Adrenergic innervation of rat sensory ganglia following proximal or distal painful sciatic neuropathy: distinct mechanisms revealed by anti-NGF treatment

Sympathetic axons invade dorsal root ganglia (DRG) following nerve injury, and activity in the resulting pericellular axonal 'baskets' may underlie painful sympathetic-sensory coupling. Sympathetic sprouting into the DRG may be stimulated by nerve growth factor (NGF). To test this hypothesis, we investigated the effect of daily anti-NGF administration on pain and on sprouting in the DRG induced by chronic sciatic constriction injury (CCI) or L5 spinal nerve ligation (SNL). These models have been shown to differ subtly in the onset of pain behaviours and adrenergic sprouting, and we now demonstrate a fundamental difference in the way sympathetic axons invade the DRG: after CCI, perivascular noradrenergic collaterals sprouted into the DRG in a manner dependent upon peripherally derived NGF. In contrast, after SNL, regenerating sympathetic axons were diverted towards the DRG from the spinal nerve by the obstructing ligature, and this effect was only moderately impeded by anti-NGF. The differential dependence on anti-NGF suggests that adrenergic innervation of the DRG after SNL and CCI may reflect regenerative and collateral sprouting, respectively. Pain behaviour was similarly affected: anti-NGF completely prevented CCI-induced thermal hyperalgesia and mechanoallodynia, but the same treatment only partly relieved these symptoms following SNL. These differences emphasize that although CCI and SNL may result in similar behavioural abnormalities, the underlying mechanisms may be governed by distinct processes, differentially dependent on peripheral NGF. These mechanistic differences will have to be considered in the development of appropriate treatment strategies for neuropathic pain produced by different types of pathology.     

Effect of lumbar 5 ventral root transection on pain behaviors: a novel rat model for neuropathic pain without axotomy of primary sensory neurons

A peripheral nerve injury often causes neuropathic pain but the underlying mechanisms remain obscure. Several established animal models of peripheral neuropathic pain have greatly advanced our understanding of the diverse mechanisms of neuropathic pain. A common feature of these models is primary sensory neuron injury and the commingle of intact axons with degenerating axons in the sciatic nerve. Here we investigated whether neuropathic pain could be induced without sensory neuron injury following exposure of their peripheral axons to the milieu of Wallerian degeneration. We developed a unilateral lumbar 5 ventral root transection (L5 VRT) model in adult rats, in which L5 ventral root fibers entering the sciatic nerve were sectioned in the spinal canal. This model differs from previous ones in that DRG neurons and their afferents are kept uninjured and intact afferents expose to products of degenerating efferent ventral root fibers in the sciatic nerve and the denervated muscles. We found that the L5 VRT produced rapid (24 h after transection), robust and prolonged (56 days) bilateral mechanical allodynia, to a similar extent to that in rats with L5 spinal nerve transection (L5 SNT), cold allodynia and short-term thermal hyperalgesia (14 days). Furthermore, L5 VRT led to significant inflammation as demonstrated by infiltration of ED-1-positive monocytes/macrophages in the DRG, sciatic nerve and muscle fibers. These findings demonstrated that L5 VRT produced behavioral signs of neuropathic pain with high mechanical sensitivity and thermal responsiveness, and suggested that neuropathic pain can be induced without damage to sensory neurons. We propose that neuropathic pain in this model may be mediated by primed intact sensory neurons, which run through the milieu of Wallerian degeneration and inflammation after nerve injury. The L5 VRT model manifests the complex regional pain syndrome in some human patients, and it may provide an additional dimension to dissect out the mechanisms underlying neuropathic pain.     

Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury

Complete sciatic nerve injury reduces substance P (SP) expression in primary sensory neurons of the L4 and L5 dorsal root ganglia (DRG), due to loss of target-derived nerve growth factor (NGF). Partial nerve injury spares a proportion of DRG neurons, whose axons lie in the partially degenerating nerve, and are exposed to elevated NGF levels from Schwann and other endoneurial cells involved in Wallerian degeneration. To test the hypothesis that SP is elevated in spared DRG neurons following partial nerve injury, we compared the effects of complete sciatic nerve transection (CSNT) with those of two types of partial injury, partial sciatic nerve transection (PSNT) and chronic constriction injury (CCI). As expected, a CSNT profoundly decreased SP expression at 4 and 14 days postinjury, but after PSNT and CCI the levels of preprotachykinin (PPT) mRNA, assessed by in situ hybridization, and the SP immunoreactivity (SP-IR) of the L4 and L5 DRGs did not decrease, nor did dorsal horn SP-IR decrease. Using retrograde labelling with fluorogold to identify spared DRG neurons, we found that the proportion of these neurons expressing SP-IR 14 days after injury was much higher than in neurons of normal DRGs. Further, the highest levels of SP-IR in individual neurons were detected in ipsilateral L4 and L5 DRG neurons after PSNT and CCI. We conclude that partial sciatic nerve injury elevates SP levels in spared DRG neurons. This phenomenon might be involved in the development of neuropathic pain, which commonly follows partial nerve injury.     

Intrathecal miR‐183 delivery suppresses mechanical allodynia in mononeuropathic rats

Members of the miR‐183 family are unique in that they are highly abundant in sensory organs. In a recent study, significant downregulation was observed for miR‐96 and miR‐183 in the L5 dorsal root ganglion (DRG) 2 weeks after spinal nerve ligation (SNL). In this study, we focused on miR‐183, which is the most regulated member of the miR‐183 family, to look at the specific role on neuropathic pain. Persistent mechanical allodynia was induced with the L5 SNL model in 8‐week‐old male Sprague‐Dawley rats. Paw withdrawal thresholds in response to mechanical stimuli were assessed with Von Frey filaments. Expression of miR‐183 in the L5 DRG was assessed with quantitative real‐time polymerase chain reaction (qPCR) analysis. Lentivirions expressing miR‐183 were injected intrathecally into SNL rats. Changes in mechanical allodynia were assessed with Von Frey filaments. In addition, changes in the predicted target genes of miR‐183 were assessed with qPCR. L5 SNL produced marked mechanical allodynia in the ipsilateral hindpaws of adult rats, beginning at postoperative day 1 and continuing to day 14. L5 SNL caused significant downregulation of miR‐183 in adult DRG cells. Intrathecal administration of lentivirions expressing miR‐183 downregulated SNL‐induced increases in the expression of Nav1.3 and brain‐derived neurotrophic factor (BDNF), which correlated with the significant attenuation of SNL‐induced mechanical allodynia. Our results show that SNL‐induced mechanical allodynia is significantly correlated with the decreased expression of miR‐183 in DRG cells. Replacement of miR‐183 downregulates SNL‐induced increases in Nav1.3 and BDNF expression, and attenuates SNL‐induced mechanical allodynia.     

Sympathetic Fiber Sprouting in Chronically Compressed Dorsal Root Ganglia Without Peripheral Axotomy

Sympathetic axonal sprouting in axotomized dorsal root ganglia (DRG) has been shown to be a major phenomenon implicated in neuropathic pain. However, it is not known whether sympathetic sprouting can occur in pathologic ganglia without peripheral axotomy. We thus examined presence and density of sympathetic axonal sprouting within DRG of rats subjected to a persistent compressive injury by inserting a stainless steel metal rod into L(4) and L(5) lumbar intervertebral foramen. Sympathetic axons were identified by immunohistochemical staining with anti-tyrosine hydroxylase antibodies. Results indicate that progressive increase in sympathetic axonal sprouting occurred in the bilateral DRGs between postoperative days 2 and 28. The sympathetic fiber density was greater on the lesion side than the contralateral side. In conclusion, chronic compressive injury of the DRG results in sympathetic sprouting in the non-axotomized ganglion and may partially contribute to the development and maintenance of certain pathological pain states.     

Wallerian degeneration in the optic nerve of the wabbler-lethal (wl/wl) mouse.

Optic nerve pathology was studied in C57BL/6J wabbler-lethal (wl/wl) and control (+/+) mice at postnatal age of 4 weeks (P28). Qualitative light and ultrastructural pathology in wl/wl animals conformed to the criteria of primary axonal (Wallerian) degeneration. Most optic nerve axons in mutant animals appeared normal, as did oligodendroglia, the degree of myelination, the integrity and maturity of vascular elements, astroglia, and most myelin. Still, degenerating axons surrounded by somewhat normal myelin and axons with thickened myelin sheaths were prevalent in wl/wl mice. Dysmyelination or hypomyelination was not evident. At P28, pathology appeared more prominent in large diameter fibers. In the optic nerve of wl/wl mice, axonal degeneration preceded myelin disruption, adding this nerve to other previously reported systems undergoing Wallerian degeneration in this mutant.     

The effects of pyronin on sprouting and regeneration of mouse motor nerves

Administration to mice of a 0.1% solution of pyronin G in their drinking water caused an acceleration both of axonal sprouting from nodes of Ranvier in partly denervated gluteus maximus muscles, and of motor nerve regeneration following a crush to the soleus nerve. Sprouting from soleus motor nerve terminals in response to botulinum toxin-induced paralysis was, however, unaffected. Removal of degenerating axons following nerve section was also accelerated by pyronin treatment. Pyronin is therefore likely to act upon the process of Wallerian degeneration, rather than upon intact motor nerves directly.     

Very Slow Retrograde and Wallerian Degeneration in the CNS of C57BL/Ola Mice

Wallerian degeneration following peripheral nerve transection in C57BL/Ola mice is very slow in comparison to other strains of mice. We show that following optic nerve transection, the axons of retinal ganglion cells in C57BL/Ola mice undergo very slow Wallerian degeneration and that retrograde degeneration of the ganglion cell bodies is much slower than in other strains of mice. The results suggest that the gene product affecting Wallerian degeneration in the peripheral nervous system (PNS) also confers a greater resistance to degeneration on central nervous system (CNS) neurons.     

CXCL10/CXCR3 Signaling in the DRG Exacerbates Neuropathic Pain in Mice

Chemokines and receptors have been implicated in the pathogenesis of chronic pain.Here,we report that spinal nerve ligation(SNL)increased CXCR3 expression in dorsal root ganglion(DRG)neurons,and intra-DRG injection of Cxcr3 shRNA attenuated the SNL-induced mechanical allodynia and heat hyperalgesia.SNL also increased the m RNA levels of CXCL9,CXCL10,and CXCL11,whereas only CXCL10 increased the number of action potentials(APs)in DRG neurons.Furthermore,in Cxcr3^-/-mice,CXCL10 did not increase the number of APs,and the SNL-induced increase of the numbers of APs in DRG neurons was reduced.Finally,CXCL10 induced the activation of p38 and ERK in ND7-23 neuronal cells and DRG neurons.Pretreatment of DRG neurons with the P38 inhibitor SB203580 decreased the number of APs induced by CXCL10.Our data indicate that CXCR3,activated by CXCL10,mediates p38 and ERK activation in DRG neurons and enhances neuronal excitability,which contributes to the maintenance of neuropathic pain.     

Sympathetic sprouting in the dorsal root ganglion after spinal nerve ligation: evidence of regenerative collateral sprouting

It is well documented that there is an increase in the number of sympathetic fibers within the dorsal root ganglion (DRG) after a peripheral nerve injury. The present study examined the numbers and distribution of sympathetic fibers in the DRG and their sprouting routes by utilizing various surgical manipulations and retrograde tracing and immunohistochemical staining methods in spinal nerve-ligated neuropathic rats. The appearance of many double immunostained fibers with antibodies to tyrosine hydroxylase (TH) and growth associated protein-43 (GAP-43) in the L5 DRG 1 week after L5 spinal nerve ligation, indicated sprouting of sympathetic fibers. The confined location of early sprouting sympathetic fibers in the distal half of the L5 DRG confirmed that sprouting fibers come primarily from the injured spinal nerve. A second cut proximal to the previously ligated L5 spinal nerve -- a process which would transect the regenerating sympathetic fibers extending from the injury site -- did not change the density of sympathetic fibers in the L5 DRG. When retrograde tracers (fast blue and diamidino yellow) were injected into the L5 spinal nerve and DRG, respectively, the number of double-labeled sympathetic postganglionic neurons was greatly increased after spinal nerve ligation, suggesting the increased number of sympathetic neurons projecting to both the spinal nerve and DRG. All these results indicate that many sympathetic fibers in the DRG are regenerating branches that are sprouting from the proximal part of the injured spinal nerve (regenerative collateral sprouting).