Inhalative as well as Intravenous Administration of H2S Provides Neuroprotection after Ischemia and Reperfusion Injury in the Rats' Retina.

BACKGROUND: Neuronal ischemia-reperfusion injury (IRI), such as it can occur in glaucoma or strokes, is associated with neuronal cell death and irreversible loss of function of the affected tissue. Hydrogen sulfide (H2S) is considered a potentially neuroprotective substance, but the most effective route of application and the underlying mechanism remain to be determined. METHODS: Ischemia-reperfusion injury was induced in rats by a temporary increase in intraocular pressure (1 h). H2S was then applied by inhalation (80 ppm at 0, 1.5, and 3 h after reperfusion) or by intravenous administration of the slow-releasing H2S donor GYY 4137. After 24 h, the retinas were harvested for Western blotting, qPCR, and immunohistochemical staining. Retinal ganglion cell survival was evaluated 7 days after ischemia. RESULTS: Both inhalative and intravenously delivered H2S reduced retinal ganglion cell death with a better result from inhalative application. H2S inhalation for 1.5 h, as well as GYY 4137 treatment, increased p38 phosphorylation. Both forms of application enhanced the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and inhalation showed a significant increase at all three time points. H2S treatment also reduced apoptotic and inflammatory markers, such as caspase-3, intracellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS). The protective effect of H2S was partly abolished by the ERK1/2 inhibitor PD98059. Inhalative H2S also reduced the heat shock response including heme oxygenase (HO-1) and heat shock protein 70 (HSP-70) and the expression of radical scavengers such as superoxide dismutases (SOD1, SOD2) and catalase. CONCLUSION: Hydrogen sulfide acts, at least in part, via the mitogen-activated protein kinase (MAPK) ERK1/2 to reduce apoptosis and inflammation. Both inhalative H2S and intravenous GYY 4137 administrations can improve neuronal cell survival.

Hydrogen Sulfide Reduces Ischemia and Reperfusion Injury in Neuronal Cells in a Dose- and Time-Dependent Manner.

BACKGROUND: The ischemia-reperfusion injury (IRI) of neuronal tissue, such as the brain and retina, leads to possible cell death and loss of function. Current treatment options are limited, but preliminary observations suggest a protective effect of hydrogen sulfide (H2S). However, the dosage, timing, and mechanism of inhaled H2S treatment after IRI requires further exploration. METHODS: We investigated possible neuroprotective effects of inhaled H2S by inducing retinal ischemia-reperfusion injury in rats for the duration of 1 h (120 mmHg), followed by the administration of hydrogen sulfide (H2S) for 1 h at different time points (0, 1.5, and 3 h after the initiation of reperfusion) and at different H2S concentrations (120, 80, and 40 ppm). We quantified the H2S effect by conducting retinal ganglion cell counts in fluorogold-labeled animals 7 days after IRI. The retinal tissue was harvested after 24 h for molecular analysis, including qPCR and Western blotting. Apoptotic and inflammatory mediators, transcription factors, and markers for oxidative stress were investigated. Histological analyses of the retina and the detection of inflammatory cytokines in serum assays were also performed. RESULTS: The effects of inhaled H2S were most evident at a concentration of 80 ppm administered 1.5 h after IRI. H2S treatment increased the expression of anti-apoptotic Bcl-2, decreased pro-apoptotic Bax expression, reduced the release of the inflammatory cytokines IL-1ß and TNF-α, attenuated NF-κB p65, and enhanced Akt phosphorylation. H2S also downregulated NOX4 and cystathionine ß-synthase. Histological analyses illustrated a reduction in TNF-α in retinal ganglion cells and lower serum levels of TNF-α in H2S-treated animals after IRI. CONCLUSION: After neuronal IRI, H2S mediates neuroprotection in a time- and dose-dependent manner. The H2S treatment modulated transcription factor NF-κB activation and reduced retinal inflammation.

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.

BACKGROUND: The 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. METHODS: Retinal 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-ß1 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. RESULTS: Ischemia-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. CONCLUSION: The 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.

Argon mediates protection by interleukin-8 suppression via a TLR2/TLR4/STAT3/NF-κB pathway in a model of apoptosis in neuroblastoma cells in vitro and following ischemia-reperfusion injury in rat retina in vivo.

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 anti-inflammatory 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-light-chain-enhancer' of activated B-cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3) but not STAT5 or cAMP-response element-binding protein (CREB) phosphorylation and DNA-binding activity. Argon treatment attenuated apoptosis by preservation of mitochondrial membrane potential and decline in reactive oxygen species (ROS) generation. NF-κB and STAT3 inhibition, as well as TLR2 and TLR4 inhibition reversed argon's effects on IL-8 mRNA expression. Argon attenuated rotenone-induced IL-8 protein and mRNA expression in vitro. Inhibition of TLR2 and 4 attenuated argon's protective effect in vivo reducing IRI driven retinal IL-8 expression. IL-8 expression was found in the retina in co-localization with Müller cells and retinal ganglion cells. Argon mediates its neuroprotective effects by TLR-mediated regulation of transcription factors NF-κB and STAT3, thus decreasing interleukin-8 expression in vitro and in vivo. These findings may open up new opportunities to effectively treat cerebral ischemia and reperfusion injury through the inhalation of argon. Argon exerts its protective effects in vitro and in vivo via toll-like receptors TLR2 and TLR4 signaling, followed by alteration of downstream enzymes. In conclusion, argon mediates its beneficial effects by suppression of STAT3 and NF-κB phosphorylation and subsequent suppression of interleukin IL-8 protein expression. These novel findings may open up opportunities for argon as a therapeutic agent, particularly in the treatment of neuronal injury. Cover image for this issue: doi: 10.1111/jnc.13334.

Carbon monoxide treatment reduces microglial activation in the ischemic rat retina.

PURPOSE: Ischemia and reperfusion (I/R) injury damages retinal neurons. Retinal injury is accompanied by activation of microglia, which scavenge the dead or dying neurons, but increasing evidence now indicates that amoeboid-shaped microglia cells activated in the brain after ischemia have neurotoxic and damaging properties in their own right. A previous study showed that postconditioning with carbon monoxide (CO) protects retinal ganglion cells (RGCs) after I/R through anti-apoptotic and anti-inflammatory mechanisms. The present study was designed to investigate and quantify the activation of retinal microglia after I/R with and without CO postconditioning. METHODS: Adult Sprague-Dawley rats underwent retinal ischemia by increasing the ocular pressure to 120 mmHg for 1 h through a needle inserted into the anterior chamber. Reperfusion was induced by removing the needle. After I/R, one group of animals was kept in a CO (250 ppm) atmosphere for 1 h; the other group was kept in room air (Air). At 1, 2, 3, and 7 days after I/R, the eyes were enucleated and fixed. Intracardiac blood was analyzed for systemic effects of CO or I/R. Retinal cross sections were taken from the middle third of the eye and were stained with anti-Iba-1. Microglia cells were graded as amoeboid or ramified phenotypes according to morphologic criteria. Retinal thicknesses were determined. RESULTS: Evaluation of retinal tissue revealed a significant reduction of amoeboid microglia cells after I/R + CO when compared to the I/R + Air group. The peak number of amoeboid microglia was observed at day 2 post-I/R + Air. This rise was attenuated by CO postconditioning (815 versus 572 cells/mm2 for I/R + Air versus I/R + CO, respectively; p = 0.005). CO reduced and further postponed the peak in the numbers of amoeboid and ramified microglia cells in ischemic eyes and prevented microglial activation in the contralateral eyes. I/R-induced leucocytosis was inhibited by CO inhalation. The reduction of retinal thickness after I/R was more serious after Air inhalation when compared to the CO group. CONCLUSIONS: Numerous activated microglia cells appear in the inner retina after I/R, and CO-treatment significantly attenuates this glial response. Antagonism of microglial activation may be a further neuroprotective effect of CO, apart from its direct anti-apoptotic capacity.

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 today's 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-κB) 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-κB and hypoxia-inducible factor 1 α-activity were analysed by DNA-binding ELISA, expression of NF-κB-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-κB) 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 µg ml) 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 α transcription factor. DNA-binding and transcriptional NF-κB-activity were inhibited by propofol. Neuronal protection and inhibition of TLR-4-NF-κB signalling were not consistently seen with midazolam or ketamine. In vivo, propofol treatment preserved rat retinal ganglion cell densities (cells mm, saline 1504 ±â€Š251 vs propofol 2088 ±â€Š144, P = 0.0001), which was accompanied by reduced neuronal caspase-3, TLR-4 and NF-κB expression. CONCLUSION: Propofol, but neither midazolam nor ketamine, provides neuroprotection to injured neuronal cells via inhibition of TLR-4-NF-κB-dependent signalling.

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.

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 (p75). 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-κB activity by DNA-binding ELISA and luciferase assay. The role of p75 was studied using the p75-blocking peptide TAT-pep5 and siRNA knockdown. The effect of isoflurane ±p75 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-κB DNA-binding and transcriptional activity of NF-κB (relative Luminescence Units: OGD 500 [499/637] versus OGD + isoflurane 1478 [1363/1643], P = 0.001). Pharmacological inhibition or siRNA knockdown of p75 counteracted the aggravating effects of isoflurane. Isoflurane increased RGC damage in vivo (IRI 1479 RGC/mm(2) [1311/1697] versus IRI + isoflurane 1170 [1093/1211], P = 0.03), which was counteracted by p75-inhibition via TAT-pep5 (P = 0.02). CONCLUSIONS: Isoflurane but not sevoflurane or desflurane postexposure aggravates neurotoxicity in preinjured neurons via activation of p75 and NF-κB. 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.

Neuroprotection Is in the Air-Inhaled Gases on Their Way to the Neurons.

Cerebral injury is a leading cause of long-term disability and mortality. Common causes include major cardiovascular events, such as cardiac arrest, ischemic stroke, and subarachnoid hemorrhage, traumatic brain injury, and neurodegenerative as well as neuroinflammatory disorders. Despite improvements in pharmacological and interventional treatment options, due to the brain's limited regeneration potential, survival is often associated with the impairment of crucial functions that lead to occupational inability and enormous economic burden. For decades, researchers have therefore been investigating adjuvant therapeutic options to alleviate neuronal cell death. Although promising in preclinical studies, a huge variety of drugs thought to provide neuroprotective effects failed in clinical trials. However, utilizing medical gases, noble gases, and gaseous molecules as supportive treatment options may offer new perspectives for patients suffering neuronal damage. This review provides an overview of current research, potentials and mechanisms of these substances as a promising therapeutic alternative for the treatment of cerebral injury.

Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect.

The noble gas argon has the potential to protect neuronal cells from cell death. So far, this effect has been studied in treatment after acute damage. Preconditioning using argon has not yet been investigated. In this study, human neuroblastoma SH-SY5Y cells were treated with different concentrations of argon (25%, 50%, and 74%; 21% O2, 5% CO2, balance nitrogen) at different time intervals before inflicting damage with rotenone (20 µM, 4 hours). Apoptosis was determined by flow cytometry after annexin V and propidium iodide staining. Surface expressions of Toll-like receptors 2 and 4 were also examined. Cells were also processed for analysis by western blot and qPCR to determine the expression of apoptotic and inflammatory proteins, such as extracellular-signal regulated kinase (ERK1/2), nuclear transcription factor-κB (NF-κB), protein kinase B (Akt), caspase-3, Bax, Bcl-2, interleukin-8, and heat shock proteins. Immunohistochemical staining was performed for TLR2 and 4 and interleukin-8. Cells were also pretreated with OxPAPC, an antagonist of TLR2 and 4 to elucidate the molecular mechanism. Results showed that argon preconditioning before rotenone application caused a dose-dependent but not a time-dependent reduction in the number of apoptotic cells. Preconditioning with 74% argon for 2 hours was used for further experiments showing the most promising results. Argon decreased the surface expression of TLR2 and 4, whereas OxPAPC treatment partially abolished the protective effect of argon. Argon increased phosphorylation of ERK1/2 but decreased NF-κB and Akt. Preconditioning inhibited mitochondrial apoptosis and the heat shock response. Argon also suppressed the expression of the pro-inflammatory cytokine interleukin-8. Immunohistochemistry confirmed the alteration of TLRs and interleukin-8. OxPAPC reversed the argon effect on ERK1/2, Bax, Bcl-2, caspase-3, and interleukin-8 expression, but not on NF-κB and the heat shock proteins. Taken together, argon preconditioning protects against apoptosis of neuronal cells and mediates its action via Toll-like receptors. Argon may represent a promising therapeutic alternative in various clinical settings, such as the treatment of stroke.

Retrospective investigation of >400 patients undergoing thoracic endovascular aortic repair with or without cerebrospinal fluid drainage.

OBJECTIVES: The aim of this study was to analyse the risks and benefits of cerebrospinal fluid drainage (CSFD) placement in patients undergoing thoracic endovascular aortic repair. METHODS: Between 2009 and 2020, 411 patients underwent thoracic endovascular aortic repair in 1 institution where 236 patients (57%) received a preoperative CSFD. Patient and outcome characteristics were retrospectively analysed and compared between patients with and without preoperative CSFD placement. RESULTS: Preoperative CSFD was performed significantly more frequently in elective patients, especially those undergoing distal stent graft extension following frozen elephant trunk-stent placement (P < 0.001). Significantly fewer CSFD was placed in patients with acute aortic injury (P < 0.001). The incidence of permanent spinal cord ischaemia (SCI) was higher in patients without preoperative CSFD [10 patients (2%) vs 1 patient (0.2%), P = 0.001]. Postoperative CSFD was placed in 3 patients (0.7%). Severe CSFD-associated complications affected 2 patients (0.5%) namely, a subdural spinal haematoma causing permanent paraplegia in one of those 2 patients. CONCLUSIONS: CSFS placement is associated with low procedural risk and can potentially help to prevent SCI. However, the SCI incidence is most likely also associated with other preoperative factors including the patient's haemodynamics. Hence, a general recommendation for placing a preoperative CSFD cannot be made when relying on the present evidence.

Angiopoietin-2 is associated with capillary leak and predicts complications after cardiac surgery.

BACKGROUND: Patients undergoing cardiac surgery are prone to numerous complications. Increased vascular permeability may be associated with morbidity and mortality due to hemodynamic instability, fluid overload, and edema formation. We hypothesized that markers of endothelial injury and inflammation are associated with capillary leak, ultimately increasing the risk of postoperative complications. METHODS: In this prospective, observational, multidisciplinary cohort study at our tertiary academic medical center, we recruited 405 cardiac surgery patients. Patients were assessed daily using body impedance electrical analysis, ultrasound, sublingual intravital microscopy, and analysis of serum biomarkers. Multivariable models, as well as machine learning, were used to study the association of angiopoietin-2 with extracellular water as well as common complications after cardiac surgery. RESULTS: The majority of patients underwent coronary artery bypass grafting, valvular, or aortic surgeries. Across the groups, extracellular water increased postoperatively (20 ± 6 preoperatively to 29 ± 7L on postoperative day 2; P < 0.001). Concomitantly, the levels of the biomarker angiopoietin-2 rose, showing a strong correlation based on the time points of measurements (r = 0.959, P = 0.041). Inflammatory (IL-6, IL-8, CRP) and endothelial biomarkers (VE-Cadherin, syndecan-1, ICAM-1) suggestive of capillary leak were increased. After controlling for common risk factors of edema formation, we found that an increase of 1 ng/mL in angiopoietin-2 was associated with a 0.24L increase in extracellular water (P < 0.001). Angiopoietin-2 showed increased odds for the development of acute kidney injury (OR 1.095 [95% CI 1.032, 1.169]; P = 0.004) and was furthermore associated with delayed extubation, longer time in the ICU, and a higher chance of prolonged dependence on vasoactive medication. Machine learning predicted postoperative complications when capillary leak was added to standard risk factors. CONCLUSIONS: Capillary leak and subsequent edema formation are relevant problems after cardiac surgery. Levels of angiopoietin-2 in combination with extracellular water show promising potential to predict postoperative complications after cardiac surgery. TRIAL REGISTRATION NUMBER: German Clinical Trials Registry (DRKS No. 00017057), Date of registration 05/04/2019, www.drks.de.

Argon reduces microglial activation and inflammatory cytokine expression in retinal ischemia/reperfusion injury.

We previously found that argon exerts its neuroprotective effect in part by inhibition of the toll-like receptors (TLR) 2 and 4. The downstream transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NF-κB) are also affected by argon and may play a role in neuroprotection. It also has been demonstrated that argon treatment could mitigate brain damage, reduce excessive microglial activation, and subsequently attenuate brain inflammation. Despite intensive research, the further exact mechanism remains unclear. In this study, human neuroblastoma cells were damaged in vitro with rotenone over a period of 4 hours (to mimic cerebral ischemia and reperfusion damage), followed by a 2-hour post-conditioning with argon (75%). In a separate in vivo experiment, retinal ischemia/reperfusion injury was induced in rats by increasing intraocular pressure for 1 hour. Upon reperfusion, argon was administered by inhalation for 2 hours. Argon reduced the binding of the transcription factors signal transducer and activator of transcription 3, nuclear factor kappa B, activator protein 1, and nuclear factor erythroid 2-related factor 2, which are involved in regulation of neuronal damage. Flow cytometry analysis showed that argon downregulated the Fas ligand. Some transcription factors were regulated by toll-like receptors; therefore, their effects could be eliminated, at least in part, by the TLR2 and TLR4 inhibitor oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). Argon treatment reduced microglial activation after retinal ischemia/reperfusion injury. Subsequent quantitative polymerase chain reaction analysis revealed a reduction in the pro-inflammatory cytokines interleukin (IL-1α), IL-1ß, IL-6, tumor necrosis factor α, and inducible nitric oxide synthase. Our results suggest that argon reduced the extent of inflammation in retinal neurons after ischemia/reperfusion injury by suppression of transcription factors crucial for microglial activation. Argon has no known side effects or narcotic properties; therefore, therapeutic use of this noble gas appears ideal for treatment of patients with neuronal damage in retinal ischemia/reperfusion injury. The animal experiments were approved by the Commission for Animal Care of the University of Freiburg (approval No. 35-9185.81/G14-122) on October 19, 2012.

Expression of HO1 and PER2 can predict the incidence of delirium in trauma patients with concomitant brain injury.

Intensive care unit (ICU)-acquired delirium is associated with adverse outcome in trauma patients with concomitant traumatic brain injury (TBI), but diagnosis remains challenging. Quantifying circadian disruption by analyzing expression of the circadian gene period circadian regulator 2 (PER2) and heme oxygenase 1 (HO1), which determines heme turnover, may prove to be potential diagnostic tools. Expression of PER2 and HO1 was quantified using qPCR from blood samples 1 day and 7 days after trauma. Association analysis was performed comparing mRNA expression levels with parameters of trauma (ISS-injury severity score), delirium, acute kidney injury (AKI) and length of ICU stay. 48 polytraumatized patients were included (equal distribution of TBI versus non-TBI) corrected for ISS, age and gender using a matched pairs approach. Expression levels of PER2 and HO1 were independent of age (PER2: P = 0.935; HO1: P = 0.988), while expression levels were significantly correlated with trauma severity (PER2: P = 0.009; HO1: P < 0.001) and longer ICU length of stay (PER2: P = 0.018; HO1: P < 0.001). High expression levels increased the odds of delirium occurrence (PER2: OR = 4.32 [1.14-13.87]; HO1: OR = 4.50 [1.23-14.42]). Patients with TBI showed a trend towards elevated PER2 (OR = 3.00 [0.84-9.33], P = 0.125), but not towards delirium occurrence (P = 0.556). TBI patients were less likely to develop AKI compared to non-TBI (P = 0.022). Expression levels of PER2 and HO1 correlate with the incidence of delirium in an age-independent manner and may potentially improve diagnostic algorithms when used as delirium biomarkers.Trial registration: German Clinical Trials Register (Trial-ID DRKS00008981; Universal Trial Number U1111-1172-6077; Jan. 18, 2018).

Diagnosing capillary leak in critically ill patients: development of an innovative scoring instrument for non-invasive detection.

BACKGROUND: The concomitant occurrence of the symptoms intravascular hypovolemia, peripheral edema and hemodynamic instability is typically named Capillary Leak Syndrome (CLS) and often occurs in surgical critical ill patients. However, neither a unitary definition nor standardized diagnostic criteria exist so far. We aimed to investigate common characteristics of this phenomenon with a subsequent scoring system, determining whether CLS contributes to mortality. METHODS: We conducted this single-center, observational, multidisciplinary, prospective trial in two separately run surgical ICUs of a tertiary academic medical center. 200 surgical patients admitted to the ICU and 30 healthy volunteers were included. Patients were clinically diagnosed as CLS or No-CLS group (each N = 100) according to the grade of edema, intravascular hypovolemia, hemodynamic instability, and positive fluid balance by two independent attending physicians with > 10 years of experience in ICU. We performed daily measurements with non-invasive body impedance electrical analysis, ultrasound and analysis of serum biomarkers to generate objective diagnostic criteria. Receiver operating characteristics were used, while we developed machine learning models to increase diagnostic specifications for our scoring model. RESULTS: The 30-day mortility was increased among CLS patients (12 vs. 1%, P = 0.002), while showing higher SOFA-scores. Extracellular water was increased in patients with CLS with higher echogenicity of subcutaneous tissue [29(24-31) vs. 19(16-21), P < 0.001]. Biomarkers showed characteristic alterations, especially with an increased angiopoietin-2 concentration in CLS [9.9(6.2-17.3) vs. 3.7(2.6-5.6)ng/mL, P < 0.001]. We developed a score using seven parameters (echogenicity, SOFA-score, angiopoietin-2, syndecan-1, ICAM-1, lactate and interleukin-6). A Random Forest prediction model boosted its diagnostic characteristics (AUC 0.963, P < 0.001), while a two-parameter decision tree model showed good specifications (AUC 0.865). CONCLUSIONS: Diagnosis of CLS in critically ill patients is feasible by objective, non-invasive parameters using the CLS-Score. A simplified two-parameter diagnostic approach can enhance clinical utility. CLS contributes to mortality and should, therefore, classified as an independent entity. TRIAL REGISTRATION: German Clinical Trials Registry (DRKS No. 00012713), Date of registration 10/05/2017, www.drks.de.

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 anti-inflammatory effects of E-α-(p-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone are mediated via HO-1 induction.

Inflammation plays a central role in the pathophysiology of many diseases. The inducible enzyme heme oxygenase-1 (HO-1) protects cells against inflammation and can be induced by electrophilic compounds like the chalcones (1,3-diphenylprop-2-enones) from the class of α,ß-unsaturated carbonyl compounds. We hypothesized that the synthetic chalcone E-α-(p-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) exerts anti-inflammatory effects in RAW264.7, Jurkat lymphocytes and HK-2 cells via HO-1 induction. RAW264.7 cells were treated with lipopolysaccharide prior to E-α-p-OMe-C6H4-TMC treatment. Subsequently, HO-1 protein induction and activity were analyzed, as well as expression of pro- and anti-inflammatory mediators, transcription factors and mitogen-activated protein kinases to evaluate the possible molecular mechanism. These results were confirmed in human cell lines (Jurkat T-lymphocytes and HK-2 epithelial cells). We found that the E-α-p-OMe-C6H4-TMC exerts significant anti-inflammatory effects in a dose dependent manner, showing no toxic effects in LPS-treated RAW264.7 macrophages. E-α-p-OMe-C6H4-TMC induced HO-1 and SOD-1 protein expression and HO-1 enzyme activity, reduced the upregulation of COX-2 and iNOS, while inducing the translocation of Nrf2. NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment accompanied by the downregulation of proinflammatory cytokines IL-1ß, IL-6 and MCP-1. Pretreatment with E-α-p-OMe-C6H4-TMC revealed significant changes in phosphorylation of ERK and p38, but not JNK. These anti-inflammatory effects of E-α-p-OMe-C6H4-TMC were approved in Jurkat and HK-2 cells, furthermore revealing a downregulation of IL-8 and IL-10. In conclusion, it is tempting to speculate about E-α-p-OMe-C6H4-TMC as a new and non-toxic agent, inducing HO-1 in cells. This opens up new opportunities regarding the development of therapeutic agents using beneficial effects of HO-1 and its products.

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.