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, 202 (1), 145-56

Inhibition of Astroglial Nuclear Factor kappaB Reduces Inflammation and Improves Functional Recovery After Spinal Cord Injury

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Inhibition of Astroglial Nuclear Factor kappaB Reduces Inflammation and Improves Functional Recovery After Spinal Cord Injury

Roberta Brambilla et al. J Exp Med.

Abstract

In the central nervous system (CNS), the transcription factor nuclear factor (NF)-kappaB is a key regulator of inflammation and secondary injury processes. After trauma or disease, the expression of NF-kappaB-dependent genes is highly activated, leading to both protective and detrimental effects on CNS recovery. We demonstrate that selective inactivation of astroglial NF-kappaB in transgenic mice expressing a dominant negative (dn) form of the inhibitor of kappaB alpha under the control of an astrocyte-specific promoter (glial fibrillary acidic protein [GFAP]-dn mice) leads to a dramatic improvement in functional recovery 8 wk after contusive spinal cord injury (SCI). Histologically, GFAP mice exhibit reduced lesion volume and substantially increased white matter preservation. In parallel, they show reduced expression of proinflammatory chemokines and cytokines, such as CXCL10, CCL2, and transforming growth factor-beta2, and of chondroitin sulfate proteoglycans participating in the formation of the glial scar. We conclude that selective inhibition of NF-kappaB signaling in astrocytes results in protective effects after SCI and propose the NF-kappaB pathway as a possible new target for the development of therapeutic strategies for the treatment of SCI.

Figures

Figure 1.
Figure 1.
Biochemical characterization of GFAP-IκBα-dn mice. (A) RT-PCR analysis of GFAP-IκBα-dn (IκBα-dn) expression in the brain (Br), spinal cord (SC), sciatic nerve (SN), heart (Ht), kidney (Kd), lung (Ln), liver (Lv), and spleen (Sp). β-Actin was amplified as a control. Samples lacking RT (−RT) were amplified as controls for genomic DNA contamination. Genomic DNA from a TG animal and water were used as positive (+) and negative (−) controls for the PCR reaction, respectively. (B) Inhibition of NF-κB DNA binding activity exclusively in astrocytes from GFAP-IκBα-dn (IκBα-dn) mice. Cultures were treated with TNF-α (+, 10 ng/ml for 30 min) or medium alone (−). (C) Induction of NF-κB DNA binding activity exclusively in neurons from GFAP-IκBα-dn (IκBα-dn) mice. Cultures were treated with TNF-α (+, 10 ng/ml for 30 min) or medium alone (−). (D) NF-κB supershift in TNF-α–stimulated WT astrocytes. Nuclear extracts from WT astrocytes, treated with TNF-α (+, 10 ng/ml for 30 min) or medium alone (−) were preincubated with anti-p65 or -p50 antibodies. The supershifted p65–p50 dimer is detected in the top left (ss). Binding specificity to the NF-κB consensus sequence was demonstrated by displacement of the 32P-labeled NF-κB oligonucleotide in the presence of 100-fold excess cold NF-κB and by a lack of displacement in the presence of 100-fold excess cold AP-1.
Figure 2.
Figure 2.
Time course of NF-κB activation after SCI in WT and GFAP-IκBα-dn mice. NF-κB activation was evaluated by Western blot analysis with an antibody that specifically recognizes the activated form of p65 (p65*). The arrowhead indicates p65* migrating at exactly 65 kD. 20 μg of proteins/sample were loaded, and blots were probed for β-actin as a control. A representative blot is shown (n = 3). nv, naive.
Figure 3.
Figure 3.
Behavioral and histological analysis of WT and GFAP-IκBα-dn mice 8 wk after SCI. (A) Evaluation of hindlimb locomotor function. WT (n = 12) and GFAP-IκBα-dn (IκBα-dn; n = 12) mice were tested 1 d after SCI and weekly thereafter for 8 wk. Motor behavior was scored under blind conditions with the BMS. ^, P < 0.04; *, P < 0.001 versus WT, as determined by one-way ANOVA and the Tukey test. (B) GFAP labeling (green), fluorescent Nissl (red), and DAPI (blue) staining of spinal cord sections 8 wk after SCI. (C) Lesion volume assessment of the two genotypes (n = 5). (right) A three-dimensional reconstruction of the lesion. (D) White matter sparing. Volume of myelinated white matter was measured in WT (n = 5) and GFAP-IκBα-dn (n = 5) mice on Luxol-stained sections. Contoured areas with darker Luxol staining delineate intact white matter. *, P < 0.02, as determined by the Student's t test. Morphometric analyses shown in C and D were conducted under blind conditions. Bars, 450 μm.
Figure 4.
Figure 4.
Immunostaining for MBP and TuJ1 in WT and GFAP-IκBα-dn spinal cords 8 wk after SCI. WT (A and B) and GFAP-IκBα-dn (C and D) sections of the spinal cord were double labeled for MBP (red; B, D, and F) and TuJ1 (green; A, C, and E). White arrows in E and F show colocalization of MBP with TuJ1 in myelinated axons. Bars: (A–D) 180 μm; (E and F) 50 μm.
Figure 5.
Figure 5.
Neurocan and phosphacan immunohistochemistry 8 wk after SCI. WT (A and C) and GFAP-IκBα-dn (B and D) sections of the spinal cord were double labeled for GFAP (red) and either neurocan (green; A and B) or phosphacan (green; C and D). Bars, 450 μm.
Figure 6.
Figure 6.
Expression of TGF-β and decorin in WT and GFAP-IκBα-dn mice after SCI. (A) Gene expression of TGF-β1, -β2, and -β3 6 h and 1 d after SCI was measured by RPA. Semiquantitative measurements were obtained by normalizing to GAPDH and L32. Results are expressed as the percentage of WT 6 h after SCI and represent the mean ± SEM (n = 4). *, P < 0.05, as determined by one-way ANOVA and the Tukey test. (B) Decorin expression 3 d and 1 wk after SCI was measured by real-time RT-PCR and normalized to β-actin. Results are expressed as the percentage of WT 3 d after SCI and represent the mean ± SEM (n = 3). Samples were also analyzed by agarose gel electrophoresis. −, negative control for the PCR.
Figure 7.
Figure 7.
Chemokine expression in WT and GFAP-IκBα-dn mice after SCI. (A) Gene expression of RANTES, CXCL10, and CCL2 6 h and 1 d after SCI was measured by RPA. Semiquantitative measurements were obtained by normalizing to GADPDH and L32. Results are expressed as the percentage of WT 6 h after SCI and represent the mean ± SEM (n = 4). *, P < 0.05 as determined by one-way ANOVA and the Tukey test. (B–D) Colocalization of CXCL10 immunostaining (green) with GFAP (red) in WT (B) and GFAP-IkBa-dn mice (C) 3 d after SCI. Bars, 20 μm.
Figure 8.
Figure 8.
Expression of TNF-α and IL-6 in WT and GFAP-IκBα-dn mice after SCI. Gene expression of TNF-α and IL-6 6 h and 1 d after SCI was measured by RPA. Semiquantitative measurements were obtained by normalizing to GADPDH and L32. Results are expressed as the percentage of WT 6 h after SCI and represent the mean ± SEM (n = 4). *, P < 0.05, as determined by one-way ANOVA and the Tukey test.

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