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Xenon Improves Neurologic Outcome and Reduces Secondary Injury Following Trauma in an In Vivo Model of Traumatic Brain Injury*

Campos-Pires, Rita MD1; Armstrong, Scott P. PhD1; Sebastiani, Anne MD2; Luh, Clara PhD2; Gruss, Marco MD3; Radyushkin, Konstantin MD4; Hirnet, Tobias2; Werner, Christian MD, PhD2; Engelhard, Kristin MD, PhD2; Franks, Nicholas P. PhD5; Thal, Serge C. MD2; Dickinson, Robert PhD1

doi: 10.1097/CCM.0000000000000624
Neurologic Critical Care

Objectives: To determine the neuroprotective efficacy of the inert gas xenon following traumatic brain injury and to determine whether application of xenon has a clinically relevant therapeutic time window.

Design: Controlled animal study.

Setting: University research laboratory.

Subjects: Male C57BL/6N mice (n = 196).

Interventions: Seventy-five percent xenon, 50% xenon, or 30% xenon, with 25% oxygen (balance nitrogen) treatment following mechanical brain lesion by controlled cortical impact.

Measurements and Main Results: Outcome following trauma was measured using 1) functional neurologic outcome score, 2) histological measurement of contusion volume, and 3) analysis of locomotor function and gait. Our study shows that xenon treatment improves outcome following traumatic brain injury. Neurologic outcome scores were significantly (p < 0.05) better in xenon-treated groups in the early phase (24 hr) and up to 4 days after injury. Contusion volume was significantly (p < 0.05) reduced in the xenon-treated groups. Xenon treatment significantly (p < 0.05) reduced contusion volume when xenon was given 15 minutes after injury or when treatment was delayed 1 or 3 hours after injury. Neurologic outcome was significantly (p < 0.05) improved when xenon treatment was given 15 minutes or 1 hour after injury. Improvements in locomotor function (p < 0.05) were observed in the xenon-treated group, 1 month after trauma.

Conclusions: These results show for the first time that xenon improves neurologic outcome and reduces contusion volume following traumatic brain injury in mice. In this model, xenon application has a therapeutic time window of up to at least 3 hours. These findings support the idea that xenon may be of benefit as a neuroprotective treatment in patients with brain trauma.

1Anaesthetics, Pain Medicine and Intensive Care Section, Department of Surgery and Cancer, Imperial College London, London, United Kingdom.

2Department of Anaesthesiology, Medical Center of Johannes Gutenberg University, Mainz, Germany.

3Department of Anaesthesiology, Klinikum Hanau, Hanau, Germany.

4Mouse Behavioral Outcome Unit, Focus Program Translational Neurosciences, Johannes Gutenberg University, Mainz, Germany.

5Department of Life Sciences, Imperial College London, London, United Kingdom.

* See also p. 250.

Drs. Campos-Pires, Armstrong, and Sebastiani contributed equally.

Supported by European Society for Anaesthesiology, Brussels, Belgium, the Association of Anaesthetists of Great Britain & Ireland, London, United Kingdom, The Royal College of Anaesthetists, London, United Kingdom, the Royal Centre for Defence Medicine, Birmingham, United Kingdom, the Medical Research Council, London and Deutscher Akademischer Austauschdienst, German Academic Exchange Service, Bonn, Germany.

Dr. Campos-Pires is the recipient of a doctoral training award from the Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. Dr. Armstrong was the recipient of a studentship from the Medical Research Council, London, United Kingdom. Mr. Hirnet is funded by the German Federal Ministry of Education and Research, Berlin, Germany (grant BMBF 01EO1003). Dr. Gruss received grant support from the University Giessen, Germany (support in 2005 used for some preliminary experiments). Dr. Radyushkin has disclosed that the mouse behavior experiments were conducted on the basis of scientific service within a collaboration between Drs. Dickinson and Radyushkin. Dr. Dickinson covered all costs regarding mouse behavior experiments. Dr. Engelhard served as board member for Fresenius Kabi and lectured for Abbvie. Dr. Franks has disclosed being a named inventor on a number of patents relating to the use of xenon as a neuroprotectant. (There are no current plans to commercialize these patents, although there may be in the future. If there are, then he may have a financial interest in their exploitation.) The remaining authors have disclosed that they do not have any potential conflicts of interest.

For information regarding this article, E-mail: r.dickinson@imperial.ac.uk

Traumatic brain injury (TBI) affects both young and elderly populations throughout the world and results in a significant global healthcare burden. In developed countries, TBI is the main cause of death and disability in those who are younger than 45 years (1), with falls and motor-vehicle crashes being the leading causes (2, 3). In the United States, between the years 2002 and 2006, on average 1.7 million people per year suffered a TBI (4). Of these, 1.4 million were treated in hospital emergency departments, 275,000 required hospitalization and survived, but 52,000 died (4). TBI causes 30% of all injury-related deaths in the United States (4). Current clinical practice for patients with TBI is largely supportive and centered on nonspecific endpoints, such as management of tissue oxygenation, cerebral perfusion pressure, and intracranial pressure (5–8). At present, there are no specific pharmacological neuroprotective treatments for TBI (9–12). Given the high economic costs associated with TBI, there is a need for neuroprotective treatments that can minimize or attenuate the brain damage following TBI and promote a faster and more complete recovery. The current study evaluates the neuroprotective efficacy of xenon in the rodent controlled cortical impact (CCI) brain trauma model.

Blunt-trauma TBI is characterized by a “primary injury” determined by the initial mechanical force, followed by a “secondary injury” which begins soon after the initial trauma and continues to develop in the following hours and days. The biological processes involved in the development of secondary injury are complex and involve multiple injury cascades (13, 14); however, glutamate excitotoxicity, involving overactivation of N-methyl-D-aspartate (NMDA) receptors, is thought to play a key role (15–17).

The noble gas xenon has been used as a general anesthetic since the 1950s (18, 19), but its molecular targets were unknown (20). Following the discovery that xenon is an NMDA receptor antagonist (21–24), xenon was shown to be neuroprotective in a number of in vitro and in vivo models of ischemic injury (25–34), and xenon is currently undergoing clinical trials as a treatment for ischemic brain injury (35, 36) and postoperative delirium (37). Information on xenon neuroprotection against TBI is much more limited. In the present study, we use the rodent CCI brain trauma model to test the following hypotheses: that xenon protects against secondary injury development, that xenon improves neurologic outcome after trauma, and that xenon has a clinically relevant therapeutic time window.

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MATERIALS AND METHODS

Adult male C57BL/6N mice aged 2.5 months, mean weight 24 ± 3 g (SEM, n = 196), were obtained from Charles River Laboratory (Sulzfeld, Germany). Animals were cared for in compliance with the institutional guidelines of the Johannes Gutenberg University (Mainz). All experiments were approved by the Animal Ethics Committee of the Landestuntersuchungsamt Rheinland-Pfalz (protocol number: G12-1–010).

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TBI

Animals were anesthetized with 3.5% sevoflurane in an air/oxygen mixture (40% O2/60% N2) supplied via a facemask in spontaneously breathing animals. Core temperature was monitored and maintained at 37°C for the duration of the surgery by means of a rectal probe and feedback-controlled heating pad (Hugo Sachs, March-Hugstetten, Germany). Traumatic injury was performed using the CCI model, as described previously (38), by an experimenter blinded to the treatment groups. Animals were fixed in a stereotactic frame (Kopf Instruments, Tujunga, CA), and a 4 × 5 mm craniotomy window was created using a saline-cooled high-speed drill, along the coronal (anterior) and lambdoid (posterior) sutures and laterally as close as possible to the temporalis muscle insertion. The bone flap was lifted exposing the dura above the right parietal cortex, between the sagittal, lambdoid, and coronal sutures. The tip of a custom-built CCI device (L. Kopacz, Mainz, Germany) was positioned above the intact dura in the center of the craniotomy window (1 mm from sagittal suture and 1 mm from lambdoid suture). The angle of the impactor, typically 25 degrees from the sagittal plane, was adjusted such that the tip was perpendicular to the dural surface. An impactor tip of diameter 3 mm, impact velocity of 8 m/s, and impact duration of 150 ms were used. In the xenon pre- and postexposure experiments shown in Figure 1 (n = 12 animals), a penetration depth of 1.5 mm was used, and in all other experiments (n = 184 animals), the penetration depth was 1.0 mm. Our pneumatically controlled CCI device is similar in design to that used by Smith et al (39) and Fox et al (40) who developed the CCI model in mice, and our impact variables are similar to those found by those workers to result in moderate injury. Following CCI injury, the craniotomy was closed with the bone flap, fixed with tissue glue (Histoacryl; Braun-Melsungen, Melsungen, Germany), and the skin sutured. Animals were returned to their individual home cages in a heated incubator (33°C, 35% humidity; IC8000; Draeger, Luebeck, Germany) and allowed to recover for 15 minutes before treatment; during this time, they were breathing room air.

Figure 1

Figure 1

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Experimental Groups, Randomizing and Blinding

Animals were randomly assigned to receive xenon (75%, 50%, or 30%) with 25% oxygen (balance nitrogen) or 75% nitrogen:25% oxygen (control gas). The experimenter performing the CCI surgery was blinded to the treatment. A separate experimenter, also blinded to treatment, performed the behavioral tests. Treatment was begun 15 minutes, 1 hour, 3 hours, or 6 hours after injury. Animals were allowed to survive 24 hours, 5 days, or 1 month. In the short-term (24 hr) survival experiments, we aimed to have 9–10 animals in each xenon-treatment group. In some experiments (e.g., time delay experiments), it was not possible to perform all experiments on the same day. In these cases, we had additional control groups that were pooled resulting in larger numbers of mice in the control groups. In the 5-day survival experiment, we had 12 animals in each group; at 24 hours neuroscore, data from 24-hour survival and 5-day survival experiments were pooled. Three animals died shortly after CCI injury and postmortem showed evidence of intracerebral hemorrhage. One animal in the 5-day survival group became unresponsive and immobile on day 4 and was euthanized, postmortem indicated intracerebral hemorrhage. Due to technical issues with the impact device (movement of the impactor during CCI, affecting impact variables), 4 animals were excluded from the dose-response analysis (one in control group, one in 30% xenon group, and two in 50% xenon group). In our long-term (1 mo) survival experiments, we had 20 animals each in the xenon and control treatment groups and 10 animals in the sham-surgery group, all animals survived and performed RotaRod test, and three animals (one in each group) failed to perform the CatWalk-XT test.

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Xenon or Control Gas Administration

Gas treatments were administered to spontaneously breathing animals in a series of custom made xenon exposure chambers linked in a closed circuit, for a total duration of 3 hours. Gas concentrations inside the circuit were monitored continuously, via a xenon meter (model 439 EX; Nyquist, Macclesfield, UK) and an oxygen meter (Oxydig; Draeger) included in the circuit. Carbon dioxide was removed from the system by soda lime pellets. Additional volumes of gases were added into the system as necessary to maintain their respective concentrations. Gases were circulated at 700 mL/min by a small animal ventilator (Inspira ASV; Harvard Apparatus, Holliston, MA). Chambers were housed inside an incubator heated to 33°C (Model IC8000; Draeger). In the therapeutic time window experiments, the start of the 3 hours of xenon-treatment was delayed 15 minutes, 1 hour, 3 hours, and 6 hours after CCI injury.

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Physiologic Measurements

Systolic and diastolic blood pressure and heart rate were measured using a Kent Coda noninvasive blood pressure monitor (EMKA Technologies, Paris, France). Temperature measurements were made using an infra-red thermometer (Thermoworks model TW2; Thermoworks, Linton, UT).

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Histologic Evaluation

At the end of the observation period (24 hr), and immediately following the final neuroscore, animals were anesthetized with sevoflurane and euthanized by cervical dislocation. The brain was carefully removed, frozen on powdered dry ice, and stored at −80°C. Frozen brains were embedded in Optimal Cutting Temperature mounting media (Cell Path, Newton, Powys, UK) and cut in the coronal plane with a cryostat tissue slicer (Leica CM3050; Leica Microsystems, Milton Keynes, UK). For each brain, a total of 16–18 sections (10-μm thickness) were collected on Superfrost Plus microscope slides (Thermo Fisher Scientific, Loughborough, UK) every 500 μm. For the quantification of contusion volume, slices were stained with cresyl violet (Thermo Fisher Scientific). Slices were imaged with a digital camera (Scopetek DCM510; Scopetek Opto-Electric, Hangzhou, China) attached to a stereomicroscope (Wild model M8, Heerbrugg, Switzerland). The contusion was evident from a clear difference in the intensity of the cresyl staining. The area of the contusion was measured using image-analysis software (Scopephoto 3.1; Scopetek Opto-Electric) by an investigator blinded to the experimental groups. Contusion volume was calculated by multiplying contusion areas, A, by the distance between brain sections, d, (500 μm), according to the following formula:

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Quantification of Functional Outcome

1) Neuroscore: Functional outcome before and after CCI injury was determined using a 15-point neurologic outcome score (41, 42). The neuroscore consists of tasks evaluating locomotor ability, vestibulomotor function, and general behavior (Table 1). The neuroscore was performed in real time, before surgery, and repeated at 24-hour intervals following CCI-injury, with the final test applied immediately before the animals were euthanized (either 24 hr or 5 d after injury). Long-term outcomes after 1 month were assessed using RotaRod and CatWalk-XT tests. 2) RotaRod: Mice were placed on the RotaRod (Ugo Basile, Comerio, Italy). The RotaRod was accelerated linearly from 4 to 40 rev/min over 5 minutes, and the time the mouse remained on the drum was recorded. Three trials were performed with the longest time on the drum used. 3) CatWalk-XT is an automated gait-analysis system (Noldus Information Technology, Wageningen, The Netherlands), consisting of a glass plate with dim light illuminating the glass from the side. In a darkened environment (< 1 lux of illumination), light is reflected downward when the animal’s paws contact the glass surface. Images of the footprints are recorded by a video camera under the walkway. The images from each trial are processed and analyzed on a PC by CatWalk-XT software. Three consecutive runs were performed, and the mean values for the gait variables for each animal were obtained.

Table 1

Table 1

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Statistics

We assessed significance using either a Student t test or one-way analysis of variance (ANOVA), with Bonferroni post hoc test. In the 5-day survival experiments, we used two-way ANOVA with repeated measures with Bonferroni post hoc test. Factor 1 was treatment (xenon or control) and factor 2 was the time after injury (e.g., 1, 2, 3, 4, or 5 d). We therefore used repeated-measures ANOVA with factor 1 as the repeated factor. We used two-tailed hypothesis testing with p values of less than 0.05 taken to indicate a significant difference between groups. The sample sizes (n) are indicated in the figure legends. From preliminary experiments with short-term outcomes (24 hr), we estimated that a sample size of ~10 animals in each treatment group would give a statistical power of ~0.80 (α = 0.05). A post hoc power analysis of the results indicate that at a level of 0.05, the power of the experiments is on average 0.83 (range, 0.55–0.98). Sample sizes for long-term behavioral experiments were estimated from previous experience with these tests. Where error bars are shown, these are the SEM. Statistical tests were implemented using SigmaPlot software (Systat, Point Richmond, CA).

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RESULTS

Xenon Pretreatment and Posttreatment Is Neuroprotective

We investigated the effect of xenon administration on neurologic outcome and contusion volume when xenon was given both before and after, but not during, brain trauma (Fig. 1). Treatment with 75% xenon:25% oxygen for 2 hours immediately before the trauma and then for another 2 hours after the trauma resulted in a significant improvement (40 ± 11%, p < 0.05) in functional neurologic outcome 24 hours after trauma (Fig. 1A). Contusion volume was reduced by 43% ± 7% (p < 0.01) following xenon treatment (Fig. 1B).

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Xenon Posttreatment Is Neuroprotective

We next determined whether xenon was effective when given only after TBI. We found that 75% xenon:25% oxygen administered 15 minutes after injury for a period of 3-hour duration resulted in a 36% ± 6% (p < 0.05) improvement in neurologic outcome score 24 hours after injury (Fig. 2A). We found that xenon treatment after injury significantly reduced contusion volume, by 19% ± 7% (p < 0.05), compared with untreated controls (Fig. 2B).

Figure 2

Figure 2

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Delayed Xenon Treatment Is Neuroprotective

Having shown that xenon was effective when given immediately after injury, we determined the therapeutic time window in which xenon remained effective. We investigated the effect of delaying the start of the xenon treatment until 1, 3, or 6 hours after injury. We found that xenon treatment begun 15 minutes or 1 hour after injury significantly (p < 0.05) improved the neurologic outcome score 24 hours after injury by 33% and 46%, respectively (Fig. 3). In the groups where xenon treatment was delayed for 3 or 6 hours, there was a trend to improved neuroscore (27% and 26%, respectively), but this did not reach statistical significance. We investigated the effect of delayed xenon treatment on contusion volume. We found that xenon treatment starting 15 minutes, 1 hour, or 3 hours after injury significantly (p < 0.05) reduced total injury by ~20% (Fig. 4), but delaying treatment until 6 hours after injury made the xenon treatment ineffective (5.0% ± 0.2% reduction).

Figure 3

Figure 3

Figure 4

Figure 4

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Concentration Dependence of Xenon’s Neuroprotective Effect

We performed a series of experiments where animals were treated with 30% xenon:25% oxygen (balance nitrogen), 50% xenon:25% oxygen (balance nitrogen), 75% xenon:25% oxygen, or 75% nitrogen:25% oxygen, for 3 hours, starting 15 minutes after injury. Xenon reduced injury significantly (p < 0.05) at all concentrations tested (Fig. 5), by 19% ± 1% at a concentration of 30%, by 17% ± 1% at a concentration of 50%, and by 19% ± 2% at a concentration of 75% xenon. Neurologic outcome (data not shown) was significantly improved by treatment with 30% xenon or 75% xenon, by 45% ± 10% (p < 0.05) and 67% ± 15% (p < 0.001), respectively. Treatment with 50% xenon resulted in a trend to reduction in neuroscore (24% ± 5%), but this did not reach significance.

Figure 5

Figure 5

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Effect of Xenon Treatment on Physiologic Variables

In order to determine whether xenon treatment affected the physiology of the animals, we measured blood pressure, heart rate, and temperature in a cohort of animals that received CCI injury followed by 3 hours of treatment with either 75% xenon:25% oxygen or control gas, 75% nitrogen:25% oxygen, starting 15 minutes after injury. There was no significant difference in physiologic variables between the xenon-treated or control gas–treated animals (Table 2).

Table 2

Table 2

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Effect of Xenon up to 5 Days Postinjury

Neurologic function was measured at 1, 2, 3, 4, and 5 days after injury (Fig. 6). Neurologic outcome in the xenon-treated groups improved significantly (p < 0.05) compared with the control group, up to 4 days after injury. On day 5, there was a trend for improvement in neurologic outcome score in the xenon-treated group compared with the control group, but this difference was not significant (p = 0.06).

Figure 6

Figure 6

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Effect of Xenon 1 Month After Injury

In order to establish whether xenon’s protective effect persisted, a cohort of animals (n = 50) were allowed to survive 1 month after traumatic injury. Two groups underwent CCI surgery followed by treatment with either 75% xenon:25% oxygen or 75% nitrogen:25% oxygen, starting 15 minutes after injury, for 3-hour duration, a third group underwent sham surgery including anesthesia, skin incision with exposure of skull, but no craniotomy or TBI impact. The duration of anesthesia and time between skin incision and suturing was identical to the CCI surgery group. The sham group received treatment with 75% nitrogen:25% oxygen for 3 hours. One month after injury, we examined performance on the RotaRod to determine whether this revealed a locomotor deficit (Fig. 7). There was no difference in RotaRod performance between the control gas–treated TBI group or the sham-surgery group or the xenon-treated TBI group, indicating that the RotaRod is not a suitable outcome at this time point. We tested the animals using the CatWalk-XT gait-analysis system to determine whether more subtle measures of locomotor function would reveal a deficit. There was a significant (p < 0.05) deficit in locomotor running speed in the control TBI group (43 ± 3 cm/s) compared with the sham-surgery group (53 ± 3 cm/s). Xenon treatment improved the deficit in running speed (50 ± 2 cm/s); there was no significant difference (p = 0.65) between the xenon-treated TBI group and the sham-surgery group (Fig. 8A). We next examined the base-of-support (BOS) for front and hind paws (Fig. 8B). The BOS measures distance between midpoints of the front paws or rear paws during locomotion. There was no change in the BOS of the front paws. However, there was a trend to increase in the BOS of the rear paws in control TBI group (33 ± 4 mm) compared with sham-operated animals (24 ± 4 mm), but this was not significant. Xenon treatment prevented the increase in BOS; there was no significant difference between the xenon-treated TBI group (25 ± 4 mm) and the sham-surgery group. The increase in BOS observed in the rear paws is consistent with the observation in the short-term neuroscore results that faults in rear paw placement occur. We observed a trend toward a reduction in cadence (Fig. 8C) in the TBI control group compared with sham-surgery group (p = 0.06). This trend was absent in the xenon-treated TBI group. To determine whether there were specific locomotor deficits in particular limbs, we examined the swing speed for each limb (Fig. 8D). Swing speed is calculated from stride length and time between paw placements for each limb. We found that there was a locomotor deficit for each paw in the TBI control group compared with sham-surgery group. This was significant (p < 0.05) for all paws except the hind left which showed a trend (p = 0.06). Interestingly, the deficit in the right hind paw was the largest (22% ± 2% reduction), and this was highly significant (p < 0.01). Xenon treatment prevented the locomotor deficit; in all cases, the xenon-treated TBI group was not significantly different to the sham-surgery group.

Figure 7

Figure 7

Figure 8

Figure 8

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DISCUSSION

The findings presented here are the first to show that xenon is neuroprotective in an animal model of TBI. We show that xenon treatment improves functional neurologic outcome and reduces secondary injury development, even when commencement of the xenon treatment is delayed 1–3 hours following trauma. Our findings are consistent with previous in vitro studies showing that xenon is neuroprotective in an in vitro model of TBI (43, 44), using mouse organotypic hippocampal slice cultures subjected to focal mechanical injury and with cell injury quantified by propidium-iodide fluorescence. Such in vitro models are useful in screening putative neuroprotectants and understanding their mechanism of action (44), but they cannot model behavioral neurologic outcome. Hence, it is important to test putative neuroprotectants such as xenon in preclinical models that allow assessment of improvements in functional neurologic outcome at clinically relevant time points.

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Experimental Model

We investigated the neuroprotective efficacy of the noble gas xenon in an in vivo mouse model of TBI. We used male animals to exclude effects of the estrous cycle and any potential confounding neuroprotective effects of female sex hormones. The rodent CCI model we used has been widely used in preclinical TBI studies (45–52) and is highly reproducible. In our experiments with xenon, we chose to investigate the effects of xenon on both acute outcome 1–5 days after injury and long-term outcome 1 month after injury.

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Xenon Pretreatment and Posttreatment

In both in vitro and in vivo models of ischemic injury, there is evidence that xenon treatment before injury protects neuronal and cardiac tissue (53–61). We reasoned that giving xenon both before and after traumatic injury would be protective. Our results show that 75% xenon:25% oxygen given for 2 hours immediately before injury and then for a further 2 hours after trauma significantly improves neurologic outcome (p < 0.05) and reduces contusion volume (p < 0.01) 24 hours after injury. It is possible that xenon could be used before traumatic injury, for example, in neurosurgical procedures, to minimize damage to neighboring healthy tissue. Nevertheless, in an accidental TBI, such as a motor-vehicle crash, xenon pretreatment would obviously not be possible.

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Xenon Posttreatment

The main objective of our study was to evaluate xenon’s potential as a treatment for TBI in clinically relevant settings. We therefore determined the effect of xenon treatment, when xenon was given only after the trauma. We chose a xenon treatment time of 3 hours based on previous studies with xenon in in vivo ischemic injury models (30, 62). We chose 15 minutes postinjury as the earliest start time for xenon treatment, based on a scenario that a patient with TBI could receive medical attention at the scene of injury within 15–20 minutes. Our results show that giving xenon for 3 hours after injury results in a significant (p < 0.05) reduction in contusion volume when xenon was given at concentrations of 30%, 50%, or 75%.

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Therapeutic Time Window for Xenon Treatment

One of the most important considerations for a potential treatment for TBI is whether there is a clinically relevant time window within which treatment is effective. We investigated the effect of delaying xenon treatment until 1, 3, or 6 hours after injury. When xenon treatment was delayed up to 3 hours after injury, there was a significant (p < 0.05) reduction in contusion volume, but delaying treatment for 6 hours did not result in reduction of contusion. Our findings also show a significant (p < 0.05) improvement in neurologic outcome when treatment is delayed up to at least 1 hour after injury.

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Effect of Xenon up to 5 Days After Trauma

The untreated TBI animals showed an improvement in neurologic outcome over the course of 5 days. This underlying improvement in control neuroscore may reflect both a recovery in locomotor function and a component of learning on successive test days. Nevertheless, on each measurement day, the xenon-treated group had a better neurologic score, by 42% on average, compared with the untreated group. The improvement with xenon treatment was significant (p < 0.05) up to day 4 after injury. The neurologic severity score measurement we used has been shown to be a good predictor of long-term outcome and to correlate with injury severity assessed by MRI (42).

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Locomotor Outcomes 1 Month After Injury

In patients with TBI, long-term impairments have the greatest financial and social cost. In order that our results have clinical relevance, we examined the effect of xenon treatment on behavioral outcomes 1 month after traumatic injury. Patients with TBI frequently report postural instability that persists years after the trauma (63, 64). Studies using analysis of gait in patients with TBI have revealed deficits compared with healthy controls. The CatWalk-XT gait-analysis system has been used to measure deficits in a variety of rodent brain-injury models (65–68). Gait analysis 1 month after injury revealed significant (p < 0.05) deficits in the TBI group, in locomotor speed, and in the swing speed of all four limbs. Remarkably, none of these deficits were present in the xenon-treated TBI group. We also observed a trend to increase in BOS of hind limbs in the TBI group that was absent in the xenon-treated group. These findings are of particular clinical relevance because deficits in walking speed and BOS are observed in patients with TBI (69–71). A recent systematic review of studies in patients with TBI found that in all studies that reported speed, TBI patients walked more slowly than healthy controls (72). Our results indicate that xenon treatment may prevent these persistent locomotor deficits in patients with TBI.

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Clinical Relevance of These Findings

Xenon has many properties of an ideal general anesthetic (20), but its widespread use has been limited, in part, due to its cost. Since the discovery that xenon was an NMDA receptor antagonist (24) and the subsequent demonstration that xenon has neuroprotective properties in models of ischemic injury (25–34), there has been a resurgence of interest in the use of xenon as a neuroprotectant. Clinical trials are currently evaluating xenon in different types of ischemic injury (e.g., neonatal asphyxia and brain damage after cardiac arrest) (35, 36). In this study, we have shown for the first time that xenon is protective against blunt TBI in the CCI rodent model. Our results show that xenon treatment reduces secondary injury and improves neurologic outcome both acutely and 1 month after injury and that xenon has a clinically relevant therapeutic time window of up to 3 hours. The fact that xenon is effective at a concentration of 30% is particularly relevant to the treatment of patients with severe TBI where it may be necessary to give increased concentrations of oxygen. The duration of xenon treatment we used is relatively short (3 hr) and it is possible, indeed likely, that extending this treatment time would result in a greater degree of neuroprotection and/or a longer therapeutic time window. A recent clinical study in patients with cardiac arrest has shown that it is practical to give xenon to patients in the ICU for up to 24 hours (36). Our findings support the idea that xenon could provide a realistic first-line treatment for patients with brain trauma and further research in this area is warranted.

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ACKNOWLEDGMENTS

We thank Frida Kornes, laboratory manager, Department of Anesthesiology, Medical Center of Johannes Gutenberg University, Mainz, Germany, for technical support; Prof. William Wisden, professor of molecular neuroscience, Department of Life Sciences, Imperial College London, for the use of the Leica cryostat; and Carina Freidrich, medical student, Johannes Gutenberg University, Mainz, Germany, and Dr. Valentina Ferretti, postdoctoral researcher, Imperial College London, for advice on cryosectioning and histology.

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Keywords:

brain injury; head trauma; inert gases; neuroprotection; xenon

© 2015 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins