Transgenic inhibition of glial NF-kappa B reduces pain behavior and inflammation after peripheral nerve injury

Abstract

The transcription factor nuclear factor kappa B (NF-kappaB) is a key regulator of inflammatory processes in reactive glial cells. We utilized a transgenic mouse model (GFAP-IkappaBalpha-dn) where the classical NF-kappaB pathway is inactivated by overexpression of a dominant negative (dn) form of the inhibitor of kappa B (IkappaBalpha) in glial fibrillary acidic protein (GFAP)-expressing cells, which include astrocytes, Schwann cells, and satellite cells of the dorsal root ganglion (DRG) and sought to determine whether glial NF-kappaB inhibition leads to a reduction in pain behavior and inflammation following chronic constriction injury (CCI) of the sciatic nerve. As expected, following CCI nuclear translocation, and hence activation, of NF-kappaB was detected only in the sciatic nerve of wild type (WT) mice, and not in GFAP-IkappaBalpha-dn mice, while upregulation of GFAP was observed in the sciatic nerve and DRGs of both WT and GFAP-IkappaBalpha-dn mice, indicative of glial activation. Following CCI, mechanical and thermal hyperalgesia were reduced in GFAP-IkappaBalpha-dn mice compared to those in WT, as well as gene and protein expression of CCL2, CCR2 and CXCL10 in the sciatic nerve. Additionally, gene expression of TNF, CCL2, and CCR2 was reduced in the DRGs of transgenic mice compared to those of WT after CCI. We can therefore conclude that transgenic inhibition of NF-kappaB in GFAP-expressing glial cells attenuated pain and inflammation after peripheral nerve injury. These findings suggest that targeting the inflammatory response in Schwann cells and satellite cells may be important in treating neuropathic pain.

Figure 1

(A) RT-PCR analysis of GFAP-IκBα-dn transgene expression in WT and GFAP-IκBα-dn mice. Each gene was normalized to β-actin expression as a loading control for the PCR reaction. (B) Nuclear translocation of activated p65 in sciatic nerve of WT and GFAP-IκBα-dn mice 1 day after CCI. Green: GFAP; Red: phospho-p65; Blue: DAPI (scale bar = 10 μm). TG: transgenic mice; DRG: dorsal root ganglion; S.N.: sciatic nerve; S.C.: spinal cord.

Figure 2

GFAP immunoreactivity in spinal cord, sciatic nerve and DRG of WT and GFAP-IκBα-dn mice under naïve conditions and 3 days after CCI (scale bar = 100 μm).

Figure 3

Nociceptive responses in the hindpaws in WT (n=6) and GFAP-IκBα-dn mice (n=10) after CCI. WT mice had greater mechanical hyperalgesia, as measured by the decrease in withdrawal thresholds in the ipsilateral (A) and contralateral (B) hindpaws. Curves are significantly different (p<0.05, two-way ANOVA). WT mice had greater thermal hyperalgesia, as measured by decreases in withdrawal latencies in the (C) ipsilateral hindpaw and (D) contralateral hindpaws. Curves are significantly different (p<0.05, two-way ANOVA). Bonferroni post hoc analysis: *p<0.001, **p<0.01, ***p<0.05, for WT ipsilateral hindpaw vs. GFAP-IκBα-dn ipsilateral hindpaw; ^#p<0.05 for WT contralateral hindpaw vs GFAP-IκBα-dn contralateral hindpaw.

Figure 4

Gene expression of IL-1β and TNF in the ipsilateral sciatic nerve of WT mice and GFAP-IκBα-dn mice following CCI. For each gene, results are expressed as folds of WT naïve mice ± S.E.M., after normalization to β-actin. Four animals/group/time point were analyzed. *p<0.001, ^#p<0.05 compared to corresponding naïve mice, one-way ANOVA, Tukey test.

Figure 5

Gene expression of CCL2, CCR2, and CXCL10 in the ipsilateral sciatic nerve of WT and GFAP-IκBα-dn mice following CCI. For each gene, results are expressed as folds of WT naïve ± S.E.M., after normalization to β-actin. Four animals/group/time points were analyzed. *p<0.01, **p<0.05 compared to WT 1 day, one-way ANOVA, Tukey test.

Figure 6

Immunostaining for CCL2 (green) and GFAP (red) in the ipsilateral sciatic nerve of WT and GFAP-IκBα-dn mice. CCI induced increased immunoreactivity for both GFAP and CCL2. On day 1, greater CCL2 immunoreactivity was present in WT mice compared to GFAP-IκBα-dn (scale bar = 50 μm).

Figure 7

Immunostaining for CCR2 (green) and GFAP (red) in the ipsilateral sciatic nerve of WT and GFAP-IκBα-dn mice. CCI induced increased immunoreactivity for both GFAP and CCL2. On day 1 and day 3, greater CCR2 immunoreactivity was present in WT compared to GFAP-IκBα-dn mice (scale bar = 50 μm).

Figure 8

Immunostaining for CXCL10 (green) and GFAP (red) in the ipsilateral sciatic nerve of WT and GFAP-IκBα-dn mice. CCI induced increased immunoreactivity for both GFAP and CXCL10. On day 3, greater CXCL10 immunoreactivity was present in WT mice compared to GFAP-IκBα-dn (scale bar = 50 μm).

Figure 9

Gene expression of CCL2, CCR2, and TNF in the ipsilateral DRGs of WT mice and GFAP-IκBα-dn mice. For each gene, results are expressed as fold of corresponding WT naïve ± S.E.M., after normalization to β-actin. Four animals/group/time point were analyzed. *p<0.05 vs WT 1d; one-way ANOVA, Tukey test.