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(Stroke. 1999;30:1271-1278.)
© 1999 American Heart Association, Inc.
Original Contributions |
Differential Time-Course Decreases in Nonselective, µ-, 
-, and 
-Opioid Receptors After Focal Cerebral Ischemia in Mice
From the University of Caen, CNRS UMR 6551, Centre CYCERON, Caen, France.
Correspondence to F. Dauphin, CNRS UMR 6551, Centre CYCERON, Boulevard H. Becquerel, BP 5229, 14074 Caen Cedex, France. E-mail dauphin{at}cyceron.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Background and Purpose—Neuroprotection studies have demonstrated the involvement of opioids in ischemia, and we have previously demonstrated alterations in Bmax of opioidergic receptors after 2 post-MCAO time points in mice.
Methods—In the present study, we have investigated in a
detailed manner the postischemic time course of variations
in [3H]diprenorphine (nonselective),
[3H]DAMGO (µ), [3H]DADLE (
), and
[3H]U69593 (
) relative binding densities after focal
cerebral ischemia (0 to 48 hours) in mice.
Results—In frontoparietal cortices, our results demonstrate
decreases in (1) receptor densities at 1 to 3 hours after MCAO, (2)
µ and nonselective binding sites at 6 to 12 hours after MCAO, and (3)
receptor densities between 6 and 24 hours after MCAO. In the
rostral part of the infarct border zone, a decrease in -receptors
was found concomitant with the extension of the infarct core;
conversely, the decrease in -receptors appeared before (6 to 12
hours) macroscopic histological damage, which occurred
between 12 hours and 24 hours after MCAO in the caudal part of this
area. In this frontier, µ- and especially -binding sites were
decreased later (12 to 48 hours after MCAO).
Conclusions—These differential alterations in opioidergic
receptors could be due to the selective sublocalization of receptors,
postsynaptically on cortical interneurons for µ- and -receptors
versus presynaptically on cortical afferent pathways for the
subtype. Further, our results suggest that - and
µ-opioidergic receptors could be markers of infarct extension and
neuronal death; the study of [3H]diprenorphine and
selective binding sites argues in favor of the use of receptor-specific
ligands. Finally, the relative preservation of -receptors might be
correlated with the neuroprotective role of -agonists, as previously
reported.
Key Words: autoradiography • cerebral infarction • receptors, opioid • mice
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Naloxone has been advanced as a potential neuroprotective, though much-debated, agent in experimental1 2 3 and clinical investigations4 5 6 ; on the basis of these findings, the implication of the opioidergic system in cerebral ischemia has been considerably studied. Several investigations have unequivocally reported the neuroprotective role of
-receptor agonists in models of
global and focal cerebral ischemia7 8 (for review,
see Reference 99 ). Despite these numerous studies, few investigations
have been carried out to analyze with precision the evolution
of the opioidergic system following cerebral ischemia (for
review, see Reference 99 ). Moreover, the existing studies have been
performed essentially with models of global cerebral ischemia,
and/or with ligands that do not discriminate between the 3 subtypes of
opioidergic receptors, namely, µ, , and .
We have recently reported differential alterations in µ, , and
maximal binding capacities (Bmax), rather than
affinities (Kd), not only in infarcted
cortices but also in penumbral areas, 6 hours and 24 hours after
permanent middle cerebral artery occlusion (MCAO) in the mouse.10 In this
previous study, we demonstrated that the µ and
Bmax values were decreased earlier than
Bmax values in infarcted cortices. Likewise, µ
and Bmax were significantly decreased at 6
hours after ischemia in the ipsilateral temporal auditory
cortex, which is typically a penumbral cortex, since the consolidated
infarct progressively recruits this adjacent tissue between 6 hours and
24 hours after MCAO. However, one criticism of our previous study is
that it was performed at only 2 time points following the induction of
ischemia; a more precise analysis of the time-related
evolution of opioidergic receptor densities following stroke is
necessary, notably for the determination of the therapeutic window for
opioidergic drugs. Consequent to our previous study in which we
reported only alterations in the Bmax values
after cerebral ischemia in mice,10 we have
quantified in the present study the relative binding densities of
the total population of opioidergic receptors with a nonselective
ligand (diprenorphine) in comparison with µ-, -, and
-opioidergic receptors labeled with 3 selective ligands at 0 hours,
1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours after MCAO
in mice. We report here the evolution of the 4 populations of receptors
not only in the infarct core but also in the penumbral areas in
parallel with the growth of the ischemic lesion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Middle Cerebral Artery Occlusion
All studies were conducted in accordance with the French legislation and European directives. Eighty OF1 mice (body weight, 17±2 grams, mean±SD, Iffa Credo, France) were kept in thermoregulated (22±2°C), humidity-controlled (55±10%) facilities under a 12-hour light/12-hour dark cycle (light on between 8 AM and 8 PM) and were allowed free access to food and water. Permanent focal cerebral ischemia was achieved by MCAO as described by Welsh et al,11 with the following modifications. Mice were anesthetized with chloral hydrate (500 mg/kg, IP), and maintained normothermic (37.5±0.5°C) by means of a heating blanket (Homeothermic Blanket Control Unit, Harvard Apparatus Limited). Under an operating microscope (Leica M715), the scalp was incised between the eye and ear; the temporal muscle was electrocoagulated (Aesculap TB50) and partially removed. The left middle cerebral artery (MCA) was identified, a craniotomy was carried out with a high-speed drill (Technobox-810), and the dura was incised. The MCA was occluded by bipolar electrocoagulation, and the scalp was sutured. For the sham-operated animals, the experimental procedures were similar, except that the MCA was not coagulated.
Autoradiography
Euthanasia was performed by vertebral dislocation 24 hours after
surgery for the sham-operated group (n=12), or at 0 hours (control
group, n=8), 1 hour (n=10), 3 hours (n=11), 6 hours (n=10), 12 hours
(n=9), 24 hours (n=10), and 48 hours (n=10) after MCAO. The brains were
rapidly removed, frozen in isopentane at -40°C, and stored at
-80°C. Coronal brain sections (15 µm) were obtained with a
cryomicrotome (Kryostat 1720 Digital, Leitz) and mounted onto
gelatin-coated slides. Autoradiographic experiments were
performed with [3H]diprenorphine (Amersham),
[3H]DAMGO, [3H]DADLE
and [3H]U69,593 (DuPont NEN) (specific
activities of 31, 53, 32, and 46 Ci/mmol, respectively) for
nonselective, µ, , and binding sites, respectively. Based on
their respective affinities, radioligands were used at
concentrations of 0.5 nmol/L for
[3H]diprenorphine, 2 nmol/L for
[3H]DAMGO, and 1 nmol/L for
[3H]DADLE and
[3H]U69,593 in appropriate buffers and
according to protocols described elsewhere.12 13 14
Nonspecific binding was determined by incubation of adjacent brain
slices in the presence of 10 µmol/L levorphanol (Sigma). Brain
sections were coexposed with standards
([3H]-microscales, Amersham) on
tritium-sensitive films (Hyperfilm, Amersham) for 8 weeks at -20°C.
Thereafter, the films were developed (LX 24, Kodak), rinsed with water,
and fixed (AL 24, Kodak). Relative densities of receptors (expressed in
femtomoles per milligram of tissue equivalent) were quantified with a
computer-assisted image analyzer (BIOCOM RAG 200) using a
5-order polynomial relationship between optical density and
radioactivity.
Histological Analysis
Macroscopic histological analysis was
performed on 12 brain sections per animal, adjacent to those used for
autoradiography, and stained by the hematoxylin-eosin
technique. Infarcted areas were delineated by the relative paleness of
histological staining in the ischemic tissue.
Regions of interest were determined through the use of a
stereotaxic atlas of the mouse brain,15 and
infarcted volumes were calculated by the integration of infarcted areas
as quantified with a computer-assisted image analyzer (BIOCOM
RAG 200) on each brain slice.
Statistical Analyses
For each ligand, a multiparametric analysis of
variance (MANOVA) was first undertaken (Statview 4.5, Abacus Concepts
Inc) with 3 factors: brain region, side (ipsilateral versus
contralateral, repeated factor) and time after MCAO. Because such an
analysis revealed a highly significant (P<0.001)
principal effect for 1 or more of these 3 factors and interactions
between them, a second-order analysis was performed to
determine the individual effect of each parameter, the last
stage of this segmentation being obtained by a 1-way ANOVA with time
after MCAO as the principal factor, followed by post hoc
multicomparison tests (Fisher protected least significant difference
[PLSD] test), for each ipsilateral and contralateral region of
interest. Infarct volumes, calculated for each post-MCAO time, were
compared between groups through the use of a 1-way ANOVA and the post
hoc Fisher PLSD test. For all statistical analyses, the
significance level was accepted to be P<0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Histological Alterations
No histological alterations were seen in control mice (0 hours); the sham-operated group was not significantly different from the control group (P=0.69, Fisher PLSD test; Table 1
). Histological
analysis revealed that the infarct core corresponded
essentially to the motor and somatosensory frontoparietal cortices,
which were totally infarcted at 1 to 3 hours after MCAO. At this time,
a limited volume of peri-infarcted tissue was situated in the
anterior motor frontal and temporal auditory cortices. The temporal
auditory cortex was progressively recruited by the infarct between 6
hours and 24 hours after MCAO (Figure 1).
Selective necroses and swelling appeared to increase between 1 hour and
24 hours after MCAO, especially in the border zone in which the
extension of infarct occurs. At these later time points, the infarcted
volume reached a maximal value (21±4 mm3)
(Table 1). In other regions, such as the entorhinal and cingular
cortices, no alterations could be observed.
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, and # indicate a significant
difference with sham and control groups, 1-hour group, and between
groups, respectively (P<0.05, ANOVA and Fisher PLSD
test).
Opioid Receptor Alterations
For each ligand, the MANOVA revealed a highly significant
(P<0.001) effect for the region of interest, side, and
post-MCAO time factors; interactions between these factors were also
revealed. The signal-to-noise ratio was measured to evaluate any
putative influence on the variations that had been detected. For each
ligand, noise was low (µ, 3±1%; , 3±0.4%; and , 7±2%;
mean±SD, in percent of the total signal measured in ipsilateral and
contralateral cortices of the sham and control groups), and thus
background noise was unlikely to significantly influence the
quantification of opioid receptors, and in turn, the variations
observed consequently to focal cerebral ischemia.
Alterations of Opioid Binding Sites in the Infarct Core
In the cortices infarcted early, namely, the motor and
somatosensory frontoparietal cortices, the number of
[3H]diprenorphine binding sites was
significantly decreased (
-25% to -50% versus sham and control
groups) at intermediate time points (6 to 12 hours), except in
layers I-II-III of the anterior somatosensory frontoparietal cortex,
where the decrease was observed as soon as 1 hour after MCAO (-21%
and -24% versus sham and control groups, respectively) (Figures 2 and 3).
These decreases of binding sites in the superficial layers of the
cortex were not dependent on the rostrocaudal level and appeared at
intermediate time points (between 6 and 24 hours after MCAO; Figure 1
), when compared with - and -receptors (Figures 2 and 3).
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µ-Opioid receptor densities were significantly decreased (-30% to
-60%, versus sham and control groups) in all layers of the
frontoparietal cortices between 6 and 12 hours after MCAO (Figures 2 and 3). The -opioid receptor population was
significantly decreased as early as at 1 hour after MCAO in all layers
of the intermediate frontoparietal cortex (-24% to -36% versus
control group), and in layers I-II-III (-32% and -51% versus sham
and control groups, respectively) and IV (-39% versus control group)
of the anterior somatosensory frontoparietal cortices. In the deep
layers (V-VI) of anterior (-35%, 3 to 6 hours, versus sham and
control groups), and in all layers of the posterior somatosensory
frontoparietal cortices (-26% to -58%, 3 to 6 hours, versus sham
and control groups), -receptor decreases were evidenced between 3
and 6 hours after MCAO. In the anterior and posterior motor
frontoparietal cortices, significant decreases in -binding densities
appeared at 3 hours after MCAO in superficial layers (I-II-III, -32%
to -38%; IV, -23% to -34%, versus sham and control groups); and
at 1 and 6 hours in deep layers of the anterior (-21% versus control
group) and posterior (-30% to -38% versus sham and control groups)
cortices, respectively. In infarcted areas, -receptor densities were
not decreased at the early time points (1 to 6 hours), except in layers
I-II-III of the intermediate (-24% and -48%, 6 hours versus sham
and control groups, respectively) and posterior (-24%, 1 hour versus
control group) somatosensory frontoparietal cortices.
After 12 hours of ischemia, the decreases in -receptor
densities were highly significant in the motor (P<0.001;
-48% to -57% versus sham and control groups) and somatosensory
(P<0.001; -52% to -91% versus sham and control groups)
frontoparietal cortices (Figures 2 and 3). At later time points
(24 to 48 hours), an important decrease in nonselective and selective
binding sites (diprenorphine, -46% to -73%; µ, -39% to -58%;
, -49% to -74%; and , -21% to -45%, 24 to 48 hours,
versus sham and control groups) appeared in these cortices, with a
similar profile of binding changes for all cortical layers (Figure 2).
In the frontoparietal cortices, the multicomparison test showed that
µ-, and especially -, opioidergic receptors were decreased earlier
(6 to 12 hours and 1 to 6 hours after MCAO, respectively) than
-opioidergic receptors (6 to 24 hours after MCAO) (eg, Figure 3). Furthermore, the decreases in µ-receptors displayed
comparable temporal profiles after 6 to 12 hours MCAO in the motor and
the somatosensory cortices, and the profile was independent of the
rostrocaudal level studied (Figures 2 and 3). In contrast, the
-receptors subtypes were first decreased (1 hour after MCAO) in the
anterior and intermediate levels of somatosensory frontoparietal
cortices (Figures 2 and 3) and thereafter in the anterior and
posterior motor frontoparietal cortices and all rostrocaudal levels of
the somatosensory frontoparietal cortices (3 hours after MCAO) (Figures 2 and 3). Thus, the extension of the -receptor decreases
followed a ventrodorsal and rostrocaudal progression which corresponds
well with the extension of histological damage.
Alterations of Opioidergic Binding Sites in the Infarct Border
Zone
In peri-infarcted areas, such as anterior frontal motor cortices,
-binding sites were first decreased in layers I-II-III and V-VI (3
hours, -40% versus sham and control groups; and 6 hours, -37%
versus control group, respectively), and thereafter in layer IV (12
hours, -50% versus control group).
[3H]Diprenorphine, µ- and -binding sites
were also decreased but only after 12 hours in the superficial layers
(-50%; -40% to -59% versus sham and control groups; -32%
versus control group, respectively) in the same region (Figure 2). The decreases in -receptor densities, observed between 3
and 12 hours after MCAO, were found to be concomitant with the early
extension of the histological damage noted in this
area. In contrast, -receptor densities were decreased 6 hours after
MCAO in layers I-II-III (-37% versus control group) and 12 hours
after MCAO in both layers IV (~-50% versus sham and control groups)
and V-VI (-44% versus control group) of the temporal auditory cortex
and in striatal cortex (~-40% versus sham and control groups)
(Figures 2 and 4); such decreases
were evidenced before the appearance of macroscopic
histological damage, which was seen only between 12 and
24 hours after MCAO in these areas (Figure 1). Similarly,
[3H]diprenorphine and µ-binding sites were
decreased at 12 hours after MCAO in layers I-II-III (-44% and -30%
versus sham and control groups, for nonselective and µ-receptors,
respectively) and IV (-40% and -27% versus sham or control animals,
for nonselective and µ-receptors, respectively). -Receptor
densities were not significantly decreased in the temporal auditory
cortex, except an early decrease (-23% versus control group, 3 hours
after MCAO) in layer IV and a delayed decrease in layers V-VI of this
cortical area (-45% versus control group, 48 hours after MCAO)
(Figures 2 and 4). No alteration of any of the
opioidergic binding sites were observed in other ipsilateral cortical
areas, such as the cingular, retrosplenial, and entorhinal cortices, or
in cortical regions of the contralateral hemisphere.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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The present study highlights significant alterations in opioidergic binding site densities, not only in the infarct core but also in penumbral tissues, after focal cerebral ischemia in the mouse. Moreover, when compared with the 2 post-MCAO time-point analyses performed previously,10 the extensive analysis of opioidergic binding sites after MCAO demonstrates that, in fact, (1) µ and
subtypes have differential time-related
decreases in the infarct core, (2) time-related decreases in
-opioidergic receptors occur along with the appearance of the
growing infarct (1 to 3 hours after MCAO), and (3) -receptors show
little decrease before 24 hours after MCAO in infarcted cortices.
Furthermore, a time course was observed for the nonselective binding
sites that differs from those of µ-, -, and -receptors.
Alterations of Opioidergic Binding Sites in Infarct Core
In infarcted areas, the early decreases of not just - but also
µ- and nonselective opioid binding sites are in accordance with our
previous report in which we demonstrated a marked decrease in and
µ Bmax values 6 hours after MCAO in the mouse
brain.10 As previously postulated by many
authors,16 17 18 we attribute these changes to the
irreversible neuronal damage observed in the frontoparietal cortices at
early time points.19 Because apoptotic and
DNA-damaged neurons have been observed in the acute stage after focal
cerebral ischemia,19 20 21 22 phenomena that evolve
toward necrosis23 24 25 and complete disappearance of
neurons at delayed time points,19 23 one might hypothesize
that the early decrease in opioid receptor densities is due to such
progressive neuronal damage and cell death. Our results demonstrate
selective alterations with the 3 subtypes of opioidergic receptors;
-receptors were decreased later than µ- and -receptors, in
accordance with our previous study.10 This delayed
alteration in binding sites could be related to a cell-specific
localization of opioidergic receptors. As previously reported for other
specific presynaptic and postsynaptic proteins such as synaptophysin
and MAP-2,26 27 28 one could hypothesize that µ- and
-receptors, classically described as neuronal postsynaptic
receptors29 30 (although some reports have
described µ-, -, and -receptors as both presynaptic and
postsynaptic receptors as a function of the brain region studied and
neurotransmitter coexpressed with the opioids),31 32 33
are localized on cortical interneurons and are more sensitive to
cerebral ischemia than subcortical afferent pathways on which
-receptors are localized presynaptically.34 A second
hypothesis, in which the varied alterations described above could be
due to an upregulation of the -opioid receptors localized on glial
cells, seems unlikely, because many studies have demonstrated, through
the use of immunocytochemistry and electron microscopy, that only a few
opioidergic receptors (<20%) are expressed by glial
cells.34 35 36 Based on our present observations, the
precocious alteration in µ-, and especially -, receptors could
constitute markers of neuronal death and infarct extension through the
use of specific ligands. The relative preservation of -receptor
densities during the first hours after MCAO (until 6 hours after MCAO),
which is similar to the therapeutic window of opportunity for
-agonists (significant decrease of infarct volume with treatment
initiation delayed until 6 hours after MCAO;7 8 for
review, see Reference 99 ), supports the use of -agents as
neuroprotective drugs.
Alterations of Opioidergic Binding Sites in Infarct Border
Zone
The anterior frontal motor and temporal auditory cortices have
been defined as a border zone (or penumbra) because they are not
infarcted at early time points (1 to 6 hours) but are progressively
recruited by the expansion of the ischemic lesion. In the
anterior frontal motor cortex as well as in frontoparietal cortices,
the decreases in -opioidergic receptor densities were concomitant
with the appearance of histological damage, as seen
with the hematoxylin-eosin staining technique. Furthermore, the
decreases in [3H]diprenorphine, µ-, and
-binding sites, which occur 12 hours after MCAO, could delineate the
neuronal loss in this cortical area. Conversely, in the temporal
auditory cortex, the decreases in (at 6 hours after MCAO, layers
I-II-III; at 12 hours after MCAO, layers IV and V-VI and striatal
cortex), µ (layers I-II-III and V-VI) and
[3H]diprenorphine binding densities (layers
I-II-III and IV) at 12 hours after MCAO, occurred just before (6 hours
after MCAO) or concomitant with (12 hours after MCAO; Figure 2)
the macroscopically evident histological changes. The
changes in relative binding densities noted in the present
study occurred at the same post-MCAO time point as decreases in
Bmax values (6 hours) observed in our previous
study10 that examined only 2 post-MCAO time points (6 and
24 hours). In contrast, the decrease in µ relative binding densities
appeared slightly later (12 to 24 hours versus 6 hours) than decreases
in µ Bmax values observed
previously10 ; this difference between the present
result and our previous report10 illustrates the precise
evolution of opioid receptors that could not be demonstrated with an
experimental paradigm with only 2 time points. Two nonexclusive
hypotheses could be advanced to explain the precocious alterations in
opioidergic receptor densities: (1) a selective neuronal loss, as
reported in the hippocampus after global cerebral
ischemia16 18 and in the infarct border zone after
focal ischemia in the mouse,20 or (2)
downregulation mechanisms linked to neuronal dysfunction in hypoxic
neurons37 or in the infarct border zone after focal
cerebral ischemia.38 39 Moreover, it seems that in
the temporal auditory cortex, the alteration in µ- and
[3H]diprenorphine receptors were relatively
similar, and occurred at intermediate time points (12 to 24 hours after
MCAO). This similarity could be explained by (1) the
heterogeneity of decreases in the 3 subtypes of
opioidergic receptor as a function of post-MCAO time (early or
intermediate for - and µ-receptor decreases and delayed or
nonsignificant for the subtype) and (2) the important proportion
(30% in the cortex) of µ-receptors with regard to the overall
population of opioidergic receptors.40 41 42 The
nonselective decreases in opioidergic binding sites observed at later
time points are likely due to a neuronal death throughout the infarcted
cortices. An exception would be the temporal auditory cortex, in which
the density of -receptors was not altered in layers I-II-III, and
only at 48 hours after MCAO in layers V-VI; this finding is of
interest, because the pharmacological strategy for neuroprotection
principally targets penumbral tissue, in which there remain potentially
viable cells.
Conclusion
In summary, our results demonstrate an involvement of the
opioidergic system in focal ischemic processes. The rapid
decrease in -binding sites indicates that this receptor subtype is
particularly susceptible in the acute phase of cerebral
ischemia, and later on, µ- and all other opioidergic
receptors are decreased in chronically infarcted tissues. The selective
alteration in -receptors argue for the in vivo use of specific
-receptor ligands as markers of early neuronal death in opposition
to nonselective ligands, such as diprenorphine, which are
less-sensitive markers of the infarct extension. The relative
preservation of -binding densities might be correlated with the
neuroprotective effect of -agonists, known to be effective even when
the treatment is induced 6 hours after MCAO.7 8 9 Our
present observation would thus further speak in favor of the use of
agents as neuroprotective agents.
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Acknowledgments |
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Studies were supported by the University of Caen, the Centre National de la Recherche Scientifique, and the "Fondation pour la Recherche Médicale." The authors wish to thank Drs Alan R. Young and Simon Roussel for their expert comments and F. Mézenge and D. Divoux for their kind technical help. The investigations were performed within the framework of the IFR 47 "Neuroimagerie fonctionnelle."
Received January 28, 1999; accepted March 8, 1999.
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Editorial Comment
Anesthesiology/Critical Care Medicine The Johns Hopkins University Baltimore, Maryland
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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The use of opioid-based drugs as neuroprotectants has been of interest for many years. Of the various subclasses, the
-receptor
agonists appear to hold the most promise for cerebral ischemia.
However, the window of opportunity during which opioid receptor subtype
ligands can bind in cortex during focal ischemia has not been
well defined.
In the accompanying article, Boutin et al performed a highly systematic
investigation of the temporal sequence and topology of the loss of
opioid receptor ligand binding during permanent focal ischemia
in the mouse. Ligand binding for µ-, -, and -opioid receptors
as well as a nonselective opioid ligand was quantified at 1, 3, 6, 12,
24, and 48 hours of permanent occlusion of the distal middle cerebral
artery, which restricts injury to cerebral cortex. In general,
significant reductions of -ligand binding were detected at shorter
ischemic durations than reductions of µ-ligand binding, which
in turn preceded reductions of -ligand binding. In the
ischemic border region where infarction developed more slowly,
the same temporal sequence of subtype ligand binding tended to be
preserved, although the decrease in binding was delayed by several
hours and the loss of -ligand binding was less prominent than that
for the -ligand. The authors conclude that -ligand binding may be
an early marker of neuronal injury and that neuroprotection shown by
others with -agonists theoretically may have a longer therapeutic
window.
It is speculated that the better preservation of -ligand binding may
be related to an enrichment in presynaptic membranes. It is also
possible that neurons expressing -receptors are less vulnerable to
ischemia because of activation of these receptors during
ischemia by endogenous agonists. Because
exogenously administered agonists provide protection, further work is
needed to discern whether neurons expressing -receptors are
inherently less vulnerable to ischemia, or whether -agonists
act by modulating neurotransmitter release and thereby protect
postsynaptic neurons. In either case, the present findings that
loss of -receptor is delayed 12 to 24 hours after the onset of
ischemia is encouraging because it suggests a relatively long
therapeutic window.
Received January 28, 1999; accepted March 8, 1999.
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