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 Previous Article
The Journal of Neuroscience, November 15, 1998, 18(22):9564-9571
Attenuated Neurotransmitter Release and Spreading Depression-Like
Depolarizations after Focal Ischemia in Mutant Mice with Disrupted Type
I Nitric Oxide Synthase Gene
Masao
Shimizu-Sasamata2,
Prince
Bosque-Hamilton1,
Paul L.
Huang3,
Michael A.
Moskowitz2, and
Eng H.
Lo1
1 Neuroprotection Research Laboratory, Departments of
Neurology and Radiology, 2 Stroke and Neurovascular
Regulation Laboratory, Departments of Neurosurgery and Neurology, and
3 Cardiovascular Research Center, Department of Medicine,
Massachusetts General Hospital, Harvard Medical School, Charlestown,
Massachusetts 02129
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ABSTRACT |
Nitric oxide (NO) plays a complex role in the pathophysiology of
cerebral ischemia. In this study, mutant mice with disrupted type I
(neuronal) NO synthase (nNOS) were compared with wild-type littermates
after permanent focal ischemia. Cerebral blood flow in the central and
peripheral zones of the ischemic distribution were measured with laser
doppler flowmetry. Simultaneously, microdialysis electrodes were used
to measure extracellular amino acid concentrations and DC potential in
these same locations. Blood flow was reduced to <25 and 60% of
baseline levels in the central and peripheral zones, respectively;
there were no differences in nNOS mutants versus wild-type mice. Within
the central ischemic zone, DC potentials rapidly shifted to  20 mV in
all mice. In the ischemic periphery, spreading depression (SD)-like
waves of depolarization were observed. SD-like events were
significantly fewer in the nNOS mutant mice. Concurrent with these
hemodynamic and electrophysiological perturbations, extracellular
elevations in amino acids occurred after ischemia. There were no
detectable differences between wild-type and mutant mice in the
ischemic periphery. However, in the central zone of ischemia,
elevations in glutamate and GABA were significantly lower in the nNOS
mutants. Twenty-four hour infarct volumes in the nNOS mutant mice were
significantly smaller than in their wild-type littermates. Overall, the
number of SD-like depolarizations and the integrated efflux of
glutamate were significantly correlated with infarct size. These
results suggest that NO derived from the nNOS isoform contributes to
tissue damage after focal ischemia by amplifying excitotoxic amino acid
release in the core and deleterious waves of SD-like depolarizations in
the periphery.
Key words:
ischemia; excitotoxicity; knock-out mice; microdialysis; NO; spreading depression
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INTRODUCTION |
Nitric oxide (NO) is a highly
diffusible gas, and biomathematical models have suggested that it can
act at distances far from the original sites of release (Gally et al.,
1990 ; Wood and Garthwaite, 1994). Recently, it has been shown that NO
can diffuse away from the original synapse where it is released and can
increase neurotransmitter efflux in adjacent synapses (Schuman and
Madison, 1994). In synaptosomal preparations, NMDA-stimulated glutamate
release was significantly attenuated by inhibition of NO synthase (NOS)
(Montague et al., 1994). In hippocampal slices, NO donors increased and
NOS inhibitors decreased NMDA-stimulated release of glutamate,
respectively (Jones et al., 1995). Similar results were obtained in
cerebellar slices, in which NO potentiated NMDA-stimulated aspartate
release (Dickie et al., 1992). Recently, we used microdialysis to show
that NMDA-stimulated glutamate release was attenuated in mutant mice
with a disrupted type I (neuronal) NOS (nNOS) gene compared with
wild-type mice (Kano et al., 1998). These results suggest that NO can
act as an amplifier of neurotransmitter release in vivo.
Under conditions of cerebral ischemia in which NO release is elevated
(Malinski et al., 1993), NO might serve to potentiate the efflux of
glutamate, thus acting as a molecular amplifier of acute excitotoxicity.
In focal ischemia, the center of the lesion comprises tissue with dense
ischemia (core), whereas the peripheral zones comprise tissue with mild
to moderate reductions in blood flow (penumbra) (Astrup et al., 1981).
Maturation of the ischemic infarct is related to expansion of the core
and collapse of the penumbra. It has been suggested that penumbral
collapse is mediated by waves of spreading depression (SD)-like
depolarizations (Hossman, 1996). Because these SD-like events involve
elevations in extracellular glutamate (Fabricius et al., 1993; Marranes
et al., 1988), it is reasonable to hypothesize that after focal
ischemia, NO amplifies the excitotoxic release of glutamate and
mediates the occurrence of SD-like events in the ischemic periphery.
In the present study, we investigate this hypothesis by comparing the
ischemic profiles of SD-like events and glutamate release in wild-type
mice versus mutant mice with disrupted nNOS genes. The central and
peripheral zones of focal ischemia are examined separately, and these
measurements are correlated with final infarct size.
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MATERIALS AND METHODS |
Animal model. Male and female mutant mice with
disruptions in the gene encoding for type I or nNOS were used together
with their wild-type littermates (Huang et al., 1993). Body weights were in the 20-27 gm range. Some of these mice were genotyped to
confirm disruption of the gene in the mutants.
Mice were anesthetized with  -chloralose (40 mg · kg 1 · hr1,
i.v.). A catheter was inserted into the femoral artery for continuous measurement of mean arterial blood pressure (Maclab/8S, AD
Instruments), and the trachea was cannulated. Thereafter, the mice were
artificially ventilated with a mechanical ventilator (SAR-830/P,
CWE) using a mixture of 70% N2O and 30%
O2. Ventilation parameters were set at optimal rates that
have been previously determined: 0.15-0.2 sec inspiration time,
110/min respiratory rate, and 250-350 ml/min inspiratory flow. Once
the animals were stabilized, oxygen concentrations in the flow mix were
adjusted to obtain arterial PO2 levels of ~140-160 mmHg. End-tidal CO2 was continuously monitored
with a microcapnometer (Columbus Instruments) and maintained at
4.8-5.5%, which corresponds to PCO2 levels of
~30-40 mmHg. Arterial blood gases and pH were measured at the end of
each experiment. Rectal core temperatures were maintained at
36.9-37.1°C using a thermostatically controlled heating pad.
After obtaining baseline recordings of hemodynamic, microdialysis, and
electrical status (Fig. 1) for at least
30 min, focal cerebral ischemia was induced by permanent occlusion of
the middle cerebral artery (Chiamulera et al., 1992). Briefly, a 2 mm
craniotomy was drilled in the inferior portion of the temporal bone to
expose the right middle cerebral artery, which was then cauterized at a
level just superior to the inferior cortical vein. In this model, the
lenticulostriate arteries were left intact; thus only cortical ischemia
should be obtained. This approach was adapted from that used to obtain
focal cortical ischemia in rats (Brint et al., 1988). Hemodynamic,
microdialysis, and electrical data were collected for 90 min after
ischemia. After that, all catheters were removed, and mice were allowed
to return to their cages for a 24 hr recovery period before being
killed to quantify infarct volumes (see below).
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Figure 1.
Schematic showing the location of microdialysis
probes and electrodes and laser doppler flow probes
(LDF) in relation to the zones of focal cortical
ischemia induced via occlusion of the distal middle cerebral artery in
mice.
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In vivo microdialysis. Microdialysis probes (0.25 mm outer
diameter, 1 mm membrane length; Applied Neuroscience, London, UK) were
stereotactically inserted into the cortex in locations that corresponded to what had been previously determined in pilot studies to
be either central or core areas of the ischemic focus (from bregma: 3.5 mm lateral, 1.5 mm caudal, 1 mm depth), or peripheral zones of the
ischemic distribution (from bregma: 1.5 mm lateral, 1.5 mm caudal, 1 mm
depth). The correct location of these probes was confirmed in all
experiments by observing the levels of blood flow reduction that were
measured via laser doppler flowmetry (Fig. 1; also see below). The
probes were perfused with artificial CSF at a rate of 2 µl/min.
Samples were collected at 10 min fractions resulting in 20 µl sample
volumes. Microdialysis samples were then analyzed with reversed phase
HPLC to quantify concentrations of various amino acid neurotransmitters
and neuromodulators, as previously described (Lo et al., 1998a,b).
L-Glu and GABA were measured because they represent the
major excitatory and inhibitory transmitters in mammalian brain (Hertz,
1979; Tossman et al., 1986). D-Ser and Gly were measured
because they are the primary neuromodulators at the NMDA receptor site
(Wroblenski et al., 1989; Kemp and Leeson, 1993), and NMDA-mediated
toxicity is a major pathological mechanism after cerebral ischemia
(Choi, 1992). Tau was assessed because there are data suggesting that
it may be a neuroprotective inhibitory neuromodulator (Lehmann et al., 1984; Schurr et al., 1987). Phosphoethanolamine (PEA) was
assessed because it is a lipid component that may reflect membrane
damage and recycling (Hagberg et al., 1985). L-Ser was
examined as a "control" enantiomer for D-Ser. Finally,
L-Ala was examined because it is considered a
non-neuroactive amino acid (Hagberg et al., 1985).
Surface electrical recordings. Steady-state DC potentials
and electrocorticograms were recorded with the electrode that was built
into the microdialysis probe (see above). An Ag/AgCl reference electrode was placed subcutaneously in the scalp. Electrical signals were captured (Axoprobe 1A, Axon Instruments) and analyzed with the
Maclab data system (Maclab/8S, AD Instruments). The characteristics of
these combination microdialysis probes and electrodes have been
previously described (Obrenovitch et al., 1993).
Laser doppler flowmetry. Regional cerebral blood flow was
assessed at the microdialysis site by a laser doppler flowmeter (Periflux PF2B, Perimed). The flow probe (0.6 mm diameter) was placed
onto the cortex adjacent to the microdialysis probe and away from large
surface vessels (Fig. 1). Steady-state baseline values were recorded
before middle cerebral artery occlusion, and blood flow data were then
expressed as percentage of the preocclusion baseline.
Determination of infarct volume. Mice were killed to
quantify infarct volumes at 24 hr after focal ischemia. Brains were
removed and frozen in isopentane (35°C). Coronal brain sections (20 µm thick) were cut with a cryostat, collected at 1 mm intervals, and
stained with hematoxylin-eosin. Infarct areas were analyzed with a
digital image analyzer, and infarct volumes were calculated by
integrating along five equally spaced coronal sections. Two methods of
calculating infarct volume were used. In the first method, infarcted
areas were directly integrated. In the second method, effects of edema
and brain swelling were normalized with a standard formula whereby
noninfarcted areas were subtracted from contralateral hemispheric areas
(Lin et al., 1992). This approach yields an "indirect" measurement
of infarct volume.
Data analysis. Data are reported as mean ± SEM.
Continuous measurements over time (blood pressure, laser doppler flow,
and microdialysis) were examined with two-way repeated measures ANOVA for comparisons between nNOS mutants and their wild-type littermates. Additionally, post hoc Fisher's PLSD analyses were
performed at ischemic time points to compare wild-type versus nNOS
mutant mice. Microdialysis measurements of extracellular amino acid
concentrations were also analyzed in terms of total integrated efflux
over the course of ischemia as well as maximal absolute release during that time. Electrical recordings were analyzed in terms of the degree
of DC depolarization and the number of transient cortical SD-like
events after ischemia. Differences in these parameters between mutants
and wild-type mice were analyzed by two-tailed Student's t
tests. Comparisons of infarct sizes between wild-type and mutant mice
were also performed with two-tailed Student's t tests.
Linear regressions were performed to assess the correlations between
infarct sizes and the electrophysiological and microdialysis measurements. p < 0.05 was considered significant.
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RESULTS |
Physiological parameters and cerebral blood flow
Arterial pH and blood gases were within the normal range for all
mice (Table 1). Mean arterial blood
pressure remained at ~80-90 mmHg throughout all experimental
periods, and there were no differences between the nNOS mutants and
their wild-type littermates (Fig. 2).
Laser doppler flowmetry showed that occlusion of the middle cerebral
artery reduced blood flow in the central zone of ischemia <25% of
baseline levels (Fig. 3A). In
the ischemic periphery, blood flow was reduced to ~50-60% of
baseline (Fig. 3B). There were no differences in the levels
of ischemic blood flow reduction in wild-type mice versus nNOS mutant
mice.
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Figure 2.
Mean arterial blood pressures remained between 80 and 90 mmHg throughout the experimental measurement period. There were
no differences between the nNOS mutants and their wild-type
littermates.
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Figure 3.
Laser doppler flowmeter probes showed that focal
ischemia resulted in severe blood flow deficits within the central
zones of the ischemic distribution (A) and mild
to moderate blood flow deficits in the ischemic periphery
(B). There were no differences in the degree of
blood flow reductions between wild-type and nNOS mutant mice.
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Neurotransmitter and neuromodulator release
Before cerebral ischemia, there were no differences in baseline
concentrations of amino acid neurotransmitters and neuromodulators between wild-type and nNOS mutant mice. After onset of focal ischemia, large and rapid elevations in neurotransmitters (L-Glu and
GABA) occurred in the central zones of ischemia in wild-type mice (Fig. 4). However, in the NOS mutants, ischemic
efflux of L-Glu and GABA were significantly reduced (Fig.
4, Table 2). For other amino acids,
including the NMDA receptor site modulators D-Ser and Gly,
the inhibitory modulator Tau, and the lipid component PEA, focal
ischemia induced statistically comparable levels of release in
wild-type and mutant mice (Fig. 4). Similarly, the non-neuroactive
amino acids L-Ser and L-Ala showed slow
progressive accumulations after ischemia in all mice (Fig. 4).
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Figure 4.
Microdialysis measurements of extracellular amino
acid concentrations in the center of the ischemic zone showed large
elevations in almost all amino acids. Two-way repeated measures ANOVA
showed that elevations in L-Glu (F = 5.645; p = 0.04) and GABA (F = 7.422; p = 0.03) were significantly lower in nNOS
mutant mice ( ) compared with wild-type littermates ( ). See Table
2 for quantitative efflux parameters. *p < 0.05 via post hoc Fisher's PLSD analyses.
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In the ischemic periphery, amino acid efflux was considerably lower
compared with that found in the central ischemic zone. Significant
efflux over baseline was only detected for L-Glu, GABA,
Tau, and PEA (Fig. 5). No elevations in
D-Ser, Gly, L-Ser, and L-Ala were
observed. There were no differences in efflux between nNOS mutants and
their wild-type littermates.
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Figure 5.
Microdialysis measurements of extracellular amino
acid concentrations in the peripheral zones of the ischemic
distribution showed small elevations in L-Glu, GABA, Tau,
and PEA. No changes in D-Ser, Gly, L-Ser, and
L-Ala were observed after ischemic onset. There were no
significant differences in response between wild-type () and mutant
() mice.
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Ischemic depolarizations
In the central zone of ischemia, a rapid negative shift in DC
potential was observed, consistent with anoxic depolarization (Fig.
6A). There were no
differences between the magnitude of these depolarizations in wild-type
mice and nNOS mutant mice (approximately 20 mV; Table
3). In contrast, electrical recordings in
the ischemic periphery revealed the presence of multiple SD-like
depolarizations (Fig. 6B). These waves of
depolarization typically involved transient negative DC shifts of
~6-8 mV. There were no differences in the magnitude of these
depolarizations in wild-type versus nNOS mutant mice. However, the
number of SD-like depolarizations was significantly reduced in the nNOS
mutants compared with their wild-type littermates (Table 3).
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Figure 6.
Representative examples of electrophysiological DC
potential measurements after onset of focal ischemia. A,
In the central zones of ischemia, rapid negative shifts in DC potential
occurred. B, In contrast, transient SD-like events were
present in the ischemic periphery. The data shown here are from a
wild-type mouse. Vertical lines at the beginning of the
traces indicate start and completion of the electrocoagulation
procedure for middle cerebral artery occlusion.
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Infarct size and correlations with amino acid efflux and
SD-like events
Permanent occlusion of the distal middle cerebral artery resulted
in focal infarction at 24 hr involving the cortex without any
detectable striatal damage in all mice. Infarct volumes in the nNOS
mutant mice were significantly smaller compared with their wild-type
littermates (Fig. 7A,B). The
sizes of the infarcts were linearly correlated with the number of
SD-like events measured in the ischemic periphery (r = 0.704; p < 0.01). Infarct sizes were also linearly
correlated with the cumulative efflux of L-Glu (r = 0.786; p < 0.05) and GABA
(r = 0.637; p < 0.05) within the central zones of focal ischemia. However, there were no significant correlations between infarct size and efflux of L-Glu or
GABA within the ischemic periphery.
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Figure 7.
Cortical infarct size after permanent focal
cerebral ischemia measured with hematoxylin-eosin staining 24 hr after
occlusion. A, Direct infarct areas measured on
representative axial slices. B, Infarct volumes were
calculated with both "direct" and "indirect" approaches (see
Materials and Methods). Infarct volumes in nNOS mutant mice were
significantly smaller compared with their wild-type littermates.
*p < 0.05 between wild-type and mutant mice.
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DISCUSSION |
It has been previously shown that mutant mice with disruptions in
the genes encoding for nNOS were significantly protected against
cerebral injury after focal (Huang et al., 1994) and global (Panahian
et al., 1996) ischemia. In the present study, we showed that reduced
cerebral infarction after permanent focal ischemia in these nNOS mutant
mice was significantly correlated with (1) decreased efflux of
glutamate within the center of the ischemic zone and (2) reduced
numbers of SD-like depolarizations in the ischemic periphery.
NO plays a complex role in the pathophysiology of cerebral ischemia
(Dawson, 1994; Iadecola et al., 1994; Iadecola, 1997; Huang and Lo,
1998). Depending on the redox milieu, it can either manifest itself as
the nitrosonium ion NO+ or the free radical
NO· (Lipton et al., 1993). The former molecular species can act
in a beneficial manner after cerebral ischemia by downregulating deleterious NMDA currents (Lei et al., 1992), whereas the latter species can contribute to free radical damage to cellular membranes, proteins, and DNA (Beckman, 1994). Another reason for the multiplicity of action of NO involves the many potential sources of NO in brain. Three isoforms of NOS have been described: type I or nNOS, type II or
inducible NOS (iNOS), and type III or endothelial NOS (eNOS). NO
derived from nNOS and iNOS isoforms can play detrimental roles in
cerebral ischemia (Iadecola, 1997). Indeed, mutant mice with disrupted
nNOS or iNOS genes suffer from smaller infarcts after focal ischemia
compared with wild-type mice (Huang et al., 1994; Iadecola et al.,
1997). In contrast, mutant mice with disrupted eNOS genes have larger
infarcts after focal ischemia (Huang et al., 1996). The protective role
for eNOS may involve hemodynamic mechanisms of compensatory
vasodilation and collateral recruitment that sustain peripheral zones
of the ischemic distribution (Lo et al., 1996). Additionally, NO can
also have effects on platelet response and the inflammatory cascade
(Moncada et al., 1991). Based on these multiple actions of NO, it is
not surprising that somewhat variable outcomes have been obtained by
pharmacological inhibition of NO synthesis in various experimental
models of focal cerebral ischemia (Buisson et al., 1992; Dawson et al.,
1992; Yamamoto et al., 1992; Yoshida et al., 1994).
Our working hypothesis here is that, in addition to these actions, NO
also serves as an amplifier of acute excitotoxicity after cerebral
ischemia. This hypothesis is based on a wide spectrum of data showing
that NO can potentiate neurotransmitter release in locally defined
neuronal networks (Gally et al., 1990; Dickie et al., 1992; Montague et
al., 1994; Schuman and Madison, 1994; Jones et al., 1995; Kano et al.,
1998). In the present study, excitotoxic efflux of glutamate after
focal cerebral ischemia was significantly reduced in mutant mice with
disrupted nNOS genes. However, these effects were only present in the
central zones of ischemia, and no differences between mutant and
wild-type mice were seen in the ischemic periphery. The most likely
reason for these results is that, although nNOS is considered a
constitutive enzyme, it can be significantly upregulated after cerebral
ischemia (Huang and Lo, 1998). In a mouse model of focal ischemia,
upregulation of nNOS takes place primarily in the ischemic core (Hara
et al., 1997). Direct measurements of NO in a cat model of focal
ischemia showed that large elevations in NO concentration only occurred in the ischemic core and not in the ischemic periphery (Ohta et al.,
1996). Therefore, it is conceivable that after focal ischemia, nNOS is
upregulated in the ischemic core, and the subsequent surge of NO then
serves to amplify the efflux of excitotoxic glutamate in these regions.
These effects of NO were specific for neurotransmitters only
(L-Glu and GABA); no differences in ischemic efflux were seen for the neuromodulators (D-Ser, Gly, Tau, and PEA).
The scenario described above leaves unanswered the question of why
there were reduced numbers of SD-like depolarizations but no
differences in glutamate efflux in the ischemic periphery of the nNOS
mutants compared with wild-type mice. If ischemic SD-like events are
indeed mediated by glutamate (Marranes et al., 1988; Fabricius et al.,
1993), and ischemic glutamate efflux is amplified by NO, then we should
expect to see reduced glutamate release in the peripheral zones of
focal ischemia as well. One possible explanation might be that the
reduced SD-like depolarizations simply reflected the reduction in
overall ischemic injury in the nNOS mutants (Ijima et al., 1992) and
merely represented an epiphenomenon not directly related to the
presence or absence of nNOS at all. Alternatively, it is possible that
amplification of gluatamte efflux within the ischemic periphery may
have occurred at the synaptic level without massive spillover into
extracellular space. Consistent with this possibility, the release of
gluatamte was 10-fold lower in the ischemic periphery compared with the
ischemic center. In this case, subtle differences within the penumbra
between nNOS mutant mice and their wild-type littermates would not be detectable with the microdialysis technique (Benveniste and Hansen, 1991).
There were significant differences in the profiles of neurotransmitter
and neuromodulator efflux in the ischemic center versus ischemic
periphery. In the center, massive efflux of almost all amino acids
occurred. In the ischemic periphery, however, only the
neurotransmitters (L-Glu and GABA) and the neuromodulators (Tau and PEA) were released. No changes in the non-neuroactive amino
acids (L-Ser and L-Ala) were detected.
Elevations in the extracellular concentrations of non-neuroactive amino
acids may reflect decreased extracellular space attributable to
astrocytic swelling (Kimelberg, 1995). These data suggest that severe
tissue injury with massive astrocytic swelling occurs only in the
ischemic core and not the ischemic periphery. The NMDA receptor site
modulators D-Ser and Gly also showed similar profiles,
being significantly released only in the ischemic center and not the
ischemic periphery. Recently, it has been suggested that
D-Ser is primarily localized within astrocytes and may be
released after stimulation of AMPA receptors that are present on
astrocytic cell membranes (Schell et al., 1997). One might speculate
that release of D-Ser in the center of the ischemic zone
occurs via astrocytic swelling and/or AMPA receptor activation. In the
latter scenario, the lack of differences in D-Ser release
between wild-type mice and nNOS mutants may be explained by the fact
that nNOS activation is closely linked to the neuronal NMDA receptor
rather than the AMPA receptor. In any case, D-Ser and Gly
significantly potentiate NMDA currents and can therefore contribute to
further excitotoxic injury after cerebral ischemia.
Although reductions in L-Glu efflux in the nNOS mutants
would be consistent with decreased excitotoxicity after ischemia, it is
important to note that GABA efflux is also significantly reduced. The
similarity between ischemic efflux profiles of L-Glu and
GABA underscore the fact that both amino acids are considered classical
neurotransmitters that are primarily released via calcium-dependent vesicular systems (Hertz, 1979; Tossman et al., 1986). The
interpretation of the changes in GABA efflux may be more difficult.
GABA, via its inhibitory actions, has been proposed to be potentially
neuroprotective after cerebral ischemia (Sternau et al., 1989; Lyden
and Hedges, 1992). However, it is known that high concentrations of
GABA can be depolarizing (Alger and Nicoll, 1982; Staley et al., 1995; van den Pol et al., 1996), and large doses of GABA have been shown to
be exacerbate hypoxic injury in neuronal cell culture models (Muir et
al., 1996).
There are a few limitations associated with the present study. First,
our measurements were restricted to the acute phase of ischemia
only. Therefore, we were unable to determine whether differences in
excitotoxic glutamate efflux in the nNOS mutants persisted as the
infarct matured over time. However, it has been shown in larger animal
models of focal ischemia that sustained elevations in glutamate
accompany the propagation of cerebral injury; i.e., excitotoxic
mechanisms are likely to contribute to the collapse of the ischemic
penumbra over time (Matsumoto et al., 1993). Therefore, it is possible
that NO can continue to amplify excitotoxic glutamate efflux even
during prolonged ischemia. Second, we were only able to obtain
measurements at single locations, whereas the spatial profile of the
ischemic penumbra is clearly three-dimensional. Nevertheless, the
appropriateness of our selected locations was confirmed with blood flow
and electrophysiological measurements. Therefore, it is likely that our
data were representative of the overall events in the central and
peripheral zones of ischemia. Finally, our correlations between
decreased infarct size and reductions in L-Glu efflux or
decreased SD-like events in the ischemic periphery cannot unequivocally
prove causality. Further experiments at the synaptic or vesicular level
may be required.
In conclusion, we showed that reduced infarctions after permanent focal
ischemia in nNOS mutant mice were significantly correlated with (1)
attenuated efflux of glutamate in the center of the ischemic distribution and (2) reduced numbers of SD-like depolarizations in the
ischemic periphery. These results are consistent with the idea that in
addition to its other multiple actions after cerebral ischemia, NO can
act as an amplifier of acute excitotoxicity.
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FOOTNOTES |
Received June 26, 1998; revised Aug. 28, 1998; accepted Sept. 4, 1998.
This work was supported in part by National Institutes of Health Grants
R29 NS32806 and R01 NS37074 (to E.H.L.), R01 NS33335 (to P.L.H.), and
P50 NS10828 (to M.A.M.) and an Established Investigatorship from the
American Heart Association (to P.L.H.).
Correspondence should be addressed to Dr. Eng H. Lo, Neuroprotection
Research Laboratory, Departments of Neurology and Radiology, Harvard
Medical School, Massachusetts General Hospital East Building 149, Room
2322, Charlestown, MA 02129.
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Abstract
Introduction
