The Carbon monoxide releasing molecule ALF-186 mediates anti-inflammatory and neuroprotective effects via the soluble guanylate cyclase ß1 in rats’ retinal ganglion cells after ischemia and reperfusion injury

Abstract

BackgroundThe endogenously produced gaseous molecule carbon monoxide is able to promote organ protection after ischemia-reperfusion injuries (IRI). The impact of carbon monoxide releasing molecules (CORM) regarding inflammation in neuronal tissues has not been studied in detail. In this investigation, we aimed to analyze the effects of the CORM ALF-186 on neuro-inflammation and hypothesized that the soluble guanylate cyclase (sGC) is playing a decisive role.MethodsRetinal ischemia-reperfusion injury was performed for 60 min in Sprague-Dawley rats. Thereafter, the CORM ALF-186 (10 mg/kg) in the presence or absence of the sGC inhibitor ODQ was injected via a tail vein. Retinal tissue was harvested 24 h later to analyze mRNA or protein expression of sGC-β1 subunit, transcription factors NF-κB and CREB, the inflammatory cytokines TNF-α and IL-6, as well as the heat shock proteins (HSP) HSP-70 and HSP-90. Immunohistochemistry was performed on frozen sections of the retina. The overall neuroprotective effect of ALF-186 was assessed by counting fluorogold-pre-labeled retinal ganglion cells (RGC) 7 days after IRI.ResultsIschemia-reperfusion mediated loss of vital RGC was attenuated by the administration of ALF-186 after injury. ALF-186 treatment after IRI induced sGC-ß1 leading to a decreased NF-κB and CREB phosphorylation. Consecutively, ALF-186 mitigated IRI induced TNF-α and IL-6 expression in the retina and in the rats’ serum. Moreover, ALF-186 attenuated heat shock protein 70 (Hsp-70) while increasing Hsp-90. The sGC-inhibitor ODQ attenuated the anti-inflammatory effects of ALF-186 and increased retinal loss of ganglion cells. These results were confirmed by immunohistochemistry.ConclusionThe CORM ALF-186 protected RGC from IRI induced loss. Furthermore, ALF-186 reduced IRI mediated neuroinflammation in the retina and in the serum by activating sGC. Inhibition of sGC stopped the beneficial and protective effects of ALF-186. ALF-186 may present a promising therapeutic alternative in treating inflammation after neuronal IRI.

Neuroprotection and neuroregeneration of retinal ganglion cells after intravitreal carbon monoxide release

“…In the first part of this study, we demonstrated that even topical CORM injection into the vitreous body leads to sustained neuroprotective effects in the context of retinal IRI, which has not yet been shown. As recently detected with intravenous application of ALF-186 after IRI [ 17 , 21 ], intravitreal ALF-186 spreads its effects on ischemic RGC in a comparable way and effective power. Intravitreal injection of ALF-186 immediately after reperfusion reduced the extent of RGC loss, corresponding to higher RGC density.…”

“…Intracellular signaling after IRI+ALF intravitreally likewise involved the gene suppression of NF-κB and TNF-α as well as the induction of p38 and HSP 90, as previously described and discussed in detail after intravenous ALF application [ 17 , 21 ]. In both studies, corresponding changes on the level of protein were displayed; we thus decided not to confirm our mRNA results reported here by western blots, although the later would be methodically more valuable.…”

“…This data correspond to the decreased caspase-3 expression in retinae after IRI+ALF compared to IRI+iALF or IRI+PBS, representing the anti-apoptotic capacity of ALF. As the mRNA results correspond excellent between intravitreal and intravenous administration [ 17 , 21 ], protein cleavage of Caspase-3 was not further analyzed in this study. CO’s ability to protect RGC from injury by inhibiting caspase-3-dependent apoptosis was recently reported by Chen et al [ 42 ] and in previous studies from our group [ 13 , 14 , 17 , 21 ].…”

“…The application of CO-releasing molecules (CORM) represents a valuable alternative to CO inhalation because they can be administered in a streamlined way to biological systems, thereby significantly reducing toxic side effects to enhance safety. Pre- and postconditioning approaches with the molybdenum-based, water-soluble CORM ALF-186 have recently shown neuroprotective properties after ischemia [ 17 , 20 , 21 ]. Therefore, it is reasonable to explore the administration of CO directly into the vitreous, a common therapeutic route in ophthalmology.…”

Regenerative Potential of Carbon Monoxide in Adult Neural Circuits of the Central Nervous System

“…Intravitreal injection of CORM ALF-186 significantly upregulated GAP43 mRNA expression and RGC regeneration in rat ischemic/reperfusion vascular injury [22]. Moreover, ALF-186 also exerts neuroprotective effects by reducing the expression of inflammatory mediators, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), tumor necrosis factor-α (TNF-α) and IL-6 via the soluble guanylate cyclase β1 in rat RGCs after ischemic/reperfusion vascular injury [69]. In this ischemic model, ALF-186 upregulates Hsp90 mRNA and protein levels [69], possibly affecting the intrinsic mechanism of axon regeneration.…”