1. Role of Subunit-Selective N-Methyl-D-Aspartate Receptors in
Neurodegeneration Caused by Oxygen-Glucose Deprivation
Abstract
N-methyl-D-aspartate receptor (NMDAR) subtype-specific antagonists have
recently been tested as potential neurodegenerative drug therapies, especially for those
who have been afflicted with stroke or other physically traumatic brain injuries.
Specifically, the NR2A- and NR2B- NMDAR subtypes play differential roles in the adult
forebrain, where strokes typically occur. Thus, our study examines a NR2A-specific
antagonist, TCN-201, and a NR2B-specific antagonist, Ro25-6981, and their potential
neuroprotective benefits against delayed neuronal death induced by OGD. Our findings
show that TCN-201 is most neuroprotective when given after OGD at higher
concentrations (1µM or 5µM). Ro25-6981 was found to be the most protective at the
lower dose (1µM) when administered after OGD. As a whole, the present work serves
as a pharmacological basis for discovering the mechanisms governing the functions of
the NMDAR subtypes in protecting against delayed neuronal death and for progressing
toward more effective neurodegenerative therapies.
Introduction
In our present society, brain traumas caused by stroke, seizures, concussions, or
neurodegenerative diseases of the central nervous system are leading agents of
morbidity and mortality in men and women alike. In fact, in U.S. citizens, the third most
frequent cause for mortality is cerebral ischemia (stroke) and the fourth, Alzheimer’s
disease [4]. Brain traumas set off cascades that lead to a prolonged period of neuronal
death, especially within the hippocampus. The hippocampus is composed of three main
regions: the CA1 and CA3 pyramidal neurons and the dentate gyrus (DG). Forebrain

2. ischemia often causes delayed loss of CA1 pyramidal neurons, while the DG and the
CA3 pyramidal neurons, for the most part, remain resistant and intact [2]. Delayed
neuronal death occurs when glutamate accumulation leads to the over-activation of
postsynaptic glutamate receptors and a large intracellular calcium influx, triggering
enzymes to permanently damage cellular structures [8].
Currently, our knowledge of NMDAR involvement in neuronal apoptosis is largely
unresolved [3]. Many studies have shown that the over-activation of the NMDAR
subtype of glutamate receptors is the primary step leading to neuronal injury after brain
trauma incidents [8]. Accordingly,
NMDAR antagonists have been suggested as potentially effective
treatments. While some studies have found that NMDA receptors antagonists
do, in fact, protect against neuronal damage [7], other studies have not found their
effectiveness in decreasing neuronal death [5]. Although the role of NMDA receptors in
neurodegeneration has been widely studied and strongly backed, mechanisms
underlying the receptors’ function, specifically their subtypes, in cell death, remains in
question.
The NMDAR, a tetrameric protein, is typically composed of one NR1 subunit and
one or more NR2 subunits [1]. Because each type of NR2 subunit takes on a distinct
molecular structure, the NR2 subunits exhibit different chemical properties. In fact, the
type of NR2 subunit forming the NMDAR may dictate the role the receptor has on
excitotoxic brain damage.The NR2A- and NR2B- subunits are located in the adult
forebrain, where strokes typically transpire; moreover, the activation of the NR2A-
containing NMDAR have been found to encourage neuronal survival while NR2B-

3. containing NMDAR have been shown to promote excitoxicity, thereby increasing
neuronal apoptosis [1], however, further data is necessary to support this theory. While
further scientific findings are necessary in order to support this theory, testing the
proposed effectiveness of these NMDAR agents may help us pinpoint optimal stroke
therapies. We studied the NMDAR subunit- specific therapies, Ro25-6981 and TCN-
201.
Ro25-6951 is a high affinity, selective, activity-dependent blocker of NMDAR,
containing NR2B subunits [10]. Studies have found that Ro25-6981 is neuroprotective
against glutamate toxicity and OGD [10, 11, 12]. Thus, we chose to test whether this
NR2B-specific antagonist can protect against delayed neuronal death caused by OGD.
In order to test the effectiveness of NR2A-specific antagonists in similar conditions, we
chose TCN-201 as a potential neuroprotectant. TCN-201 (3-chloro-4-fluoro-N-[4-[[2-
(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide) is a sulphonamide
derivative that has been identified as a potential NR2A-selective antagonist; TCN-201 is
a NR2A-selective inhibitor that can be used to assess the function of NR2A subunits in
excitotoxic neuronal damage [9]. In one study testing the effectiveness of subtype
selective NMDAR antagonists against cell death and synapse loss-- a protective
mechanism enabling cells to maintain equilibrium in situations of excess excitatory
input-- induced by exposure to HIV-1 transactivator of transcription (Tat), TCN-201 was
found to prevent synapse loss and recovery and have no effect on cell death [13]. As
TCN-201 is a relatively newly identified neurodegenerative treatment, there have been
insufficient findings on the potential benefits of this NR2A-selective antagonist.
Therefore, the goal of our study is to observe whether subunit-specific NMDAR

4. antagonists can protect against delayed neuronal cell death caused by OGD. As many
studies have shown that TCN-201 and Ro25-6981 are effective protective agents [10,
11, 12, 13], we hypothesized that these subunit-specific antagonists will reduce
neuronal damage caused by OGD.
Methods
Hippocampal slices from Sprague-Dawley rats between 8-11 days old were
cultured on semi-permeable inserts (Millicell CM, Millipore, Billerica, Massachusetts,
USA) with 1 mL of culture media (HBSS, 1M HEPES, dH20, 1X MEM, 1M NaHCO3, and
horse serum). We added NaOH to the media to adjust the pH to 7.4. Slices were
allowed to recover for 6 days before being treated with 1uM or 10uM concentrations of
TCN-201 or Ro25-6981. Because we wanted to test the effects of each drug
concentration before OGD (Pre-OGD) or after OGD (Post-OGD), we followed slightly
different procedures for the Pre- and Post- OGD slice cultures. The main difference
between the pre- and post- slice cultures is that the Pre-OGD slices were treated with
the specified drug concentrations 30 minutes before being exposed to OGD, while the
post-OGD slices were treated immediately after OGD. Besides this differing variable in
the times the slices were treated with specified concentrations of TCN-201 or Ro25-
6981, the other steps that follow are controlled in both pre- and post- hippocampal slice
cultures.
For each 6-well plate, there was one control well. The control slices (OGD with
no treatment), received a vehicle (HBSS) treatment [7]. The other slices were treated
with 1 mL of the drugged media solution either pre- or post-OGD. To ensure that the
slices were fully benefitting from the neuroprotective treatment, 10µL of the drugged

5. media solution was applied topically to each slice in every well. We exposed the slice
cultures to OGD by moving each semi-permeable insert to wells with glucose-free
artificial cerebrospinal fluid (CaCl2•2H2O, MgSO4 Anhydrous, KCl, KH2PO4, NaHCo3,
NaCl, NaH2PO4, HEPES, Mannitol). Afterwards, the slices were returned to their normal
solutions to rest and repair. Twenty-four hours post-OGD, we measured the amount of
delayed neuronal death in all slice cultures.
To measure neuronal death in the cell layers, 1µM propidium iodide (PI) was
applied to each hippocampal slice 10 min prior to imaging with a rhodamine filter (Zeiss
Microscopes-Axioscop2 FS Plus, Thornwood, New York, USA) [7]. Slice cultures were
imaged and analyzed with the PTI Imagemaster software. The fluorescence intensity of
the 18 regions of interest in the Ca1, Ca3, and DG cell layers for each slice were
measured. Statistical analysis was performed using Student’s T-test with a p-value of
less than 0.05 as significant.
Results
In order to test the potency of the antagonists, TCN-201 and Ro25-6981, we
induced in-vitro ischemia in the hippocampal slices. Due to the susceptibility of the
slices to bacterial infections, we took careful precautions to keep all equipment and
ourselves sterile. Even still, a few of the inserts got contaminated, making it difficult to
accurately measure neuronal death due solely to the OGD.
Slice cultures receiving the NR2B-selective NMDAR antagonist, Ro25-6981,
responded better when treated post-OGD at lower concentrations. The pre-OGD
treatment groups exhibited no significant levels of neuroprotection, whereas the post-
OGD treatment group at 1µM was most neuroprotective. Compared to the 10µM post-

6. OGD hippocampal slices, the 1µM post-OGD slices experienced a lesser percentage of
neuronal death. Furthermore, the 1uM pre-OGD slice cultures experienced less cell
death than did the 10µM pre-OGD slice cultures. Overall, the slices that were treated
with 1µM Ro25-6981 post-OGD had the least percent cell death, indicating optimal
neuroprotection post-OGD at a lower concentration.
Figure 1. The NR2B-selective NMDAR antagonist, Ro25-6981, is most neuroprotective when
administered at a low-dose post-OGD. *P<0.05, Student’s t-test compared with control OGD.
*
*

7. Interestingly, slices treated with the NR2A-selective NMDAR antagonist, TCN -
201, before OGD had less cell death than did slices drugged after OGD. At all
concentrations (500 nM, 1µM, 5µM), the pre-OGD treatment was a more effective
protecting agent. Of the pre-OGD treatments, the 1µM and 5µM dosages were more
effectual than the 500nM dose (65%, 61%, and 82% cell death, respectively).

8. Figure 3. The NR2A-selective NMDAR antagonist, TCN-201, is most neuroprotective when administered
higher concentrations pre-OGD. *P<0.05, Student’s t-test compared with control OGD.
* *

9. Discussion

10. Discussion
Our experiment aimed to test the neuroprotective potential of the NMDAR
subunit-specific antagonists, TCN-201 and Ro25-6981, in rat hippocampal slices
exposed to in vitro ischemic conditions, resembling a stroke. As other studies have
shown, TCN-201 and Ro25-6981 do, in fact, have a role in reducing occurrences of
neuronal death after ischemic neuronal injury [10, 11, 12, 13]. Our findings, however,
exhibit significant associations among the effectiveness of the subunit-selective
antagonists, drug doses, and the time the treatment is given. The NR2B-selective
NMDAR antagonist, Ro25-6981, was most neuroprotective when administered at a low
dose immediately after OGD. Interestingly, the TCN-201 treated culture slices
demonstrated better neuroprotection at higher concentrations (1µM and 5µM). For each
concentration, the pre-OGD slices were more resistant to cell death, and overall, 1µM
and 5µM pre-OGD TCN-201 treatments were the most neuroprotective.
While many NMDAR antagonists have failed as treatments for stroke [14], recent
studies have suggested that targeting the NMDAR may be the key to successful
antistroke drug developments [7]. A study conducted on in-vivo rat models with focal
ischemic stroke described NR2B-specific antagonists as effective neuroprotective
agents at or close to the time of ischemia [1]. Similarly, our findings for the Ro25-6981
antagonist display therapeutic potency immediately after OGD. In contrast, TCN-201
seemed to be more neuroprotective 30 minutes before ischemia. If the conclusions of
Liu et al [1] can be applied to NR2A-specific antagonists, then our time interval of 30
minutes between the Ro25-6981 drug treatment and exposure to OGD may have been

11. too long before the injury for the treatment to have taken maximum efficacy. A possible
future study would be to examine various time frames close to the induction of stroke
conditions and to see the effect of each time interval on reducing delayed neuronal
death for both NR2A- and NR2B- containing receptors.
The major setback in our study was the unidentified contaminant that caused
data from some slices to be unusable. Because each well had 3 hippocampal slices, we
were still able to obtain sufficient data for each drug at each concentration, given pre- or
post-OGD; however, with less hippocampal slices to analyze, the SEM for percent cell
death were not as representative or accurate. To prevent bias during analysis of the
fluorescent images, the analyzer was blinded to the details of the study.
Conclusion
Our study shows that TCN-201 and Ro25-6981 are effective neuroprotective
agents. Here, we see that when slices are treated with TCN-201 pre-OGD at the higher
concentrations (1µM and 5µM), the least percentage of delayed neuronal death
occurred. On the other hand, slice cultures treated with Ro25-6981 survived best when
the drug was administered post-OGD at the lowest concentration (1µM). These findings
indicate that the strategy of blocking the NR2A- and NR2B-subtype containing NMDAR
as a therapeutic agent to reduce delayed neuronal death following OGD has noteworthy
potential. Further studies are needed to find the most effective time frame and
concentration for NMDAR subtype antagonists to function at their peak, which may
revolutionize neurodegenerative therapies.

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