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DREADDed microglia in pain: Implications for spinal inflammatory signaling in male rats.

ABSTRACT: The absence of selective pharmacological tools is a major barrier to the in vivo study of microglia. To address this issue, we developed a Gq- and Gi-coupled Designer Receptor Exclusively Activated by a Designer Drug (DREADD) to enable selective stimulation or inhibition of microglia, respectively. DREADDs under a CD68 (microglia/macrophage) promoter were intrathecally transfected via an AAV9 vector. Naïve male rats intrathecally transfected with Gq (stimulatory) DREADDs exhibited significant allodynia following intrathecal administration of the DREADD-selective ligand clozapine-N-oxide (CNO), which was abolished by intrathecal interleukin-1 receptor antagonist. Chronic constriction injury-induced allodynia was attenuated by intrathecal CNO in male rats intrathecally transfected with Gi (inhibitory) DREADDs. To explore mechanisms, BV2 cells were stably transfected with Gq or Gi DREADDs in vitro. CNO treatment induced pro-inflammatory mediator production per se from cells expressing Gq-DREADDs, and inhibited lipopolysaccharide- and CCL2-induced inflammatory signaling from cells expressing Gi-DREADDs. These studies are the first to manipulate microglia function using DREADDs, which allow the role of glia in pain to be conclusively demonstrated, unconfounded by neuronal off-target effects that exist for all other drugs that also inhibit glia. Hence, these studies are the first to conclusively demonstrate that in vivo stimulation of resident spinal microglia in intact spinal cord is a) sufficient for allodynia, and b) necessary for allodynia induced by peripheral nerve injury. DREADDs are a unique tool to selectively explore the physiological and pathological role of microglia in vivo.

SUBMITTER: Grace PM

PROVIDER: S-EPMC5916033 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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DREADDed microglia in pain: Implications for spinal inflammatory signaling in male rats.

Grace Peter M PM Wang Xiaohui X Strand Keith A KA Baratta Michael V MV Zhang Yingning Y Galer Erika L EL Yin Hang H Maier Steven F SF Watkins Linda R LR

Experimental neurology 20180309

The absence of selective pharmacological tools is a major barrier to the in vivo study of microglia. To address this issue, we developed a Gq- and Gi-coupled Designer Receptor Exclusively Activated by a Designer Drug (DREADD) to enable selective stimulation or inhibition of microglia, respectively. DREADDs under a CD68 (microglia/macrophage) promoter were intrathecally transfected via an AAV9 vector. Naïve male rats intrathecally transfected with Gq (stimulatory) ...[more]

PMID: 29530713

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Project description:BackgroundBurn injury (BI) pain consists of inflammatory and neuropathic components and activates microglia. Nicotinic alpha 7 acetylcholine receptors (α7AChRs) expressed in microglia exhibit immunomodulatory activity during agonist stimulation. Efficacy of selective α7AChR agonist GTS-21 to mitigate BI pain and spinal pain-mediators was tested.MethodsAnesthetized rats after hind-paw BI received intraperitoneal GTS-21 or saline daily. Allodynia and hyperalgesia were tested on BI and contralateral paw for 21 days. Another group after BI receiving GTS-21 or saline had lumbar spinal cord segments harvested (day 7 or 14) to quantify spinal inflammatory-pain transducers or microglia activation using fluorescent marker, ionized calcium-binding adaptor protein (Iba1).ResultsBI significantly decreased allodynia withdrawal threshold from baseline of ~9-10 to ~0.5-1 g, and hyperalgesia latency from ~16-17 to ~5-6 seconds by day 1. Both doses of GTS-21 (4 or 8 mg/kg) mitigated burn-induced allodynia from ~0.5-1 to ~2-3 g threshold (P = .089 and P = .010), and hyperalgesia from ~5-6 to 8-9 seconds (P < .001 and P < .001) by day 1. The GTS-21 group recovered to baseline pain threshold by day 15-17 compared to saline-treated, where the exaggerated nociception persisted beyond 15-17 days. BI significantly (P < .01) increased spinal cord microgliosis (identified by fluorescent Iba1 staining), microglia activation (evidenced by the increased inflammatory cytokine), and pain-transducer (protein and/or messenger RNA [mRNA]) expression (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], nuclear factor-kappa B [NF-κB], interleukin-6 [IL-6], Janus-associated kinase signal transducer and activator of transcription 3 [JAK-STAT3], and/or N-methyl-D-aspartate receptor [NMDAR]). GTS-21 mitigated pain-transducer changes. The α7AChR antagonist methyllycaconitine nullified the beneficial effects of GTS-21 on both increased nociception and pain-biomarker expression.ConclusionsNonopioid, α7AChR agonist GTS-21 elicits antinociceptive effects at least in part by decreased activation spinal-cord pain-inducers. The α7AChR agonist GTS-21 holds promise as potential therapeutic adjunct to decrease BI pain by attenuating both microglia changes and expression of exaggerated pain transducers.

Dataset Information

DREADDed microglia in pain: Implications for spinal inflammatory signaling in male rats.

Publications

DREADDed microglia in pain: Implications for spinal inflammatory signaling in male rats.

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