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2A-Adrenoceptor Subtype
*Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel; Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg; Department of Anaesthesia and Critical Care, University of Wuerzburg Hospitals, Germany; Hôpital Beaujon, Assistance Publique des Hôpitaux de Paris; and ||INSERM U 676 & Service de Neuropédiatrie, Hôpital Robert Debré, Paris, France
Address correspondence to Andrea Paris, MD, Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Schwanenweg 21, D-24105 Kiel, Germany. Address e-mail to paris{at}anaesthesie.uni-kiel.de.
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2-adrenoceptor subtypes to elucidate the contribution of 2-adrenoceptor subtypes to the neuroprotective properties of dexmedetomidine in a model of perinatal excitotoxic brain injury. On postnatal Day 5, wild-type mice and mice lacking 2A-adrenoceptor (2A-KO) or 2C-adrenoceptor subtypes (2C-KO) were randomly assigned to receive dexmedetomidine (3 µg/kg) or phosphate-buffered saline intraperitoneally. Thirty minutes after the intraperitoneal injection, the glutamatergic agonist ibotenate (10 µg) was intracerebrally injected, producing transcortical necrosis and white matter lesions that mimic perinatal human hypoxic-like lesions. Quantification of the lesions was performed on postnatal Day 10 by histopathologic examination. Dexmedetomidine reduced mean lesion size in the cortex of wild-type mice and 2C-KO mice by 44% and 49%, respectively. Ibotenate-induced white matter lesions were reduced by 71% (wild-type mice) and 75% (2C-KO mice) after pretreatment with dexmedetomidine. In contrast, in 2A-KO mice, dexmedetomidine did not protect against the cortical excitotoxic insult, and white matter lesions were even more pronounced (82% increase of mean lesion size). Dexmedetomidine provides potent neuroprotection in a model of perinatal excitotoxic brain damage. This effect was completely abolished in 2A-KO mice, suggesting that the neuroprotective effect is mediated via the 2A-adrenoceptor subtype. |
Introduction |
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Intracerebral injection of ibotenate, a glutamatergic agonist, in newborn mice has been used as a well-characterized murine model of excitotoxic brain lesions, displaying striking similarities to human perinatal hypoxic brain lesions (6). Ibotenate administration on postnatal Day 5, when all neurons have completed their migration into the neocortex, produces a spectrum of cortical and white matter injuries, resembling cortical damage observed more frequently in full-term babies (4) and mimicking certain types of periventricular leukomalacias most often observed in premature neonates (3). In this murine model, 2-adrenoceptor agonists, such as dexmedetomidine, have been shown to be neuroprotective and provided a dose-dependent protection against cortical and white matter lesions (7).
Three subtypes of 2-adrenoceptors, termed 2A, 2B, and 2C, were initially revealed by pharmacological means and were cloned from several species, including mice and humans (8). Dexmedetomidine is a highly specific 2-adrenoceptor agonist with a high affinity to each of the 2-adrenoceptor subtypes. The 2A-adrenoceptor subtype has been reported to be the predominant subtype in the brain and is involved in a variety of physiological functions including antinociceptive, sedative, hypotensive, hypothermic, and behavioral actions of 2-adrenoceptor agonists (9,10). Stimulation of 2B-adrenoceptors in vascular smooth muscle leads to vasoconstriction, which causes the initial hypertension after the administration of 2-adrenoceptor agonists (11). In addition, the 2B-adrenoceptor subtype is involved in mediation of the antinociceptive action of nitrous oxide (12) and in central thermoregulation (13). The 2C-adrenoceptor subtype has been shown to modulate dopaminergic neurotransmission, as well as various behavioral responses, and to induce hypothermia (14). In addition, this subtype contributes to spinal antinociception of the imidazoline moxonidine in mice (15).
To further characterize which of the adrenoceptor subtypes mediates the neuroprotective effect of 2-adrenoceptor agonists, we studied the effect of dexmedetomidine in a murine model of perinatal excitotoxic brain injury in vivo in mice carrying targeted deletions of the 2A-adrenoceptor or 2C-adrenoceptor genes (8).
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-amino-3-hydroxy-5-methyl-4-isoxazole-propionate and kainate receptors. Dexmedetomidine (Orion Pharmos, Turku, Finland) was diluted in 0.1 M of phosphate-buffered saline (PBS), and ibotenate was diluted in PBS containing 0.02% acetic acid.
Wild-type mice and transgenic mice lacking 2A-adrenoceptor or 2C-adrenoceptor subtypes were used for the experiments. All mouse lines used in this study were maintained on a C57BL6J/OlaHsd background. For this purpose, the 2A and 2C deletions were backcrossed for >10 generations with C57BL6 wild-type mice. The generation of mouse lines lacking single 2-adrenoceptor subtypes has been previously described (11,16). Pregnant mice were housed in groups in a specified pathogen-free facility given free access to water and food, with a 12-h light-dark cycle. The experimental protocol received approval from the IRB of the Hôpital Robert Debré (Paris, France) and complied with the guidelines of the Institut National de la Santé et de la Recherche Médicale.
Pups of both sexes from at least 2 different litters per genotype were used for experiments on postnatal Day 5. Dexmedetomidine 5 µL or PBS 5 µL was intraperitoneally injected 30 min before a stereotactic intracerebral injection of ibotenate. As previously described (6,7), pups were anesthetized using ether inhalation and maintained under a warming lamp. Intracerebral injection of ibotenate was performed using a 26-gauge needle on a 50-µL Hamilton syringe mounted on a calibrated microdispenser. The needle was inserted 2 mm under the external surface of the scalp skin in the frontoparietal area of the right hemisphere, 2 mm from the midline in the lateral-medial plane, and 3 mm from the junction of the sagittal and lambdoid sutures in the rostrocaudal plane. Histological analysis from previous experiments in >1500 animals confirmed that this technique results in highly reproducible injections (6,7). Two 1-µL boluses were injected 30 s apart into the cortex and into the periventricular white matter. The needle was left in place for 30 s after the second bolus. After the injection, the pups were allowed to return to their dams.
Wild-type mice, transgenic mice lacking 2A-adrenoceptor subtypes (2A-KO), and mice lacking 2C-adrenoceptor subtypes (2C-KO) were used. In all experimental groups, a stereotactic intracerebral injection of ibotenate (10 µg) was given on postnatal Day 5. Thirty minutes before intracerebral injection, pups were randomly assigned to receive an intraperitoneal injection (5 µL) of dexmedetomidine (3 µg/kg of body weight) or PBS. Eight to 32 pups from at least 2 different litters were used in each experimental group. The numbers of pups used in each experimental group are given in Figure 1.
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On postnatal Day 10, surviving pups were killed by decapitation. Brains were removed, fixed in 4% formalin for 7 days, and embedded in paraffin subsequently. Coronal serial sections, 15-µm thick, were cut along the entire brain from the frontal to the occipital pole, and every third section was stained with cresyl violet. In theory, neocortical and white matter lesions can be defined based on the maximal length in three orthogonal axes: the lateral-medial axis (in the coronal plane), the radial axis (also in the coronal plane, from the pial surface to the lateral ventricle), and the frontooccipital axis (in the sagittal plane). In preliminary studies, there was a high correlation between the maximal size of the lateral-medial, radial, and frontooccipital diameters of the ibotenate-induced lesions (6). Based on these findings, the entire brain was serially sectioned in the coronal plane. This permitted an accurate and reproducible measurement of the maximal sagittal frontooccipital diameter of the lesion. The number of sections demonstrating the lesion, multiplied by 15 µm, served as an index of the lesion size. Lesion size determination was performed in a blinded manner by an investigator unaware of the experimental group to which the animal was assigned.
Data are presented as mean ± sem. Kruskal-Wallis nonparametric analysis of variance was used to analyze effects between different genotypes given the same pretreatment (dexmedetomidine or PBS, respectively). The Mann-Whitney test was used to compare pups treated with ibotenate + dexmedetomidine with pups of the same genotype but treated with ibotenate + PBS. A value of P < 0.05 was considered statistically significant.
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The stereotactic, intracerebral injection of ibotenate caused dramatic cortical lesions characterized by loss of neurons affecting all cortical layers and large periventricular white matter cysts (Fig. 2).
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In wild-type pups injected with intraperitoneal PBS and intracerebral ibotenate, the mean cortical lesion size was 898 ± 56 µm (Fig. 1). Comparable cortical lesion size was found in the brains of 2A-KO mice (816 ± 55 µm) and 2C-KO mice (815 ± 64 µm) pretreated with PBS. When compared with PBS-pretreated controls, pretreatment with dexmedetomidine significantly reduced mean cortical lesion size in wild-type mice by 44% (500 ± 39 µm; P < 0.05) and in 2C-KO mice by 49% (418 ± 69 µm; P < 0.05). In contrast, dexmedetomidine did not significantly reduce mean cortical lesion size in 2A-KO mice (772 ± 71 µm).
In pups pretreated with PBS, ibotenate-induced white matter lesion size did not differ between groups and was 590 ± 52 µm in wild-type mice, 384 ± 60 µm in 2A-KO mice, and 530 ± 57 µm in 2C-KO mice. When compared with PBS-pretreated controls, pretreatment with dexmedetomidine significantly reduced mean white matter lesion size in wild-type mice by 71% (170 ± 44 µm; P < 0.05) and in 2C-KO mice by 75% (134 ± 39 µm; P < 0.05). In contrast, pretreatment with dexmedetomidine did not afford neuroprotection but significantly increased mean white matter lesion size in 2A-KO mice by 82% (701 ± 97 µm; P < 0.05).
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Discussion |
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2C-adrenoceptor subtypes, dexmedetomidine potently protects the developing brain from transcortical necrosis and white matter lesions induced by intracerebral injection of the glutamatergic agonist ibotenate in newborn mice. In contrast, dexmedetomidine did not prevent excitotoxic cortical neuronal loss and produced even more pronounced white matter cystic lesions in mice lacking the 2A-adrenoceptor subtype.
Dexmedetomidine has neuroprotective properties in various experimental designs, mainly conducted as models of cerebral ischemia in adult animals (17). Moreover, it was effective in a model of excitotoxic neuronal injury protecting against kainic acid-induced neuronal damage (18). However, there are only few models investigating neuroprotective properties of 2-adrenoceptor agonists in developing brains that are highly susceptible to neuronal damage. In neonatal rats, dexmedetomidine (19) attenuated hypoxic-ischemic brain injury. Previous studies in our murine model of perinatal excitotoxic brain injury revealed that clonidine and dexmedetomidine provided potent neuroprotection (7). However, the respective contribution of 2-adrenoceptor subtypes has not yet been determined. Therefore, we studied wild-type mice and transgenic mice lacking single 2-adrenoceptor subtypes (11,16) to elucidate the contributions of different 2-adrenoceptor subtypes to the neuroprotective effect of dexmedetomidine in this model of excitotoxic perinatal brain injury. We confirmed the neuroprotective properties of dexmedetomidine in wild-type mice and 2C-KO mice. In contrast, the neuroprotective effects of dexmedetomidine were completely abolished in 2A-KO mice, suggesting that the neuroprotective properties of dexmedetomidine are mediated by the 2A-adrenoceptor subtype.
These results are in accordance with the results of Ma et al. (19). In vitro dexmedetomidine did not exert a protective effect on neuronal injury (exposure to oxygen and glucose deprivation) in mixed neuronal-glial cultures derived from transgenic mice (D79N) expressing dysfunctional 2A-adrenoceptors. In a model of neonatal hypoxia-ischemia in vivo, the dose-dependent protection against brain matter loss by dexmedetomidine was reversed by an 2A-adrenoceptor subtype-preferring antagonist (BRL44408) (19). However, 2-adrenoceptor antagonists lack high subtype specificity. Thus, the results of the present in vivo study in 2A-KO mice present an important confirmation of the crucial role of the 2A-adrenoceptor subtype in mediating neuroprotective effects of 2-adrenoceptor agonists.
We used a well-characterized murine model of excitotoxic brain injury displaying remarkable histopathological similarities to human neuronal injuries described after hypoxia occurring in human fetuses and neonates (6). Several drugs including 2-adrenoceptor agonists have been shown to be neuroprotective in this model and provided insight into the pathophysiology of neuronal damage and neuroprotection in the developing brain (7). However, the exact mechanisms of 2-adrenoceptor-mediated neuroprotection still remain unclear. Studies using models of cerebral ischemia showed that a massive release of glutamate and catecholamines during ischemia may play a key role in ischemic neurologic damage (17,20). An in vivo positive correlation between circulating norepinephrine and neurologic outcome was revealed after cerebral ischemia (17,21). 2-adrenoceptors modulate neurotransmitter release in the central and peripheral sympathetic nervous system (16), and genetic mouse models indicate that the 2A-adrenoceptor subtype is the major subtype regulating sympathetic neurotransmission (8,16), thus offering a possible explanation for the neuroprotective properties of dexmedetomidine. Furthermore, in vitro 2-adrenoceptor agonists were able to reduce hypoxia-evoked glutamate release from hippocampal slices (22). In contrast, in an in vivo model of cerebral ischemia, dexmedetomidine failed to suppress brain norepinephrine and glutamate concentrations (23). However, these findings cannot be simply extrapolated to our model of excitotoxic brain lesions. Even if a secondary release of catecholamines and glutamate, triggered by excitotoxic brain damage in our model, seems likely, the initial intracerebral injection of the glutamatergic analog ibotenate may directly activate local N-methyl-d-aspartate receptors (7). Thus, the mechanism of neuroprotection by 2-adrenoceptor agonists may differ depending on experimental design.
There are some limitations to our study. We did not investigate mice lacking 2B-adrenoceptor subtypes. At present, 2B-adrenoceptor–deficient mice cannot be maintained on a congenic C57BL6 background because of embryonic lethality of homozygous 2B-KO mice (Hein and Brede, unpublished observation). To avoid developmental effects or influences of mixed genetic backgrounds, we chose not to include 2B-KO mice in this study. Each subtype has a distinct tissue distribution. In the brain, the 2A-adrenoceptor is the predominant receptor subtype and can be found ubiquitously. Disruption of the 2A-adrenoceptor gene resulted in a 90% reduction in total 2-adrenoceptor binding in mouse brains (16). Residual binding can primarily be attributed to the 2C-adrenoceptor subtype found in basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex, whereas the distribution of the 2B-adrenoceptor subtype in the brain is quite limited and mainly restricted to the thalamic nuclei (8,9,16); however, species differences have been described. Moreover, the 2A-adrenoceptor is the major subtype regulating sympathetic neurotransmission and mediating sedative and antinociceptive effects (8,9,16). Therefore, it seems unlikely that 2B-adrenoceptors contribute substantially to the neuroprotective effect of 2-adrenoceptor agonists. Consistently, in the current study, the neuroprotective action of dexmedetomidine was completely abolished in 2A-KO mice, indicating that 2B-adrenoceptors are not involved in neuroprotection.
In the current study, it was found that white matter lesions were even more pronounced after dexmedetomidine in 2A-KO mice. The reason for this finding is unclear. The dexmedetomidine dose we used provided potent neuroprotective effects on excitotoxic brain injury in previous investigations (7). Dexmedetomidine displayed a U-shaped dose-response curve showing decreasing neuroprotective properties with large doses (7,18,19). After permanent occlusion of the middle cerebral artery in the rat, large doses of dexmedetomidine were reported to worsen outcome (24). However, studies in neuronal and glial co-cultures from D79N transgenic mice expressing dysfunctional 2A-adrenoceptors did not report any direct neurotoxic-like effects of dexmedetomidine (19). Studies in 2A-deficient mice have shown that dexmedetomidine resulted in a pronounced initial hypertension accompanied by a reduction in heart rate, whereas the hypotensive effect of dexmedetomidine was completely absent (16). 2A-adrenoceptors are the predominant subtypes involved in the sedative, antinociceptive, and hypothermic effects of dexmedetomidine (9,16). Consequently, dexmedetomidine may cause complex changes in physiological pathways possibly harmful or beneficial in cases of neuronal damage. Additionally, previous studies in this model of neonatal excitotoxic brain injury strongly suggest that ibotenate-induced white matter and cortical lesions develop independently from each other and may differ with respect to their pathophysiology (25).
Another limitation and a common criticism of studies in transgenic mice is the possible induction of compensatory mechanisms that are not operational in wild-type mice, and that may mask the functional results of a targeted mutation. Accordingly, such influences cannot be excluded in the present study. However, studies using pharmacological approaches and studies in transgenic mice can complement each other, although both strategies have their own drawbacks. The fact that the results of our study are in accordance with other studies using pharmacological approaches (19) clearly suggests a key role for the 2A-adrenoceptor subtype in the neuroprotective action of dexmedetomidine.
In conclusion, in a model of neonatal excitotoxic brain damage, dexmedetomidine exerts potent neuroprotection in wild-type and 2C-KO mice but not in 2A-KO mice, suggesting that this effect is mediated via the 2A-adrenoceptor subtype.
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