Inactivation of astroglial NF-kappa B promotes survival of retinal neurons following ischemic injury

Abstract

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF-kappaB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia-reperfusion in wild-type (WT) and in GFAP-IkappaBalpha-dn transgenic mice, where the NF-kappaB classical pathway is suppressed specifically in astrocytes. The GFAP-IkappaBalpha-dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro-inflammatory genes, encoding Tnf-alpha, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO-synthase in the retinas of GFAP-IkappaBalpha-dn mice. These data suggest that certain NF-kappaB-regulated pro-inflammatory and redox-active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

Figure 1

Inhibition of NF-κB DNA binding activity in retinas from GFAP-IκBα-dn mice 24 hours after IR injury A. Representative gel shift analysis of NF-κB DNA and nuclear protein combination in retinas of ischemic and sham operated eyes of WT and GFAP-IκBα-dn mice. NF-κB DNA–binding activity in the nuclei of retinal cells of sham operated WT eyes (Lane 1), sham operated GFAP-IκBα-dn eyes (Lane 2), WT eyes 24 hours after IR injury (Lane 3), GFAP-IκBα-dn eyes 24 hours after IR injury (Lane 4), NF-κB DNA–binding activity by competition electrophoretic mobility shift assay (EMSA) with a hundredfold excess of cold NF-κB oligonucleotides (Lane 5). B. Quantification of NF-κB DNA–binding activity in retinas shown in (A). (*p<0.05, compared with the control retina, n=5, ■- wild type, □- GFAP-IκBα-dn mice)

Figure 2

Inhibition of astroglial NF-κB results in neuroprotective effects in the GCL after IR. A. Numbers of NeuN-labeled neurons in regions of central, middle and peripheral retina were compared between sham operated and ischemic eyes of wild type and GFAP-IκBα-dn animals. Values are means +/− SEM of each microscopic field. *p<0.05 (□ - wild type sham operated, ■- wild type 7 days after IR injury, - GFAP-IκBα-dn sham operated, - GFAP-IκBα-dn 7 days after IR injury) B. Percentage of GCL neurons lost at one week following IR of wild type and GFAP-IκBα-dn mice. Central, middle and peripheral regions were compared bilaterally for individual animals. (■ - wild type, □ - GFAP-IκBα-dn mice) C. In contrast to protected GFAP-IκBα-dn post-ischemic retinas that have lost an average of 9.6% of cells, neuronal density decreased significantly (37.5%, p<0.002) in WT ischemic retinas 7 days after reperfusion. D. Representative confocal images of NeuN-labeled GCLs (green) in flat-mounted retinas acquired at centre, middle and periphery from optic nerve in sham-operated controls and ischemic retinas 7 days after reperfusion.

Figure 3

Differential expression of the cytokines, the chemokines, the cell adhesion molecules and neurotrophins in WT and GFAP-IκBα-dn retinal tissues after IR. Gene expression was assessed in sham operated controls and experimental retinas following IR (24 hours after reperfusion). For each gene, results are expressed as folds of corresponding WT sham-operated eye ± SEM after normalization to β-actin. *P<0.05 with respect to corresponding WT. (■ - WT, □ - GFAP-IκBα-dn)

Figure 4

A. Tnf-α protein (green) accumulation in GCL co-localized with astrocyte marker Gfap (red) and was significantly increased in WT relative to GFAP-IκBα-dn post-ischemic retinas (24 hours after reperfusion). Schematic on the right shows localization of the field of view (framed) relative to the rest of the retina. Arrows point to RGCs, arrowheads – to astrocytes. Retinal layers: INL, inner nuclear, ONL, outer nuclear, PhRL, photoreceptors. B. Immunofluorescent labeling for Icam1 in the GCL after IR significantly co-localized with astrocyte vascular endfeet labeled with Gfap (red), but not with microglia (labeled with Cd11b, blue/cyan) in both WT and GFAP-IκBα-dn flat-mounted retinas (24 hours after reperfusion). Icam1 labeling was significantly increased in WT vs. GFAP-IκBα-dn retinas. Microglial Cd11b-positive cells in GFAP-IκBα-dn retinas appear ramified, in contrast to severely pruned and hypertrophic cells in WT post-ischemic retinas. Scale bar: 100 µm.

Figure 5

A. Differential expression of genes encoding Nos2 and NADPH-oxidase in GCL of WT and GFAP-IκBα-dn retinas following IR. Gene expression was assessed in sham-operated controls and experimentl eyes following IR (24 hours after reperfusion). For each gene, results are expressed as folds of corresponding WT sham-operated eye ± SEM after normalization to β-actin. *P<0.05 with respect to corresponding WT. B. Immunohistochemistry for the Cybb and Ncf1 proteins accumulation in GCL of WT and GFAP-IκBα-dn post-ischemic retinas (green) validated increased levels of the transcripts at the level of corresponding proteins 24 hours after reperfusion; GFAP, red. Scale bar: 25 µm. C. Protein carbonylation assayed 1 and 3 days post-reperfusion, revealed statistically significant (P<0.05) increase in oxidative damage to proteins in WT, but not in GFAP-IκBα-dn retinas.

Figure 6

A. Differential expression of genes encoding glial cell markers in sham-operated controls and experimental eyes following IR (24 hours after reperfusion). For each gene, results are expressed as folds of corresponding WT sham-operated eye ± SEM after normalization to β-actin. *P<0.05 with respect to corresponding WT. (■ - WT, □ - GFAP-IκBα-dn) B. Microglial cell (Cd11b-labeled, crimson) morphology assessed in flat-mounted retinas was similar in sham-operated WT and GFAP-IκBα-dn eyes, but changed dramatically in WT post-ischemic eyes 24 hours after reperfusion, showing both hypertrophy and presence of significant amounts of undifferentiated cells. In contrast, GFAP-IκBα-dn microglial cell morphology changed insignificantly following IR. Scale bar: 25 µm.