Nasal application of HSV encoding human preproenkephalin blocks craniofacial pain in a rat model of traumatic brain injury.

According to Centers for Disease Control and Prevention, each year, an estimated 1.7 million Americans sustain a traumatic brain injury (TBI), which frequently leads to chronic craniofacial pain. In this study we examine a gene therapy approach to the treatment of post-TBI craniofacial neuropathic pain using nasal application of a herpes simplex virus (HSV)-based vector expressing human proenkephalin (SHPE) to target the trigeminal ganglia. Mild TBI was induced in rats by the use of a modified fluid percussion model. Two days after mild TBI, following the development of facial mechanical allodynia, animals received either an intranasal application of vehicle or recombinant HSV encoding human preproenkephalin or lacZ reporter gene encoding control vector (SHZ.1). Compared with baseline response thresholds, mild TBI in SHZ.1 or vehicle-treated animals induced a robust craniofacial allodynia lasting at least 45 days. On the other hand, nasal SHPE application 2 days post-TBI attenuated facial allodynia, reaching significance by day 4-7 and maintaining this effect throughout the duration of the experiment. Immunohistochemical examination revealed strong expression of human proenkephalin in trigeminal ganglia of SHPE, but not SHZ.1-treated rats. This study demonstrates that intranasal administration of HSV-based gene vectors may be a viable, non-invasive means of treating chronic craniofacial pain, including post-TBI pain.

Gene therapy for trigeminal pain in mice.

The aim of this study was to test the efficacy of a single direct injection of viral vector encoding for encephalin to induce a widespread expression of the transgene and potential analgesic effect in trigeminal behavioral pain models in mice. After direct injection of herpes simplex virus type 1 based vectors encoding for human preproenkephalin (SHPE) or the lacZ reporter gene (SHZ.1, control virus) into the trigeminal ganglia in mice, we performed an orofacial formalin test and assessed the cumulative nociceptive behavior at different time points after injection of the viral vectors. We observed an analgesic effect on nociceptive behavior that lasted up to 8 weeks after a single injection of SHPE into the trigeminal ganglia. Control virus-injected animals showed nociceptive behavior similar to naive mice. The analgesic effect of SHPE injection was reversed/attenuated by subcutaneous naloxone injections, a µ-opioid receptor antagonist. SHPE-injected mice also showed normalization in withdrawal latencies upon thermal noxious stimulation of inflamed ears after subdermal complete Freund's adjuvant injection, indicating widespread expression of the transgene. Quantitative immunohistochemistry of trigeminal ganglia showed expression of human preproenkephalin after SHPE injection. Direct injection of viral vectors proved to be useful for exploring the distinct pathophysiology of the trigeminal system and could also be an interesting addition to the pain therapists' armamentarium.

High-dose remifentanil prevents development of thermal hyperalgesia in a neuropathic pain model.

BACKGROUND: Intraoperative nerve lesions can lead to chronic postoperative pain. There are conflicting data as to whether or not anaesthetics administered intraoperatively are beneficial. We investigated if remifentanil administered at the time of nerve injury was able to attenuate neuropathic hypersensitivity. METHODS: Rats were anaesthetized with isoflurane, endotracheally intubated, and a tail vein catheter was inserted. Rats received an i.v. infusion of either saline or low- or high-dose remifentanil (2 or 20 µg kg(-1) min(-1), respectively) for 20 min. During this time, rats received a spinal nerve L5 transection to induce neuropathic pain or a sham procedure. Behavioural tests to assess mechanical and cold allodynia and heat hyperalgesia were performed on postoperative days 1, 3, 7, 14, 21, and 28. RESULTS: Sham-operated animals exhibited no hypersensitivity regardless of the intraoperative remifentanil dose. In rats which received spinal nerve L5 transection, mechanical and cold allodynia developed with no significant differences between treatment groups. However, thermal hyperalgesia was reduced in rats given high-dose remifentanil: mean (standard deviation) area under the curve 426 (53) compared with 363 (34) and 342 (24) in saline or low-dose remifentanil treated rats, respectively (P<0.05). CONCLUSIONS: High-dose remifentanil administered at the time of transection of the spinal nerve at L5 prevents subsequent thermal hyperalgesia.

Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins.

The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1-1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.

Herpes virus-based recombinant herpes vectors: gene therapy for pain and molecular tool for pain science.

This paper reviews work by Yeomans and Wilson in the area of herpes vector-mediated gene transfer to sensory neurons. Beginning in 1997, these researchers have published a number of papers describing and exploiting this technology in altering the phenotype of pain-sensing neurons (nociceptors). Their initial work, continuing to the present, inserted a transgene cassette encoding the human preproenkephalin gene into the thymidine kinase locus under control of a cytomegalovirus promoter. This vector induced enkephalin expression selectively in the nociceptors innervating the tissue onto which it was applied, producing a profound analgesic and antihyperalgesic in acute and chronic pain models in both rodents and non-human primates. An improved version of this vector is now in clinical trials. In addition to inducing the de novo expression of foreign transgenes, this group also investigated the utility of herpes vectors in altering the endogenous genome of nociceptors. Thus, they inserted antisense sequences for genes of interest in the physiology of these neurons and successfully and selectively knocked down expression of several proteins known or thought to be involved in various pain states, including calcitonin gene-related peptide and mu-opioid receptors. They also used similar techniques to investigate the involvement of acid-sensing ion channels and Nav1.7 sodium channel in different pain states. These experiments uniquely allowed for spatially and temporally selective investigations into the function of these proteins in pain, highly valuable information in target validation for therapy development.

Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Nav1.7 sodium channels in primary afferents.

Induction of peripheral inflammation increases the expression of the Nav1.7 sodium channel in sensory neurons, potentially increasing their excitability. Peripheral inflammation also produces hyperalgesia in humans and an increase in nociceptive responsiveness in animals. To test the relationship between these two phenomena we applied a recombinant herpes simplex-based vector to the hindpaw skin of mice, which encoded both green fluorescent protein (GFP) as well as an antisense sequence to the Nav1.7 gene. The hindpaw was subsequently injected with complete Freund's adjuvant to induce robust inflammation. Application of the vector, but not a control vector encoding only GFP, prevented an increase in Nav1.7 expression in GFP-positive neurons and prevented development of hyperalgesia in both C and Adelta thermonociceptive tests. These results provide clear evidence of the involvement of an increased expression of the Nav1.7 channel in nociceptive neurons in the development of inflammatory hyperalgesia.

Effects of systemic morphine on responses of primates to first or second pain sensations.

Despite evidence that systemic morphine preferentially attenuates second pain sensations that are presumed to result from activation of unmyelinated (C) nociceptors, most animal models of nociception elicit sensations that result from or are dominated by activation of myelinated (A-delta) nociceptors. Therefore, methods were developed to directly compare the effects of morphine on late (second) pain sensations and early onset (first) pain sensations in an animal model. In order to establish appropriate stimulus parameters, human psychophysical experiments compared characteristics of sensations evoked by brief (pulsed) thermal stimulation and ramp-and-hold thermal stimulation. Brief (500 msec) contact of a pre-heated thermode with the skin produced late pain sensations with peripheral conduction velocities in the range of C afferents, as estimated by latencies from stimulation of proximal and distal sites on the leg. The sensations evoked by brief contact increased with successive contacts (pulses) at 0.4 Hz, demonstrating temporal summation of sensation intensity. Pretreatment of the skin with capsaicin enhanced the late pain sensations from pulsed stimulation. In contrast, peak sensations evoked by ramp-and-hold thermal stimulation were evoked at similar latencies from disparate sites on the leg, and capsaicin pretreatment of the skin did not increase the magnitude of these sensations. The pulsed and ramp-and-hold forms of stimulation were used in a paradigm designed to test for differential effects of systemic morphine on operant responses of non-human primates. Low doses of morphine reduced operant responding to pulsed thermal contact, while higher doses were required to affect responses to ramp-and-hold thermal stimulation. The low doses of morphine did not suppress non-nociceptive (intertrial) motor responses, indicating that motor inhibition was not responsible for the effects on escape responses to pulsed stimulation. Measurements of skin temperature 10 cm from the site of stimulation showed that morphine had no effect on baseline temperature but attenuated changes in skin temperature that were elicited by pulsed and by ramp-and-hold stimulation. This effect of morphine on skin temperature responses could not account for the reduction of operant responsivity to thermal stimulation. These results support previous findings that systemic morphine preferentially attenuates second pain sensations, and a new animal model of morphine-sensitive thermal nociception is established. These findings demonstrate the importance of defining the sources of afferent input and the response measures in experiments which attempt to measure antinociceptive effects of pharmacological agents.

Peripheral and central p38 MAPK mediates capsaicin-induced hyperalgesia.

The stress-activated mitogen-activated protein kinase (MAPK) p38 is emerging as an important mediator of pain. The present study examined the possible involvement of peripheral and spinal p38 MAPK in capsaicin-induced thermal hyperalgesia. Topical capsaicin produced phosphorylation of p38 MAPK in the skin from the affected hindpaw as well as the corresponding lumbar spinal cord in a time dependent manner. Topical capsaicin produced robust C-fiber mediated thermal hyperalgesia that was inhibited by systemic, local peripheral, or central intrathecal pre-treatment with the p38 MAPK inhibitor, SD-282. Intraperitoneal SD-282 (10-60 mg/kg) significantly and dose-dependently attenuated capsaicin-induced C-fiber mediated thermal hyperalgesia. Similarly, 0.1-5mg/kg subcutaneous SD-282 in the hindpaw dose-dependently attenuated capsaicin-induced thermal hyperalgesia. Intrathecal administration of 1microg SD-282 was also anti-hyperalgesic in this model. Functionally, SD-282 decreased capsaicin-induced release of calcitonin gene related peptide in an in vitro skin release assay, consistent with a role for p38 MAPK in peripheral nerve function. These results suggest that p38 MAPK plays a role in the development of hyperalgesic states, exerting effects both centrally in the spinal cord and peripherally in sensory C fibers.

Antinociception induced by microinjection of substance P into the A7 catecholamine cell group in the rat.

Stimulation of neurons in the ventromedial medulla produces antinociception that is mediated in part by indirect activation of pontospinal noradrenergic neurons. Substance P-containing neurons located in the ventromedial medulla project to the A7 catecholamine cell group and may serve as an excitatory link between these two cell groups. Thus, the antinociception induced by stimulation of the neurons in ventromedial medulla may be mediated by substance P released from these projections which activates spinally projecting noradrenergic neurons in the A7 cell group. This hypothesis was tested by determining whether microinjection of various doses of substance P into the A7 cell group of the rat could induce antinociception. The results indicated that substance P induced dose-dependent antinociception that was more pronounced in the hindlimb ipsilateral to the microinjections. This observation is consistent with anatomical observations that noradrenergic A7 neurons project predominantly to the ipsilateral spinal cord dorsal horn. Moreover, the antinociceptive effects of substance P microinjection appear to be mediated at least in part by activation of spinally projecting noradrenergic neurons in the A7 cell group, because intrathecal injections of the alpha-2 noradrenergic antagonists yohimbine and idazoxan blocked these antinociceptive effects. The results of these experiments support the hypothesis that the antinociception induced by stimulation of neurons in the ventromedial medulla is mediated in part by activation of substance P-containing neurons that project to, and activate, spinally projecting noradrenergic neurons located in the A7 catecholamine cell group.

Differential antinociceptive effects of spinal opioids on foot withdrawal responses evoked by C fibre or A delta nociceptor activation.

1. Intrathecal application of mu, delta, and kappa opioids attenuate responses on several tests of animal nociception. However, the potency of these opioids differ depending on which tests were used. One factor contributing to these discrepancies is that different types of noxious stimuli activate different sets of nociceptor types, which may be differentially sensitive to opiate inhibition. To examine this hypothesis, we used a recently developed behavioural test which allows for differential assessment of nociception evoked by the activation of myelinated (A delta) and unmyelinated C thermonociceptors. 2. Administration of a kappa-selective agonist was ineffective on either type of response. Delta1 drugs were slightly more potent on C fibre-mediated responses than on A delta-mediated responses. 3. Intrathecal mu and delta2 drugs were antinociceptive on both A delta and C nociceptor-mediated responses. However, unlike the delta1 effects, the dose-response curves for mu and delta2 drugs were significantly more steep for A delta than for C fibre-mediated responses, potentially indicating differences in the mechanisms by which the drugs act on these 2 response types.

Purification of adrenal chromaffin cells increases antinociceptive efficacy of xenotransplants without immunosuppression.

We have found that immunosuppression is necessary for the survival of xenogeneic adrenal medullary transplants. Because chromaffin cells are essentially nonimmunogenic, it is likely that the highly immunogenic "passenger" cells in the transplant preparation bring about rejection. This article describes a procedure that produces an essentially pure preparation of chromaffin cells for transplantation. Bovine adrenal medullary cells were isolated and differentially plated, resulting in a semipurified preparation of chromaffin cells. Ferromagnetic beads were added to the cell suspension, some of which were phagocytized by endothelial cells, which allowed their removal by exposure to a magnet. The remaining cells were then exposed to ferromagnetic beads coated with isolectin B4 from Griffonia simplicifolia and once again to a magnetic field. The "semipurified" preparation contained approximately 90% chromaffin cells, whereas the "highly purified" preparation was > 99.5% chromaffin cells as determined immunohistochemically. The immunogenicity of the two cell preparations was assessed in vitro by determining their capacity to evoke lymphocyte proliferation. Rat spleen lymphocytes were mixed with either a highly purified or semipurified population of bovine chromaffin cells. The results of this assay demonstrated that the highly purified preparation was a much weaker stimulant of lymphocyte proliferation than was the semipurified preparation and may demonstrate better graft survival in vivo. Transplantation via intrathecal catheter of either 80,000 or 250,000 cells from the highly or partially purified preparations onto the lumbar spinal cord of nonimmunosuppressed and non-nicotine-stimulated rats produced a cell number-dependent antinociception for both A(delta) and C fiber-mediated thermonociception at 6 days after transplantation. After 6 days and up to 28 days, only the "highly purified" preparation showed antinociception. These results suggest that nearly complete purification of bovine chromaffin cells minimizes immunorejection of xenogeneic transplants of these cells.

Projections of substance P-immunoreactive neurons located in the ventromedial medulla to the A7 noradrenergic nucleus of the rat demonstrated using retrograde tracing combined with immunocytochemistry.

Stimulation of neurons located in the ventromedial medulla (VMM), including the nucleus raphe magnus (RMg), produces antinociception which appears to be mediated in part by activation of spinally-projecting noradrenergic neurons located in the A7 catecholamine nucleus. Although the identity of the VMM neurons that project to the A7 nucleus is not known, there is indirect evidence that these neurons contain substance P. This possibility was examined by injecting the retrograde tracer Fluoro-Gold into the A7 nucleus and determining whether substance P-immunoreactive neurons in the VMM were labeled with Fluoro-Gold. The results of these experiments demonstrated that numerous substance P-immunoreactive cells in the RMg, gigantocellular reticular nucleus pars alpha and the paragigantocellular reticular nucleus were retrogradely labeled by an injection of Fluoro-Gold into the A7 nucleus. These observations indicate that substance P-containing neurons in these areas of the VMM project to the A7 nucleus. Thus, the antinociception induced by stimulation of the VMM may be mediated by activation of substance P-containing neurons that project to and activate spinally projecting noradrenergic neurons in the A7 nucleus.

Low but not high rate noxious radiant skin heating evokes a capsaicin-sensitive increase in spinal cord dorsal horn release of substance P.

Some kinds of nociception appear to be partially mediated by the release of substance P (SP) in the spinal cord dorsal horn from terminals of primary afferent nociceptors. Only some nociceptors contain and release SP however. Specifically, SP appears to be released by unmyelinated (C) nociceptive afferents when activated by noxious stimulation to the skin, but does not appear to be contained in cutaneous myelinated (A delta) nociceptive afferents. We have proposed a model of nociception in rats that uses different rates of noxious skin heating to allow for differential assessment on behavioral responses mediated by the activation of A delta or C fiber nociceptors. As one means of testing the validity of this model we have examined the effects of using high and low rate noxious skin heating on the dorsal horn release of substance P-like immunoreactivity (SPLI) in decerebrate/spinal transected animals. Consistent with the model, low rate skin heating evokes a significant increase in dorsal horn SPLI release indicating C fiber mediation, whereas high rate skin heating did not evoke SP release, indicating mediation by afferents other than C afferents, i.e. A delta nociceptive afferents. Also consistent with behavioral effects, topical application of capsaicin, which sensitizes C nociceptors, increased the SPLI release evoked by low but not high rate skin heating. These data provide additional evidence that foot withdrawals evoked by low rate skin heating are mediated by C fiber activation, whereas foot withdrawals evoked by high rate skin heating are evoked by A delta fiber activation.

Comparisons of dose-dependent effects of systemic morphine on flexion reflex components and operant avoidance responses of awake non-human primates.

Electromyographic activity and the force of reflex and operant responses were recorded following administration of morphine. Low doses facilitated reflex responses to input from A-delta afferents but not from A-beta input. Higher doses inhibited A-delta responses but not A-beta responses. Operant avoidance responses to visual cues were unchanged. Thus, depending on the dose, nociceptive reflexes were facilitated or inhibited, without associated effects on non-nociceptive input or on motor output.

The function of noradrenergic neurons in mediating antinociception induced by electrical stimulation of the locus coeruleus in two different sources of Sprague-Dawley rats.

Although noradrenergic neurons in the nucleus locus coeruleus are known to project to the spinal cord, these neurons appear to innervate different regions of the spinal cord in Sprague-Dawley rats obtained from two different vendors. Recent anatomical studies demonstrated that the noradrenergic neurons in the locus coeruleus in Sasco Sprague-Dawley rats primarily innervate the ventral horn, whereas Harlan Sprague-Dawley rats have coeruleospinal projections that terminate in the dorsal horn of the spinal cord. This report describes the results of behavioral experiments that were designed to determine the functional significance of these anatomical differences. Electrical stimulation of neurons in the locus coeruleus produced antinociception in both Harlan and Sasco rats. The antinociception in Harlan rats was readily reversed by intrathecal injection of yohimbine, a selective alpha 2-adrenoceptor antagonist, or by phentolamine, a non-selective alpha 2-adrenoceptor antagonist. In contrast, these antagonists did not alter the antinociception produced by locus coeruleus stimulation in Sasco rats. Finally, the alpha 2-antagonist, idazoxan, did not alter the antinociceptive effect of locus coeruleus stimulation in either group of rats. These observations indicate that coeruleospinal noradrenergic neurons in Harlan and Sasco Sprague-Dawley rats have different physiological functions. Thus, electrical stimulation of noradrenergic neurons in the locus coeruleus that innervate the spinal cord dorsal horn (Harlan rats) produces antinociception, but stimulation of coeruleospinal noradrenergic neurons that project to the ventral horn (Sasco rats) does not produce antinociception. It is likely that genetic differences between these outbred stocks of rats account for the fundamental differences in the projections of coeruleospinal neurons and their function in controlling nociception.

The noradrenergic innervation of the spinal cord: differences between two substrains of Sprague-Dawley rats determined using retrograde tracers combined with immunocytochemistry.

We have recently described the spinal cord terminations of noradrenergic neurons located in the A5, A6 and A7 cell groups. However, recent reports from another laboratory, using similar experimental methods, have described results that are profoundly different. The present experiments were designed to determine whether these discrepant results are due to fundamental differences between the substrains of rats used in the conflicting experiments. To this end, retrograde tract tracing experiments were done using Sprague-Dawley rats from either Sasco, Inc. or Harlan Sprague-Dawley, Inc. The results indicate that noradrenergic neurons in the pontine catecholamine cell groups exhibit remarkably different spinal cord projections in these two substrains of Sprague-Dawley derived rats.

Ameroid rings for gradual chronic constriction of the sciatic nerve in rats: contribution of different nerves to neuropathic pain.

Mononeuropathy was induced by placing an ameroid ring around the sciatic nerve and was compared with chronic constriction injury (CCI) of the sciatic nerve [Pain 33 (1988) 87] in rats. Mechanical allodynia was assessed and the role of sciatic and saphenous afferents (Adelta and C) in thermal hyperalgesia investigated. A shorter duration of mechanical allodynia in ameroid rats as compared to CCI rats was observed. Thermal hyperalgesia was observed in the saphenous innervated skin of the hindpaw for Adelta and C nociceptors in ameroid and for Adelta nociceptors only in CCI rats, respectively. The sciatic innervated skin showed a thermal hypoalgesia with a fast onset for Adelta afferents and a slower onset for C afferents in CCI and ameroid rats. The duration of both thermal hypo- and hyperalgesia was longer in ameroid rats. We conclude that ameroid rings are a useful tool for the investigation of long-duration hyperalgesic effects of nerve injury, as the effects were more stable and seen for a longer time (>8 weeks) as compared to the CCI model. The uninjured saphenous afferents, in particular C fibers, mediate thermal hyperalgesia after chronic constriction of the sciatic nerve using an ameroid ring.

Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: a microdialysis study.

Animal studies have documented a critical role for cytokines in cell signaling events underlying inflammation and pain associated with tissue injury. While clinical reports indicate an important role of cytokines in inflammatory pain, methodological limitations have made systematic human studies difficult. This study examined the utility of a human in vivo bioassay combining microdialysis with multiplex immunoassay techniques for measuring cytokine arrays in tissue. The first experiment measured cytokines in interstitial fluid collected from non-inflamed and experimentally inflamed skin (UVB). The effects of noxious heat on cytokine release were also assessed. The second experiment examined whether anti-hyperalgesic effects of the COX-inhibitor ibuprofen were associated with decreased tissue levels of the pro-inflammatory cytokines IL-1 beta and IL-6. In the first experiment, inflammation significantly increased IL-1 beta, IL-6, IL-8, IL-10, G-CSF, and MIP-1 beta. Noxious heat but not experimental inflammation significantly increased IL-7 and IL-13. In the second experiment, an oral dose of 400 and 800 mg ibuprofen produced similar anti-hyperalgesic effects suggesting a ceiling effect. Tissue levels of IL-1 beta and IL-6 were not affected after the 400mg dose but decreased significantly (44+/-32% and 38+/-13%) after the 800 mg dose. These results support the utility of explored method for tracking cytokines in human tissue and suggest that anti-hyperalgesic and anti-inflammatory effects of ibuprofen are at least partially dissociated. The data further suggest that high clinical doses of ibuprofen exert anti-inflammatory effects by down-regulating tissue cytokine levels. Explored human bioassay is a promising tool for studying the pathology and pharmacology of inflammatory and chronic pain conditions.

Genetic therapy for pain management.

Two approaches to genetic therapy for the management of chronic pain have recently been investigated in animal models of pain. First, transgene-mediated delivery of antinociceptive molecules to the cerebrospinal fluid has been performed with engineered cell lines transplanted to the subarachnoid space and with recombinant adenoviruses that transduce pia mater cells. Second, the phenotype of nociceptive neurons has been altered by recombinant herpes viruses overexpressing antinociceptive peptides or reducing expression of endogenous nociceptive molecules. Both approaches attenuate or reverse persistent nociceptive states, suggesting use in the development of genetic therapy for pain management in humans.