Dexmedetomidine produces its neuroprotective effect via the α2A-adrenoceptor subtype

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Abstract

Which of the three α2-adrenoceptor subtypes of α2A, α2B, or α2C mediates the neuroprotective effect of dexmedetomidine was examined in cell culture as well as in an in vivo model of neonatal asphyxia. Dexmedetomidine dose-dependently attenuated neuronal injury (IC50=83±1 nM) in neuronal-glial co-cultures derived from wild-type mice; contrastingly, dexmedetomidine did not exert neuroprotection in injured cells from transgenic mice (D79N) expressing dysfunctional α2A-adrenoceptors. An α2A-adrenoceptor subtype-preferring antagonist 2-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL44408) completely reversed dexmedetomidine-induced neuroprotection, while other subtype-preferring antagonists 2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239) (α2B) and rauwolscine2C) had no significant effect on the neuroprotective effect of dexmedetomidine in neuronal-glial co-cultures. Dexmedetomidine also protected against exogenous glutamate induced cell death in pure cortical neuron cultures assessed by flow cytometry and reduced both apoptotic and necrotic types of cell death. Likewise this neuroprotective effect was antagonised by BRL44408 but not ARC239 or rauwolscine. Dexmedetomidine exhibited dose-dependent protection against brain matter loss in vivo (IC50=40.3±6.1 μg/kg) and improved the neurologic functional deficit induced by the hypoxic-ischemic insult. Protection by dexmedetomidine against hypoxic-ischemic-induced brain matter loss was reversed by the α2A-adrenoceptor subtype-preferring antagonist BRL44408; neither ARC239 nor rauwolscine reversed the neuroprotective effect of dexmedetomidine in vivo. Our data suggest that the neuroprotective effect of dexmedetomidine is mediated by activation of the α2A adrenergic receptor 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.

References (59)

  • E. MacDonald et al.

    Gene targeting—homing in on alpha 2-adrenoceptor-subtype function

    Trends Pharmacol. Sci.

    (1997)
  • M. Matsumoto et al.

    The α2 adrenergic agonist, dexmedetomidine, selectively attenuates ischemia-induced increases in striatal norepinephrine concentrations

    Brain Res.

    (1993)
  • F.J. Northington et al.

    Early neurodegeneration after hypoxia-ischemia in neonatal rat is necrosis while delayed neuronal death is apoptosis

    Neurobiol. Dis.

    (2001)
  • T. Roohey et al.

    Animal models for the study of perinatal hypoxic-ischemic encephalopathy: a critical analysis

    Early Hum. Dev.

    (1997)
  • B. Schutte et al.

    Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells

    J. Neurosci. Methods

    (1998)
  • S.P. Svensson et al.

    Heterologous expression of the cloned guinea pig α2A, α2B and α2C adrenoceptor subtypes. Radioligand binding and functional coupling to a CAMP-responsive reporter gene

    Biochem. Pharmacol.

    (1996)
  • S. Uhlen et al.

    [3H]RS79948-197 binding to human, rat, guinea pig and pig alpha2A-, alpha2B- and alpha2C-adrenoceptors. Comparison with MK912, RX821002, rauwolscine and yohimbine

    Eur. J. Pharmacol.

    (1998)
  • S.Z. Yuan et al.

    Hypoxic-ischaemic brain damage in immature rats: effects of adrenoceptor modulation

    Eur. J. Pediatr. Neurol.

    (2001)
  • B.C. Calzada et al.

    Alpha-adrenoceptor subtypes

    Pharmacol. Res.

    (2001)
  • D.W. Choi

    Limitations of in vitro models of ischemia

    Prog. Clin. Biol. Res.

    (1990)
  • D.W Choi et al.

    Glutamate neurotoxicity in cortical cell culture

    J. Neurosci.

    (1987)
  • K. Engelhard et al.

    The effect of the α2-agonist dexmedetomidine and the N-methyl-d-aspartate antagonist S(+)-ketamine on the expression of apoptosis-regulating proteins after incomplete cerebral ischemia and reperfusion in rats

    Anesth. Analg.

    (2003)
  • Giombini M., Ohashi Y., Sanders R.D., Maze M., Fujinaga M., 2002. Antinociceptive effect of dexmedetomidine, an...
  • J.M. Gidday et al.

    Nitric oxide mediates cerebral ischemic tolerance in a neonatal rat model of hypoxic preconditioning

    J. Cereb. Blood Flow Metab.

    (1999)
  • M.Y. Globus et al.

    Direct evidence for acute and massive norepinephrine release in the hippocampus during transient ischemia

    J. Cereb. Blood Flow Metab.

    (1989)
  • M.P. Goldberg et al.

    N-methyl-d-aspartate receptors mediate hypoxic neuronal injury in cortical culture

    J. Pharmacol. Exp. Ther.

    (1987)
  • L.G. Harsing et al.

    Evidence that two stereochemically different α2-adrenoceptors modulate norepinephrine release in rat cerebral cortex

    J. Pharmacol. Exp. Ther.

    (1991)
  • L. Hein

    Transgenic models of α2-adrenergic receptor subtype function

    Physiol. Biochem. Pharmacol.

    (2001)
  • L. Hein et al.

    Two functionally distinct α2-adrenergic receptors regulate sympathetic neurotransmission

    Nature

    (1999)
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