Retinal ischemia and reperfusion injuries (R-IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti-apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK-1/2 dependent regulation of heat-shock proteins. Inhalation of Argon (75 Vol%) was performed after R-IRI on the rats 0 left eyes for 1 h immediately or with delay. Abbreviations used: ChAT, choline acetyltransferase; CO 2 , carbon dioxcide; DAPI, 4 0 , 6-diamino-2-phenylindole dihydrochloride hydrate; DMSO, dimethylsulfoxid; ERK, extracellular regulated protein kinase; FG, Fluorogold; GFAP, glial fibrillary acidic protein; HO-1, hemeoxygenase-1; HSP, heat-shock protein(s); Iba1, ionized calcium binding adaptor molecule 1; JNK, c-Jun N-terminale kinase; MAPK, mitogen activated protein kinases; NF-jB, nuclear factor j B; PBS, phosphate buffered saline; PD98059, ERK inhibitor; RGC, retinal ganglion cell(s); R-IRI, retinal ischemia/reperfusion injury.

Argon has recently come into scientific focus as a neuroprotective agent. The underlying neuroprotective mechanism remains unknown although toll-like receptors were recently suggested to play an important role. We hypothesized that TLR-associated downstream transcription factors are responsible for argon's effects, leading to anti-apoptotic and antiinflammatory properties. Apoptosis was induced in human neuroblastoma cells. Immediately afterwards, argon treatment (75 Vol% for 2 h) was initiated. Cells were analyzed, measuring mitochondrial membrane potential, reactive-oxygen-species, annexin-V/propidium iodide staining, transcription factor phosphorylation and binding activity as well as protein and mRNA expression of interleukins. Argon's in vivo effects were analyzed by quantification of retinal ganglion cell density, mRNA expression, serum cytokine analysis and immunohistochemistry after retinal ischemia reperfusion injury (IRI) in rats. Argon diminished rotenone-induced kappa-lightchain-enhancer' of activated B-cells (NF-jB) and signal transducer and activator of transcription 3 (STAT3) but not Cerebral ischemia and reperfusion injury -for example, stroke -is associated with destruction of neuronal tissue and subsequent increased mortality or functional disability. At a molecular level inflammation, oxidative stress and apoptosis play an important role in this detrimental process (Ahmad et al. 2014). As a result of the fact that neurons have a limited potential to regenerate, ischemia and reperfusion injury is a major cause of morbidity and mortality (Ng et al. 2011 Abbreviations used: DMSO, dimethylsulfoxid; ERK-1/2, extracellular regulated kinase-1/2; IL, interleukin; IRI, ischemia reperfusion injury; NF-jB, kappa-light-chain-enhancer' of activated B-cells; STAT3, signal transducer and activator of transcription 3.

PurposeRetinal ischemia and reperfusion injuries (IRI) permanently affect neuronal tissue and function by apoptosis and inflammation due to the limited regenerative potential of neurons. Recently, evidence emerged that the noble gas Argon exerts protective properties, while lacking any detrimental or adverse effects. We hypothesized that Argon inhalation after IRI would exert antiapoptotic effects in the retina, thereby protecting retinal ganglion cells (RGC) of the rat's eye.MethodsIRI was performed on the left eyes of rats (n = 8) with or without inhaled Argon postconditioning (25, 50 and 75 Vol%) for 1 hour immediately or delayed after ischemia (i.e. 1.5 and 3 hours). Retinal tissue was harvested after 24 hours to analyze mRNA and protein expression of Bcl-2, Bax and Caspase-3, NF-κB. Densities of fluorogold-prelabeled RGCs were analyzed 7 days after injury in whole-mounts. Histological tissue samples were prepared for immunohistochemistry and blood was analyzed regarding systemic effects of Argon or IRI. Statistics were performed using One-Way ANOVA.ResultsIRI induced RGC loss was reduced by Argon 75 Vol% inhalation and was dose-dependently attenuated by lower concentrations, or by delayed Argon inhalation (1504±300 vs. 2761±257; p<0.001). Moreover, Argon inhibited Bax and Bcl-2 mRNA expression significantly (Bax: 1.64±0.30 vs. 0.78±0.29 and Bcl-2: 2.07±0.29 vs. 0.99±0.22; both p<0.01), as well as caspase-3 cleavage (1.91±0.46 vs. 1.05±0.36; p<0.001). Expression of NF-κB was attenuated significantly. Immunohistochemistry revealed an affection of Müller cells and astrocytes. In addition, IRI induced leukocytosis was reduced significantly after Argon inhalation at 75 Vol%.ConclusionImmediate and delayed Argon postconditioning protects IRI induced apoptotic loss of RGC in a time- and dose-dependent manner, possibly mediated by the inhibition of NF-κB. Further studies need to evaluate Argon's possible role as a therapeutic option.

The study was registered in the German Clinical Trials Register (DRKS 00006961). https://drks-neu.uniklinik-freiburg.de/drks_web/.