Forced retraction of spinal root injury enhances activation of p38 MAPK cascade in infiltrating macrophages

Root‐rupture injury is a type of preganglionic brachial plexus injury resulting from traction force, where a small section of the spinal root is usually left behind. We have established experimental models of both root‐rupture injury with traction force and rhizotomy without traction force in rats and we examined the activation of microglia/macrophages in both conditions. LGP107 and LGP96, which are rat homologs of lysosome‐associated membrane proteins, were most useful as immunohistochemical markers of mononuclear phagocytes. The metabolic activation of macrophages was analyzed by immunohistochemistry with a series of antibodies against tumor necrosis factor‐alpha (TNF‐alpha), cathepsin B, p38 mitogen‐activated protein kinase (MAPK), and mitogen‐activated kinase kinase 3 (MKK3). Both root‐rupture injury and rhizotomy rapidly induced the aggregation of numerous macrophages from the injured dorsal root to the dorsal funiculus and TNF‐alpha was highly expressed by the macrophages in the injured dorsal root at 48 h. Activation of p38 MAPK was preferentially observed in the macrophages at the ruptured dorsal root; however, only slight activation of p38 MAPK was observed at the rhizotomized dorsal root. These findings suggest that traction injury of the spinal root might induce activation of the p38 MAPK cascade and production of TNF‐alpha in the infiltrating macrophages, both of which might participate in aggravation of the root injury.     

Effects of glial cell line-derived neurotrophic factor intrathecal injection on spinal dorsal horn glial fibrillary acidic protein expression in a rat model of neuropathic pain

Glial fibrillary acidic protein (GFAP) is a specific astrocytic marker in the central nervous system. Few studies on the effects of glial cell line-derived neurotrophic factor (GDNF) intrathecal injection on GFAP expression exist in the literature. The present study determined GFAP expression in rat spinal dorsal horn following a spinal nerve ligation (SNL). The effects of GDNF intrathecal injection on GFAP expression were examined to gather experimental evidence on the mechanisms underlying neuropathic pain. Following L5-6 SNL, male Sprague-Dawley rats were randomly divided into four groups: normal control, sham-operated, SNL, and GDNF. Each group was further divided into three subgroups (n = 10) according to the times of sacrifice: 3, 7, and 14 days after surgery. Compared with the normal control and the sham-operated groups, GFAP expression in the SNL group increased at day 3 after surgery and lasted until 14 days after. GFAP expression was significantly less in the GDNF group compared with the SNL group which lasted until 14 days after surgery, suggesting that rat spinal dorsal horn GFAP expression contributes to SNL-induced neuropathic pain. The mechanisms underlying GDNF alleviation of neuropathic pain were shown to be related to the GDNF inhibition of GFAP expression in the spinal dorsal horn.     

Galanin and its receptor system promote the repair of injured sciatic nerves in diabetic rats

Various studies have reported that galanin can promote axonal regeneration of dorsal root ganglion neuronsin vitro and inhibit neuropathic pain. However, little is known about its effects on diabetic peripheral neuropathy, andin vivo experimental data are lacking. We hypothesized that repeated applications of exogenous galanin over an extended time frame may also repair nerve damage in diabetic peripheral neuropathy, and relieve pain in vivo. We found that neuropathic pain occurred in streptozotocin-induced diabetic rats and was more severe after sciatic nerve pinch injury at 14 and 28 days than in diabetic sham-operated rats. Treatment with exogenous galanin alleviated the neuropathic pain and promoted sciatic nerve regeneration more effectively in diabetic rats than in non-diabetic rats after sciatic nerve pinch injury. This was accompanied by changes in the levels of endogenous galanin, and its receptors galanin receptor 1 and galanin receptor 2 in the dorsal root ganglia and the spinal dorsal horn when compared with nerve pinch normal rats. Our results show that application of exogenous galanin daily for 28 days can promote the regeneration of injured sciatic nerves, and alleviate neuropathic pain in diabetic rats.     

Regulation of neuronal and glial galectin-1 expression by peripheral and central axotomy of rat primary afferent neurons

Galectin-1 (Gal1) is an endogenously-expressed protein important for the embryonic development of the full complement of primary sensory neurons and their synaptic connections in the spinal cord. Gal1 also promotes axonal regeneration following peripheral nerve injury, but the regulation of Gal1 by axotomy in primary afferent neurons has not yet been examined. Here, we show by immunohistochemistry and in situ hybridization that Gal1 expression is differentially regulated by peripheral nerve injury and by dorsal rhizotomy. Following peripheral nerve injury, the proportion of Gal1-positive DRG neurons was increased. An increase in the proportion of large-diameter DRG neurons immunopositive for Gal1 was paralleled by an increase in the depth of immunoreactivity in the dorsal horn, where Gal1-positive terminals are normally restricted to laminae I and II. Dorsal rhizotomy did not affect the proportions of neurons containing Gal1 mRNA or protein, but did deplete the ipsilateral dorsal horn of Gal1 immunoreactivity, indicating that it is transported centrally by dorsal root axons. Dorsal rhizotomy also resulted in an increase in Gal1 mRNA the nerve peripheral to the PNS-CNS interface (likely within Schwann cells and/or macrophages), and to a lesser extent within deafferented spinal cord regions undergoing Wallerian degeneration. This latter increase was notable in the dorsal columns and along the prior trajectories of myelinated afferents into the deeper dorsal horn. These results show that neuronal and glial expressions of Gal1 are tightly correlated with regenerative success. Thus, the differential expression pattern of Gal1 following peripheral axotomy and dorsal rhizotomy suggests that endogenous Gal1 may be a factor important to the regenerative response of injured axons.     

Dynamic expression of Slit1–3 and Robo1–2 in the mouse peripheral nervous system after injury

The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors.Although we previously reported that Slit1–3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system,how this expression changes after injury has not been well studied.Herein,we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve.At 14 days after injury,quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1–3 and Robo1–2 in L4–5 spinal cord and dorsal root ganglia,as well as the sciatic nerve.Immunohistochemical analysis was performed to examine Slit1–3,Robo1–2,neurofilament heavy chain,F4/80,and vimentin in L4–5 spinal cord,L4 dorsal root ganglia,and the sciatic nerve.Co-expression of Slit1–3 and Robo1–2 in L4 dorsal root ganglia was detected by in situ hybridization.In addition,Slit1–3 and Robo1–2 protein expression in L4–5 spinal cord,L4 dorsal root ganglia,and sciatic nerve were detected by western blot assay.The results showed no significant changes of Slit1–3 or Robo1–2 mRNA expression in the spinal cord within 14 days after injury.In the dorsal root ganglion,Slit1–3 and Robo1–2 mRNA expression were initially downregulated within 4 days after injury;however,Robo1–2 mRNA expression returned to the control level,while Slit1–3 mRNA expression remained upregulated during regeneration from 4–14 days after injury.In the sciatic nerve,Slit1–3 and their receptors Robo1–2 were all expressed in the proximal nerve stump;however,Slit1,Slit2,and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury.Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury.Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge.Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury.Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration,and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury.This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No.30/3203) on April 12,2014.     

Sciatic nerve injury in adult rats causes distinct changes in the central projections of sensory neurons expressing different glial cell line‐derived neurotrophic factor family receptors

Most small unmyelinated neurons in adult rat dorsal root ganglia (DRG) express one or more of the coreceptors targeted by glial cell line‐derived neurotrophic factor (GDNF), neurturin, and artemin (GFRα1, GFRα2, and GFRα3, respectively). The function of these GDNF family ligands (GFLs) is not fully elucidated but recent evidence suggests GFLs could function in sensory neuron regeneration after nerve injury and peripheral nociceptor sensitization. In this study we used immunohistochemistry to determine if the DRG neurons targeted by each GFL change after sciatic nerve injury. We compared complete sciatic nerve transection and the chronic constriction model and found that the pattern of changes incurred by each injury was broadly similar. In lumbar spinal cord there was a widespread increase in neuronal GFRα1 immunoreactivity (IR) in the L1‐6 dorsal horn. GFRα3‐IR also increased but in a more restricted area. In contrast, GFRα2‐IR decreased in patches of superficial dorsal horn and this loss was more extensive after transection injury. No change in calcitonin gene‐related peptide‐IR was detected after either injury. Analysis of double‐immunolabeled L5 DRG sections suggested the main effect of injury on GFRα1‐ and GFRα3‐IR was to increase expression in both myelinated and unmyelinated neurons. In contrast, no change in basal expression of GFRα2‐IR was detected in DRG by analysis of fluorescence intensity and there was a small but significant reduction in GFRα2‐IR neurons. Our results suggest that the DRG neuronal populations targeted by GDNF, neurturin, or artemin and the effect of exogenous GFLs could change significantly after a peripheral nerve injury. J. Comp. Neurol. 518:3024–3045, 2010. © 2010 Wiley‐Liss, Inc.     

Astrocytic reactions in spinal gray matter following sciatic axotomy

Astrocytic responses following unilateral sciatic nerve axotomy were examined in the spinal gray matter. Using an antiserum to glial fibrillary acidic protein (GFAP), immunoreactive astrocytes were studied in both dorsal and ventral gray matter at intervals from 2 days through 34 days post-axotomy. In all axotomized animals, increased numbers of strongly immunoreactive astrocytes were present in the gray matter ipsilateral to the surgery. Such astrocytes were absent from the contralateral intact side and from gray matter bilaterally in adjacent spinal segments not involved in formation of the sciatic nerve. These GFAP-positive astrocytes occurred not only in association with large motor neurons in the ventral gray matter but also in association with central processes of dorsal root ganglion neurons in the dorsal gray matter. The response was quite rapid, being discernible both dorsally and ventrally as early as the second post-operative day. This increased GFAP immunoreactivity persisted throughout the entire observation period, with the perikarya of large ventral motor neurons appearing to become surrounded or encapsulated by the immunoreactive processes. A further alteration noted at the longest post-operative intervals was the presence in the ventral gray matter of astrocytes appearing to be binucleate. The data obtained indicate that the astrocytic response is not related solely to reactions in motor neurons and, furthermore, the rapidity with which it develops in the dorsal gray matter suggests that its induction is not dependent upon transganglionic degeneration, which others have reported to occur weeks after peripheral nerve injury.     

Substance P: Depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons

The substance P content, glutamic acid decarboxylase and choline acetyltransferase activities and the level of [3H]diprenorphine binding were measured in various regions of the lumbar spinal cord of rats after unilateral section of the sciatic nerve or after dorsal rhizotomy. Sciatic nerve section produced a 75--80% depletion of substance P in the dorsal horn but did not change the substance P content of the ventral horn. The onset of substance P depletion occurred within 7 days and was maintained for 2 months. The substance P content of the dorsal root ganglia and both the peripheral and central branches of primary sensory neurons was also reduced after sciatic nerve section. Glutamic acid decarboxylase and choline acetyltransferase activity were unchanged; however, a small decrease in opiate receptor binding occurred 1 month after nerve section. Dorsal rhizotomy produced an 80% depletion of substance P in the dorsal horn. In addition, the substance P content of the ventral horn was significantly reduced. Glutamic acid decarboxylase activity in the dorsal horn was unaffected by dorsal rhizotomy whereas opiate receptor binding was reduced by 40%. From these studies it appears that peripheral nerve injury results in the degeneration of primary sensory neurons which contain and release substance P as neurotransmitter.     

Correlation between putative inhibitory molecules at the dorsal root entry zone and failure of dorsal root axonal regeneration

The molecular mechanisms involved in preventing regenerating dorsal root axons from entering the spinal cord at the dorsal root entry zone (DREZ) are obscure. We used immunohistochemistry, in situ hybridization, and electron microscopy to study axonal regeneration after dorsal rhizotomy in adult rats and its relationship to cellular changes and the distribution of putative growth inhibitory molecules in this region. Astrocyte processes, ending as bulb-shaped expansions, grew up to 700 microm into the basal lamina tubes of injured roots, where regenerating axons were also present. Some of these axons approached or reached the DREZ but grew no further; others turned back toward the ganglion, suggesting the presence of repulsive cues in or near the DREZ. Tenascin-C mRNA and protein and CSPG stub immunoreactivity were strongly upregulated in the roots after rhizotomy, but were only weakly expressed in the DREZ. Tenascin-R immunoreactivity was confined to CNS tissue, and unaffected by rhizotomy. Large, rounded GFAP-negative, NG2-immunoreactive cells, a few of which were OX42 positive, were found in the DREZ following rhizotomy. Astrocyte processes projecting into the roots were tenascin-R and NG2 negative. Hence, only NG2-expressing cells and tenascin-R were appropriately situated to inhibit regeneration through the DREZ.     

The astrocytic barrier to axonal regeneration at the dorsal root entry zone is induced by rhizotomy

After dorsal rhizotomy, sensory axons fail to regenerate beyond the astrocytic glia limitans at the dorsal root entry zone (DREZ) but this inhibition can be overcome with the delivery of exogenous neurotrophin-3. We investigated whether axonal inhibition at the DREZ is constitutive or induced after dorsal rhizotomy. Primary afferent neurones from enhanced green fluorescent protein-expressing mice were transplanted into adult rat dorsal root ganglia in the presence or absence of dorsal rhizotomy. In the absence of dorsal rhizotomy mouse axons freely extended into the rat central nervous system. After host dorsal rhizotomy, mouse axons were unable to cross the DREZ. However, in rats that received a dorsal rhizotomy concomitant with intrathecal neurotrophin-3, the mouse axons were able to cross the DREZ. These results indicate that, under normal circumstances, the adult DREZ is permissive to the regeneration of adult sensory axons and that it only becomes inhibitory once dorsal root axons have been injured and astrocytes at the DREZ have become reactive.     

miR-30c promotes Schwann cell remyelination following peripheral nerve injury

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination fol-lowing peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.     

A comparative histological analysis of two models of nerve root avulsion injury in the adult rat

D. J. Chew, T. Carlstedt and P. J Shortland (2011) Neuropathology and Applied Neurobiology 37, 613–632 A comparative histological analysis of two models of nerve root avulsion injury in the adult rat Aims: This study has investigated the reliability of the artificial surgical model dorsal root rhizotomy (DRR), to the surgical tearing of the roots, avulsion, that occurs clinically. Root avulsion of the limb nerves is common in high‐impact motor vehicle accidents and results in paraesthesia, paralysis and intractable pain. Limited treatment options are largely due to a lack of basic research on underlying mechanisms, and few animal models. We assess this limitation by histologically assessing the spatial and temporal injury profile of dorsal root avulsion (DRA) and DRR within the spinal cord. Methods: Rats underwent DRR, DRA or sham surgery to the L3–L6 dorsal roots unilaterally. At 1, 2, 14, and 28 days post injury, immunohistochemical density staining was used to characterize the progression of spinal cord trauma. Neuronal (NeuN) and vascular degeneration (RECA‐1), inflammatory infiltrate (ED1, anti‐neutrophil), gliosis (Iba1, GFAP) and apoptosis (TUNEL) were assessed. Results: Unilateral DRA produced a prolonged and bilateral glial and inflammatory response, and vascular degeneration compared to transient and unilateral effects after DRR. Transsynaptic neurodegeneration after DRA was greater than after DRR, and progressed across 28 days coinciding with gliosis and macrophage infiltration. Conclusions: Rhizotomy leads to a milder representation of the spinal cord trauma that occurs after ‘true’ avulsion injury. We recommend DRA be used in the future to more reliably model clinical avulsion injury. Avulsion is an injury with a chronic profile of degenerative and inflammatory progression, and this theoretically provides a window of clinical therapeutic opportunity in treatment of secondary trauma progression.     

脂膜微囊趋向于鼠脑创伤区聚集机制的实验研究

目的研究脂膜微囊向脑损伤位点靶向性聚集的特点，探讨其向鼠脑损伤区靶向性聚集的机制。方法建立脑损伤动物实验模型，制备脂膜微囊悬液。将脑损伤模型建立成功的28只大鼠随机分为以下几组，损伤当日组、伤后24h组、48h组、72h组、7d组、10d组、14d组、21d组、28d组；分别于损伤后不同时间，由尾静脉注射脂膜微囊悬液，鼠脑标本用油红O染色，研究脂膜微囊在脑损伤后的不同时间在损伤灶周围聚集的特点，并采用双重免疫组化染色，研究增生细胞核抗原（PCNA）及胶原纤维酸性蛋白（GFAP）阳性细胞在损伤灶周围分布的特点，讨论其与脂膜微囊靶向性聚集的关系。结果早期即可在损伤灶周围及损伤灶中发现脂膜微囊，且脂膜微囊密度逐渐增加，48h后可在损伤区的周围发现有微囊以丛集方式聚集；在病损10d左右，微囊的密度最大并聚集成环状。损伤后的第2—3周微囊密度下降至一稳定的水平；GFAP，PCNA双重染色发现了它们各自的密度变化曲线，均在损伤后48h达高峰；GFAP阳性细胞的数量及分布范围远较PCNA范围大，且二者均阳性的细胞数量很少。结论脂膜微囊可靶向性聚集于脑损伤位点周围，不同的时间点微囊的密度不同，损伤后第10天其密度达高峰，脂膜微囊靶向性聚集的机制复杂，与血脑屏障破坏引起的血源性细胞渗出有关，也与反应性星形细胞有关。     

Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo

In contrast to injuries in the central nervous system, injured peripheral neurons will regenerate their axons. However, axotomized motoneurons progressively lose their ability to regenerate their axons, following peripheral nerve injury often resulting in very poor recovery of motor function. A decline in neurotrophic support may be partially responsible for this effect. The initial upregulation of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) by Schwann cells of the distal nerve stump after nerve injury has led to the speculation that they are important for motor axonal regeneration. However, few experiments directly measure the effects of exogenous BDNF or GDNF on motor axonal regeneration. This study provided the first direct and quantitative evidence that long-term continuous treatment with exogenous GDNF significantly increased the number of motoneurons which regenerate their axons, completely reversing the negative effects of chronic axotomy. The beneficial effect of GDNF was not dose-dependent. A combination of exogenous GDNF and BDNF on motor axonal regeneration was significantly greater than either factor alone, and this effect was most pronounced following long-term continuous treatment. The ability of GDNF, either alone or in combination with BDNF, to increase the number of motoneurons that regenerated their axons correlated well with an increase in axon sprouting within the distal nerve stump. Thus long-term continuous treatment with neurotrophic factors, such as GDNF and BDNF, can be used as a viable treatment to sustain motor axon regeneration.     

脑缺血诱发星形胶质细胞粒细胞集落刺激因子受体和胶质细胞源性神经营养因子的表达

目的 探讨粒细胞集落刺激因子(G-CSF)在缺血脑保护中的作用机制.方法 制作大鼠大脑中动脉栓塞模型,7 d后断头取脑做冰冻切片,用免疫荧光双标的方法观察缺血周边区与假手术组相同部位G-CSF受体、胶质细胞源性神经营养因子(GDNF)和微管相关蛋白2(MAP2)、神经胶质原酸性蛋白(GFAP)的共表达情况.结果 G-CSF受体和GDNF在正常大鼠脑内广泛表达于神经元,不表达于星形胶质细胞;但在缺血周边区,星形胶质细胞亦部分表达G-CSF受体和GDNF.在正常脑组织,大部分G-CSF受体阳性的细胞也表达GDNF.结论 脑缺血可诱导缺血周边区星形胶质细胞表达G-CSF受体和GDNF,推测缺血后的内源性神经保护作用可能与缺血周边区星形胶质细胞的G-CSF受体表达以及GDNF产生有关.     

Cooperative regulation of calcitonin gene-related peptide levels in rat sensory neurons via their central and peripheral processes.

Calcitonin gene-related peptide (CGRP) is present in both motor and sensory neurons and transported in the somatofugal direction. CGRP levels in sensory neurons are assumed to be regulated by NGF supplied from their peripheral targets. In cultured sensory neurons, however, a basal level of CGRP persists even without NGF. This suggests that some additional factors may be involved in regulation of CGRP levels of sensory neurons. The present study shows that chronic section of the sciatic nerve in the rat reduces CGRP levels in the lumbar dorsal root ganglia (DRG), whereas section of dorsal roots increases CGRP levels in the DRG. This increased CGRP level by dorsal rhizotomy was associated with enhancement of the CGRP mRNA expression in the DRG. Thus, CGRP expression in DRG appears to be regulated reciprocally via their central and peripheral processes. When the sciatic nerve had been cut 1 week previously, however, dorsal rhizotomy no longer increased CGRP levels in the lumbar DRG. Therefore, stimulation of CGRP synthesis in the DRG by dorsal rhizotomy may require the integrity of the peripheral processes. When NGF had been infused into the central stump of the cut sciatic nerve, dorsal rhizotomy again increased CGRP levels in the DRG, despite prior section of the peripheral processes. We conclude that CGRP expression in sensory neurons may be regulated by cooperative action of some factors derived via their central processes and NGF supplied from the peripheral targets.