Glial NF-κB inhibition alters neuropeptide expression after sciatic nerve injury in mice

Abstract

We utilized a transgenic mouse model where nuclear factor kappa B (NF-κB) is selectively inhibited in glial fibrillary acidic protein (GFAP) expressing cells. The transgene, GFAP-IκBα-dn, overexpresses a dominant negative form of the inhibitor of NF-κB (IκBα) under the control of the GFAP promoter. In the present work, we sought to understand the impact of glial NF-κB inhibition on the expression of pain mediating sensory neuropeptides galanin and calcitonin gene related peptide (CGRP) in a model of neuropathic pain in mice. Chronic constriction injury (CCI) of the left sciatic nerve was performed on wild type (WT) and GFAP-IκBα-dn transgenic mice. RT-PCR and immunohistological staining were performed in sciatic nerve and/or L4-L5 DRG tissue for galanin, CGRP and macrophage marker CD11b. GFAP-IκBα-dn mice had less mechanical and thermal hyperalgesia compared to WT mice post-CCI. After CCI, we observed galanin upregulation in DRG and sciatic nerve, which was less in GFAP-IκBα-dn mice. CGRP gene expression in the DRG increased transiently on day 1 post-CCI in WT but not in GFAP-IκBα-dn mice, and no evidence of CGRP upregulation in sciatic nerve post-CCI was found. After CCI, upregulation of CD11b in sciatic nerve was less in GFAP-IκBα-dn mice compared to WT mice, indicative of less macrophage infiltration. Our results showed that glial NF-κB inhibition reduces galanin and CGRP expression, which are neuropeptides that correlate with pain behavior and inflammation after peripheral nerve injury.

Fig. 1

(A) Withdrawal thresholds for the affected paw (hindpaw) at baseline (day 0), day 1 and day 3 post-CCI in WT and GFAP-IκBα-dn mice. (B) Withdrawal latencies to radiant heat of the affected paw at baseline (day 0), day 1 and day 3 post-CCI in WT and GFAP-IκBα-dn mice. Values are expressed as mean±SEM of 6–10 animals per group. *p<0.001, **p<0.05, vs. corresponding day 0; ^#p<0.001 vs. corresponding WT, one-way ANOVA Tukey test.

Fig. 2

Galanin and CGRP gene expression in DRGs from WT and GFAP-IκBα-dn mice following CCI. *p<0.05 vs. corresponding naïve; **p<0.05 vs. WT day 3; ^#p<0.05 vs. WT day 1, one-way ANOVA Tukey test.

Fig. 3

Immunofluorescent staining of galanin (green) in combination with GFAP (red) in the DRGs of WT and GFAP-IκBα-dn mice 3 days after CCI; dpi=days post-injury. Scale bar=50 μm.

Fig. 4

Immunofluorescent staining of galanin (green) in combination with GFAP (red) in the sciatic nerve of WT and GFAP-IκBα-dn mice 3 days after CCI; dpi=days post-injury. Scale bar=100 μm.

Fig. 5

Quantification of the numbers of galanin-positive neuron profiles in the DRG of WT and GFAP-IκBα-dn mice following CCI. DRG neuron profiles are defined as small (<600 μm²), medium (between 600 and 1200 μm²), and large (>1200 μm²) according to their cross-sectional area. Results are expressed as percent±SEM of total neuron profiles. *p<0.05, compared to corresponding naïve; ^#p<0.05, compared to WT same size (medium) neuron profiles.

Fig. 6

Immunofluorescent staining of CD11b positive cells in the sciatic nerve of WT and GFAP-IκBα-dn mice 3 days after CCI; dpi=days post-injury. Scale bar=100 μm.