A shed form of LDL receptor-related protein-1 regulates peripheral nerve injury and neuropathic pain in rodents.

Injury to the peripheral nervous system (PNS) initiates a response controlled by multiple extracellular mediators, many of which contribute to the development of neuropathic pain. Schwann cells in an injured nerve demonstrate increased expression of LDL receptor-related protein-1 (LRP1), an endocytic receptor for diverse ligands and a cell survival factor. Here we report that a fragment of LRP1, in which a soluble or shed form of LRP1 with an intact alpha-chain (sLRP-alpha), was shed by Schwann cells in vitro and in the PNS after injury. Injection of purified sLRP-alpha into mouse sciatic nerves prior to chronic constriction injury (CCI) inhibited p38 MAPK activation (P-p38) and decreased expression of TNF-alpha and IL-1beta locally. sLRP-alpha also inhibited CCI-induced spontaneous neuropathic pain and decreased inflammatory cytokine expression in the spinal dorsal horn, where neuropathic pain processing occurs. In cultures of Schwann cells, astrocytes, and microglia, sLRP-alpha inhibited TNF-alpha-induced activation of p38 MAPK and ERK/MAPK. The activity of sLRP-alpha did not involve TNF-alpha binding, but rather glial cell preconditioning, so that the subsequent response to TNF-alpha was inhibited. Our results show that sLRP-alpha is biologically active and may attenuate neuropathic pain. In the PNS, the function of LRP1 may reflect the integrated activities of the membrane-anchored and shed forms of LRP1.

The ology of neuropathy: an integrative review of the role of neuroinflammation and TNF-α axonal transport in neuropathic pain.

This 2011 Peripheral Nerve Society plenary lecture reviews the role of axonal transport in neuroimmune communication following peripheral nerve injury, linking focal changes in Schwann cell activation and release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) with subsequent activation and sensitization of ascending sensory neurons and glia which culminate in the neuropathic pain state. New data demonstrate that axonally transported (biotinylated) TNF-α activates and localizes with dorsal horn astrocytes within 96 h after injection into sciatic nerve, and that glial fibrillary acidic protein (GFAP) activation in these glial cells is diminished in TNF receptor 1 knockout mice. The pathophysiology, neuropathology and molecular biology of Wallerian degeneration are also reviewed from a perspective that links it to upregulation of proinflammatory cytokines and the development of neuropathic pain states. Finally, insights into neuroimmune communication provide rationale for new therapy based on interference with the processes of Wallerian degeneration, cytokine signaling and TNF-α protein sequestration.

Immediate anti-tumor necrosis factor-alpha (etanercept) therapy enhances axonal regeneration after sciatic nerve crush.

Peripheral nerve regeneration begins immediately after injury. Understanding the mechanisms by which early modulators of axonal degeneration regulate neurite outgrowth may affect the development of new strategies to promote nerve repair. Tumor necrosis factor-alpha (TNF-alpha) plays a crucial role in the initiation of degenerative cascades after peripheral nerve injury. Here we demonstrate using real-time Taqman quantitative RT-PCR that, during the time course (days 1-60) of sciatic nerve crush, TNF-alpha mRNA expression is induced at 1 day and returned to baseline at 5 days after injury in nerve and the corresponding dorsal root ganglia (DRG). Immediate therapy with the TNF-alpha antagonist etanercept (fusion protein of TNFRII and human IgG), administered systemically (i.p.) and locally (epineurially) after nerve crush injury, enhanced the rate of axonal regeneration, as determined by nerve pinch test and increased number of characteristic clusters of regenerating nerve fibers distal to nerve crush segments. These fibers were immunoreactive for growth associated protein-43 (GAP-43) and etanercept, detected by anti-human IgG immunofluorescence. Increased GAP-43 expression was found in the injured nerve and in the corresponding DRG and ventral spinal cord after systemic etanercept compared with vehicle treatments. This study established that immediate therapy with TNF-alpha antagonist supports axonal regeneration after peripheral nerve injury.

MMPs initiate Schwann cell-mediated MBP degradation and mechanical nociception after nerve damage.

Matrix metalloproteinases (MMPs) emerge as modulators of neuropathic pain. Because myelin protects Abeta afferents from ectopic hyperexcitability and nociception from innocuous mechanical stimuli (or mechanical allodynia), we analyzed the role of MMPs in the development of mechanical allodynia through myelin protein degradation after rat and MMP-9-/- mouse L5 spinal nerve crush (L5 SNC). MMPs were shown to promote selective degradation of myelin basic protein (MBP), with MMP-9 regulating initial Schwann cell-mediated MBP processing after L5 SNC. Acute and long-term therapy with GM6001 (broad-spectrum MMP inhibitor) protected from injury-induced MBP degradation, caspase-mediated apoptosis, macrophage infiltration in the spinal nerve and inhibited astrocyte activation in the spinal cord. The effect of GM6001 therapy on attenuation of mechanical allodynia was robust, immediate and sustained through the course of L5 SNC. In conclusion, MMPs mediate the initiation and maintenance of mechanical nociception through Schwann cell-mediated MBP processing and support of neuroinflammation.

The role of neuroinflammation in neuropathic pain: mechanisms and therapeutic targets.

Neuroinflammation is a proinflammatory cytokine-mediated process that can be provoked by systemic tissue injury but it is most often associated with direct injury to the nervous system. It involves neural-immune interactions that activate immune cells, glial cells and neurons and can lead to the debilitating pain state known as neuropathic pain. It occurs most commonly with injury to peripheral nerves and involves axonal injury with Wallerian degeneration mediated by hematogenous macrophages. Therapy is problematic but new trials with anti-cytokine agents, cytokine receptor antibodies, cytokine-signaling inhibitors, and glial and neuron stabilizers provide hope for future success in treating neuropathic pain.

Exacerbated synucleinopathy in mice expressing A53T SNCA on a Snca null background.

Alpha-Synuclein is a major component of Lewy bodies, neuronal inclusions diagnostic for Parkinson's disease (PD). While an Ala53Thr mutation in alpha-synuclein can cause PD in humans, in mice the wildtype residue at position 53 is threonine, indicating that mice are either too short-lived to develop PD, or are protected by the six other amino acid differences between the proteins in these two species. Mice carrying an Ala53Thr human SNCA transgene driven by the mouse prion promoter show a mild movement disorder and only rarely develop severe pathology by 2 years of age. To determine whether the presence of mouse alpha-synuclein affects the pathogenicity of the human protein, the transgene was crossed into mice lacking endogenous alpha-synuclein. Mice that express only human alpha-synuclein developed a neuronopathy characterized by limb weakness and paralysis with onset beginning at 16 months of age. The neuronopathy is probably due to high levels of expression of the transgene in the ventral spinal cord leading to motor neuron damage and Wallerian degeneration of the ventral roots. These data suggest mouse alpha-synuclein is protective against the deleterious effects of the human mutant protein.

Effect of acute nerve root compression on endoneurial fluid pressure and blood flow in rat dorsal root ganglia.

The objective of the current study was to test the hypothesis that crush injury to nerve root increases endoneurial fluid pressure (EFP) and decreases blood flow in the associated dorsal root ganglion (DRG). A total of 21 adult, female Sprague-Dawley rats had their left L5 nerve root and DRG exposed. The L5 nerve root was clamped for 2 s with a vascular suture clip just proximal to the DRG (compression group). Sham-operated animals without compression were used for control (control group). EFP was recorded with a servo-null micropipette system using a glass micropipette with tip diameter of 4 mum before and after 3 h of treatment. After the final measurement of EFP, DRG was excised and processed for histology. Blood flow in the DRG was continuously monitored by laser Doppler flow meter for 3 h. Three hours after treatment, EFP was 4.7+/-2.7 cm H(2)O in the compression group and 2.2+/-1.2 cm H(2)O in the control group (P<0.05). Edema was the principal pathologic findings seen consistently in the DRG from animals in the compression group. Blood flow in the compression group was reduced 10 min after compression. This reduction was statistically significant compared with that of the control (P<0.01). An acute compression to the nerve root increased endoneurial edema, increased EFP in the associated DRG, and reduced DRG blood flow. This combination of increased EFP and decreased blood flow in the DRG may result in neuronal ischemia and sensory dysfunction. These acute pathophysiologic changes may thus have a role in the pathogenesis of low back pain and sciatica due to disc herniation and spinal canal stenosis.

Differential block of N-propyl derivatives of amitriptyline and doxepin for sciatic nerve block in rats.

BACKGROUND AND OBJECTIVES: The propyl group of ropivacaine ( N -propyl-2',6'-pipecoloxylidide hydrochloride) could be responsible for conferring some sensory selectivity to this drug. Thus, adding a propyl group to experimental local anesthetics (LAs) (e.g., the tricyclic antidepressants amitriptyline and doxepin) to increase sensory selectivity may be useful. We, therefore, synthesized N -propyl amitriptyline and N -propyl doxepin and investigated a potential predominance of sensory/nociceptive block over motor block (differential block) in a rat sciatic nerve block model. In addition, tetrodotoxin (TTX), a naturally occuring Na + channel blocker, was coinjected to investigate whether it increased block duration. METHODS: A 0.2-mL test dose of N -propyl amitriptyline and N -propyl doxepin, at a concentration of 1, 2.5, 5, and 10 mM, (alone or in combination with TTX at a concentration of 20 microM) was injected by the subfascial sciatic nerve approach. Motor function and sensory function (nociception) were evaluated by the force a rat's hind limb produced when pushing against a balance and the reaction to pinch, respectively. RESULTS: N -propyl amitriptyline and N -propyl doxepin demonstrated prolonged block duration, with N -propyl amitriptyline displaying significant differential block at higher concentrations (5 and 10 mM). The combination of either of these drugs with TTX increased the potency as well as the efficacy. Neurotoxicity commenced at concentrations of 5 to 10 mM. CONCLUSIONS: Detailed histopathologic nerve toxicity evaluations are justified to determine whether N -propyl amitriptyline has potential as a more sensory-selective local anesthetic at lower concentrations or as a predominantly sensory-selective neurolytic agent at higher concentrations.

Pathogenesis of sciatic pain: role of herniated nucleus pulposus and deformation of spinal nerve root and dorsal root ganglion.

The basic pathophysiologic mechanisms related to disc herniation and sciatica are poorly understood. Recently it was demonstrated that nucleus pulposus from an intervertebral disc could induce structural and functional changes in adjacent nerve roots when applied epidurally, however, it is not known if such changes are painful. In a model for inducing disc herniation in the rat, we found that puncture of a lumbar disc with subsequent herniation of nucleus pulposus without nerve root compression, or chronic displacement of the 4th lumbar nerve root and ganglion, did not individually induce significant changes in thresholds for mechanical or thermal stimulation compared to sham-operated animals. However, the combination of disc puncture and displacement induced a reduction of the threshold for thermal stimulation, indicating hyperalgesia, that was present 2 days after surgery and gradually recovered during a 14-day period. These data and the associated description of this new model for experimental disc herniation may increase our understanding of the pathophysiologic events leading to sciatica and help in evaluating new modalities for diagnosis and treatment of disc herniation and sciatica.

Anti-inflammatory interleukin-10 therapy in CCI neuropathy decreases thermal hyperalgesia, macrophage recruitment, and endoneurial TNF-alpha expression.

The chronic constriction injury model of mononeuropathy is a direct, partial nerve injury yielding thermal hyperalgesia. The inflammation that results from this injury is believed to contribute importantly to both the neuropathological and behavioral sequelae. This study involved administering a single dose (250 ng) of interleukin-10 (IL-10), an endogenous anti-inflammatory peptide, at the site and time of a chronic constriction injury (CCI) lesion to determine if IL-10 administration could attenuate the inflammatory response of the nerve to CCI and resulting thermal hyperalgesia. In IL-10-treated animals, thermal hyperalgesia was significantly reduced following CCI (days 3, 5 and 9). Histological sections from the peripheral nerve injury site of those animals had decreased cell profiles immunoreactive for ED-1, a marker of recruited macrophages, at both times studied (2 and 5 days post-CCI). IL-10 treatment also decreased cell profiles immunoreactive for the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) at day 2, but not day 5. Qualitative light microscopic assessment of neuropathology at the lesion site did not suggest substantial differences between IL-10 and vehicle-treated sections. The authors propose that initial production of TNF-alpha and perhaps other proinflammatory cytokines at the peripheral nerve lesion site importantly influences the long-term behavioral outcome of nerve injury, and that IL-10 therapy may accomplish this by downregulating the inflammatory response of the nerve to injury.

The effect of thalidomide treatment on vascular pathology and hyperalgesia caused by chronic constriction injury of rat nerve.

Tumor necrosis factor alpha (TNF) may be involved in the pathogenic mechanisms of neuropathic pain by affecting endothelial cells and by upregulation of receptor sensitivity in afferent nerve fibers. To test the hypothesis that TNF plays a role in the vascular changes and the pain-related behavior in an experimental painful neuropathy in rats produced by tying loosely constrictive ligatures around one sciatic nerve, we investigated the effect of thalidomide, a selective blocker of TNF-production in activated macrophages. In rats in which treatment with thalidomide was started preoperatively, there was diminished mechanical allodynia and thermal hyperalgesia during the early stage of the disease. TNF immunohistochemistry revealed reduced endoneurial immunoreactivity on day 5 post surgery as compared to sham-treated animals. The pathologic vascular changes were also reduced in thalidomide-treated rats. Starting treatment with thalidomide at a time point when hyperalgesia was already present did not alter the course of the pain-related behavior. We conclude that preemptive treatment with a substance that blocks production of TNF reduces pain-related symptoms and pathologic vascular changes in the chronic constriction injury model of neuropathic pain.

Wallerian degeneration and hyperalgesia after peripheral nerve injury are glutathione-dependent.

Experimental inflammatory compression injury to the sciatic nerve (chronic constriction injury, CCI) induces Wallerian degeneration of axons and damages non-neuronal cells at the injury site in association with the development of exaggerated pain-like behavior, or hyperalgesia, to noxious thermal stimuli in the affected anatomical area. We examined whether glutathione, one of whose many functions is an important endogenous antioxidant, influenced resulting neuropathology and hyperalgesia following CCI. Dietary supplementation of the amino acid N-acetyl-cysteine (NAC), a rate-limiting component of glutathione production, beginning 1 day prior to CCI significantly diminished both Wallerian degeneration, measured by quantitative morphometry of myelinated fibers, and thermal hyperalgesia. NAC treatment raised nerve glutathione levels compared to untreated nerves, as indicated using hemeoxygenase-1 (hsp32) immunoreactivity as a marker of glutathione depletion. Because NAC is also known to have antioxidant abilities, studies simultaneously inhibited glutathione synthesis, and results demonstrated no significant reduction in resulting neuropathology or hyperalgesia. Delaying NAC administration to post-injury times consistently decreased hyperalgesia, although not significantly. This study identifies glutathione levels, and presumably oxidative stress, as important determinants of the neuropathological and behavioral consequences of nerve injury, and suggests that dietary supplementation of NAC constitutes an effective pre-emptive therapeutic strategy for situations involving painful nerve injury, such as occurs during surgery.

Anterograde TNF alpha transport from rat dorsal root ganglion to spinal cord and injured sciatic nerve.

Tumor necrosis factor-alpha (TNF alpha) is a key modulator of painful peripheral nerve injury. We have previously shown that a tracer of TNF alpha injected at the site of rat sciatic nerve injury undergoes retrograde axonal transport to the dorsal root ganglia (DRG). To further understand the role of TNF alpha in DRG, we injected rat L5 DRG with biotinylated TNF alpha, neurobiotin, or vehicle, and detected translocation of the biotin tag by avidin-biotin histochemistry. Biotinylated TNF alpha was transported intraaxonally toward the periphery of both normal and injured nerves. It also reached the dorsal horn of the spinal cord in injured rats, but not in control rats. These findings highlight a dynamic process of TNF alpha axonal transport in the peripheral neural axis, and help explain activation of central cytokines in the pathogenesis of painful neuropathy.

In vivo evaluation of quantitative percussion diagnostics for determining implant stability.

PURPOSE: To test in a rat model whether quantitative percussion diagnostics provide reliable, reproducible indications of osseointegration. MATERIALS AND METHODS: Titanium implants were placed in femurs of 36 Sprague-Dawley rats. Each animal was assigned to one of six groups defined by one of three time points (2, 4, or 8 weeks postplacement) and one of two treatments (matrix metalloproteinase [MMP] inhibitor GM6001 or control). Percussion testing was conducted three times per subject at implant placement and before sacrifice at one of the time points. For each time point, there was an experimental group that received daily intraperitoneal injections of GM6001, and a control group that received no MMP inhibitor. The percussion data consisted of loss coefficient (LC) values that characterize energy dissipation. Statistical analysis was performed on the LC values for the two animal groups using the paired Student t test to assess differences as a function of time, and the independent t test to compare mean LC for the study groups at sacrifice (α = .05). Histologic evaluation using the osteogenic CD40 protein marker was also performed. RESULTS: A nearly significant difference in mean LC at the 2-week time point was observed between the two treatments with the GM6001 group having the higher value (P = .053). There was a greater difference between the mean LC values for the 4-week GM6001 and control groups (P = .001). The histologic evidence for subjects in these two groups confirmed reduction of osteogenesis at the implant interface after administration of the MMP inhibitor. CONCLUSIONS: Lower control LC values relative to the GM6001 therapeutic group were observed, consistent with the effect MMP inhibition has on matrix remodeling at the implant bone interface. This finding in conjunction with histologic observations confirms that osseointegration can be monitored using percussion diagnostics.

Cytokine regulation of MMP-9 in peripheral glia: implications for pathological processes and pain in injured nerve.

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced in Schwann cells hours after peripheral nerve injury and controls axonal degeneration and macrophage recruitment to the lesion. Here, we report a robust (90-fold) increase in MMP-9 mRNA within 24 h after rat sciatic nerve crush (1 to 60 days time-course). Using direct injection into a normal sciatic nerve, we identify the proinflammatory cytokines TNF-alpha and IL-1beta as potent regulators of MMP-9 expression (Taqman qPCR, zymography). Myelinating Schwann cells produced MMP-9 in response to cytokine injection and crush nerve injury. MMP-9 gene deletion reduced unstimulated neuropathic nociceptive behavior after one week post-crush and preserved myelin thickness by protecting myelin basic protein (MBP) from degradation, tested by Western blot and immunofluorescence. These data suggest that MMP-9 expression in peripheral nerve is controlled by key proinflammatory cytokine pathways, and that its removal protects nerve fibers from demyelination and reduces neuropathic pain after injury.

A derivative of the plasma protease inhibitor alpha(2)-macroglobulin regulates the response to peripheral nerve injury.

Peripheral nerve injury induces endoneural inflammation, controlled by diverse cytokines and extracellular mediators. Although inflammation is coupled to axonal regeneration, fulminant inflammation may increase nerve damage and neuropathic pain. alpha(2)-Macroglobulin (alpha2M) is a plasma protease inhibitor, cytokine carrier, and ligand for cell-signaling receptors, which exists in two well-characterized conformations and in less well-characterized intermediate states. Previously, we generated an alpha2M derivative (alpha(2)-macroglobulin activated for cytokine binding; MAC) similar in structure to alpha(2)M conformational intermediates, which binds tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and inhibits endotoxin toxicity. In this study, we report that the continuum of cytokines that bind to MAC includes IL-6 and IL-18. MAC inhibited TNF-alpha-induced p38 mitogen-activated protein kinase activation and cell death in cultured Schwann cells. When administered by i.p. injection to mice with sciatic nerve crush injury, MAC decreased inflammation and preserved axons. Macrophage infiltration and TNF-alpha expression also are decreased. MAC inhibited TNF-alpha expression in the chronic constriction injury model of nerve injury. When MAC was prepared using a mutated recombinant alpha2M, which does not bind to the alpha2M receptor, low-density lipoprotein receptor-related protein-1, activity in the chronic constriction injury model was blocked. These studies demonstrate that an alpha2M derivative is capable of regulating the response to peripheral nerve injury by a mechanism that requires low-density lipoprotein receptor-related protein-1.

TNFalpha-induced MMP-9 promotes macrophage recruitment into injured peripheral nerve.

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced hours after injury to peripheral nerve. This study shows that MMP-9 gene deletion and neutralization with MMP-9 antibody reduce macrophage content in injured wild-type nerves. In mice with delayed Wallerian degeneration (WldS), MMP-9 and tumor necrosis factor alpha (TNFalpha) decline in association with the reduced macrophage recruitment to injured nerve that characterizes this strain of mice. We further determined that TNFalpha acts as an MMP-9 inducer by establishing increased MMP-9 levels after TNFalpha injection in rat sciatic nerve in vivo and primary Schwann cells in vitro. We found reduced MMP-9 expression in crushed TNFalpha knockout nerves that was rescued with exogenous TNFalpha. Finally, local application of MMP-9 on TNFalpha-/- nerves increased macrophage recruitment to the lesion. These data suggest that TNFalpha lies upstream of MMP-9 in the pathway of macrophage recruitment to injured peripheral nerve.

TNF-alpha and phosphorylation of ERK in DRG and spinal cord: insights into mechanisms of sciatica.

STUDY DESIGN: Characterize extracellular signal-regulated kinase (ERK) and its phosphorylation (pERK) in neural tissues after topical application of tumor necrosis factor-alpha (TNF-alpha) to L5 nerve root. OBJECTIVE: Identify time-course, localization, and expression of pERK. SUMMARY OF BACKGROUND DATA: TNF-alpha has a key role in disc herniation and sciatica as an inflammatory component of the nucleus pulposus. ERK is associated with neuronal signal transduction and nociception. METHODS: We studied tissue from naive rats, vehicle-treated rats, and rats receiving rat recombinant TNF-alpha using Western blots of total and phosphorylated ERK (pERK). We used immunohistochemistry of pERK with neuronal nuclear (NeuN) antibody to identify its cellular distribution. RESULTS: Topical application of TNF-alpha to rat nerve root increased pERK in ipsilateral dorsal root ganglion (DRG) neurons and glia within 5 hours. pERK was not expressed in DRG during the first hour after TNF-alpha application, nor was it seen at anytime in spinal cord dorsal horn. DRG satellite cells had increased pERK 5 hours after TNF-alpha or vehicle treatment. TNF-alpha treatment increased pERK in small- and medium-sized DRG neurons and to a lesser degree in large neurons. CONCLUSIONS: These findings suggest that ERK signaling plays a role in the activation of DRG cells following inflammatory injuries to nerve roots and further documents the importance of inflammation in the pathogenesis of painful spine disorders.