Noble gas neuroprotection: xenon and argon protect against hypoxic–ischaemic injury in rat hippocampus in vitro via distinct mechanisms

Background Noble gases may provide novel treatments for neurological injuries such as ischaemic and traumatic brain injury. Few studies have evaluated the complete series of noble gases under identical conditions in the same model. Methods We used an in vitro model of hypoxia–ischaemia to evaluate the neuroprotective properties of the series of noble gases, helium, neon, argon, krypton, and xenon. Organotypic hippocampal brain slices from mice were subjected to oxygen-glucose deprivation, and injury was quantified using propidium iodide fluorescence. Results Both xenon and argon were equally effective neuroprotectants, with 0.5 atm of xenon or argon reducing injury by 96% (P<0.0001), whereas helium, neon, and krypton were devoid of any protective effect. Neuroprotection by xenon, but not argon, was reversed by elevated glycine. Conclusions Xenon and argon are equally effective as neuroprotectants against hypoxia–ischaemia in vitro, with both gases preventing injury development. Although xenon's neuroprotective effect may be mediated by inhibition of the N-methyl-d-aspartate receptor at the glycine site, argon acts via a different mechanism. These findings may have important implications for their clinical use as neuroprotectants.

Neurological injuries resulting from hypoxiaeischaemia are leading causes of morbidity and mortality worldwide. 1e3 Hypoxiceischaemic brain injury has a variety of aetiologies including stroke, cardiac arrest, neonatal hypoxiceischaemic encephalopathy (HIE), drowning and exposure to asphyxiant gases and carbon monoxide. Many who survive a hypoxiceischaemic brain injury have persisting disability, with long-term care and rehabilitation costs. 4 Treatment options are limited to thrombolytic drugs and clot removal for ischaemic stroke, and therapeutic cooling (or hypothermia) for cardiac arrest and neonatal HIE. Currently, there are no clinically proven treatments specifically targeted at preventing or limiting neuronal death resulting from ischaemia.
There is a need to develop neuroprotective treatments for hypoxiceischaemic brain injury. Currently there is interest in the noble gases as novel treatments for ischaemic and traumatic brain injury. 5e9 Attention has focused on xenon, which has already undergone clinical trials for HIE 10e12 and out-of-hospital cardiac arrest, 13,14 but there is also interest in the use of argon and helium, which have been evaluated in in vitro and in vivo models. 15e20 Neuroprotection by particular noble gases has been reported under different conditions. 7,21e25 Few studies, however, have evaluated neuroprotection by krypton or neon, 26,27 and investigation of the whole series of noble gases in hypoxiceischaemic brain injury under the same conditions has been limited to dissociated cell cultures. 27 We report the neuroprotective efficacy of helium, neon, argon, krypton, and xenon under identical conditions using organotypic hippocampal brain-slice cultures subjected to oxygen-glucose deprivation (OGD), an experimental model of cerebral ischaemia. We tested the hypothesis that the N-methyl-D-aspartate (NMDA) receptor glycine site is involved in noble gas neuroprotection against hypoxiceischaemic brain injury in vitro.

Hippocampal organotypic slices
Experiments were performed in compliance with the Animal Welfare and Ethical Review Body of Imperial College London and the Animals (Scientific Procedures) Act of 1986. Animals (pups and their dams) were housed in individually ventilated cages in a pathogen-free facility in a 12:12 h lightedark cycle (7:00 AMe7:00 PM light) at 22 C with ad libitum access to food and water. Animals were checked at least once daily. Organotypic hippocampal slice cultures were prepared as described 24,26,28,29 from male and female 7-day-old C57BL/6 mouse pups (Harlan Ltd, Bicester, Oxfordshire, UK). Briefly, after euthanasia, brains were removed and placed in ice-cold 'preparation' medium that contained Gey's balanced salt solution, 33 mM D-glucose (Fisher Scientific, Loughborough, Leicestershire, UK) and 1% antibioticeantimycotic suspension. The hippocampi were removed, and 400 mm thick transverse slices were prepared using a McIllwain tissue chopper. Slices were transferred into ice-cold preparation medium, gently separated and then placed on tissue culture inserts (Millicell-CM; Millipore Corporation, Carrigtwohill, Co. Cork, Ireland) that were inserted into a six-well tissue culture plate. The wells contained 'growth' medium consisting of 50% (v:v) Minimal Essential Medium Eagle, 25% Hank's balanced salt solution, 25% inactivated horse serum, 2 mM L-glutamine, 32 mM D-glucose, and 1% antibioticeantimycotic suspension. Slices were incubated at 37 C in a 95% air:5% CO 2 humidified atmosphere. The growth medium was changed every 3 days. Experiments were carried out after 14 days in culture. Cell culture inserts containing four to seven slices were randomly assigned to sham, OGD control, or OGD noble gas treatment groups.

Oxygen-glucose deprivation and hyperbaric gas chamber
The growth medium was changed to serum-free 'experimental' medium consisting of 75% Minimal Essential Medium Eagle, 25% Hank's balanced salt solution, 2 mM L-glutamine, 33 mM D-glucose, 1% antibioticeantimycotic suspension, and 4.5 mM propidium iodide (PI). One hour after transfer to experimental media, slices were imaged to assess viability before OGD. Typically, slices exhibited very little PI fluorescence, an indicator of healthy slices. A small number of slices were excluded from further analysis because they failed to meet objective viability criteria at this time point (t¼0); either there were regions of dense staining, or there were more than 20 pixels at intensity levels above 80, or tissue fragments were visible, indicating compromised viability, presumably as a result of mechanical damage during slice preparation. Immediately after initial imaging, experimental medium was exchanged for 'OGD medium', 120 mM NaCl, 5 mM KCl, 1.25 mM NaH 2 PO 4 , 2 mM MgSO 4 , 2 mM CaCl 2 , 25 mM NaHCO 3 , 10 mM sucrose, 20 mM HEPES, pH 7.25 or 'sham medium,' which had the same composition, except that sucrose was replaced with 10 mM D-glucose. OGD medium was deoxygenated before use by bubbling for 45 min at 50 ml min À1 with 95% N 2 :5% CO 2 , in a Dreschel bottle using a fine-sintered glass bubbler and filter-sterilised using a 0.2 mm filter. Sham medium was treated in the same way except it was bubbled with 20% O 2 :75% N 2 :5% CO 2 . After solution exchange, culture dishes were transferred to a small chamber (Fig. 1a) that contained a high-speed fan for rapid gas mixing. The chamber was housed in an incubator at 37 C. The chamber (gas volume 0.925 L) was flushed with humidified gas (95% N 2 :5% CO 2 or 20% O 2 :75% N 2 :5% CO 2 ) for 5 min at 5 L min À1 ensuring better than 99.99% gas replacement. After flushing, the chamber was sealed for a set period of 30 min, constituting the duration of OGD (or sham treatment).
After the period of OGD, slices were removed from the chamber and medium was replaced with experimental medium (in experiments with added glycine [100 mM], this was added for the first time at this stage). Slices were returned to the chamber which was flushed with 20% O 2 :75% N 2 :5% CO 2 as before and sealed. In the helium experiments at 1.0 atm the chamber was flushed with 20% O 2 :75% He:5% CO 2 . In experiments with xenon, krypton, argon, and neon, after flushing with 20% O 2 :75% N 2 :5% CO 2 an additional 0.5 atm of noble gas was added after sealing the chamber, with helium used as control for the effects of pressure. Treatment with noble gases was started 10 min after OGD. For all gas mixtures (except during OGD), the partial pressures of oxygen and carbon dioxide were fixed at 0.2 and 0.05 atm, respectively. During OGD, the partial pressures were 0.95 atm nitrogen and 0.05 atm carbon dioxide. The chamber fan was left on for 5 min to achieve mixing of gases. After 24 h in the chamber, slices were imaged using a fluorescent microscope (see section 'Quantifying cell injury'). The experimental timeline is shown in Fig. 1b.

Quantifying cell injury
PI only enters cells with compromised cellular membranes and becomes fluorescent after binding to nucleic acids, allowing quantification of cell injury. 30e32 The PI assay does not distinguish between different cell types or grey and white matter, as would be possible with histopathology, but PI has the advantage in that real-time quantification of injury can take place in the same slices at different time points (in this case, the viability assessment at t¼0 h before injury and at t¼24 h after OGD or sham procedure). An epifluorescence microscope (Nikon Eclipse 80; Kingston upon Thames, Surrey, UK), with a low-power (2Â) objective was used to quantify PI fluorescence. A digital video camera and software (Micropublisher 3.3 RTV camera and QCapture Pro software; Qimaging Inc, Surrey, BC, Canada) were used to capture the images. Image intensity analysis of the red channel was performed using ImageJ software, 33 with the distribution of intensities plotted as a histogram over 256 intensity levels. Uninjured sham slices under control conditions, incubated in the chamber for 24 h at 37 C with 20% O 2 :75% N 2 :5% CO 2 , showed little PI fluorescence ( Fig. 1c[i]) compared with OGD injured slices ( Fig. 1c[ii]) that exhibited bright PI fluorescence. In order to determine the relative magnitude of the OGD injury we determined maximal cell death by incubating some slices in 70% ethanol overnight at 4 C (Fig. 1c[iii]). To quantify the injury we integrated the number of pixels above a threshold of 100, which provides a robust quantitative measurement of cell injury (Fig. 1d). 24 Absolute pixel values were normalised to the median value of the control OGD slices (Fig. 1e).

Statistical analysis
Data were tested for normality using the ShapiroeWilk test and found to be non-normal. Results are shown as median values with error bars representing 95% confidence intervals. We assessed significance using the KruskaleWallis test with Dunn's correction for multiple comparisons. A P-value of <0.05 was taken to indicate a significant difference between groups. OGD (brown bar), and maximal injury (dark red bar) slices at 24 h after injury or sham procedure. Slices were exposed to control gas (75% N 2 :20% O 2 :5% CO 2 ) with 0.5 atm helium for 24 h after OGD or sham procedure. The intensity histograms are the mean of 10 (sham) and 25 (OGD) slices. Pixel numbers have been normalised to the median of the OGD slices. Bars are median values, error bars are the 95% confidence interval. n¼191, sham; n¼326, OGD, n¼125 max injury. ****P<0.0001, KruskaleWallis test with Dunn's correction for multiple comparisons.

Oxygen-glucose deprivation results in sub-maximal injury
To determine the relative intensity of our OGD injury we compared uninjured sham slices with slices subjected to OGD and slices subjected to maximal injury. Compared with uninjured shams, slices subjected to OGD exhibited a bright PI fluorescence, which was sub-maximal ( Fig. 1c and d). Injury in the OGD slices was greater than sham and less than maximal injury (Fig. 1e).
Helium has no effect on hypoxiceischaemic injury We determined the effect of 1.0 atm helium (75% He:20% O 2 :5% CO 2 ) on sham and OGD slices (Fig. 2). There was no significant difference between sham slices with or without helium. Injury was low in sham slices, with median values 4.9% and 8.3% of the median value of the control OGD in the absence and presence of helium, respectively. After OGD, injury developed significantly (p < 0.0001) at 24 h compared with shams, in both the absence and presence of helium. However, there was no significant difference between OGD slices treated with helium and control OGD slices treated with 75% N 2 :20% O 2 :5% CO 2 .
Xenon and argon prevent hypoxiceischaemic injury, whereas krypton and neon have no effect As helium was without effect, we investigated the effect of 0.5 atm of the noble gases xenon, krypton, argon, and neon on OGD injury (Fig. 3). As these experiments used mild hyperbaric conditions, we used 0.5 atm helium in the control OGD to control for any effects of pressure. Sham slices exhibited very little injury (Fig. 3a[i]) compared with control OGD (Fig. 3a[ii]), whereas treatment with xenon ( Fig. 3a[iii]) or argon (Fig. 3a[iv]) after OGD reduced injury. Xenon and argon were equally effective at reducing OGD injury, both reducing injury significantly (p < 0.0001) by 96% (Fig. 3b). The OGD slices treated with xenon and argon were not significantly different to each other or to the uninjured sham group (Fig. 3b). Thus both of these noble gases can prevent injury development in this in vitro model. We found that krypton and neon were without significant effect on OGD injury (Fig. 3b).
Glycine reverses the protective effect of xenon, but not of argon, against hypoxiceischaemic injury In order to determine the role of the NMDA receptor in the protective effect of xenon and argon, we investigated the effect of glycine on neuroprotection by these noble gases. The addition of 100 mM glycine had no significant effect on control OGD injury with helium (Fig. 4). The protective effect of argon was unaffected by addition of glycine, with a 91% reduction with glycine compared with 96% reduction without glycine. In contrast, addition of glycine completely reversed the Helium at atmospheric pressure has no effect after injury or sham procedure. Slices were exposed to either control gas (75% N 2 :20% O 2 :5% CO 2 ) or helium (75% He:20% O 2 :5% CO 2 ) for 24 h after OGD or sham procedure. Pixel numbers have been normalised to the median of the control OGD slices. Bars are median values, error bars are the 95% confidence interval. n¼31, sham; n¼35, sham helium; n¼54, OGD; n¼46, helium OGD. ****p<0.0001 compared with OGD, KruskaleWallis test with Dunn's correction for multiple comparisons. Ns, not significant; OGD, oxygen-glucose deprivation. sham (white bar), OGD þ 0.5 atm xenon (red bar), OGD þ 0.5 atm argon (blue bar), OGD þ 0.5 atm neon (green bar), OGD þ 0.5 atm krypton (purple bar), OGD control (brown bar). OGD control and sham slices were exposed to 0.5 atm helium. All slices were also exposed to 1.0 atm control gas (75% N 2 :20% O 2 :5% CO 2 ) with total partial pressure 1.5 atm. Pixel numbers have been normalised to the median of the OGD þ 0.5 atm helium slices. Bars are median values, error bars are the 95% confidence interval. n¼191, sham; n¼95, OGD þ xenon; n¼52, OGD þ argon; n¼89, OGD þ neon; n¼108, OGD þ krypton; n¼326, OGD control. ****P<0.0001 compared with OGD, KruskaleWallis test with Dunn's correction for multiple comparisons. Ns, not significant; OGD, oxygenglucose deprivation. protective effect of xenon, with a 96% reduction in injury without and a 0.4% reduction in injury with added glycine. This is consistent with xenon neuroprotection being mediated by the NMDA receptor glycine binding site. These findings indicate that xenon and argon provide neuroprotection against hypoxiceischaemic injury by different mechanisms.

Discussion
Oxygen-glucose deprivation model Organotypic hippocampal slice cultures (OHSCs) were subjected to OGD, with injury quantified by PI fluorescence. This preparation retains a variety of cell types (e.g. different types of neurones and glia) with cellular organisation and synaptic connectivity similar to in vivo, 34,35 and is widely used as an intermediate between dissociated cell cultures and wholeanimal models. 20,24,26,28,36e40 We chose 30 min as the duration of OGD because we previously showed that this produced a reliable and robust injury. 24 OGD results in a diffuse global injury, and the injury produced by 30 min OGD was submaximal. The OHSC model we used has advantages and limitations. An in vitro model allows us to control the slice environment. Organotypic brain slice cultures exposed to OGD are a widely used model of cerebral hypoxiaeischaemia, 41e43 and in vitro OGD causes disruption of cellular function that is similar to hypoxiaeischaemia in vivo. 44e46 We measured cell death and neuroprotection in the hippocampal slice as a whole in order to avoid subjectivity associated with precisely defining the boundaries of CA1, CA3, and dentate gyrus. In humans with ischaemic brain injury, hippocampal subregions exhibit differential sensitivity, with CA1 being particularly vulnerable. 47 In our study, we observed qualitatively that the CA1 region appeared more sensitive to ischaemic injury, in agreement with clinical data and previous in vitro studies, 24,48 and this likely reflects the density of NMDA receptors. Nevertheless, xenon and argon appeared to reduce injury to a similar degree in different hippocampal areas as observed for other neuroprotective drugs. 24,26,42,43,49 Lack of effect of helium There are few studies of helium as a neuroprotectant, and these have produced contradictory results. Helium was found to be neuroprotective in a rat model of ischaemic brain injury; however, this was shown not to be a pharmacological effect but because of hypothermia resulting from breathing helium at room temperature (because of the high thermal conductivity of helium). 5,21,50 In an in vitro OGD model in isolated cell cultures, a detrimental effect of helium was reported. 27 We previously showed that mild hyperbaric helium (0.5 atm partial pressure at 1.5 atm) had no effect on OGD injury in hippocampal slices. 24 In the current study, we investigated the effects of 0.75 atm helium under normobaric conditions (1 atm) and found that normobaric helium had no effect on uninjured sham slices or on OGD slices after 24 h of treatment. Our treatment is given inside a temperature-controlled incubator at 37 C; hence, we can exclude the effect of hypothermia as found in the in vivo studies. In our OHSC model at 37 C, helium is devoid of any observable effect against OGD.
Xenon and argon are equally effective as neuroprotectants, whereas other noble gases are without effect In contrast to helium, both xenon and argon resulted in significant neuroprotection. Both gases prevented injury development; OGD slices treated with 0.5 atm xenon or 0.5 atm argon were not significantly different to uninjured sham slices. Interestingly, we found that xenon and argon were equally effective in the OGD model, in contrast to an in vitro model of traumatic brain injury where argon was less effective than xenon. 26 The reasons why argon appears to be as effective as xenon in this ischaemic injury model are not clear, but this suggests that different secondary injury mechanisms may be involved in ischaemic and traumatic brain injury. The comparable efficacy of xenon and argon we observe in vitro does not necessarily mean that similar long-term functional improvements will be observed for these gases in vivo. The relative efficacy of xenon and argon on clinically relevant longterm functional outcomes after hypoxiaeischaemia in vivo remains to be determined. Krypton and neon were without any protective effect, consistent with findings on isolated cell cultures subjected to OGD and OHSCs that had experienced a traumatic insult. 26,27 Neuroprotection by xenon, but not argon, involves NMDA receptor inhibition at the glycine site Xenon inhibits the NMDA receptor by competing for the binding of the co-agonist glycine, and xenon inhibition can be OGD control and sham slices were exposed to 0.5 atm helium. All slices were also exposed to 1.0 atm control gas (75% N 2 :20% O 2 :5% CO 2 ) with total partial pressure 1.5 atm. Pixel numbers have been normalised to the median of the OGD control slices. Bars are median values, error bars are the 95% confidence interval. n¼191, sham; n¼95, OGD þ xenon; n¼91, OGD þ xenon þ glycine; n¼52, OGD þ argon; n¼48, OGD þ argon þ glycine; n¼326, OGD control; n¼69, OGD control þ glycine. ****P<0.0001 compared with OGD, KruskaleWallis test with Dunn's correction for multiple comparisons. Ns, not significant; OGD, oxygenglucose deprivation; gly, glycine.
prevented by elevating glycine concentrations. 51,52 In the current study we found that addition of glycine had no effect on the control OGD injury. The simplest explanation for this observation is that the concentration of endogenous glycine is just below saturating on the concentrationeeffect curve. However, the neuroprotective effect of xenon was completely reversed by the addition of glycine, consistent with inhibition of the NMDA receptor glycine site mediating xenon's protective effect. In contrast, addition of glycine had no effect on neuroprotection by argon, indicating that argon acts via a different mechanism. The reversal of neuroprotection by xenon but not argon with added glycine is consistent with what we observed in an in vitro model of traumatic brain injury. 26 Xenon acts at other targets, such as the two poredomain potassium channel TREK-1 and the ATP-sensitive potassium (K-ATP) channel, but has no effect on N-type calcium channels 53e55 ; however, our findings indicate that inhibition of the NMDA receptor glycine site is likely to play a major role in the neuroprotective effect of xenon. 24,26,51,52,56,57 The targets mediating the neuroprotective effect of argon are less clear; we have shown that argon does not inhibit NMDA receptors or activate TREK-1 channels. 26 Nevertheless, other in vitro and in vivo studies with argon have identified activation of signalling pathways involving MEK-ERK 1/2 and PI3K/AKT, with up-regulation of heme-oxygenase-1. 17,58,59 A recent study has also identified Nrf2 and the mammalian target of rapamycin (mTOR) signalling pathway as targets for argon. 60 Although these studies clearly identify changes in these signalling pathways after argon treatment, it is not clear whether argon is acting on the upstream targets of these pathways.

Clinical relevance of our findings
There is currently much interest in the clinical use of noble gases as neuroprotectants in ischaemic brain injuries. Xenon is licenced for use as a general anaesthetic and has already undergone clinical trials in neonatal HIE, ischaemic brain damage after cardiac arrest, coronary artery bypass graft surgery, and orthopaedic surgery in the elderly. 10,11,13,14,61e63 The most recent study in ischaemic brain injury in cardiac arrest patients found reduced early white-matter damage in the xenon-treated group, 14 and a larger multi-centre study is underway. Relative to the other inert gases, xenon is more expensive and is used in closed or semi-closed circuits to conserve gas. There has been interest in evaluating other less expensive noble gases as neuroprotectants that could be given in open circuits. Helium and oxygen mixtures have been used medically for respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD) in adults and bronchiolitis and croup in children, but systematic reviews conclude that the currently available evidence does not support its use in these conditions. 64e67 Our finding that helium has no effect on hypoxiceischaemic injury in vitro is consistent with a lack of pharmacological effect of helium at normobaric pressures. The finding that helium has neuroprotective properties in animal models of hypoxiaeischaemia via the physical mechanism of inducing cooling 5,21 is an interesting observation, but more straightforward and controllable techniques for therapeutic cooling are available. Our finding that argon is neuroprotective agrees with a large body of in vitro and in vivo evidence. 7,18,68 Interestingly, in this OGD model we found 0.5 atm argon to be as effective as 0.5 atm xenon, in contrast to our earlier work with an in vitro model of traumatic brain injury where argon was less effective than xenon. Argon has been shown not to affect cerebral circulation in humans, 69 and there are proposals to evaluate argon as a neuroprotectant in patients. 6 One obstacle, perhaps, to its clinical use is an unambiguous identification of its mode of action but, on the other hand, it is the most abundant of the noble gases and the cheapest to produce. On a positive note, the fact that argon and xenon do not act by the same mechanism means that combinations of these gases may have a synergistic effect, or that argon and xenon are effective treatments for different forms of ischaemic neurological injury. The observation that in a mouse model of traumatic brain injury, xenon is able to prevent development of very late-onset traumatic brain injury-related memory deficits with reduced white matter loss in the corpus callosum is of great interest, 9 but it remains to be seen if this will translate into similar findings in humans.
In conclusion our findings that argon and xenon are equally effective neuroprotective in hypoxiceischaemic injury but act via different mechanisms will prompt further translational studies to evaluate these two noble gases as neuroprotectants, either singly or in combinations.