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YEXNR-09316; No. of pages: 8; 4C:
Experimental Neurology xx (2006) xxx – xxx
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Brain neuroprotection by scavenging blood glutamate
Alexander Zlotnik a , Boris Gurevich a , Sergei Tkachov a , Ilana Maoz b ,
Yoram Shapira a , Vivian I. Teichberg b,⁎
a
Division of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel
b
Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel
Received 27 June 2006; revised 2 August 2006; accepted 4 August 2006
Abstract
Excess glutamate in brain fluids characterizes acute brain insults such as traumatic brain injury and stroke. Its removal could prevent the
glutamate excitotoxicity that causes long-lasting neurological deficits. As blood glutamate scavenging has been demonstrated to increase the efflux
of excess glutamate from brain into blood, we tested the prediction that oxaloacetate-mediated blood glutamate scavenging causes neuroprotection
in a pathological situation such as closed head injury (CHI), in which there is a well established deleterious increase of glutamate in brain fluids.
We observed highly significant improvements of the neurological status of rats submitted to CHI following an intravenous treatment with 1 mmol
oxaloacetate/100 g rat weight which decreases blood glutamate levels by 40%. No detectable therapeutic effect was obtained when rats were
treated IV with 1 mmol oxaloacetate together with 1 mmol glutamate/100 g rat. The treatment with 0.005 mmol/100 g rat oxaloacetate was no
more effective than saline but when it was combined with the intravenous administration of 0.14 nmol/100 g of recombinant glutamateoxaloacetate transaminase, recovery was almost complete. Oxaloacetate provided neuroprotection when administered before CHI or at 60 min post
CHI but not at 120 min post CHI. Since neurological recovery from CHI was highly correlated with the decrease of blood glutamate levels
(r = 0.89, P = 0.001), we conclude that blood glutamate scavenging affords brain neuroprotection Blood glutamate scavenging may open now new
therapeutic options.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Acute brain neurodegeneration; Closed head injury; Brain glutamate levels; Blood glutamate scavenging; Oxaloacetate; Glutamate-oxaloacetate
transaminase; Neuroprotection; Neurological severity score; Spontaneous recovery
Introduction
It is nowadays well established that abnormally high
glutamate (Glu) levels in brain interstitial and cerebrospinal
fluids (ISF and CSF, respectively) are the hallmark of several
neurodegenerative conditions that result from acute events, such
as stroke, traumatic brain injury or bacterial meningitis (Castillo
et al., 1996; Johnston et al., 2001; Zauner et al., 1996) or
develop chronically in diseases such as glaucoma, amyotrophic
lateral sclerosis or HIV dementia (Ferrarese et al., 2001; Shaw et
al., 1995; Spranger et al., 1996; Spreux-Varoquaux et al., 2002).
Because excess Glu exerts neurotoxic properties, a great deal of
efforts have been made in recent years to reach a better
⁎ Corresponding author.
E-mail address: [email protected] (V.I. Teichberg).
understanding of how the brain protects itself from excess Glu,
and on ways drugs could provide neuroprotection.
Brain inherent protection is credited mainly to the presence,
both on nerve terminals and on astrocytes, of members of a large
family of Na+-dependent Glu transporters which bind and take
up Glu and guarantee that the very high concentrations of Glu
transiently present after synaptic or astrocytic release (Danbolt,
2001) are soon decreased to concentrations at which Glu exerts
neither overt excitatory nor excitotoxic activities (Sattler and
Tymianski, 2001).
So far, however, little attention has been given to the Glu
transporters present on brain blood vessels (Danbolt, 2001) and
on their role in the control of brain extracellular Glu. The
existence of a brain-to-blood efflux of Glu, though discovered
already 45 years (Berl et al., 1961) ago, has also been widely
ignored. Nevertheless, in vitro studies (O'Kane et al., 1999)
have proposed that the Glu transporters present on the
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antiluminal side of the brain capillary endothelial cells could be
responsible for the elimination of Glu from brain into blood
while in vivo studies (Hosoya et al., 1999) have detailed the
efflux of L-aspartate through the blood–brain barrier. Following
this lead, we recently established (Gottlieb et al., 2003) that a
rapid brain-to-blood Glu efflux indeed takes place since one
observes radioactive Glu appearing in blood as soon as it is
injected into rat brain lateral ventricles. We further showed that
this brain-to-blood Glu efflux can be accelerated by the creation
of a larger Glu concentration gradient between the cerebrospinal
fluid/capillary endothelial cell and blood plasma. Using two
paradigms based on the fate of radiolabeled Glu infused into
brain, we observed its increased appearance in blood and
enhanced disappearance from brain following a decrease of
blood Glu levels (Gottlieb et al., 2003). A decrease of blood Glu
levels and an increase of the driving force for the brain-to-blood
Glu efflux was achieved by exploiting the Glu scavenging
properties of the blood resident enzymes glutamate-pyruvate
transaminase (GPT) and glutamate-oxaloacetate transaminase
(GOT) which transform Glu into 2-ketoglutarate in the presence
of the respective Glu co-substrates, pyruvate and oxaloacetate
(Gottlieb et al., 2003).
Having demonstrated that the latter co-substrates, also called
blood Glu scavengers (Gottlieb et al., 2003), cause an increased
brain-to-blood Glu efflux, we have tested now the prediction
that the intravenous administration of oxaloacetate should
provide brain neuroprotection to rats submitted to an insult
resulting in the release of excess Glu in brain interstitial and
cerebrospinal fluids. In rats as in humans, traumatic brain injury
is accompanied by a large increase of Glu in brain fluids (Baker
et al., 1993; Bullock et al., 1998; Palmer et al., 1993; Richards
et al., 2003; Rose et al., 2002; Vespa et al., 1998) and there is a
tight correlation between the elevated levels of Glu and the poor
neurological outcome (Koura et al., 1998; Zauner et al., 1996;
Zhang et al., 2001). Using a rat model of traumatic brain injury,
we confirm here the prediction of the brain neuroprotective
effects of blood Glu scavenging and establish a very tight
correlation between the improvement of the neurological status
and the reduction of blood Glu levels. These observations could
have clinical implications.
and to the Guidelines for the Use of Experimental Animals of
the European Community. The experiments were approved by
the Animal Care Committees of Ben-Gurion University of the
Negev and of the Weizmann Institute. Spontaneously
breathing male Sprague Dawley rats weighing 200–300 g
were anesthetized with a mixture of isoflurane (initial inspired
concentration 2%) in 100% oxygen (1 l/min). The rectal
temperature was maintained at 37°C using a heating pad and
anesthesia was considered as sufficient for surgery when tail
reflex was abolished. Catherization of the tail vein was carried
out with a BD Neoflon 24 g catheter for allowing fluid
infusion. Catherization of the tail artery was performed to
allow blood sampling and determination of blood pressure
and heart rate. Blood samples were analyzed for pH, pO2,
pCO2, HCO3−. Glucose levels were measured with Accu-Chek
sensor comfort. After scalp infiltration with bupivacaine 0.5%,
it was incised and reflected laterally and a cranial impact of
0.5 J was delivered by a silicone-coated rod which protruded
from the center of a free-falling plate as previously described
(Shapira et al., 1988). The impact point was 1–2 mm lateral
to the midline on the skull's convexity. Following closed head
injury (CHI), the incision was sutured and rats were laid on
their left side in order to recover from anesthesia. A
neurological severity score (NSS) was evaluated 1 h after
CHI.
Neurological severity score
The NSS was determined (Shapira et al., 1988) by a
blinded observer. Points are assigned for alterations of motor
functions and behavior so that the maximal score of 25
represents severe neurological dysfunction while a score of 0
indicates an intact neurological condition. Specifically, the
following were assessed: ability to exit from a circle (3 point
scale), gait on a wide surface (3 point scale), gait on a narrow
surface (4 point scale), effort to remain on a narrow surface
(2 point scale), reflexes (5 point scale), seeking behavior
(2 point scale), beam walking (3 point scale), and beam balance
(3 point scale).
Experimental design
Materials and methods
Materials
pET28 was purchased from Novagen. L-Glutamate, oxaloacetate, NAD, glycine, and hydrazine hydrate were purchased
from Sigma. Glutamate dehydrogenase was purchased from
Roche. Glutamate-oxaloacetate transaminase (GOT) cDNA was
cloned from human hepatoma cells hepG2, expressed in E. coli
and purified by Ni-agarose chromatography. GOT assay kit was
purchased from Sigma.
Animal anesthesia
The experiments were conducted according to the
recommendations of the Declarations of Helsinki and Tokyo
The 1-h assessment of NSS was followed 15 min later by
a 30-min-long intravenous administration at a rate of 30 μl/
min/100 g of a solution containing either saline, or the
drugs to be administered. Following treatment, the animals
were returned to their cages and given free access to food
and water. At 24 h and 48 h, the assessment of NSS was
repeated.
Determination of blood/plasma Glu
Aliquots of deproteinized whole blood/plasma (200 μl
aliquot) were used for the determination of Glu concentration
by the fluorometric method of Graham and Aprison (1966). All
determinations were done at least in duplicates. The results are
expressed as mean ± SD.
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Brain water content
Brain hemispheres were removed 120 min after CHI in some
groups while in others, brain tissue samples of approximately
50 mg were excised at 24 h post CHI from a location
immediately adjacent to the area of macroscopic damage in the
left hemisphere and from a corresponding area in the right
hemisphere. These tissue samples were used for determination
of water content. Water content was determined from the
difference between wet weight (WW) and dry weight (DW).
Specifically, after WW of fresh brain tissue samples was
obtained, samples were dried in a desiccating oven at 120°C for
24 h and weighed again to obtain DW. Tissue water content (%)
was calculated as (WW − DW) × 100 / WW.
Statistical analysis
The a priori hypothesis was that the Glu concentrations in
blood samples would differ for treatment groups versus
controls. Accordingly, this comparison was made with a t-test
(differences were considered as significant when P < 0.05). The
significance of comparisons of the NSS between different
groups was assessed using analysis of variance (ANOVA) with
Bonferroni post hoc testing. The minimal level of significance
accepted was P < 0.05. Data are presented as means ± SD or
SEM when n > 8. Differences were considered as significant
when P < 0.05.
Results
Intravenous OxAc improves recovery from CHI
We examined whether OxAc-mediated blood Glu scavenging (Gottlieb et al., 2003) causes neuroprotection in a
pathological situation, such as closed head injury (CHI), in
which there is a well-established deleterious increase of Glu in
brain fluids (Baker et al., 1993; Bullock et al., 1998; Palmer
et al., 1993; Richards et al., 2003; Rose et al., 2002; Vespa et al.,
1998). We therefore submitted rats to CHI and treated them
either with intravenous OxAc, Glu or saline, as described in
Fig. 1. We also investigated the effects of an intravenous
treatment with OxAc + Glu, as the presence of elevated Glu in
blood is expected to neutralize the OxAc-mediated decrease of
blood Glu levels. Fig. 1 shows that at both 24 and 48 h post
CHI, animals treated with OxAc recovered best from CHI while
those treated with Glu recovered the least. Animals treated with
OxAc + Glu had a similar recovery as those treated with saline.
It is of interest to note that in comparison to the NSS measured
1 h post CHI, all rats showed a very significant recovery after
48 h.
To correlate the therapeutic effect of OxAc with its blood Glu
scavenging activity, we investigated the blood Glu levels before
and after CHI, as well as the outcome of the subsequent 30-minlong treatments with either OxAc or saline. Fig. 2 shows that
CHI caused by itself a 20% reduction (Student's t-test
P = 5 × 10− 4) of the blood Glu levels measured after 60 min,
and those were further reduced to 40% by the treatment with
Fig. 1. Effects of a blood Glu scavenger and of Glu on the recovery from TBI.
Rats were submitted to TBI and a NSS established after 60 min. Rats were then
injected intravenously for a total duration of 30 min (as in Fig. 3A) either with
saline (30 μl/min/100 g), OxAc (30 μmol/min/100 g) or OxAc + Glu (30 μmol/
min/100 g each). Twenty-four and forty-eight hours later, rats were submitted to
various tests to establish the NSS. The scores shown above each column
correspond to NSS averages with bars indicating the standard error of the mean.
The groups from left to right are as follows: control saline (n = 33) at 1 h and
24 h; OxAc (n = 32); Glu (n = 32); OxAc + Glu (n = 17) at 24 h and 48 h; one-way
ANOVA and Bonferroni multiple comparison tests: at 24 h: saline versus OxAc:
P < 0.05; saline versus Glu: P < 0.01; Glu versus OxAc: P < 0.001; saline versus
OxAc + Glu: P > 0.05. At 48 h: saline versus OxAc: P < 0.001; OxAc versus Glu:
P < 0.001; OxAc versus OxAc + Glu: P < 0.05.
intravenous OxAc, but not with saline. The OxAc-induced
decrease of blood Glu is transient and is soon followed by a
suggested compensatory influx into blood of Glu originating
from peripheral organs that sense the momentous decrease in
the blood Glu levels (Gottlieb et al., 2003).
Intravenous OxAc fastens recovery from CHI
To compare the time course of the spontaneous recovery
from CHI of saline- versus OxAc-treated rats, we monitored
their NSS values over 24 days. Fig. 3 shows that an extensive
recovery from the neurological deficits inflicted by CHI occurs
over this time period. However, the rate of recovery in the
OxAc-treated rats is significantly faster than that of the salinetreated rats, and the latter remain at 25 days with an overt
neurological handicap.
The recovery from CHI correlates with the decrease of
blood Glu levels
In order to demonstrate that the NSS changes are correlated
with the changes of blood Glu levels, we plotted in Fig. 4 the %
NSS improvement of individual rats assessed 24 h after CHI
versus the %decrease of their blood Glu levels measured 90 min
after CHI. A strong correlation (r = 0.89) with a high statistical
significance (P = 0.001) was observed revealing that a 40%
decrease of blood Glu levels affords an almost optimal
improvement of the NSS. Thus, low blood Glu levels that
facilitate an increased brain-to-blood Glu efflux appear to exert
a brain neuroprotective effect while high blood Glu levels are
deleterious. This conclusion is in line with the clinical
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Fig. 2. Blood Glu levels following a traumatic brain injury. Glu levels are
expressed as the percent of the basal Glu levels measured before TBI. Blood
aliquots are removed 60 min after TBI, at 75 min (i.e., 15 min of intravenous
injection of either saline 30 μl/min/100 g or 30 μmol/min/100 g OxAc) and at
90 min (end of intravenous treatment). The results are presented as mean ± standard
error of the mean (n = 12–13). Paired t-test: 0 versus 60 min saline P = 5 × 10− 4; 0
versus 60 min OxAc P = 10− 4; 60 min versus 75 min saline: P = 0.59; 60 min
versus 75 min OxAc: P = 3 × 10− 3; 75 min versus 90 min saline: P = 0.89; 75 min
versus 90 min OxAc: P = 2 × 10− 4.
observations that establish a linear correlation between CSF and
plasma Glu concentrations (Castillo et al., 1997), and a very
significant association between the high blood Glu levels and
the neurological deterioration and outcome after stroke (Castillo
et al., 1997; Serena et al., 2001) and intracerebral hemorrhage
(Castillo et al., 2002).
OxAc decreases brain edema after CHI
To assess whether the treatment with OxAc improves, in
addition to the NSS, other parameters that reflect the severity of
the inflicted CHI, we also measured, 120 min and 24 h after
CHI, the extent of brain edema in OxAc-treated versus salinetreated animals. Brain edema is partly due the presence of
excess Glu since it is decreased by glutamate receptor
antagonists (Furukawa et al., 2003; Shapira et al., 1992;
Westergren and Johansson, 1992; Westergren and Johansson,
1993). The efficient removal of brain excess Glu is thus
expected to decrease the edema. Fig. 5 shows that the OxAc
Fig. 3. Time course of the recovery from TBI in saline (n = 7) and OxAc-treated
(n = 7) rats as monitored by NSS values presented here as mean ± standard
deviation. Student's t-test for saline versus OxAc at 25 days: P = 0.006.
Fig. 4. Correlation between the decrease of blood Glu levels and the
improvement of NSS. The %blood Glu decrease and the %NSS decrease of
individual rats were calculated respectively as follows: (Glut=0 − Glut=90 min) /
Glut=0, (NSSt=1 h − NSSt=24 h) / NSSt=1 h.
treatment caused a significant reduction of brain water content
at 24 h.
The beneficial effect of OxAc was not observed in the other
groups for which the treatment includes the intravenous
administration of Glu (data not shown). Since OxAc could act
on brain edema as an osmotic agent, we measured both the
blood osmolality and Na content both before and after the 30min-long treatments with OxAc or saline. There was no change
in the saline group (303 ± 3.5 mosM (pretreatment) versus
299 ± 3 mosM (post-treatment); Na: 140 ± 1 meq/l versus
141 ± 0.8 meq/l) but a very significant increase in the OxAcgroup: (301 ± 5 mosM versus 338 ± 8 mosM; Na: 139 ± 1 meq/
l versus 164 ± 3.4 meq/l). The hypernatriema and hyperosmolarity in the OxAc-treated rats can be accounted by the fact that
OxAc is administered together with 2 Na equivalents. To
determine that the NSS improvement and edema reduction in
the OxAc-treated rats were not due to an osmotic “therapy”, we
submitted rats after CHI to a hypertonic (3% NaCl) saline
treatment. As the respective NSS values obtained at 60 min and
Fig. 5. Brain edema formation at 120 min and 24 h following TBI and treatment
with either saline 30 μl/min/100 g or 30 μmol/min/100 g OxAc. The columns
represent mean ± standard deviation; 120 min: saline (n = 6); OxAc (n = 6);
P = 0.09; 24 h: saline (n = 7); OxAc (n = 6) P = 0.008; the P values correspond to
unpaired t-tests.
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24 h post CHI were 15.6 ± 3.6 and 12.4 ± 5.3 (n = 7; P = 0.16),
we conclude that the beneficial effects of OxAc are not the
result of an osmotic therapy.
Therapeutic dose range of OxAc
The pharmacological effects described above were all
obtained following the administration of 1 mmol OxAc/100 g
rat weight. This dose produces an almost complete recovery of
rat submitted to CHI. To determine the effective dose range of
OxAc, we repeated CHI experiments while decreasing the
OxAc dose administrated down to 0.01 mmol/100 g rat weight.
Fig. 6 shows that the therapeutic effects of OxAc are dosedependent with an EC50 value around 0.01 mmol/g rat weight.
The therapeutic effect of OxAc depends on the presence of GOT
As the smaller extent of neurological recovery obtained with
the low doses of OxAc (< 1 mmol/100 g rat weight—Fig. 6)
could be due to slower rates of activation of blood resident
GOT, we investigated whether the low doses of OxAc (i.e.,
of the GOT co-substrate concentration) could be compensated by increasing blood GOT concentrations, as expected
from the Michaelis–Menten enzyme rate equation. Fig. 7
shows that it is indeed the case since an almost complete
neurological recovery could be obtained when rats were
treated IV with 0.005 mmol OxAc/100 g (a dose which by
itself has no detectable therapeutic effect) administered
together with 0.14 nmol recombinant GOT/100 g rat. This
GOT dose increases by about 30-fold the basal amount of
blood GOT.
Therapeutic time window of OxAc
Fig. 8 illustrates the fact that OxAc exerts a neuroprotective
action when administered just before submitting rats to CHI or
Fig. 6. The neurological improvement caused by OxAc after TBI is dose-dependent.
The %neurological recovery at 24 h was calculated as (NSSt=1 h − NSSt=24 h) /
NSSt=1 h. The results are presented as mean±standard deviation (n=6–8). Note that
the abscissa show the amounts of OxAc injected in mmol/100 g.
Fig. 7. Potentiation of the therapeutic effects of OxAc after TBI by the
concomitant intravenous administration of recombinant glutamate-oxaloacetate
transaminase. GOT. Rats were submitted to TBI and treated with a solution
containing 0.14 nmol GOT/100 g rat and increasing amounts of OxAc. The %
neurological recovery at 24 h was calculated as (NSSt=1 h−NSSt=24 h)/NSSt=1 h.
The results are presented as mean ± standard deviation (n = 6–8). Note that the
abscissa show the amounts of OxAc injected in μmol/100 g.
when the treatment is delayed to 60 min post CHI. It is
however no more effective when it is delayed by 2 h post
CHI. These particular time points were selected because of the
well-established duration of the excess Glu in rat brain fluids
following CHI (Faden et al., 1989; Palmer et al., 1993; Rose
et al., 2002; Stoffel et al., 2002) or stroke (Guyot et al., 2001;
Margaill et al., 1996; Phillis et al., 1996). In all cases,
following the brain insult, there is a rapid and transient
elevation of excess Glu in brain fluids that does not last more
than 2 h. Thus, one can predict that OxAc will be effective in
removing excess brain Glu only when administered before the
brain injury or during the time window of elevated Glu but
not after. The results presented in Fig. 8 are in line with this
prediction.
Fig. 8. Therapeutic time window of OxAc. Rats submitted to TBI were either
pretreated with 30 μmol/min/100 g OxAc or treated after 60 min or 120 min post
TBI with 30 μmol/min/100 g OxAc. Twenty-four and forty-eight hours later, rats
were submitted to various tests to establish the NSS. The results are presented as
NSS averages with bars indicating the standard deviation (n = 8).
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Discussion
In the present work, we tested the prediction that blood Glu
scavenging should produce brain neuroprotection in a pathological situation characterized by the presence of excess Glu in
brain fluids. CHI was chosen as a test system because the
occurrence of elevated Glu levels in brain fluids upon brain
injury has been extensively documented (Baker et al., 1993;
Bullock et al., 1998; Palmer et al., 1993; Richards et al., 2003;
Rose et al., 2002; Vespa et al., 1998). Moreover, there is a tight
correlation between the elevated levels of Glu and the poor
neurological outcome (Koura et al., 1998; Zauner et al., 1996;
Zhang et al., 2001) suggesting that the removal of excess Glu
may possibly improve the neurological outcome after CHI.
OxAc was chosen as a blood Glu scavenger because it is more
effective than pyruvate (Gottlieb et al., 2003).
As predicted from its blood Glu scavenging activity and
ability to increase the efflux of excess Glu from brain into blood
(Gottlieb et al., 2003), OxAc was found here to exert a potent
brain neuroprotection manifested by the enhanced neurological
recovery after CHI and brain edema reduction.
It could be argued that the neuroprotective effects of OxAc
are not due to its blood Glu scavenging activity but rather to a
therapeutic activity exerted within the brain. Two major
neuroprotective mechanisms could be here involved: (a)
OxAc can contribute to an improvement of NAD-linked
mitochondrial energetics via an enhancement of the malateaspartate shuttle. (b) Being a ketoacid, OxAc has the ability to
scavenge hydrogen peroxide (H2O2), one of the main reactive
oxygen species generated by traumatic brain injury (Desagher
et al., 1997).
Several rather compelling arguments, however, can be raised
to support the contention that OxAc exerts its neuroprotective
effects mainly via blood Glu scavenging.
(1) The OxAc-induced neurological recovery after CHI is
not observed in rats treated with OxAc and glutamate. This
finding is in line with the view that the presence of excess Glu in
blood prevents OxAc to effectively exert its blood Glu
scavenging action which is necessary for a therapeutic enhanced
efflux of excess Glu from brain into blood. Would OxAc exert
its therapeutic effect in brain, it is not clear why this action
would be prevented by intravenous excess Glu since the
entrance of OxAc first from blood into the brain and then further
into neurons takes place via dicarboxylate transporters such as
the NaDC-3 which are not inhibited by Glu (Burckhardt et al.,
2005; Pajor et al., 2001). One can further argue that the Gluinduced neutralization of the therapeutic effect of OxAc must be
exerted within the blood and not within the brain since Glu does
not significantly penetrate from blood into brain neither in
normal physiological conditions (Smith, 2000) nor after the
breakdown of the blood–brain barrier (Ronne Engstrom et al.,
2005).
(2) The OxAc-induced neurological recovery after CHI takes
place following the administration of OxAc within the time
window during which excess Glu is present in brain i.e., up to
2 h post CHI. Though we did not measure the kinetics of brain
Glu elevation after CHI, numerous studies demonstrate that the
excess Glu in rat brain fluid does not last more than 2 h
following either CHI or stroke (Faden et al., 1989; Farooque et
al., 1996; Guyot et al., 2001; Phillis et al., 1996; Rose et al.,
2002) but see (Alves et al., 2005). Assuming that OxAc
therapeutic action is unrelated to blood Glu scavenging but
rather to an improvement of mitochondrial energetics, the
observation that OxAc has no therapeutic effect when
administered 2 h after CHI cannot be easily reconciled with
the observation that mitochondrial dysfunction is observed for
several hours after CHI (Singh et al., 2006; Takamatsu et al.,
1998; Xiong et al., 2005).
(3) The observation that the neurological recovery after CHI
depends on the presence in blood of both OxAc and GOT (Figs.
6 and 7) is in line with two predictions: (a) that neuroprotection
can be achieved upon OxAc/GOT-mediated blood Glu
scavenging; (b) that the rate of GOT-mediated blood Glu
scavenging via transamination will not change if the product of
the OxAc and GOT concentrations is kept constant, i.e., that
lowering the OxAc concentration in the solution injected into
blood can be compensated by increasing the GOT concentration
in that solution. The tight therapeutic synergism between blood
OxAc and GOT is also difficult to reconcile with the suggestion
of an intraparenchymal site of action, as OxAc and GOT should
be both translocated from blood into brain to exert beneficial
effects. The respective kinetics of intraparenchymal entry of
OxAc (which is specifically transported via a dicarboxylate
transporter) and GOT (which cannot gain access into the brain
via an intact BBB) are probably incompatible with a synergy of
action since the BBB opening that begins at the time of the
trauma is transient, and lasts no more than 30 min (Barzo et al.,
1996), while GOT is administered after 60–90 min post trauma.
(4) The observation that the neurological recovery after CHI is
highly correlated with the decrease of blood Glu levels (Fig. 4)
is strongly in line with the concept that blood Glu scavenging
enhances the efflux of excess Glu from brain into blood and
provides thereby neuroprotection. This strong correlation
cannot be explained by an intraparenchymal site of action of
OxAc.
Though we do not rule out the possibility that OxAc exerts
some neuroprotective effects within the brain, we believe that
the major therapeutic action of OxAc is decisively linked to its
blood Glu scavenging activity.
It is of interest to note that saline-treated rats recover from
TBI to a significant extent, though neither as fast nor as much as
the OxAc-treated rats. It is a tantalizing hypothesis that the
spontaneous recovery of rats after TBI would rely on the same
mechanism as that amplified by OxAc.
OxAc is here shown to have a relatively short time window
that appears to be tightly linked to the limited duration of excess
Glu in the rat brain after CHI (Faden et al., 1989; Farooque
et al., 1996; Guyot et al., 2001; Phillis et al., 1996; Rose et al.,
2002). Since, excess Glu in human brain can be observed
after CHI for hours and even days (Baker et al., 1993; Bullock et al., 1998; Palmer et al., 1993; Richards et al., 2003; Rose
et al., 2002; Vespa et al., 1998), and is in correlation with the
poor neurological outcome (Koura et al., 1998; Zauner et al.,
1996; Zhang et al., 2001), one might possibly expect for
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A. Zlotnik et al. / Experimental Neurology xx (2006) xxx–xxx
OxAc a much longer therapeutic time window in human patients
than in rats. One might further expect that the administration of
this blood Glu scavenger may display in man a therapeutic effect
which has so far not been observed for glutamate receptor
antagonists. One possible benefit is that the enhanced brain-toblood Glu efflux produced by blood Glu scavenging is selflimiting since it slows down in parallel with the decrease of brain
Glu. Thus, it will eliminate excess Glu without preventing Glu
from exerting a role in neurorepair (Biegon et al., 2004), a factor
that has been suggested (Ikonomidou and Turski, 2002) to
account for the failure of glutamate receptor antagonists in
human clinical studies.
Acknowledgments
This work was supported in part by grants to VIT from the
Nella and Leon Benoziyo Center for Neurological Diseases; the
Irwin Green Fund for Studying the Development of the Brain, the
Yeshaya Horowitz Foundation, the Carl and Micaela EinhornDominic Institute for Brain Research, the Weizmann-Negri Fund
and the Israel Cancer Research Fund. The data obtained are part
of A.Z.'s PhD thesis. VIT is the incumbent of the Louis and
Florence Katz-Cohen Chair of Neuropharmacology.
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