Neuropathic pain after spinal cord injury (SCI) represents a difficult problem that is commonly refractory to conventional medical management. To determine if spinal release of gamma-amino butyric acid (GABA) could reduce below-level central neuropathic pain after SCI, we constructed a replication-incompetent herpes simplex virus (HSV)-based vector encoding one isoform of human glutamic acid decarboxylase (GAD67). Dorsal root ganglion (DRG) neurons transduced in vitro or in vivo by subcutaneous inoculation produced GAD and released GABA constitutively. T13 spinal cord hemisection resulted in central neuropathic pain manifested by mechanical allodynia and thermal hyperalgesia. Subcutaneous inoculation of the vector into both feet reduced both manifestations of below-level SCI pain; the vector-mediated effect was partially reversed by intrathecal bicuculline or phaclofen at doses that did not affect thresholds in normal or injured uninoculated animals. Vector-mediated GABA release attenuated the increase in spinal calcitonin gene-related peptide immunoreactivity caused by cord hemisection. These results suggest that HSV-mediated gene transfer to DRG could be used to treat below-level central neuropathic pain after incomplete SCI.

We examined the pharmacologic characteristics of herpes simplex virus (HSV) vector-mediated expression of proenkephalin in the dorsal root ganglion in a rodent model of neuropathic pain. We found that: (i). vector-mediated enkephalin produced an antiallodynic effect that was reversed by naloxone; (ii). vector-mediated enkephalin production in animals with spinal nerve ligation prevented the induction of c-fos expression in second order sensory neurons in the dorsal horn of spinal cord; (iii). the effect of vector-mediated enkephalin enhanced the effect of morphine, reducing the ED(50) of morphine 10-fold; (iv). animals did not develop tolerance to the continued production of vector-mediated enkephalin over a period of several weeks; and, (v). vector transduction continued to provide an analgesic effect despite the induction of tolerance to morphine. This is the first demonstration of gene transfer to provide an analgesic effect in neuropathic pain. The pharmacologic analysis demonstrates that transgene-mediated expression and local release of opioid peptides produce some effects that are distinct from peptide analogues delivered pharmacologically.

Background: To examine the role of inflammatory mediators in neuropathic pain, we used a replication-defective genomic herpes simplex virus (HSV)-based vector containing the coding sequence for the anti-inflammatory peptide interleukin (IL)-4 under the transcriptional control of the HSV ICP4 immediate early promoter, vector S4IL4, to express IL-4 in dorsal root ganglion (DRG) neurons in vivo.

We examined the role of spinal tumor necrosis factor-alpha (TNFa) in neuropathic pain of peripheral nerve origin. Two weeks after selective L5 spinal nerve ligation (SNL), rats exhibiting mechanical allodynia and thermal hyperalgesia showed a marked increase in full-length membrane-associated TNFa (mTNFa) in the dorsal horn of spinal cord, in the absence of detectable soluble TNFa peptide. Local release of the soluble p55 TNF receptor, achieved by herpes simplex virus vector-based gene transfer to dorsal root ganglion, resulted in a reduction of mTNFa and concomitant reductions in interleukin-1b and phosphorylated p38 MAP kinase. Subcutaneous inoculation of soluble p55 TNF receptor expressing HSV vector into the plantar surface of the hind foot ipsilateral to the ligation 1 week before SNL delayed the development of both mechanical allodynia and thermal hyperalgesia; subcutaneous inoculation into the hind foot ipsilateral to the ligation 1 week after SNL resulted in a statistically significant reduction in mechanical allodynia and thermal hyperalgesia that was apparent 1 week after inoculation. These results suggest a novel 'reverse signaling' through glial mTNFa, which may be exploited to downregulate the neuroimmune reaction in spinal cord to reduce chronic neuropathic pain.

To dissect the molecular basis of the neuroimmune response associated with the genesis of inflammatory (nociceptive) pain, we constructed a herpes simplex virus-based gene transfer vector to express the antiinflammatory cytokine interleukin-10 (IL-10), and used it to examine the effect of IL-10 expression in activated microglial cells in vitro, and in inflammatory pain in vivo. IL-10 reduced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and decreased the expression of full-length membrane spanning tumor necrosis factor-a (mTNFa) following lipopolysaccharide stimulation of microglia in vitro. IL-10 also reduced intracellular cleavage of mTNFa and release of the soluble cleavage product sTNFa. Similar effects on TNFa expression were observed when the cells were pretreated with a p38 MAPK inhibitor. In animals, injection of a dilute solution of formalin in the skin resulted in an increase in mTNFa in spinal dorsal horn, without detectable sTNFa. Local release of IL-10 achieved by gene transfer reduced the number of spontaneous flinches in the early and delayed phases of the formalin test of inflammatory pain. The effect of IL-10 on nocisponsive behavior correlated with a block in phosphorylation of p38 and reduced expression of 26 kDa mTNFa in spinal microglia. The results emphasize the key role played by membrane TNFa in the spinal neuroimmune response in pain caused by peripheral inflammation.

BackgroundHIV-associated sensory neuropathy (HIV-SN) is one of the most common forms of peripheral neuropathy, affecting about 30% of people with acquired immune deficiency syndrome (AIDS). The symptoms of HIV-SN are dominated by neuropathic pain. Glia activation in the spinal cord has become an attractive target for attenuating chronic pain. This study will investigate the role of spinal TNFα released from glia in HIV-related neuropathic pain.ResultsPeripheral gp120 application into the rat sciatic nerve induced mechanical allodynia for more than 7 weeks, and upregulated the expression of spinal TNFα in the mRNA and the protein levels at 2 weeks after gp120 application. Spinal TNFα was colocalized with GFAP (a marker of astrocytes) and Iba1 (a marker of microglia) in immunostaining, suggesting that glia produce TNFα in the spinal cord in this model. Peripheral gp120 application also increased TNFα in the L4/5 DRG. Furthermore, intrathecal administration of TNFα siRNA or soluble TNF receptor reduced gp120 application-induced mechanical allodynia.ConclusionsOur results indicate that TNFα in the spinal cord and the DRG are involved in neuropathic pain, following the peripheral HIV gp120 application, and that blockade of the glial product TNFα reverses neuropathic pain induced by HIV gp120 application.

Tolerance and dependence are common complications of long-term treatment of pain with opioids, which substantially limit the long-term use of these drugs. The mechanisms underlying these phenomena are poorly understood. Studies have implicated the midbrain periaqueductal gray (PAG) in the pathogenesis of morphine withdrawal, and recent evidence suggests that proinflammatory cytokines in the PAG may play an important role in morphine withdrawal. Here we report that chronic morphine withdrawal-induced upregulation of glial fibrillary acidic protein (GFAP), tumor necrosis factor alpha (TNFα) and phosphorylation of ERK1/2 (pERK1/2) in the caudal ventrolateral PAG (vlPAG). Microinjection of recombinant TNFα into the vlPAG followed by intraperitoneal naloxone resulted in morphine withdrawal-like behavioral signs, and upregulation of pERK1/2, expression of Fos, and phosphorylation of cAMP response element binding (pCREB) protein. We used a herpes simplex virus (HSV)-based vector expressing p55 soluble TNF receptor (sTNFR) microinjected into the PAG to examine the role of the proinflammatory cytokine TNFα in the PAG in the naloxone-precipitated withdrawal response. Microinjection of HSV vector expressing sTNFR into the PAG before the start of morphine treatment significantly reduced the naloxone-precipitated withdrawal behavioral response and downregulated the expression of GFAP and TNFα in astrocytes of the PAG. TNFR type I colocalized with neuronal pERK1/2. Microinjection of HSV vector expressing sTNFR into the PAG also significantly reduced the phosphorylation of both ERK1/2 and CREB, and reduced Fos immunoreactivity in neurons of the PAG following naloxone-precipitated withdrawal. These results support the concept that proinflammatory cytokines expressed in astrocytes in the PAG may play an important role in the pathogenesis of morphine withdrawal response.

We tested whether transfer of the gene coding for glutamic acid decarboxylase to dorsal root ganglion using a herpes simplex virus vector to achieve release of GABA in dorsal horn would attenuate nociception in this condition. Subcutaneous inoculation of a replication-defective herpes simplex virus vector expressing glutamic acid decarboxylase (vector QHGAD67) 7 days after selective L5 spinal nerve ligation reversed mechanical allodynia and thermal hyperalgesia; the antiallodynic effect lasted 6 weeks and was reestablished by reinoculation. QH-GAD67 inoculation also suppressed induction of c-Fos and phosphorylated extracellular signal-regulated kinase 1 and 2 in the spinal cord.Peripheral neuropathic pain is a common and difficult to treat concomitant of polyneuropathy or structural nerve injury. Opioids are relatively ineffective, and their use is limited by side effects. Antidepressants and anticonvulsants have demonstrated efficacy in randomized controlled trials but provide only 50% relief in less than half of patients treated. 1 Among the complex mechanisms underlying neuropathic pain, partial nerve injury results in a selective loss of GABAergic inhibitory synaptic currents in spinal cord 2 that contribute to abnormal pain sensitivity and the phenotypic features of the neuropathic pain syndrome. GABAergic agents have not been widely used in the treatment of neuropathic pain because the therapeutic window of these agents is modest and the dose is limited by side effects.Gene transfer represents a novel and useful means to target expression of peptides to focal sites within the nervous system. We have shown that transduction of sensory neurons of the dorsal root ganglion (DRG) by footpad inoculation with herpes simplex virus (HSV)-based vectors can be used to achieve a regional antinociceptive effect. HSV vectors coding for proenkephalin or the glial cell-derived neurotrophic factor produce an antihyperalgesic and antiallodynic effect in rodent models of inflammatory pain, neuropathic pain, and pain resulting from cancer in bone, 3-6 and an HSV-vector expressing glutamic acid decarboxylase (GAD) provides an analgesic effect in the central neuropathic pain syndrome resulting from spinal cord injury. 7 In this study, we examined the antinociceptive effect of GAD expressed from an HSV-based vector in the spinal nerve ligation (SNL) model of neuropathic pain in the rat. Materials and MethodsThe nonreplicating HSV vector QHGAD67, which is defective in expression of the HSV immediate early genes ICP4, ICP22, ICP27, and ICP47 and contains the human GAD67 gene under the control of the human cytomegalovirus immediate early promoter in the U L 41 locus, has been described previously. 7 The control vector QOZHG is defective in the same HSV genes but contains the green fluorescent protein and Escherichia coli lacZ reporter genes. 7Male Sprague-Dawley rats weighing 225 to 250gm underwent selective L5 SNL, as described previously, 5 with the approval of the University Committee on Use and Care of Animals. One wee...