Sex Dependent Disparities in the Central Innate Immune Response after Moderate Spinal Cord Contusion in Rat.

Subacute spinal cord injury (SCI) displays a complex pathophysiology associated with pro-inflammation and ensuing tissue damage. Microglia, the resident innate immune cells of the CNS, in concert with infiltrating macrophages, are the primary contributors to SCI-induced inflammation. However, subpopulations of activated microglia can also possess immunomodulatory activities that are essential for tissue remodeling and repair, including the production of anti-inflammatory cytokines and growth factors that are vital for SCI recovery. Recently, reports have provided convincing evidence that sex-dependent differences exist in how microglia function during CNS pathologies and the extent to which these cells contribute to neurorepair and endogenous recovery. Herein we employed flow cytometry and immunohistochemical methods to characterize the phenotype and population dynamics of activated innate immune cells within the injured spinal cord of age-matched male and female rats within the first week (7 days) following thoracic SCI contusion. This assessment included the analysis of pro- and anti-inflammatory markers, as well as the expression of critical immunomodulatory kinases, including P38 MAPK, and transcription factors, such as NFκB, which play pivotal roles in injury-induced inflammation. We demonstrate that activated microglia from the injured spinal cord of female rats exhibited a significantly diminutive pro-inflammatory response, but enhanced anti-inflammatory activity compared to males. These changes included lower levels of iNOS and TLR4 expression but increased levels of ARG-1 and CD68 in females after SCI. The altered expression of these markers is indicative of a disparate secretome between the microglia of males and females after SCI and that the female microglia possesses higher phagocytic capabilities (increased CD68). The examination of immunoregulatory kinases and transcription factors revealed that female microglia had higher levels of phosphorylated P38Thr180/Tyr182 MAPK and nuclear NFκB pp50Ser337 but lower amounts of nuclear NFκB pp65Ser536, suggestive of an attenuated pro-inflammatory phenotype in females compared to males after SCI. Collectively, this work provides novel insight into some of the sex disparities that exist in the innate immune response after SCI and indicates that sex is an important variable when designing and testing new therapeutic interventions or interpretating positive or negative responses to an intervention.

Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal.

During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs' miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.

Combined non-psychoactive Cannabis components cannabidiol and ß-caryophyllene reduce chronic pain via CB1 interaction in a rat spinal cord injury model.

The most frequently reported use of medical marijuana is for pain relief. However, its psychoactive component Δ9-tetrahydrocannabinol (THC) causes significant side effects. Cannabidiol (CBD) and ß-caryophyllene (BCP), two other cannabis constituents, possess more benign side effect profiles and are also reported to reduce neuropathic and inflammatory pain. We evaluated the analgesic potential of CBD and BCP individually and in combination in a rat spinal cord injury (SCI) clip compression chronic pain model. Individually, both phytocannabinoids produced dose-dependent reduction in tactile and cold hypersensitivity in male and female rats with SCI. When co-administered at fixed ratios based on individual A50s, CBD and BCP produced enhanced dose-dependent reduction in allodynic responses with synergistic effects observed for cold hypersensitivity in both sexes and additive effects for tactile hypersensitivity in males. Antinociceptive effects of both individual and combined treatment were generally less robust in females than males. CBD:BCP co-administration also partially reduced morphine-seeking behavior in a conditioned place preference (CPP) test. Minimal cannabinoidergic side effects were observed with high doses of the combination. The antinociceptive effects of the CBD:BCP co-administration were not altered by either CB2 or µ-opioid receptor antagonist pretreatment but, were nearly completely blocked by CB1 antagonist AM251. Since neither CBD or BCP are thought to mediate antinociception via CB1 activity, these findings suggest a novel CB1 interactive mechanism between these two phytocannabinoids in the SCI pain state. Together, these findings suggest that CBD:BCP co-administration may provide a safe and effective treatment option for the management of chronic SCI pain.

Attenuation of SCI-Induced Hypersensitivity by Intensive Locomotor Training and Recombinant GABAergic Cells.

The underlying mechanisms of spinal cord injury (SCI)-induced chronic pain involve dysfunctional GABAergic signaling and enhanced NMDA signaling. Our previous studies showed that SCI hypersensitivity in rats can be attenuated by recombinant rat GABAergic cells releasing NMDA blocker serine-histogranin (SHG) and by intensive locomotor training (ILT). The current study combines these approaches and evaluates their analgesic effects on a model of SCI pain in rats. Cells were grafted into the spinal cord at 4 weeks post-SCI to target the chronic pain, and ILT was initiated 5 weeks post-SCI. The hypersensitivity was evaluated weekly, which was followed by histological and biochemical assays. Prolonged effects of the treatment were evaluated in subgroups of animals after we discontinued ILT. The results show attenuation of tactile, heat and cold hypersensitivity in all of the treated animals and reduced levels of proinflammatory cytokines IL1ß and TNFα in the spinal tissue and CSF. Animals with recombinant grafts and ILT showed the preservation of analgesic effects even during sedentary periods when the ILT was discontinued. Retraining helped to re-establish the effect of long-term training in all of the groups, with the greatest impact being in animals with recombinant grafts. These findings suggest that intermittent training in combination with cell therapy might be an efficient approach to manage chronic pain in SCI patients.

Analgesic effect of recombinant GABAergic precursors releasing ω-conotoxin MVIIA in a model of peripheral nerve injury in rats.

Development of chronic pain has been attributed to dysfunctional GABA signaling in the spinal cord. Direct pharmacological interventions on GABA signaling are usually not very efficient and often accompanied by side effects due to the widespread distribution of GABA receptors in CNS. Transplantation of GABAergic neuronal cells may restore the inhibitory potential in the spinal cord. Grafted cells may also release additional analgesic peptides by means of genetic engineering to further enhance the benefits of this approach. Conopeptides are ideal candidates for recombinant expression using cell-based strategies. The omega-conopeptide MVIIA is in clinical use for severe pain marketed as FDA approved Prialt in the form of intrathecal injections. The goal of this study was to develop transplantable recombinant GABAergic cells releasing conopeptide MVIIA and to evaluate the analgesic effect of the grafts in a model of peripheral nerve injury-induced pain. We have engineered and characterized the GABAergic progenitors expressing MVIIA. Recombinant and nonrecombinant cells were intraspinally injected into animals after the nerve injury. Animals were tested weekly up to 12 weeks for the presence of hypersensitivity, followed by histochemical and biochemical analysis of the tissue. We observed beneficial effects of the grafted cells in reducing hypersensitivity in all grafted animals, especially potent in the recombinant group. The level of pain-related cytokines was reduced in the grafted animals and correlation between these pain markers and actual behavior was indicated. This study demonstrated the feasibility of recombinant cell transplantation in the management of chronic pain.

Cannabinoid receptor agonists from Conus venoms alleviate pain-related behavior in rats.

Cannabinoid (CB) receptor agonists show robust antinociceptive effects in various pain models. However, most of the clinically potent CB1 receptor-active drugs derived from cannabis are considered concerning due to psychotomimetic side effects. Selective CB receptor ligands that do not induce CNS side effects are of clinical interest. The venoms of marine snail Conus are a natural source of various potent analgesic peptides, some of which are already FDA approved. In this study we evaluated the ability of several Conus venom extracts to interact with CB1 receptor. HEK293 cells expressing CB1 receptors were treated with venom extracts and CB1 receptor internalization was analyzed by immunofluorescence. Results showed C. textile (C. Tex) and C. miles (C. Mil) samples as the most potent. These were serially subfractionated by HPLC for subsequent analysis by internalization assays and for analgesic potency evaluated in the formalin test and after peripheral nerve injury. Intrathecal injection of C. Tex and C. Mil subfractions reduced flinching/licking behavior during the second phase of formalin test and attenuated thermal and mechanical allodynia in nerve injury model. Treatment with proteolytic enzymes reduced CB1 internalization of subfractions, indicating the peptidergic nature of CB1 active component. Further HPLC purification revealed two potent antinociceptive subfractions within C. Tex with CB1 and possible CB2 activity, with mild to no side effects in the CB tetrad assessment. CB conopeptides can be isolated from these active Conus venom-derived samples and further developed as novel analgesic agents for the treatment of chronic pain using cell based or gene therapy approaches.

Development of a Phantom Limb Pain Model in Rats: Behavioral and Histochemical Evaluation.

Therapeutic strategies targeting phantom limb pain (PLP) provide inadequate pain relief; therefore, a robust and clinically relevant animal model is necessary. Animal models of PLP are based on a deafferentation injury followed by autotomy behavior. Clinical studies have shown that the presence of pre-amputation pain increases the risk of developing PLP. In the current study, we used Sprague-Dawley male rats with formalin injections or constriction nerve injury at different sites or time points prior to axotomy to mimic clinical scenarios of pre-amputation inflammatory and neuropathic pain. Animals were scored daily for PLP autotomy behaviors, and several pain-related biomarkers were evaluated to discover possible underlying pathological changes. Majority displayed some degree of autotomy behavior following axotomy. Injury prior to axotomy led to more severe PLP behavior compared to animals without preceding injury. Autotomy behaviors were more directed toward the pretreatment insult origin, suggestive of pain memory. Increased levels of IL-1ß in cerebrospinal fluid and enhanced microglial responses and the expression of NaV1.7 were observed in animals displaying more severe PLP outcomes. Decreased expression of GAD65/67 was consistent with greater PLP behavior. This study provides a preclinical basis for future understanding and treatment development in the management of PLP.

Intensive Locomotor Training Provides Sustained Alleviation of Chronic Spinal Cord Injury-Associated Neuropathic Pain: A Two-Year Pre-Clinical Study.

Neuropathic pain often accompanies the functional deficits associated with spinal cord injury (SCI) and further reduces a patient's quality of life. Clinical and pre-clinical research is beginning to highlight the beneficial role that rehabilitative therapies such as locomotor training can have not only on functional recovery but also on chronic pain management. Our group has previously developed an intensive locomotor training (ILT) treadmill protocol on rats that reduced SCI neuropathic pain symptoms for at least 3 months. We have extended these findings in the current study to evaluate the ability of regular ILT regimen over a 2 year period post-SCI to maintain neuropathic pain reduction. To assess this, the rat clip compression SCI model (T7/8) was used and treadmill training was initiated starting 4 weeks after SCI and continuing through the duration of the study. Results showed continued suppression of SCI neuropathic pain responses (reduced mechanical, heat, and cold hypersensitivity throughout the entire time course of the study). In contrast, non-exercised rats showed consistent and sustained neuropathic pain responses during this period. In addition, prolonged survival and improved locomotor outcomes were observed in rats undergoing ILT as the study longevity progressed. Potential contributory mechanisms underlying beneficial effects of ILT include reduced inflammation and restoration of anti-nociceptive inhibitory processes as indicated by neurochemical assays in spinal tissue of remaining rats at 2 years post-SCI. The benefits of chronic ILT suggest that long-term physical exercise therapy can produce powerful and prolonged management of neuropathic pain, partly through sustained reduction of spinal pathological processes.

Paclitaxel-induced peripheral neuropathy is caused by epidermal ROS and mitochondrial damage through conserved MMP-13 activation.

Paclitaxel induces peripheral neuropathy as a side effect of cancer treatment. The underlying causes are unclear, but epidermal, unmyelinated axons have been shown to be the first to degenerate. We previously utilized an in vivo zebrafish model to show that the epidermal matrix-metalloproteinase 13 (MMP-13) induces degeneration of unmyelinated axons, whereas pharmacological inhibition of MMP-13 prevented axon degeneration. However, the precise functions by which MMP-13 is regulated and affects axons remained elusive. In this study, we assessed mitochondrial damage and reactive oxygen species (ROS) formation as possible inducers of MMP-13, and we analyzed MMP-13-dependent damage. We show that the small ROS, H2O2, is increased in basal keratinocytes following treatment with paclitaxel. Cytoplasmic H2O2 appears to derive, at least in part, from mitochondrial damage, leading to upregulation of MMP-13, which in turn underlies increased epidermal extracellular matrix degradation. Intriguingly, also axonal mitochondria show signs of damage, such as fusion/fission defects and vacuolation, but axons do not show increased levels of H2O2. Since MMP-13 inhibition prevents axon degeneration but does not prevent mitochondrial vacuolation, we suggest that vacuolization occurs independently of axonal damage. Finally, we show that MMP-13 dysregulation also underlies paclitaxel-induced peripheral neuropathy in mammals, indicating that epidermal mitochondrial H2O2 and its effectors could be targeted for therapeutic interventions.

Mutually beneficial effects of intensive exercise and GABAergic neural progenitor cell transplants in reducing neuropathic pain and spinal pathology in rats with spinal cord injury.

Spinal cord injury (SCI) produces both locomotor deficits and sensory dysfunction that greatly reduce the overall quality of life. Mechanisms underlying chronic pain include increased neuro-inflammation and changes in spinal processing of sensory signals, with reduced inhibitory GABAergic signaling a likely key player. Our previous research demonstrated that spinal transplantation of GABAergic neural progenitor cells (NPCs) reduced neuropathic pain while intensive locomotor training (ILT) could reduce development of pain and partially reverse already established pain behaviors. Therefore, we evaluate the potential mutually beneficial anti-hypersensitivity effects of NPC transplants cells in combination with early or delayed ILT. NPC transplants were done at 4 weeks post-SCI. ILT, using a progressive ramping treadmill protocol, was initiated either 5 days post-SCI (early: pain prevention group) or at 5 weeks post-SCI (delayed: to reverse established pain) in male Sprague Dawley rats. Results showed that either ILT alone or NPCs alone could partially attenuate SCI neuropathic pain behaviors in both prevention and reversal paradigms. However, the combination of ILT with NPC transplants significantly enhanced neuropathic pain reduction on most of the outcome measures including tests for allodynia, hyperalgesia, and ongoing pain. Immunocytochemical and neurochemical analyses showed decreased pro-inflammatory markers and spinal pathology with individual treatments; these measures were further improved by the combination of either early or delayed ILT and GABAergic cellular transplantation. Lumbar dorsal horn GABAergic neuronal and process density were nearly restored to normal levels by the combination treatment. Together, these interventions may provide a less hostile and more supportive environment for promoting functional restoration in the spinal dorsal horn and attenuation of neuropathic pain following SCI. These findings suggest mutually beneficial effects of ILT and NPC transplants for reducing SCI neuropathic pain.

Experimental Gene Therapy with Serine-Histogranin and Endomorphin 1 for the Treatment of Chronic Neuropathic Pain.

The insufficient pain relief provided by current pharmacotherapy for chronic neuropathic pain is a serious medical problem. The enhanced glutamate signaling via NMDA receptors appears to be one of the key events in the development of chronic pain. Although effective, clinical use of systemic NMDA antagonists is limited by adverse effects such as hallucinations and motor dysfunction. Opioids are also potent analgesics but their chronic use is accompanied by tolerance and risk of addiction. However, combination of NMDA antagonists and opioids seems to provide a stable pain relieve at subthreshold doses of both substances, eliminating development of side effects. Our previous research showed that combined delivery of NMDA antagonist Serine histrogranin (SHG) and endomorphin1 (EM1) leads to attenuation of acute and chronic pain. The aim of this study was to design and evaluate an analgesic potency of the gene construct encoding SHG and EM1. Constructs with 1SHG copy in combination with EM1, 1SHG/EM1, and 6SHG/EM1 were intraspinally injected to animals with peripheral nerve injury-induced pain (chronic constriction injury, CCI) or spinal cord injury induced pain (clip compression model, SCI) and tactile and cold allodynia were evaluated. AAV2/8 particles were used for gene delivery. The results demonstrated 6SHG/EM1 as the most efficient for alleviation of pain-related behavior. The effect was observed up to 8 weeks in SCI animals, suggesting the lack of tolerance of possible synergistic effect between SHG and EM1. Intrathecal injection of SHG antibody or naloxone attenuated the analgesic effect in treated animals. Biochemical and histochemical evaluation confirmed the presence of both peptides in the spinal tissue. The results of this study showed that the injection of AAV vectors encoding combined SHG/EM constructs can provide long term attenuation of pain without overt adverse side effects. This approach may provide better treatment options for patients suffering from chronic pain.

Antinociceptive effects of topical mepivacaine in a rat model of HIV-associated peripheral neuropathic pain.

BACKGROUND: A consequence of HIV infection is sensory neuropathy, a debilitating condition that degrades the quality of life of HIV patients. Furthermore, life-extending antiretroviral treatment may exacerbate HIV sensory neuropathy. Analgesics that relieve other neuropathic pains show little or no efficacy in ameliorating HIV sensory neuropathy. Thus, there is a need for analgesics for people with this particular pain. While lidocaine is used in the management of painful peripheral neuropathies, another local anesthetic mepivacaine, with a potentially improved bioavailability, could be utilized for the management of HIV neuropathic pain. METHODS: The efficacy of topical anesthetics was evaluated in a preclinical rodent model of painful peripheral neuropathy induced by epineural administration of the HIV envelope protein gp120 delivered using saturated oxidized cellulose implanted around the sciatic nerve. Beginning at 2 weeks following gp120 administration, the effects of local anesthetics topically applied via gauze pads were tested on heat and mechanical hyperalgesia in the hind paw. Rats were tested using several concentrations of mepivacaine or lidocaine during the following 2 weeks. RESULTS: By 2 weeks following epineural gp120 implantation, the ipsilateral hind paw developed significant hypersensitivity to noxious pressure and heat hyperalgesia. A short-lasting, concentration-dependent amelioration of pressure and heat hyperalgesia was observed following topical application of mepivacaine to the ipsilateral plantar hind paw. By contrast, topical lidocaine ameliorated heat hyperalgesia in a concentration-dependent manner but not pressure hyperalgesia. Equipotent concentrations of mepivacaine and lidocaine applied topically to the tail of mice significantly increased tail withdrawal latencies in the tail flick test, demonstrating that both local anesthetics attenuate responding to a brief noxious stimulus. CONCLUSION: These findings showed that mepivacaine, rather than lidocaine, consistently attenuated two distinct symptoms of neuropathic pain and suggest that topical formulations of this local anesthetic could have utility in the alleviation of clinical HIV neuropathic pain.

Analgesic Effect of Recombinant GABAergic Cells in a Model of Peripheral Neuropathic Pain.

Chronic neuropathic pain represents a clinically challenging state with a poor response to current treatment options. Long-term management of chronic pain is often associated with the development of tolerance, addiction, and other side effects, reducing the therapeutic value of treatment. Alternative strategies based on cell therapy and gene manipulation, balancing the inhibitory and excitatory events in the spinal cord, may provide sustained pain relief in the long term. Transplantation of GABAergic cells has been successfully used to enhance inhibition and to restore physiological spinal pain processing. However, since the underlying mechanism of chronic pain development involves changes in several pain-signaling pathways, it is essential to develop an approach that targets several components of pain signaling. Recombinant cell therapy offers the possibility to deliver additional analgesic substances to the restricted area in the nervous system. The current study explores the analgesic potential of genetically modified rat embryonic GABAergic cells releasing a peptidergic NMDA receptor antagonist, Serine(1)-histogranin (SHG). Overactivation of glutamate NMDA receptors contributes to the hyperexcitability of spinal neurons observed in chronic pain models. Our approach allows us to simultaneously target spinal hyperexcitability and reduced inhibitory processes. Transplantable cells were transduced by viral vectors encoding either one or six copies of SHG cDNAs. The analgesic potential of recombinant cells after their intraspinal transplantation was evaluated in a model of peripheral nerve injury. Enhanced reduction of hypersensitivity to thermal and mechanical stimuli was observed in animals treated by recombinant cells compared to the nonrecombinant group. The recombinant peptide was detected in the spinal tissue, suggesting its successful production by transplanted cells. Our results demonstrate the feasibility of using recombinant cells releasing adjunct analgesic peptides in the therapy of neuropathic pain.

Recombinant neural progenitor transplants in the spinal dorsal horn alleviate chronic central neuropathic pain.

Neuropathic pain induced by spinal cord injury (SCI) is clinically challenging with inadequate long-term treatment options. Partial pain relief offered by pharmacologic treatment is often counterbalanced by adverse effects after prolonged use in chronic pain patients. Cell-based therapy for neuropathic pain using GABAergic neuronal progenitor cells (NPCs) has the potential to overcome untoward effects of systemic pharmacotherapy while enhancing analgesic potency due to local activation of GABAergic signaling in the spinal cord. However, multifactorial anomalies underlying chronic pain will likely require simultaneous targeting of multiple mechanisms. Here, we explore the analgesic potential of genetically modified rat embryonic GABAergic NPCs releasing a peptidergic NMDA receptor antagonist, Serine-histogranin (SHG), thus targeting both spinal hyperexcitability and reduced inhibitory processes. Recombinant NPCs were designed using either lentiviral or adeno-associated viral vectors (AAV2/8) encoding single and multimeric (6 copies of SHG) cDNA. Intraspinal injection of recombinant cells elicited enhanced analgesic effects compared with nonrecombinant NPCs in SCI-induced pain in rats. Moreover, potent and sustained antinociception was achieved, even after a 5-week postinjury delay, using recombinant multimeric NPCs. Intrathecal injection of SHG antibody attenuated analgesic effects of the recombinant grafts suggesting active participation of SHG in these antinociceptive effects. Immunoblots and immunocytochemical assays indicated ongoing recombinant peptide production and secretion in the grafted host spinal cords. These results support the potential for engineered NPCs grafted into the spinal dorsal horn to alleviate chronic neuropathic pain.

Viral vectors encoding endomorphins and serine histogranin attenuate neuropathic pain symptoms after spinal cord injury in rats.

BACKGROUND: The treatment of spinal cord injury (SCI)-induced neuropathic pain presents a challenging healthcare problem. The lack of available robust pharmacological treatments underscores the need for novel therapeutic methods and approaches. Due to the complex character of neuropathic pain following SCI, therapies targeting multiple mechanisms may be a better choice for obtaining sufficient long-term pain relief. Previous studies in our lab showed analgesic effects using combinations of an NMDA antagonist peptide [Ser1]histogranin (SHG), and the mu-opioid peptides endomorphins (EMs), in several pain models. As an alternative to drug therapy, this study evaluated the analgesic potential of these peptides when delivered via gene therapy. RESULTS: Lentiviruses encoding SHG and EM-1 and EM-2 were intraspinally injected, either singly or in combination, into rats with clip compression SCI 2 weeks following injury. Treated animals showed significant reduction in mechanical and thermal hypersensitivity, compared to control groups injected with GFP vector only. The antinociceptive effects of individually injected components were modest, but the combination of EMs and SHG produced robust and sustained antinociception. The onset of the analgesic effects was observed between 1-5 weeks post-injection and sustained without decrement for at least 7 weeks. No adverse effects on locomotor function were observed. The involvement of SHG and EMs in the observed antinociception was confirmed by pharmacologic inhibition using intrathecal injection of either the opioid antagonist naloxone or an anti-SHG antibody. Immunohistochemical analysis showed the presence of SHG and EMs in the spinal cord of treated animals, and immunodot-blot analysis of CSF confirmed the presence of these peptides in injected animals. In a separate group of rats, delayed injection of viral vectors was performed in order to mimic a more likely clinical scenario. Comparable and sustained antinociceptive effects were observed in these animals using the SHG-EMs combination vectors compared to the group with early intervention. CONCLUSIONS: Findings from this study support the potential for direct gene therapy to provide a robust and sustained alleviation of chronic neuropathic pain following SCI. The combination strategy utilizing potent mu-opioid peptides with a naturally-derived NMDA antagonist may produce additive or synergistic analgesic effects without the tolerance development for long-term management of persistent pain.

Attenuation of persistent pain-related behavior by fatty acid amide hydrolase (FAAH) inhibitors in a rat model of HIV sensory neuropathy.

Distal sensory neuropathies are a hallmark of HIV infections and can result in persistent and disabling pain despite advances in antiretroviral therapies. HIV-sensory neuropathic (HIV-SN) pain may be amenable to cannabinoid treatment, but currently available agonist treatments are limited by untoward side effects and potential for abuse in this patient population. Fatty acid amide hydrolase (FAAH) inhibitors may offer an alternative approach by inhibiting the degradation of endocannabinoids with purportedly fewer untoward CNS side effects. In order to evaluate this potential approach in the management of HIV-SN pain, the recombinant HIV envelope protein gp120 was applied epineurally to the rat sciatic nerve to induce an HIV-SN-like pain syndrome. Two distinct FAAH inhibitory compounds, URB597 and PF-3845 were tested, and contrasted with standard antinociceptive gabapentin or vehicle treatment, for attenuation of tactile allodynia, cold allodynia, and mechanical hyperalgesia. Both FAAH inhibitors markedly reduced cold and tactile allodynia with limited anti-hyperalgesic effects. Peak antinociceptive effects produced by both agents were more modest than gabapentin in reducing tactile allodynia with similar potency ranges. URB597 produced comparable cold anti-allodynic effects to gabapentin, and the effects of both FAAH inhibitors were longer lasting than gabapentin. To assess the contribution of cannabinoid receptors in these antinociceptive effects, CB1 antagonist AM251 or CB2 antagonist SR144528 were tested in conjunction with FAAH inhibitors. Results suggested a contribution of both CB1- and CB2-mediated effects, particularly in reducing tactile allodynia. In summary, these findings support inhibition of endocannabinoid degradation as a promising target for management of disabling persistent HIV-SN pain syndromes.

An Intensive Locomotor Training Paradigm Improves Neuropathic Pain following Spinal Cord Compression Injury in Rats.

Spinal cord injury (SCI) is often associated with both locomotor deficits and sensory dysfunction, including debilitating neuropathic pain. Unfortunately, current conventional pharmacological, physiological, or psychological treatments provide only marginal relief for more than two-thirds of patients, highlighting the need for improved treatment options. Locomotor training is often prescribed as an adjunct therapy for peripheral neuropathic pain but is rarely used to treat central neuropathic pain. The goal of this study was to evaluate the potential anti-nociceptive benefits of intensive locomotor training (ILT) on neuropathic pain consequent to traumatic SCI. Using a rodent SCI model for central neuropathic pain, ILT was initiated either 5 d after injury prior to development of neuropathic pain symptoms (the "prevention" group) or delayed until pain symptoms fully developed (∼3 weeks post-injury, the "reversal" group). The training protocol consisted of 5 d/week of a ramping protocol that started with 11 m/min for 5 min and increased in speed (+1 m/min/week) and time (1-4 minutes/week) to a maximum of two 20-min sessions/d at 15 m/min by the fourth week of training. ILT prevented and reversed the development of heat hyperalgesia and cold allodynia, as well as reversed developed tactile allodynia, suggesting analgesic benefits not seen with moderate levels of locomotor training. Further, the analgesic benefits of ILT persisted for several weeks once training had been stopped. The unique ability of an ILT protocol to produce robust and sustained anti-nociceptive effects, as assessed by three distinct outcome measures for below-level SCI neuropathic pain, suggests that this adjunct therapeutic approach has great promise in a comprehensive treatment strategy for SCI pain.

Selective antinociceptive effects of a combination of the N-methyl-D-aspartate receptor peptide antagonist [Ser(1)]histogranin and morphine in rat models of pain.

Numerous rather than a few analgesic endogenous neuropeptides are likely to work in concert in vivo in ameliorating pain. Identification of effective neuropeptide combinations would also facilitate the development of gene or cell-based analgesics. In this study, opioid peptides endomorphin-1 (EM-1) and endomorphin-2 (EM-2) and the peptide histogranin analogue [Ser(1)]histogranin (SHG), which possess activity as an N-methyl-d-aspartate (NMDA) receptor antagonist, were intrathecally (i.t.) injected alone and in combination in rat models of acute and persistent pain. None of the peptides when injected alone altered hind paw responses of uninjured rats to acute noxious stimulation. EM-1 and EM-2 showed divergent efficacies in the persistent pain models. For example, EM-1 injected alone was antinociceptive in rats with neuropathic pain, whereas EM-2 demonstrated no efficacy. Demonstration of synergism was also divergent across the models. For example, while SHG combined with EM-1 did not alter the efficacy of EM-1 in rats with neuropathic pain, SHG significantly increased the efficacy of EM-1 in the formalin test. By contrast, the potency and efficacy of the peptides alone and combinations were much less than those of the reference analgesic morphine. Furthermore, morphine combined with the clinically used NMDA receptor antagonist ketamine showed synergism across a broad range of pain states. While the current set of neuropeptides could serve as a basis for analgesic therapeutics, there could be other neuropeptides with greater efficacy and potency and broader therapeutic application.

Effects of repeated dosing with mechanistically distinct antinociceptive ligands in a rat model of neuropathic spinal cord injury pain.

A lack of efficacy of some analgesic drugs has been previously described in rats with neuropathic spinal cord injury (SCI) pain. It has been suggested that repeated dosing in these animals over time may eventually lead to efficacy. However, it is also possible that efficacy may diminish over time with repeated dosing. This study evaluated the efficacy of various drugs upon repeated dosing over time in a rat model of SCI pain. Four weeks following an acute spinal cord compression at the mid-thoracic level, rats developed decreased hind paw withdrawal threshold, suggestive of below level neuropathic hypersensitivity. Either cannabinoid (CB) receptor agonist CP 55,940, the anticonvulsant carbamazepine or gabapentin, the antidepressant amitriptyline or vehicle was administered over a period of 7 days. Neither carbamazepine nor amitriptyline demonstrated efficacy either after a single or repeated dosing. Beginning with a 50% efficacious dose of gabapentin, the effect of gabapentin in SCI rats neither increased nor decreased over the treatment period. The antinociceptive effect of CP 55,940 was maintained for the entire treatment period, which was mediated by CB1 but not CB2 receptors. The current data suggest that sustained antinociception can be obtained with some drugs in rats with neuropathic SCI pain. Furthermore, the current data do not substantiate the notion that repeated treatment with initially ineffective drugs will eventually lead to efficacy; treatments that are not acutely effective are unlikely to demonstrate clinical efficacy.

Fatty acid amide hydrolase (FAAH) inhibitors exert pharmacological effects, but lack antinociceptive efficacy in rats with neuropathic spinal cord injury pain.

Amelioration of neuropathic spinal cord injury (SCI) pain is a clinical challenge. Increasing the endocannabinoid anandamide and other fatty acid amides (FAA) by blocking fatty acid amide hydrolase (FAAH) has been shown to be antinociceptive in a number of animal models of chronic pain. However, an antinociceptive effect of blocking FAAH has yet to be demonstrated in a rat model of neuropathic SCI pain. Four weeks following a SCI, rats developed significantly decreased hind paw withdrawal thresholds, indicative of below-level cutaneous hypersensitivity. A group of SCI rats were systemically treated (i.p.) with either the selective FAAH inhibitor URB597 or vehicle twice daily for seven days. A separate group of SCI rats received a single dose (p.o.) of either the selective FAAH inhibitor PF-3845 or vehicle. Following behavioral testing, levels of the FAA N-arachidonoylethanolamide, N-oleoyl ethanolamide and N-palmitoyl ethanolamide were quantified in brain and spinal cord from SCI rats. Four weeks following SCI, FAA levels were markedly reduced in spinal cord tissue. Although systemic treatment with URB597 significantly increased CNS FAA levels, no antinociceptive effect was observed. A significant elevation of CNS FAA levels was also observed following oral PF-3845 treatment, but only a modest antinociceptive effect was observed. Increasing CNS FAA levels alone does not lead to robust amelioration of below-level neuropathic SCI pain. Perhaps utilizing FAAH inhibition in conjunction with other analgesic mechanisms could be an effective analgesic therapy.