Carbon Monoxide Abrogates Ischemic Insult to Neuronal Cells via the Soluble Guanylate Cyclase-cGMP Pathway

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Abstract

Purpose: Carbon monoxide (CO) is an accepted cytoprotective molecule. The extent and mechanisms of protection in neuronal systems have not been well studied. We hypothesized that delivery of CO via a novel releasing molecule (CORM) would impart neuroprotection in vivo against ischemia-reperfusion injury (IRI)-induced apoptosis of retinal ganglion cells (RGC) and in vitro of neuronal SH-SY5Y-cells via activation of soluble guanylate-cyclase (sGC). Methods: To mimic ischemic respiratory arrest, SH-SY5Y-cells were incubated with rotenone (100 nmol/L, 4 h) +/- CORM ALF186 (10-100 mu mol/L) or inactivated ALF186 lacking the potential of releasing CO. Apoptosis and reactive oxygen species (ROS) production were analyzed using flow-cytometry (Annexin V, mitochondrial membrane potential, CM-H(2)DCFDA) and Western blot (Caspase-3). The impact of ALF186 +/- respiratory arrest on cell signaling was assessed by measuring expression of nitric oxide synthase (NOS) and soluble guanylate-cyclase (sGC) and by analyzing cellular cGMP levels. The effect of ALF186 (10 mg/kg iv) on retinal IRI in Sprague-Dawley rats was assessed by measuring densities of fluorogold-labeled RGC after IRI and by analysis of apoptosis-related genes in retinal tissue. Results: ALF186 but not inactivated ALF186 inhibited rotenone-induced apoptosis (Annexin V positive cells: 25 +/- 2% rotenone vs. 14 +/- 1% ALF186+rotenone, p<0.001; relative mitochondrial membrane potential: 17 +/- 4% rotenone vs. 55 +/- 3% ALF186+rotenone, p<0.05). ALF186 increased cellular cGMP levels (33 +/- 5 nmol/L vs. 23 +/- 3 nmol/L; p<0.05) and sGC expression. sGC-inhibition attenuated ALF186-mediated protection (relative mitochondrial membrane potential: 55 +/- 3% ALF186+rotenone vs. 20 +/- 1% ODQ+ALF186+rotenone, p<0.05). ALF186 protected RGC in vivo (IRI 1255 +/- 327 RGC/mm(2) vs. ALF186+IRI 2036 +/- 83; p<0.05) while sGC inhibition abolished the protective effects of ALF186 (ALF186+IRI 2036 +/- 83 RGC/mm(2) vs. NS-2028+ALF186+IRI 1263 +/- 170, p<0.05). Conclusions: The CORM ALF186 inhibits IRI-induced neuronal cell death via activation of sGC and may be a useful treatment option for acute ischemic insults to the retina and the brain.
Original languageUnknown
Pages (from-to)Online
JournalPLoS ONE
Volume8
Issue number4
Publication statusPublished - 1 Jan 2013

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