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

Abstract

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.

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

“…A modification of Bennett and Xie's [46] sciatic nerve (SCN) Chronic constriction injury (CCI) was used after BL hindpaw threshold assessment, as detailed previously [47] . Briefly, following isoflurane anesthesia (induction at 3.0 vol.% followed by 1.5 vol.%-2.0 vol.% in oxygen, 2.0 L/min), the dorsal left thigh was shaved and cleaned using 70% Ethanol (EtOH) that was air dried prior to surgery.…”

“…Mice were habituated to the testing environment for ~45 min within the first 4 h of the light cycle, for four periods over the course of one week prior to BL hindpaw assessment. Hindpaw threshold responses to light mechanical stimuli were assessed by adopting principles of the von Frey fiber test originally developed for the rat [49] , and modified for the mouse, as recently described in detail [47] . Hindpaw assessment occurred within the first 2 h of the light cycle in testing groups of 4-6, with testers blind to experimental conditions.…”

“…The total number of positive and negative responses were then entered into the computer software program, PsychoFit (http://psych.colorado.edu/~lharvey: RRID: SCR_015381) to determine the absolute withdrawal threshold (50% paw withdrawal threshold), as previously described [50] . The PsychoFit program fits a Gaussian integral psychometric function to the observed withdrawal rates for each monofilament using a maximum-likelihood fitting method [47,51] . The interpolated 50% withdrawal thresholds were then used for statistical analysis.…”

“…Tissue collection was conducted in six cohorts of mice (8 mice in each cohort, N of 1 from each experimental condition) as previously described [47,51] and modified as described here. Immediately following behavioral analysis on Day 13 post-surgery (Day 3 post-injection), mice were deeply anesthetized under isoflurane (10 min in 5% isoflurane and in oxygen at 2 L/min), followed by rapid transcardial perfusion with ice cold 0.1 M phosphate buffered saline (PBS; pH = 7.4; flow rate 10 mL/min).…”

Ginsenoside Rf relieves mechanical hypersensitivity, depression‐like behavior, and inflammatory reactions in chronic constriction injury rats

“…IL‐10 is considered to be a powerful anti‐inflammatory cytokine that inhibits the actions of many proinflammatory cytokines including tumor necrosis factor and IL‐1β (Lobo‐Silva, Carriche, Castro, Roque, & Saraiva, ; Vanderwall et al, ). Chronic ginsenoside Rf treatment enhanced the level of antinociceptive IL‐10 in the DRG but not in the spinal cord of CCI rats (Figure c,d), suggesting that IL‐10 pathway is involved in antinociception of ginsenoside to a greater extent at the DRG level compared with the spinal cord.…”