Postconditioning with Inhaled Carbon Monoxide Counteracts Apoptosis and Neuroinflammation in the Ischemic Rat Retina

<div><h3>Purpose</h3><p>Ischemia and reperfusion injury (I/R) of neuronal structures and organs is associated with increased morbidity and mortality due to neuronal cell death. We hypothesized that inhalation of carbon monoxide (CO) after I/R injury (‘postconditioning’) would protect retinal ganglion cells (RGC).</p> <h3>Methods</h3><p>Retinal I/R injury was performed in Sprague-Dawley rats (n = 8) by increasing ocular pressure (120 mmHg, 1 h). Rats inhaled room air or CO (250 ppm) for 1 h immediately following ischemia or with 1.5 and 3 h latency. Retinal tissue was harvested to analyze Bcl-2, Bax, Caspase-3, HO-1 expression and phosphorylation of the nuclear transcription factor (NF)-κB, p38 and ERK-1/2 MAPK. NF-κB activation was determined and inhibition of ERK-1/2 was performed using PD98059 (2 mg/kg). Densities of fluorogold prelabeled RGC were analyzed 7 days after injury. Microglia, macrophage and Müller cell activation and proliferation were evaluated by Iba-1, GFAP and Ki-67 staining.</p> <h3>Results</h3><p>Inhalation of CO after I/R inhibited Bax and Caspase-3 expression (Bax: 1.9±0.3 vs. 1.4±0.2, p = 0.028; caspase-3: 2.0±0.2 vs. 1.5±0.1, p = 0.007; mean±S.D., fold induction at 12 h), while expression of Bcl-2 was induced (1.2±0.2 vs. 1.6±0.2, p = 0.001; mean±S.D., fold induction at 12 h). CO postconditioning suppressed retinal p38 phosphorylation (p = 0.023 at 24 h) and induced the phosphorylation of ERK-1/2 (p<0.001 at 24 h). CO postconditioning inhibited the expression of HO-1. The activation of NF-κB, microglia and Müller cells was potently inhibited by CO as well as immigration of proliferative microglia and macrophages into the retina. CO protected I/R-injured RGC with a therapeutic window at least up to 3 h (n = 8; RGC/mm²; mean±S.D.: 1255±327 I/R only vs. 1956±157 immediate CO treatment, vs. 1830±109 1.5 h time lag and vs. 1626±122 3 h time lag; p<0.001). Inhibition of ERK-1/2 did not counteract the CO effects (RGC/mm²: 1956±157 vs. 1931±124, mean±S.D., p = 0.799).</p> <h3>Conclusion</h3><p>Inhaled CO, administered after retinal ischemic injury, protects RGC through its strong anti-apoptotic and anti-inflammatory effects.</p> </div

Effects of carbon monoxide postconditioning on retinal Bax and Bcl-2 mRNA and protein expression.

<p>(<b>A</b>) Fold induction of Bax mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR (n = 8 per group; mean±SD; * p = 0.028, 0.024 and 0.016 I/R vs. I/R+CO at 12, 48 and 72 h). (<b>B</b>) Representative Western blot images (of n = 4) analyzing the suppression of retinal Bax protein expression by carbon monoxide postconditioning. Densitometric analysis of n = 4 western blots (mean±SD; * p = 0.028, <0.001 and <0.001 I/R vs. I/R+CO at 12, 24 and 48 h). (<b>C</b>) Retinal expression of Bcl-2 mRNA (n = 8 per group; mean±SD; * p = 0.001, 0.011 and 0.038 I/R vs. I/R+CO at 12, 24 and 48 h). (<b>D</b>) Representative Western blot images (of n = 4) analyzing the induction of retinal Bcl-2 protein expression by carbon monoxide postconditioning. Densitometric analysis of n = 4 western blots (mean±SD; * p<0.001 I/R vs. I/R+CO at 48 h).</p

Effect of carbon monoxide postconditioning on retinal expression of HO-1 mRNA and HO-1 protein.

<p>(<b>A</b>) Fold induction of HO-1 mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR (n = 8 per group; mean±SD; * p<0.001 I/R vs. I/R+CO at 12, logarithmic scale). (<b>B</b>) Representative Western blot images (of n = 4) analyzing the suppression of retinal HO-1 protein expression by carbon monoxide postconditioning. Densitometric analysis of n = 4 western blots (mean±SD; * p<0.001 I/R vs. I/R+CO at 12 and 48 h).</p

Diagram depicting the proposed mechanism of CO-mediated protective effects on RGC after I/R injury.

<p>CO postconditioning after retinal I/R strongly inhibits the inflammatory response by suppressing NF-κB activation, abolishing the infiltration of proliferating microglia and macrophages and inhibiting the activation of Müller cells. Inflammatory oxidative cellular stress is reduced, indicated by inhibition of HO-1 expression. CO attenuates RGC apoptosis as indicated by reduced Caspase-3 activity and Bax expression and by increased Bcl-2 expression. Apoptosis- and inflammation-related MAPK pathways are regulated in favor of anti-apoptosis and anti-inflammation. Overall, these anti-inflammatory and anti-apoptotic effects lead to higher RGC survival and neuroprotection (→ = activation; ⊣ = inhibition).</p

Effects of carbon monoxide postconditioning, inhibiting p38 MAPK while activating ERK-1/2 MAPK phosphorylation.

<p>(<b>A</b>) Representative Western blot images (of n = 4) analyzing the influence of carbon monoxide postconditioning on the phosphorylation of retinal p38 MAPK. Densitometric analysis of n = 4 western blots (mean±SD; * p = 0.023 and <0.001 I/R vs. I/R+CO at 24 and 48 h). (<b>B</b>) Representative Western blot images (of n = 4) analyzing the influence of carbon monoxide postconditioning on the phosphorylation of retinal ERK-1/2 MAPK. Densitometric analysis of n = 4 western blots (mean±SD; * p<0.001 I/R vs. I/R+CO at 24 and 48 h).</p

Effect of carbon monoxide postconditioning on ischemia reperfusion (I/R) injury in RGC.

<p>(<b>A</b>) Representative images (of n = 8) from flat mounts with flourogold-labeled RGC 7 days after I/R injury and CO treatment immediately, 1.5 h and 3 h after initiation of reperfusion. (<b>B</b>) Quantification of retinal ganglion cell density [cells/mm²] 7 days after I/R injury (n = 8 per group; mean±S.D.; * p<0.001 I/R vs. I/R+CO immediate, vs. I/R+CO 1.5 h and vs. I/R+CO 3 h; IR+CO immediate vs. I/R+CO 3 h).</p

Real time polymerase chain reaction primers.

<p>Genes of interest and the corresponding assay IDs of real time polymerase chain reactions primers used (HMOX1 = HO-1, heme oxygenase 1; BAX = Bax, Bcl-2-associated X protein; BCL2 = Bcl-2, B-cell lymphoma 2; CASP3 = Caspase-3; GAPDH = glyceraldehyde 3-phosphate dehydrogenase).</p

Effect of carbon monoxide postconditioning on phosphorylation of ERK-1/2 in Thy-1 positive RGC.

<p>Representative images of immunhistochemical staining against p-ERK-1/2 and Thy-1 in the retina, showing increased phosphorylation of ERK-1/2 in the GCL after I/R and I/R+CO. Only after I/R+CO, ERK-1/2 is phosphorylated in the RGC (white arrows: Thy-1 positive RGC), whereas after I/R alone, p-ERK-1/2 is evident mainly in other cells of the GCL (*: p-ERK-1/2 positive cells). Scale bar 100 µm and 50 µm (in “Detail GCL” pictures). Abbreviations: GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer, DAPI = 4′,6-diamidino-2-phenylindole.</p

Effects of carbon monoxide postconditioning on NF-κB protein expression, phosphorylation and NF-κB DNA-binding.

<p>(<b>A</b>) Representative Western blot images (of n = 4) analyzing the influence of carbon monoxide postconditioning on expression and phosphorylation of retinal NF-κB p65. Densitometric analysis of n = 4 western blots (mean±SD; * p<0.001 I/R vs. I/R+CO at 24 h). (<b>B</b>) Representative EMSA (of n = 4) of NF-κB DNA binding. Lanes 1–16: individual experiments at 12, 24, 48 and 72 hours after carbon monoxide postconditioning, lanes 17 and 18: supershift analysis shows specificity of NF-κB, lane 19: self competition with unlabeled NF-κB, lane 20: non-self competition with unlabeled AP-1, lane 21: positive control, achieved by induction of SY5Y cell line exposed to PMA/Ionomycin. Densitometric analysis of n = 4 EMSA (mean±SD; * p = 0.016 I/R vs. I/R+CO at 48 h). (Abbreviations: NF-κB Ab = nuclear factor κB antibody, c-fos Ab = c-fos antibody, AP-1 = activator protein 1).</p

Effect of carbon monoxide postconditioning on retinal expression of caspase-3 mRNA and caspase-3 cleavage.

<p>(<b>A</b>) Fold induction of caspase-3 mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR (n = 8 per group; mean±SD; * p = 0.007, 0.002, <0.001 and 0.006 I/R vs. I/R+CO at 12, 24, 48 and 72 h). (<b>B</b>) Representative Western blot images (of n = 4) analyzing the suppression of retinal cleavage of caspase-3 protein by carbon monoxide postconditioning. Densitometric analysis of n = 4 western blots (mean±SD; p = 0.042 and 0.028 I/R vs. I/R+CO at 24 and 48 h).</p