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Comparative Study
. 2009 Jul;110(2):765-78.
doi: 10.1111/j.1471-4159.2009.06190.x. Epub 2009 May 26.

Transgenic inhibition of astroglial NF-kappa B leads to increased axonal sparing and sprouting following spinal cord injury

Roberta Brambilla  1 , Andres Hurtado, Trikaldarshi Persaud, Kim Esham, Damien D Pearse, Martin Oudega, John R Bethea
Affiliations
Comparative Study

Transgenic inhibition of astroglial NF-kappa B leads to increased axonal sparing and sprouting following spinal cord injury

Roberta Brambilla et al. J Neurochem. 2009 Jul.

Abstract

We previously showed that Nuclear Factor kappaB (NF-kappaB) inactivation in astrocytes leads to improved functional recovery following spinal cord injury (SCI). This correlated with reduced expression of pro-inflammatory mediators and chondroitin sulfate proteoglycans, and increased white matter preservation. Hence we hypothesized that inactivation of astrocytic NF-kappaB would create a more permissive environment for axonal sprouting and regeneration. We induced both contusive and complete transection SCI in GFAP-Inhibitor of kappaB-dominant negative (GFAP-IkappaBalpha-dn) and wild-type (WT) mice and performed retrograde [fluorogold (FG)] and anterograde [biotinylated dextran amine (BDA)] tracing 8 weeks after injury. Following contusive SCI, more FG-labeled cells were found in motor cortex, reticular formation, and raphe nuclei of transgenic mice. Spared and sprouting BDA-positive corticospinal axons were found caudal to the lesion in GFAP-IkappaBalpha-dn mice. Higher numbers of FG-labeled neurons were detected immediately rostral to the lesion in GFAP-IkappaBalpha-dn mice, accompanied by increased expression of synaptic and axonal growth-associated molecules. After transection, however, no FG-labeled neurons or BDA-filled axons were found rostral and caudal to the lesion, respectively, in either genotype. These data demonstrated that inhibiting astroglial NF-kappaB resulted in a growth-supporting terrain promoting sparing and sprouting, rather than regeneration, of supraspinal and propriospinal circuitries essential for locomotion, hence contributing to the improved functional recovery observed after SCI in GFAP-IkappaBalpha-dn mice.

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Conflict of interest statement

The authors have no conflicting financial interests.

Figures

Figure 1
Figure 1. Immunostaining for GFAP and CD11b in spinal cords of WT and GFAP-IκBα-dn mice 10 weeks after contusive SCI
Longitudinal spinal cord sections of WT (A, B, C) and GFAP-IκBα-dn (D, E, F) mice, cut along the horizontal plane, were double labeled for GFAP (green) and CD11b (red). Micrographs were taken at the lesion site (A, D), immediately below (B, E), and caudally to the lesion site (C, F). Scale bar: 50 μm.
Figure 2
Figure 2. Determination of FG-labeled neurons following retrograde tracing with FG in WT and GFAP-IκBα-dn mice 10 weeks after contusive SCI
(A) Schematic representation of the FG injection sites located bilaterally to the midline 3 mm below the injury site. Micrographs represent FG-positive cells in the spinal cord and reticular formation of the brain stem. Panels B and C summarize the total number of FG-positive cell bodies counted in spinal cord (B) and whole brain (C). A brakedown of FG-positive counts in the various nuclei and regions of the brain is provided in panels D, E and F. *P<0.05, Student’s T test. N= 9 per group. LC: locus ceruleus; PAG: periaqueductal gray matter; VCN: vestibulocerebellar nucleus; SPVN: spinal vestibular nucleus; NTS: nucleus of the solitary tract; Misc: miscellaneous.
Figure 3
Figure 3. Fluorescent labeling of CST axons following anterograde tracing with BDA of WT and GFAP-IκBα-dn spinal cords 10 weeks after contusive SCI
CST axons which anterogradely transported BDA were identified by fluorescent labeling with streptavidin conjugated with Alexa-488 in longitudinal sections of the spinal cord cut along the horizontal plane. WT mice (A) did not show any labeled axon below the injury site, contrary to GFAP-IκBα-dn mice (B), that showed a significant presence of labeled CST fibers through and below the lesion site (C, D, E). The contour in A and B delineates the lesion area. Scale bars: A, B = 500 μm. C, D, E = 80 μm
Figure 4
Figure 4. Co-labeling of BDA-positive CST axons with GAP-43 in GFAP-IκBα-dn spinal cords 10 weeks after contusive SCI
The high power micrograph shows the presence of CST fibers (green; BDA/Streptavidin-Alexa 488), which co-express the growth-associated molecule GAP-43 (red) far below the lesion site in GFAP-IκBα-dn mice. No double-labeled fibers were found in WT mice. Scale bar: 20 μm.
Figure 5
Figure 5. Immuno-fluorescent labeling of serotonergic raphespinal projections in WT and GFAP-IκBα-dn spinal cords 10 weeks after contusive SCI
Raphespinal axons were identified by immunolabeling with an anti-5-HT antibody in longitudinal sections of the spinal cord cut along the horizontal plane. WT mice (A–D) showed minimal 5-HT labeling below the injury site, contrary to GFAP-IκBα-dn mice (E–H), who showed a significant presence of 5-HT-labeled raphespinal projections fibers throughout and below the lesion site (H, I). Convoluted and arborized fibers in GFAP-IκBα-dn mice are an anatomical indication of collateral regenerative sprouting (I). The contour in A and E delineates the lesion area. Scale bars: A, E = 500 μm; B, C, D = 50 μm; F, G, H, I: 80 μm.
Figure 6
Figure 6. Differential expression of neuronal-specific molecules in WT and GFAP-IκBα-dn mice 10 weeks mice following contusive SCI
SNAP-25, Synaptotagmin and GAP-43 gene expression was assessed in the brainstem, motor cortex and spinal cord of WT and transgenic animals 8 weeks after SCI. For each gene, results are expressed as % of corresponding WT ± SEM after normalization to β-actin. Six animals per group were analyzed. *P<0.05 with respect to corresponding WT, one-way ANOVA, Tukey test.
Figure 7
Figure 7. Fluorescent labeling of CST axons following anterograde tracing with BDA of WT and GFAP-IκBα-dn spinal cords 10 weeks after spinal cord transcection
CST axons which anterogradely transported BDA were identified by fluorescent labeling with streptavidin conjugated with Alexa-488 (green) in longitudinal sections of the spinal cord cut along the horizontal plane. Both WT (A) and GFAP-IκBα-dn mice (D) did not show any labeled axon below the injury site. BDA labeling was observed only above the lesion site (B, C, E, F). Panels G (WT) and H (GFAP-IκBα-dn) show the complete transection of the spinal cord surrounded by intense GFAP immunofluorescence (red). The contour in A and D delineates the lesion area. Scale bars: 500 μm.
See this image and copyright information in PMC

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