Argon inhalation for 24 hours after onset of permanent focal cerebral ischemia in rats provides neuroprotection and improves neurologic outcome

Abstract

Objectives:

We tested the hypothesis that prolonged inhalation of 70% argon for 24 hours after in-vivo permanent or temporary stroke provides neuroprotection, and improves neurologic outcome and overall recovery after 7 days.

Design:

Controlled, randomized, double-blinded laboratory study

Setting:

Animal research laboratories

Subjects:

Adult Wistar male rats (n=110)

Interventions:

Rats were subjected to permanent or temporary focal cerebral ischemia via middle cerebral artery occlusion, followed by inhalation of 70% argon or nitrogen in 30% oxygen for 24 hours. On postoperative day 7, a 48-point neuroscore and histologic lesion size were assessed.

Measurements and Main Results:

After argon inhalation for 24 hours immediately following severe permanent ischemia induction, neurologic outcome (neuroscore, p=0.034), overall recovery (body weight, p=0.02), and infarct volume (total infarct volume p=0.0001, cortical infarct volume p=0.0003, subcortical infarct volume p=0.0001) were significantly improved. When 24-hour argon treatment was delayed for 2 hours after permanent stroke induction, or until after post-ischemic reperfusion treatment, neurologic outcomes remained significantly improved (neuroscore, p=0.043 and p=0.014, respectively), as was overall recovery (body weight, p=0.015), compared to nitrogen treatment. However, infarct volume and 7-day mortality were not significantly reduced when argon treatment was delayed.

Conclusions:

Neurologic outcome (neuroscore), overall recovery (body weight), and infarct volumes were significantly improved after 24-hour inhalation of 70% argon administered immediately after severe permanent stroke induction. Neurologic outcome and overall recovery were also significantly improved even when argon treatment was delayed for 2 hours or until after reperfusion.

Introduction

Cerebral ischemic stroke remains one of the most common causes of death and disability worldwide. Despite extensive efforts to develop new treatment strategies, timely reperfusion remains the only effective intervention for ischemic stroke. Although reperfusion within a certain time window can reduce infarct size and improve clinical outcome, “cerebral reperfusion injury” [1] can occur, exacerbating brain injury. Unfortunately, only about 10% of stroke patients are eligible for reperfusion attempts, but not all are successfully reperfused. Thus, there is a critical need for additional treatment options for this large population of patients with severe permanent ischemic stroke.

Recently, the noble gases xenon and argon have gained substantial attention due to their neuroprotective potential. Argon, the third most abundant element in our atmosphere, is inexpensive and, if neuroprotective, could improve stroke outcomes. Further, unlike xenon, argon has no sedative properties and hence, is less likely to confound neurologic status. Finally, ease of application (via face mask) and lack of obvious toxicity would extend early argon treatment from arrival of first responders through the acute stroke phase, ie, 24-72 hours after ischemia onset. Since the intensive care unit or hospital setting would allow controlled long-term administration, clinical translation seems feasible if preclinical evidence for protection against ischemic injury is sufficient.

Although regarded as chemically inert, argon exerts biologic effects, but specific mechanisms of action are not fully understood. In-vitro and in-vivo studies in several models of hypoxic/ischemic insults [2–5], as well as studies in organ systems [6–8], suggest neuroprotective potential for argon in the ischemic brain. Two argon studies using in-vivo models of cerebral ischemia via middle cerebral artery occlusion (MCAO) showed inconsistent results [9, 10]. Duration of treatment and observation windows were short in both, and neither study examined outcome parameters after permanent ischemia. Here, we analyzed the extent to which argon affords neuroprotection and improves functional outcome after administration for 24 hours post onset of permanent stroke.

Materials and methods

Middle Cerebral Artery Occlusion

This study was approved by the Duke University Animal Care and Use Committee. Male Wistar rats weighing 250-300 g (10-12 weeks old; n=110) were housed in adequately spaced cages with a 12-hour light/dark cycle, with light from 8 am to 8 pm. Animals had free access to water but were fasted for 12 hours before surgery to standardize the glycemic state. Rats were anesthetized with 5% isoflurane in 30% O₂/70% N₂, endotracheally intubated, and mechanically ventilated with 1.5% isoflurane in 30% O₂/70% N₂. Pericranial temperature was maintained at 37.5°C±0.2°C by surface heating or cooling during anesthesia. Rats were prepared for middle cerebral artery occlusion (MCAO) as previously described [11, 12] with modification. A midline ventral cervical skin incision was made, and the right common carotid artery was identified. The external carotid artery was then isolated, ligated, and divided, and the internal carotid artery was dissected distally until the origin of the pterygopalatine artery was visualized.

To achieve different stroke severities, we used commercially available nylon monofilaments (Doccol Corporation, Sharon, MA, USA) with 0.38-mm diameter silicon tips (for severe stroke w/wo reperfusion) or 0.27-mm diameter tips (for moderate permanent stroke). To achieve MCAO, we inserted the coated tip into the external carotid artery stump and advanced it 19-20 mm from the carotid artery bifurcation into the internal carotid artery. For temporary stroke, the filament was removed after 90 minutes MCAO (tMCAO) to allow reperfusion. For permanent ischemia (pMCAO), the filament was not removed. Wounds were closed, isoflurane was discontinued, and rats were extubated.

Argon Exposure System

The argon exposure system included a 5-liter acrylic animal chamber, injection ports for introducing fresh 30% oxygen and either 70% argon or 70% nitrogen into the circulating gas-flow, and oxygen, argon, and nitrogen mass-flow controllers (Model GFC17; Aalborg Inc, Orangeburg, NY, USA). These components were connected in series in a closed loop. Oxygen and carbon dioxide concentrations were monitored with in-chamber sensors for the 24 hour treatment period (Model 5120k; Datex Ohmeda, Louisville, CO, USA) and a pressure release vent prevented over-pressurization in the chamber. The mass-flow controllers maintained total gas flow continuously at a rate of approximately 1 L/min in the closed exposure system. Ultra-pure quality gases were purchased from Praxair, USA. Finally, the temperature in the box was monitored and maintained at ambient room temperature (Table 1).

Table 1.

Body temperature of rats before stroke and after post-stroke 24-hour gas treatment

Temperature	Nitrogen	Argon
in vivo (rectal)
Pre-ischemia	37.2 ± 0.03	37.2 ± 0.02
Post 24-hour gas treatment	38.2 ± 0.3	38.4 ± 0.2
ambient box
In-box temperature	22.5 ± 0.3	22.5 ± 0.3
In-box O2 concentration	30% ± 1%	30% ± 1%

Values are expressed as mean ± SD. Body temperatures are given in °C. Body temperatures were significantly increased after stroke and 24-hour treatment with argon or nitrogen in 30% oxygen. The slightly higher temperature in argon animals did not reach statistical significance (p = 0.07; n = 16/group). Ambient box temperature equaled room temperature, and oxygen concentration was stable at 30% throughout. Airflow in the box was 1 L/min, and was continuously supplied and controlled by mass-flow controllers.

Neurologic Assessment

Immediately after anesthesia emergence from stroke-induction surgery, a screening neurologic assessment was performed using a 4-point scoring system (Bederson score) [13]. This assessment confirmed successful stroke and includes forelimb flexion, resistance to lateral push, and circling behavior. The scoring scale (0-3) reflects basic neurologic deficits. On postoperative day (POD) 7, rats underwent neurologic examination to evaluate sensorimotor function. This previously described neurologic scoring system [14] incorporates the major elements of the Bederson and Garcia scoring systems [15], and evaluates general status, simple motor deficit, complex motor deficit, and sensory deficit. Score totals range from 0 (no deficit) to 48 (maximal deficit). Values from this scoring system correlate with total infarct volume in rat [14, 16, 17]. All observers were blinded to group assignment and study design, and were trained and certified in performing these assessments.

Histologic Assessment

After final neurologic scoring on POD 7, rats were anesthetized with 5% isoflurane and decapitated, and brains were harvested for hematoxylin and eosin staining and consecutive lesion size histology [18, 19]. Details of this protocol are provided in the supplemental material (supplemental digital content 1).

Statistical Analysis

After confirming normal distribution using the D’Agostino Pearson omnibus normality test, neurobehavioral and histologic data were analyzed using the t-test. P < 0.05 was considered statistically significant. Values are reported as mean ± SD. All statistical analyses were performed with GraphPad Prism software.

Experimental Design

Experiment 1:

Severe stroke via permanent ischemia (pMCAO) without reperfusion; 24-hour gas treatment started immediately after stroke-induction surgery.

Rats were subjected to pMCAO. After awakening from anesthesia, they were quickly evaluated using the Bederson score to confirm successful stroke, ie, presence of neurologic deficit, and then randomized to immediately begin 24-hour exposure to either 70% argon/30% oxygen (Group A) or 70% nitrogen/30% oxygen (Group B).

Experiment 2:

Moderate stroke via permanent ischemia (pMCAO) without reperfusion; 24-hour gas treatment started 2 hours after surgery.

Rats were subjected to pMCAO. After awakening from anesthesia, they were quickly evaluated using the Bederson score to confirm successful stroke, and randomized to begin 24-hour exposure to 70% argon/30% oxygen (Group A) or 70% nitrogen/30% oxygen (Group B) 2 hours after stroke surgery.

Experiment 3:

Temporary focal ischemia (tMCAO) with reperfusion at 90 minutes; 24-hour gas treatment started immediately after reperfusion.

Rats were subjected to 90 minutes MCAO followed by reperfusion. After awakening from anesthesia, they were quickly evaluated using the Bederson score to confirm successful stroke, and randomized to begin 24-hour exposure to 70% argon/30% oxygen (Group A) or 70% nitrogen/30% oxygen (Group B) immediately after reperfusion.

Food and water were provided in the treatment chamber. After gas exposure, rats were returned to their home cages for 7 days, followed by outcome assessment.

Results

Mortality and Physiologic Values

A total of 110 rats were entered into the experimental study protocol. Three died before group assignment, and autopsy revealed intracranial hemorrhage. Thus, 107 animals were included in the study: 35 in Experiment 1, 35 in Experiment 2, and 37 in Experiment 3. The mortality rate was similar in all experiments though less in the reperfusion experiment. Death occurred in all cases within 48 hours, and all autopsies revealed malignant strokes and herniation (more in the permanent stroke groups). We found a significant increase in body temperature after 24 hours of treatment in both groups (Table 1). We chose 30% as the supplemental oxygen concentration in order to: 1) achieve a maximal argon concentration, 2) avoid potential oxygen toxicity over the 24-hour treatment period, and 3) simulate a common clinical practice of administering oxygen via nasal cannula to an inspired oxygen fraction of about 30%.

Experiment 1:

Severe stroke, permanent ischemia (pMCAO) without reperfusion; 24-hour gas treatment started immediately after stroke-induction surgery (Fig. 1).

Figure 1:

(A) 48-point neuroscore on day 7 after severe permanent stroke induction, (pMCAO) [argon vs nitrogen neuroscore 10 ± 3.5 vs 14 ± 5; n=12/group; p=0.034] (B) edema-corrected infarct sizes after permanent stroke, 24-hour gas treatment, and 7 days recovery [argon vs nitrogen total infarct volume: 161 ± 75 mm³ vs 302 ± 61 mm³; p=0.0001; cortical infarct volume: 104 ± 60.5 mm³ vs 194 ± 41 mm³; p=0.0003; subcortical infarct volume: 58 ± 21 mm³ vs 109 ± 29 mm³, p=0.0001] (C) overall recovery based on weight at baseline before stroke and after 24-hour gas treatment and 7 days recovery [argon vs nitrogen weight: 249 ± 48 g vs 206 ± 38 g; p=0.02].

On POD 7 after induction of severe permanent ischemia, neuroscore was significantly improved after 24-hour exposure to argon vs nitrogen (p=0.034; Fig. 1A). Cortical and subcortical infarct volumes were substantially reduced in the argon vs nitrogen group (total infarct volume: p=0.0001; cortical infarct volume: p=0.0003; subcortical infarct volume: p=0.0001; Fig. 1B). Overall recovery, based on weight, was also significantly improved in the argon group (Fig. 1C). Mortality was similar between groups: 6/18 animals died in the argon group vs 5/17 in the nitrogen group. In both groups, animals died 24-48 hours after stroke induction.

Experiment 2:

Moderate stroke via permanent ischemia (pMCAO) without reperfusion; 24-hour gas treatment started 2 hours after stroke-induction surgery (Fig. 2).

Figure 2:

(A) 48-point neuroscore on day 7 after moderate, permanent stroke induction (pMCAO) [argon vs nitrogen neuroscore 9 ± 4 vs 14 ± 6; n=11/group; p=0.043] (B) edema-corrected infarct sizes after permanent stroke, delayed 24-hour gas treatment, and 7 days recovery; (C) overall recovery based on weight at baseline before stroke and after delayed 24-hour gas treatment and 7 days recovery.

This experiment simulates a common clinical delay in first response and treatment of stroke. In this scenario, neurologic outcome was again significantly improved after argon exposure (p=0.043; Fig. 2A). However, infarct size was not reduced nor weight recovery improved in the argon group (Fig. 2B, 2C), indicating some loss of neuroprotection due to the 2-hour delay in treatment. In the argon group, 5/16 rats died vs 8/19 in the nitrogen group; however, this difference did not reach statistical significance.

Experiment 3:

Temporary focal ischemia (tMCAO) with reperfusion; 24-hour gas treatment started immediately after reperfusion (Fig. 3).

Figure 3:

(A) 48-point neuroscore on day 7 after temporary stroke induction (tMCAO) with reperfusion [argon vs nitrogen neuroscore: 14 ± 5 vs 18 ± 2; n=16/group; p=0.014] (B) edema-corrected infarct sizes after temporary stroke with reperfusion, 24-hour gas treatment, and 7 days recovery; (C) overall recovery based on weight at baseline before stroke and after temporary stroke with reperfusion, 24-hour gas treatment, and 7 days recovery [argon vs nitrogen weight: 247 ± 58 g vs 203 ± 37 g; p=0.015].

Based on our findings in Experiments 1 and 2, we hypothesized that reperfusion at 90 minutes potentiates neuroprotective effects of 24-hour exposure to argon after ischemic stroke. In Experiment 3, neurologic outcome and weight recovery were significantly improved after 24-hour treatment with argon vs nitrogen post reperfusion (p=0.014; Fig. 3A and p=0.015; Fig. 3C, respectively). Infarct size, however, was not significantly improved after argon exposure in this scenario (Fig. 3B). Again, there was no difference in 7-day mortality between groups: 3/19 deaths in the argon group vs 2/18 in the nitrogen group.

4. Discussion

This is the first in-vivo study, to our knowledge, that demonstrated argon’s neuroprotective potential after permanent, non-reperfused cerebral ischemia in rats. Rats exposed to 24 hours inhalation of 70% argon/30% oxygen vs 70% nitrogen/30% oxygen demonstrated significantly improved neurologic outcome in both of our permanent, non-reperfused stroke experiments, but most importantly, in our opinion, in our severe permanent cerebral ischemia model. When argon administration was delayed 2 hours after permanent stroke onset or instituted after reperfusion, neurologic outcome was still improved but infarct size was not reduced.

These results are in contrast to the few reported studies on argon and stroke, which administered argon for shorter periods (~1 hour) and at lower concentration (~50%) in reperfusion models with much shorter observation times (24- and 48-hour outcome) [9, 10]. For example, David et al [9] showed no difference in neurologic outcome between room air and argon after ischemia with reperfusion. Likewise Ryang et al [10] showed decreased infarct size but no change in neurologic score after administering argon at 50% for the second hour of ischemia. Our reperfusion studies (Experiment 3) show improved neurologic outcome at 7 days after 24-hour treatment with 70% argon, but minimal improvement in infarct size, and no deleterious effect at either cortical or subcortical levels (Fig.3). The effect of reperfusion after stroke on reducing argon’s neuroprotective properties is not clear. Reperfusion itself can cause neuronal injury due to hyperperfusion and hemorrhagic transformation [20]. Further, despite successful recanalization with thrombolysis or thrombectomy, incomplete reperfusion may still occur within the microvasculature [21]. In the end, reperfusion injury may simply override the protective effects of argon.

Based on argon’s apparent protective effect in permanent stroke, prolonged argon treatment may affect microcirculation and collateral blood flow, and may contribute to keeping microvascular beds open, especially in the penumbra of the infarct. David et al showed recently that argon has a concentration-dependent synergy with the catalytic and thrombolytic efficiency of tPA in vitro [22]. Indeed, at concentrations higher than 50 vol%, argon increases the catalytic and thrombolytic efficiency of endogenous tPA, which could contribute to restoring or maintaining perfusion, particularly in the microvasculature, based on its effects on formation/degradation of micro-clots in in-vivo stroke. Paradoxically, improved microcirculatory function with argon may not necessarily lead to reduced infarct size after delayed argon treatment, despite improved neurologic function, if argon is mainly improving microvascular function. Argon, therefore, should be administered acutely before the micro-clot load has increased.

Argon could also influence the occurrence of spreading depolarizations (SDs), a major contributor to expansion of ischemia due to exacerbated tissue energy demand. Recent data suggest that neuronal hemichannel pannexin 1 forms a complex with P2X7 that facilitates SDs [23] and consecutive spreading ischemia and cytotoxic edema [24]. Though speculative at this point, it is intriguing to hypothesize, for future mechanistic studies, that a high concentration of an inert gas in the neuronal membrane changes membrane fluidity and interferes with such pore formation. Indeed, if argon can inhibit SDs and in this way exert its neuroprotective effect, a 2-hour delay in treatment would allow SDs, which cluster in the first hours after stoke onset [25], to take their toll, and could in part explain the partial loss of protection that we observed in Experiment 2.

The discrepancy between improved neurologic outcome and the unaltered overall lesion size assessed in this experiment must be further studied. The value of histologic lesion size as a clinically relevant outcome predictor remains controversial, as correlation is at best modest. The severity of neurologic impairment also depends on the actual location of the lesion, whereas size may be less important [26]. We did not observe a significant effect of argon on subcortical damage, but it would be interesting to know whether white matter is more protected and whether such protection could explain the improved neurologic outcome despite overall unaltered histology.

As David et al reported [9], we also noted slightly elevated body temperature in our animals at 24 hours after stroke induction. However, this observation was similar in both argon- and nitrogen-treated groups. Slight hyperthermia is a well-known side effect of stroke in humans [27, 28] that seems to promote clot lysis and support the effect of tPA after reperfusion therapy [29, 30]. Since the chamber air for both groups was continuously refreshed, monitored, and maintained at ambient room temperature (Table 1), we conclude that the higher body temperature in both treatment groups in our study is attributable directly to the stroke. It is not yet clear whether slight hyperthermia promotes or potentially diminishes argon’s protective effect. However, Broad et al [31] reported that in a piglet model of perinatal hypoxia, argon combined with hypothermia boosts brain protection compared to hypothermia alone, and that adding argon to cooling increases whole-brain ATP and phosphocreatine/inorganic phosphate levels at 48 hours, and reduces the secondary rise in white, but not gray, matter lactate levels at 24 and 48 hours. Such preservation of energy substrates would support the hypothesis that argon enhances mitochondrial function at low oxygen tensions [32].

Other molecular changes that may underlie neuroprotective properties of argon in vivo are 1) enhanced HIF1 alpha downstream effects [2, 36], 2) interaction with Toll-like receptors 2 and 4 [33], and 3) activation of the ERK1/2 pathway [34], engaging downstream effects of heme oxygenase-1 [35], which can initiate neuroprotection.

The current study was designed to demonstrate argon’s neuroprotective effects on clinically relevant outcome parameters after permanent ischemia. Our results in in-vivo stroke are consistent with the protective effects of argon in several other disease models including cardiac arrest [4, 5], global ischemia [2], and organ transplantation [8]. However, while some have advocated for clinical studies [36] due to the ease of argon administration and generally low assumed toxicity [37], we must interject a word of caution. Although argon holds promise as a valuable tool for protecting the brain from cerebral ischemia, or at least extending the current treatment windows, important parameters such as treatment duration, time of application, accompanying oxygen concentration, effects of sex and age, and potential temperature effects must be systematically studied and defined before we can justify and appropriately design clinical trials.

Supplementary Material

Supplemental Data File (.doc, .tif, pdf, etc.)

Supplemental Digital Content Legend 1:

Histologic Assessment Methods Description

A detailed method description of histologic stroke lesion size assessment and data analysis is provided.