT-3 may play a local role in the responses to the SCI in primates. Especially, the increase of NT-3 level occurred continuously in both the cPG and the spinal cord pointed to a possible transportation of NT-3 to the cord following SCI.     

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。     

神经营养因子(NGF、NT-3、BDNF及CNTF)和受体trkA、trkB、trkC在正常大鼠脊髓和胳鼠脊髓移植体的免疫组织化学研究

本研究采用免疫组织化学方法观察了NGF家族（NGF、NT－3、BDNF）和非NGF家族的CNTF以及NGF家族因子受体trkA、trkB、trkC在正常大鼠脊髓腰段的分布和胎鼠脊髓移植体在移植后4周的表达。在正常大鼠脊髓腰段,各神经营养因子及受体反应物主要存在于脊髓灰质,特别是前角的运动神经元。但在脊髓后角的分布稍有不同,BDNF阳性细胞的胞浆染色较深,胞核不染色;而NT－3的胞核染色较胞浆为深,核仁不染色。在胎鼠脊髓移植体,NGF、BDNF、CNTF和NT－3及受体trkA、trkB、trkC均有不同程度的染色。本实验结果揭示了在正常大鼠脊髓神经元内各神经营养因子及受体的表达,提示神经营养因子除具有靶源性来源以外,还有神经元自分泌的产物。而在胎鼠移植体内和其周围组织神经营养因子及其受体的表达,可能是在移植体内的移植细胞自分泌和成鼠脊髓损伤的刺激所引起的,这在移植体的存活和发育中有重要作用。     

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.     

Progesterone up-regulates neuronal brain-derived neurotrophic factor expression in the injured spinal cord

Progesterone (PROG) provides neuroprotection to the injured central and peripheral nervous system. These effects may be due to regulation of myelin synthesis in glial cells and also to direct actions on neuronal function. Recent studies point to neurotrophins as possible mediators of hormone action. Here, we show that the expression of brain-derived neurotrophic factor (BDNF) at both the mRNA and protein levels was increased by PROG treatment in ventral horn motoneurons from rats with spinal cord injury (SCI). Semiquantitative in situ hybridization revealed that SCI reduced BDNF mRNA levels by 50% in spinal motoneurons (control: 53.5+/-7.5 grains/mm(2) vs. SCI: 27.5+/-1.2, P<0.05), while PROG administration to injured rats (4 mg/kg/day during 3 days, s.c.) elicited a three-fold increase in grain density (SCI+PROG: 77.8+/-8.3 grains/mm(2), P<0.001 vs. SCI). In addition, PROG enhanced BDNF immunoreactivity in motoneurons of the lesioned spinal cord. Analysis of the frequency distribution of immunoreactive densities (chi(2): 812.73, P<0.0001) showed that 70% of SCI+PROG motoneurons scored as dark stained whereas only 6% of neurons in the SCI group belonged to this density score category (P<0.001). PROG also prevented the lesion-induced chromatolytic degeneration of spinal cord motoneurons as determined by Nissl staining. In the normal intact spinal cord, PROG significantly increased BDNF inmunoreactivity in ventral horn neurons, without changes in mRNA levels. Our findings suggest that PROG enhancement of endogenous neuronal BDNF could provide a trophic environment within the lesioned spinal cord and might be part of the PROG activated-pathways to provide neuroprotection.     

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.     

Effects of electro-acupuncture on NT-4 expression in spinal dorsal root ganglion and associated segments of the spinal dorsal horn in cats subjected to adjacent dorsal root ganglionectomy

It is well known that neuroplasticity occurs in the central nervous system in response to injury. Electro-acupuncture (EA) may also promote neuroplasticity. But little is known about the underlying molecular mechanisms for the beneficial effects of EA. This study investigated the effects of EA on neurotrophin-4 (NT-4) expression in L(6) spinal dorsal root ganglion (DRG) and associated segments of the spinal dorsal horn in cats subjected to unilateral removal of L(1)-L(5) and L(7)-S(2) DRG. NT-4 protein was normally present in the cytoplasm of the L(6) DRG neurons and L(3) and L(6) spinal dorsal horn neurons and glia. Adjacent ganglionectomy leads to a significant decrease in NT-4 expression in the L(6) DRG, but no change in the spinal dorsal horn. Following EA treatment a significant increase occurred in the L(6) DRG at 14 days post-operation (dpo) as well as the L(6) cord segment at 7 and 14 dpo. These findings pointed to a possible association between NT-4 expression and EA promoted spinal cord plasticity in adult cats subjected to partial ganglionectomy.     

Expression of neurotrophins and their receptors tropomyosin-related kinases (Trk) under tension-stress during distraction osteogenesis

The localization and expression of neurotrophins and their receptors during distraction osteogenesis was investigated in 72 male rat femurs (11 weeks old) to further clarify the concurrence of cellular and molecular events of new bone formation. After osteotomy, a 7-day lag phase was followed by distraction at the rate of 0.25 mm/12 h for 21 days (distraction phase), and a 7-day consolidation phase. The localization of neurotrophins (NGF, BDNF and NT-3) and their receptors tropomyosinrelated kinases (TRKA, TRKB and TRKC) by immunostaining showed positive staining in bone forming cells in each stage, although the presence and staining intensity varied by cell type and phase. The expressions of NGF, BDNF and NT-3 by real-time polymerase chain reaction (real-time PCR) showed that the peak of the mRNA expression of NGF occurred 10 days after distraction. NT-3 increased during bone extension, but decreased when distraction stopped. In contrast, BDNF continued to increase gradually throughout the distraction and consolidation phases. These findings suggest that neurotrophins and their receptors may play different roles in endochondral and intramembranous ossification in distraction osteogenesis. The tension stress caused by distraction may stimulate the expression of neurotrophins and their receptors, and promote osteogenesis.     

BDNF-exercise interactions in the recovery of symmetrical stepping after a cervical hemisection in rats

Clinical evidence indicates that motor training facilitates functional recovery after a spinal cord injury (SCI). Brain-derived neurotrophic factor (BDNF) is a powerful synaptic facilitator and likely plays a key role in motor and sensory functions. Spinal cord hemisection decreases the levels of BDNF below the injury site, and exercise can counteract this decrease [Ying Z, Roy RR, Edgerton VR, Gomez-Pinilla F (2005) Exercise restores levels of neurotrophins and synaptic plasticity following spinal cord injury. Exp Neurol 193:411-419]. It is not clear, however, whether the exercise-induced increases in BDNF play a role in mediating the recovery of locomotion after a SCI. We performed a lateral cervical ( approximately C4) hemisection in adult rats. Seven days after hemisection, the BDNF inhibitor trkB IgG was injected into the cervical spinal cord below the lesion ( approximately C5-C6). Half of the rats were exposed to voluntary running wheels for 14 days. Locomotor ability was assessed by determining the symmetry between the contralateral (unaffected) vs. the ipsilateral (affected) forelimb at the most optimum treadmill speed for each rat. Sedentary and exercised rats with BDNF inhibition showed a higher level of asymmetry during the treadmill locomotion test than rats not treated with the BDNF inhibitor. In hemisected rats, exercise normalized the levels of molecules important for synaptic function, such as cyclic AMP response element binding protein (CREB) and synapsin I, in the ipsilateral cervical enlargement, whereas the BDNF blocker lessened these exercise-associated effects. The results indicate that BDNF levels play an important role in shaping the synaptic plasticity and in defining the level of recovery of locomotor performance after a SCI.     

腺病毒介导的神经营养素-3基因能够在大鼠脊髓前角运动神经元内过表达神经营养素-3

目的观察腺病毒介导的神经营养素-3(NT-3)基因在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元的过表达。方法在坐骨神经内直接注射含有绿色荧光蛋白(GFP)基因(报告基因)的NT-3基因重组腺病毒(Ad-NT-3-GFP),7d后应用免疫荧光组织化学染色技术,在荧光显微镜下观察脊髓前角运动神经元的NT-3过表达。结果 GFP表达组(对照组)和NT-3加GFP表达组两组动物的L4和L5脊髓段横切片上,有绿色荧光蛋白阳性标记的细胞。在NT-3加GFP表达组,还可以观察到NT-3阳性标记的细胞,这种细胞能与绿色荧光蛋白阳性标记的细胞重合,是过表达NT-3的前角运动神经元。与GFP表达组的前角运动神经元形态比较,NT-3加GFP表达组的过表达NT-3的前角运动神经元呈现更富有分支的突起。结论腺病毒介导的NT-3基因能够在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元内过表达NT-3,这为下一歩应用NT-3基因治疗策略修复实验性脊髓损伤提供初歩的实验资料。     

Recovery of hindlimb locomotion after incomplete spinal cord injury in the cat involves spontaneous compensatory changes within the spinal locomotor circuitry

After incomplete spinal cord injury (SCI), compensatory changes occur throughout the whole neuraxis, including the spinal cord below the lesion, as suggested by previous experiments using a dual SCI paradigm. Indeed, cats submitted to a lateral spinal hemisection at T10-T11 and trained on a treadmill for 3-14 wk re-expressed bilateral hindlimb locomotion as soon as 24 h after spinalization, a process that normally takes 2-3 wk when a complete spinalization is performed without a prior hemisection. In this study, we wanted to ascertain whether similar effects could occur spontaneously without training between the two SCIs and within a short period of 3 wk in 11 cats. One day after the complete spinalization, 9 of the 11 cats were able to re-express hindlimb locomotion either bilaterally (n = 6) or unilaterally on the side of the previous hemisection (n = 3). In these 9 cats, the hindlimb on the side of the previous hemisection (left hindlimb) performed better than the right side in contrast to that observed during the hemispinal period itself. Cats re-expressing the best bilateral hindlimb locomotion after spinalization had the largest initial hemilesion and the most prominent locomotor deficits after this first SCI. These results provide evidence that 1) marked reorganization of the spinal locomotor circuitry can occur without specific locomotor training and within a short period of 3 wk; 2) the spinal cord can reorganize in a more or less symmetrical way; and 3) the ability to walk after spinalization depends on the degree of deficits and adaptation observed in the hemispinal period.     

大鼠脊髓全横断后脊髓内BDNF表达的变化

为了探讨大鼠脊髓全横断后不同时相(3、7、14、21d)脊髓内BDNF表达的变化,我们采用免疫组织化学ABC法和阳性神经元计数,来检测脊髓全横断后不同时相BDNF的表达。结果发现:(1)BDNF免疫阳性产物主要位于腹角运动神经元,胞浆着色;(2)脊髓全横断后脊髓腹角BDNF阳性细胞数3d开始增多,7d达高峰(P<0.05),14d恢复正常(P>0.05)。提示BDNF表达的变化可能与脊髓可塑性有关。     

Foreperiod duration and motor preparation during childhood

To assess the role of brain-derived neurotrophic factor (BDNF) in nociceptive processing after chronic lateral spinal cord hemisection injury (SCI) at T13, we studied the effects of BDNF on evoked activity of dorsal horn wide dynamic range (WDR) neurons. Evoked responses of WDR cells (n=34 total) at L3–L5 were characterized electrophysiologically after spinal administration of vehicle, or BDNF (10 μg). In hemisected animals, application of BDNF to the surface of the cord resulted in reductions in evoked activity in 24 of 32 cells (75%), and enhancement of evoked activity in eight of 32 (25%) cells. Phosphate-buffered saline-receiving animals demonstrated evoked response rates of between 75 and 93 Hz, while BDNF(−) cells had evoked rates from between 20 and 41 Hz, and BDNF(+) activities were between 80 and 119 Hz, significant changes of 76 and 124%, respectively. Effects were bilateral and differences in sidedness were not observed. These results further implicate BDNF in nociceptive processing, but suggest a complex role after chronic SCI.     

一种新颖的神经胶质细胞-嗅鞘细胞

嗅鞘细胞(OECs)移植治疗脊髓损伤(SC I)已显示出良好的应用前景,不同来源的OECs体外培养时,在不同发育阶段和不同生长环境中具有不同的生物学特性。OECs具有特异性的标志蛋白,如S-100、p75NTR、GFAP等。体外培养的OECs分泌NGF、BDNF、NT-3/4、GDNF等多种促进神经细胞生长、分化和神经纤维再生的神经营养因子。将OECs植入脊髓损伤部位,该细胞能分裂增殖,并可抑制胶质瘢痕的形成,拮抗Nogo等神经再生抑制性因子的活性,包绕再生轴突形成髓鞘。由于OECs具有上述独特的生物学功能,被认为是继神经干细胞和雪旺氏细胞之后可用于移植治疗脊髓损伤的一种新颖的神经胶质细胞。     

外源性NGF对大鼠坐骨神经切断缝合术后脊髓和背根节CGRP表达的影响

观察外源性神经生长因子(NGF)对坐骨神经切断立即缝合后大鼠脊髓和背根节(DRG)内不同时间点降钙素基因相关肽(CGRP)表达的影响。成年SD雄性大鼠随机分为NGF组、生理盐水(NS)组、假手术组与正常对照组。实验组动物右侧坐骨神经切断后立即缝合,每天给予NGF(400单位/kg腹腔注射),动物分别存活1、3、5、7、14、21、28d,免疫组织化学方法结合图像分析检测相应节段脊髓和背根节内CGRP的表达。结果表明,NGF处理组术侧背根节与脊髓后角内的CGRP表达明显高于同时间点的NS对照组,平均光密度值相比P<0.05。但各组中脊髓前角运动神经元内的CGRP表达没有明显变化(P>0.05)。结果提示外源性的NGF能明显增加损伤后背根节感觉神经元与损伤侧脊髓后角的CGRP表达,对CGRP在脊髓前角运动神经元内的表达没有明显影响。     

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.     

Neurotrophin and Neurotrophin Receptors in Vascular Smooth Muscle Cells: Regulation of Expression in Response to Injury

The neurotrophins, a family of related polypeptide growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3 and NT-4/5 promote the survival and differentiation of distinctive sets of embryonic neurons. Here we define a new functional role for neurotrophins, as autocrine or local paracrine mediators of vascular smooth muscle cell migration. We have identified neurotrophins, and their cognate receptors, the trk tyrosine kinases, in human and rat vascular smooth muscle cells in vivo. In vitro, cultured human smooth muscle cells express BDNF; NT-3; and trk A, B, and C Similarly, rat smooth muscle cells expressed all three trk receptors as well as all four neurotrophins. Moreover, NGF induces cultured human smooth muscle cell migration at subnanomolar concentrations. In the rat aortic balloon deendothelialization model of vascular injury, the expression of NGF, BDNF, and their receptors trk A and trk B increased dramatically in the area of injury within 3 days and persisted during the formation of the neointima. In human coronary atherosclerotic lesions, BDNF, NT-3, and NT-4/5, and the trk B and trk C receptors could be demonstrated in smooth muscle cells. These findings suggest that neurotrophins play an important role in regulating the response of vascular smooth muscle cells to injury.