Neuropharmacology 155 (2019) 173–184

Contents lists available at ScienceDirect

Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

The effect of pyruvate on the development and progression of post-stroke T

depression: A new therapeutic approach
Dmitry Franka,1, Ruslan Kutsa,1, Philip Tsenterb, Benjamin F. Gruenbaumc, Yulia Grinshpuna,
Vladislav Zvenigorodskyd, Ilan Shelefd, Dmitry Natanela, Evgeny Brotfaina, Alexander Zlotnika,
Matthew Boykoa,∗
a
Department of Anesthesiology and Critical Care, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
b
Division of Internal Medicine, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
c
Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USA
d
Department of Radiology, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

HIGHLIGHTS

• Post-stroke neurological deficit with concurrent elevation in glutamate levels were demonstrated, with peak glutamate levels 24 h after MCAO.
• Treatment with pyruvate led to reduced glutamate levels 24 h after MCAO and improved neurologic recovery.
• Rats demonstrated post-stroke depressive behavior that was improved by the administration of pyruvate.
• There was less anxiety-like behavior in post-stroke rats treated with placebo in comparison to the post-stroke rats treated with pyruvate or sham operated rats.
• Our main conclusion was that glutamate scavenging with pyruvate appears to be an effective as a method in providing neuroprotection following stroke and as a
therapeutic option for the treatment of PSD by reducing the consequent elevations in CNS glutamate levels.

ARTICLE INFO ABSTRACT

Keywords: Post-stroke depression (PSD) is a common and serious complication following stroke. Both stroke and depression
Glutamate scavenging have independently been associated with pathologically elevated glutamate levels in the brain's extra-cerebral
Pyruvate fluid (ECF). Here we evaluate an alternative therapeutic approach to PSD with pyruvate. Rats were randomly
Post-stroke depression assigned into one of 3 groups: Middle Cerebral Artery Occlusion (MCAO) plus pyruvate treatment, MCAO plus
Neuroprotection
placebo treatment, and sham operated rats. Post-MCAO depressive and anxiety-like behavior was assessed, along
Antidepressants
with neurological status, brain infarct zone, brain edema, blood brain barrier (BBB) breakdown, cerebrospinal
Anxiety
fluid and blood glutamate levels. Anxiety-like behavior and levels of blood alanine and α-ketoglutarate were
measured in naïve rats treated with pyruvate, as a control. Post-stroke neurological deficit with concurrent
elevation in glutamate levels were demonstrated, with peak glutamate levels 24 h after MCAO. Treatment with
pyruvate led to reduced glutamate levels 24 h after MCAO and improved neurologic recovery. Pyruvate treat-
ment reduced lesion volume, brain edema and the extent of BBB permeability 24 h post-MCAO. Naïve rats
treated with pyruvate showed increased levels of α-ketoglutarate. Rats demonstrated post-stroke depressive
behavior that was improved by the administration of pyruvate. There was less anxiety-like behavior in post-
stroke rats treated with placebo in comparison to the post-stroke rats treated with pyruvate or sham operated
rats. Glutamate scavenging with pyruvate appears to be an effective as a method in providing neuroprotection
following stroke and as a therapeutic option for the treatment of PSD by reducing the consequent elevations in
CNS glutamate levels.

Corresponding author.Ben-Gurion University of the Negev Faculty of Health Sciences, Anesthesiology and Critical Care, Soroka University Medical Center, Sderot
∗

Yitshak Reger 151, Beer Sheva, 84101, Israel.

E-mail address: matthewboykoresearch@gmail.com (M. Boyko).
1
Equal contribution.

https://doi.org/10.1016/j.neuropharm.2019.05.035
Received 21 January 2019; Received in revised form 26 May 2019; Accepted 30 May 2019
Available online 31 May 2019
0028-3908/ © 2019 Elsevier Ltd. All rights reserved.
D. Frank, et al.
Abbreviations
(PSD) Post-stroke depression
(ECF) extra-cerebral fluid
(MCAO) Middle Cerebral Artery Occlusion
(BBB) blood brain barrier
(CNS) central nervous system
(NMDA) N-methyl-D-aspartate
(BGC) blood glutamate scavenging
(MRI) magnetic resonance imaging
1. Introduction
Post-stroke depression (PSD) is a common and serious complication
of stroke. While the exact prevalence rate of PSD is difficult to define, it
is estimated that it can be seen in up to 30–35% of patients, ranging
from 20 to 60% (Dafer et al., 2008; Lenzi et al., 2008), with a peak
prevalence between six months and two years (Dafer et al., 2008).
Despite its significant impact on functional recovery and quality of life
following stroke, this psychiatric consequence is often overlooked and
untreated.
Stroke is accompanied by a sharp three to four hundred-fold in-
crease in the brain's extra-cerebral fluid (ECF) and cerebrospinal fluid
(CSF) glutamate concentrations (Benveniste et al., 1984; Castillo et al,
1996, 1997, 2002; Guyot et al., 2001). The glutamate spreads and
subsequently causes neuronal damage in areas well beyond the in-
farcted tissue (Han et al., 2008). A toxic excess of glutamate in the
brain's ECF stimulates glutamate receptors, which in turn leads to
swelling of the cell, apoptosis, and neuronal death. These increased
glutamate levels have been strongly correlated with poor neurological
outcomes (Koura et al., 1998; Zauner et al., 1996; Zhang et al., 2001).
Similarly, there is a growing body of evidence that the glutama-
tergic system is central to the development of many mood disorders,
including anxiety and depression (Altamura et al., 1995; Hashimoto
et al., 2007; Kucukibrahimoglu et al., 2009; Levine et al., 2000; Mauri
et al., 1998; Mitani et al., 2006). There are a number of studies re-
porting alterations in glutamate levels in blood (Mauri et al., 1998) and
CSF (Levine et al., 2000) in patients with major depression. There is
also a positive correlation between plasma glutamate levels and se-
verity of depressive symptoms in patients with major depression
(Mitani et al., 2006). In a study of postmortem tissue from the human
frontal cortex, glutamate levels were elevated in subjects with a history
of depression compared with controls (Hashimoto et al., 2007).
Clinical studies have demonstrated that the N-methyl-D-aspartate
(NMDA) receptor antagonist ketamine, which interferes with glutamate
receptor activation, has rapid antidepressant effects in patients suf-
fering from treatment-resistant major depressive disorder
(Diazgranados et al., 2010). As such, the U.S. Food and Drug Admin-
istration recently approved esketamine for treatment-resistant depres-
sion (Kim et al., 2019). Animal models have demonstrated the anti-
depressive effects of interfering with glutamate activation at a variety
of receptors (Hashimoto, 2011). However, while agents such as NMDA
receptor antagonists showed initial promise for neuroprotection in an-
imal models (Lee et al., 1999; McCulloch, 1992), studies with glutamate
antagonists have failed to demonstrate clinical neuroprotective efficacy
in human clinical studies (Ikonomidou and Turski, 2002; Muir, 2006).
An alternative method of reducing glutamate is to reduce excess
toxic glutamate, rather than antagonize the receptors. One method of
eliminating excess glutamate from the brain's interstitial fluids is by
utilizing the naturally occurring brain-to-blood glutamate efflux via the
endothelial transport systems (Teichberg, 2007; Teichberg et al., 2009).
Glutamate co-substrates, pyruvate and oxaloacetate, via blood resident
enzymes glutamate-oxaloacetate transaminase and glutamate-pyruvate
transaminase convert glutamate into its inactive form 2-ketoglutarate
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Neuropharmacology 155 (2019) 173–184
(MRS) magnetic resonance spectroscopy
(ICA) Internal carotid artery
(CCA) common carotid artery
(NAD) Nicotinamide adenine dinucleotide
(PWI) perfusion weighted imaging
(ADC) apparent diffusion coefficient
(CBF) cerebral blood flow
(RICH) Ratios of Ipsilateral and Contralateral Cerebral
Hemispheres
(NSS) neurological severity score
(Gonzalez et al., 2005; Gray et al., 2014; Leibowitz et al., 2012). This
method of reducing excess glutamate, known as blood glutamate
scavenging (BGC), has been shown to be effective in various animal
studies (Leibowitz et al., 2012). BGC is achieved by activating several
mechanisms including the catalyzation of the enzymatic process in-
volved in glutamate metabolism, the redistribution of glutamate into
tissue, and as an acute stress response (Leibowitz et al., 2012).
We have previously demonstrated that a decrease in blood gluta-
mate concentrations in the injured brain results in improved neurolo-
gical deficit (Zlotnik et al., 2012), reduced cerebral edema (Zlotnik
et al., 2007), reduced infarct zone (Boyko et al., 2011c), and reduced
BBB breakdown (Boyko et al., 2012a). Additional studies, based on the
intravenous injection of pyruvate, demonstrated faster and greater
neurological recovery after closed head injury in a rat model (Zlotnik
et al., 2007). Pyruvate may provide neuroprotection by other me-
chanisms as well, including via hydroxyl radical scavenging, stimu-
lating NADPH-dependent peroxide scavenging systems, inhibiting poly
(ADP-ribose) polymerase activity, and by serving as an alternative en-
ergy source for brain cells (Leibowitz et al., 2012).
The activity of BGS in stimulating the brain-to-blood glutamate ef-
flux is a self-limiting process; as excess glutamate levels decrease to
concentrations below the threshold of activation of the brain vascu-
lature glutamate transporters (i.e below their Km values), the process of
glutamate efflux slows and eventually stops. Thus, BGS preserves the
physiological effects of glutamate in regulating the metabolic and
electrolyte balance, maintains neuronal integrity, and exerts beneficial
effects in neurologic repair after brain injury. This method maintains a
balance between eliminating the undesirable effects of excess glutamate
and preserving its positive effects that are necessary to sustain life
(Teichberg et al., 2009).
The objective of this study was to investigate the efficacy of long-
term pyruvate treatment on post-stroke depression, as determined by
neurologic status and performance on several behavioral tests.
Additionally, this study examines this treatment modality on neurolo-
gical outcomes as determined by the volume of infarcted zone, blood
brain barrier (BBB) breakdown, real-time monitoring of brain ECF
concentrations via biosensors and by concentration of blood glutamate.
The therapeutic effects were also examined by magnetic resonance
imaging (MRI) techniques, which included in vivo MRI spectroscopy,
MRI monitoring of BBB disruption and MRI evaluation of infarcted zone
and brain edema.
2. Methods
2.1. Animals
The experiments were conducted in accordance with the re-
commendation of the Declarations of Helsinki and Tokyo and to the
Guidelines for the use of Experimental Animals of the European
Community. The experiments were approved by the Animal Care
Committee of Ben-Gurion University of Negev, Israel. A total of one
hundred-forty male Sprague-Dawley rats (Harlan Laboratories, Israel)
were used in this experiment. All rats weighed between 300 and 400 g.
D. Frank, et al.
Purina Chow and water were made available ad libitum. The tem-
perature in the room was maintained at 22 °C, with a 12 h light–dark
cycle.
2.2. Experimental design
Eighty rats were randomly assigned into one of 3 groups: Middle
Cerebral Artery Occlusion (MCAO) plus pyruvate treatment (n = 30
rats), MCAO plus placebo treatment (n = 30), and sham operated rats
(n = 20). Rats who died or still had neurological deficits after 4 weeks
were excluded from the study to avoid the effect of a motor deficit on
rat behavioral performance. The final number of animals in each group
was 23, 20, and 20, respectively.
After induction of anesthesia, BD Neoflon 24G catheters had been
introduced into the tail artery for blood pressure monitoring and blood
sampling. Neurological status was tested before surgery (baseline) and
at 1 h, 24 h, 1 week, 2 week and 4 weeks following stroke. Magnetic
resonance imaging (MRI) with magnetic resonance spectroscopy (MRS)
was performed 0 h, 3 h, 24 h and 1 week following stroke. Based on data
from the MRI/MRS, we evaluated the development of the infarct zone,
brain edema, BBB breakdown, and ischemic penumbra.
Rats were assessed for depressive behavior via a sucrose preference
test at 0, 1, 2, 3 and 6 months post-MCAO, and assessed for anxiety-like
behavior with the Vogel and elevated plus maze tests, at 0, 1, 3 and 6
months post-MCAO.
Sixty separate rats were also assigned to three additional groups for
monitoring CSF and blood glutamate levels 24 h and 1-week post-
stroke, respectively: MCAO plus pyruvate treatment (n = 19 rats),
MCAO plus placebo treatment (n = 22), and naive rats (n = 19).
Thirty-two additional rats were used as a control group. In 16 of
these naïve rats, the Vogel and plus maze tests were performed at
baseline and then again after 1 month of pyruvate treatment. In the
other 16 naïve rats, blood samples for alanine and α-ketoglutarate were
collected at baseline and then again after 1 month of treatment with
pyruvate.
2.3. Middle Cerebral Artery Occlusion (MCAO) procedure
Rats were anesthetized with a mixture of isoflurane (4% for in-
duction, 2% for surgery, 1.3% for maintenance) in 24% oxygen (2 L/
min) without tracheotomy, and allowed to breathe spontaneously. Rats
were anesthetized for 35–40 min during each procedure. There were no
differences in the time allotted for anesthesia between groups in order
to control the effects of isoflurane, pO2, or pCO2. The anesthesia was
considered sufficient for surgery when the tail reflex was abolished.
Physiological parameters, including mean arterial pressure, heart rate
and O2 saturation of arterial blood were monitored. A heating plate was
used to maintain a core body temperature of 37 °C, measured via a
probe placed in the rats’ rectum. Body temperature was kept constant
between rats, thus minimizing any effect of hypothermia or hy-
perthermia on neurological outcome and neurological injury. Blood
samples were collected to measure arterial blood gas tensions (pCO2,
pO2, and HCO3), pH, and concentrations of glucose, hemoglobin, and
electrolytes (Na, K, and Cl). Measurements of physiological parameters
were recorded before anesthesia and after recovery from anesthesia and
surgery. The body temperature before surgery was measured at 8:00 to
9:00 a.m. in all animals and at 24 h after the onset of MCAO. The MCA
was occluded in our modified technique by inserting the catheter di-
rectly through the ICA (Boyko et al., 2010).
Following a careful dissection and exposure of the common and
internal carotid artery, and after separation of these arteries from the
vagus nerve, the ICA was permanently blocked (a 4-0 silk suture was
tied loosely around ICA just above the CCA bifurcation) proximal to the
filament insertion point and temporarily blocked distal to the filament
insertion point. The purpose of the proximal ligation was to occlude the
ICA while the additional distal ligation reduced the bleeding around the
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Neuropharmacology 155 (2019) 173–184
filament and secured it in place. A heat-blunted monofilament was in-
troduced via the ICA into the circle of Willis, effectively occluding the
MCA. The silk suture in the ICA was fastened around the intraluminal
thread to prevent bleeding. The suture was inserted approximately
18–18.5 mm from the bifurcation of the CCA until a sensation of mild
resistance was reached to occlude the MCA. The thread was then fixed
in position by tying a silk filament over the ICA, just above the pter-
ygopalatine artery. Rats were allowed to fully recover after the surgery
to prevent any potential motor impairment that could impact the per-
formance of the behavior tests (Boyko et al., 2010).
2.4. Drugs and doses
Pyruvate was purchased from Sigma Israel Chemicals (Rehovot,
Israel) and was stored at 2–4 °C until use and was dissolved in drinking
water immediately prior to administration. A fresh solution was pre-
pared every 8 h. In rats that received pyruvate, doses of 180 mg/kg/day
(in three divided doses) was administered in their drinking water for 30
days. Doses of pyruvate were chosen based on our previous work from
MRS data, that demonstrated that doses of 180 mg/kg/day were op-
timal to produce a blood and brain glutamate reduction of about
25–35% (under publication). Equal volumes of drinking water without
pyruvate were given to the placebo groups.
2.5. Determination of blood glutamate
Whole blood (200 μl aliquot) was de-proteinized by adding an equal
volume of ice-cold 1 M perchloric acid and then centrifuging at
10 000×g for 10 min at 4 °C. The pellet was discarded and the super-
natant was collected, adjusted to pH 7.2, with 2 M K2CO3, and stored at
−80 °C for later analysis, if needed. Glutamate concentration was
measured using the fluorometric method of Graham and Aprison. A
60 μl aliquot from the perchloric acid supernatant was added to 90 μl of
a 0.3 M glycine; 0.25 M hydrazine hydrate buffer adjusted to pH 8.6
with 1 M H2SO4 and containing 11.25 U of glutamate dehydrogenase in
10 mM Nicotinamide adenine dinucleotide (NAD). After incubation for
30–45 min at room temperature, the fluorescence was measured at
460 nm with excitation at 350 nm. A glutamate standard curve was
established with concentrations ranging from 0 to 6 μM. All determi-
nations were done at least in duplicates (Boyko et al., 2012b).
2.6. Determination of CSF glutamate
Fresh CSF (110 μl) was mixed with perchloric acid (25 μl) of 0.3 M,
and then centrifuged at 10 000×g for 10 min at 4 °C. The pellet was
discarded and the supernatant was collected, adjusted to pH 7.2 with
12.5 μl of 2 M K2CO3 and stored at −80 °C for later analysis (Boyko
et al., 2012a). Analysis was performed by fluorometric method as de-
scribed above for blood samples.
2.7. Determination of alanine
Whole blood (200 μl aliquot) was collected into biochemical mi-
crotubes and centrifuging at 13 000×g for 10 min at 4 °C. Serum sam-
ples was deproteinized with a 10 kDa MWCO spin filter. Samples was
collected into Eppendorf microtubes and stored at −80 °C for later
analysis. For fluorometric analyzes, we used 50 μl of the sample for
each reaction (well). Samples were diluted to optimal concentration for
readings within the linear range of the standard curve. Alanine con-
centration was performed using the coupled enzymatic assay, according
to the manufacturer's instructions (Sigma; catalog #: MAK001 - Alanine
Assay Kit https://www.sigmaaldrich.com/content/dam/sigma-aldrich/
docs/Sigma/Bulletin/1/mak001bul.pdf), as previously described (Cao
et al., 2013). We used a blank sample for each sample by omitting the
Alanine Converting Enzyme in the Reaction Mix. After incubation for
60 min at 37 °C, the fluorescence intensity was measured at λex = 535/
D. Frank, et al.
λem = 587 nm. An alanine standard curve was established with con-
centrations ranging from 0 to 10 nmole/well.
2.8. Determination of α-ketoglutarate
Whole blood (200 μl aliquot) was collected into biochemical mi-
crotubes and centrifuging at 13 000×g for 10 min at 4 °C. Serum sam-
ples was deproteinized with a 10 kDa MWCO spin filter. Samples was
collected into Eppendorf microtubes and stored at −80 °C for later
analysis. For fluorometric analyzes, we used 50 μl of the sample for
each reaction (well). Samples were diluted to optimal concentration for
readings within the linear range of the standard curve. The α-ketoglu-
tarate concentration was performed using the coupled enzymatic assay
according to the manufacturer's instructions (Sigma; catalog #:
MAK054 α-Ketoglutarate Assay Kit https://www.sigmaaldrich.com/
content/dam/sigma-aldrich/docs/Sigma/Bulletin/1/mak054bul.pdf),
as previously described (Bergmeyer et al., 1974). We used a blank
sample for each sample by omitting the α-Ketoglutarate Converting
Enzyme in the Reaction Mix. After incubation for 30 min at 37 °C, the
fluorescence intensity was measured at λex = 535/λem = 587 nm. A
α-Ketoglutarate standard curve was established with concentrations
ranging from 0 to 10 nmole/well.
2.9. Imaging protocol (MRS/MRI)
MRS was used to measure brain glutamate concentrations (Cai et al.,
2012), and MRI was used for the determination of Ktrans, DWI, T2, and
perfusion-weighted imaging (PWI). The experimental procedure was
performed as described previously at 1 h, 24 h, and 1 week after MCAO.
Measurements were performed in injured hemispheres and symmetric
area of the contralateral hemisphere in the penumbra area in close
proximity to the necrotic core. Animals were maintained under general
anesthesia (1.5% isoflurane in oxygen). A tail vein catheter was in-
troduced and connected to a syringe containing a solution of Gd-DTPA
(Dotarem, 0.5 mmoL/ml Guerbet, France). A 3T MRI was used (Ingenia,
Philips Medical Systems, Best, The Netherlands) using an eight-channel
receive-only coil. Localising T2w TSE sequences were acquired in sa-
gittal and coronal planes with TR/TE = 3000/80 msec, turbo
factor = 15, water-fat shift = 1.6 pixels, resolution
(freq × phase × slice) = 0.47 × 0.41 × 2.0 mm and one average for a
scan time of 1:00 min. In the axial direction the scan parameters were
TR/TE = 3000/80 msec, turbo factor = 14, water-fat shift = 1.6 pixels,
resolution (freq × phase × slice) = 0.37 × 0.33 × 2.0 mm. Four
averages were acquired for a scan time of 4:54 min. Diffusion tensor
imaging in 6 directions was performed in the axial direction using a
multi-shot STEAM spin-echo, echo-planar sequence with TR/TM/
TE = 1355/15.0/143 msec, SENSE reduction factor = 1.5, turbo
factor = 19, b = 1000 s/mm2, resolution (freq × phase × slice) =
0.55 × 0.55 × 2.0 mm with spectrally-selective fat suppression. Five
signal averages were acquired for a scan time of 8:40 min. T1 perme-
ability studies were performed using a segmented 3D T1w-FFE se-
quence with 50 dynamics for a total scan time of 25:52 min. The scan
parameters were TR/TE = 16/4.9 msec, turbo factor = 48, SENSE
factor 1.5, resolution (freq × phase × slice) = 0.30 × 0.37 × 2.0 mm,
tip angle = 80 and two signal averages for a scan time of 31 s/dynamic.
Three calibration scans with identical resolution preceeded the dy-
namic sequence with tip angles 50, 100 and 150. The contrast agent
was injected after the 5th dynamic scan. T2 perfusion studies were
carried out using a dynamic, single-shot gradient-echo epi sequence
with spectrally-selective fat suppression. The scan parameters were TR/
TE = 1300/40 msec, resolution (freq × phase × slice) = 0.64 ×
0.69 × 2.0 mm, and one signal average giving a scan time of 1.3sec/
dynamic. A total of 150 dynamics were acquired for a scan time of
3:19 min. For PWI we used a typical multimodal stroke MRI protocol
consists of PWI adjusted to clinical MRI 3T scanner technique (Ulmer S
et al., 2008). The contrast agent (Dotarem – Gadoteric acid 0.5 mmol/
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Neuropharmacology 155 (2019) 173–184
ml) was injected after the 5th dynamic (0.03 ml Gd in 0.4 ml saline).
The Intellispace Portal workstation (V5.0.0.20030, Philips Medical
Systems, Best, The Netherlands) was used for the post-processing of the
permeability and perfusion studies. The MRS acquisitions were per-
formed using a multi-voxel, 2D PRESS acquisition. A VOI
5.0 × 10.0 mm was graphically placed in a FOV of 40 × 40 mm divided
into 16 × 16. The scan parameters were TR/TE = 2000/32 msec,
SENSE = 2 × 2 (AP × RL), slab thickness = 10 mm for an acquisition
resolution (AP × RL × FH) of 2.5 × 2.5 × 10.0 mm. Second-order
shimming were used yielding a water line width of approximately
25 Hz. Water suppression was achieved by applying two bandwidth
selective rf pulses. The acquisition bandwidth was 2000 Hz giving a
spectral resolution of 0.98 Hz. A total of 23 averages were acquired for
a scan time of 38:48 min. The spectra was analyzed using the Philips
SpectroView software. To identify and assess the peak of glutamate
from glutamine we used modern methods (Hancu, 2009; Prescot et al.,
2012).
2.10. MR image Analysis
Image analysis was performed by an expert, who was blind to the
group assignment. Quantitative CBF and apparent diffusion coefficient
(ADC) maps, in units of square millimeters per second, were generated
in Philips software package (Ingenia, Philips Medical Systems, Best, The
Netherlands) and subsequently analyzed using Image J software 1.50i
version (http://rsb.info.nih.gov/ij/), as previously described (Boyko
et al., 2019; Reid et al., 2012).
These thresholds were used to identify all pixels with abnormal CBF
and ADC characteristics on each slice. The viability thresholds were
0.53 × 10-3mm2/s for ADC images (Boyko et al., 2019) and for CBF
maps (Bardutzky et al., 2005): 0–6 mL/100 g/min was associated with
damage brain tissue and called infarct core, 0–15 mL/100 g/min was
associated with damage brain tissue and include 2 abnormal zones-in-
farct core and penumbra, 0–70 mL/100 g/min was associated with
damage brain tissue and include 3 abnormal zones: infarct core, pe-
numbra and benign oligemia.
Calculation of infarcted zone was performed by the Ratios of
Ipsilateral and Contralateral Cerebral Hemispheres (RICH) method. The
calculation of the lesion volume with the correction for tissue swelling
by the RICH technique was done using the following formula:
Infarct size × Contralateral hemisphere size
Corrected infarct size =
Ipsilateral hemisphere size
The infarcted brain volume was measured as a percentage of the
total brain (Boyko et al, 2013, 2019).
Calculation of brain edema was performed by the RICH method. The
calculation of brain edema by the RICH technique was done by com-
paring the contralateral and ipsilateral hemispheres, and performed
using the following formula:
Brain edema
Volume of the right hemisphere Volume of the left hemisphere
=
Volume of the left hemisphere
The infarcted brain volume was measured as a percentage of the
total brain (Boyko et al, 2011a, 2013).
2.11. Examination of neurologic status
Two observers, who were blinded as to which surgical procedure
each rat had received, tested the animals for neurological deficits fol-
lowing MCAO. All animals were tested according to the advanced
neurological severity score (NSS), as previously described (Boyko et al.,
2011b). The testing procedure did not take more than 7–10 min per rat.
The rat's neurological status was examined as baseline before MCAO
and then at 1 h, 24 h, 1 week, 2 weeks and 4 weeks following MCAO.
D. Frank, et al. Neuropharmacology 155 (2019) 173–184

(caption on next page)

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D. Frank, et al. Neuropharmacology 155 (2019) 173–184

Fig. 1. The neuro-behavioral profile for post-stroke rats treated with pyruvate and post-stroke rats treated with placebo compared to naïve rats. a. Sucrose preference
test: At 1-month post-MCAO there was a significant difference in sucrose preference for all three groups (p < 0.01). There was also a significant difference between
post-stroke rats given placebo and post-stroke rats given pyruvate at 2 and 3 months post-MCAO (p < 0.01). By month 6, there were no differences between all 3
groups. The data are measured in ml and expressed as a mean percentage ± SD. b. Neurologic severity score: Neurological severity deficit at one week after MCAO was
significantly greater for the 20 post-stroke rats treated with placebo than for the 23 post-stroke rats treated with pyruvate (median = 1.5 vs. 0.96, p < 0.05). c-h.
Elevated plus maze: There was a significant difference in the time spent on the open arms for post-stroke rats treated with pyruvate compared to sham operated rats at
1 month (p < 0.01) and post-stroke rats treated with placebo compared to either post-stroke rats treated with pyruvate or sham operated rats at 1, 3, and 6 months
post-MCAO (p < 0.01). There was a significant difference in open arm entries in post-stroke rats treated with placebo compared to either post-stroke rats treated
with pyruvate or sham operated rats at 1 (p < 0.01 and p < 0.05, respectively), 3 (p < 0.01), and 6 (p < 0.01) months post-MCAO. There was also a difference
found for post-stroke rats treated with pyruvate compared to sham operated rats at 3 (p < 0.05) and 6 (p < 0.01) months post-MCAO. There was a significant
difference in platform entries by sham operated rats compared to post-stroke rats treated with placebo at 1 (p < 0.01) and 3 (p < 0.01) months post-MCAO and
compared to post-stroke rats treated with pyruvate at 1 (p < 0.01), 3 (p < 0.01), and 6 (p < 0.01) months post-MCAO. A significant difference was also found
between post-stroke rats treated with pyruvate and post-stroke rats treated with placebo at 3 months (p < 0.05). There was a significant difference in the time spent
on the platform by post-stroke rats treated with pyruvate compared to both post-stroke rats treated with placebo and sham operated rats at 1, 3, and 6 months post-
MCAO (p < 0.01). A significant difference was also found between post-stroke rats treated with placebo and sham operated rats at 3 months (p < 0.01). The data
are measured in seconds and expressed as a mean percentage of the baseline ± SEM. i-j. Vogel test: At 1-month post-MCAO, there was a significant difference
between post-stroke rats treated with placebo (p < 0.05) and post-stroke rats treated with pyruvate (p < 0.01) compared to sham operated rats. At 3- and 6-months
post-stroke rats treated with placebo scored significantly different than both rats treated with pyruvate (p < 0.05) and sham operated rats (p < 0.01). Number of
shocks were estimated as a percentage from baseline. The data are measured in seconds or count and expressed as a mean percentage of the baseline ± SEM.

Due to a fast recovery after Isoflurane anesthesia, rats were fully awake
1 h after surgery.
2.12. Elevated plus maze test
The plus maze was situated in a darken room and consisted of two
open and two closed arms (each of dimensions 16 × 46 cm). It was
constructed from black plastic and positioned 100 cm above the floor.
The closed arms, opposite to one another, had a surrounding wall of
height 40 cm. Experiments were recorded by a video camera (CC TV
Panasonic, Japan) suspended approximately 200 cm above the center of
the plus maze. 5% ethanol was used to clean the maze prior to the
introduction of each animal. Rats were tested on the maze in a rando-
mized order. Each rat was placed in the center of the plus maze facing
one of the open arms, and the rat's behavior was videotaped for 5 min
for future analysis.
The number of entries into the various arms and time spent in arms
and in the center of the elevated plus maze was recorded with a video
camera (CC TV Panasonic, Japan) and subsequently analyzed using
Ethovision XT software (Noldus, Wageningen, Netherlands) (Boyko
et al., 2013). The elevated plus maze test was performed at 0, 1, 3 and 6
months.
2.13. Conflict drinking test (Vogel test)
Rats were water-deprived before testing. Food was available in the
home cage at all times. After 24 h of water deprivation, rats were ha-
bituated to the testing cages to assess licking behavior and allowed to
drink for 15 min (training session), with an additional 15 min drinking
in their home cages. Water deprivation then continued for another 24 h.
On the test day, rats were placed in the testing cages. When the rats
licked the water spigot on the bottle, they receiving an electric shock of
0.5 mA with pulse duration of 0.2 s. The number of punished responses
was automatically recorded for each rat during the 5 min of testing
(Belcheva et al., 1997). The Vogel test was performed at 0, 1, 3 and 6
months.
2.14. Sucrose preference test
Following the adaptation procedure, the rats were deprived of water
and food for12 h. The sucrose preference test was conducted at 9:00
a.m. The rats were housed in individual cages and given free access to
the two bottles containing 100 mL of sucrose solution (1%, w/v)
and100 ml of water, respectively. After 4 h, the volume (ml) of both the
consumed sucrose solution and water was recorded, and sucrose pre-
ference was calculated as sucrose preference (%) = sucrose consump-
tion (ml)/(sucrose consumption (ml) + water consumption
(ml)) × 100%. The sucrose preference test was performed at 0, 1, 2, 3
and 6 months.
2.15. Statistical analysis
Statistical analysis was performed with the SPSS 22 package (SPSS
Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was used, con-
sidering the number of rats in each group for deciding the appropriate
test for the comparisons between the different parameters. For non-
parametric data, we used the transformation or applied the appropriate
tests suitable for non-parametric data. The significance of comparisons
between groups had been determined using the Kruskal–Wallis fol-
lowed by Mann–Whitney (for nonparametric data) and one-way
ANOVA with Bonferroni post hoc test or the Student's t-tests (for
parametric data). Mortality rate was analyzed with chi-square, Fisher's
exact test. Results were considered statistically significant when
P < 0.05, and highly significant when P < 0.01.
3. Results
3.1. Survival rate
The eighty original rats were divided randomly into three groups:
MCAO plus pyruvate treatment (n = 30 rats), MCAO plus placebo
treatment (n = 30), and sham operated rats (n = 20). After rats that
were excluded, as noted above, the final number of animals in each
group was 23, 20, and 20, respectively. The mortality rate was lower in
the rats treated with pyruvate compared to the control group, although
the difference was not statistically significant (10% vs 13.3%, respec-
tively).

The sucrose preference test was performed as described previously
(Boyko et al., 2015). The rats were allowed to consume1% (w/v) by
placing two bottles of sucrose solution in each cage for 24 h. The ra-
tionale for the two bottles is that the appearance of an additional bottle
in the rat's cage during the initial part of the sucrose preference test may
frighten the rat. Afterwards, one of the bottles was replaced with water
for 24 h. As such, this design may help avoid the effects of neophobia.
3.2. Sucrose preference test
Performance over time on the sucrose preference test post-MCAO is
illustrated in Fig. 1a. At 1-month post-MCAO there was a significant
difference in sucrose preference for all three groups (64.4% ± 9.2 and
80.1% ± 7.3 vs. 95.6% ± 6.1, p < 0.01). There was also a significant
difference between post-stroke rats given placebo and post-stroke rats

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(caption on next page)

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D. Frank, et al. Neuropharmacology 155 (2019) 173–184

Fig. 2. The effect of long-term treatment with pyruvate using MRS techniques. a. Brain glutamate: 24 h post-MCAO, both groups, post-stroke rats treated with
pyruvate (p < 0.05) and post-stroke rats treated with placebo (p < 0.01) demonstrated increased brain glutamate concentrations compared to sham operated rats.
The data is presented as mean ± SEM. b. Brain edema: Post-stroke rats treated with either placebo or pyruvate demonstrated increased brain edema 24 h post-MCAO
compared to sham operated rats. The data are measured as a percentage to the contralateral hemisphere and expressed as mean ± SEM. c-d. Lesion volume: Post-
stroke rats treated with either placebo (p < 0.01) or pyruvate (p < 0.05) demonstrated increased lesion volume 24 h post-MCAO compared to sham operated rats.
Furthermore, there was a significant increase in lesion volume 1 week following MCAO in the rats treated with placebo compared to sham operated rats (p < 0.01).
The data are measured as a percentage to the contralateral hemisphere and expressed as mean ± SEM. e-f. BBB breakdown: Post-stroke rats treated with either
placebo (p < 0.01) or pyruvate (p < 0.05) demonstrated increased BBB permeability 24 h post-MCAO compared to sham operated rats. Moreover, there was a
significant increase in BBB permeability 1 week following MCAO in the rats treated with placebo compared to sham operated rats (p < 0.05). The data are measured
as a percentage to the contralateral hemisphere and expressed as mean ± SEM. g-h. Cerebral blood flow: There was a significant increase in CBF in all rats who
underwent MCAO 3 h after injury in the second, third, fourth, and fifth brain sections. There was a significant increase in CBF 3 h post-MCAO in the injured
hemisphere as a percentage to the contralateral hemisphere compared to sham operated rats. The data are measured as a percentage to the contralateral hemisphere
and expressed as mean ± SEM.

given pyruvate at 2 (68.4% ± 4.8 and 92.4% ± 3.6, p < 0.01) and 3
(76.1% ± 5.4 and 92.6% ± 4.6, p < 0.01) months post-MCAO. By
month 6, there were no differences between all 3 groups.
3.3. Neurological severity score
Neurological deficit at one week after MCAO was significantly
greater for the 20 post-stroke rats treated with placebo than for the 23
post-stroke rats treated with pyruvate (median = 1.5 vs. 0.96,
p < 0.05, Fig. 1b). Two weeks after MCAO administration, there was
also less neurological deficit in rats treated with pyruvate than in rats
treated with placebo, but the difference wasn't statistically significant.
3.4. The elevated plus maze
Performance over time on the elevated plus maze post-MCAO is il-
lustrated in Fig. 1c–h, broken down into time spent on open arms, open
arm entries, platform entries and time spent on the platform.
At 1-month post-MCAO, there was a significant difference in the
time spent on the open arms between post-stroke rats treated with
placebo (214% ± 27, p < 0.01) and post-stroke rats treated with
pyruvate (193% ± 22, p < 0.01) compared to sham operated rats
(93% ± 15). Additionally, there was a significant difference for post-
stroke rats treated with placebo compared to either post-stroke rats
treated with pyruvate or sham operated rats at 3 months (319% ± 23
vs. 124% ± 22 and 85% ± 11, p < 0.01), and 6 (261% ± 20 and
95% ± 15 vs. 102% ± 10, p < 0.01) months post-MCAO.
There was a significant difference in open arm entries in post-stroke
rats treated with placebo compared to either post-stroke rats treated
with pyruvate or sham operated rats at 1 (138% ± 18 vs. 69% ± 5
and 124% ± 21, p < 0.01 and p < 0.05, respectively), 3
(284% ± 24 vs. 48% ± 7 and 112% ± 21, p < 0.01), and 6
(228% ± 14 vs. 50% ± 7 and 126% ± 17, p < 0.01) months post-
MCAO. There was also a difference found for post-stroke rats treated
with pyruvate compared to sham operated rats at 3 (p < 0.05) and 6
(p < 0.01) months post-MCAO.
There was a significant difference in platform entries by sham op-
erated rats compared to post-stroke rats treated with placebo at 1
(114% ± 9 vs. 80% ± 8, p < 0.01) and 3 (107% ± 11 vs.
40% ± 6, p < 0.01) months post-MCAO and compared to post-stroke
rats treated with pyruvate at 1 (114% ± 9 vs. 76% ± 4, p < 0.01), 3
(107% ± 11 vs. 70% ± 4, p < 0.01), and 6 (119% ± 11 vs.
78% ± 4, p < 0.01) months post-MCAO. A significant difference was
also found between post-stroke rats treated with pyruvate and post-
stroke rats treated with placebo at 3 months (p < 0.05).
There was a significant difference in the time spent on the platform
by post-stroke rats treated with pyruvate compared to both post-stroke
rats treated with placebo and sham operated rats at 1 (150% ± 12 vs.
78% ± 7 and 89% ± 12, p < 0.01), 3 (164% ± 9 vs. 17% ± 2 and
94% ± 13, p < 0.01), and 6 (180% ± 13 vs. 93% ± 7 and
91% ± 12, p < 0.01) months post-MCAO. A significant difference
was also found between post-stroke rats treated with placebo and sham
operated rats at 3 months (p < 0.01).
In naïve rats treated with 1 month of pyruvate, there were no sig-
nificant differences in performance on the elevated plus maze compared
to baseline.
3.5. The Vogel conflict test
Performance over time on the Vogel conflict test post-MCAO is il-
lustrated in Fig. 1i and j. At 1-month post-MCAO, there was a sig-
nificant difference between post-stroke rats treated with placebo
(185% ± 80, p < 0.05) and post-stroke rats treated with pyruvate
(155% ± 50, p < 0.01) compared to sham operated rats (96% ± 70).
At 3- and 6-months post-stroke rats treated with placebo scored sig-
nificantly different than both rats treated with pyruvate (245% ± 69
and 204% ± 65 vs 129% ± 43 and 90% ± 26, p < 0.05) and sham
operated rats (245% ± 69 and 204% ± 65 vs. 129% ± 84 and
123% ± 85, p < 0.01).
In naïve rats treated with 1 month of pyruvate, there were no sig-
nificant differences in performance on the Vogel test compared to
baseline.
3.6. Determination of brain glutamate by MRS
Glutamate levels by Chemical Exchange Saturation Transfer (CEST)
imaging were estimated as a ratio to creatinine and expressed as a
percentage to the contralateral hemisphere. 24 h post-MCAO, both
groups, post-stroke rats treated with pyruvate (169.9% ± 15.6,
p < 0.05) and post-stroke rats treated with placebo (253.5% ± 15.8,
p < 0.01) demonstrated increased brain glutamate concentrations
compared to sham operated rats (92.7% ± 6.8, Fig. 2a). There was a
trend towards higher brain glutamate in rats treated with placebo
compared to pyruvate or sham operated rats 118.7% ± 6 vs.
106.9% ± 5.6 and 99.6% ± 5.6), although the difference was not
statistically significant.
3.7. Determination of brain edema by MRI
Post-stroke rats treated with either placebo (5.69% ± 1.19,
p < 0.01) or pyruvate (3.78% ± 0.68, p < 0.05) demonstrated in-
creased brain edema 24 h post-MCAO compared to sham operated rats
(0.32% ± 0.62, Fig. 2b). There were no significant differences in brain
edema between the 3 experimental groups one-week post-MCAO.
3.8. Determination of lesion volume by MRI
Post-stroke rats treated with either placebo (5.07% ± 0.98,
p < 0.01) or pyruvate (3.96% ± 0.86, p < 0.05) demonstrated in-
creased lesion volume 24 h post-MCAO compared to sham operated rats
(0.72% ± 0.89, Fig. 2c). Furthermore, there was a significant increase
in lesion volume 1 week following MCAO in the rats treated with pla-
cebo compared to sham operated rats (5.78% ± 1.01 vs.
1.47% ± 0.68, p < 0.01, Fig. 2d). Post-stroke rats treated with

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pyruvate had no statistical difference in lesion volume compared with pyruvate on glutamate scavenging for these disorders in a rat model.
sham operated rats (3.9% ± 0.9 vs. 1.47% ± 0.68). First, a significant elevation of glutamate levels in ischemic brain
tissue was demonstrated using non-invasive MRS imaging and invasive
3.9. Determination of BBB breakdown by MRI CSF measurements, methods established in previous trials. Peak levels

Post-stroke rats treated with either placebo (2.55% ± 0.45,

p < 0.01) or pyruvate (1.96% ± 0.41, p < 0.05) demonstrated in-
creased BBB permeability 24 h post-MCAO compared to sham operated
rats (0.42% ± 0.38, Fig. 2e). Moreover, there was a significant in-
crease in BBB permeability 1 week following MCAO in the rats treated
with placebo compared to sham operated rats (1.83% ± 0.28 vs.
0.47% ± 0.21, p < 0.05, Fig. 2f). Post-stroke rats treated with pyr-
uvate had no statistical difference in BBB permeability compared with
sham operated rats (0.81% ± 0.38 vs. 0.47% ± 0.21).

3.10. Determination cerebral blood flow by MRI

An independent-samples t-test indicated that the regions of reduced

CBF below 6 mL/100 g/min were associated with damaged brain tissue
(p < 0.05). CBF at each brain section, as a percentage to the con-
tralateral section, is illustrated in Fig. 2g. There was a significant in-
crease in CBF in all rats who underwent MCAO 3 h after injury in the
second (4.38% ± 1.7 vs. −0.15% ± 0.44, p < 0.05), third
(3.4% ± 1.1 vs. 0.54% ± 0.14, p < 0.05), fourth (6.64% ± 1.24 vs.
0.17% ± 0.17, p < 0.01), and fifth (7.76% ± 1.19 vs.
0.09% ± 0.32, p < 0.01) brain sections. There was also a significant
increase in CBF 3 h post-MCAO in the injured hemisphere as a per-
centage to the contralateral hemisphere compared to sham operated
rats (5.55% ± 0.78 vs. 0.17% ± 0.1, p < 0.01, Fig. 2h).

3.11. CSF glutamate concentrations

There was a significant increase in CSF glutamate concentration

24 h post-MCAO in both post-stroke rats treated with placebo (23 μM/
L ± 4.4) and post-stroke rats treated with pyruvate (11.1 μM/
L ± 2.6), compared to sham operated rats (2.3 μM/L ± 1, p < 0.01,
Fig. 3a). Furthermore, CSF glutamate concentrations were significantly
lower in post-stroke rats treated with pyruvate compared to post-stroke
rats treated with placebo (p < 0.05).

3.12. Blood glutamate concentrations

There was a significant decrease in concentrations of glutamate in

the blood 1-week post-MCAO in rats treated with pyruvate
(74.7%, ± SD 22.5) compared to post-stroke rats treated with placebo
(98.1%, ± SD 20.6) and sham operated rats (101%, ± SD 16.1,
p < 0.01, Fig. 3b).

3.13. Blood serum α-Ketoglutarate concentrations

In naïve rats treated with pyruvate for 1 month, concentration of α-
ketoglutarate in blood serum was significantly increased compared to
baseline levels (113%, 4.4 vs. 100% ± 2.6, p < 0.05, Fig. 3c).
3.14. Blood serum alanine concentrations
In naïve rats treated with pyruvate for 1 month, concentration of
alanine in blood serum was increased compared to baseline
(103%, ± 2.7 vs. 100% ± 2.5, Fig. 3c) although the difference was not
statistically significant.
4. Discussion
These studies demonstrated some important findings about the
connection between post-stroke elevation of glutamate levels, devel-
opment of post-stroke anxiety and depression, and the impact of
Fig. 3. In-vivo effects of pyruvate on blood and brain glutamate levels in a rat
model of MCAO. a. Concentrations of glutamate in CSF: There was a significant
increase in CSF glutamate concentration 24 h post-MCAO in both post-stroke
rats treated with placebo and post-stroke rats treated with pyruvate, compared
to sham operated rats (p < 0.01). Furthermore, CSF glutamate concentrations
were significantly lower in post-stroke rats treated with pyruvate compared to
post-stroke rats treated with placebo (p < 0.05). The data are measured in μM/
L and presented as mean ± SEM. b. Concentrations of glutamate in blood: There
was a significant decrease in concentrations of glutamate in the blood 1-week
post-MCAO in rats treated with pyruvate compared to post-stroke rats treated
with placebo and sham operated rats (p < 0.01). The data are measured as
μM/l and expressed as a percentage of the baseline ± SD. c. Serum blood
concentrations of alanine and α-Ketoglutarate: In naïve rats treated with pyruvate
for 1 month, concentration of α-ketoglutarate in blood serum was significantly
increased compared to baseline levels (p < 0.05). While concentration of
alanine in blood serum was increased compared to baseline, the difference was
not statistically significant. The data is presented as average percentage of
baseline ± SEM.

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of glutamate were recorded 24 h after ischemia was induced by MCAO.
Gradually, glutamate levels began to decline and returned to near-
normal levels 1 week after ischemia induction. The net amount of ex-
posure in the brain to glutamate during the first week post-stroke was
recorded as markedly higher than in a perfused brain.
Second, MRS and CSF measuring confirmed that pyruvate served as
a useful method to reduce brain glutamate levels. Despite differences in
the estimates of glutamate reduction 24 h post-ischemia induction
(approximately 33% by MRS imaging and approximately 50% by CSF
measurement), there is a clear efficacy of pyruvate as a blood glutamate
scavenger.
Third, the development of post-stroke mood disorders was demon-
strated in the rats treated with placebo, as suggested by previous trials
(Boyko et al., 2013; Soares et al., 2016). Although mood disorders can
be difficult to measure, the data suggests that the significantly elevated
post-stroke glutamate levels are responsible for the development of the
mood disorders. The disorders normalized when the glutamate sca-
venger pyruvate was administered.
In the sucrose preference test, post-stroke rats given placebo showed
significantly less voluntary sucrose consumption during the first three
months compared to naïve rats, which is a sign of depressive behavior.
The ischemic rats treated with pyruvate showed less of a reduction in
voluntary sucrose consumption than the rats given placebo. After a
month of observation, the rats given pyruvate demonstrated behavior
that did not differ from the sham operated rats, emphasizing the ef-
fectiveness of pyruvate in eliminating signs of depressive behavior
following stroke.
On the other hand, we received unexpected results from the Vogel
and elevated plus maze tasks, aimed to evaluate anxiety in post-stroke
rats. These results revealed less anxiety behavior in post-stroke rats
treated with placebo in comparison to sham operated rats. When the
stroke rats were treated with the blood glutamate scavenger, pyruvate,
there was an increase in anxiety level. Given the concurrent reduction
of depressive behavior with this increase in anxious behavior after
pyruvate application, it does not appear that errors were made that
produced the conflicting results. The results are consistent with our
procedural models, and we propose two possible explanations for this
seemingly-paradoxical post-stroke behavior:
1) It is possible that the post-stroke rats in our experiment developed
severe depression that could lead to a loss of fear and anxiety in
itself. Severe depression can include a reduction of anxiety and
behavior that lacks fear, given the depressed individual's apathy for
the value of life (Dutton and Karakanta, 2013; Painuly et al., 2005;
Piko and Pinczés, 2014). Therefore, post-stroke rats treated with
placebo may have experienced reduced fear, demonstrated through
less anxiety behaviors, as a result of severe depression. The group
treated with pyruvate showed less anxious behavior than the group
treated with placebo, but more anxious behavior than the sham
operated group. These results confirm pyruvate administration for
depression which, when severe, includes reduction in fear and
consequently reduction in anxiety.
2) It is also possible that post-stroke, rats may have developed not only
depression but bipolar disorder with manic states as well. Bipolar is
a less common though well-known post-stroke or post-brain injury
complication, consisting of periods of mania and depression with
euthymic moods between episodes (Ferro et al., 2009; Rege et al.,
1993; Santos et al., 2011). Depressive episodes consist of persistent
sadness, fatigue, eating disturbances, sleep disturbances, suicidal
thoughts, guilt and social withdrawal, while manic episodes consist
of hyperactivity, elevated mood or agitation, racing thoughts,
reckless behavior, little need for sleep, and sometimes psychosis
(Association, 2013). Rats and mice brought to a manic state through
pharmacological methods, environment and genetic modification
displayed reduced anxiety and elevated hedonia (Logan and
McClung, 2016). A manic state explains the reduced anxiety and
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Neuropharmacology 155 (2019) 173–184
elevated risk behavior seen in the Vogel and elevated plus maze
tasks, and the depressed state would account for the increase in
depressive behaviors demonstrated in the sucrose preference test for
the post-stroke rats on placebo. Treatment with pyruvate, could
reduce the effect of mania, allowing anxiety-like behavior to become
more prominent. A related possibility is that many of the rats treated
with placebo were in a manic state after the stroke, and the de-
pression rate according to the sucrose test for the control group
would be higher without including the manic rats.
It is important to note, however, that although depression following
brain insults, such as stroke and traumatic brain injury, has clearly been
established and depends on the severity and location of the brain insult
(Boyko et al., 2013; Ifergane et al., 2018; Kuts et al., 2019), there is no
clear consensus in the literature about the development of anxiety-like
behavior following brain insult. Evidence of the development of an-
xiety-like behavior following stroke or brain injury has been inconstant
in our laboratory. Further studies examining the effects of BGS on an-
xiety-like behavior are warranted.
Pyruvate treatment reduced lesion volume, brain edema, and the
extent of BBB permeability compared to the placebo-treated group, as
measured 24 h after ischemia. In addition, lesion volume and BBB
permeability were less pronounced in the pyruvate-treated group than
in the placebo group. Although those measurements remained higher
than in the naïve group one week following the ischemia induction,
they were not statistically different, confirming the success of pyruvate
for neuroprotection. This improvement aligns with previously pub-
lished data suggesting glutamate reduction in the brain's ECF after
pyruvate administration (Boyko et al, 2011c, 2012a). A significant
decrease of NSS was recorded in the group administered pyruvate 1
week after stroke induction compared to the placebo group. The pyr-
uvate-treated group displayed reduction of depression as a result of the
neuroprotective effects of pyruvate to reduce excess glutamate.
One of the limitations of our study was that we used only one
protocol of pyruvate treatment with a fixed pyruvate dose and mea-
suring the subsequent level of reduced brain glutamate. In future stu-
dies, levels of brain glutamate reduction should be varied to better
determine the impact of pyruvate glutamate scavenging.
Despite the mood stabilization and neuroprotective effects achieved
by the reduction of CNS glutamate with oral pyruvate administration,
and despite the use of native mechanisms of removing glutamate from
the CNS, the existence of possible harmful effects of prolonged elevated
blood ketoglutarate and alanine (due to exaggeration of pyruvate with
glutamate enzymatic reaction by pyruvate administration) were not
excluded by this study. Additional studies should include a control
group of naïve rats treated with pyruvate supplementation, along with
prolonged monitoring of ketoglutarate and alanine blood levels, while
measuring neurological and behavior outcomes.
In conclusion, glutamate scavenging by oral pyruvate administra-
tion seems to be an efficient method for reducing post-stroke patholo-
gically elevated CNS glutamate levels and for the treatment of post-
stroke depression (unipolar disorder) and manic disorders (bipolar
disorder). Additionally, glutamate scavenging by oral pyruvate ad-
ministration displays neuroprotective effects of subsequent post-stroke
reduction of lesion volume, brain edema and BBB breakdown. The
impact of oral pyruvate admission on post-stroke anxiety disorder and
the exclusion of possible harmful effects of prolonged blood ketoglu-
tarate and alanine elevation should be further investigated.
Acknowledgements/conflict of interest disclosure/compliance
with ethical standards
We thank Dr. Nataly Zueva, Division of Internal Medicine, Soroka
Medical Center, Ben-Gurion University, Beer-Sheva, Israel, Dr. Svetlana
Li, Department of Radiology, Soroka University Medical Center, Ben-
Gurion University, Beer-Sheva, Israel and Dr. Ruslan Biliar, Department
D. Frank, et al.
of Urology, Soroka Medical Center, Ben-Gurion University, Beer-Sheva,
Israel for their outstanding help in behavioral examination and analysis
of results by computer software. We thank Valeria Frishman, laboratory
assistant and Prof. Amos Douvdevani Head, Research Lab., from the
Department of Clinical Biochemistry, Soroka Medical Center, Ben-
Gurion University, for her help with the biochemical analysis. We thank
Dr. Kaziev Eleonora, Dr. Ibrahim Al Saif, Dr. Elena Braun, Dr. Tomer
Kotek and the all staff at the Department of Anesthesiology and Critical
Care, Soroka University Medical Center, for their support and helpful
discussions. We thank PhD candidate Ohad Stoler and Prof. Ilya
Fleidervish Head, Research Lab., from Department of Physiology and
Cell Biology, Faculty of Health Sciences, Ben–Gurion University, Beer
Sheva 84105, Israel for her help with the electrophysiological assess-
ment. The data obtained are part of D.F.‘s PhD thesis. This research was
supported by the Israel Science Foundation (grant No. 1490/15)
awarded to Matthew Boyko and Alexander Zlotnik. Authors Matthew
Boyko and Alexander Zlotnik declare that they do not have a conflict of
interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.neuropharm.2019.05.035.
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