Effects of prenatal opioid and alcohol exposures on immune and serotonin factors in human placenta.

PURPOSE: Opioids and alcohol impact critical serotonin (5-HT) function in the developing placenta and fetus through the actions of immune proinflammatory factors. Yet, possible convergent effects of opioids and alcohol on human placental toll-like receptor 4 (TLR4) activation and subsequent 5-HT homeostasis remain entirely unknown. The purpose of this study was to examine the effect of prenatal exposure to opioids with or without prenatal alcohol exposure (PAE) on the expression of key placental immune and serotonin signaling factors in human placental tissue obtained from a well-characterized prospective cohort. METHODS: Data were collected from a subset of participants enrolled in the prospective pre-birth Ethanol, Neurodevelopment, Infant, and Child Health (ENRICH-1) cohort. Women were recruited and classified into four study groups: 1) PAE (n = 20); 2) those taking medications for opioid use disorder (MOUD; n = 28), 3) concurrent PAE and MOUD (n = 20); and 4) controls (HC; n = 20) based on prospective, repeated self-report, and biomarker analysis. Placenta samples underwent tissue processing to identify mRNA for TLR4, nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), serotonin transporter (SERT), tryptophan hydroxylase (TPH1), indoleamine 2,3-Dioxygenase 1 (IDO) as well as protein concentrations of TLR4, IL-1ß, TNF-α, SERT. To consider the association between study group and mRNA/protein expression of our targets, multivariable regression models were developed with inclusion of a priori selected covariates. RESULTS: There was a significant negative association between PAE and SERT mRNA (ß = -0.01; p < 0.01) and a positive association with TPH1 mRNA expression (ß = 0.78; p < 0.05). In addition, there was a negative association between MOUD and TNF-α protein expression (ß = -0.12; p < 0.05). CONCLUSIONS: This study provides the first evidence that PAE may inhibit SERT expression while simultaneously promoting increased TPH1 protein expression in human placenta. This may result in increased 5-HT in fetal circulation known to affect neurodevelopment. Our data suggest that opioids and alcohol may disturb the bidirectional, dynamic interaction between the placental immune and serotonin system. Given the implication for brain development and health across the life-span further investigation of these critical mechanisms in well-defined cohorts is required.

Peripheral versus central mechanisms of the cannabinoid type 2 receptor agonist AM1710 in a mouse model of neuropathic pain.

The CB2 R 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 CB1 R, 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 CB1 R. 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 CB1 R (-/-), (+/-), (+/+) 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 CB1 R is dispensable for either peripheral or central anti-allodynic actions of AM1710 in neuropathic mice. Cannabinoid CB2 R agonists produce heightened spinal IL-10 which may be clinically relevant to successfully treat neuropathic pain.

The LFA-1 antagonist BIRT377 reverses neuropathic pain in prenatal alcohol-exposed female rats via actions on peripheral and central neuroimmune function in discrete pain-relevant tissue regions.

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)-1ß, 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-1ß 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-1ß, 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.

Chorioamnionitis in Rats Precipitates Extended Postnatal Inflammatory Lymphocyte Hyperreactivity.

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 ß1, 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.

Cytokines in Pain: Harnessing Endogenous Anti-Inflammatory Signaling for Improved Pain Management.

Current pain therapeutics offer inadequate relief to patients with chronic pain. A growing literature supports that pro-inflammatory cytokine signaling between immune, glial, and neural cells is integral to the development of pathological pain. Modulation of these communications may hold the key to improved pain management. In this review we first offer an overview of the relationships between pro-inflammatory cytokine and chemokine signaling and pathological pain, with a focus on the actions of cytokines and chemokines in communication between glia (astrocytes and microglia), immune cells (macrophages and T cells), and neurons. These interactions will be discussed in relation to both peripheral and central nervous system locations. Several novel non-neuronal drug targets for controlling pain are emerging as highly promising, including non-viral IL-10 gene therapy, which offer the potential for substantial pain relief through localized modulation of targeted cytokine pathways. Preclinical investigation of the mechanisms underlying the success of IL-10 gene therapy revealed the unexpected discovery of the powerful anti-nociceptive anti-inflammatory properties of D-mannose, an adjuvant in the non-viral gene therapeutic formulation. This review will include gene therapeutic approaches showing the most promise in controlling pro-inflammatory signaling via increased expression of anti-inflammatory cytokines like interleukin-10 (IL-10) or IL-4, or by directly limiting the bioavailability of specific pro-inflammatory cytokines, as with tumor necrosis factor (TNF) by the TNF soluble receptor (TNFSR). Approaches that increase endogenous anti-inflammatory signaling may offer additional opportunities for pain therapeutic development in patients not candidates for gene therapy. Promising novel avenues discussed here include the disruption of lymphocyte function-associated antigen (LFA-1) activity, antagonism at the cannabinoid 2 receptor (CB2R), and toll-like receptor 4 (TLR4) antagonism. Given the partial efficacy of current drugs, new strategies to manipulate neuroimmune and cytokine interactions hold considerable promise.

Preclinical chorioamnionitis dysregulates CXCL1/CXCR2 signaling throughout the placental-fetal-brain axis.

In the United States, perinatal brain injury (PBI) is a major cause of infant mortality and childhood disability. For a large proportion of infants with PBI, central nervous system (CNS) injury begins in utero with inflammation (chorioamnionitis/CHORIO) and/or hypoxia-ischemia. While studies show CHORIO contributes to preterm CNS injury and is also a common independent risk factor for brain injury in term infants, the molecular mechanisms mediating inflammation in the placental-fetal-brain axis that result in PBI remain a gap in knowledge. The chemokine (C-X-C motif) ligand 1 (CXCL1), and its cognate receptor, CXCR2, have been clinically implicated in CHORIO and in mature CNS injury, although their specific role in PBI pathophysiology is poorly defined. Given CXCL1/CXCR2 signaling is essential to neural cell development and neutrophil recruitment, a key pathological hallmark of CHORIO, we hypothesized CHORIO would upregulate CXCL1/CXCR2 expression in the placenta and fetal circulation, concomitant with increased CXCL1/CXCR2 signaling in the developing brain, immune cell activation, neutrophilia, and microstructural PBI. On embryonic day 18 (E18), a laparotomy was performed in pregnant Sprague Dawley rats to induce CHORIO. Specifically, uterine arteries were occluded for 60min to induce placental transient systemic hypoxia-ischemia (TSHI), followed by intra-amniotic injection of lipopolysaccharide (LPS). Pups were born at E22. Placentae, serum and brain were collected along an extended time course from E19 to postnatal day (P)15 and analyzed using multiplex electrochemiluminescence (MECI), Western blot, qPCR, flow cytometry (FC) and diffusion tensor imaging (DTI). Results demonstrate that compared to sham, CHORIO increases placental CXCL1 and CXCR2 mRNA levels, concomitant with increased CXCR2+ neutrophils. Interestingly, pup serum CXCL1 expression in CHORIO parallels this increase, with sustained elevation through P15. Analyses of CHORIO brains reveal similarly increased CXCL1/CXCR2 expression through P7, together with increased neutrophilia, microgliosis and peripheral macrophages. Similar to the placenta, cerebral neutrophilia was defined by increased CXCR2 surface expression and elevated myeloperoxidase expression (MPO), consistent with immune cell activation. Evaluation of microstructural brain injury at P15 with DTI reveals aberrant microstructural integrity in the callosal and capsular white matter, with reduced fractional anisotropy in superficial and deep layers of overlying cortex. In summary, using an established model of CHORIO that exhibits mature CNS deficits mimicking those of preterm survivors, we show CHORIO induces injury throughout the placental-fetal-brain axis with a CXCL1/CXCR2 inflammatory signature, neutrophilia, and microstructural abnormalities. These data are concomitant with abnormal cerebral CXCL1/CXCR2 expression, and support temporal aberrations in CXCL1/CXCR2 and neutrophil dynamics in the placental-fetal-brain axis following CHORIO. These investigations define novel targets for directed therapies for infants at high risk for PBI.

Prenatal alcohol exposure is a risk factor for adult neuropathic pain via aberrant neuroimmune function.

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-1ß) 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-1ß. 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.

Prenatal alcohol exposure potentiates chronic neuropathic pain, spinal glial and immune cell activation and alters sciatic nerve and DRG cytokine levels.

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 ß-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)-1ß, 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.

Chronic Sciatic Neuropathy in Rat Reduces Voluntary Wheel-Running Activity With Concurrent Chronic Mechanical Allodynia.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Intrathecal cannabilactone CB(2)R agonist, AM1710, controls pathological pain and restores basal cytokine levels.

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-1ß 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-1ß, 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-1ß, 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-1ß and TNF-α protein quantification from lumbar spinal and DRG homogenates was determined, and revealed increased DRG IL-1ß 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.

Release of plasmid DNA-encoding IL-10 from PLGA microparticles facilitates long-term reversal of neuropathic pain following a single intrathecal administration.

PURPOSE: 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. METHODS: PLGA microparticles encapsulating pDNA-IL-10 were developed and assessed both in vitro and in vivo. RESULTS: 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. CONCLUSIONS: 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.

Enduring reversal of neuropathic pain by a single intrathecal injection of adenosine 2A receptor agonists: a novel therapy for neuropathic pain.

Previous studies of peripheral immune cells have documented that activation of adenosine 2A receptors (A(2A)Rs) 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 A(2A)R agonists on neuropathic pain using the chronic constriction injury (CCI) model. A single intrathecal injection of the A(2A)R 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 A(2A)R 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 A(2A)R 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 A(2A)Rs 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.

Immunogenicity of intrathecal plasmid gene delivery: cytokine release and effects on transgene expression.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

PEGylation of brain-derived neurotrophic factor for preserved biological activity and enhanced spinal cord distribution.

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.

Interleukin-6 mediates low-threshold mechanical allodynia induced by intrathecal HIV-1 envelope glycoprotein gp120.

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.

Intrathecal interleukin-10 gene therapy attenuates paclitaxel-induced mechanical allodynia and proinflammatory cytokine expression in dorsal root ganglia in rats.

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.

Intrathecal gene transfer by adeno-associated virus for pain.

Chronic pain is among the most prevalent medical problems, affecting more than half of patients with advanced cancer and many with other common diseases. Current analgesics often fail to provide satisfactory symptom relief and frequently cause severe side effects. Intrathecal (IT) gene transfer is an attractive method for pain research in rodent models, because it allows targeting of a wide variety of secretable peptides and proteins to the spinal cord, an important neural center for the processing of nociceptive signals. The potential of IT gene transfer for improving opioid therapy and for validating new analgesic targets, such as cytokines involved in spinal glial activation, is discussed. The IT space has been notoriously resistant to efficient gene transfer, limiting therapeutic gene expression to less than 2 weeks with most vector systems. Recent progress with adeno-associated virus (AAV) technology allowed efficient long-term gene expression, facilitating studies reflective of the chronic nature of many pain states. AAV is one of the most advanced gene therapy vectors currently undergoing clinical trials for a variety of disorders. In patients, AAV vectors could be administered intrathecally by a lumbar puncture, a safe procedure routinely performed at the bedside. AAV vectors may therefore become an important tool for translational studies to validate newly identified therapeutic targets in clinical pain states.

Controlling pathological pain by adenovirally driven spinal production of the anti-inflammatory cytokine, interleukin-10.

Gene therapy for the control of pain has, to date, targeted neurons. However, recent evidence supports that spinal cord glia are critical to the creation and maintenance of pain facilitation through the release of proinflammatory cytokines. Because of the ability of interleukin-10 (IL-10) to suppress proinflammatory cytokines, we tested whether an adenoviral vector encoding human IL-10 (AD-h-IL10) would block and reverse pain facilitation. Three pain models were examined, all of which are mediated by spinal pro-inflammatory cytokines. Acute intrathecal administration of rat IL-10 protein itself briefly reversed chronic constriction injury-induced mechanical allodynia and thermal hyperalgesia. The transient reversal caused by IL-10 protein paralleled the half-life of human IL-10 protein in the intrathecal space (t(1/2) approximately 2 h). IL-10 gene therapy both prevented and reversed thermal hyperalgesia and mechanical allodynia, without affecting basal responses to thermal or mechanical stimuli. Extra-territorial, as well as territorial, pain changes were reversed by this treatment. Intrathecal AD-h-IL10 injected over lumbosacral spinal cord led to elevated lumbosacral cerebrospinal fluid (CSF) levels of human IL-10, with far less human IL-10 observed in cervical CSF. In keeping with IL-10's known anti-inflammatory actions, AD-h-IL10 lowered CSF levels of IL-1, relative to control AD. These studies support that this gene therapy approach provides an alternative to neuronally focused drug and gene therapies for clinical pain control.

Controlling neuropathic pain by adeno-associated virus driven production of the anti-inflammatory cytokine, interleukin-10.

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.

Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation.

Activated glial cells (microglia and astroglia) in the spinal cord play a major role in mediating enhanced pain states by releasing proinflammatory cytokines and other substances thought to facilitate pain transmission. In the present study, we report that intrathecal administration of minocycline, a selective inhibitor of microglial cell activation, inhibits low threshold mechanical allodynia, as measured by the von Frey test, in two models of pain facilitation. In a rat model of neuropathic pain induced by sciatic nerve inflammation (sciatic inflammatory neuropathy, SIN), minocycline delayed the induction of allodynia in both acute and persistent paradigms. Moreover, minocycline was able to attenuate established SIN-induced allodynia 1 day, but not 1 week later, suggesting a limited role of microglial activation in more perseverative pain states. Our data are consistent with a crucial role for microglial cells in initiating, rather than maintaining, enhanced pain responses. In a model of spinal immune activation by intrathecal HIV-1 gp120, we show that the anti-allodynic effects of minocycline are associated with decreased microglial activation, attenuated mRNA expression of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-1beta-converting enzyme, TNF-alpha-converting enzyme, IL-1 receptor antagonist and IL-10 in lumbar dorsal spinal cord, and reduced IL-1beta and TNF-alpha levels in the CSF. In contrast, no significant effects of minocycline were observed on gp120-induced IL-6 and cyclooxygenase-2 expression in spinal cord or CSF IL-6 levels. Taken together these data highlight the importance of microglial activation in the development of exaggerated pain states.