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Previous reports show that moderate prenatal alcohol exposure (PAE) poses a risk factor for developing neuropathic pain following adult-onset peripheral nerve injury in male rats. Recently, evidence suggests that immune-related mechanisms underlying neuropathic pain in females are different compared to males despite the fact that both sexes develop neuropathy of similar magnitude and duration following chronic constriction injury (CCI) of the sciatic nerve. Data suggest that the actions of peripheral T cells play a greater role in mediating neuropathy in females. The goal of the current study is to identify specificity of immune cell and cytokine changes between PAE and non-PAE neuropathic females by utilizing a well-characterized rodent model of sciatic nerve damage, in an effort to unmask unique signatures of immune-related factors underlying the risk of neuropathy from PAE. Cytokines typically associated with myeloid cell actions such as interleukin (IL)-1β, tumor necrosis factor (TNF), IL-6, IL-4 and IL-10 as well as the neutrophil chemoattractant CXCL1, are examined. In addition, transcription factors and cytokines associated with various differentiated T cell subtypes are examined (anti-inflammatory FOXP3, proinflammatory IL-17A, IL-21, ROR-γt, interferon (IFN)-γ and T-bet). Lymphocyte function associated antigen 1 (LFA-1) is an adhesion molecule expressed on peripheral immune cells including T cells, and regulates T cell activation and extravasation into inflamed tissue regions. A potential therapeutic approach was explored with the goal of controlling proinflammatory responses in neuroanatomical regions critical for CCI-induced allodynia by blocking LFA-1 actions using BIRT377. The data show profound development of hindpaw allodynia in adult non-PAE control females following standard CCI, but not following minor CCI, while minor CCI generated allodynia in PAE females. The data also show substantial increases in T cell-associated proinflammatory cytokine mRNA and proteins, along with evidence of augmented myeloid/glial activation (mRNA) and induction of myeloid/glial-related proinflammatory cytokines, CCL2, IL-1β and TNF in discrete regions along the pain pathway (damaged sciatic nerve, dorsal root ganglia; DRG, and spinal cord). Interestingly, the characteristic anti-inflammatory IL-10 protein response to nerve damage is blunted in neuropathic PAE females. Moreover, T cell profiles are predominantly proinflammatory in neuropathic Sac and PAE females, augmented levels of Th17-specific proinflammatory cytokines IL-17A and IL-21, as well as the Th1-specific factor, T-bet, are observed. Similarly, the expression of RORγt, a critical transcription factor for Th17 cells, is detected in the spinal cord of neuropathic females. Blocking peripheral LFA-1 actions with intravenous (i.v.) BIRT377 reverses allodynia in Sac and PAE rats, dampens myeloid (IL-1β, TNF, CXCL1)- and T cell-associated proinflammatory factors (IL-17A and RORγt) and spinal glial activation. Moreover, i.v. BIRT377 treatment reverses the blunted IL-10 response to CCI observed only in neuropathic PAE rats and elevates FOXP3 in pain-reversed Sac rats. Unexpectedly, intrathecal BIRT377 treatment is unable to alter allodynia in either Sac or PAE neuropathic females. Together, these data provide evidence that: 1) fully differentiated proinflammatory Th17 cells recruited at the sciatic nerve, DRGs and lumbar spinal cord may interact with the local environment to shape the immune responses underlying neuropathy in female rats, and, 2) PAE primes peripheral and spinal immune responses in adult females. PAE is a risk factor in females for developing peripheral neuropathy after minor nerve injury.

This review addresses underlying physiological, cellular, and molecular factors that alter the developing fetal brain stress circuits and responses of the hypothalamic-pituitary-adrenal (HPA) axis caused by maternal stress and prenatal alcohol exposure (PAE). An emphasis is placed on the contribution of the placenta following maternal stress separately, and as a co-occurrence with PAE. Altered fetal HPA axis ultimately results in dysregulation of the brain stress-response system long after birth and possibly lifelong. Additional consideration of the role of placentally-derived endocrine and sex hormones, as well as a brief discussion of epigenetic mechanisms of altered placental expression of genes encoding the glucocorticoid receptor and the enzymes 11β-HSD that rapidly convert glucocorticoids into its active or inactive forms are reviewed. Data highlighting the strong, reciprocal interactions between the neuroimmune and neuroendocrine systems during fetal development that are impacted by maternal stress and PAE are considered, emphasizing the role of the placenta as a key contributor to the dysregulation of these systems. In view of the maternal-placental-fetal interface, important physiological, cellular, and molecular factors underlying later life dysregulated stress responses are additionally considered. Literature from animal models of PAE and maternal stress is reviewed that support clinical observations of the effect of maternal stress and alcohol exposure during fetal development on later-life adult stress responses and associated mood dysregulation. An appreciation of dysregulated stress responses in individuals with fetal alcohol spectrum disorders (FASD) are addressed given the greater prevalence of adult dysregulated stress responses and a greater co-occurrence of mood disorders in individuals diagnosed with FASD.

The CB2R agonist AM1710, examined in animal models of peripheral neuropathy, is effective in controlling aberrant light touch sensitivity, referred to as mechanical allodynia. However, nonspecific binding of AM1710 to CB1R, either peripherally or centrally, could be partially responsible for the analgesic effects of AM1710. Thus, we sought to determine in mice whether spinal (intrathecal; i.t.) or peripheral AM1710 administration could lead to anti‐allodynia by reducing the protein expression of spinal and dorsal root ganglia (DRG) proinflammatory cytokines and elevating the anti‐inflammatory cytokine interleukin‐10 (IL‐10) in the absence of CB1R. Macrophage cell cultures were examined to characterize AM1710‐mediated suppression of the proinflammatory cytokine tumor necrosis factor‐alpha (TNF‐α). Either i.p. or i.t. AM1710 reversed CCI‐induced mechanical allodynia to sham levels in CB1R (−/−), (+/−), (+/+) mice. CCI‐induced neuropathy decreased IL‐10 immunoreactivity (IR) in the dorsal root ganglia (DRG) and the dorsal horn of the spinal cord, with i.t. AM1710 restoring basal IL‐10 IR. CCI‐induced elevations in proinflammatory cytokine IR were decreased within the spinal cord only after i.t. AM1710 in all mouse genotypes. Meanwhile, within DRG tissue from neuropathic mice, proinflammatory cytokines were decreased following either i.p. or i.t. AM1710. Analysis of cultured supernatants revealed AM1710 decreased TNF‐alpha protein. We conclude that CB1R is dispensable for either peripheral or central anti‐allodynic actions of AM1710 in neuropathic mice. Cannabinoid CB2R agonists produce heightened spinal IL‐10 which may be clinically relevant to successfully treat neuropathic pain., Either i.p. or i.t. AM1710 reversed CCI‐induced mechanical allodynia in CB1R (−/−), (+/−), (+/+) mice similar to sham control threshold levels. The absence of the CB1R following CCI‐induced neuropathy revealed greater significant increases in IL‐10 immunoreactivity (IR) in the dorsal root ganglia (DRG) and the dorsal horn of the spinal cord in (−/−) and (+/−) mice that was further elevated following i.t. and i.p. AM1710. The absence of CB1R may allow for enhanced tissue/axon damage‐associated signaling to the DRG and spinal cord, resulting in endogenous compensatory increases in IL‐10 expression.

The role of environmental stressors, particularly exposure to air pollution, in the development of neurodegenerative disease remains underappreciated. We examined the neurological effects of acute ozone (O(3)) exposure in aged mice, where increased blood–brain barrier (BBB) permeability may confer vulnerability to neuroinflammatory outcomes. C57BL/6 male mice, aged 8–10 weeks or 12–18 months were exposed to either filtered air or 1.0 ppm O(3) for 4 h; animals received a single IP injection of sodium fluorescein (FSCN) 20 h postexposure. One-hour post-FSCN injection, animals were transcardially perfused for immunohistochemical analysis of BBB permeability. β-amyloid protein expression was assessed via ELISA. Flow cytometric characterization of infiltrating immune cells, including neutrophils, macrophages, and microglia populations was performed 20 h post-O(3) exposure. Flow cytometry analysis of brains revealed increased microglia “activation” and presentation of CD11b, F4/80, and MHCII in aged animals relative to younger ones; these age-induced differences were potentiated by acute O(3) exposure. Cortical and limbic regions in aged brains had increased reactive microgliosis and β-amyloid protein expression after O(3) insult. The aged cerebellum was particularly vulnerable to acute O(3) exposure with increased populations of infiltrating neutrophils, peripheral macrophages/monocytes, and Ly6C(+) inflammatory monocytes after insult, which were not significantly increased in the young cerebellum. O(3) exposure increased the penetration of FSCN beyond the BBB, the infiltration of peripheral immune cells, and reactive gliosis of microglia. Thus, the aged BBB is vulnerable to insult and becomes highly penetrable in response to O(3) exposure, leading to greater neuroinflammatory outcomes.

Preterm birth is an important cause of perinatal brain injury (PBI). Neurological injury in extremely preterm infants often begins in utero with chorioamnionitis (CHORIO) or inflammation/infection of the placenta and concomitant placental insufficiency. Studies in humans have shown dysregulated inflammatory signaling throughout the placental-fetal brain axis and altered peripheral immune responses in children born preterm with cerebral palsy (CP). We hypothesized that peripheral immune responses would be altered in our well-established rat model of CP. Specifically, we proposed that isolated peripheral blood mononuclear cells (PBMCs) would be hyperresponsive to a second hit of inflammation throughout an extended postnatal time course. Pregnant Sprague-Dawley dams underwent a laparotomy on embryonic day 18 (E18) with occlusion of the uterine arteries (for 60 min) followed by intra-amniotic injection of lipopolysaccharide (LPS, 4 μg/sac) to induce injury in utero. Shams underwent laparotomy only, with equivalent duration of anesthesia. Laparotomies were then closed, and the rat pups were born at E22. PBMCs were isolated from pups on postnatal day 7 (P7) and P21, and subsequently stimulated in vitro with LPS for 3 or 24 h. A secreted inflammatory profile analysis of conditioned media was performed using multiplex electrochemiluminescent immunoassays, and the composition of inflammatory cells was assayed with flow cytometry (FC). Results indicate that CHORIO PBMCs challenged with LPS are hyperreactive and secrete significantly more tumor necrosis factor α (TNFα) and C-X-C chemokine ligand 1 at P7. FC confirmed increased intracellular TNFα in CHORIO pups at P7 following LPS stimulation, in addition to increased numbers of CD11b/c immunopositive myeloid cells. Notably, TNFα secretion was sustained until P21, with increased interleukin 6, concomitant with increased expression of integrin β1, suggesting both sustained peripheral immune hyperreactivity and a heightened activation state. Taken together, these data indicate that in utero injury primes the immune system and augments enhanced inflammatory signaling. The insidious effects of primed peripheral immune cells may compound PBI secondary to CHORIO and/or placental insufficiency, and thereby render the brain susceptible to future chronic neurological disease. Further understanding of inflammatory mechanisms in PBI may yield clinically important biomarkers and facilitate individualized repair strategies and treatments.

A growing body of evidence indicates that prenatal alcohol exposure (PAE) may predispose individuals to secondary medical disabilities later in life. Animal models of PAE reveal neuroimmune sequelae such as elevated brain astrocyte and microglial activation with corresponding region-specific changes in immune signaling molecules such as cytokines and chemokines. The aim of this study was to evaluate the effects of moderate PAE on the development and maintenance of allodynia induced by chronic constriction injury (CCI) of the sciatic nerve in adult male rat offspring. Because CCI allodynia requires the actions of glial cytokines, we analyzed lumbar spinal cord glial and immune cell surface markers indicative of their activation levels, as well as sciatic nerve and dorsal root ganglia (DRG) cytokines in PAE offspring in adulthood. While PAE did not alter basal sensory thresholds before or after sham manipulations, PAE significantly potentiated adult onset and maintenance of allodynia. Microscopic analysis revealed exaggerated astrocyte and microglial activation, while flow cytometry data demonstrated increased proportions of immune cells with cell surface major histocompatibility complex II (MHCII) and β-integrin adhesion molecules, which are indicative of PAE-induced immune cell activation. Sciatic nerves from CCI rats revealed that PAE potentiated the proinflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor-alpha (TNFα) protein levels with a simultaneous robust suppression of the anti-inflammatory cytokine, IL-10. A profound reduction in IL-10 expression in the DRG of PAE neuropathic rats was also observed. Taken together, our results provide novel insights into the vulnerability that PAE produces for adult-onset central nervous system (CNS) pathological conditions from peripheral nerve injury.

Aim The majority of preclinical studies investigating aberrant glial-neuroimmune actions underlying neuropathic pain have focused on male rodent models. Recently, studies have shown peripheral immune cells play a more prominent role than glial cells in mediating pathological pain in females. Here, we compared the onset and duration of allodynia in males and females, and the anti-allodynic action of a potentially novel therapeutic drug (BIRT377) that not only antagonizes the action of lymphocyte function-associated antigen-1 (LFA-1) to reduce cell migration in the periphery, but may also directly alter the cellular inflammatory bias. Methods Male and female mice were subjected to peripheral nerve injury chronic constriction injury (CCI) applying two methods, using either 4-0 or 5-0 chromic gut suture material, to examine potential sex differences in the onset, magnitude and duration of allodynia. Hindpaw sensitivity before and after CCI and application of intravenous BIRT377 was assessed. Peripheral and spinal tissues were analyzed for protein (multiplex electrochemiluminescence technology) and mRNA expression (quantitative real-time PCR). The phenotype of peripheral T cells was determined using flow cytometry. Results Sex differences in proinflammatory CCL2 and IL-1β and the anti-inflammatory IL-10 were observed from a set of cytokines analyzed. A profound proinflammatory T cell (Th17) response in the periphery and spinal cord was also observed in neuropathic females. BIRT377 reversed pain, reduced IL-1β and TNF, and increased IL-10 and transforming growth factor (TGF)-β1, also an anti-inflammatory cytokine, in both sexes. However, female-derived T cell cytokines are transcriptionally regulated by BIRT377, as demonstrated by reducing proinflammatory IL-17A production with concurrent increases in IL-10, TGF-β1 and the anti-inflammatory regulatory T cell-related factor, FOXP3. Conclusion This study supports that divergent peripheral immune and neuroimmune responses during neuropathy exists between males and females. Moreover, the modulatory actions of BIRT377 on T cells during neuropathy are predominantly specific to females. These data highlight the necessity of including both sexes for studying drug efficacy and mechanisms of action in preclinical studies and clinical trials.

Background Clinical studies show that prenatal alcohol exposure (PAE) results in effects that persist into adulthood. Experimental animal models of moderate PAE demonstrate that young adults with PAE display potentiated sensitivity to light touch, clinically termed allodynia, following sciatic nerve chronic constriction injury (CCI) that coincides with heightened spinal glial, spinal macrophage, and peripheral immune responses. However, basal touch sensitivity and corresponding glial and leukocyte activation are unaltered. Therefore, the current study explored whether the enduring pathological consequences of moderate PAE on sensory processing are unmasked only following secondary neural insult. Methods In middle-aged (1 year) Long Evans rats that underwent either prenatal saccharin exposure (control) or moderate PAE, we modified the well-characterized model of sciatic neuropathy, CCI, to study the effects of PAE on neuro-immune responses in adult offspring. Standard CCI manipulation required 4 chromic gut sutures, while a mild version applied a single suture loosely ligated around one sciatic nerve. Spinal glial immunoreactivity was examined using immunohistochemistry. The characterization and functional responses of leukocyte populations were studied using flow cytometry and cell stimulation assays followed by quantification of the proinflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Data were statistically analyzed by ANOVA and unpaired t tests. Results The current report demonstrates that mild CCI generates robust allodynia only in PAE rats, while the pathological effects of PAE following the application of a standard CCI are revealed by enhanced allodynia and elevated spinal glial activation. Additionally, mild CCI increases spinal astrocyte activation but not microglia, suggesting astrocytes play a larger role in PAE-induced susceptibility to aberrant sensory processing. Leukocyte populations from PAE are altered under basal conditions (i.e., prior to secondary insult), as the distribution of leukocyte populations in lymphoid organs and other regions are different from those of controls. Lastly, following in vitro leukocyte stimulation, only PAE augments the immune response to antigen stimulation as assessed by heightened production of TNF-α and IL-1β. Conclusions These studies demonstrate PAE may prime spinal astrocytes and peripheral leukocytes that contribute to enduring susceptibility to adult-onset neuropathic pain that is not apparent until a secondary insult later in life.

Studies show that spinal (intrathecal; i.t.) interleukin-10 (IL-10) gene therapy reverses neuropathic pain in animal models, and co-administration with the mannose receptor (MR; CD206) ligand d-mannose (DM) greatly improves therapeutic efficacy. However, the actions of endogenous IL-10 may be required for enduring pain control observed following i.t. IL-10 gene therapy, potentially narrowing the application of this non-viral transgene delivery approach. Here, we show that i.t. application of naked plasmid DNA expressing the IL-10 transgene co-injected with DM (DM/pDNA-IL-10) for the treatment of peripheral neuropathic pain in IL-10 deficient (IL-10 KO) mice results in a profound and prolonged bilateral pain suppression. Neuropathic pain is induced by unilateral sciatic chronic constriction injury (CCI), and while enduring relief of light touch sensitivity (mechanical allodynia) in both wild type (WT) and IL-10 KO mice was observed following DM/pDNA-IL-10 co-therapy, transient reversal from allodynia was observed following i.t. DM alone. In stably pain-relieved IL-10 KO mice given DM/pDNA-IL-10, mRNA for the IL-10 transgene is detected in the cauda equina and ipsilateral dorsal root ganglia (DRG), but not the lumbar spinal cord. Further, DM/pDNA-IL-10 application increases anti-inflammatory TGF-β1 and decreases pro-inflammatory TNF mRNA in the ipsilateral DRG compared to allodynic controls. Additionally, DM/pDNA-IL-10 treated mice exhibit decreased spinal pro-inflammatory mRNA expression for TNF, CCL2 (MCP-1), and for the microglial-specific marker TMEM119. Similarly, DM/pDNA-IL-10 treatment decreases immunoreactivity for the astrocyte activation marker GFAP in lumbar spinal cord dorsal horn. Despite transient reversal and early return to allodynia in DM-treated mice, lumbar spinal cord revealed elevated TNF, CCL2 and TMEM119 mRNA levels. Both MR (CD206) and IL-10 receptor mRNAs are increased in the DRG following CCI manipulation independent of injection treatment, suggesting that pathological conditions stimulate upregulation and availability of relevant receptors in critical anatomical regions required for the therapeutic actions of the DM/pDNA-IL-10 co-therapy. Taken together, the current report demonstrates that non-viral DM/pDNA-IL-10 gene therapy does not require endogenous IL-10 for enduring relief of peripheral neuropathic pain and does not require direct contact with the spinal cord dorsal horn for robust and enduring relief of neuropathic pain. Spinal non-viral DM/pDNA-IL-10 co-therapy may offer a framework for the development of non-viral gene therapeutic approaches for other diseases of the central nervous system.

Animal models of peripheral neuropathy produced by a number of manipulations are assessed for the presence of pathologic pain states such as allodynia. Although stimulus-induced behavioral assays are frequently used and important to examine allodynia (ie, sensitivity to light mechanical touch; von Frey fiber test), other measures of behavior that reflect overall function are not only complementary to stimulus-induced responsive measures, but are also critical to gain a complete understanding of the effects of the pain model on quality of life, a clinically relevant aspect of pain on general function. Voluntary wheel-running activity in rodent models of inflammatory and muscle pain is emerging as a reliable index of general function that extends beyond stimulus-induced behavioral assays. Clinically, reports of increased pain intensity occur at night, a period typically characterized with reduced activity during the diurnal cycle. We therefore examined in rats whether alterations in wheel-running activity were more robust during the inactive phase compared with the active phase of their diurnal cycle in a widely used rodent model of chronic peripheral neuropathic pain, the sciatic nerve chronic constriction injury (CCI) model.In adult male Sprague Dawley rats, baseline (BL) hindpaw threshold responses to light mechanical touch were assessed using the von Frey test before measuring BL activity levels using freely accessible running wheels (1 hour/day for 7 sequential days) to quantify the distance traveled. Running wheel activity BL values are expressed as total distance traveled (m). The overall experimental design was after BL measures, rats underwent either sham or CCI surgery followed by repeated behavioral reassessment of hindpaw thresholds and wheel-running activity levels for up to 18 days after surgery. Specifically, separate groups of rats were assessed for wheel-running activity levels (1 hour total/trial) during the onset (within first 2 hours) of either the (1) inactive (n = 8/group) or (2) active (n = 8/group) phase of the diurnal cycle. An additional group of CCI-treated rats (n = 8/group) was exposed to a locked running wheel to control for the potential effects of wheel-running exercise on allodynia. The 1-hour running wheel trial period was further examined at discrete 20-minute intervals to identify possible pattern differences in activity during the first, middle, and last portions of the 1-hour trial. The effect of neuropathy on activity levels was assessed by measuring the change from their respective BLs to distance traveled in the running wheels.Although wheel-running distances between groups were not different at BL from rats examined during either the inactive phase of the diurnal cycle or active phase of the diurnal cycle, sciatic nerve CCI reduced running wheel activity levels compared with sham-operated controls during the inactive phase. In addition, compared with sham controls, bilateral low-threshold mechanical allodynia was observed at all time points after surgical induction of neuropathy in rats with free-wheel and locked-wheel access. Allodynia in CCI compared with shams was replicated in rats whose running wheel activity was examined during the active phase of the diurnal cycle. Conversely, no significant reduction in wheel-running activity was observed in CCI-treated rats compared with sham controls at any time point when activity levels were examined during the active diurnal phase. Finally, running wheel activity patterns within the 1-hour trial period during the inactive phase of the diurnal cycle were relatively consistent throughout each 20-minute phase.Compared with nonneuropathic sham controls, a profound and stable reduction of running wheel activity was observed in CCI rats during the inactive phase of the diurnal cycle. A concurrent robust allodynia persisted in all rats regardless of when wheel-running activity was examined or whether they ran on wheels, suggesting that acute wheel-running activity does not alter chronic low-intensity mechanical allodynia as measured using the von Frey fiber test. Overall, these data support that acute wheel-running exercise with limited repeated exposures does not itself alter allodynia and offers a behavioral assay complementary to stimulus-induced measures of neuropathic pain.

During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB(2)R), leads to effective anti-allodynia and modifies a variety of spinal microglial and astrocyte responses. Here, following spinal administration of the CB(2)R compound, AM1241, we examined immunoreactive alterations in markers for activated p38 mitogen-activated protein kinase, interleukin-1β (IL-1β), the anti-inflammatory cytokine, interleukin-10 (IL-10) as well as degradative endocannabinoid enzymes, and markers for altered glial responses in neuropathic rats. In these studies, the dorsal horn of the spinal cord and dorsal root ganglia were examined. AM1241 produced profound anti-allodynia with corresponding immunoreactive levels of p38 mitogen-activated kinase, IL-1β, IL-10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls. In contrast, spinal AM1241 did not suppress the increased microglial responses observed in neuropathic rats. The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity. These data reveal expression profiles that support the actions of intrathecal AM1241 control pathological pain through anti-inflammatory mechanisms by modulating critical glial factors, and additionally decrease expression levels of endocannabinoid degradative enzymes.

Previous studies of peripheral immune cells have documented that activation of adenosine 2A receptors (A2ARs) decrease proinflammatory cytokine release and increase release of the potent anti-inflammatory cytokine, interleukin-10 (IL-10). Given the growing literature supporting that glial proinflammatory cytokines importantly contribute to neuropathic pain and that IL-10 can suppress such pain, we evaluated the effects of intrathecally administered A2AR agonists on neuropathic pain using the chronic constriction injury (CCI) model. A single intrathecal injection of the A2AR agonists 4-(3-(6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-ynyl)piperidine-1-carboxylic acid methyl ester (ATL313) or 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido adenosine HCl (CGS21680), 10–14 d after CCI versus sham surgery, produced a long-duration reversal of mechanical allodynia and thermal hyperalgesia for at least 4 weeks. Neither drug altered the nociceptive responses of sham-operated controls. An A2AR antagonist [ZM241385 (4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl)phenol)] coadministered intrathecally with ATL313 abolished the action of ATL313 in rats with neuropathy-induced allodynia but had no effect on allodynia in the absence of the A2AR agonist. ATL313 attenuated CCI-induced upregulation of spinal cord activation markers for microglia and astrocytes in the L4–L6 spinal cord segments both 1 and 4 weeks after a single intrathecal ATL313 administration. Neutralizing IL-10 antibodies administered intrathecally transiently abolished the effect of ATL313 on neuropathic pain. In addition, IL-10 mRNA was significantly elevated in the CSF cells collected from the lumbar region. Activation of A2ARs after intrathecal administration may be a novel, therapeutic approach for the treatment of neuropathic pain by increasing IL-10 in the immunocompetent cells of the CNS.

Summary We recently described a non-viral gene therapy paradigm offering long-term resolution of established neuropathic pain in several animal models. Here, the requirements for long term therapeutic effects are described, and evidence is provided for a mechanism of action based on immunological priming of the intrathecal space. Long-term pain reversal was achieved when two intrathecal injections of various naked plasmid DNA doses were separated by 5 hr to 3 days. We demonstrate that an initial DNA injection, regardless of whether a transgene is included, leads to an accumulation of phagocytic innate immune cells. This accumulation coincides with the time in which subsequent DNA injection efficacy is potentiated. We demonstrate the ability of non-coding DNA to induce short term pain reversal that is dependent on endogenous interleukin-10 (IL-10) signaling. Long term efficacy requires the inclusion of an IL-10F129S transgene in the second injection. Blockade of IL-10, via neutralizing antibody, either between the two injections or following the second injection induces therapeutic failure. These results demonstrate that this gene therapy paradigm utilizes an initial “priming” injection of DNA to induce accumulation of phagocytic immune cells, allowing for potentiated efficacy of a subsequent “therapeutic” DNA injection in a time and dose dependent manner.

Glia have emerged as key contributors to pathological and chronic pain mechanisms. On activation, both astrocytes and microglia respond to and release a number of signalling molecules, which have protective and/or pathological functions. Here we review the current understanding of the contribution of glia to pathological pain and neuroprotection, and how the protective, anti-inflammatory actions of glia are being harnessed to develop new drug targets for neuropathic pain control. Given the prevalence of chronic pain and the partial efficacy of current drugs, which exclusively target neuronal mechanisms, new strategies to manipulate neuron–glia interactions in pain processing hold considerable promise.

Multiple Sclerosis (MS) is an autoimmune inflammatory disease that presents clinically with a range of symptoms including motor, sensory, and cognitive dysfunction as well as demyelination and lesion formation in brain and spinal cord. A variety of animal models of MS have been developed that share many of the pathological hallmarks of MS including motor deficits (ascending paralysis), demyelination and axonal damage of central nervous system (CNS) tissue. In recent years, neuropathic pain has been recognized as a prevalent symptom of MS in a majority of patients. To date, there have been very few investigations into sensory disturbances in animal models of MS. The current work contains the first assessment of hind paw mechanical allodynia (von Frey test) over the course of a relapsing-remitting myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE) rat model of MS and establishes the utility of this model in examining autoimmune induced sensory dysfunction. We demonstrate periods of both decreased responsiveness to touch that precedes the onset of hind limb paralysis, and increased responsiveness (allodynia) that occurs during the period of motor deficit amelioration traditionally referred to as symptom remission. Furthermore, we tested the ability of our recently characterized anti-inflammatory IL-10 gene therapy to treat the autoimmune inflammation induced behavioral symptoms and tissue histopathological changes. This therapy is shown here to reverse inflammation induced paralysis, to reduce disease associated reduction in sensitivity to touch, to prevent the onset of allodynia, to reverse disease associated loss of body weight, and to suppress CNS glial activation associated with disease progression in this model.

Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked DNA can result in long-term expression of secreted proteins. Plasmids expressing either secreted alkaline phosphatase (SEAP) or human interleukin-10 (hIL-10) were injected into the i.t. space in rats, and transgene products were repeatedly measured in the cerebrospinal fluid (CSF). Both SEAP and hIL-10 were maximal at 1 and 2 days after the injection and still detectable at 4 months. The utilization of a plasmid having two features that are hypothesized to increase gene expression (matrix attachment regions (MARs) and lack of CpG dinucleotides) resulted in a significant increase in gene expression. Reinjection of SEAP or hIL-10 plasmids after 4 months significantly increased protein levels at 1 and 14 days after the reinjection. SEAP was uniformly distributed between the DNA delivery site (approximately vertebral level T13) and the lumbar puncture site (L5/L6 inter-vertebral space), was reduced at the cisterna magna, and was detectable, though at much lower levels, in serum. These data suggest that naked DNA has the potential to be used as a therapeutic tool for applications that require long-term release of transgenes into the CSF.

Microglia and/or astrocytes play a significant role in the creation and maintenance of exaggerated pain states with inflammatory and/or neuropathic etiologies. The chemokine, fractalkine, has several functions, including the newly recognized role of mediating neuropathic pain conditions. Although constitutively expressed and released during inflammation, increased release of fractalkine binds to and activates microglia glia leading to pathological pain. We review the critical role of fractalkine in neuron-to-glial communication after peripheral nerve injury and inflammation and explore anti-inflammatory cytokines like interleukin-10 as a novel and effective approach for clinical pain control.

Paclitaxel is a commonly used cancer chemotherapy drug that frequently causes painful peripheral neuropathies. The mechanisms underlying this dose-limiting side effect are poorly understood. Growing evidence supports that proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), released by activated spinal glial cells and within the dorsal root ganglia (DRG) are critical in enhancing pain in various animal models of neuropathic pain. Whether these cytokines are involved in paclitaxel-induced neuropathy is unknown. Here, using a rat neuropathic pain model induced by repeated systemic paclitaxel injections, we examined whether paclitaxel upregulates proinflammatory cytokine gene expression, and whether these changes and paclitaxel-induced mechanical allodynia can be attenuated by intrathecal IL-1 receptor antagonist (IL-1ra) or intrathecal delivery of plasmid DNA encoding the anti-inflammatory cytokine, interleukin-10 (IL-10). The data show that paclitaxel treatment induces mRNA expression of IL-1, TNF, and immune cell markers in lumbar DRG. Intrathecal IL-1ra reversed paclitaxel-induced allodynia and intrathecal IL-10 gene therapy both prevented, and progressively reversed, this allodynic state. Moreover, IL-10 gene therapy resulted in increased IL-10 mRNA levels in lumbar DRG and meninges, measured 2 weeks after initiation of therapy, whereas paclitaxel-induced expression of IL-1, TNF, and CD11b mRNA in lumbar DRG was markedly decreased. Taken together, these data support that paclitaxel-induced neuropathic pain is mediated by proinflammatory cytokines, possibly released by activated immune cells in the DRG. We propose that targeting the production of proinflammatory cytokines by intrathecal IL-10 gene therapy may be a promising therapeutic strategy for the relief of paclitaxel-induced neuropathic pain.

Spinal cord glia (microglia and astrocytes) contribute to enhanced pain states. One model that has been used to study this phenomenon is intrathecal (i.t.) administration of gp120, an envelope glycoprotein of HIV-1 known to activate spinal cord glia and thereby induce low-threshold mechanical allodynia, a pain symptom where normally innocuous (non-painful) stimuli are perceived as painful. Previous studies have shown that i.t. gp120-induced allodynia is mediated via the release of the glial pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF), and interleukin-1beta (IL-1). As we have recently reported that i.t. gp120 induces the release of interleukin-6 (IL-6), in addition to IL-1 and TNF, the present study tested whether this IL-6 release in spinal cord contributes to gp120-induced mechanical allodynia and/or to gp120-induced increases in TNF and IL-1. An i.t. anti-rat IL-6 neutralizing antibody was used to block IL-6 actions upon its release by i.t. gp120. This IL-6 blockade abolished gp120-induced mechanical allodynia. While the literature predominantly documents the cascade of pro-inflammatory cytokines as beginning with TNF, followed by the stimulation of IL-1, and finally TNF plus IL-1 stimulating the release of IL-6, the present findings indicate that a blockade of IL-6 inhibits the gp120-induced elevations of TNF, IL-1, and IL-6 mRNA in dorsal spinal cord, elevation of IL-1 protein in lumbar dorsal spinal cord, and TNF and IL-1 protein release into the surrounding lumbosacral cerebrospinal fluid. These results would suggest that IL-6 induces pain facilitation, and may do so in part by stimulating the production and release of other pro-inflammatory cytokines.

The present experiments examined the role of spinal proinflammatory cytokines [interleukin-1β (IL-1)] and chemokines (fractalkine) in acute analgesia and in the development of analgesic tolerance, thermal hyperalgesia, and tactile allodynia in response to chronic intrathecal morphine. Chronic (5 d), but not acute (1 d), intrathecal morphine was associated with a rapid increase in proinflammatory cytokine protein and/or mRNA in dorsal spinal cord and lumbosacral CSF. To determine whether IL-1 release modulates the effects of morphine, intrathecal morphine was coadministered with intrathecal IL-1 receptor antagonist (IL-1ra). This regimen potentiated acute morphine analgesia and inhibited the development of hyperalgesia, allodynia, and analgesic tolerance. Similarly, intrathecal IL-1ra administered after the establishment of morphine tolerance reversed hyperalgesia and prevented the additional development of tolerance and allodynia. Fractalkine also appears to modulate the effects of intrathecal morphine because coadministration of morphine with intrathecal neutralizing antibody against the fractalkine receptor (CX3CR1) potentiated acute morphine analgesia and attenuated the development of tolerance, hyperalgesia, and allodynia. Fractalkine may be exerting these effects via IL-1 because fractalkine (CX3CL1) induced the release of IL-1 from acutely isolated dorsal spinal cordin vitro. Finally, gene therapy with an adenoviral vector encoding for the release of the anti-inflammatory cytokine IL-10 also potentiated acute morphine analgesia and attenuated the development of tolerance, hyperalgesia, and allodynia. Taken together, these results suggest that IL-1 and fractalkine are endogenous regulators of morphine analgesia and are involved in the increases in pain sensitivity that occur after chronic opiates.

Background Peri-spinal subarachnoid (intrathecal; i.t.) injection of non-viral naked plasmid DNA encoding the anti-inflammatory cytokine, IL-10 (pDNA-IL-10) suppresses chronic neuropathic pain in animal models. However, two sequential i.t. pDNA injections are required within a discrete 5 to 72-hour period for prolonged efficacy. Previous reports identified phagocytic immune cells present in the peri-spinal milieu surrounding the i.t injection site that may play a role in transgene uptake resulting in subsequent IL-10 transgene expression. Methods In the present study, we aimed to examine whether factors known to induce pro-phagocytic anti-inflammatory properties of immune cells improve i.t. IL-10 transgene uptake using reduced naked pDNA-IL-10 doses previously determined ineffective. Both the synthetic glucocorticoid, dexamethasone, and the hexose sugar, D-mannose, were factors examined that could optimize i.t. pDNA-IL-10 uptake leading to enduring suppression of neuropathic pain as assessed by light touch sensitivity of the rat hindpaw (allodynia). Results Compared to dexamethasone, i.t. mannose pretreatment significantly and dose-dependently prolonged pDNA-IL-10 pain suppressive effects, reduced spinal IL-1β and enhanced spinal and dorsal root ganglia IL-10 immunoreactivity. Macrophages exposed to D-mannose revealed reduced proinflammatory TNF-α, IL-1β, and nitric oxide, and increased IL-10 protein release, while IL-4 revealed no improvement in transgene uptake. Separately, D-mannose dramatically increased pDNA-derived IL-10 protein release in culture supernatants. Lastly, a single i.t. co-injection of mannose with a 25-fold lower pDNA-IL-10 dose produced prolonged pain suppression in neuropathic rats. Conclusions Peri-spinal treatment with D-mannose may optimize naked pDNA-IL-10 transgene uptake for suppression of allodynia, and is a novel approach to tune spinal immune cells toward pro-phagocytic phenotype for improved non-viral gene therapy.

Intrathecal administration of the human immunodeficiency virus-1 envelope glycoprotein, gp120, activates astrocytes and microglia to release products that induce thermal hyperalgesia and mechanical allodynia. Both pain states are disrupted by intrathecal CNI-1493, a p38 mitogen-activated protein (MAP) kinase inhibitor. Whether CNI-1493, or any other p38 MAP kinase inhibitor, can cross the blood-brain barrier to affect spinal cord function is unknown. Given that several such drugs are in clinical trials, it is of interest to determine whether they may be potentially useful in treating centrally mediated pain. The aim of the present studies was to determine whether systemic CNI-1493 could block intrathecal gp120-induced thermal hyperalgesia and/or mechanical allodynia. Because p38 MAP kinase inhibition would be expected to prevent proinflammatory cytokine release and/or signal transduction, we sought to determine from the same animals the likely mechanism by which CNI-1493 blocks gp120-induced pain states. These studies show that systemic CNI-1493 blocks intrathecal gp120-induced thermal hyperalgesia and mechanical allodynia. Because CNI-1493 did not block proinflammatory cytokine release, this may suggest disruption at the level of signal transduction. These studies provide the first evidence that systemic p38 MAP kinase inhibitors can prevent centrally mediated exaggerated pain states. Thus, CNI-1493 may provide a novel therapeutic approach for the treatment of pain. © 2001 by the American Pain Society

Perispinal (intrathecal) injection of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 creates exaggerated pain states. Decreases in response thresholds to both heat stimuli (thermal hyperalgesia) and light tactile stimuli (mechanical allodynia) are rapidly induced after gp120 administration. gp120 is the portion of HIV-1 that binds to and activates microglia and astrocytes. These glial cells have been proposed to be key mediators of gp120-induced hyperalgesia and allodynia because these pain changes are blocked by drugs thought to affect glial function preferentially. The aim of the present series of studies was to determine whether gp120-induced pain changes involve proinflammatory cytokines [interleukin-1β (IL-1) and tumor necrosis factor-α (TNF-α)], substances released from activated glia. IL-1 and TNF antagonists each prevented gp120-induced pain changes. Intrathecal gp120 produced time-dependent, site-specific increases in TNF and IL-1 protein release into lumbosacral CSF; parallel cytokine increases in lumbar dorsal spinal cord were also observed. Intrathecal administration of fluorocitrate (a glial metabolic inhibitor), TNF antagonist, and IL-1 antagonist each blocked gp120-induced increases in spinal IL-1 protein. These results support the concept that activated glia in dorsal spinal cord can create exaggerated pain states via the release of proinflammatory cytokines.

We have shown that chronic cold stress strongly interacts with corticosterone (B) to determine subsequent regulation of the hypothalamo-pituitary-adrenal (HPA) axis responses to novel stress. These studies, using the same 2 sets of rats, show that chronic cold also interacts with B and testosterone on signals of energy balance. The two groups of rats differed in weight by 20% and in age by 2 weeks (44-59 days of age). Adrenalectomized rats, replaced with varying doses of B, were exposed to cold or served as controls. Food intake and body weight during the experiments and hormones, metabolites and fat depots were measured on day 5. B, but not cold, affected food intake in the younger rats; by contrast, cold, but not B, affected food intake in the older rats. Testosterone was higher in older control rats and was markedly depressed by cold; younger rats had lower testosterone that was minimally affected by cold. Weight gain decreased in all rats at room temperature with increasing B, whereas they all lost weight in cold independently of B. Cold stimulated and B inhibited interscapular brown adipose tissue DNA content (reflecting sympathetic stimulation of thermogenesis). B stimulated insulin, whereas cold inhibited leptin and insulin; B also increased white adipose tissue weight gain in controls and inhibited its loss in cold. Leptin was unrelated to white adipose tissue depots in older control rats but was strongly related to these stores in younger rats and in all rats in cold. We conclude that: 1. By decreasing signals that act centrally to inhibit food intake (insulin, leptin and testosterone) cold allows B to stimulate food intake; 2. B inhibits weight gain although it causes accrual of fat; 3. Cold, probably through sympathetic stimulation of white adipose tissue, causes fat loss which is modulated by the inhibitory effect of B on sympathetic outflow; and, 4. The slope of the relationship between fat depot size and leptin becomes flatter in cold, possibly because of increased sympathetic outflow to these depots.

To determine the effects of both corticosterone (B) and chronic stressors on acute ACTH responses to restraint, young male rats were exposed to streptozotocin-induced diabetes, cold (5-7 degreesC) or intracerebroventricular (icv) neuropeptide Y (NPY) for 5 d and then exposed to restraint within 2 h after lights on. Two groups of rats were studied: intact and adrenalectomized replaced with B pellets that maintained plasma B in the normal mean 24-h range of intact rats. In addition to ACTH and B responses to restraint on d 5, body weight, food intake, fat depots, glucose and other hormones were measured to determine the role of stress-induced elevations in B on energy balance. ACTH responses to restraint were normal in intact rats subjected to diabetes or cold. By contrast, there was no ACTH or B response to restraint in NPY-infused intact rats. All 3 groups of chronically stimulated adrenalectomized rats with clamped B had facilitated ACTH responses to restraint compared to their treatment controls. Overall food intake increased in all groups of stressed rats; however, augmented intake occurred only during the light in intact rats and equally in the light and dark in B-clamped rats. White adipose depot weights were decreased by both diabetes and cold and increased by NPY in intact rats; the decreases with cold and increases with NPY were both blunted and changes in fat stores were not significant in adrenalectomized, B-clamped rats. We conclude that: 1. diabetes- and cold-induced facilitation of restraint-induced afferent input to hypothalamic control of the hypothalamo-pituitary-adrenal (HPA) axis is opposed in intact rats by the elevated feedback signal of B secretion; 2. NPY does not induce facilitation of afferent stress pathways; 3. chronic stimulation of the HPA axis induces acute hyperresponsiveness of hypothalamic neurons to restraint provided that the afferent input of this acute stimulus is not prevented by B feedback; 4. stimulus-induced elevations in B secretion result in day-time feeding; 5. insensitivity of both caloric efficiency and white fat stores to chronic stress in adrenalectomized, B-clamped rats results from loss of normally variable B levels.

Introduction: Current research indicates that chronic peripheral neuropathic pain includes a role for glia and the actions of proinflammatory factors. This review briefly discusses the glial and cytokine responses that occur following peripheral nerve damage in support of utilizing anti-inflammatory cytokine interleukin-10 (IL-10) therapy to suppress chronic peripheral neuropathic pain. Spinal Nonviral Interleukin-10 Gene Therapy: IL-10 is one of the most powerful endogenous counter-regulators of proinflammatory cytokine function that acts in the nervous system. Subarachnoid (intrathecal) spinal injection of the gene encoding IL-10 delivered by nonviral vectors has several advantages over virally mediated gene transfer methods and leads to profound pain relief in several animal models. Nonviral gene delivery: Lastly, data are reviewed that nonviral deoxyribonucleic acid (DNA) encapsulated by a biologically safe copolymer, poly(lactic-co-glycolic) acid (PLGA), thought to protect DNA, leads to significantly improved therapeutic gene transfer in animal models, which additionally and significantly extends pain relief. Conclusions: The impact of these early studies exploring anti-inflammatory genes emphasizes the exceptional therapeutic potential of new biocompatible intrathecal nonviral gene delivery approaches such as PLGA microparticles. Ultimately, ongoing expression of therapeutic genes is a viable option to treat chronic neuropathic pain in the clinic.

Under normal conditions, acute pain processing consists of well-characterized neuronal signaling events. When dysfunctional pain signaling occurs, pathological pain ensues. Glial activation and their released factors participate in the mediation of pathological pain. The use of cannabinoid compounds for pain relief is currently an area of great interest for both basic scientists and physicians. These compounds, bind mainly either the cannabinoid receptor subtype 1 (CB1R) or cannabinoid receptor subtype 2 (CB2R) and are able to modulate pain. Although cannabinoids were initially only thought to modulate pain via neuronal mechanisms within the central nervous system, strong evidence now supports that CB2R cannabinoid compounds are capable of modulating glia, (e.g. astrocytes and microglia) for pain relief. However, the mechanisms underlying cannabinoid receptor-mediated pain relief remain largely unknown. An emerging body of evidence supports that CB2R agonist compounds may prove to be powerful novel therapeutic candidates for the treatment of chronic pain.

Spinal glial and proinflammatory cytokine actions are strongly implicated in pathological pain. Spinal administration of the anti-inflammatory cytokine interleukin (IL)-10 abolishes pathological pain and suppresses proinflammatory IL-1β and tumor necrosis factor alpha (TNF-α). Drugs that bind the cannabinoid type-2 receptor (CB(2)R) expressed on spinal glia reduce mechanical hypersensitivity. To better understand the CB(2)R-related anti-inflammatory profile of key anatomical nociceptive regions, we assessed mechanical hypersensitivity and protein profiles following intrathecal application of the cannabilactone CB(2)R agonist, AM1710, in 2 animal models; unilateral sciatic nerve chronic constriction injury (CCI), and spinal application of human immunodeficiency virus-1 glycoprotein 120 (gp120), a model of peri-spinal immune activation. In CCI animals, lumbar dorsal spinal cord and corresponding dorsal root ganglia (DRG) were evaluated by immunohistochemistry for expression of IL-10, IL-1β, phosphorylated p38-mitogen-activated-kinase (p-p38MAPK), a pathway associated with proinflammatory cytokine production, glial cell markers, and degradative endocannabinoid enzymes, including monoacylglycerol lipase (MAGL). AM1710 reversed bilateral mechanical hypersensitivity. CCI revealed decreased IL-10 expression in dorsal spinal cord and DRG, while AM1710 resulted in increased IL-10, comparable to controls. Adjacent DRG and spinal sections revealed increased IL-1β, p-p38MAPK, glial markers, and/or MAGL expression, while AM1710 suppressed all but spinal p-p38MAPK and microglial activation. In spinal gp120 animals, AM1710 prevented bilateral mechanical hypersensitivity. For comparison to immunohistochemistry, IL-1β and TNF-α protein quantification from lumbar spinal and DRG homogenates was determined, and revealed increased DRG IL-1β protein levels from gp120, that was robustly prevented by AM1710 pretreatment. Cannabilactone CB(2)R agonists are emerging as anti-inflammatory agents with pain therapeutic implications.

Interleukin-10 (IL-10) is an anti-inflammatory molecule that has achieved interest as a therapeutic for neuropathic pain. In this work, the potential of plasmid DNA-encoding IL-10 (pDNA-IL-10) slowly released from biodegradable microparticles to provide long-term pain relief in an animal model of neuropathic pain was investigated.PLGA microparticles encapsulating pDNA-IL-10 were developed and assessed both in vitro and in vivo.In vitro, pDNA containing microparticles activated macrophages, enhanced the production of nitric oxide, and increased the production of IL-10 protein relative to levels achieved with unencapsulated pDNA-IL-10. In vivo, intrathecally administered microparticles embedded in meningeal tissue, induced phagocytic cell recruitment to the cerebrospinal fluid, and relieved neuropathic pain for greater than 74 days following a single intrathecal administration, a feat not achieved with unencapsulated pDNA. Therapeutic effects of microparticle-delivered pDNA-IL-10 were blocked in the presence of IL-10-neutralizing antibody, and elevated levels of plasmid-derived IL-10 were detected in tissues for a prolonged time period post-injection (28 days), demonstrating that therapeutic effects are dependent on IL-10 protein production.These studies demonstrate that microparticle encapsulation significantly enhances the potency of intrathecally administered pDNA, which may be extended to treat other disorders that require intrathecal gene therapy.

The anti-inflammatory cytokine interleukin-10 (IL-10) shows promise for the treatment of neuropathic pain, but for IL-10 to be clinically useful as a short-term therapeutic its duration needs to be improved. In this study, IL-10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL-10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL-10 products for subsequent in vivo testing. PEGylation of IL-10 by acylation yielded a highly PEGylated product with a 35-fold in vitro biological activity reduction. PEGylation of IL-10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of approximately 3-fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL-10 increased the concentration of IL-10 in the cerebrospinal fluid over time. Relative to unmodified IL-10, the 20 kDa PEG-IL-10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short-term treatment or, in conjunction with other approaches, the long-term treatment of enhanced pain states.

One method for the delivery of therapeutic proteins to the spinal cord is to inject nonviral gene vectors including plasmid DNA into the cerebrospinal fluid (CSF) that surrounds the spinal cord (intrathecal space). This approach has produced therapeutic benefits in animal models of disease and several months of protein expression; however, there is little information available on the immune response to these treatments in the intrathecal space, the relevance of plasmid CpG sequences to any plasmid-induced immune response, or the effect of this immune response on transgene expression.In the present study, coding or noncoding plasmids were delivered to the intrathecal space of the lumbar spinal region in rats. Lumbosacral CSF was then collected at various time points afterwards for monitoring of cytokines and transgene expression.This work demonstrates, for the first time, increased tumor necrosis factor-alpha and interleukin-1 in response to intrathecal plasmid vector injection and provides evidence indicating that this response is largely absent in a CpG-depleted vector. Transgene expression in the CSF is not significantly affected by this immune response. Expression after intrathecal plasmid injection is variable across rats but correlates with the amount of tissue associated plasmid and is increased by disrupting normal CSF flow.The data obtained in the present study indicate that plasmid immunogenicity may affect intrathecal plasmid gene therapy safety but not transgene expression in the CSF. Furthermore, the development of methods to prevent loss of plasmid via CSF flow out of the central nervous system through the injection hole and/or natural outflow routes may increase intrathecal plasmid gene delivery efficacy.

Traditional approaches to treating chronic neuropathic pain largely focus on manipulations directly altering neuronal activity or neuron-to-neuron communication. Recently, however, it has become clear that glial cells (including microglia and astroglia) play a significant role in pain expression in a variety of neuropathic pain models. Multiple aspects of the inflammatory response of glial cells, commonly observed in neuropathic pain conditions, have been implicated in pain expression. Thus, glial cell inflammation has emerged as a potential therapeutic target in neuropathic pain. Our laboratory has been exploring the use of an anti-inflammatory cytokine, interleukin-10 (IL-10), to control glial inflammatory activation thereby controlling neuropathic pain. IL-10 protein delivery is limited by a short half-life and an inability to cross into the central nervous system from the periphery, making a centrally delivered gene therapy approach attractive. We have recently characterized a non-viral gene therapy approach using two injections of naked DNA to achieve long-term (>3 months) control of neuropathic pain in a peripheral nerve injury model. Timing and dose requirements leading to long-term pain control are discussed in this review, as is recent work using microparticle-encapsulated DNA to achieve long-term therapeutic efficacy with a single injection.

Brain-derived neurotrophic factor (BDNF) was covalently attached to polyethylene glycol (PEG) in order to enhance delivery to the spinal cord via the cerebrospinal fluid (intrathecal administration). By varying reaction conditions, mixtures of BDNF covalently attached to one (primary), two (secondary), three (tertiary), or more (higher order) PEG molecules were produced. The biological activity of each resulting conjugate mixture was assessed with the goal of identifying a relationship between the number of PEG molecules attached to BDNF and biological activity. A high degree of in vitro biological activity was maintained in mixtures enriched in primary and secondary conjugate products, while a substantial reduction in biological activity was observed in mixtures with tertiary and higher order conjugates. When a biologically active mixture of PEG-BDNF was administered intrathecally, it displayed a significantly improved half-life in the cerebrospinal fluid and an enhanced penetration into spinal cord tissue relative to native BDNF. Results from these studies suggest a PEGylation strategy that preserves the biological activity of the protein while also improving the half-life of the protein in vivo. Furthermore, PEGylation may be a promising approach for enhancing intrathecal delivery of therapeutic proteins with potential for treating disease and injury in the spinal cord.

It is recently become clear that activated immune cells and immune-like glial cells can dramatically alter neuronal function. By increasing neuronal excitability, these non-neuronal cells are now implicated in the creation and maintenance of pathological pain, such as occurs in response to peripheral nerve injury. Such effects are exerted at multiple sites along the pain pathway, including at peripheral nerves, dorsal root ganglia, and spinal cord. In addition, activated glial cells are now recognized as disrupting the pain suppressive effects of opioid drugs and contributing to opioid tolerance and opioid dependence/withdrawal. While this review focuses on regulation of pain and opioid actions, such immune-neuronal interactions are broad in their implications. Such changes in neuronal function would be expected to occur wherever immune-derived substances come in close contact with neurons.

Spinal glial activation and consequent interleukin-1 (IL-1) release are implicated in pain facilitation induced by inflammation/damage to skin and peripheral nerves. It is unclear whether pain facilitation induced at deep tissue sites also depends on these. We investigated whether spinal IL-1 and/or glial activation mediates bilateral allodynia induced by repeated unilateral intramuscular injections of acidic saline to rats. Given the prominent role of spinal IL-1 in various bilateral pain models, we predicted that intrathecal IL-1 receptor antagonist (IL-1ra) would suppress bilateral allodynia in this model as well. Surprisingly, neither single nor repeated intrathecal injections of IL-1ra affected allodynia, measured by the von Frey test, induced by prior intramuscular acidic saline compared with vehicle-injected controls. In addition, we tested the effect of 2 additional intrathecal manipulations that are broadly efficacious in suppressing glially mediated pain facilitation: (1) a glial metabolic inhibitor (fluorocitrate) and (2) the anti-inflammatory cytokine, interleukin-10 (IL-10). Like IL-1ra, fluorocitrate and IL-10 each failed to reverse allodynia. Finally, we observed no significant activation of glial cells, as assessed by immunohistochemistry of glial activation markers, in the lumbar spinal cord in response to intramuscular acidic saline. Taken together, the present data suggest that acidic saline-induced bilateral allodynia is created independently of glial activation. Perspective From converging lines of evidence, the current studies suggest that persistent bilateral allodynia induced by repeated intramuscular acidic saline is not mediated by spinal IL-1 and/or spinal glial activation. As such, this might represent the first evidence for pain facilitation occurring in the absence of glial involvement.

The present series of experiments examined whether the complement cascade might play a key role in the expression of mechanical allodynia. Soluble complement receptor 1 (sCR1) was used to block the activation of the membrane attack pathway of the complement cascade. In doing so, sCR1 prevents the formation of the biologically active end products C3a, C5a, and membrane attack complexes (MACs). Intrathecal sCR1 had no effect on the behavioral responses of control groups. In contrast, blockade of this pathway abolished the expression of mechanical allodynia induced by peripheral nerve inflammation (sciatic inflammatory neuropathy model), partial sciatic nerve injury (chronic constriction injury model), and intrathecal injection of human immunodeficiency virus type 1 gp120, a viral envelope protein that activates glia. The fact that enhanced nociception was prevented or reversed in all 3 paradigms suggests that complement might be broadly involved in spinally mediated pain enhancement. The mechanisms whereby complement activation might potentially affect the functioning of microglia, astrocytes, and neurons are discussed. The complement cascade has not been previously implicated in spinal sensitization. These data suggest that complement activation within the spinal cord might contribute to enhanced pain states and provide additional evidence for immune regulation of pain transmission.

Despite many decades of drug development, effective therapies for neuropathic pain remain elusive. The recent recognition of spinal cord glia and glial pro-inflammatory cytokines as important contributors to neuropathic pain suggests an alternative therapeutic strategy; that is, targeting glial activation or its downstream consequences. While several glial-selective drugs have been successful in controlling neuropathic pain in animal models, none are optimal for human use. Thus the aim of the present studies was to explore a novel approach for controlling neuropathic pain. Here, an adeno-associated viral (serotype II; AAV2) vector was created that encodes the anti-inflammatory cytokine, interleukin-10 (IL-10). This anti-inflammatory cytokine is known to suppress the production of pro-inflammatory cytokines. Upon intrathecal administration, this novel AAV2-IL-10 vector was successful in transiently preventing and reversing neuropathic pain. Intrathecal administration of an AAV2 vector encoding beta-galactosidase revealed that AAV2 preferentially infects meningeal cells surrounding the CSF space. Taken together, these data provide initial support that intrathecal gene therapy to drive the production of IL-10 may prove to be an efficacious treatment for neuropathic pain.

Glia are now recognized as important contributors in pathological pain creation and maintenance. Spinal cord glia exhibit extensive gap junctional connectivity, raising the possibility that glia are involved in the contralateral spread of excitation resulting in mirror image pain. In the present experiments, the gap junction decoupler carbenoxolone was administered intrathecally after induction of neuropathic pain in response to sciatic nerve inflammation (sciatic inflammatory neuropathy) or partial nerve injury (chronic constriction injury). In both neuropathic pain models, a low dose of carbenoxolone reversed mirror image mechanical allodynia, while leaving ipsilateral mechanical allodynia unaffected. Ipsilateral thermal hyperalgesia was briefly attenuated. Critically, blockade of mechanical allodynia and thermal hyperalgesia was not observed in response to intrathecal glycyrrhizic acid, a compound similar to carbenoxolone in all respects but it does not decouple gap junctions. Thus, blockade of mechanical allodynia and thermal hyperalgesia by carbenoxolone does appear to reflect an effect on gap junctions. Examination of carbenoxolone's effects on intrathecal human immunodeficiency virus type 1 gp120 showed that blockade of pain facilitation might result, at least in part, via suppression of interleukin-1 and, in turn, interleukin-6. These data provide the first suggestion that spread of excitation via gap junctions might contribute importantly to inflammatory and traumatic neuropathic pain. Perspective The current studies provide evidence for involvement of gap junctions in spinal cord pain facilitation. Intrathecal carbenoxolone, a gap junction decoupler, reversed neuropathy-induced mirror image pain and intrathecal gp120-induced allodynia. In addition, it decreased gp120-induced proinflammatory cytokines. This suggests gap junction activation might lead to proinflammatory cytokine release by distantly activated glia.

Snakebites are a relevant public health problem in Central and South America. Snake bite envenomations cause intense pain, not relieved by anti-venom. The fangs of many species are short, causing subcutaneous injection. Fangs of larger species inflict subcutaneous or intramuscular envenomation. To understand pain induced by subcutaneous venom, this study examined spinal mechanisms involved in pain-enhancing effects of subcutaneous Lys49 and Asp49 secretory phospholipase-A2 (sPLA2), two components of Bothrops asper snake venom showing highly different enzymatic activities. Unilateral intraplantar sPLA2-Lys49 (catalytically inactive) or sPLA2-Asp49 (catalytically active) into rat hindpaws each induced mechanical hyperalgesia (Randall–Selitto test), whereas only catalytically active sPLA2-Asp49 caused mechanical allodynia (von Frey test). Effects induced by both sPLA2s were inhibited by intrathecal fluorocitrate, a reversible glial metabolic inhibitor. In support, immunohistochemical analysis revealed activation of dorsal horn astrocytes and microglia after intraplantar injection of either sPLA2. Spinal proinflammatory cytokines, nitric oxide, and prostanoids each appear to be involved in the pain-enhancing effects of these sPLA2s. Blockade of interleukin-1 (IL1) inhibited hyperalgesia induced by both sPLA2s, while leaving allodynia unaffected. Blockade of tumor necrosis factor reduced responses to sPLA2-Asp49. An inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7-NI), inhibited hyperalgesia induced by both sPLA2s, without interfering with allodynia induced by sPLA2-Asp49. On the other hand, l-N6-(1-iminoethyl)lysine (l-NI), an inhibitor of the inducible nitric oxide synthase, did not alter any sPLA2-induced effect. Lastly, celecoxib, an inhibitor of cyclooxygenase-2, attenuated sPLA2 actions. These data provide the first evidence of spinal mediators involved in pain facilitation induced by subcutaneous venoms. International Association for the Study of Pain//IASP/Estados Unidos Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/[0596/02-7]/CAPES/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[99/08432-8]/FAPESP/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[00/12303-8]/FAPESP/Brasil UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Snakebites constitute a serious public health problem in Central and South America, where species of the lancehead pit vipers (genus Bothrops) cause the majority of accidents. Bothrops envenomations are very painful, and this effect is not neutralized by antivenom treatment. Two variants of secretory phospholipases A2 (sPLA2), corresponding to Asp49 and Lys49 PLA2s, have been isolated from Bothrops asper venom. These sPLA2s induce hyperalgesia in rats following subcutaneous injection. However, venom in natural Bothrops bites is frequently delivered intramuscularly, thereby potentially reaching peripheral nerve bundles. Thus, the present series of experiments tested whether these sPLA2s could exert pain-enhancing effects following administration around healthy sciatic nerve. Both were found to produce mechanical allodynia ipsilateral to the injection site; no thermal hyperalgesia was observed. As no prior study has examined potential spinal mechanisms underlying sPLA2 actions, a series of anatomical and pharmacological studies were performed. These demonstrated that both sPLA2s produce activation of dorsal horn astrocytes and microglia that is more prominent ipsilateral to the site of injection. As proinflammatory cytokines and nitric oxide have each been previously implicated in spinally mediated pain facilitation, the effect of pharmacological blockade of these substances was tested. The results demonstrate that mechanical allodynia induced by both sPLA2s is blocked by interleukin-1 receptor antagonist, anti-rat interleukin-6 neutralizing antibody, the anti-inflammatory cytokine interleukin-10, and a nitric oxide synthesis inhibitor (l-NAME). As a variety of immune cells also produce and release sPLA2s during inflammatory states, the data may have general implications for the understanding of inflammatory pain. International Association for the Study of Pain//IASP/Estados Unidos Coordenação de Aperfeiçoamento de Pessoal de nivel Superior/[BEX0596/02-7]/CAPES/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[99/08432-8]/FAPESP/Brasil National Institutes of Health/[DA015642]/NIH/Estados Unidos National Institutes of Health/[NS40696]/NIH/Estados Unidos National Institutes of Health/[NS38020]/NIH/Estados Unidos National Institutes of Health/[DA015656]/NIH/Estados Unidos National Institutes of Health/[AI51093]/NIH/Estados Unidos UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)