San Sebastian 2004 Session 3-3
 

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Neuroprotection with Xenon / Neuroprotección con Xenon.

Prof. Mervyn Maze, London, U.K.

Is xenon a neuroprotective agent against acute neuronal injury?

Introduction:

 Professor Franks discovered that xenon potently inhibits the NMDA subtype of the glutamate receptor non-competitively, with little effect on GABAA receptors or non-NMDA glutamatergic receptors.1 Because of the pivotal role which activation of this receptor subtype plays in the propagation of acute neuronal injury2 we undertook a series of studies to determine whether xenon could act as an effective neuroprotectant.

Results of Studies:

We used a neuronal-glial co-culture system in which release of the cytosolic enzyme, lactate dehydrogenase (LDH), into the culture medium, is indicative of neuronal injury [1]. We confirmed that the neurones and not the glia were the source of the LDH. In this system xenon exerts a concentration-dependent neuroprotective effect vs excitotoxicity-induced injury by each of NMDA and glutamate, with IC50 values of 19%, and 28%, respectively [1]. Using the same co-culture model we extended these studies to injury produced by deprivation of either oxygen alone or oxygen + glucose and showed that xenon is protective with IC50 values of 10% and 36%, respectively [1,2].

In an in vivo excitotoxicity model of brain injury in rats, xenon concentration-dependently reduced neuronal degeneration in the arcuate nucleus of the hypothalamus, monitored either histologically [1] or immunohistochemically [3], following subcutaneous administration of the excitotoxin, N-methyl DL-aspartate (NMA).

We subsequently extended these studies to assess whether the functional disability associated with acute neuronal injury could be prevented by xenon [4,5]. Mice were subjected to 60 minutes of right middle cerebral artery occlusion (MCAO) using an intraluminal filament while receiving either xenon (Group 1) or another putative neuroprotectant nitrous oxide (N2O; Group 2) or a combination of xenon and N2O (Group 3). After 24 hours, the mice underwent functional neurological assessment following which they were sacrificed to determine cerebral infarct size. Compared to Group 2, the rats in Group 1 had a significant reduction in total infarct size and displayed significant improvement in functional outcome on a general neurological deficits scale. Mice in Group 3 had intermediate values between those in Groups 1 and 2.

Perinatal cerebral hypoxic-ischaemia remains a frequent cause of chronic neurological morbidity occurring with a frequency of between 2-4 per 1000 full-term newborn infants. Various biochemical pathways contribute to the development of hypoxic-ischaemic (HI) brain injury, including oxygen-free radical formation, release of excitatory neurotransmitters and consequent elevation of intracellular calcium, culminating in excitotoxic neuronal death. Because similar pathogenic mechanisms have been invoked in the development of acute brain injuries in adults (e.g., stroke, and head injury) models of neonatal asphyxia have proven a useful model system to explore possible therapies for these devastating conditions. Therefore, we implemented an in vivo neonatal rat model of HI.3 In brief, seven-day old postnatal rat pups underwent right common carotid artery ligation after which the pups were exposed to 8% oxygen in either nitrogen or xenon for 90 min at 37°C. On the 7th day after hypoxia/ischaemia, rats were sacrificed and their brains removed, and the right hemisphere was separated from the left and weighed. The ratio of the right hemispheric weight to that of the left (R/L ratio) was deduced and is akin to “infarct size” for MCAO studies. For each group a cohort of animals was kept alive for 30 days in order to undergo neuromotor functional assessment using the methodology described earlier [5]. Xenon prevented the brain shrinkage and functional deficit (Figure 10); this protective effect was present even when xenon is administered up to four hours after HI. In contrast to other anaesthetics that require anaesthetic or supra-anaesthetic doses to act as a neuroprotectant, xenon exerts its neuroprotection at sub-anaesthetic concentrations. The neuroprotectant effect Text Box: Figure 10:TOP Brain sections from animals that have suffered 90 minutes of hypoxia-ischemia injury (centre) show gross anatomical deterioration compared to control animals (left). The brain slices on the right are from animals that have suffered the same hypoxia-ischemia but have been breathing 70% xenon during the hypoxic period. BOTTOM Effect of 70% xenon on neurological functions. Thirty days after the insult, rats were evaluated for neuromotor function (Left) using a panel that included assays of prehensile traction, strength, and balance beam performance (graded on a 0-9 scale) and (Right) balance on a Rotarod, a standard test of balance and neuromotor function.   is unrelated to any possible hypothermic action as brain temperatures were unaffected by the presence of xenon when the animals were kept at an ambient temperature of 37oC.

Although a direct reduction in NMDA-mediated excitotoxicity is a likely mechanism for the neuroprotective action of xenon, inflammation is a known mechanism that modulates both functional and histologic outcome following cerebral injury4. Migration of leukocytes to injured areas is a key inflammatory feature in disease progression within the brain. Leukocyte migration is facilitated by the expression of cell adhesion molecules (AM) on endothelial cells and the ability to change their expression can mitigate disease progression. Xenon (up to 75%) exerted no meaningful change of LPS-induced upregulation of AMs in an in vitro model involving mice brain endothelial cells [6]. We investigated whether xenon can reduce the inflammatory response occurring on cardiopulmonary bypass (CPB), as reflected by the upregulation of cytokines5. Under the same xenon conditions that produced attenuation of the neurocognitive deficit of CPB, there was no change in the cytokine response [7]. Therefore, we can conclude that xenon does not produce its neuroprotective through an anti-inflammatory action.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Exploring Clinical Extrapolations:

Text Box: Figure 11: Twelve days after cardiopulmonary bypass surgery animals were subjected to a probe trial, a test of spatial memory. The time spent in the quadrant in which a platform had been removed is taken as a measure of spatial memory. Animals in the Sham and CPB+Xe groups have longer times spent in the correct quadrant, indicating good spatial memory function. The results are mean ± SD (n =10). *p < 0.05, **p < 0.01 when compared with the values in CPB group. (i) Despite successful surgical correction of their cardiac problems, patients often experience clinically significant postoperative neurocognitive deficits (PONCD), the long-term consequences of which include a higher mortality rate and lower quality of life. Up to 50% of patients exhibit signs of cognitive decline, defined as memory and intellectual impairments in the remote post-surgical period6. The neurological damage caused by cardiac surgery with cardiopulmonary bypass (CPB) represents a major medico-social problem due to increasing prevalence of cardiac surgical procedures, and because patients may be at a productive vocational stage of their lives. Their illness burdens both acute and chronic health as well as social care resources. Although multifactorial in origin, a pivotal step in the pathogenesis of PONCD following cardiac surgery is thought to be excitotoxicity initiated by activation of the NMDA subtype of the glutamate receptor; indeed, the only successful treatment involved perioperative administration of remacemide, an NMDA receptor antagonist (although the clinical development of this compound was abandoned because of a poor kinetic profile)7.

          CPB provides an invaluable testing ground for neuroprotective agents, both because this clinical setting is an indication for the use of neuroprotective agents, and because positive data can be extended to stroke, trauma and perinatal asphyxia – all of which represent huge burdens for the NHS and society as a whole. Before we undertake such a study in humans, we first needed to perform functional outcome studies using a rat cardiopulmonary bypass model of neurological injury [5]. Physiological factors known to affect neurological function (such as blood pressure, temperature, glucose control etc.), were monitored and maintained within normal limits; neuromotor skills, visuospatial memory and spatial memory were assessed for up to 12 days after rats were subjected to CPB in the presence of either xenon or nitrogen (delivered through an oxygenator in the extracorporeal circuit) at a concentration of 65%. As a positive control for an NMDA receptor antagonist we used MK801 (dizolcipine), one of the most potent drugs for neuroprotection8. Neurocognitive dysfunction following CPB was attenuated by xenon; the neuroprotection provided by xenon proved to be superior to that seen with dizolcipine (Figure 11).

(ii) Several NMDA receptor antagonists that have been shown to be effective in animal models have had to be abandoned in clinical trials because of their own inherent neurotoxicity (characterised by a specific lesion in the retrosplenial cortex). Not only does xenon not share the toxicity attendant on the use of other NMDA receptor antagonists [3], but it even protects against the development of the neurotoxicity when co-administered with other NMDA receptor antagonists9.

(iii) Another important consideration for the clinical application of xenon as a neuroprotectant is the effect that other contemporaneously delivered neuroprotectant strategies may have on its efficacy. Because of the recent data suggesting that hypothermia can be protective against acute neuronal injury in neonatal HI, we undertook a series of studies exploring the interaction between xenon and hypothermia. Cultured neurons injured by oxygen-glucose deprivation (OGD) were protected by a combination of interventions with xenon (20%) and hypothermia (35oC) which, when administered alone, were not efficacious. The xenon IC50 concentration for the neuroprotection vs  OGD-induced injury decreased from 40% at 37oC to just 12% at 33oC (Figure 12). Such a synergistic interaction with hypothermia may be a unique feature of xenon because it is not present with other neuroprotectants with activity at the NMDA receptor such as gavestinel. A combination of xenon and hypothermia administered 4 h after hypoxic-ischemic injury in neonatal rats provided synergistic neuroprotection assessed by morphological criteria, by hemispheric weight, and by neuromotor functional studies 30 days after the injury. In both in vitro and in vivo models, the protective mechanism involved an anti-apoptotic action with increased expression of BclXL, and decreased expression of Bax, important anti- and pro-apoptotic factors, respectively . If extrapolatable to humans, these data suggest that low (subanesthetic) concentrations of xenon in combination with mild hypothermia may provide a safe and effective therapy for perinatal asphyxia.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

References from our Laboratory

[1]          Wilhelm S, Ma D, Maze M, Franks NP. Effects of xenon on in vitro and in vivo models of neuronal injury. Anesthesiology 2002;96:1485-91.

[2]          Ma D, Hossain M, Rajakumaraswamy N, Franks NP, Maze M. Combination of xenon and isoflurane produces a synergistic protective effect against oxygen-glucose deprivation injury in a neuronal-glial co-culture model. Anesthesiology 2003;99:748-51.

[3]          Ma D, Wilhelm S, Maze M, Franks NP. Neuroprotective and neurotoxic properties of the 'inert' gas, xenon. Br. J. Anaesth. 2002;89:739-46.

[4]          Homi HM, Yokoo N, Ma D, Warner DS, Franks NP, Maze M, Grocott HP. The neuroprotective effect of xenon administration during transient middle cerebral artery occlusion in mice. Anesthesiology 2003;99:876-81.

[5]          Ma D, Yang H, Lynch J, Franks NP, Maze M, Grocott HP. Xenon attenuates cardiopulmonary bypass-induced neurologic and neurocognitive dysfunction in the rat. Anesthesiology 2003;98:690-8.

[6]          Yamamoto H, Takata M, Marczin N, Franks NP, Maze M Xenon`s Effect on Adhesion Molecule Expression in Inflammation Model of Mouse Brain Endothelial Cell. A-477 2003 ASA Meeting Abstracts.

[7]          Clark JA, Ma D, Homi HH, Mathew JP, Maze M, Grocott HP. Xenon and the Inflammatory Response to  Cardiopulmonary Bypass in the Rat  Anaesthesia. Submitted to Anesthesiology.

 

Other references

1.              de Sousa SL, Dickinson R, Lieb WR, Franks NP. Contrasting synaptic actions of the inhalational general anesthetics isoflurane and xenon. Anesthesiology 2000;92:1055-66.

2.              Nicotera and Lipton J. Excitotoxins in neuronal apoptosis and necrosis. Cereb. Blood Flow Metab. 1999;19:583-91.

3.              Rice III JE, Vannucci RC, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in rats. Ann. Neurol. 1981;9:131-41.

4.              del Zoppo G, Ginis I, Hallenbeck JM et al. Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol 2000;10:95-112.

5.              Wan S, Marchant A, DeSmet JM et al. Human cytokine responses to cardiac transplantation and coronary artery bypass grafting. J. Thorac. Cardiovasc Surg. 1996;111:469-77.

6.              Newman MF, Grocott HP, Mathew JP, White WD, Landolfo K, Reves JG, Laskowitz DT, Mark DB, Blumenthal JA. Neurologic Outcome Research Group and the Cardiothoracic Anesthesia Research Endeavors (CARE) Investigators of the Duke Heart Center. Report of the substudy assessing the impact of neurocognitive function on quality of life 5 years after cardiac surgery. Stroke 2001;32:2874-81.

7.              Arrowsmith JE, Harrison MJ, Newman SP, Stygall J, Timberlake N, Pugsley WB. Neuroprotection of the brain during cardiopulmonary bypass: a randomized trial of remacemide during coronary artery bypass in 171 patients. Stroke 1998;29:2357-62

8.              Levene M. Role of excitatory amino acid antagonists in the management of birth asphyxia. Biol. Neonate 1992;62:248-51.

9.              Nagata A, Nakao Si S, Nishizawa N, Masuzawa M, Inada T, Murao K, et al. Xenon inhibits but N2O enhances ketamine-induced c-Fos expression in the rat posterior cingulate and retrosplenial cortices. Anesth. Analg. 2001; 92: 362-8.