Felix Ulbrich

Argon inhalation attenuates retinal apoptosis after ischemia/reperfusion injury in a time- and dose-dependent manner in rats.

Purpose Retinal 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 rats eye. Methods IRI 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. Results IRI 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%. Conclusion Immediate 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 Argons possible role as a therapeutic option.

Neuroprotective effects of Argon are mediated via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1 in retinal ganglion cells

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′ left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat‐shock proteins −70, −90 and heme‐oxygenase‐1, mitogen‐activated protein kinases (p38, JNK, ERK‐1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova. Argon significantly reduced the R‐IRI‐affected heat‐shock protein expression (p < 0.05). While Argon significantly induced ERK‐1/2 expression (p < 0.001), inhibition of ERK‐1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme‐oxygenase‐1 (p < 0.05). R‐IRI‐induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK‐1/2 activation in Müller cells. We conclude, that Argon treatment protects R‐IRI‐induced apoptotic loss of RGC via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1. We proposed the following possible mechanism for Argon‐mediated neuroprotection: Argon exerts its protective effects via an induction of an ERK with subsequent suppression of the heat shock response. In conclusion, ischemia and reperfusion injuries and subsequent neuronal apoptosis are attenuated. These novel findings may open up new opportunities for Argon as a therapeutic option, especially since Argon is not toxic.

Isoflurane but not sevoflurane or desflurane aggravates injury to neurons in vitro and in vivo via p75NTR-NF-ĸB activation.

BACKGROUND:General anesthesia in patients with or at risk for neuronal injury remains challenging due to the controversial influence of volatile anesthetics on neuronal damage. We hypothesized that isoflurane, sevoflurane, and desflurane would exert variable degrees of neurotoxicity in vitro and in vivo via activation of the p75 neurotrophin receptor (p75NTR). METHODS:SH-SY5Y cells were exposed to oxygen–glucose deprivation (OGD, 16 hours), preceded or followed by incubation with isoflurane, sevoflurane, or desflurane (1.2 minimal alveolar concentration, 2 hours). Neuronal cell death was analyzed by flow cytometry (mitochondrial membrane potential, Annexin V/propidium iodide [AV/Pi]) and quantification of lactate dehydrogenase release. We analyzed NF-&kgr;B activity by DNA-binding ELISA and luciferase assay. The role of p75NTR was studied using the p75NTR-blocking peptide TAT-pep5 and siRNA knockdown. The effect of isoflurane ±p75NTR inhibition on retinal ischemia-reperfusion injury (IRI) in adult Sprague-Dawley rats was assessed by analyzing retinal ganglion cell (RGC) density. RESULTS:Isoflurane but not sevoflurane or desflurane postexposure aggravated OGD-induced neuronal cell death (AV/Pi positive cells: OGD 41.1% [39.0/43.3] versus OGD + isoflurane 48.5% [46.4/63.4], P = 0.001). Isoflurane significantly increased NF-&kgr;B DNA-binding and transcriptional activity of NF-&kgr;B (relative Luminescence Units: OGD 500 [499/637] versus OGD + isoflurane 1478 [1363/1643], P = 0.001). Pharmacological inhibition or siRNA knockdown of p75NTR counteracted the aggravating effects of isoflurane. Isoflurane increased RGC damage in vivo (IRI 1479 RGC/mm2 [1311/1697] versus IRI + isoflurane 1170 [1093/1211], P = 0.03), which was counteracted by p75NTR-inhibition via TAT-pep5 (P = 0.02). CONCLUSIONS:Isoflurane but not sevoflurane or desflurane postexposure aggravates neurotoxicity in preinjured neurons via activation of p75NTR and NF-&kgr;B. These findings may have implications for the choice of volatile anesthetic being used in patients with or at risk for neuronal injury, specifically in patients with a stroke or history of stroke and in surgical procedures in which neuronal injury is likely to occur, such as cardiac surgery and neurovascular interventions.

Argon Mediates Anti-Apoptotic Signaling and Neuroprotection via Inhibition of Toll-Like Receptor 2 and 4.

Purpose Recently, the noble gas argon attracted significant attention due to its neuroprotective properties. However, the underlying molecular mechanism is still poorly understood. There is growing evidence that the extracellular regulated kinase 1/2 (ERK1/2) is involved in Argon´s protective effect. We hypothesized that argon mediates its protective effects via the upstream located toll-like receptors (TLRs) 2 and 4. Methods Apoptosis in a human neuroblastoma cell line (SH-SY5Y) was induced using rotenone. Argon treatment was performed after induction of apoptosis with different concentrations (25, 50 and 75 Vol% in oxygen 21 Vol%, carbon dioxide and nitrogen) for 2 or 4 hours respectively. Apoptosis was analyzed using flow cytometry (annexin-V (AV)/propidiumiodide (PI)) staining, caspase-3 activity and caspase cleavage. TLR density on the cells’ surface was analyzed using FACS and immunohistochemistry. Inhibition of TLR signaling and extracellular regulated kinase 1/2 (ERK1/2) were assessed by western blot, activity assays and FACS analysis. Results Argon 75 Vol% treatment abolished rotenone-induced apoptosis. This effect was attenuated dose- and time-dependently. Argon treatment was accompanied with a significant reduction of TLR2 and TLR4 receptor density and protein expression. Moreover, argon mediated increase in ERK1/2 phosphorylation was attenuated after inhibition of TLR signaling. ERK1/2 and TLR signaling inhibitors abolished the anti-apoptotic and cytoprotective effects of argon. Immunohistochemistry results strengthened these findings. Conclusion These findings suggest that argon-mediated anti-apoptotic and neuroprotective effects are mediated via inhibition of TLR2 and TLR4.

Propofol, but not ketamine or midazolam, exerts neuroprotection after ischaemic injury by inhibition of Toll-like receptor 4 and nuclear factor kappa-light-chain-enhancer of activated B-cell signalling: A combined in vitro and animal study.

BACKGROUND Propofol, midazolam and ketamine are widely used in todays anaesthesia practice. Both neuroprotective and neurotoxic effects have been attributed to all three agents. OBJECTIVE To establish whether propofol, midazolam and ketamine in the same neuronal injury model exert neuroprotective effects on injured neurones in vitro and in vivo by modulation of the Toll-like receptor 4-nuclear factor kappa-light-chain-enhancer of activated B cells (TLR-4-NF-&kgr;B) pathway. DESIGN AND SETTING Cell-based laboratory (n = 6 repetitions per experiment) and animal (n = 6 per group) studies using a neuronal cell line (SH-SY5Y cells) and adult Sprague-Dawley rats. INTERVENTIONS Cells were exposed to oxygen-glucose deprivation before or after treatment using escalating, clinically relevant doses of propofol, midazolam and ketamine. In animals, retinal ischaemia (60 min) was induced followed by reperfusion and randomised treatment with saline or propofol. MAIN OUTCOME MEASURES Neuronal cell death was determined using flow-cytometry (mitochondrial membrane potential) and lactate dehydrogenase (LDH) release. Nuclear factor NF-&kgr;B and hypoxia-inducible factor 1 &agr;-activity were analysed by DNA-binding ELISA, expression of NF-&kgr;B-dependent genes and TLR-4 by luciferase-assay and flow-cytometry, respectively. In animals, retinal ganglion cell density, caspase-3 activation and gene expression (TLR-4, NF-&kgr;B) were used to determine in vivo effects of propofol. Results were compared using ANOVA (Analysis of Variance) and t test. A P value less than 0.05 was considered statistically significant. RESULTS Post-treatment with clinically relevant concentrations of propofol (1 to 10 &mgr;g ml−1) preserved the mitochondrial membrane potential in oxygen-glucose deprivation-injured cells by 54% and reduced LDH release by 21%. Propofol diminished TLR-4 surface expression and preserved the DNA-binding activity of the protective hypoxia-inducible factor 1 &agr; transcription factor. DNA-binding and transcriptional NF-&kgr;B-activity were inhibited by propofol. Neuronal protection and inhibition of TLR-4-NF-&kgr;B signalling were not consistently seen with midazolam or ketamine. In vivo, propofol treatment preserved rat retinal ganglion cell densities (cells mm−2, saline 1504 ± 251 vs propofol 2088 ± 144, P = 0.0001), which was accompanied by reduced neuronal caspase-3, TLR-4 and NF-&kgr;B expression. CONCLUSION Propofol, but neither midazolam nor ketamine, provides neuroprotection to injured neuronal cells via inhibition of TLR-4-NF-&kgr;B-dependent signalling.

Argon: a novel therapeutic option to treat neuronal ischemia and reperfusion injuries?

Neuronal injury and neuroprotection: Ischemia and reperfusion injuries in neuronal cells such as acute ischemic stroke - represent the third leading cause of death in the world. Current therapeutic concepts mainly aim to re-establish cerebral blood flow within a time window of less than 3 hours with the goal of limiting secondary brain injury. Secondary brain injury of the “penumbra” develops exponentially, causing severe mental and physical disabilities. The resulting morbidity and mortality in patients of all ages leads to an enormous economic burden. Neurons are well-known to possess a limited potential to recover and regenerate their basic function once damaged. Therefore the protection and especially the therapy of neuronal injuries in cells and organ systems has emerged as a major focus of research in recent years. The aim is to maximize neuronal function not only by restoring sufficient blood flow to the areas at risk, but also by protecting neuronal cells with various novel interventions. Many of these interventions, such as intravascular cooling, focus on the reduction of neuronal metabolism in an effort to induce protection.

The CORM ALF-186 Mediates Anti-Apoptotic Signaling via an Activation of the p38 MAPK after Ischemia and Reperfusion Injury in Retinal Ganglion Cells

Purpose Ischemia and reperfusion injury may induce apoptosis and lead to sustained tissue damage and loss of function, especially in neuronal organs. While carbon monoxide is known to exert protective effects after various harmful events, the mechanism of carbon monoxide releasing molecules in neuronal tissue has not been investigated yet. We hypothesize that the carbon monoxide releasing molecule (CORM) ALF-186, administered after neuronal ischemia-reperfusion injury (IRI), counteracts retinal apoptosis and its involved signaling pathways and consecutively reduces neuronal tissue damage. Methods IRI was performed in rat´s retinae for 1 hour. The water-soluble CORM ALF-186 (10 mg/kg) was administered intravenously via a tail vein after reperfusion. After 24 and 48 hours, retinal tissue was harvested to analyze mRNA and protein expression of Bcl-2, Bax, Caspase-3, ERK1/2, p38 and JNK. Densities of fluorogold pre-labeled retinal ganglion cells (RGC) were analyzed 7 days after IRI. Immunohistochemistry was performed on retinal cross sections. Results ALF-186 significantly reduced IRI mediated loss of RGC. ALF-186 treatment differentially affected mitogen-activated protein kinases (MAPK) phosphorylation: ALF-186 activated p38 and suppressed ERK1/2 phosphorylation, while JNK remained unchanged. Furthermore, ALF-186 treatment affected mitochondrial apoptosis, decreasing pro-apoptotic Bax and Caspase-3-cleavage, but increasing anti-apoptotic Bcl-2. Inhibition of p38-MAPK using SB203580 reduced ALF-186 mediated anti-apoptotic effects. Conclusion In this study, ALF-186 mediated substantial neuroprotection, affecting intracellular apoptotic signaling, mainly via MAPK p38. CORMs may thus represent a promising therapeutic alternative treating neuronal IRI.

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

Purpose Retinal ischemia induces apoptosis leading to neurodegeneration and vision impairment. Carbon monoxide (CO) in gaseous form showed cell-protective and anti-inflammatory effects after retinal ischemia-reperfusion-injury (IRI). These effects were also demonstrated for the intravenously administered CO-releasing molecule (CORM) ALF-186. This article summarizes the results of intravitreally released CO to assess its suitability as a neuroprotective and neuroregenerative agent. Methods Water-soluble CORM ALF-186 (25 μg), PBS, or inactivated ALF (iALF) (all 5 μl) were intravitreally applied into the left eyes of rats directly after retinal IRI for 1 h. Their right eyes remained unaffected and were used for comparison. Retinal tissue was harvested 24 h after intervention to analyze mRNA or protein expression of Caspase-3, pERK1/2, p38, HSP70/90, NF-kappaB, AIF-1 (allograft inflammatory factor), TNF-α, and GAP-43. Densities of fluorogold-prelabeled retinal ganglion cells (RGC) were examined in flat-mounted retinae seven days after IRI and were expressed as mean/mm2. The ability of RGC to regenerate their axon was evaluated two and seven days after IRI using retinal explants in laminin-1-coated cultures. Immunohistochemistry was used to analyze the different cell types growing out of the retinal explants. Results Compared to the RGC-density in the contralateral right eyes (2804±214 RGC/mm2; data are mean±SD), IRI+PBS injection resulted in a remarkable loss of RGC (1554±159 RGC/mm2), p<0.001. Intravitreally injected ALF-186 immediately after IRI provided RGC protection and reduced the extent of RGC-damage (IRI+PBS 1554±159 vs. IRI+ALF 2179±286, p<0.001). ALF-186 increased the IRI-mediated phosphorylation of MAP-kinase p38. Anti-apoptotic and anti-inflammatory effects were detectable as Caspase-3, NF-kappaB, TNF-α, and AIF-1 expression were significantly reduced after IRI+ALF in comparison to IRI+PBS or IRI+iALF. Gap-43 expression was significantly increased after IRI+ALF. iALF showed effects similar to PBS. The intrinsic regenerative potential of RGC-axons was induced to nearly identical levels after IRI and ALF or iALF-treatment under growth-permissive conditions, although RGC viability differed significantly in both groups. Intravitreal CO further increased the IRI-induced migration of GFAP-positive cells out of retinal explants and their transdifferentiation, which was detected by re-expression of beta-III tubulin and nestin. Conclusion Intravitreal CORM ALF-186 protected RGC after IRI and stimulated their axons to regenerate in vitro. ALF conveyed anti-apoptotic, anti-inflammatory, and growth-associated signaling after IRI. CO’s role in neuroregeneration and its effect on retinal glial cells needs further investigation.

The Molecular Pathway of Argon-Mediated Neuroprotection

The noble gas argon has attracted increasing attention in recent years, especially because of its neuroprotective properties. In a variety of models, ranging from oxygen-glucose deprivation in cell culture to complex models of mid-cerebral artery occlusion, subarachnoid hemorrhage or retinal ischemia-reperfusion injury in animals, argon administration after individual injury demonstrated favorable effects, particularly increased cell survival and even improved neuronal function. As an inert molecule, argon did not show signs of adverse effects in the in vitro and in vivo model used, while being comparably cheap and easy to apply. However, the molecular mechanism by which argon is able to exert its protective and beneficial characteristics remains unclear. Although there are many pieces missing to complete the signaling pathway throughout the cell, it is the aim of this review to summarize the known parts of the molecular pathways and to combine them to provide a clear insight into the cellular pathway, starting with the receptors that may be involved in mediating argons effects and ending with the translational response.