Spinal Nerve Transection Research Articles

Peripheral nerve injury up-regulates expression of interactor protein for cytohesin exchange factor 1 (IPCEF1) mRNA in rat dorsal root ganglion

Interaction protein for cytohesin exchange factors 1 (IPCEF1) is a recently identified protein that binds to cytohesin 2 and might participate in membrane receptor trafficking by enhancing the activity of cytohesin 2 in cultured cells. However, whether IPCEF1 is involved in this process in vivo is unknown. Here, we report that IPCEF1 messenger RNA (mRNA) is expressed in the dorsal root ganglions (DRGs) of adult rats. Moreover, peripheral nerve injury induced by fifth spinal nerve ligation and transection or sciatic nerve transection significantly up-regulated expression of IPCEF1 mRNA in the injured DRGs. Because peripheral nerve injury leads to changes in membrane receptor trafficking in the injured DRG, the correlation of IPCEF1 mRNA expression with nerve injury input in the injured DRG suggests that IPCEF1 might participate in the mechanisms that underlie nerve injury-induced membrane receptor trafficking in the DRG under neuropathic pain conditions.

Spinal Microglial Expression and Mechanical Hypersensitivity in a Postoperative Pain Model: Comparison with a Neuropathic Pain Model

Postoperative pain control contributes to quality of life. Activation of spinal cord microglia after peripheral nerve injury contributes to mechanical hypersensitivity. The contribution of spinal cord microglia to hypersensitivity after surgery, however, is not well understood. Here, the authors evaluated whether inhibition of spinal microglia reduced postoperative mechanical hypersensitivity, and if so, whether the effect differed from that in a rat neuropathic pain model. Male Sprague-Dawley rats underwent either unilateral plantar hind paw incision (postoperative pain model) or L5 spinal nerve transection (neuropathic pain model), and the development of mechanical hypersensitivity was assessed using von Frey filaments. The microglial inhibitor minocycline was intraperitoneally administered daily for either 3 or 7 days. Spinal microglial activation was evaluated by OX42 immunohistochemistry. We also tested the effect of intrathecal administration of a p38 mitogen-activated protein kinase inhibitor, SB203580. In the postoperative pain model, minocycline did not suppress mechanical hypersensitivity, but did inhibit an increase in spinal OX42 expression. In contrast, in the neuropathic pain model, minocycline reduced mechanical hypersensitivity in a dose-related manner and inhibited spinal OX42 expression. SB203580 attenuated hypersensitivity in the neuropathic pain model, but not in the postoperative pain model. The results of the present study suggest that spinal OX42 expression has a more important role in the development of neuropathic pain than in postoperative pain, and that an increase in spinal OX42 expression does not contribute to postoperative mechanical hypersensitivity.

Effects of recombinant human erythropoietin on neuropathic pain and cerebral expressions of cytokines and nuclear factor-kappa B

The effect of recombinant human erythropoietin (rhEPO) on neuropathic pain remains unclear. This study aimed to determine the effects of preemptive administration of rhEPO on the behavioural changes and neuroinflammatory responses in a rat model of neuropathic pain. Fifty rats were randomly allocated into five groups, sham-operation treated with saline and L5 spinal nerve transection treated with different doses of rhEPO (0 [saline], 1000, 3000, or 5000 U x kg(-1), respectively). The rats were intraperitoneally treated from 1 day before surgery to post-surgery day 7. The mechanical (paw pressure thresholds, PPT) and thermal thresholds (paw withdrawal latencies, PWL) were measured on post-surgery days 1, 3, and 7. The contralateral brain was obtained on post-surgery day 7 to determine the expressions of tumour necrosis factor (TNF-alpha), interleukin (IL)-1beta, IL-6, L-10, and nuclear factor-kappa B (NF-kappaB) activity. There were significant decreases in PPT and PWL after L5 spinal nerve transection (P < 0.001). Compared with the saline group, the rhEPO 3000 and 5000 U x kg(-1) groups resulted in significant increases in PPT and PWL (P < 0.001) and reduced the cerebral expressions of TNF-alpha, IL-1beta, IL-6, and NF-kappaB activity associated with the increase in IL-10 (rhEPO3000 group, P < 0.05, and rhEPO5000 group, P < 0.001, respectively). Administration of rhEPO 1000 U x kg(-1) had no significant effects on these variables. Preemptive rhEPO dose-dependently attenuated the mechanical and thermal hyperalgesia in L5 spinal nerve transection rats, which correlated with the decreased cerebral expressions of TNF-alpha, IL-1beta, and IL-6 via downregulating NF-kappaB activity and the increased expression of IL-10.

Modulation of delta opioid receptor expression with herpes simplex viral vectors attenuates allodynia and hyperalgesia in a rodent model of neuropathic pain

The treatment of neuropathic pain remains a clinical challenge with a need for identification of new therapeutic targets and strategies. We tested the hypothesis that herpes simplex viral vectors can be used to modulate expression of delta opioid receptors for the treatment of neuropathic-pain behavior and to enhance opioid analgesia in a mouse L5 spinal nerve transection model. On day 7 post-nerve transection mechanical allodynia and thermal hyperalgesia was verified and a recombinant herpes simplex virus type 1 encoding the cDNA sequences for the sense delta opioid receptor (NPDOR), antisense delta opioid receptor (NPADOR), or E.

Post pressure response of skin blood flowmotions in anesthetized rats with spinal cord injury

Pressure ulcer is a common complication developed in persons with spinal cord injury (SCI) when prolonged unrelieved pressure was applied to the body/skin and underlying tissues. The objective of this study is to assess the hyperemic response of the skin blood flowmotions in anesthetized rats with spinal cord injury subjected to prolonged pressure using spectral analysis based on wavelets transform of the periodic oscillations of the cutaneous laser Doppler flowmetry (LDF) signal. A total of twenty-eight Sprague–Dawley rats were used in this study, of which 14 were normal rats and the other 14 were spinal cord injured rats with transection of the T1 spinal nerves. External pressure of 13.3 kPa (100 mmHg) was applied to the trochanter area of rats via a specifically designed indentors. The loading duration was 6 h. LDF measurement was monitored for 20 min prior to and after the prescribed compression period. Five frequency intervals were identified (0.01–0.05 Hz, 0.05–0.15 Hz, 0.15–0.4 Hz, 0.4–2 Hz and 2–5 Hz) corresponding to endothelial related metabolic, neurogenic, myogenic, respiratory and cardiac origins. The absolute amplitude of oscillations of each particular frequency interval and the normalized amplitude were calculated for quantitative assessments. Comparisons of hyperemic response were performed between SCI rats and normal ones. The results showed that the normalized amplitude in the frequency interval II (0.05–0.15 Hz) was significantly lower on SCI rats than that in normal ones ( p < 0.01). Also, decreased reactive hyperemic response was observed in rats suffered from spinal cord injury.

Localization of the Endocannabinoid-Degrading Enzyme Fatty Acid Amide Hydrolase in Rat Dorsal Root Ganglion Cells and Its Regulation after Peripheral Nerve Injury

Fatty acid amide hydrolase (FAAH) is a degradative enzyme for a group of endogenous signaling lipids that includes anandamide (AEA). AEA acts as an endocannabinoid and an endovanilloid by activating cannabinoid and vanilloid type 1 transient receptor potential (TRPV1) receptors, respectively, on dorsal root ganglion (DRG) sensory neurons. Inhibition of FAAH activity increases AEA concentrations in nervous tissue and reduces sensory hypersensitivity in animal pain models. Using immunohistochemistry, Western blotting, and reverse transcription-PCR, we demonstrate the location of the FAAH in adult rat DRG, sciatic nerve, and spinal cord. In naive rats, FAAH immunoreactivity localized to the soma of 32.7 +/- 0.8% of neurons in L4 and L5 DRG. These were small-sized (mean soma area, 395.96 +/- 5.6 mum(2)) and predominantly colabeled with peripherin and isolectin B4 markers of unmyelinated C-fiber neurons; 68% colabeled with antibodies to TRPV1 (marker of nociceptive DRG neurons), and <2% colabeled with NF200 (marker of large myelinated neurons). FAAH-IR was also present in small, NF200-negative cultured rat DRG neurons. Incubation of these cultures with the FAAH inhibitor URB597 increased AEA-evoked cobalt uptake in a capsazepine-sensitive manner. After sciatic nerve axotomy, there was a rightward shift in the cell-size distribution of FAAH-immunoreactive (IR) DRG neurons ipsilateral to injury: FAAH immunoreactivity was detected in larger-sized cells that colabeled with NF200. An ipsilateral versus contralateral increase in both the size and proportion of FAAH-IR DRG occurred after spinal nerve transection injury but not after chronic inflammation of the rat hindpaw 2 d after injection of complete Freund's adjuvant. This study reveals the location of FAAH in neural tissue involved in peripheral nociceptive transmission.

Relationship between neuronal nitric oxide synthase and NADPH-diaphorase in the dorsal root ganglia during neuropathic pain.

Changes in nitric oxide (NO) production in the dorsal root ganglia (DRG) may contribute to allodynia after nerve injury. It is known that the histochemistry of NADPH-diaphorase (NADPH-d) is known to be not always coincident with NOS. This study was conducted to investigate the relationship between nNOS and NADPH-d expression in the DRG in a spinal nerve injury model of neuropathic pain, and to elucidate role that NO plays in neuropathic pain. nNOS immunohistochemistry and/or NADHP-d histochemistry were conducted in the DRG of a spinal nerve transection model of neuropathic pain, and the pain behavior was then measured by a von Frey filament test of the hindpaws of wild type and nNOS knock-out mice. nNOS immunoreactive neurons and NADPH-d stained neurons were not always identical. Additionally NADPH-d increased, but nNOS did not increase significantly in the DRG after spinal nerve transection. Neuropathic pain behavior increased in the hindpaw of nNOS(-/-) mice after spinal nerve transection, but was lower than that of wild type mice after spinal nerve transection. nNOS immunoreactive neurons and NADPH-d stained neurons were not always identical in the DRG, and a novel NADPH-d positive source may be involved in neuropathic pain after spinal nerve transection. Changes in nNOS expression in the DRG were not the primary cause of neuropathic pain behavior in a spinal nerve transection model of neuropathic pain.

Interaction of glia activation and neurotransmission in hippocampus of neuropathic rats treated with mirtazapine.

Recent studies have characterized a central nervous system neuroimmune effect in the analgesic mechanisms of antidepressants. Our study investigated the effect of a novel antidepressant mirtazapine on pro-inflammatory cytokines expression and astrocytic activation in hippocampus of neuropathic rats. L5 spinal nerve transection was made to produce mononeuropathic model. Mirtazapine was orally administered to rats that had been operated on daily for 14 days. Adrenergic or serotonergic receptor antagonist was intraperitoneally administered to examine their ability of blocking antinociceptive effect. In the region of hippocampus, TNFalpha and IL-1beta levels were assayed and the activity of astrocytes was immunostained with GFAP (glial fibrillary acidic protein) antibody. As a result, mirtazapine significantly inhibited the hyperalgesia produced by surgery, which was partially reversed by adrenergic and serotonergic antagonist. After surgery, the hippocampus demonstrated elevated TNFalpha and IL-1beta levels, concomitant with reactive astrocytes. Mirtazapine markedly reduced hippocampal cytokines production or astrocytic activation, which was blocked by both adrenergic and serotonergic antagonists. Our results indicate a possible role of hippocampal pro-inflammatory cytokines and glia in the pathogenesis of neuropathic pain. A potential analgesic mechanism of mirtazapine may be inhibiting the above neuroimmune action through affecting adrenergic and serotonergic system.

Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIV-associated neuropathic pain

To elucidate the mechanisms underlying peripheral neuropathic pain in the context of HIV infection and antiretroviral therapy, we measured gene expression in dorsal root ganglia (DRG) of rats subjected to systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) and concomitant delivery of HIV-gp120 to the rat sciatic nerve. L4 and L5 DRGs were collected at day 14 (time of peak behavioural change) and changes in gene expression were measured using Affymetrix whole genome rat arrays. Conventional analysis of this data set and Gene Set Enrichment Analysis (GSEA) was performed to discover biological processes altered in this model. Transcripts associated with G protein coupled receptor signalling and cell adhesion were enriched in the treated animals, while ribosomal proteins and proteasome pathways were associated with gene down-regulation. To identify genes that are directly relevant to neuropathic mechanical hypersensitivity, as opposed to epiphenomena associated with other aspects of the response to a sciatic nerve lesion, we compared the gp120 + ddC-evoked gene expression with that observed in a model of traumatic neuropathic pain (L5 spinal nerve transection), where hypersensitivity to a static mechanical stimulus is also observed. We identified 39 genes/expressed sequence tags that are differentially expressed in the same direction in both models. Most of these have not previously been implicated in mechanical hypersensitivity and may represent novel targets for therapeutic intervention. As an external control, the RNA expression of three genes was examined by RT-PCR, while the protein levels of two were studied using western blot analysis.

Spinal Microglial and Perivascular Cell Cannabinoid Receptor Type 2 Activation Reduces Behavioral Hypersensitivity without Tolerance after Peripheral Nerve Injury

Cannabinoids induce analgesia by acting on cannabinoid receptor (CBR) types 1 and/or 2. However, central nervous system side effects and antinociceptive tolerance from CBR1 limit their clinical use. CBR2 exist on spinal glia and perivascular cells, suggesting an immunoregulatory role of these receptors in the central nervous system. Previously, the authors showed that spinal CBR2 activation reduces paw incision hypersensitivity and glial activation. This study tested whether CBR2 are expressed in glia and whether their activation would induce antinociception, glial inhibition, central side effects, and antinociceptive tolerance in a neuropathic rodent pain model. Rats underwent L5 spinal nerve transection or sham surgery, and CBR2 expression and cell localization were assessed by immunohistochemistry. Animals received intrathecal injections of CBR agonists and antagonists, and mechanical withdrawal thresholds and behavioral side effects were assessed. Peripheral nerve transection induced hypersensitivity, increased expression of CR3/CD11b and CBR2, and reduced ED2/CD163 expression in the spinal cord. The CBR2 were localized to microglia and perivascular cells. Intrathecal JWH015 reduced peripheral nerve injury hypersensitivity and CR3/CD11b expression and increased ED2/CD163 expression in a dose-dependent fashion. These effects were prevented by intrathecal administration of the CBR2 antagonist (AM630) but not the CBR1 antagonist (AM281). JWH015 did not cause behavioral side effects. Chronic intrathecal JWH015 treatment did not induce antinociceptive tolerance. These data indicate that intrathecal CBR2 agonists may provide analgesia by modulating the spinal immune response and microglial function in chronic pain conditions without inducing tolerance and neurologic side effects.

Propentofylline-induced astrocyte modulation leads to alterations in glial glutamate promoter activation following spinal nerve transection

We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.

The Effect of Pentoxifylline on Existing Hypersensitivity in a Rat Model of Neuropathy

Using a rat L5 spinal nerve transection model we previously showed that pentoxifylline prevents hyperalgesia through antiinflammation in the prefrontal brain. In this study, we examined efficacy when applied after injury. We examined the effect of pentoxifylline on existing mechanical allodynia, observing glial activation and proinflammatory cytokine expression in the lumbar spinal cord, when given 7 days after L5 spinal nerve transection. There was no effect from pentoxifylline on existing hypersensitivity, glial activation, and cytokine expression when applied after L5 spinal nerve transection. Pentoxifylline administered intraperitoneally on day 7 postsurgery failed to alleviate existing hypersensitivity, or reduce glial activation and cytokine expression.

Repeated administration of mirtazapine inhibits development of hyperalgesia/allodynia and activation of NF-κB in a rat model of neuropathic pain

Antidepressants have been widely used to treat neuropathic pain for many years. However, the mechanisms of their analgesic actions are little known and remain controvertible. Recent studies indicate that cytokines in central nervous system (CNS) play a critical role in the pathological states of pain. The present study was designed to explore the effects and most appropriate dosage of mirtazapine in treating neuropathic pain and its possible neuroimmune mechanisms. L5 spinal nerve transection was done to produce hyperalgesia in rats. Mirtazapine (10, 20 and 30 mg/kg, respectively) was orally administered daily for 14 days, beginning from the 5th day after nerve transection. Mechanical and thermal hyperalgesia was measured using Von-Frey filament and Hargreaves tests before and after the surgery. Rats were then sacrificed on days 3, 7, 14, 21 post-administration. The inflammatory cytokines production such as TNFalpha, IL-1beta, IL-10 and nuclear factor kappa B (NF-kappaB) activity in brain was quantified using enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assay (EMSA). We found that mirtazapine (20 and 30 mg/kg) can markedly attenuate mechanical and thermal hyperalgesia produced by nerve transection, most significantly on the 14th day. The elevated TNFalpha, IL-1beta and NF-kappaB in brain were accordingly reduced, while the expression of increased IL-10 were even stimulated after repeated mirtazapine administration. Our data could conclude that mirtazapine suppressed neuropathic pain partially through inhibiting cerebral proinflammatory cytokines production and NF-kappaB activation in CNS.

Local application of capsaicin alleviates mechanical hyperalgesia after spinal nerve transection

Whether modulation of C afferent fiber activities could relieve peripheral neuropathic pain was tested. After establishment of neuropathic pain induced by L5 and 6 spinal nerve transection (SNT), the sciatic nerve was treated with 2% capsaicin at the level of the midthigh. Mechanical hyperalgesia (von Frey filaments) was significantly alleviated from 7 days to 4 weeks after capsaicin treatment, but cold allodynia (acetone) was unchanged. Immunohistochemical studies showed a significant increase in the number of calcitonin gene-related peptide (CGRP)-positive neurons, but not TRPV1-positive neurons in intact L4 dorsal root ganglia after SNT. Capsaicin treatment decreased TRPV1- and CGRP-positive neurons in L4 DRG of the treated side, but not the opposite side. These results suggest that local application of capsaicin onto the sciatic nerve can alleviate mechanical hyperalgesia, but not cold allodynia, in a peripheral neuropathic pain model and the pain alleviation may result from a decrease of TRPV1- and CGRP-positive sensory neurons of which fibers pass through the sciatic nerve.

Resolution of Inflammation-Induced Axonal Mechanical Sensitivity and Conduction Slowing in C-Fiber Nociceptors

The present study is an in vivo investigation into the time course of inflammation-induced axonal mechanical sensitivity (AMS) in intact C-fiber axons. After induction of a localized neuritis in the rat sciatic nerve, AMS developed in C-fiber axons at 1 (18.2%) and 4 weeks (11.6%). By 8 weeks, AMS was virtually absent (2.1%). AMS was also tested in intact L5 neurons after L4 spinal nerve transection, which induces a diffuse inflammation within the sciatic nerve. At 1 week, AMS developed in 10% of neurons. No AMS was observed in unoperated animals. The localized neuritis also caused changes in L5 dorsal root conduction velocities (CVs). CVs decreased at 1 week (-7.7%) and 4 weeks (-17.6%) and returned to normal by 8 weeks. L4 transection similarly reduced CVs (-13.7%) of L5 dorsal root axons. There were no significant changes among any groups in the proportion or rate of ongoing activity. These results demonstrate that the axonal changes due to neuritis are not permanent. Therefore, in patients with persistent movement-induced radiating limb pain with few clinically apparent signs of nerve damage, there may be a persisting inflammatory lesion affecting the nerve. Nerve inflammation, or neuritis, causes axonal mechanical sensitivity, which is the neural substrate for radiating limb pain induced by movement. This study examined the time course of induced axonal mechanical sensitivity and conduction velocity changes in intact C-fiber axons after nerve inflammation. The results suggest that treatment to reduce nerve inflammation may be beneficial to patients with radiating pain.

IL-1β signaling is required for mechanical allodynia induced by nerve injury and for the ensuing reduction in spinal cord neuronal GRK2

Many neurotransmitters involved in pain perception transmit signals via G protein-coupled receptors (GPCRs). GPCR kinase 2 (GRK2) regulates agonist-induced desensitization and signaling of multiple GPCRs and interacts with downstream molecules with consequences for signaling. In general, low GRK2 levels are associated with increased responses to agonist stimulation of GPCRs. Recently, we reported that in mice with reduced GRK2 levels, inflammation-induced mechanical allodynia was increased. In addition, mice with impaired interleukin (IL)-1β signaling did not develop mechanical allodynia after L5 spinal nerve transection (SNT). We hypothesized that in the L5 SNT model mechanical allodynia would be associated with reduced neuronal GRK2 levels in the spinal cord dorsal horn and that IL-1β signaling would be required to induce both the decrease in GRK2 and mechanical allodynia. We show here that in wild type (WT) mice L5 SNT induces a bilateral decrease in neuronal GRK2 expression in the lumbar spinal cord dorsal horn, 1 and 2 weeks after L5 SNT. No changes in GRK2 were observed in the thoracic segments. Moreover, spinal cord GRK2 expression was not decreased in IL-1R −/− mice after L5 SNT. These data show that IL-1β signaling is not only required for the development of mechanical allodynia, but also to reduce neuronal GRK2 expression. These results suggest a functional relation between the L5 SNT-induced IL-1β-mediated decrease in GRK2 and development of mechanical allodynia.

A synthetic kainoid, (2 S,3 R,4 R)-3-carboxymethyl-4-(phenylthio)pyrrolidine-2-carboxylic acid (PSPA-1) serves as a novel anti-allodynic agent for neuropathic pain

In spite of prominent progress in basic pain research, neuropathic pain remains a significant medical problem, because it is often poorly relieved by conventional analgesics. Thus this situation encourages us to make more sophisticated efforts toward the discovery of new analgesics. We previously showed that i.t. administration of acromelic acid-A (ACRO-A), a Japanese mushroom poison, provoked prominent tactile pain (allodynia) at an extremely low dose of 1 fg/mouse. In the present study we synthesized ACRO-A analogues (2 S,3 R,4 R)-3-carboxymethyl-4-phenoxypyrrolidine-2-carboxylic acid (POPA-2) and (2 S,3 R,4 R)-3-carboxymethyl-4-(phenylthio)pyrrolidine-2-carboxylic acid (PSPA-1) chemically and examined their ability to induce allodynia in conscious mice. Whereas POPA-2 induced allodynia at extremely low doses from 1 to 100 fg/mouse, similar to ACRO-A, PSPA-1 did not induce allodynia; rather, it inhibited the ACRO-A-induced allodynia with an ID 50 value (95% confidence limits) of 2.19 fg/mouse (0.04–31.8 fg/mouse). Furthermore, PSPA-1 relieved neuropathic pain produced by L5 spinal nerve transection on day 7 after the operation in a dose-dependent manner from 1 to 100 pg/mouse. In contrast, it did not affect thermal or mechanical nociception or inflammatory pain. PSPA-1 reduced the increase in neuronal nitric oxide synthase activity in the spinal cord of neuropathic pain mice assessed by NADPH-diaphorase histochemistry and blocked the allodynia induced by N-methyl- d-aspartate. These results demonstrate that PSPA-1 may represent a novel class of anti-allodynic agents for neuropathic pain acting by blocking the glutamate-nitric oxide pathway.

Depletion of vesicular zinc in dorsal horn of spinal cord causes increased neuropathic pain in mice

Zinc enriched (ZEN) neurons and terminals are abundant in the rodent spinal cord. Zinc ions have been suggested to modulate the excitability of primary afferent fibers believed to be important in nociceptive transmission. To test the hypothesis that vesicular zinc concentration is related to neuropathic pain we applied Chung's rodent pain model on BALB/c mice, and traced zinc transporter 3 (ZnT3) proteins and zinc ions with immunohistochemistry and autometallography (AMG), respectively. Under anesthesia the left fifth lumbar spinal nerve was ligated in male mice in order to produced neuropathic pain. The animals were then sacrificed 5 days later. The ZnT3 immunoreactivity was found to have decreased significantly in dorsal horn of fourth, fifth, and sixth lumbar segments. In parallel with the depressed ZnT3 immunoreactivity the amount of vesicular zinc decreased perceptibly in superficial gray matters of especially layer I-IV of the same segments. The transection-induced reduction of vesicular zinc in ZEN terminals of the dorsal horn was synchronic to reduced pain threshold, as measured by von Frey method. In a separate study, we observed intensive zinc selenite precipitation in somata of the smaller spinal ganglion cell, but 5 days after spinal nerve transection zinc precipitation was also found in the lager ganglion cells. The present results indicate that zinc may be involved in pain mechanism in the spinal ganglion level. These results support the hypothesis that vesicular zinc might have a modulatory role for neuropathic pain. Thus, increased pain sensitivity might be related to reduce vesicular zinc level in the dorsal spinal gray matter.

Reprint of “Efficacy of propentofylline, a glial modulating agent, on existing mechanical allodynia following peripheral nerve injury” [Brain Behav. Immun. 21 (2007) 238–246

Increasing evidence points to a role for spinal neuroimmune dysregulation (glial cell activation and cytokine expression) in the pathogenesis of chronic pain. Suppression of astrocytic and microglial activation with the methylxanthine derivative, propentofylline, pre-emptively attenuates the development of nerve injury-induced allodynia. Currently, we investigated the ability of systemic propentofylline to reverse existing, long-term allodynia after nerve injury—a clinically relevant paradigm. Rats received L5 spinal nerve transection or sham surgery and the development of mechanical allodynia was assessed daily for 2 weeks, at which time injured rats exhibited robust responses to non-noxious von Frey filaments. On days 14–27, rats received either saline or 101 mg/kg propentofylline by intraperitoneal (i.p.) injection. On day 28 or 42 (after a 14-day drug washout period), lumbar spinal cord sections were processed for assessment of astrocytic glial fibrillary acidic protein (GFAP) and microglial OX-42 (antibody against CR3/CD11b). Propentofylline treatment to nerve injured rats resulted in significant reversal of allodynia that lasted throughout the 14-day washout period. Spinal microglial activation was observed at days 28 and 42 post-injury at the protein level, in the absence of mRNA level changes. Less robust increases in GFAP immunoreactivity were observed at days 28 and 42 post-transection. Interestingly, propentofylline treatment suppressed microglial activation at both time points in this paradigm. Taken together, our results highlight the clinical potential of the glial modulating agent, propentofylline, for the treatment of neuropathic pain as well as a role for microglia in the long-term maintenance of allodynia.

Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical image analysis and flow cytometry

Microgliosis is implicated in the pathophysiology of several neurological disorders, including neuropathic pain. Consequently, perturbation of microgliosis is a mechanistic and drug development target in neuropathic pain, which highlights the requirement for specific, sensitive and reproducible methods of microgliosis measurement. In this study, we used the spinal microgliosis associated with L5 spinal nerve transection and minocycline-induced attenuation thereof to: (1) evaluate novel software based semi-quantitative image analysis paradigms for the assessment of immunohistochemical images. Microgliosis was revealed by immunoreactivity to OX42. Several image analysis paradigms were assessed and compared to a previously validated subjective categorical rating scale. This comparison revealed that grey scale measurement of the proportion of a defined area of spinal cord occupied by OX42 immunoreactive cells is a robust image analysis paradigm. (2) Develop and validate a flow cytometric approach for quantification of spinal microgliosis. The flow cytometric technique reliably quantified microgliosis in spinal cord cell suspensions, using OX42 and ED9 immunoreactivity to identify microglia.The results suggest that image analysis of immunohistochemical revelation of microgliosis reliably detects the spinal microgliosis in response to peripheral nerve injury and pharmacological attenuation thereof. In addition, flow cytometry provides an alternative approach for quantitative analysis of spinal microgliosis elicited by nerve injury.