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.

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.

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.

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.

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.

Mitigation of direct neurotoxic effects of lidocaine and amitriptyline by inhibition of p38 mitogen-activated protein kinase in vitro and in vivo.

BACKGROUND: Local anesthetic-induced direct neurotoxicity (paresthesia, failure to regain normal sensory and motor function) is a potentially devastating complication of regional anesthesia. Local anesthetics activate the p38 mitogen-activated protein kinase (MAPK) system, which is involved in apoptotic cell death. The authors therefore investigated in vitro (cultured primary sensory neurons) and in vivo (sciatic nerve block model) the potential neuroprotective effect of the p38 MAPK inhibitor SB203580 administered together with a clinical (lidocaine) or investigational (amitriptyline) local anesthetic. METHODS: Cell survival and mitochondrial depolarization as marker of apoptotic cell death was assessed in rat dorsal root ganglia incubated with lidocaine or amitriptyline either with or without the addition of SB203580. Similarly, in a sciatic nerve block model, the authors assessed wallerian degeneration by light microscopy to detect a potential mitigating effect of MAPK inhibition. RESULTS: Lidocaine at 40 mm/approximately 1% and amitriptyline at 100 microm reduce neuron count, but coincubation with the p38 MAPK inhibitor SB203580 at 10 mum significantly reduces cytotoxicity and the number of neurons exhibiting mitochondrial depolarization. Also, wallerian degeneration and demyelination induced by lidocaine (600 mm/approximately 15%) and amitriptyline (10 mm/approximately 0.3%) seem to be mitigated by SB203580. CONCLUSIONS: The cytotoxic effect of lidocaine and amitriptyline in cultured dorsal root ganglia cells and the nerve degeneration in the rat sciatic nerve model seem, at least in part, to be mediated by apoptosis but seem efficiently blocked by an inhibitor of p38 MAPK, making it conceivable that coinjection might be useful in preventing local anesthetic-induced neurotoxicity.

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.

Erythropoietin reduces Schwann cell TNF-alpha, Wallerian degeneration and pain-related behaviors after peripheral nerve injury.

Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain-like behaviors. These effects are mediated in large part by pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF-alpha has been implicated in the development of pain-related behaviors, we measured TNF-alpha mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF-alpha mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF-alpha in Schwann cells, axoplasm and macrophages. In rhEpo-treated animals, TNF-alpha immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF-alpha in CCI by blocking expression of TNF-alpha in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF-alpha expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF-alphain vitro. These results indicated that rhEpo regulates TNF-alpha by multiple mechanisms; rhEpo regulates TNF-alpha mRNA expression by Schwann cells but also may directly counteract TNF-alpha signaling pathways that lead to injury, chronic pain and/or death.

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.

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.

Ephedrine blocks rat sciatic nerve in vivo and sodium channels in vitro.

BACKGROUND: The sympathomimetic drug ephedrine has been used intrathecally as the sole local anesthetic for labor and delivery. Because ephedrine may be a useful adjuvant to local anesthetics, the authors investigated the local anesthetic properties of ephedrine in a rat sciatic nerve block model and the underlying mechanism in cultured cells stably expressing Na channels. METHODS: After approval of the animal protocol, the sciatic nerves of anesthetized rats were exposed by lateral incision of the thighs, 0.2 ml ephedrine at 0.25, 1, 2.5, or 5% and/or bupivacaine at 0.125% was injected, and the wound was closed. Motor and sensory/nociceptive functions were evaluated by the force achieved by pushing against a balance and the reaction to pinch, respectively. The whole cell configuration of the patch clamp technique was used to record Na currents from human embryonal kidney cells stably transfected with Nav1.4 channels. RESULTS: The nociception blockade was significantly longer than the motor blockade at test doses of 2.5 and 5% of ephedrine, or when 1% ephedrine was combined with 0.125% bupivacaine (analysis of variance with repeated measures, P < 0.001, n = 8/group). In vitro, the 50% inhibitory concentrations of ephedrine at -150 and -60 mV were 1,043 +/- 70 and 473 +/- 13 mum, respectively. High-frequency stimulation revealed a use-dependent block of 18%, similar to most local anesthetics. CONCLUSIONS: Because ephedrine's properties are at least partly due to Na channel blockade, detailed histopathologic investigations are justified to determine the potential of ephedrine as an adjuvant to clinically used local anesthetics.

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.

The histologic effects of pulsed and continuous radiofrequency lesions at 42 degrees C to rat dorsal root ganglion and sciatic nerve.

STUDY DESIGN: Experimental histologic study of the effects of radiofrequency (RF) or convective heating of the rat dorsal root ganglion or sciatic nerve to 42 degrees C. OBJECTIVE: To determine whether treatment causes neuropathologic changes in an effort to explore the mechanisms and safety of pulsed RF pain therapy. SUMMARY OF BACKGROUND DATA: Clinical data suggest that low temperature pulsed RF energy delivered to the DRG is a safe and effective form of therapy for low back pain. However, the mechanism by which this treatment modifies pain is unclear. METHODS: A total of 118 Sprague-Dawley rats were divided into five groups for different RF and thermal treatments. All treatments increased tissue temperature to 42 degrees C. Treatments of the DRG included pulsed RF, continuous RF, and conductive heat. The generator output was increased until 42 degrees C was obtained in the tissue and was then maintained for 120 seconds. As a positive control, some rat sciatic nerves were treated with continuous RF lesions at 80 degrees C. Animals were killed for histologic study at 2, 7, or 21 days after treatment. Tissue was fixed in gluteraldehyde and embedded in plastic resin for detailed light microscopic neuropathologic evaluation. RESULTS: The methods used to heat the tissue to 42 degrees C caused no significant difference in pathology. However, subclinical changes included endoneurial edema caused by alterations in the function of the blood-nerve barrier, fibroblast activation, and collagen deposition. Tissue returned to normal conditions by 7 days in nerve and 21 days in the DRG. These minor structural changes observed at the light microscopic level in normal animals do not exclude the possibility that there would be nonstructural changes in gene expression or cytokine upregulation in injured tissue. Lesions at 80 degrees C caused consistent thermal injury characterized by Wallerian degeneration of nerve fibers. CONCLUSIONS: The data support the hypothesis that pulsed RF treatment does not rely on thermal injury of neurologic tissue to achieve its effect.

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.

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.

Matrix metalloproteinase-9 promotes nerve growth factor-induced neurite elongation but not new sprout formation in vitro.

Matrix metalloproteinase-9 (MMP-9) is a basal-lamina-degrading protease that we have recently shown to be localized in regenerating sciatic nerve. We now demonstrate that MMP-9 colocalizes with growth-associated protein GAP-43 in regenerating nerves in vivo and is involved in vitro in axonal sprouting. By using a PC12 cell model for neuronal sprouting, we analyzed the effects of recombinant MMP-9, MMP-9-neutralizing antibody, and a broad-spectrum MMP inhibitor (Ro 31-9790) on sprout formation, elongation, and branching. Quantitative phase-contrast microscopy showed that MMP-9 elongated neuronal sprouts by 67% and increased their branching by 14% but did not change the number of sprouts relative to nerve growth factor (NGF) treatment. Double immunofluorescence for GAP-43, a marker for growth cones, and alpha-tubulin, a marker for axonal microtubules, showed that MMP-9-treated cells had increased distribution of alpha-tubulin but no effect on GAP-43. Western blot analyses of cell lysates demonstrated that the NGF-induced increase in GAP-43 was unchanged with MMP-9 treatment or inhibition, confirming that MMP-9 had no effect on new sprout formation. However, Ro 31-9790 reduced GAP-43 levels to those seen in untreated cells, suggesting that an MMP other than MMP-9 is important for sprout formation. Finally, phosphorylated neurofilament M (NFM-p), a marker for regenerative elongation, was induced with MMP-9 treatment and was inhibited by the anti-MMP-9 antibody treatment, confirming the role of MMP-9 in axonal elongation. NFM-p colocalized with MMP-9 in regenerating sciatic nerve fibers. These findings suggest that MMP-9 regulates neurite extension in regenerating peripheral nerve fibers and, therefore, might be of therapeutic value in promoting regeneration in vivo.

TNF-alpha and TNF-alpha receptor type 1 upregulation in glia and neurons after peripheral nerve injury: studies in murine DRG and spinal cord.

OBJECTIVES: The purpose of the current study was to evaluate changes in tumor necrosis factor-alpha (TNF-alpha ) and TNF-alpha receptor 1 (p55 receptor) using double fluorescent immunohistochemistry in glial and neural cells in the dorsal root ganglion and spinal cord after sciatic nerve injury in mice. SUMMARY OF BACKGROUND DATA.: TNF-alpha is a primary mediator of the inflammatory response and is primarily synthesized and released in the nervous system by macrophages and Schwann cells following peripheral nerve injury. TNF-alpha is also released from astrocytes and microglia in the central nervous system, where it plays a crucial role in the pathophysiology of injury. METHODS: Sixteen female mice were used. Under anesthesia, the left sciatic nerve was crushed. At 3, 5, and 14 days after surgery, the spinal cord at the level of L5 and the left L5 DRG were removed and processed for immunohistochemistry. Tissue sections were double stained with antibodies to either glial fibrillary acidic protein (GFAP; marker for astrocytes or satellite cells) or NeuN (marker for neurons), and TNF or p55 receptor. RESULTS.: In the dorsal root ganglion, GFAP-immunoreactive (IR) satellite cells became evident after injury and were also immunoreactive for both TNF-alpha and p55 receptor. Dorsal root ganglion neurons expressed p55 receptor after injury. TNF-alpha and GFAP-IR satellite cells surrounded p55-IR neurons. Furthermore, the number of GFAP-IR astrocytes dramatically increased in the spinal cord after nerve injury, and some astrocytes were also TNF-alpha -IR and p55 receptor-IR. TNF-alpha -1R astrocytes were seen around p55 receptor-IR neurons. CONCLUSIONS: These data demonstrate that upregulation of glial TNF-alpha is associated with the expression of the p55 receptor on adjacent neurons. This association may have induced the expression of several cytokines and immediate early genes in dorsal root ganglion and spinal cord neurons via the TNF signaling pathway. These findings may be related to the pathogenesis of neuropathic pain.