Hyperhomocysteinemia leads to exacerbation of ischemic brain damage: Role of GluN2A NMDA receptors

Article abstract of DOI:10.1016/j.nbd.2019.03.012

Hyperhomocysteinemia has been implicated in several neurodegenerative disorders including ischemic stroke. However, the pathological consequences of ischemic insult in individuals predisposed to hyperhomocysteinemia and the associated etiology are unknown. In this study, we evaluated the outcome of transient ischemic stroke in a rodent model of hyperhomocysteinemia, developed by subcutaneous implantation of osmotic pumps containing L-homocysteine into male Wistar rats. Our findings show a 42.3% mortality rate in hyperhomocysteinemic rats as compared to 7.7% in control rats. Magnetic resonance imaging of the brain in the surviving rats shows that mild hyperhomocysteinemia leads to exacerbation of ischemic injury within 24 h, which remains elevated over time. Behavioral studies further demonstrate significant deficit in sensorimotor functions in hyperhomocysteinemic rats compared to control rats. Using pharmacological inhibitors targeting the NMDAR subtypes, the study further demonstrates that inhibition of GluN2A-containing NMDARs significantly reduces ischemic brain damage in hyperhomocysteinemic rats but not in control rats, indicating that hyperhomocysteinemia-mediated exacerbation of ischemic brain injury involves GluN2A-NMDAR signaling. Complementary studies in GluN2A-knockout mice show that in the absence of GluN2A-NMDARs, hyperhomocysteinemia-associated exacerbation of ischemic brain injury is blocked, confirming that GluN2A-NMDAR activation is a critical determinant of the severity of ischemic damage under hyperhomocysteinemic conditions. Furthermore, at the molecular level we observe GluN2A-NMDAR dependent sustained increase in ERK MAPK phosphorylation under hyperhomocysteinemic condition that has been shown to be involved in homocysteine-induced neurotoxicity. Taken together, the findings show that hyperhomocysteinemia triggers a unique signaling pathway that in conjunction with ischemia-induced pathways enhance the pathology of stroke under hyperhomocysteinemic conditions.

Full text of DOI:10.1016/j.nbd.2019.03.012

Accepted Manuscript
Hyperhomocysteinemia leads to exacerbation of ischemic brain
damage: Role of GluN2A NMDA receptors
Ankur Jindal, Sathyanarayanan Rajagopal, Lucas Winter, Joshua
W. Miller, Donald W. Jacobsen, Jonathan Brigman, Andrea M.
Allan, Surojit Paul, Ranjana Poddar
PII:
S0969-9961(18)30325-5
https://doi.org/10.1016/j.nbd.2019.03.012
YNBDI 4424
DOI:
Reference:
To appear in:
Neurobiology of Disease
Received date:
Revised date:
Accepted date:
26 July 2018
19 February 2019
14 March 2019
Please cite this article as: A. Jindal, S. Rajagopal, L. Winter, et al., Hyperhomocysteinemia
leads to exacerbation of ischemic brain damage: Role of GluN2A NMDA receptors,
Neurobiology of Disease, https://doi.org/10.1016/j.nbd.2019.03.012
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ACCEPTED MANUSCRIPT
Hyperhomocysteinemia leads to exacerbation of ischemic brain damage: role of GluN2A
NMDA receptors
#
Ankur Jindal*, Sathyanarayanan Rajagopal*, Lucas Winter*, Joshua W Miller , Donald W
$
ψ
ψ
Jacobsen , Jonathan Brigman , Andrea M Allan , Surojit Paul* and Ranjana Poddar*
ψ
*
Department of Neurology, Department of Neurosciences, University of New Mexico Health
Sciences Center, 1 University of New Mexico, Albuquerque, NM – 87131
#
Department of Nutritional Sciences, Rutgers University, 65 Dudley Road, Room 107
New Brunswick, NJ 08901
$
Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic,
Cleveland, OH 44195
¶
Corresponding Author:
Ranjana Poddar, PhD, University of New Mexico Health Sciences Center, Department of
Neurology, 1, University of New Mexico, Albuquerque, NM – 87131
Tel: (505) 272-5859
e-mail: rpoddar@salud.unm.edu
Abbreviated title: Hyperhomocysteinemia and GluN2A-NMDAR in stroke injury
1

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ABSTRACT
Hyperhomocysteinemia has been implicated in several neurodegenerative disorders
including ischemic stroke. However, the pathological consequences of ischemic insult in
individuals predisposed to hyperhomocysteinemia and the associated etiology are unknown. In
this study, we evaluated the outcome of transient ischemic stroke in a rodent model of
hyperhomocysteinemia, developed by subcutaneous implantation of osmotic pumps containing
L-homocysteine into male Wistar rats. Our findings show a 42.3% mortality rate in
hyperhomocysteinemic rats as compared to 7.7% in control rats. Magnetic resonance imaging of
the brain in the surviving rats shows that mild hyperhomocysteinemia leads to exacerbation of
ischemic injury within 24h, which remains elevated over time. Behavioral studies further
demonstrate significant deficit in sensorimotor functions in hyperhomocysteinemic rats
compared to control rats. Using pharmacological inhibitors targeting the NMDAR subtypes, the
study further demonstrates that inhibition of GluN2A-containing NMDARs significantly reduces
ischemic brain damage in hyperhomocysteinemic rats but not in control rats, indicating that
hyperhomocysteinemia-mediated exacerbation of ischemic brain injury involves GluN2A-
NMDAR signaling. Complementary studies in GluN2A-knockout mice show that in the absence
of GluN2A-NMDARs, hyperhomocysteinemia-associated exacerbation of ischemic brain injury
is blocked, confirming that GluN2A-NMDAR activation is a critical determinant of the severity
of ischemic damage under hyperhomocysteinemic conditions. Furthermore, at the molecular
level we observe GluN2A-NMDAR dependent sustained increase in ERK MAPK
phosphorylation under hyperhomocysteinemic condition that has been shown to be involved in
homocysteine-induced
neurotoxicity.
Taken
together,
the
findings
show
that
2

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hyperhomocysteinemia triggers an unique signaling pathway that in conjunction with ischemia-
induced pathways enhance the pathology of stroke under hyperhomocysteinemic conditions.
Key words: hyperhomocysteinemia; GluN2A-NMDA receptors (also known as NR2A-NMDA
receptors); GluN2B-NMDA receptors (also known as NR2B-NMDA receptors); ERK MAPK,
GluN2A-NMDAR knockout mice; middle cerebral artery occlusion; ischemic brain injury;
magnetic resonance imaging; behavioral studies;
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Abbreviations: NMDAR, N-methyl-D-aspartate receptors; ERK MAPK, extracellular-regulated
kinase mitogen activated protein kinase; GluN2A-KO, GluN2A-NMDAR knockout mice, WT,
wild type; MCAO, middle cerebral artery occlusion; MRI, magnetic resonance imaging; ADC,
apparent diffusion coefficient; FA, fractional anisotropy;
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INTRODUCTION
Cerebral ischemic stroke is one of the leading causes of death and long-term disability
worldwide (Chin and Vora, 2014; Johnston et al., 2009). Prevalence of comorbidities further
worsen the clinical outcome, creating additional complications to any treatment approach for
stroke (Fisher et al., 2009). A better understanding of stroke pathology under comorbid
conditions is therefore of paramount importance to develop treatment strategies to reduce both
stroke-induced brain damage as well as the synergistic effect induced by comorbid conditions.
Hyperhomocysteinemia, a common metabolic disorder characterized by systemic elevation
of homocysteine is considered to be a risk factor for neurological disorders including ischemic
stroke (Hankey and Eikelboom, 2001; Seshadri et al., 2002; Zoccolella et al., 2006).
Epidemiological studies have linked mild (15-30 µM) and moderate (30-100 µM)
hyperhomocysteinemia to nutritional deficiency of folate, vitamin B12 and vitamin B6, renal
impairment and hypothyroidism, while severe hyperhomocysteinemic conditions (100-500 µM)
are caused by genetic mutations and impaired activity of key enzymes in the homocysteine
metabolic pathway (Austin et al., 2004; Obeid and Herrmann, 2006; Refsum et al., 1998;
Seshadri et al., 2002). Although vitamin supplementation has considerably reduced mild to
moderate
hyperhomocysteinemia
in
the
general population,
the prevalence of
hyperhomocysteinemia in the elderly population is still significantly high worldwide (Janson et
al., 2002; Ramos et al., 2005; Robles et al., 2005; Selhub et al., 1999). This has been attributed to
low nutritional absorption of essential B-vitamins, decreased metabolic function with advanced
age, insufficient renal / hepatic function and medications involving vitamin B6 and folate
antagonists (Hankey and Eikelboom, 2001; McCully, 2007; Refsum et al., 1998; Zoccolella et
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al., 2006). Thus, it is conceivable that predisposition to hyperhomocysteinemia could contribute,
at least in part, to the severity of neurological disorders in the elderly. However, the outcome of
an ischemic insult under hyperhomocysteinemic conditions is still elusive.
In previous studies, we and others have reported that homocysteine is an agonist of the N-
methyl-D-aspartate subtype of glutamate receptors (NMDARs) that is known to be involved in
excitiotoxicity and ischemic brain damage, caused by excessive release of glutamate during an
insult (Ganapathy et al., 2011; Jara-Prado et al., 2003; Kruman et al., 2000; Lipton et al., 1997;
Poddar and Paul, 2009; Poddar and Paul, 2013). Our recent findings also show that treatment of
cultured neurons with homocysteine leads to cell death through activation of a unique signaling
cascade, which is independent of excitotoxic cell death mediated through GluN2B subunit
containing NMDARs (GluN2B-NMDAR), suggesting a role of GluN2A subunit containing
NMDARs (GluN2A-NMDAR) in homocysteine-induced neurotoixicty (Poddar et al., 2017a;
Poddar and Paul, 2009; Poddar and Paul, 2013). These findings further show that homocysteine-
induced neurotoxicity involves sustained increase in extracellular-regulated kinase mitogen
activated protein kinase (ERK MAPK) phosphorylation, which involves a rapid initial increase
followed by a delayed larger increase. This later increase in ERK MAPK phosphorylation is
predominantly responsible for homocytsiene-dependent neuronal cell death (Poddar and Paul,
2
013). In addition to our findings, a more recent study performed in neuronal cultures indicates
that homocysteine-induced increase in native NMDAR current preferentially involves GluN2A-
NMDAR stimulation (Sibarov et al., 2016). This raises the possibility that homocysteine-induced
intracellular signaling mechanisms act in concert with glutamate-mediated excitotoxic pathways
to influence the pathology of stroke under hyperhomocysteinemic conditions.
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A significant goal of the current study is to unravel whether hyperhomocysteinemia is a
critical determinant of the severity of ischemic brain damage. To address this issue, we evaluated
the pathophysiology and behavioral outcome of stroke in an animal model of
hyperhomocysteinemia. Using non-invasive magnetic resonance imaging (MRI) approach and a
battery of behavioral tests, we show that hyperhomocysteinemia exacerbates ischemic brain
damage resulting in severe functional deficits. Pharmacological inhibition of GluN2A-NMDAR
significantly reduces the extent of brain damage and functional deficits in the
hyperhomocysteinemic rats, suggesting that exacerbation of ischemic brain injury under
hyperhomocysteinemic conditions is mediated through GluN2A-NMDAR signaling. Consistent
with this interpretation, hyperhomocysteinemia fails to exacerbate ischemic brain injury in
GluN2A-knockout mice (GluN2A-KO), confirming the role of GluN2A-NMDARs in promoting
ischemic brain injury under hyperhomocysteinemic conditions. The role of GluN2A-NMDAR
signaling in exacerbation of ischemic brain injury under hyperhomocysteinemic condition
strongly suggests that a single therapeutic approach targeting the glutamate/excitotoxic pathway
may not be universally applicable to all ischemic stroke patients.
MATERIALS AND METHODS
Materials and reagents: L-homocysteine thiolactone hydrochloride, cytosine D-
arabinofuranoside, glycine and Hoechst 33342 were obtained from Sigma-Aldrich (St. Louis,
MO, USA). Selective pharmacological inhibitors were: Ro 256981 was obtained from Tocris,
(Bristol, UK) and NVP-AAM077 ([(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-
tetrahydro-quinoxalin-5-yl)-methyl]phosphonic acid) was a gift from Dr. Yves P Auberson,
Novartis, (Basel, Switzerland). Anti-phospho ERK 1/2 (Thr202/Tyr204) monoclonal antibody
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(
pERK) and the anti-rabbit and anti-mouse horse-radish-peroxidase conjugated secondary
antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-ERK2 polyclonal
antibody (ERK) was obtained from Santacruz Biotechnology (Dallas, TX). Bicinchoninic acid
(BCA) protein estimation kit and West Pico supersignal chemiluminescence reagents for
immunoblotting were obtained from Pierce (Rockland, IL). All reagents required for tissue
culture studies were obtained from Life Technologies (Carlsbad, CA). Male Wistar rats were
purchased from Envigo (Placentia, CA, USA). Rats were maintained in a 12-h light/dark
vivarium (light off at 18.00 h) and with access to food and water ad libitum. Female time
pregnant Sprague-Dawley rats were purchased from Envigo (Livermore, CA, USA) for
establishing rat primary neuron cultures. GluN2A-NMDAR knockout mice (GluN2A-KO) was
generated as previously described (Sakimura et al., 1995). They were obtained from Dr. Andrew
Holmes, NIH/NIAAA, (Rockville, MD) and bred in the animal facility of University of New
Mexico. Time pregnant female wild type (WT) and GluN2A-KO mice were generated at the
animal facility of University of New Mexico for establishing mice neuronal cultures.
All
experiments were performed in accordance with protocols approved by the Institutional Animal
Care Committee of University of New Mexico and were in compliance with the ARRIVE
guidelines.
Development of hyperhomocysteinemic rat and mouse models: L-homocysteine (200
mM) was freshly prepared by alkali hydrolysis of L-homocysteine thiolactone hydrochloride
followed by neutralization, and maintained in 0.02mM of N-Tris(hydroxymethyl)methyl-2-
aminoethanesulfonic acid buffer pH 7.4 as described earlier (Poddar et al., 2001). Osmotic
pumps (Alzet; 2ML1, flow rate 10 µl/hr) containing either 2 ml of freshly prepared 200 mM L-
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homocysteine (for hyperhomocysteinemia) or normal saline (for control) were surgically
implanted subcutaneously on the back and posterior to the scapulae of male Wistar rats under
anesthesia (2% isoflurane in medical grade oxygen). The incision was closed after implantation
and the rats were allowed to recover in their cage. For both WT and GluN2A-KO mice, osmotic
pumps (Alzet; 2001, flow rate 1µl/hr) containing either 200 µl of freshly prepared 200 mM L-
homocysteine (for hyperhomocysteinemia) or normal saline (for control) were implanted
similarly.
Measurement of total plasma homocysteine in rats and mice: Blood was obtained
from rats (retro-orbital) and mice (cardiac puncture) under anesthesia with 2% isoflurane in
medical grade oxygen, before installation of osmotic pumps (0 day) and at various time points
after installation of osmotic pumps (rats: 3, 5 and 7 day; mice: 3 and 5 day). Blood was collected
in standardized Vacutainer venous blood collection EDTA-tubes and centrifuged at 3,500 rpm
for 15 min. The plasma obtained was analyzed for total plasma homocysteine levels using high
performance liquid chromatography with post-column fluorescence detection as described earlier
(Gilfix et al., 1997; Jacobsen et al., 1994; Miller et al., 2013).
Induction of transient focal cerebral ischemia: Middle cerebral artery occlusion
(
MCAO) was performed on both male rats and mice. For rat studies, control and
hyperhomocysteinemic male Wistar rats (8-9 weeks, 290-295 g) were subjected to MCAO under
anesthesia (2% isoflurane in medical grade oxygen), using the intraluminal method as described
in our previous studies (Candelario-Jalil et al., 2004; Deb et al., 2013; Poddar et al., 2017b).
Briefly, the right common carotid artery (CCA) was exposed through an incision made in the
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ventral midline neck region. To prevent improper insertion of the occluding filament, both the
external carotid artery and pterygopalatine branch of the internal carotid artery were clipped. A
silicon-rubber-coated monofilament (403756, Doccol) was inserted into the internal carotid
artery through an incision made in the CCA, 2 mm proximal from the bifurcation of the CCA,
and advanced 18-19 mm from the bifurcation, to occlude the origin of the anterior cerebral,
middle cerebral, and posterior communicating arteries. The incision was closed and the rats were
allowed to recover from anesthesia. A quick assessment of neurological deficit was done
immediately prior to reperfusion and only those aninmals that had more than 50% of
contralateral forelimb flexion and walked in circles to the contralateral side were considered for
the study (Longa et al., 1989). Following 60 min of occlusion, the rats were anesthetized and the
filament was gently retracted to allow reperfusion. The incision was closed and the rats were
allowed to recover from anesthesia. A subset of control and hyperhomocysteinemic rats received
a single intravenous injection of GluN2A-NMDAR selective inhibitor NVP-AAM077 (1.2
mg/kg body weight) through the femoral vein, and another subset received a single
intraperitoneal injection of GluN2B-NMDAR inhibitor Ro 256981 (6 mg/kg body weight)
(Chaperon et al., 2003; Fox et al., 2006). Both NVP-AAM077 and Ro 256981 were administered
at the onset of ischemic insult. The rats were then subjected to MRI on days 1, 3, and 14 post-
MCAO and a series of behavioral tests was performed between Day 7-9, post-MCAO.
For biochemical studies control and hyperhomocysteinemic rats were subjected to sham
surgery or MCAO for 60 min followed by reperfusion for 0, 3, 6, 12 or 24h. A sub-group of
hyperhomocysteinemic rats were injected with NVP-AAM077 (1.2 mg/kg body weight) through
the femoral vein at the onset of MCAO and sacrificed after 3h reperfusion. Rats were decapitated
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at each of the above specified time points and the cortex from the ipsilateral hemisphere
0
dissected on ice, briefly sonicated in Laemmli sample buffer, boiled at 100 C for 10 min, and
processed for immunoblotting analyses (Deb et al., 2013).
For studies using WT and GluN2A KO mice (12-14 weeks, 26.5 – 27.5 g), MCAO was
performed by the intraluminar method as described above. A silicon-rubber-coated
monofilament (602234, Doccol) was advanced from an incision in the CCA through the internal
carotid artery to a length of 10-11 mm from the bifurcation, occluding the middle cerebral artery.
After 30 min of MCAO, the filament was removed to allow reperfusion, and the incision was
closed. The mice were then subjected to MRI 24h after the ischemic insult.
Quantitation of infarct size and structural integrity by MRI: Multimodal MRI of rat
brain that includes relaxation time imaging and diffusion imaging was performed at 1, 3 and 14
days after MCAO and reperfusion. The rats were placed in a dedicated holder, and positioned in
the isocenter of a 4.7-Tesla MRI scanner (Bruker Biospin) that is equipped with a 40-cm bore
and a gradient of 660 mT/m at rise time within 120 μsec. To obtain good signal-to-noise ratio, a
small-bore linear RF coil (inner diameterꢀ=ꢀ72 mm) was used for signal excitation, and a single
tuned surface coil (RAPID Biomedical, Rimpar, Germany) was used for signal detection (Poddar
et al., 2017b; Sood et al., 2009; Taheri and Sood, 2007; Yang et al., 2013). For mice, the MRI
was performed only at day 1. The mice were placed in its dedicated holder of inner diameter
7
2mm in the 4.7-Tesla MRI scanner with a 40-cm bore size and a gradient rise time / maximum
speed of 9000 T/m/s. During MRI, the rats and mice were kept anesthetized with 2% isoflurane
in medical grade oxygen. Respiration and heart rate were monitored continuously and body
0
temperature was maintained at 37.0ꢀ±ꢀ0.5 C.
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For rats, T2-weighted images were acquired with a rapid acquisition with relaxation
enhancement (RARE) sequence of Repetition Time (TR)/Echo Time (TE)ꢀ=ꢀ5,000 ms/56 ms,
Field of View (FOV)ꢀ=ꢀ4 cmꢀ×ꢀ4 cm, slice thicknessꢀ=ꢀ1 mm, inter-slice distanceꢀ=ꢀ1.1 mm,
number of slicesꢀ=ꢀ12, matrixꢀ=ꢀ256ꢀ×ꢀ256 and number of averageꢀ=ꢀ3. For mice, T2 weighted
images were acquired with a RARE sequence of Repetition Time / Echo Timeꢀ=ꢀ5,000 ms/56 ms,
Field of View =ꢀ4 cmꢀ×ꢀ4 cm, slice thicknessꢀ=ꢀ1 mm, number of slicesꢀ=ꢀ12, matrixꢀ=ꢀ256ꢀ×ꢀ256
and number of averageꢀ=ꢀ3. The infarcted area was determined from T2 maps derived from the
T2-weighted images by comparing regions of hyperintensity (infarcted or damaged area) and
hypointensity (noninfarcted or undamaged area). An observer blinded to the experimental
conditions evaluated the volume of ischemic brain damage from the T2 maps. For each slice,
regions of hypointensity were highlighted on the ipsilateral side and the area measured. The total
area on the contralateral side was also determined. The areas of hypointensity for the ipsilateral
side and contralateral side were obtained by adding all slices together and the respective volumes
were calculated by multiplying each sum by 1 (thickness of each section). The percentage of
infarction volume was calculated as follows: [(volume of contralateral side − noninfarcted
volume of the lesioned side)/volume of contralateral side] × 100 (Swanson et al., 1990).
Multi-slice, multi-shot, diffusion-weighted echo-planar imaging (Repetition Time / Echo
2
Timeꢀꢀ=ꢀ3,800 ms/38 ms; b-valuesꢀ=ꢀ600 and 1,900 s/mm in 30 directions; Field of View
ꢀ=ꢀ4 cmꢀ×ꢀ4 cm, slice thicknessꢀ=ꢀ1 mm, matrixꢀ=ꢀ256ꢀ×ꢀ256) was performed to assess tissue
architecture. Quantitative apparent diffusion coefficient (ADC) maps were calculated on a voxel-
wise basis, with a linear least squares fit on the logarithm of the signal intensity versus the b-
value for each diffusion direction. Based on the ADC maps, fractional anisotropy (FA) maps
were generated using ParaVision 5.1 (Bruker Biospin MRI, Bellerica, MA, USA). Both the ADC
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and FA values were computed for each slice and averaged over all the slices for the tissue. For
MRI study, thirteen rats were subjected to MCAO in the control group, out of which one died
within 24h. MRI scans were too noisy for one rat on day 1, one rat on day 3 and two rats on day
1
4. In the hyperhomocysteinemic group, MCAO was performed on twenty-six rats, out of which
eleven died within 24 h. MRI scan was too noisy for one rat on day 14. These animals were
therefore excluded from the MRI study on those days. In addition, ADC and FA values were not
computed for one NVP-AAM077 treated hyperhomocysteinemic rat, as the processed maps were
too noisy.
Behavioral Studies: All rats were subjected to a battery of behavioral tests on day 7
(CatWalk), day 8 (cylinder and rotarod tests) and day 9 (adhesive test) after MCAO to evaluate
normal gait, motor coordination and sensory motor functions. Habituation and training (3 days)
was performed for one week before MCAO. The first two trainings days were before osmotic
pump implantation and the third training day was after pump implantation. An experienced
observer blinded to the experimental conditions evaluated all behavioral parameters.
Catwalk: An automated quantitative gait analysis system (Catwalk XT 10.5, Noldus) was
used to assess deficits in normal gait in the control, hyperhomocysteinemic and NVP-AAM077
treated hyperhomocysteinemic rats as described earlier (Parkkinen et al., 2013; Poddar et al.,
2
017b; Wang et al., 2008). Briefly, rats were trained to walk on a glass platform or walkway (1.3
m long and 90 mm wide) with a fluorescent light reflected internally in the glass floor as the rats
cross the walkway, scattering at points where the paws touch the glass. A camera was positioned
5
6 cm below the walkway with intensity threshold set to 0.15, camera gain set to 17 and the
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maximum allowed speed variation set to 60%. Pixels below the light intensity of 16 units on a 0 -
2
55 arbitrary scale were filtered out. A trial was regarded as successful if the animal did not have
a maximum speed variation greater than 60% or did not stop on the runway. If an animal failed
to complete a trial within 10 sec, walked backwards, or reared during the run, an additional re-
run was performed. The camera recorded three such complete or successful runs across the
walkway and the average of the three runs is reported. An experienced observer, blinded to the
experimental group, labeled each paw on the recorded video and paw-related parameters were
analyzed. The steps were automatically labeled as right fore paw (RF), right hind paw (RH), left
fore paw (LF), and left hind paw (LH), where the right represents the non-impaired side and the
left represents the impaired or affected side. Faulty labels caused by tail, whiskers, or genitalia
were corrected. Automated analysis of wide range of parameters was performed: (A) The
2
maximal contact area (expressed in mm ), which is the paw area contacted at the moment of
2
maximal paw-floor contact during stance was measured; (B) The print area (expressed in mm ),
which is the total floor area contacted by the paw during the stance phase was measured; (C) The
print position (expressed in cm), which is the space relationship or distance between the former
fore paw position to the consecutive hind paw position of the same side during one crossing of
the walkway was evaluated. One control rat and one hyperhomocyteinemic rat were excluded
from the study as they paused repeatedly during the run.
Cylinder
test:
Control,
hyperhomocysteinemic
and
NVP-AAM077
treated
hyperhomocysteinemic rats were subjected to cylinder test to assess the post-stroke asymmetry
in fore-limb use (Balkaya et al., 2013; Poddar et al., 2017b). The rats were placed individually
inside a transparent plexiglass cylinder (diameter 20 cm, height 45 cm). A vertical exploration
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movement with either the left or right forelimb along the wall was scored as contact of each paw
with the glass wall for a total period of 2 min. Simultaneous contact by both paws was scored
separately. Two trials (10 min rest between trials) were recorded and the percentage use of the
affected fore limb was calculated (Liu et al., 2013; Schaar et al., 2010). Six control rats and one
hyperhomocysteinemic rat were excluded from the study, as they did not participate in the
vertical exploration of the cylinder.
Accelerated Rotarod test: Control, hyperhomocysteinemic and NVP-AAM077 treated
hyperhomocysteinemic rats were subjected to the accelerated Rotarod test to evaluate
impairment of motor co-ordination and balance following stroke (Bouet et al., 2007; Dunham
and Miya, 1957; Poddar et al., 2017b; Schaar et al., 2010). Rats were placed on a rotating
cylindrical rod accelerating from 0 to 50 rpm for a period of 180 sec, and their latency to fall was
recorded. The mean of four runs (10 min rest between trials) was used for statistical analysis.
One hyperhomocysteinemic rat was excluded from the study as the rat jumped off the rod
repeatedly.
Adhesive removal test: Sensorimotor deficits following stroke was assessed by
performing the adhesive removal test in control, hyperhomocysteinemic and NVP-AAM077
treated hyperhomocysteinemic rats. A small adhesive patch (7 mm ×3 mm) was applied to the
contralateral forepaw of each rat with equal pressure and the time taken to notice the presence of
the patch (time to contact) and to remove it (time to remove) was recorded (Bouet et al., 2009;
Poddar et al., 2017b). The time taken to notice the patch was recorded from the moment of
placing the patch until the paw was shaken or touched by mouth. The time taken to remove the
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patch was recorded from the moment the mouth touched the paw to the time the patch was
removed. The trial ended after the adhesive patch was removed or a maximum latency of 3 min.
The mean of three trials (10 min rest between tests) was used for statistical analysis.
Cresyl violet staining: On day 14 following MCAO, control, hyperhomocysteinemic and
NVP-AAM077 treated hyperhomocysteinemic rats were anesthetized with isoflurane and
perfused intracardialy with 4% paraformaldehyde in 0.01M phosphate buffered saline pH 7.2
(PBS). Brains were removed, cryoprotected in 30% sucrose in PBS and then frozen in Optimal
Cutting Temperature compound (OCT) kept in dry ice. Cresyl Violet Acetate staining was
performed on 12µm cryosections. The fat was removed from the sections by immersing them in
ethanol followed by chloroform. The sections were then re-hydrated by sequential exposure to
decreasing concentrations of ethanol (100%, 95%, 70% and 0%) followed by staining with
Cresyl Violet Acetate solution (5% Cresyl Violet acetate in glacial acetic acid-sodium acetate
buffer, pH 3.7) for 8 min. The sections were differentiated in 0.1% glacial acetic acid in 70%
ethanol for 30 sec followed by sequential dehydration in 95% and 100% ethanol and clearing
with xylene. Finally, the sections were mounted with Cytoseal (DPX) and Multi Area Time
Lapse images were obtained for the whole section using an Olympus microscope (10x objective).
Primary Neuron culture preparation and stimulation: Embryos obtained from time
pregnant (1) Sprague Dawley female rats (16-17 day gestation), (2) WT mice and (3) GluN2A-
KO mice (15-16 day gestation) were used to establish primary cortical neuronal cultures as
described earlier (Poddar et al., 2017a; Poddar and Paul, 2009; Poddar and Paul, 2013). The
neuron cultures were maintained in culture for 12-14 days and then treated with freshly prepared
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L-homocysteine (50 µM) in Hank’s balanced salt solution containing 50 µM of glycine (Lipton
et al., 1997; Poddar et al., 2017a; Poddar and Paul, 2009; Poddar and Paul, 2013) for the
specified time periods. In some of the neuron cultures obtained from rat embryos
pharmacological inhibitors (NVP-AAM007, 30 nM; or Ro 25-6981 1 µM) were added 10 min
before exposure to homocysteine. At the specified time points cells were harvested and cell
lysates were processed for immunoblotting studies.
Oxygen glucose deprivation (OGD) and Hoechst DNA staining: For OGD challenge,
neurons were placed in an anaerobic chamber (Coy Laboratories; 95:5% of N2:CO2 gas mixture
0
and maintained at 37 C) and incubated in balanced salt solution lacking glucose (116 mM NaCl,
5
.4 mM KCl, 1 mM NaH2PO4, 1.8 mM CaCl2, 26.2 mM NaHCO3, 5 mM HEPES, 0.01 mM
glycine, pH 7.4) as described earlier (Deb et al., 2013). Some cultures were exposed to L-
homocysteine (50 µM) during the OGD insult in the absence or presence of NVP-AAM077 (30
nM; 3h). At specified time periods (1, 2, and 3h) during OGD cells were removed from the
chamber and lysed for immunoblot analysis. In some experiments after a 2h insult (OGD alone
or OGD + L-homocysteine), the OGD medium was replaced with the original culture medium
0
and then incubated at 37 C for an additional 22h in a humidified atmosphere (95:5% of air/CO2
mixture). Cells were then subjected to staining with Hoechst 33342 dye for 15 min, washed
extensively with PBS, and analyzed using fluorescent microscopy to assess nuclear damage.
Imaging was performed with a Zeiss Axiovert 200M fluorescence microscope with attached
AxioCam CCD camera using 20· objective lens (Carl Zeiss, Thornwood, NY, USA). To
quantitatively assess the percentage of pyknotic nuclei, a total of 1000 cells were counted for
each set of experiments (Poddar and Paul, 2009; Poddar and Paul, 2013).
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Immunoblotting: Equal protein from total cell lysates obtained from rat and mice
neuronal cultures as well as from brain lysates were resolved by SDS-PAGE (7.5%), and
subjected to immunoblotting procedure as described earlier (Deb et al., 2013; Poddar and Paul,
2
009). Blots were analyzed with anti-pERK and anti-ERK antibodies according to manufacturers
recommendation. Signals from immune complexes in the blots were developed using West Pico
supersignal chemiluminescence reagents and then captured on X-ray films. Densitometric
analysis of the images was performed using Image J software.
Experimental design and statistical analysis: Data involving multiple groups were
analyzed using one-way or two-way analysis of variance (ANOVA) as indicated and where
assessment was performed across multiple days, the data was analyzed using repeated measure
ANOVA (SPSS 24.0 software). Post-hoc analysis was done by Bonferroni’s or Newman-Keuls
multiple comparison tests. Vehicle vs. hyperhomocysteinemic groups or hyperhomocysteinemic
vs. NVP- AAM077 treated hyperhomocysteinemic groups were considered as between group
factor and days post-stroke as repeated factor. Analysis of two-group comparison was done using
the Student’s t-test. Data in the text and figures are expressed as means ± SEM and differences
were considered statistically significant when p < 0.05. Pearson’s r was used for effect size
calculation for all t-test data (Mukaka, 2012). Power calculations were run using G* power
(3.1.9.2). For the one-way ANOVA, using an effect size of 0.60, an alpha of .05 and n = 13 per
treatment group produced 83% power. A sensitivity test using a one-way ANOVA with n = 13
and a power of 80% at an alpha level of .05 indicates that we have sufficient sample size to
detect an effect size of 0.57 and for the repeated measure ANOVA we can detect an effect size of
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0
.256 with 80% power. Achieved power for the repeated measures ANOVA (2 between and 3
repeated measures) with an effect size of 0.7, an alpha of .05, and n = 12 per treatment group
produced a power of 99%. For the lowest significant t-test the effect size was 0.507, the
calculated power was determined to be 76% with the given sample sizes n = 10 for vehicle and n
=
12 for hyperhomocysteinemia. Thus, there is sufficient power in the data set to justify the
conclusions.
RESULTS
Hyperhomocysteinemia exacerbates ischemic brain injury in rats
In initial studies, blood samples were obtained from control and hyperhomocysteinemic
rats at different time intervals as outlined in Figure 1A, for analysis of total plasma
homocysteine. Figure 1B shows that total plasma homocysteine level prior to pump implantation
is 8.8 ± 0.76 µM. This basal level does not change significantly in the control rats with the
installation of saline containing pumps. However, installation of L-homocysteine containing
pumps increases total plasma homocysteine level to 23.2 ± 0.52 µM (p < 0.0001; r = 0.969)
within 3 days that remains sustained for the time period of the study (day 5: 23.57 ± 0.57 µM; p
<
0.0001; r = 0.9663, day 7: 20.52 ± 0.88; p < 0.0001, r = 0.954).
In subsequent experiments control and hyperhomocysteinemic rats were subjected to
either sham surgery or MCAO (60 min), 4 days after pump implantation, followed by
reperfusion. Infarct size and functional outcome were assessed as outlined in Figure 2A. Our
findings show that ischemic insult under hyperhomocysteinemic (HHcy) condition increases the
mortality rate substantially when compared with the control group (Fig. 2B; control: 7.69% vs.
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HHcy: 42.3%). The surviving animals were subjected to MRI 24h after MCAO to assess the
extent of ischemic brain injury. The representative T2 maps show regions of increased T2 signal
intensity in the stroked hemisphere of both control and hyperhomocysteinemic rats indicating
ischemic lesion (Fig. 2C). The spatial distribution of ischemic lesion in hyperhomocysteinemic
rats extends to both anterior and posterior cortex, regions that are unaffected in the control rats.
Figure 2C further show that in the absence of an ischemic insult hyperhomocysteinemia by itself
does not cause any brain lesion (compare control sham vs. hyperhomocysteinemic sham).
Quantitative analysis of lesion volume in control and hyperhomocysteinemic rats subjected to
MCAO (Fig. 2D) shows a significant increase in infarct size under hyperhomocysteinemic
condition (control: 18.36 ± 2.85% vs. HHcy: 37.41 ± 2.33%; p < 0.0001, r = 0.716).
Long-term progression of ischemic damage in hyperhomocysteinemic rats
Longitudinal evaluation of ischemic lesion volume in control and hyperhomocysteinemic
rats up to 14 days post-MCAO show significant group effect [F (1, 22) = 26.39, p < 0.0001] and
day effect [F (2, 44) = 17.702, p < 0.0001] between the two groups. However, treatment by day
interaction is not significant. The representative T2 maps and quantitative measurement of lesion
volume from the T2 maps (Fig. 3A) show that the infarct size remains significantly higher in the
hyperhomocysteinemic rats at both day 3 (control: 21.94 ± 2.74% vs. HHcy: 38.63 ± 2.75%) and
day 14 (control: 13.81 ± 2.77% vs. HHcy: 27.16 ± 2.18%) after MCAO. The representative ADC
and FA maps and the quantitative analysis of ADC and FA values, evaluated at 14-day post-
3
3
2
MCAO, show significant increase in the ADC value (control: 1.01 x 10 ± 0.01 x 10 mm /sec vs.
3
3
2
HHcy: 1.41 x 10 ± 0.06 x 10 mm /sec; p = 0.002; r = 0.587) with concomitant decrease in FA
value (control: 0.3 ± 0.02 vs. HHcy: 0.25 ± 0.01; p = 0.049; r = 0.405) in the
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hyperhomocysteinemic rats (Fig. 3B and C). This pattern of increased mean diffusion with
decreased directional diffusion as observed from the ADC and FA data respectively suggests that
an ischemic insult under hyperhomocysteinemic condition accelerates tissue breakdown resulting
in greater loss of structural integrity and orientation of the brain tissue in the infarcted area. For
histopathological confirmation of the ischemic lesion observed using MRI at 14-day post-MCAO
brain sections from both control and hyperhomocysteinemic rats were processed for cresyl violet
staining. The representative photomicrographs presented in Figure 3D shows that the
characteristic pattern of lesion observed by cresyl violet staining is comparable to the ischemic
lesion detectable in T2 maps (Fig. 3A).
Exacerbation of ischemic brain damage in hyperhomocysteinemic rats is dependent on
GluN2A-NMDAR
To delineate the role of GluN2A- and GluN2B-NMDAR subunits in exacerbation of
ischemic brain injury under hyperhomocysteinemic condition we evaluated the effect of NVP-
AAM077, which preferentially inhibits GluN2A-NMDARs (Auberson et al., 2002; Chaperon et
al., 2003), and Ro 25-6981, the selective inhibitor of GluN2B-NMDARs (Fischer et al., 1997;
Mutel et al., 1998). Both control and hyperhomocysteinemic rats were subjected to 60min
MCAO followed by reperfusion (24h) and NVP-AAM077 or Ro 256981 was injected at the
onset of the ischemic insult. The representative T2 maps from control and
hyperhomocysteinemic rats treated with NVP-AAM077 (Fig. 4A) and quantitative anslysis of
mean lesion volume (Fig. 4B, C) by two-way ANOVA followed by t-test reveals a significant
effect of HHcy [F (1, 45)= 13.5, p=0.001] and NVP-AAM077 [F(1,45) = 15.4, p = 0.001] and an
interaction between NVP-AAM077 and HHcy [F(1,45) 9.01, p = 0.004]. The interaction
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demonstrates that inhibition of GluN2A-NMDARs with NVP-AAM077 fails to reduce the
infarct size in the control rats (Fig. 4B; control: 18.65 ± 2.89% vs. control + NVP: 16.07 ± 2.8%;
p = 0.530; r = 0.137), which has also been observed in an earlier study (Liu et al., 2007). In
contrast, inhibition of GluN2A-NMDARs in hyperhomocysteinemic rats reduces mortality
(HHcy: 42.3% vs. HHcy + NVP-AAM077: 15.4%) as well as ischemic infarct size (Fig. 4C;
HHcy: 37.41 ± 2.33% vs. HHcy + NVP AAM077: 17.98 ± 3.24%; p < 0.0001; r = 0.714) to a
level that is comparable with the infarct size observed in the control rats (p = 0.8744; r = 0.035).
On the other hand, analysis of mean lesion volume following inhibition of GluN2B-NMDARs
with Ro 256981 (Fig. 4A, D, E) reveals a significant effect of HHcy [F(1,39) = 62.3, p = 0.001]
and an effect of Ro 256981 [F(1,39) = 8.5, p = 0.006]. The findings show that inhibition of
GluN2B-NMDARs significantly reduces infarct size in the control rats (Fig. 4D; control: 18.65 ±
2
.89% vs. control + Ro 256981: 7.46 ± 1.24%; p = 0.002; r = 0.618) and is consistent with earlier
findings (Gogas, 2006; Liu et al., 2007; Parsons et al., 1998). Inhibition of GluN2B-NMDARs in
hyperhomocysteinemic rats also results in a small but non-significant decrease in infarct size
(Fig. 4E: HHcy: 37.41±2.33% vs. HHcy + Ro 256981: 32.48 ± 4.32%; p = 0.286; r = 0.237). The
inability of the GluN2A-NMDAR inhibitor to reduce ischemic lesion volume in the control rats
suggest that in the absence of any underlying comorbidity, the progression of ischemic brain
damage is primarily mediated through GluN2B-NMDAR signaling. However, in the presence of
the comorbid condition of hyperhomocysteinemia GluN2A-NMDAR activation plays an
additional role in the exacerbation of the ischemic brain damage.
In subsequent studies, ischemic lesion volume in hyperhomocysteinemic rats treated with
or without NVP-AAM077 was evaluated up to 14 days post-MCAO to assess the late
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manifestation of brain injury following treatment. Longitudinal evaluation of the infarct size
show significant group difference [F (1, 23) = 26.891, p < 0.0001] and day effect [F (2, 46) = 14.891,
p < 0.0001] between NVP-AAM077 treated and untreated hyperhomocysteinemic rats. However,
treatment by day interaction is not significant. The representative T2 maps and post hoc analysis
of the lesion volume (Fig. 5A) show that NVP-AAM077 treated group has significantly smaller
lesion size at both day 3 (HHcy: 38.63 ± 2.75% vs. HHcy + NVP-AAM077: 19.61 ± 4.48%) and
day 14 (HHcy: 27.16 ± 2.18% vs. HHcy + NVP-AAM077: 10.76 ± 2.40%) after MCAO.
Evaluation of the structural integrity of the brain tissue in the infarcted area at day 14 show a
3
3
2
significant decrease in ADC value (HHcy: 1.41 x 10 ± 0.06 x 10 mm /sec vs. HHcy + NVP-
3
3
2
AAM077: 1.16 x 10 ± 0.05 x 10 mm /sec; p = 0.003; r = 0.574) and concomitant increase in FA
value (HHcy: 0.25 ± 0.01 vs. HHcy + NVP-AAM077: 0.32 ± 0.01; p = 0.002; r = 0.599)
following treatment with NVP-AAM077, reflecting reduced tissue breakdown and less
accumulation of extracellular water in the residual stroke cavity (Fig. 5B, C). These findings
indicate that the effect of early treatment with NVP-AAM077 is not transient.
Inhibition of GluN2A-NMDARs reduces behavioral deficits following ischemia in
hyperhomocysteinemic rats
We next investigated the effect of ischemic brain injury on post-stroke behavioral
impairment
in
the
control,
hyperhomocysteinemic
and
NVP-AAM077
treated
hyperhomocysteinemic rats. Assessment of gait parameters using CatWalk, one week after
stroke reveals significant differences between the treatment groups for maximum contact area [F
=
2, 33)
4.956, p = 0.0131], print area [F (2, 33) = 5.776, p = 0.007] and print position [F (2, 33)
=
(
6
.129, p = 0.005] by one-way ANOVA. Post hoc analyses further show that the maximum
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contact area of the affected forepaw in hyperhomocysteinemic rats is significantly reduced when
compared to control rats (Fig. 6A; control: 1.09 ± 0.056 vs. HHcy: 0.8 ± 0.082; p < 0.05). In
contrast, treatment with NVP-AAM077 significantly increases the maximum contact area of the
affected paw when compared to the untreated hyperhomocysteinemic group (Fig. 6A; HHcy: 0.8
±
0.082 vs. HHcy + NVP-AAM077: 1.11 ± 0.092; p < 0.05). Similarly, the print area is
significantly lesser for hyperhomocysteinemic rats when compared with the control rats (Fig. 6B;
control: 1.42 ± 0.063 vs. HHcy: 1.06 ± 0.097; p < 0.05). However, a significant improvement in
print area is observed following treatment with NVP-AAM077 (Fig. 6B; HHcy: 1.06 ± 0.097 vs.
HHcy + NVP-AAM077: 1.41 ± 0.092; p < 0.05). In addition, the print position of the affected
side is significantly less in the hyperhomocysteinemic rats when compared to the control rats
(Fig. 6C; control: 1.4 ± 0.19 vs. HHcy: 0.72 ± 0.15; p < 0.05), which returns to control levels
following treatment with NVP-AAM077 (Fig. 6C; HHcy: 0.72 ± 0.15 vs. HHcy + NVP-
AAM077: 1.37 ± 0.097; p < 0.05).
Evaluation of the spontaneous usage of the affected forelimb using cylinder test show
significant difference between the treatment groups [F (2, 29) = 22.98, p < 0.0001]. Figure 6D
shows that the usage of the affected forepaw in hyperhomocysteinemic rats is significantly less
as compared to control rats (control: 43.73 ± 1.61% vs. HHcy: 24.24 ± 2.37%; p < 0.0001), and
treatment with NVP-AAM077 results in significant improvement in spontaneous usage of the
affected forepaw in hyperhomocysteinemic rats (HHcy: 24.24 ± 2.37% vs. HHcy + NVP-
AAM077: 40.51 ± 1.86; p < 0.0001).
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Evaluation of motor coordination and balance using accelerated rotarod show significant
difference between the treatment groups [F (2, 34) = 5.813, p = 0.006]. Rotarod performance of
each group show that hyperhomocysteinemic rats are significantly less efficient in maintaining
balance on the rotarod as compared to the control rats (Fig. 6E; control: 118.5 ± 5.31 sec vs.
HHcy: 96.99 ± 4.11 sec; p < 0.05), while treatment with NVP-AAM077 significantly enhances
the performance of hyperhomocysteinemic rats (Fig. 6E; HHcy: 96.99 ± 4.11 sec vs. HHcy +
NVP-AAM077: 112.51 ± 4.9 sec; p < 0.05).
Evaluation of sensory motor deficit using adhesive test show significant group
differences for both the time to make contact [F (2, 35) = 26.42, p < 0.0001] and the time to
remove [F (2, 35) = 42.57, p < 0.0001] the adhesive tape from the contralateral forepaw. Figure 6F
and G shows that hyperhomocysteinemic rats take significantly longer time to contact (control:
3
0.61 ± 4.69s vs. HHcy: 76.82 ± 5.45s; p < 0.0001) and remove (control: 54.53 ± 6.51s vs.
HHcy: 127.86 ± 7.29s; p < 0.0001) the tape when compared to the control rats. On the other
hand, treatment of hyperhomocysteinemic rats with NVP-AAM077 significantly reduces both
the time to contact (HHcy: 76.82 ± 5.45s vs. HHcy + NVP-AAM077: 31.18 ± 5.68s; p < 0.0001)
and remove (HHcy: 127.86 ± 7.29s vs. HHcy + NVP-AAM077: 44.24 ± 7.59s; p < 0.0001) the
tape, indicating an improvement in both sensory and motor skill.
Genetic deletion of GluN2A-NMDARs mitigates ischemic brain injury in
hyperhomocysteinemic mice
We next examined whether deletion of endogenous GluN2A-NMDARs could attenuate
the exacerbation of ischemic brain damage observed under hyperhomocysteinemic condition. In
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initial studies osmotic pumps containing L-homocysteine were installed in both WT and
GluN2A-KO mice and blood samples were collected on day 0 (before pump installation) and
days 3 and 5 after pump implantation for analysis of plasma homocysteine level (outlined in
figure 7A). Figure 7B shows that total plasma homocysteine in WT mice increases from 6.6 ±
0
.73 µM (day 0) to 18.97 ± 1.68 µM on day 3 (p < 0.01) and remains elevated at 16.68 ± 1.73
µM on day 5 (p < 0.01). In GluN2A-KO mice, the total plasma homocysteine level also increases
from 5.72 ± 0.33 µM (day 0) to 18.46 ± 2.55 µM on day 3 (p < 0.01) that remain elevated at
1
5.92 ± 2.09 µM on day 5 (p < 0.01). In subsequent studies, WT and GluN2A-KO mice
implanted with saline pump (control) or homocysteine pump (hyperhomocysteinemic) were
subjected to MCAO (30 min) 4 days after pump implantation. This was followed by reperfusion
and assessment of infarct size by MRI 24h after MCAO. The mild ischemic insult in the WT and
GluN2A-KO control mice did not result in any mortality within the time period of our study,
whereas in hyperhomocysteinemic WT mice 28.5% mortality is observed in the same time
period. Ischemic insult in the hyperhomocysteinemic GluN2A-KO mice also did not result in any
mortality during this period. The representative T2 maps (Fig. 7C) and quantitative analysis of
lesion volumes in the surviving mice (Fig. 7D) by two way ANOVA followed by t-test reveals a
significant effect of genotype [F(1,15)-12.2, p = 0.003] and HHcy [F(1,15) = 13.02, p = 0.003],
as well as an interaction between genotype and HHcy [F(1,15)= 14.13, p = 0.002]. The
interaction shows that ischemic insult in both the WT control mice and GluN2A-KO control
mice result in a small infarct size that is limited to the striatum (WT control: 13.52 ± 3.13% vs.
GluN2A-KO control: 14.85 ± 3.8%, p = 0.82; r = 0.077) and is consistent with earlier findings
(Deb et al., 2013). However, ischemic insult in the hyperhomocysteinemic WT mice results in
significant exacerbation of the ischemic brain damage that encompasses the striatum and the
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cortex (WT control: 13.52 ± 3.12% vs. WT HHcy: 50.28 ± 6.54%; p = 0.0001; r = 0.864). These
findings are in agreement with our previous results in rats (Fig 2C and D), suggesting that
regardless of species, hyperhomocysteinemic condition exacerbates ischemic brain injury.
However, such exacerbation of ischemic brain injury is not observed in hyperhomocysteinemic
GluN2A-KO mice (GluN2A-KO control: 14.85 ± 3.8% vs. GluN2A-KO HHcy: 14.1 ± 1.77; p =
0
.867; r = 0.088), confirming a role of GluN2A-NMDARs in hyperhomocysteinemia-induced
exacerbation of ischemic brain injury.
Mechanistic basis of GluN2A-NMDAR dependent exacerbation of ischemic brain injury
under hyperhomocysteinemic condition.
To examine the role of GluN2A-NMDARs in homocysteine induced delayed ERK MAPK
phosphorylation in initial studies cortical neuronal cultures were treated with L-homocysteine
(50 µM, 4h) in the presence or absence of NVP-AAM077 or Ro 25-6981. Figure 8A shows
significant increase in ERK MAPK phosphorylation 4h after homocysteine treatment, which is
consistent with our earlier findings (Poddar and Paul, 2013). Incubation of neurons with NVP-
AAM077 blocks homocysteine-induced ERK MAPK phosphorylation. In contrast, Ro 25-6981
has no effect on the homocysteine-mediated ERK MAPK phosphorylation. In subsequent studies
neuron cultures obtained from WT and GluN2A-KO mice were exposed to L-homocysteine
treatment (50 µM, 4h). Figure 8B shows that homocysteine treatment leads to significant
increase in ERK MAPK phosphorylation in the neuronal cultures obtained from WT mice, while
it remains unchanged in the neurons obtained from GluN2A-KO mice, when compared to the
respective untreated controls. These findings confrm the role of GluN2A-NMDAR in
homocysteine-induced delayed ERK MAPK phosphorylation that has been shown to be
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7

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responsible for homocysteine-induced neuronal cell death (Poddar and Paul, 2009; Poddar and
Paul, 2013).
We next exposed neuronal cultures to OGD (1, 2 and 3h) to mimic ischemic injury, for
assessment of ERK MAPK phosphorylation profile in the absence or presence of homocysteine.
Immunoblot analysis shows that OGD insult alone leads to a rapid but transient increase in ERK
MAPK phosphorylation by 1h (Fig. 8C), In contrast, OGD in the presence of homocysteine leads
to sustained increase in ERK MAPK phosphorylation throughout the time course examined (Fig.
8
C). To evaluate the role of GluN2A-NMDAR in the sustained ERK MAPK phosphorylation,
neurons were exposed to OGD and L-homocysteine in the presence of NVP-AAM077 for 3h.
Figure 8D shows that treatment with NVP-AAM077 inhibits ERK MAPK phosphorylation in
neurons exposed to OGD in the presence of L-homocysteine. To further determine whether the
sustained ERK MAPK phosphorylation observed following OGD in the presence of L-
homocysteine is also associated with enhanced neuronal injury, neuronal cultures were subjected
to OGD for 2 hr in the presence of homocysteine (50 µM) and then maintained in re-oxygenated
conditions for 22 hr. Quantitative analysis of the extent of cell death shows that OGD-induced
cell death increases by 2-fold in the presence of homocysteine (Fig. 8E).
We next investigated whether a mild ischemic insult under hyperhomocysteinemic
condition could lead to prolonged increase in ERK MAPK phosphorylation, similar to what we
have observed in cultured neurons exposed to OGD in the presence of homocysteine (Fig. 9A).
For these experiment both normal and hyperhomocysteinemic Wistar rats were subjected to
MCAO for 60 min followed by reperfusion for specified time periods (0, 3, 6, 12h). Immunoblot
analysis of tissue punches from the ipsilateral cortex show that, during the insult, (I60/0h RPF)
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ERK MAPK phosphorylation increases significantly in the hyperhomocysteinemic rats when
compared to control rats, and it remains sustained during reperfusion (Fig. 9A). We then
determined whether administration of NVP-AAM077 could block the sustained phosphorylation
of ERK MAPK in hyperhomocysteinemic rats at 3h of reperfusion. Figure 9B shows a
significant reduction in ERK MAPK phosphorylation in hyperhomocysteinemic rats when
treated with NVP-AAM077. The findings highlights the role of GluN2A-NMDAR in sustained
ERK MAPK phosphorylation following ischemic insult under hyperhomocysteinemic condition.
DISCUSSION
The key finding of the current study is that mild hyperhomocysteinemia leads to
exacerbation of brain damage and significant deficits in sensory motor function following a
transient cerebral ischemia. Importantly, the study also show that pharmacological inhibition of
GluN2A-NMDARs significantly reduces ischemic brain damage in hyperhomocysteinemic rats
but does not alter ischemic lesion size in rats with normal homocysteine level, indicating that
exacerbation of brain injury under hyperhomocysteinemic condition involves GluN2A-NMDAR
activation. Consistent with this interpretation, studies in GluN2A-KO mice further show that in
the absence of GluN2A-NMDARs, hyperhomocysteinemia fails to exacerbate ischemic brain
damage, validating the role of GluN2A-NMDARs in hyperhomocysteinemia-induced brain
injury during an ischemic insult. Biochemical studies further show that GluN2A-NMDAR
mediated ERK MAPK activation is a critical mediator of neuronal injury in the presence of
homocysteine that could act in concert with cytotoxic pathways activated following an ischemic
insult to exacerbate ischemic brain damage under hyperhomocysteinemic condition. To the best
of our knowledge the current study provides the first evidence that GluN2A-NMDARs, which
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9