Dexmedetomidine produces its neuroprotective effect via the α2A-adrenoceptor subtype
Introduction
Perinatal cerebral hypoxia-ischemia remains a frequent cause of chronic neurological morbidity. Estimates suggest that between 2 and 4/1000 full-term newborn infants suffer asphyxia at or shortly before birth (Vannucci and Perlman, 1997). Various biochemical pathways contribute to the development of hypoxic-ischemic brain injury, including oxygen-free radical formation, release of excitatory neurotransmitters (including glutamate and catecholamines) and consequent elevation of intracellular calcium, culminating in excitotoxic neuronal death (Vannucci and Palmer, 1997). High levels of norepinephrine exhibit detrimental effects on neuronal tissue (Stein and Cracco, 1982); furthermore, the norepinephrine surge which occurs during birth, especially in the setting of asphyxia, may compromise the defence of the fetus resulting in neonatal asphyxia (Lagercrantz and Slotkin, 1986). Because similar pathogenic mechanisms have been invoked in the development of acute or chronic brain injuries in adults (e.g., stroke, head injury or neurodegenerative diseases), models of neonatal asphyxia have proven useful in seeking effective therapies for these devastating conditions.
We, and others, have previously reported on the ameliorative effect that α2-adrenoceptor agonists, especially dexmedetomidine, exhibit in acute neuronal injury (Hoffman et al., 1991a, Hoffman et al., 1991b, Maier et al., 1993, Halonen et al., 1995, Kuhmonen et al., 1997, Jolkkonen et al., 1999, Laudenbach et al., 2002). Conversely, pretreatment with the nonselective α-adrenoceptor antagonist phentolamine is associated with a reduced ability to survive anoxia (Yuan et al., 1997), stressing the functional significance of α2-adrenoceptors in resistance to hypoxic-ischemic brain damage. The reason for the neuroprotective effect of dexmedetomidine is thought to be due to its action in attenuating the massive release of catecholamines that occurs with cerebral hypoxic-ischemia in multiple parts of the brain (Globus et al., 1988, Globus et al., 1989, Matsumoto et al., 1993); this action may be mediated by pre-synaptic α2-adrenoceptos (Harsing and Vizi, 1991).
Molecular cloning has led to the identification of three independent genes encoding α2-adrenoceptor subtypes, termed α2A, α2B, and α2C, which are ubiquitously distributed (Calzada and De Artinano, 2001). We (Lakhlani et al., 1997) and others (Callado and Stamford, 1999, Hein et al., 1999, Hein, 2001) have suggested that the α2A adrenoceptor subtype modulates the release of catecholamines; in that case, this same receptor subtype may be responsible for the neuroprotective properties of α2 agonists.
Using both in vitro and in vivo models of perinatal neuronal injury, we investigated which of the α2A-, α2B-, or α2C adrenoceptor subtypes contributes to the neuroprotective effects of dexmedetomidine. In vitro mixed glial-neuronal co-cultures exposed to combined oxygen and glucose deprivation were used to simulate the environment associated with ischemic neuronal death in vivo (Koh and Choi, 1987). This approach affords precise control of temperature, pH, and O2/CO2 tension, each of which may independently affect the degree of injury (Choi, 1990). Lactate dehydrogenase (LDH) release was used as an indicator of neuronal injury. Furthermore, pure neuronal cultures were exposed to pathological levels of glutamate and cell viability was assessed by flow cytometry using Annexin V and propidium iodide staining to distinguish between apoptotic and necrotic cell death, respectively. In vivo we then employed the unilateral common carotid artery ligation method which is a validated as a model of cerebral hypoxia-ischemia (Vannucci et al., 1999, Vannucci et al., 2001). In rat pups, this procedure exposes the neurons to an environment not dissimilar from that seen with neonatal asphyxia (Andine et al., 1990). Brain weight deficit of the ipsilateral hemisphere (either as an absolute change or as a ratio of the unaffected contralateral hemisphere) was used as a measure of brain injury and correlates with the loss of evoked responsiveness, enzymatic neuronal markers and tissue destruction as evaluated by tissue histopathology (Roohey et al., 1997). Herein we demonstrate the neuroprotective effects of dexmedetomidine in these various models and characterise the adrenoceptor subtype dependence of this effect via pharmacological and transgenic methods.
Section snippets
Materials and methods
This study conforms to the UK Animals (Scientific Procedures) Act of 1986 and the Home Office (UK) approved the study protocol.
Effect of dexmedetomidine on OGD-induced LDH release
Neuronal damage induced by 75-min duration of OGD was dose-dependently diminished by dexmedetomidine with an IC50 concentration of 0.083±0.001 μM (Fig. 3).
Effect of α2-adrenoceptor antagonists on dexmedetomidine-induced inhibition of LDH release provoked by OGD
In cells derived from BALB/c mice, dexmedetomidine at 10 μM inhibited LDH release induced by OGD to 31±2% of maximal release of LDH (P<0.05 vs. control; Fig. 4A). Both subtype nonselective α2 antagonists, i.e., yohimbine and atipamezole, blocked the effect of dexmedetomidine on OGD-induced LDH release (Fig. 4A). BRL44408, 100 μM, the α2A
Discussion
Using a primary culture of neuronal and glial cells derived from the cerebral cortex of neonatal mice, predictable neuronal injury (as reflected by the amount of LDH released into the culture medium) was induced by exposing the cells to an environment deprived of glucose and oxygen (OGD). Dexmedetomidine, administered at the start of glucose and oxygen deprivation, concentration-dependently attenuated the subsequent neuronal injury provoked by glucose and oxygen deprivation in the cortical
Acknowledgements
This work was supported by the Medical Research Council, UK and the Westminster Medical School Research Trust. Orion-Farmos provided dexmedetomidine.
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