Gezina T. M. L. Oei

Cellular Effects of Helium in Different Organs

Experimental research in cardiac and neuronal tissue has shown that besides volatile anesthetics and xenon, the nonanesthetic noble gas helium also reduces ischemia–reperfusion damage. Even though the distinct mechanisms of helium-induced organ protection are not completely unraveled, several signaling pathways have been identified. Beside the protective effects on heart and brain that are mainly obtained by different pre- and postconditioning protocols, helium also exerts effects in the lungs, the immune system, and the blood vessels. Obviously, this noble gas is biochemically not inert and exerts biologic effects, although until today the question remains open on how these changes are mediated. Because of its favorable characteristics and the lack of hemodynamic side effects, helium is suitable for use also in critically ill patients. This review covers the cellular effects of helium, which may lead to new clinical strategies of tissue salvage in ischemia–reperfusion situations, both within and outside the perioperative setting.

Helium Induces Preconditioning in Human Endothelium In Vivo

Aims:Helium protects myocardium by inducing preconditioning in animals. We investigated whether human endothelium is preconditioned by helium inhalation in vivo. Methods and Results:Forearm ischemia–reperfusion (I/R) in healthy volunteers (each group n = 10) was performed by inflating a blood pressure cuff for 20 min. Endothelium-dependent and endothelium-independent responses were measured after cumulative dose–response infusion of acetylcholine and sodium nitroprusside, respectively, at baseline and after 15 min of reperfusion using strain-gauge, venous occlusion plethysmography. Helium preconditioning was applied by inhalation of helium (79% helium, 21% oxygen) either 15 min (helium early preconditioning [He-EPC]) or 24 h before I/R (helium late preconditioning). Additional measurements of He-EPC were done after blockade of endothelial nitric oxide synthase. Plasma levels of cytokines, adhesion molecules, and cell-derived microparticles were determined. Forearm I/R attenuated endothelium-dependent vasodilation (acetylcholine) with unaltered endothelium-independent response (sodium nitroprusside). Both He-EPC and helium late preconditioning attenuated I/R-induced endothelial dysfunction (max increase in forearm blood flow in response to acetylcholine after I/R was 180 ± 24% [mean ± SEM] without preconditioning, 573 ± 140% after He-EPC, and 290 ± 32% after helium late preconditioning). Protection of helium was comparable to ischemic preconditioning (max forearm blood flow 436 ± 38%) and was not abolished after endothelial nitric oxide synthase blockade. He-EPC did not affect plasma levels of cytokines, adhesion molecules, or microparticles. Conclusion:Helium is a nonanesthetic, nontoxic gas without hemodynamic side effects, which induces early and late preconditioning of human endothelium in vivo. Further studies have to investigate whether helium may be an instrument to induce endothelial preconditioning in patients with cardiovascular risk factors.

Helium-induced cardioprotection of healthy and hypertensive rat myocardium in vivo

Helium protects healthy myocardium against ischemia/reperfusion injury by early and late preconditioning (EPC, LPC) and postconditioning (PostC). We investigated helium-induced PostC of the hypertensive heart and enhancement by addition of LPC and EPC. We also investigated involvement of signaling kinases glycogen synthase kinase 3 beta (GSK-3β) and protein kinase C-epsilon (PKC-ε). To assess myocardial cell damage, we performed infarct size measurements in healthy Wistar Kyoto (WKY rats, n=8-9) and Spontaneous Hypertensive rats (SHR, n=8-9) subjected to 25 min ischemia and 120 min reperfusion. Rats inhaled 70% helium for 15 min after index ischemia (PostC), combined with 15 min helium 24h prior to index ischemia (LPC+PostC), a triple intervention with additional 3 short cycles of 5 min helium inhalation shortly before ischemia (EPC+LPC+PostC), or no further treatment. In WKY rats, PostC reduced infarct size from 46 ± 2% (mean ± S.E.M) in the control group to 29 ± 2%. LPC+PostC or EPC+LPC+PostC reduced infarct sizes to a similar extent (30 ± 3% and 32 ± 2% respectively). In SHR, EPC+LPC+PostC reduced infarct size from 53 ± 3% in control to 39 ± 3%, while PostC or LPC+PostC alone were not protective; infarct size 48 ± 4% and 44 ± 4%, respectively. Neither PostC in WKY rats nor EPC+LPC+PostC in SHR was associated with an increase in phosphorylation of GSK-3β and PKC-ε after 15 min of reperfusion. Concluding, a triple intervention of helium conditioning results in cardioprotection in SHR, whereas a single intervention does not. In WKY rats, the triple intervention does not further augment protection. Helium conditioning is not associated with a mechanism involving GSK-3β and PKC-ε.

Hypoxia induces late preconditioning in the rat heart in vivo.

Background:Although hypoxic late preconditioning (LPC) limits ischemia-reperfusion injury in vitro, its cardioprotective effect is not established in vivo. Methods:In part 1, rats were exposed to 4 h of hypoxia (16%, 12%, 8% oxygen) before 24 h of reoxygenation. In part 2, normoxic rats received early preconditioning with sevoflurane (1 minimum alveolar concentration [MAC] for 3 × 5 min), continuous administration of 1 MAC sevoflurane, or 11 mg · kg · h propofol. Thereafter, all rats underwent 25 min of regional myocardial ischemia and 120 min of reperfusion. After reperfusion, hearts were excised for infarct staining. The expression of protein kinase C (PKC)&agr; and PKC&egr; was assessed by Western blot analysis and the expression of heme oxygenase-1 and vascular endothelial growth factor by reverse transcriptase polymerase chain reaction. Results:In normoxic control rats, infarct size was 62 ± 6% of the area at risk. Hypoxic LPC reduced infarct size (LPC16: 36 ± 11%, LPC12: 38 ± 10%, LPC8: 39 ± 11%; each P < 0.001) to approximately the same magnitude as sevoflurane-preconditioning (40 ± 8%; P < 0.001). Combined LPC16 and sevoflurane preconditioning was not superior to either substance alone. Continuous sevoflurane or propofol was not protective. The PKC inhibitor calphostin C abolished the cardioprotective effects of LPC16. PKC&egr;, but not PKC&agr;, expression was increased 6 and 28 h after hypoxic LPC. Heme oxygenase-1 and vascular endothelial growth factor were transiently up-regulated after 6 h. Conclusion:Hypoxic LPC at 8%, 12%, and 16% oxygen reduces infarct size in the rat heart in vivo. This effect is as powerful as sevoflurane-preconditioning. PKC&egr; is a key player in mediating hypoxic LPC.

Reduction of cardiac cell death after helium postconditioning in rats: transcriptional analysis of cell death and survival pathways

Helium, a noble gas, has been used safely in humans. In animal models of regional myocardial ischemia/reperfusion (I/R) it was shown that helium conditioning reduces infarct size. Currently, it is not known how helium exerts its cytoprotective effects and which cell death/survival pathways are affected. The objective of this study, therefore, was to investigate the cell protective effects of helium postconditioning by PCR array analysis of genes involved in necrosis, apoptosis and autophagy. Male rats were subjected to 25 min of ischemia and 5, 15 or 30 min of reperfusion. Semiquantitative histological analysis revealed that 15 min of helium postconditioning reduced the extent of I/R-induced cell damage. This effect was not observed after 5 and 30 min of helium postconditioning. Analysis of the differential expression of genes showed that 15 min of helium postconditioning mainly caused upregulation of genes involved in autophagy and inhibition of apoptosis versus I/R alone. The results suggest that the cytoprotective effects of helium inhalation may be caused by a switch from pro-cell-death signaling to activation of cell survival mechanisms, which appears to affect a wide range of pathways.

Effects of helium and air inhalation on the innate and early adaptive immune system in healthy volunteers ex vivo

BackgroundHelium inhalation protects myocardium, brain and endothelium against ischemia/reperfusion injury in animals and humans, when applied according to specific “conditioning” protocols. Before widespread use of this “conditioning” agent in clinical practice, negative side effects have to be ruled out. We investigated the effect of prolonged helium inhalation on the responsiveness of the human immune response in whole blood ex vivo.MethodsMale healthy volunteers inhaled 30 minutes heliox (79%He/21%O2) or air in a cross over design, with two weeks between measurements. Blood was withdrawn at T0 (baseline), T1 (25 min inhalation) and T2-T5 (1, 2, 6, 24 h after inhalation) and incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), T-cell stimuli anti-CD3/ anti-CD28 (TCS) or RPMI (as control) for 2, 4 and 24 hours or not incubated (0 h). An additional group of six volunteers inhaled 60 minutes of heliox or air, followed by blood incubation with LPS and RPMI. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ) and interleukin-2 (IL-2) was analyzed by cytometric bead array. Statistical analysis was performed by the Wilcoxon test for matched samples.ResultsIncubation with LPS, LTA or TCS significantly increased TNF-α, IL-1β, IL-6, IL-8, IFN-γ and IL-2 in comparison to incubation with RPMI alone. Thirty min of helium inhalation did not influence the amounts of TNF-α, IL-1β, IL-6, IL-8, IFN-γ and IL-2 in comparison to air. Sixty min of helium inhalation did not affect cytokine production after LPS stimulation.ConclusionsWe conclude that 79% helium inhalation does not affect the responsiveness of the human immune system in healthy volunteers.Trial registrationDutch Trial Register:http://www.trialregister.nl/ NTR2152

Helium postconditioning regulates expression of caveolin-1 and -3 and induces RISK pathway activation after ischaemia/reperfusion in cardiac tissue of rats.

Caveolae, lipid enriched invaginations of the plasma membrane, are epicentres of cellular signal transduction. The structural proteins of caveolae, caveolins, regulate effector pathways in anaesthetic-induced cardioprotection, including the RISK pathway. Helium (He) postconditioning (HePoc) is known to mimic anaesthetic conditioning and to prevent damage from myocardial infarction. We hypothesize that HePoc regulates caveolin-1 and caveolin-3 (Cav-1 and Cav-3) expression in the rat heart and activates the RISK pathway. Male Wistar rats (n=8, each group) were subjected to 25min of cardiac ischaemia followed by reperfusion (I/R) for 5, 15 or 30min (I/R 5/15/30). The HePoc groups underwent I/R with 70% helium ventilation during reperfusion (IR+He 5/15/30min). Sham animals received surgical treatment without I/R. After each protocol blood and hearts were retrieved. Tissue was obtained from the area-at-risk (AAR) and non-area-at-risk (NAAR) and processed for western blot analyses and reverse-transcription-real-time-polymerase-chain-reaction (RT-qPCR). Protein analyses revealed increased amounts of Cav-1 and Cav-3 in the membrane of I/R+He15 (AAR: Cav-1, P<0.05; Cav-3, P<0.05; both vs. I/R15). In serum, Cav-3 was found to be elevated in I/R+He15 (P<0.05 vs. I/R15). RT-qPCR showed increased expression of Cav-1 in IR+He15 in AAR tissue (P<0.05 vs. I/R15). Phosphorylation of RISK pathway proteins pERK1/2 (AAR: P<0.05 vs. I/R15) and pAKT (AAR: P<0.05; NAAR P<0.05; both vs. I/R15) was elevated in the cytosolic fraction of I/R+He15. These results suggest that 15min of HePoc regulates Cav-1 and Cav-3 and activates RISK pathway kinases ERK1/2 and AKT. These processes might be crucially involved in HePoc mediated cardioprotection.

Prolonged helium postconditioning protocols during early reperfusion do not induce cardioprotection in the rat heart in vivo: role of inflammatory cytokines

Postconditioning of myocardial tissue employs short cycles of ischemia or pharmacologic agents during early reperfusion. Effects of helium postconditioning protocols on infarct size and the ischemia/reperfusion-induced immune response were investigated by measurement of protein and mRNA levels of proinflammatory cytokines. Rats were anesthetized with S-ketamine (150 mg/kg) and diazepam (1.5 mg/kg). Regional myocardial ischemia/reperfusion was induced; additional groups inhaled 15, 30, or 60 min of 70% helium during reperfusion. Fifteen minutes of helium reduced infarct size from 43% in control to 21%, whereas 30 and 60 minutes of helium inhalation led to an infarct size of 47% and 39%, respectively. Increased protein levels of cytokine-induced neutrophil chemoattractant (CINC-3) and interleukin-1 beta (IL-1β) were found after 30 or 60 min of helium inhalation, in comparison to control. 30 min of helium increased mRNA levels of CINC-3, IL-1β, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in myocardial tissue not directly subjected to ischemia/reperfusion. These results suggest that the effectiveness of the helium postconditioning protocol is very sensitive to duration of noble gas application. Additionally, helium was associated with higher levels of inflammatory cytokines; however, it is not clear whether this is causative of nature or part of an epiphenomenon.