LFA-1 antagonist (BIRT377) similarly reverses peripheral neuropathic pain in male and female mice with underlying sex divergent peripheral immune proinflammatory phenotypes.

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-1ß 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-1ß and TNF, and increased IL-10 and transforming growth factor (TGF)-ß1, 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-ß1 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.

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.

Effects of spinal non-viral interleukin-10 gene therapy formulated with d-mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues.

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-ß1 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.

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.