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

Abstract

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.

Keywords: COX2; GFAP-positive glia; neuropathic pain; non-myelinating Schwann cells; nuclear factor κB signaling; peripheral nervous system; tetracycline transactivator system.

FIGURE 1

SNI induced long-term mechanical allodynia that was temporarily alleviated in both male and female COX2 cKO mice. Withdrawal threshold (A,B) and paw withdrawal frequency (C,D) following SNI in male (A,C) and female (B,D) COX2 cKO mice (male n = 10; female n = 10), littermate controls (male n = 10; female n = 10), and littermate animals given tamoxifen (male n = 10; female n = 10) were shown. In COX2 cKO mice and a subset of littermate controls, 16 tamoxifen injections (3 mg/40 g) were given over a 8-day period (twice daily I.P., starting at p21). SNI were performed at P60. There was no difference in basal mechanical sensitivity between any of the groups. Bonferroni post hoc analysis: *P < 0.05, **P < 0.01, ***P < 0.001, for COX2 cKO vs. control ipsilateral hind paws. Black arrows indicate the time of spared nerve injury.

FIGURE 2

(A) A diagram showing expression of eGFP in the spinal cord and sciatic nerve (longitudinal section) from GFAP-tTA eGFP reporter mice. The mice were taken off doxycycline at weaning to allow the expression of the transgene in GFAP-positive cells. (B) The native eGFP reporter (green) colocalized with immunohistochemical staining for astrocytes (GFAP, red) in the dorsal horn and with NMSCs (GFAP, red) in the sciatic nerve (transverse sections), but not with satellite cells (GFAP, red) in the DRG (N = 5). eGFP did not colocalize with any markers for microglia or macrophages (Iba-1), neurons (NeuN) or large or small axonal markers (NF200 or peripherin). All cellular and axonal markers are red and are listed on the individual images. Native eGFP is shown in green. DAPI is shown in blue. White arrows indicate colocalized cells. The scale bar is 20 μm.

FIGURE 3

SNI induced long-term mechanical allodynia which was temporarily alleviated in both male and female IKKdn mice. The mice were off doxycycline starting at weaning to allow the expression of the IKKdn in GFAP-positive cells. Withdrawal threshold (A,B) and paw withdrawal frequency (C,D) following SNI in male (A,C) and female (B,D) IKKdn mice (male n = 6; female n = 5) and IKKdn-negative littermate controls (male n = 9; female n = 9). There was no difference in basal mechanical sensitivity between the groups. Bonferroni post hoc analysis: **P < 0.01, ***P < 0.001, IKKdn vs. control ipsilateral hind paws. Black arrows indicate the time of spared nerve injury.

FIGURE 4

(A) A diagram showing expression of eGFP in the spinal cord, but not in the DRG or sciatic nerve (longitudinal section) of GFAP-tTA eGFP reporter mice following treatment with Oxy (starting at weaning). (B) Following treatment with Oxy, the native eGFP reporter (green) colocalized with immunohistochemical staining for astrocytes (GFAP, red) in the dorsal horn, but with not NMSCs (GFAP, red) in the sciatic nerve (transverse sections) or with satellite cells (GFAP, red) in the DRG (N = 5). eGFP did not colocalize with any markers for microglia or macrophages (Iba-1), neurons (NeuN) or large or small axonal markers (NF200 or peripherin). All cellular and axonal markers are shown in red and are listed on the individual images. Native eGFP is shown in green. DAPI is shown in blue. White arrows indicate colocalized cells. The scale bar is 20 μm.

FIGURE 5

Oxy did not cross the blood-brain barrier to inhibit central reporter gene expression at any time-point following surgery, while it continually suppressed peripheral transgene expression. eGFP expression (green) were detected in spinal cord sections (left panels) but not in peripheral sciatic nerve (right panels) and showed cellular colocalization with GFAP immunohistochemical staining (red) from mice on Oxy (white arrows) (starting at weaning, N = 5). The scale bar on left is 100 μm. The scale bar on right is 20 μm.

FIGURE 6

(A) Immunostaining for S100β (green), pNFκB (red), and DAPI (blue) in the sciatic nerve 4 days post-SNI. (B) Cells positive for GFAP, S100β and nuclear pNFκB were quantified (In all groups, nine images were analyzed from three different animals). The number of positive cells displayed is the mean per image ± SEM. *P < 0.05, ***P < 0.001 for the comparison of the SNI group to its naïve counterpart. #P < 0.001 comparing IKKdn 4 days post-injury off drug vs. on Oxy. White arrows indicate colocalized cells.

FIGURE 7

(A) Diagrams showing expression of eGFP from GFAP-tTA::eGFP animals with or without Oxy treatment. Mice were taken off doxycycline at weaning followed by drinking water (to allow the expression of the transgene in GFAP-positive cells) or drinking water with Oxy (to only express IKKdn in peripheral GFAP-positive glia). The red arrows indicate the regions of transgene expression and thus the regions of NFκB suppression. (B) Peripheral suppression of IKKdn by Oxy eliminated the IKKdn phenotype following SNI. Withdrawal threshold (top panels) and paw withdrawal frequency (bottom panels) following SNI in IKKdn and littermate control animals with and without administration of the transgene inhibitor Oxy (IKKdn Off n = 10; IKKdn Oxy n = 10; control Off n = 8; control Oxy n = 9). Bonferroni post hoc analysis: ***P < 0.001, for IKKdn vs. control ipsilateral hind paws. Black arrows indicate the time of spared nerve injury.