Injury-induced Mechanical Hypersensitivity Research Articles

Role of spinal phosphatases in cannabinoid-induced antinociception in a rodent neuropathic pain model

A cannabinoid receptor type 2 (CBR2) agonist administered intrathecally reduces peripheral nerve injury-induced mechanical hypersensitivity without exhibiting neurological side effects. Using in vitro rat cortical primary microglial cells, we previously observed that a CBR2 agonist induces the expression of MAP kinase phosphatase (MKP)-1 and 3 and reduces p-ERK, TNF expression and microglial migration. Herein, we investigated the hypothesis that CBR2 agonists produce analgesia by inducing spinal MKP expression and ERK dephosphorylation. We tested our hypothesis in vivo using male Sprague-Dawley rats and the L5 spinal nerve transection (L5NT) neuropathic pain model. Sham surgery was used as the control group. JWH015 (50 μg), a CBR2 agonist, was administered intrathecally by lumbar puncture four days after surgery. Mechanical withdrawal thresholds were determined with von Frey filaments, before surgery, four days after surgery and two hours after treatment. MKP-1 and pCREB expression was assessed using immunohistochemistry. Peripheral nerve injury-induced mechanical hypersensitivity, increased spinal pCREB and reduced spinal MKP-1 expression. JWH015 reversed nerve injury-induced pCREB expression and restored MKP-1 expression levels in association with its antinociceptive effects. To further investigate the functional association of MKPs with the antinociceptive effects of CBR2 agonists, we challenged the antinociceptive effects of JWH015 with triptolide (a MKP-1 and MKP-3 inhibitor). We observed that the intrathecal co-administration of triptolide (20 μg) with JWH015 blocked the antinociceptive effects of JWH015. These results suggest that CBR2 agonists may induce antinociception in neuropathic pain conditions via MKP induction. In conclusion, our results demonstrate that MKP modulation is part of the mechanism of action of CBR2 agonists. To further investigate the role of MKP in neuropathic pain and CBR2 agonist mechanisms of action we will determine the expression of spinal MKP-3 and the cellular localization of MKP-1 and MKP-3. (Supported by APS 2009 future leaders in pain research award and NIH 1R01DA025211.) This abstract will also be presented as an Oral Paper Presentation on May 8. Refer to the daily Schedule-At-A-Glance for presentation time and location.

Injury-induced mechanical hypersensitivity requires C-low threshold mechanoreceptors

Mechanical pain contributes to the morbidity associated with inflammation and trauma, but primary sensory neurons that convey the sensation of acute and persistent mechanical pain have not been identified. Dorsal root ganglion (DRG) neurons transmit sensory information to the spinal cord using the excitatory transmitter glutamate1, a process that depends on glutamate transport into synaptic vesicles for regulated exocytotic release. Here we report that a small subset of cells in the DRG expresses the low abundance vesicular glutamate transporter VGLUT3. In the dorsal horn of the spinal cord, these afferents project to lamina I and the innermost layer of lamina II, which has previously been implicated in persistent pain caused by injury2. Since the different VGLUT isoforms generally exhibit a nonredundant pattern of expression3, we used VGLUT3 knock-out (KO) mice to assess the role of VGLUT3+ primary afferents in the behavioral response to somatosensory input. The loss of VGLUT3 specifically impairs mechanical pain sensation and in particular the mechanical hypersensitivity to normally innocuous stimuli that accompanies inflammation, nerve injury and trauma. Direct recording from VGLUT3+ neurons in the DRG further identifies them as a poorly understood population of unmyelinated, low threshold mechanoreceptors (C-LTMRs)4,5. The analysis of VGLUT3 KO mice now indicates a critical role for C-LTMRs in the mechanical hypersensitivity caused by injury.

Genetic Knockout and Pharmacologic Inhibition of Neuronal Nitric Oxide Synthase Attenuate Nerve Injury-Induced Mechanical Hypersensitivity in Mice

Neuronal nitric oxide synthase (nNOS) is a key enzyme for nitric oxide production in neuronal tissues and contributes to the spinal central sensitization in inflammatory pain. However, the role of nNOS in neuropathic pain remains unclear. The present study combined a genetic strategy with a pharmacologic approach to examine the effects of genetic knockout and pharmacologic inhibition of nNOS on neuropathic pain induced by unilateral fifth lumbar spinal nerve injury in mice. In contrast to wildtype mice, nNOS knockout mice failed to display nerve injury-induced mechanical hypersensitivity. Furthermore, either intraperitoneal (100 mg/kg) or intrathecal (30 μg/5 μl) administration of L-NG-nitro-arginine methyl ester, a nonspecific NOS inhibitor, significantly reversed nerve injury-induced mechanical hypersensitivity on day 7 post-nerve injury in wildtype mice. Intrathecal injection of 7-nitroindazole (8.15 μg/5 μl), a selective nNOS inhibitor, also dramatically attenuated nerve injury-induced mechanical hypersensitivity. Western blot analysis showed that the expression of nNOS protein was significantly increased in ipsilateral L5 dorsal root ganglion but not in ipsilateral L5 lumbar spinal cord on day 7 post-nerve injury. The expression of inducible NOS and endothelial NOS proteins was not markedly altered after nerve injury in either the dorsal root ganglion or spinal cord. Our findings suggest that nNOS, especially in the dorsal root ganglion, may participate in the development and/or maintenance of mechanical hypersensitivity after nerve injury.

Activation of Src-Family Kinases in Spinal Microglia Contributes to Mechanical Hypersensitivity after Nerve Injury

Hypersensitivity to mechanical stimulation is a well documented symptom of neuropathic pain, for which there is currently no effective therapy. Src-family kinases (SFKs) are involved in proliferation and differentiation and in neuronal plasticity, including long-term potentiation, learning, and memory. Here we show that activation of SFKs induced in spinal cord microglia is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in SFK phosphorylation in the ipsilateral dorsal horn, and SFKs were activated in hyperactive microglia but not in neurons or astrocytes. Intrathecal administration of the Src-family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) suppressed nerve injury-induced mechanical hypersensitivity but not heat and cold hypersensitivity. Furthermore, PP2 reversed the activation of extracellular signal-regulated protein kinase (ERK), but not p38 mitogen-activated protein kinase, in spinal microglia. In contrast, there was no change in SFK phosphorylation in primary sensory neurons, and PP2 did not decrease the induction of transient receptor potential ion channel TRPV1 and TRPA1 in sensory neurons. Together, these results demonstrate that SFK activation in spinal microglia contributes to the development of mechanical hypersensitivity through the ERK pathway. Therefore, preventing the activation of the Src/ERK signaling cascade in microglia might provide a fruitful strategy for treating neuropathic pain.