Effects of endogenous neurotrophins on sympathetic sprouting in the dorsal root ganglia and allodynia following spinal nerve injury

Peripheral nerve injury is often complicated by a chronic pain syndrome that is difficult to treat. In animal models of peripheral nerve injury, sympathetic nerve terminals in the dorsal root ganglia (DRG) sprout to form baskets around large diameter neurons, an anatomical change that has been implicated in the induction of neuropathic pain. In the present study, we have investigated whether neurotrophins derived from peripheral sources play any roles in sympathetic sprouting and neuropathic pain in a rat model of peripheral nerve injury. After transection of the left lumbar (L) 5 spinal nerve, antisera specific to neurotrophins were injected intraperitoneally twice a week for 2 weeks. The foot withdrawal response to von Frey hairs was examined on days 1, 3, 7, 10, and 14 postlesion. After completion of behavioral tests, sympathetic sprouting in DRG was examined by tyrosine hydroxylase (TH) immunohistochemistry. The number of TH-immunoreactive (ir) fibers and baskets around large neurons within the lesioned DRG was dramatically increased in the rats treated with control normal sheep serum. Antisera specific to nerve growth factor (NGF), neurotrophin-3 (NT3), and brain-derived neurotrophic factor (BDNF) significantly reduced the sympathetic sprouting and the formation of baskets. L5 spinal nerve lesion induced a significant increase in foot withdrawal responses to von Frey hair stimuli, which was attenuated by treatment of antisera to neurotrophins with a different time sequential. The effect of BDNF antiserum occurred earlier and lasted longer than those of NGF and NT3 antisera. These results implicate that peripherally derived neurotrophins are involved in the induction of sympathetic sprouting and neuropathic pain following peripheral nerve injury.     

Painful nerve injury upregulates thrombospondin‐4 expression in dorsal root ganglia

Thrombospondin‐4 (TSP4) belongs to a family of large, oligomeric extracellular matrix glycoproteins that mediate interactions between cells and interactions of cells with underlying matrix components. Recent evidence shows that TSP4 might contribute to the generation of neuropathic pain. However, there has been no systematic examination of TSP4 expression in the dorsal root ganglia (DRG) after injury. This study, therefore, investigates whether TSP4 protein level is changed in DRG after injury following spinal nerve ligation (SNL) and spared nerve injury in rats by performing Western blotting, immunohistochemistry, and immunocytochemistry. After nerve ligation, TSP4 protein level is upregulated in the axotomized somata of the fifth lumbar (L5) DRG. There is substantial additional TSP4 in the nonneuronal compartment of the L5 DRG that does not costain for markers of satellite glia, microglia, or Schwann cells and appears to be in the interstitial space. Evidence of intracellular overexpression of TSP4 persists in neurons dissociated from the L5 DRG after SNL. These findings indicate that, following peripheral nerve injury, TSP4 protein expression is elevated in the cytoplasm of axotomized sensory neurons and in the surrounding interstitial space. © 2014 Wiley Periodicals, Inc.     

Electrical stimulation accelerates nerve regeneration and functional recovery in delayed peripheral nerve injury in rats

The present study aims to investigate the potential of brief electrical stimulation (ES; 3 V, 20 Hz, 20 min) in improving functional recovery in delayed nerve injury repair (DNIR). The sciatic nerve of Sprague Dawley rats was transected, and the repair of nerve injury was delayed for different time durations (2, 4, 12 and 24 weeks). Brief depolarizing ES was applied to the proximal nerve stump when the transected nerve stumps were bridged with a hollow nerve conduit (5 mm in length) after delayed periods. We found that the diameter and number of regenerated axons, the thickness of myelin sheath, as well as the number of Fluoro‐Gold retrograde‐labeled motoneurons and sensory neurons were significantly increased by ES, suggesting that brief ES to proximal nerve stumps is capable of promoting nerve regeneration in DNIR with different delayed durations, with the longest duration of 24 weeks. In addition, the amplitude of compound muscle action potential (gastrocnemius muscle) and nerve conduction velocity were also enhanced, and gastrocnemius muscle atrophy was partially reversed by brief ES, indicating that brief ES to proximal nerve stump was able to improve functional recovery in DNIR. Furthermore, brief ES was capable of increasing brain‐derived neurotrophic factor (BDNF) expression in the spinal cord in DNIR, suggesting that BDNF‐mediated neurotrophin signaling might be one of the contributing factors to the beneficial effect of brief ES on DNIR. In conclusion, the present findings indicate the potential of using brief ES as a useful method to improve functional recovery for delayed repair of peripheral nerve lesions.     

Effects of spinal nerve ligation on immunohistochemically identified neurons in the L4 and L5 dorsal root ganglia of the rat

This study examined the effect of spinal nerve ligation on different populations of immunohistochemically identified neurons in the dorsal root ganglia (DRG) of the rat. The optical fractionator method was used to count neurons in the ipsilateral L4 and L5 DRG 1-20 weeks after ligation of the L5 and L6 spinal nerves, sham surgery, or no surgery. One week after ligation, neurons in the L5 DRG that were labeled by IB4, a marker of unmyelinated primary afferent neurons, were largely absent. The numbers of IB4-labeled neurons then progressively increased to reach control values by 20 weeks. A smaller, sustained decrease occurred in the number of small-, medium- and large-sized neurons immunoreactive for calcitonin gene-related peptide (CGRP), a marker for peptidergic primary afferents, in the L5 DRG. There was a proportionately greater decrease in the numbers of medium- to large-sized CGRP-like immunoreactive neurons. The number of myelinated afferents in the L5 DRG, identified by their staining for neurofilament protein (N52), did not change after ligation. However, closer examination revealed a significant decrease in the numbers of large-sized neurons, coupled with an increase in the numbers of small- to medium-sized neurons, and the appearance of a novel population of very small-sized neurons labeled by N52. The numbers and cell size distributions of IB4-labeled, CGRP-like immunoreactive, and N52-labeled neurons were unchanged in the adjacent L4 DRG. Unlike the L5 DRG, injury-induced changes in the expression of various receptors, neurotransmitters and neurotrophic factors in the L4 DRG are not confounded by a change in the immunohistochemical phenotype of primary afferent neurons.     

关节炎大鼠脊髓背角内脑源性神经营养因子和促肾上腺皮质激素释放因子表达增加(英文)

研究表明 ,外周感染可导致脑源性神经营养因子 (BDNF)的过度表达 ,BDNF可影响其他神经递质的合成。采用免疫组化和原位杂交的方法观察了完全福氏佐剂所致的关节炎大鼠脊髓内 BDNF及其功能性受体酪氨酸蛋白激酶 B(trk B)的表达和促肾上腺皮质激素释放因子 (CRF) m RNA的水平。实验发现 ,在皮下注射完全福氏佐剂后 4h,脊髓腰膨大同侧背角中的 BDNF免疫活性神经元和 CRF m RNA阳性神经元数升高 ,在 2 4 h时达到峰值 ,在 7d时仍维持在较高水平。实验提示 ,关节炎大鼠脊髓内的 BDNF和 CRF可能参与了慢性痛反应。     

Distribution of RET immunoreactivity in the rodent spinal cord and changes after nerve injury

RET (for “rearranged during transfection”) is a transmembrane tyrosine kinase signaling receptor for members of the glial cell line‐derived neurotrophic factor (GDNF) family of ligands. We used RET immunohistochemistry (IHC), double‐labeling immunofluorescence (IF), and in situ hybridization (ISH) in adult naïve and nerve‐injured rats to study the distribution of RET in the spinal cord. In the dorsal horn, strong RET‐immunoreactive (‐ir) fibers were abundant in lamina II‐inner (II_i), although this labeling was preferentially observed after an antigen‐unmasking procedure. After dorsal rhizotomy, RET‐ir fibers in lamina II_i completely disappeared from the dorsal horn, indicating that they were all primary afferents. After peripheral axotomy, RET‐ir in primary afferents decreased in lamina II_i and appeared to increase slightly in laminae III and IV. RET‐ir was also observed in neurons and dendrites throughout the dorsal horn. Some RET‐ir neurons in lamina I had the morphological appearance of nociceptive projection neurons, which was confirmed by the finding that 53% of RET‐ir neurons in lamina I colocalized with neurokinin‐1. GDNF‐ir terminals were in close proximity to RET‐ir neurons in the superficial dorsal horn. In the ventral horn, RET‐ir was strongly expressed by motoneurons, with the strongest staining in small, presumably γ‐motoneurons. Increased RET expression following peripheral axotomy was most pronounced in α‐motoneurons. The expression and regulation pattern of RET in the spinal cord are in line with its involvement in regenerative processes following nerve injury. The presence of RET in dorsal horn neurons, including nociceptive projection neurons, suggests that RET also has a role in signal transduction at the spinal level. This role may include mediating the effects of GDNF released from nociceptive afferent fibers. J. Comp. Neurol. 500:1136–1153, 2007. © 2006 Wiley‐Liss, Inc.     

Substance P and calcitonin gene-related peptide expression in dorsal root ganglia in sciatic nerve injury rats

The neuropeptides, substance P and calcitonin gene-related peptide, have been shown to be involved in pain transmission and repair of sciatic nerve injury. A model of sciatic nerve defect was prepared by dissecting the sciatic nerve at the middle, left femur in female Sprague Dawley rats. The two ends of the nerve were encased in a silica gel tube. L5 dorsal root ganglia were harvested 7, 14 and 28 days post sciatic nerve injury for immunohistochemical staining. Results showed that substance P and cal- citonin gene-related peptide expression increased significantly in dorsal root ganglion of rats with sci- atic nerve injury. This increase peaked at 7 days, declined at 14 days, and reduced to normal levels by 28 days post injury. The findings indicate that the neuropeptides, substance P and calcitonin gene- related peptide, mainly increased in the early stages after sciatic nerve injury.     

Depression of Ca2+/calmodulin‐dependent protein kinase II in dorsal root ganglion neurons after spinal nerve ligation

The enzyme calcium/calmodulin‐dependent protein kinase II (CaMKII) is associated with memory and its α isoform is critical for development of activity‐induced synaptic changes. Therefore, we hypothesized that CaMKII is involved in altered function of dorsal root ganglion (DRG) neurons after neuronal injury. To test this hypothesis, Sprague–Dawley rats were made hyperalgesic by L5 and L6 spinal nerve ligation (SNL), and changes in total phosphorylated and unphosphorylated CaMKII (tCaMKII) and phosphorylated form of its α isoform (pCaMKIIα) were analyzed using immunochemistry in different subpopulations of DRG. SNL did not induce any changes in tCaMKII between experimental groups, while the overall percentage of pCaMKIIα‐positive neurons in injured L5 DRG SNL (24.8%) decreased significantly when compared to control (41.7%). SNL did not change the percentage of pCaMKIIα/N52 colabeled neurons but decreased the percentage of N52‐negative nonmyelinated neurons that expressed pCaMKIIα from 27% in control animals to 11% after axotomy. We also observed a significant decrease in the percentage of small nonpeptidergic neurons labeled with IB4 (37.6% in control vs. 4.0% in L5 SNL DRG), as well as a decrease in the percentage of pCaMKIIα/IB4 colabeled neurons in injured L5 DRGs (27% in control vs. 1% in L5 DRG of SNL group). Our results show that reduction in pCaMKIIα levels following peripheral injury is due to the loss of IB4‐positive neurons. These results indicate that diminished afferent activity after axotomy may lead to decreased phosphorylation of CaMKIIα. J. Comp. Neurol. 518:64–74, 2010. © 2009 Wiley‐Liss, Inc.     

川芎嗪对紫杉醇致神经病理性疼痛大鼠的疗效及其对坐骨神经中神经生长因子表达的影响

目的探讨川芎嗪对紫杉醇致神经病理性疼痛大鼠的疗效及其对坐骨神经中神经生长因子(NGF)表达的影响。方法成年雄性SD大鼠30只随机分为A组(对照组,n=6)、B组(n=8)、C组(n=8)、D组(n=8)。以首次给药为d1,实验前一日为d0,各组分别在d1、d3、d5、d7腹腔注入2.0 mg/(kg·d)紫杉醇。于d1~d14,B组腹腔注入川芎嗪200 mg/(kg·d),C组口服普瑞巴林3 mg/(kg·d),D组同时腹腔注射川芎嗪和口服普瑞巴林,剂量同B组、C组。分别于d0和d7、d12、d16、d20测定大鼠的机械缩足反射阈值(MWT)和热缩足潜伏期(TWL)。采用透射电镜观察各组大鼠坐骨神经的超微结构,用免疫组化法检测大鼠坐骨神经中NGF的表达。结果 4组大鼠d0时后肢MWT差异无统计学意义(均P0.05);D组大鼠d7~d20时后肢平均MWT与A组及C组相比明显升高(均P0.05);B、C组大鼠d12~d20时后肢MWT与A组相比明显升高(均P0.05);B组d12、d16时MWT明显高于C组(均P0.05)。4组大鼠d0时后肢TWL差异无统计学意义(均P0.05);B、C、D组大鼠d7~d20时后肢TWL与A组相比明显升高(均P0.05);D组大鼠d7~d20时后肢TWL与C组相比明显升高(均P0.05);C组大鼠d16、d20时后肢TWL与B组相比明显升高(均P0.05)。透射电镜下,A组大鼠的坐骨神经纤维髓鞘板层结构明显松散,呈网状,严重脱髓鞘;B组、C组、D组大鼠的坐骨神经纤维髓鞘板层结构较松散,明显脱髓鞘,但较A组明显减轻,且B组、D组脱髓鞘程度较C组更轻。与A组比较,B组和C组大鼠坐骨神经中NGF表达减少不明显(均P0.05),D组大鼠坐骨神经中NGF表达明显减少(P0.05)。结论川芎嗪能有效防治紫杉醇所致的大鼠神经病理性疼痛,疗效与普瑞巴林相似并有协同作用,作用机理可能是减低坐骨神经NGF的表达,减轻神经损伤。     

Responses of spinal neurones to cutaneous and dorsal root stimuli in rats with mechanical allodynia after contusive spinal cord injury

The firing of neurones in spinal segments adjacent to a contusive T13 spinal cord injury was characterised in anaesthetised rats. Three groups of rats were examined: (1) allodynic spinally injured, (2) non-allodynic spinally injured and (3) normal, uninjured. Spinal cord field potentials evoked by electrical dorsal root stimulation and the responses of 207 dorsal horn neurones to mechanical stimuli applied to the skin were studied. Within the lesioned spinal segment few active neurones were encountered and field potentials were absent. Depolarising field potentials recorded rostral to the lesion were reduced in both allodynic and non-allodynic animals compared to uninjured controls, while those recorded in caudal segments were enhanced in allodynic animals. Neuronal recordings revealed that allodynia was associated with exaggerated responses, including afterdischarges, to innocuous and noxious mechanical stimuli in a proportion of wide dynamic range, but not low threshold, neurones. These changes were observed both rostral and caudal to the site of injury. The results suggest that an increased responsiveness of some dorsal horn neurones in segments neighbouring a contusive spinal cord injury may contribute to the expression of mechanical allodynia. It is proposed that a relative lack of inhibition underlies altered cell responses.     

Study on molecular mechanism for improving neural regeneration after repair of sciatic nerve defect in rat by acellular nerve allograft

Objective: Discuss the molecular mechanism for improving neural regeneration after repair of sciatic nerve defect in rat by acellular nerve allograft (ANA). Methods: Randomly divide 36 Wistar rats into six groups as normal control group, autografting group, and bridging groups of 2, 4, 8, 12 weeks, six rats for each group. Observe the expression of brain‐derived neurotrophic factor (BDNF) in L₄ spinal cord and anterior tibial muscle at the injury site, calcitonin gene‐related peptide (CGRP) protein as well as mRNA, respectively. 12w after operation, histopathological observation was performed. Results: 2w after ANA bridging the sciatic nerve defect in rats, it was observed that the expression level of BDNF in spinal cord at the injury site and CGRP protein increased, reaching the peak level at 4w, lasting till 8w, then decreased but still significantly higher than that in normal control group at 12w, and was not significantly different compared with that in autografting group. However, the expression level of BDNF in anterior tibial muscle decreased gradually within the initial 4w, then increased progressively, reaching normal level at 12w, and was not significantly different compared with that in autografting group. The expression of BDNF mRNA and CGRPmRNA was essentially the same. 12w after operation, there was nerve regeneration in bridging group of 12w and autografting group. Conclusions: ANA possessed fine histocompatibility, and might substitute autograft to repair long‐segment defect of sciatic nerve in rats. This action might be related to upregulation of protein and mRNA expression for BDNF and CGRP in spinal cord. Synapse, 2012. © 2011 Wiley Periodicals, Inc.     

单一腰6脊神经根结扎延长大鼠神经病理性疼痛发展期的研究

目的：腰5和腰6脊神经根共同结扎是目前最常见的神经病理性疼痛鼠模型,但结扎后大鼠的机械触诱发痛会在24～72 h内迅速发展,时间窗过短,本课题组探索和研究了一种新型的神经病理性疼痛大鼠模型—单一腰6脊神经根结扎鼠模型。方法将36只大鼠随机分为空白对照组、腰5和腰6脊神经根结扎组、单一腰6脊神经根结扎组及假手术组,除单一腰6脊神经根结扎组18只外,其余3组每组6只大鼠;分别检测其50％机械缩爪阈值（50％PWT）。另取54只大鼠,随机分为空白对照组、腰5和腰6脊神经根结扎组、腰6脊神经根结扎组,各组大鼠只数分别为6只、24只、24只。采用蛋白免疫印迹法检测脊髓背角胶质纤丝酸性蛋白（ GFAP）在结扎术后第1 d、第7 d、第14 d和第28 d的表达。结果单一腰6脊神经根结扎组产生了长期发展的机械触诱发痛（1～14 d）;且大鼠同侧脊髓背角GFAP表达随之升高。结论单一腰6脊神经根结扎鼠模型可以作为研究神经病理性疼痛发展期具体机制的有效模型。     

Satellite glial cell proliferation in the trigeminal ganglia after chronic constriction injury of the infraorbital nerve

We have examined satellite glial cell (SGC) proliferation in trigeminal ganglia following chronic constriction injury of the infraorbital nerve. Using BrdU labeling combined with immunohistochemistry for SGC specific proteins we positively confirmed proliferating cells to be SGCs. Proliferation peaks at approximately 4 days after injury and dividing SGCs are preferentially located around neurons that are immunopositive for ATF‐3, a marker of nerve injury. After nerve injury there is an increase GFAP expression in SGCs associated with both ATF‐3 immunopositive and immunonegative neurons throughout the ganglia. SGCs also express the non‐glial proteins, CD45 and CD163, which label resident macrophages and circulating leukocytes, respectively. In addition to SGCs, we found some Schwann cells, endothelial cells, resident macrophages, and circulating leukocytes were BrdU immunopositive. GLIA 2013;61:2000–2008     

Brain‐derived neurotrophic factor exerts antinociceptive effects by reducing excitability of colon‐projecting dorsal root ganglion neurons in the colorectal distention‐evoked visceral pain model

The mechanism underlying visceral pain is still largely unclear. Recently, much attention has focused on a potential modulatory role of brain‐derived neurotrophic factor (BDNF) in visceral pain. In the present study, we investigated the expression of BDNF in dorsal root ganglia (DRG) primary sensory neurons and its role in a colorectal distention (CRD)‐induced model of visceral pain. Results obtained from enzyme‐linked immunosorbant assay (ELISA) revealed that BDNF protein was upregulated after CRD. An abdominal withdrawal reflex (AWR) assay confirmed that BDNF played an antinociceptive role in this model. Application of BDNF directly to DRG neurons decreased their hypersensitivity when evoked by CRD. Pretreatment with k252a partially blocked the effect of BDNF. These findings suggest that BDNF might be a novel analgesic agent for the treatment of visceral pain. © 2012 Wiley Periodicals, Inc.     

Effects of cyclooxygenase 2 inhibitor on growth-associated protein 43 and nerve growth factor expression in dorsal root ganglion during neuropathic pain development

BACKGROUND： Inflammatory responses in injured nerves have been recognized as important factors for initially sensitizing nociceptive neurons. Cyclooxygenase （COX） is the rate-limiting enzyme in prostaglandin synthesis, and COX-2 inhibitor is involved in mechanisms of analgesia and anti-inflammation. OBJECTIVE： To investigate the effects of COX-2 inhibitor on thermal and mechanical hyperalgesia, as well as expression of growth associated protein 43 （GAP-43） and nerve growth factor （NGF） in dorsal root ganglion, in a rat model of neuropathic pain due to chronic constriction injury. DESIGN, TIME AND SETTING： A randomized, controlled, comparison study that was performed at the Surgical Department and Pathological Laboratory, Second Affiliated Hospital of Shantou University Medical College from September 2006 to September 2007. MATERIALS： COX-2 inhibitor, Iornoxicam, was purchased from Nycomed Pharmaceutical （Austria）; rabbit anti-GAP-43, and rabbit anti-NGF polyclonal antibodies were purchased from Boster, Wuhan, China. METHODS： A total of 50 adult, Wistar rats were randomly assigned to four groups： normal control （n = 5）, model （n = 15）, normal saline control （n = 15）, and Iornoxicam treatment （n =15）. With exception of the control group, the sciatic nerve of all rats was loosely ligated to establish a model of chronic constriction injury. The model rats were divided into three subgroups according to varying post-operative survival periods： 3, 7 and 14 days （n = 5）, respectively. Rats in the Iornoxicam treatment group were intraperitoneally injected with 1.3 mg/kg lornoxicam every 12 hours throughout the entire experimental procedure. Rats in the normal saline control group were intraperitoneally injected with 1.3 mL/kg saline. MAIN OUTCOME MEASURES： Immunohistochemistry revealed expression of GAP-43 and NGF in the L5 dorsal root ganglions. Mechanical withdrawal threshold and thermal withdrawal latency were used to observe neurological behavioral changes in rats. RESULTS： The relative gray values of GAP-43- and NGF-positive neurons in the model group were remarkably increased compared with the normal control rats （P 〈 0.01）, while the relative gray values in the Iomoxicam treatment group were significantly less than the model and normal saline control groups （P 〈 0.01）. Mechanical withdrawal threshold and thermal withdrawal latency gradually decreased with increasing injury time in the model, normal saline control, and Iornoxicam treatment groups, and were significantly less than the normal control group （P 〈 0.05）. In addition, mechanical withdrawal threshold and thermal withdrawal latency were significantly greater in the Iornoxicam treatment group compared with the model and normal saline control groups （P 〈 0.05）. CONCLUSION： Intraperitoneal injection of the COX-2 inhibitor Iornoxicam attenuated mechanical and thermal hyperalgesia induced by sciatic nerve chronic constriction injury and inhibited the increased expression of GAP-43 and NGF.     

Role of phosphatidylinositol 3-kinase in neuronal survival and axonal outgrowth of adult mouse dorsal root ganglia explants

Adult ganglionic peripheral neurons have lost dependence on target-derived neurotrophin signaling for survival and regeneration after injury. To understand the mechanisms required to sustain such processes at maturity, we are studying neuronal survival and axonal outgrowth of adult mouse dorsal root ganglia (DRG) explants. We have here examined the role of phosphatidylinositol 3-kinase (PI3-K) activity. Both neuronal survival and axonal outgrowth of spontaneously growing preparations were decreased significantly by the PI3-K inhibitor LY294002 as was the increased outgrowth caused by nerve growth factor or glial cell line-derived factor. Inhibition of PI3-K activity promoted neuronal cell death to the same extent in the presence as in the absence of a growth factor, whereas inhibition of mitogen-activated protein kinase, MAPK, lacked effect. Using a compartmentalized system, it could be shown that only axonal outgrowth was decreased when the outgrowth region only was exposed to LY294002. Already-formed growth cones showed morphological changes within 5-10 min after exposure to LY294002. Akt (PKB) is one downstream effector of PI3-K. Immunofluorescence revealed the presence of activated Akt in DRG cell bodies and in axonal growth cones. Immunoreactivity was decreased by PI3-K inhibition. The results suggest that Akt is constitutively active in adult DRG neurons, and that PI3-K mediated processes are involved in neuronal survival of one or more DRG neuronal subpopulations and also in axonal elongation. The possible significance of Akt signaling for these effects is discussed.