Nuclear Factor κB-COX2 Pathway Activation in Non-myelinating Schwann Cells Is Necessary for the Maintenance of Neuropathic Pain in vivo

Chronic neuropathic pain leads to long-term changes in the sensitivity of both peripheral and central nociceptive neurons. Glial fibrillary acidic protein (GFAP)-positive glial cells are closely associated with the nociceptive neurons including astrocytes in the central nervous system (CNS), satellite glial cells (SGCs) in the sensory ganglia, and non-myelinating Schwann cells (NMSCs) in the peripheral nerves. Central and peripheral GFAP-positive cells are involved in the maintenance of chronic pain through a host of inflammatory cytokines, many of which are under control of the transcription factor nuclear factor κB (NFκB) and the enzyme cyclooxygenase 2 (COX2). To test the hypothesis that inhibiting GFAP-positive glial signaling alleviates chronic pain, we used (1) a conditional knockout (cKO) mouse expressing Cre recombinase under the hGFAP promoter and a floxed COX2 gene to inactivate the COX2 gene specifically in GFAP-positive cells; and (2) a tet-Off tetracycline transactivator system to suppress NFκB activation in GFAP-positive cells. We found that neuropathic pain behavior following spared nerve injury (SNI) significantly decreased in COX2 cKO mice as well as in mice with decreased glial NFκB signaling. Additionally, experiments were performed to determine whether central or peripheral glial NFκB signaling contributes to the maintenance of chronic pain behavior following nerve injury. Oxytetracycline (Oxy), a blood-brain barrier impermeable analog of doxycycline was employed to restrict transgene expression to CNS glia only, leaving peripheral glial signaling intact. Signaling inactivation in central GFAP-positive glia alone failed to exhibit the same analgesic effects as previously observed in animals with both central and peripheral glial signaling inhibition. These data suggest that the NFκB-COX2 signaling pathway in NMSCs is necessary for the maintenance of neuropathic pain in vivo

Modulation of the autonomic nervous system and behaviour by acute glial cell G q protein-coupled receptor activation in vivo: Glial GPCR signalling in physiology and behaviourin vivo

Glial fibrillary acidic protein (GFAP)-expressing cells (GFAP+ glial cells) are the predominant cell type in the central and peripheral nervous systems. Our understanding of the role of GFAP+ glial cells and their signalling systems in vivo is limited due to our inability to manipulate these cells and their receptors in a cell type-specific and non-invasive manner. To circumvent this limitation, we developed a transgenic mouse line (GFAP-hM3Dq mice) that expresses an engineered Gq protein-coupled receptor (Gq-GPCR) known as hM3Dq DREADD (designer receptor exclusively activated by designer drug) selectively in GFAP+ glial cells. The hM3Dq receptor is activated solely by a pharmacologically inert, but bioavailable, ligand (clozapine-N-oxide; CNO), while being non-responsive to endogenous GPCR ligands. In GFAP-hM3Dq mice, CNO administration increased heart rate, blood pressure and saliva formation, as well as decreased body temperature, parameters that are controlled by the autonomic nervous system (ANS). Additionally, changes in activity-related behaviour and motor coordination were observed following CNO administration. Genetically blocking inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ increases in astrocytes failed to interfere with CNO-mediated changes in ANS function, locomotor activity or motor coordination. Our findings reveal an unexpectedly broad role of GFAP+ glial cells in modulating complex physiology and behaviour in vivo and suggest that these effects are not dependent on IP3-dependent increases in astrocytic Ca2+

The Expression of Transcription Factors Mecp2 and CREB Is Modulated in Inflammatory Pelvic Pain

Early activation of transcription factors is one of the epigenetic mechanisms contributing to the induction and maintenance of chronic pain states. Previous studies identified the changes in a number of nociception-related genes, such as calcitonin gene-related peptide (CGRP), substance P (SP), and brain-derived neurotropic factor (BDNF) in the pelvic organs after transient colonic inflammation. The gene and protein expression of these neuropeptides could be modulated by transcription factors Methyl-CpG-binding protein 2 (Mecp2) and cAMP response element-binding protein (CREB). In this study, we aimed to evaluate time-dependent changes in the expression levels of Mecp2 and CREB in the lumbosacral (LS) spinal cord and sensory ganglia after inflammation-induced pelvic pain in rat. Adult Sprague-Dawley rats were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce transient colonic inflammation. LS (L6-S2) spinal cord segments and respective dorsal root ganglias (DRGs) were isolated from control and experimental animals at 1, 2, 6, 24 h and 3 days post-TNBS treatment. Immunohistochemical (IHC) labeling and Western blotting experiments were performed to assess the expression of Mecp2, CREB and their phosphorylated forms. Total Mecp2 expression, but not phosphorylated p-Mecp2 (pS421Mecp2) expression was detected in the cells of the spinal dorsal horn under control conditions. Colonic inflammation triggered a significant decrease in the number of Mecp2-expressing neurons in parallel with elevated numbers of pS421Mecp2-expressing cells at 2 h and 6 h post-TNBS. The majority of Mecp2-positive cells (80 ± 6%) co-expressed CREB. TNBS treatment caused a transient up-regulation of CREB-expressing cells at 1 h post-TNBS only. The number of cells expressing phosphorylated CREB (pS133CREB) did not change at 1 h and 2 h post-TNBS, but was down-regulated by three folds at 6 h post-TNBS. Analysis of DRG sections revealed that the number of Mecp2-positive neurons was up-regulated by TNBS treatment, reaching three-fold increase at 2 h post-TNBS, and eight-fold increase at 6 h post-TNBS (p ≤ 0.05 to control). These data showed early changes in Mecp2 and CREB expression in the dorsal horn of the spinal cord and sensory ganglia after colonic inflammation, suggesting a possible contribution Mecp2 and CREB signaling in the development of visceral hyperalgesia and pelvic pain following peripheral inflammation

Targeting sympathetic glia for treating cardiovascular diseases

Gq G protein-coupled receptor (Gq-GPCR) signaling in glial fibrillary acidic protein-expressing (GFAP+) glia is essential for neuron-glia interaction in the Central Nervous System (CNS). However, the exploration of the roles of Gq-GPCR signaling in peripheral GFAP+ glia has just begun. Our recent study showed that GFAP+ glia in the sympathetic ganglia, namely satellite glial cells (SGCs), positively modulate sympathetic-regulated cardiac functions following their Gq-GPCR activation. In this research highlight, we discuss the significance of satellite glial modulation of sympathetic nerve activity (SNA) in both physiology and in diseases. We also present a new experimental strategy for manipulating satellite glial signaling in the sympathetic ganglia using adeno-associated virus (AAV). The success of targeted viral transduction in ganglionic SGCs suggest a strong therapeutic potential of targeting sympathetic glia for the treatment of cardiovascular diseases (CVDs)

Differential transcriptomic changes in the central nervous system and urinary bladders of mice infected with a coronavirus

Multiple sclerosis (MS) often leads to the development of neurogenic lower urinary tract symptoms (LUTS). We previously characterized neurogenic bladder dysfunction in a mouse model of MS induced by a coronavirus, mouse hepatitis virus (MHV). The aim of the study was to identify genes and pathways linking neuroinflammation in the central nervous system with urinary bladder (UB) dysfunction to enhance our understanding of the mechanisms underlying LUTS in demyelinating diseases. Adult C57BL/6 male mice (N = 12) received either an intracranial injection of MHV (coronavirus-induced encephalomyelitis, CIE group), or sterile saline (control group). Spinal cord (SC) and urinary bladders (UB) were collected from CIE mice at 1 wk and 4 wks, followed by RNA isolation and NanoString nCounter Neuroinflammation assay. Transcriptome analysis of SC identified a significantly changed expression of >150 genes in CIE mice known to regulate astrocyte, microglia and oligodendrocyte functions, neuroinflammation and immune responses. Two genes were significantly upregulated (Ttr and Ms4a4a), and two were downregulated (Asb2 and Myct1) only in the UB of CIE mice. Siglec1 and Zbp1 were the only genes significantly upregulated in both tissues, suggesting a common transcriptomic link between neuroinflammation in the CNS and neurogenic changes in the UB of CIE mice

Up-regulation of <i>Zbp1</i> and <i>Siglec1</i> genes in both, SC and UB, samples from CIE mice.

The two genes, Zbp1 (A and B) and Siglec1 (C and D), were upregulated in both, SC (B and D) and UB (A and C), specimens in CIE mice. *p ≤0.05, **p≤0.01, ***p ≤0.001.</p

Cell type- and pathway-specific changes in the expression of the genes in the UB of control and CIE mice.

A, A multi-dimensional scaling plot of the UB samples shows a different profile of gene expression in the CIE group at 1 wk post-infection, but less significant changes in gene expression between the control and CIE 4 wks group post-infection with a coronavirus. The coordinates on the axis are arbitrary. This plot is presented in a way of displaying similarities and differences between data points. Samples that are clustered together generally have similar patterns of expression. Global significance score (GSS) -based gene changes in the UB signaling pathways in CIE mice at 1 wk (B) and 4 wks (C) post-infection. D, Heat map of the UB genes clustered based on their expression levels in different cell types.</p

Expression of the genes changed only in the UB of CIE mice.

Two genes, Ttr (A) and Ms4a4a (B) were upregulated only in UB samples of CIE 1 wk mice. Additional two genes, Asb2 (C) and Myct1 (D) were downregulated only in UB samples from CIE 1 wk group. The same significant changes were observed for all respective genes in UB samples from CIE 4 wks groups. N = 4 for each group. *p ≤0.05, **p≤0.01.</p

Cell type- and signaling pathway-specific changes in gene expression of SC specimens from control and CIE mice.

A, A multi-dimensional scaling plot of the SC samples shows group-specific clustering of the genes in the control (Ctrl) group, and in both experimental groups (CIE 1 wk, and CIE 4 wks). The coordinates on the axis are arbitrary. This plot is presented in a way of displaying similarities and differences between data points. Samples that are clustered together generally have similar patterns of expression. Gene changes in top 10 signaling pathways in the SC cord of CIE mice at 1 wk (B) and 4 wks (C) post-infection. D, Heat map of the genes clustered based on their expression levels in different cell types (SC specimens).</p

Expression of the genes in spinal cord specimens in CIE 1 wk group based on signaling pathways.

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