Published online before print July 10, 2008, doi: 10.1161/STROKEAHA.107.506048
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(Stroke. 2008;39:2544.)
© 2008 American Heart Association, Inc.
Original Contributions |
Effectiveness of PSD95 Inhibitors in Permanent and Transient Focal Ischemia in the Rat
From the Toronto Western Hospital Research Institute (H.-S.S., Y.F., L.T., M.A., A.L., J.P.F., M.T.), Toronto, Ontario, Canada; the Departments of Physiology (H.-S.S., M.T.) and Surgery (M.T.), University of Toronto, Toronto, Ontario, Canada; the Departments of Biology (T.A.D.), Biomedical Sciences (T.A.D., P.B.B., R.A.T.), and Psychology (C.L.R.), University of Prince Edward Island, Charlottetown, PEI, Canada; the Programme in Brain and Behaviour (M.W.S.), Hospital for Sick Children, Toronto, Ontario, Canada; and the Brain Research Center and Department of Medicine (Y.L., Y.T.W.), Vancouver Hospital and Health Sciences Center, University of British Columbia, Vancouver, BC, Canada.
Correspondence to Michael Tymianski, MD, PhD, Toronto Western Hospital Research Institute, Suite 4W-435, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8. E-mail mike.tymianski{at}uhn.on.ca
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Abstract |
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Background and Purpose— Postsynaptic density-95 inhibitors reduce ischemic brain damage without inhibiting excitatory neurotransmission, circumventing the negative consequences of glutamatergic inhibition. However, their efficacy in permanent ischemia and in providing permanent neuroprotection and neurobehavioral improvement in a practical therapeutic window is unproven. These were tested here under conditions that included fever, which is a common occurrence in clinical stroke.
Methods— Six studies were performed in unfasted Sprague-Dawley rats. Two involved permanent pial vessel occlusion in male and female rats. Two involved permanent middle cerebral artery occlusion, which induced severe hyperthermia, and 2 involved transient middle cerebral artery occlusion. Animals were treated with a single intravenous injection of postsynaptic density-95 inhibitors (Tat-NR2B9c[SDV] or Tat-NR2B9c[TDV]) 1 hour or 3 hours after stroke. Infarct volumes and neurobehavior were assessed in a blinded manner at 24 hours (pial vessel occlusion and permanent middle cerebral artery occlusion) or at 62 days (transient middle cerebral artery occlusion).
Results— Postsynaptic density-95 inhibitors dramatically reduced infarct size in male and female animals exposed to pial vessel occlusion (>50%), in hyperthermic animals with fever exceeding 39°C exposed to permanent middle cerebral artery occlusion (approximately 50%), and at 62 days poststroke in animals exposed to transient middle cerebral artery occlusion (approximately 80%). Effectiveness of postsynaptic density-95 inhibitors was achieved without the drugs affecting body temperature. In transient middle cerebral artery occlusion, a single dose of postsynaptic density-95 inhibitor given 3 hours after stroke onset permanently maintained reduced infarct size and improved neurobehavior.
Conclusions— Postsynaptic density-95 inhibitors administrated 3 hours after stroke onset reduced infarct volumes and improved long-term neurobehavioral functions in a wide therapeutic window. This raises the possibility that they may have future clinical usefulness.
Key Words: behavior • cerebral ischemia • hyperthermia • middle cerebral artery occlusion • N-methyl-D-aspartate receptor • postsynaptic density
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Introduction |
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Despite its significant socioeconomic toll, stroke has not yet been significantly impacted by neuroprotective drug therapy, and thrombolytic agents remain the mainstay of treatment for patients that qualify. Many failed neuroprotectants targeted N-methyl-D-aspartate glutamate receptors (NMDARs), which mediate excitatory neurotransmission in the central nervous system. NMDARs also mediate excitotoxicity and ischemic brain damage, but blocking them has failed as a clinical stroke therapy.1 NMDAR blockers inhibit essential excitatory neurotransmission, producing systemic and psychotropic consequences that limit their usefulness.1 These consequences may be circumvented by blocking neurotoxic NMDAR signaling such as free radical production by neuronal nitric oxide synthase (nNOS) without blocking NMDARs. We recently achieved this by perturbing the interactions of NMDARs with the submembrane scaffolding postsynaptic density-95 (PSD95) protein, which also binds nNOS.2 We then developed PSD95 inhibitors, peptides that disrupt PSD95/NMDAR and PSD95/nNOS interactions.3,4 Treatment with PSD95 inhibitors before or 1 hour after transient middle cerebral artery occlusion (MCAO) significantly reduced infarct volumes at 24 hours.3 However, it remains unknown whether they have efficacy in permanent ischemia, whether the neuroprotection is permanent, whether it translates to neurobehavioral improvement, or whether treatment can be instituted in a practical time window poststroke.
To test PSD95 inhibitors as putative neuroprotectants in stroke, we presently examined their efficacy in reducing stroke damage in both permanent and temporary MCAO, in posttreatment paradigms of up to 3 hours poststroke, using histological and neurobehavioral correlates. Although no single study suffices to translate a stroke treatment from rodent studies to humans, our data indicate for the first time that PSD95 inhibitors significantly reduced infarct size even in permanent MCAO compounded by hyperthermia and significantly spared sensory and motor function as well as emotional and cognitive abilities over the long term.
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Materials and Methods |
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Postsynaptic Density-95 Inhibitors
Three synthetic peptides were fused to the cell membrane protein transduction domain of the HIV-1-Tat protein (YGRKKRRQRRR; Tat) to render them cell-permeant5 (Advanced Protein Technology Centre, Hospital for Sick Kids, Toronto, Ontario, Canada). One comprising the C-terminal 9 residues of the NMDA NR2B subunit, KLSSIESDV, termed Tat-NR2B9c(SDV)3; a variant comprising KLSSIETDV, termed Tat-NR2B9c(TDV); and the inactive control comprising KLSSIEADA, termed Tat-NR2B9c(ADA). The latter is incapable of binding PSD954 and does not affect excitotoxic vulnerability3 or infarct size.3 The peptides were administered intravenously in saline over 4 to 5 minutes by blinded individuals.
In some experiments, the selective NR2B subunit blocker, (R,S)-
-(4-ydroxyphenyl)-βmethyl-4-(phenylmethyl)-1-piperidinepropanol (RO 25–6981)6 (Sigma; 6 mg/kg in saline), was used as positive control.
Animals
Adult male Sprague-Dawley rats (250 to 300 g; Charles River Laboratories, Sherbrooke, Quebec, Canada) were used according to procedures approved by the institutional animal care committees. All experiments were performed on unfasted animals. Free overnight access to both food and water resulted in higher morning blood glucose levels than in animals that had free overnight access to water but not food (5.50±0.08 mmol/L; n=12 versus 3.71±0.21 mmol/L; n=8, respectively; t18=9.134; P<0.001).
Surgical Preparation
For permanent pial vessel occlusion (PVO), rats were anesthetized with 100 mg/kg ketamine, 2 mg/kg acepromazine, and 50 mg/kg xylazine. For permanent and temporary middle cerebral artery occlusion (pMCAO and tMCAO, respectively), anesthesia was induced and maintained with 3.5% and 0.8% halothane, respectively, in 2:1 nitrous oxide and oxygen. Rats were intubated and ventilated (60 strokes/min, tidal volume of 30 to 35 mL). Mean arterial blood pressure, blood gases, pH, and glucose were monitored with a left femoral artery catheter. Drug delivery was through the femoral vein.
Core, Brain, and Temporalis Temperature Measurements/Control
Core body temperature (CT) was monitored from 20 hours pre- to 24 hours post-pMCAO using intraperitoneal transmitters (Mini-Mitter/Respironics). To measure brain temperatures after pMCAO, the skull was exposed and 1-mm holes were drilled bilaterally at (stereotactic coordinates from bregma): anteroposterior 3.3 mm, mediolateral 4.0 mm, dorsal–ventral 3.0 mm. A 19-gauge cannula was inserted 5 mm and secured with dental glue. Brain temperature was recorded at 0, 1, 2, 4, and 24 hours using a thermocouple probe in the cannula. Temporalis muscle temperatures were measured with the same probe inserted into the muscle. Cage temperatures were recorded continuously using an external probe (Tektronix). In some experiments, cage temperature was continuously adjusted based on CT using a feedback-controlled cooling fan (Igloo KoolMate18) activated whenever CT exceeded 37.1°C.
Permanent Distal Middle Cerebral Artery Pial Vessel Occlusion
The right external carotid artery was cannulated with PE10 tubing. The skull was exposed in the midline. A 6- to 8-mm cranial window was made over the right somatosensory cortex (2 mm caudal and 5 mm lateral to bregma). The pial arteries were visualized by injecting 10 to 20 µL of Patent Blue Violet (10 mmol/L; Sigma) in saline into the external carotid artery. The 3 pial arteriolar middle cerebral artery branches were cauterized and dye injections were repeated to ensure the blockage. As described,7 this produces a reproducible infarction in the cortex underlying the coagulated pial arteries.
Middle Cerebral Artery Occlusion (permanent and transient)
The left middle cerebral artery was occluded by the intraluminal suture method of Longa et al.8 In brief, a 3–0 monofilament nylon suture tip (0.33 to 0.36 mm diameter, 0.5 to 0.6 mm length) coated with poly-L-lysine was introduced into the internal carotid artery through the external carotid artery, effectively occluding the middle cerebral artery. For tMCAO, the suture was withdrawn after 90 minutes and the external carotid artery ligated.3 For pMCAO, the suture was left ligated in situ. Sham animals underwent identical procedures without MCAO. All physiological parameters were similar between groups (Supplemental Table I
, available online at http://stroke.ahajournals.org). CT was maintained at 36.8°C to 37.2°C using a heat lamp.
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Neurobehavioral Evaluation
After pMCAO, behavioral scoring was performed at 2 hours and 24 hours using the postural reflex and the forelimb placing tests as used previously3 (normal score, 0; maximal score, 12).
Before term tMCAO experiments, drug effects on baseline motor function (Rotorod performance),9 arousal/anxiety (novel object exploration),10 and sensorimotor function (sticky label task)11 were determined. Rotorod performance was evaluated at both 40 and 220 minutes postinjection (75 mm diameter; Stoelting) at 12 rpm/min. Time spent exploring a novel object (5x5-cm rubber block) was measured beginning at 150 minutes, and latency to attend to a sticky substance (honey) applied to one forepaw was measured at 180 minutes after drug administration.
A strict postoperative care protocol12 ensured the long-term survival (62 days) necessary to carry out the neurobehavioral tests described subsequently.
Tests of Physical Outcomes and Sensorimotor Function
All rats were weighed and tested for sensorimotor function over 56 days using a modified 5-task bilateral sensorimotor battery.13 Performance on each of 11 assessments gave a cumulative deficit score for each rat (maximum unilateral deficit score of 7) scale. On postoperative day (POD) 5, 10, and 15, rats were tested for exploration and number of rotations in an open field arena (5-minute trial).
Motor agility was tested on a horizontal ladder (1.2-m ladder with equi-spaced rungs 2.5 cm apart). Latency (seconds) to reach the home cage and number/type of paw slips were recorded.
On POD 10, rats were tested in a straight alley swim task and scored for head/body angle (0: normal, 1: 45° angle, or 2: 90° angle), forelimb paddling, and escape latency. The apparatus was a 115x20x18-cm fiberglass tank.
Tests of Emotionality
Rats were tested on 3 days (5-minute trial) in an open-field arena to determine state of arousal and ability to habituate to a novel environment and in the elevated plus maze to assess anxiety/emotionality. The maze consisted of 4 arms (2 open, 2 closed: 15 cm width and 60 cm length) extending from a central platform elevated 1.5 m.
Tests of Cognition
The Barnes Circular Maze is a test of spatial learning and memory.14 The apparatus was a white circular platform (1.2 m diameter; elevation 1.0 m) with 18 equally sized (9 cm) and spaced holes around the perimeter. An escape box was located under one of the holes, its location remaining constant for all trials. The animal had to locate and enter the escape box with bright light and white noise as negative reinforcement. Rats were acclimated to the maze on POD 35 and latency to find the goal box was assessed on POD 36 to 38 (maximum trial latency of 180 seconds). Between trials, the rat remained within the goal box and the maze was rotated to a preselected direction to eliminate the use of odor cues on subsequent trials. Each rat was given 4 trials per day with the goal box remaining constant on each test day. One week later, a retest was administered in which each rat received 4 additional trials with the goal box located at the same position.
On POD 56 to 59, rats were tested for spatial learning and memory in the Morris water maze.15 A circular tank (1.5 m diameter) filled with water (approximately 21°C) made opaque by nontoxic white paint (Reeves & Poole) was divided into 4 quadrants with a submerged clear Plexiglas escape platform in one quadrant. Extramaze cues were held constant throughout the experiment. Performance was recorded using Ethovision software (Noldus Information Technology Inc). Rats were given 4 trials per day for each of 4 test days (60 seconds per trial, 60-second intertrial interval). A 60-second probe trial was administered 24 hours after the last test day, and time spent in the target quadrant was recorded.
Infarct Volume Evaluation
Twenty-four hours after pMCAO or PVO, the animals were anesthetized, brains were removed, sliced into 8 1-mm thick coronal sections, and incubated for 30 minutes in 2% triphenyltetrazolium chloride (Sigma) in saline at 37°C.16 For tMCAO, animals underwent transcardiac perfusion with heparinized saline followed by 4% wt/vol paraformaldehyde in phosphate-buffered saline. Brains were extracted, processed, and stained with hematoxylin and eosin. Infarct areas were digitally traced on standard templates containing 8 coronal sections and thus corrected for brain edema or shrinkage.17 Infarct volumes were derived from the infarct areas.
Data Analysis
All animal surgeries, drug infusions, behavioral assessments, and infarct volume determinations were performed by individuals blinded to the treatment group. Data are expressed in mean±SEM. Differences were analyzed using analysis of variance (ANOVA) or repeated-measures ANOVA on ranks. The Bonferroni correction was used for pairwise comparisons. In some experiments (as in Figure 5), when comparisons by analysis of variance and Bonferroni revealed no differences between the Tat-NR2B9c(ADA)-treated and saline-treated control groups, these were pooled for comparison with Tat-NR2B9c(SDV) by Student t test.
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Results |
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Effects of Postsynaptic Density-95 Inhibitors on Core Body Temperature
Core temperature was measured in 6 animals from 20 hours pre- to 24 hours postintravenous injection of 3 nM/g of Tat-NR2B9c(SDV), the highest dose used in the present study. Controls underwent sham surgery with a saline infusion (vehicle, n=4). All animals exhibited a mild (approximately 0.5°C) transient (10 to 15 hours) CT elevation after termination of anesthesia. However, Tat-NR2B9c(SDV) had no impact on CTs at any time as compared with shams (Figure 1Ai, ii).
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Effect of Permanent Middle Cerebral Artery Occlusion on Core Body, Brain, and Temporalis Muscle Temperature
Spontaneous hyperthermia is recognized in experimental MCAO.18 For the pMCAO experiments, the occluding filament was fashioned according to Abraham et al18 (tip diameter: 0.33 to 0.36 mm), which caused the animals’ CT to rise rapidly after pMCAO, peaking at approximately 39.5°C at approximately 2 hours and remaining at or above 39°C for the next 24 hours (Figure 1Aiv). Cooling the animals’ cage whenever CT rose above 37.1°C had no impact on CT (n=8; Figure 1Av) as compared with animals that underwent pMCAO but whose cage was maintained at room temperature (n=8; Figure 1Aiv). This resistance to cooling by ambient temperature reduction suggests that the hyperthermia post-pMCAO was not due to a loss of thermoregulatory ability but rather to an alteration in the animal’s temperature set point.
Because brain temperature relates to ischemic damage, we determined whether CT was reflective of brain temperature in this study. CT, brain, and temporalis muscle temperatures were measured in animals that underwent pMCAO. CT measurements reflected brain and temporalis muscle temperatures to within 0.5°C in animals receiving either saline (n=6; Figure 1Avi) or 3 nM/g of Tat-NR2B9c(SDV) (n=6; Figure 1Avi). Thus, CT was used for the remainder of the study.
Effect of Postsynaptic Density-95 Inhibitors on Permanent Middle Cerebral Artery Occlusion Infarction Volume
First, we used the PVO model that produces a small infarction (Figure 2A–B) without altering CT as compared with sham surgery animals (Figure 1Ai, iii). The animals were treated with vehicle (saline), control peptide Tat-NR2B9c(ADA), or low (0.3 nM/g) or high (3 nM/g) doses of Tat-NR2B9c(SDV) at 1 hour after PVO. Treatment with either vehicle or Tat-NR2B9c(ADA) resulted in cortical infarcts underlying the PVO, occupying approximately 9% to 10% of the hemisphere (Figure 2C). This was reduced by approximately 60% by Tat-NR2B9c(SDV) (3 nM/g; Figure 2C–D).
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PSD95 inhibitors reduce nNOS activity,3 but studies from nNOS-null mice suggested that nNOS deletion may not protect against stroke in females.19 Thus, Tat-NR2B9c(SDV) was re-evaluated in the PVO model as stated previously in both male and female rats. Treatment with Tat-NR2B9c(SDV) (3 nM/g) 1 hour after stroke dramatically reduced infarcts in both genders (Figure 2E–F) suggesting that female rats subjected to acute treatment with PSD95 inhibitors react differently than female nNOS knockout mice with chronic nNOS deletions.
Next, we evaluated the PSD95 inhibitors in pMCAO. All animals exhibited hyperthermia with CT >39.5°C in the first 8 hours and remaining at approximately 39°C thereafter (Figure 1Bi–iv). The PSD95 inhibitors had no impact on CT (no differences between controls and treatment groups in either peak or mean CT elevation; ANOVA, P>0.15 for each).
Saline-treated animals sustained large hemispheric infarcts that, at 24 hours, occupied the majority of the cortical surface and deep structures (Figure 3C). However, treatment with Tat-NR2B9c(SDV) 1 hour after pMCAO (0.3 to 3.0 nM/g) attenuated the total infarct volume by as much as 40% (Figure 3Ai, C) and the cortical component by approximately 45% (Figure 3Aii, C). Similarly, Tat-NR2B9c(TDV), a previously untested PSD95 inhibitor, also reduced hemispheric infarction volume by approximately 35% and cortical infarction by 40% to 45% (Figure 3Ai, Aii, C). To test the reproducibility of these findings a second, confirmatory study was performed by a different team blinded to the first study. Similar results were obtained, confirming that both the Tat-NR2B9c(SDV) and Tat-NR2B9c(TDV) peptides have a similar effect in reducing both hemispheric and cortical infarction volumes (Figure 3Bi–ii, C).
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Efficacy of Postsynaptic Density-95 Inhibitor Administered 3 Hours Postischemia Onset
The pMCAO in saline-treated animals produced severe neurological deficits, which lasted for the entire observation period (composite neurological score 
11; not shown) and did not improve by 24 hours. The severity of functional impairment and high mortality rate after pMCAO is a limitation that renders long-term studies difficult as recognized by the STAIR committee.20 Because longer poststroke recovery times than 24 hours were necessary to evaluate the PSD95 inhibitors, we carried out additional experiments in a model of transient (90 minutes) MCAO.
We first tested Tat-NR2B9c(SDV) treatment administered 3 hours after ischemia onset. As a control, we used Ro 25-6981, a, selective NR2B antagonist with efficacy against excitotoxicity and oxygen–glucose deprivation in vitro and stroke in vivo.6 Ro 25-6981 (6 mg/kg) was infused intravenously 30 minutes before or 3 hours after stroke onset, whereas Tat-NR2B9c(SDV) was infused at 0.03 to 3 nMol/g over 5 minutes, starting 3 hours after ischemia onset. Infarct volumes were evaluated at 24 hours. The dose range of Tat-NR2B9c(SDV) was based on the assumption of equal distribution throughout the animal. A dose of 3 nM/g (approximately 3 µmol/L) approximates the IC50 of Tat-NR2B9c(SDV) inhibiting NR2-PSD95 interactions and exceeds the IC50 for inhibiting PSD95–nNOS interactions.4
Tat-NR2B9c(SDV) treatment 3 hours after ischemia onset reduced infarct volumes by 50% or more at all doses tested (ANOVA, F=8.087, P<0.001). This effect was similar to that obtained with Ro 25–6891 applied 30 minutes before ischemia, but not when Ro 25–6891 was given 3 hours after ischemia. There were no significant differences between infarct volumes obtained with the different doses of Tat-NR2B9c(SDV) (ANOVA, F=0.718, P=0.552), raising the possibility that even lower doses may have efficacy (Figure 4Ai–ii).
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Postsynaptic Density-95 Inhibitors Offer Long-Term Neuroprotection and Improve Functional Recovery
Next, using the 90-minute tMCAO, we determined whether PSD95 inhibitors impart permanent neuroprotection or simply delay damage and whether they improve neurological function. The inhibitors had no neurobehavioral effects of their own, because treatment of naive rats with Tat-NR2B9c(SDV) (0.5, 5, and 50 nM/g) had no effect on Rotorod performance or the sticky label task at any dose. Novel object exploration latencies were increased at 50 nM/g (not shown).
The efficacy of a single treatment with 3 nM/g Tat-NR2B9c(SDV) at 3 hours post-tMCAO onset was evaluated over a 62-day survival period. Physiological parameters during surgeries were similar between groups (Supplemental Table I
D). All animals survived the 62 days. On euthanization, animals treated with Tat-NR2B9c(SDV) had reduced infarct volumes relative to Tat-NR2B9c(ADA) and saline controls by approximately 75% (Figure 4B–C; F[2,27]=5.72, P=0.0085), indicating permanent histological neuroprotection.
During the 62-day period, the animals were subjected to physical, sensorimotor, emotive, and cognitive tests to assess neurological outcomes. Tat-NR2B9c(SDV)-treated rats returned to presurgery weights approximately 1 week earlier than saline- or Tat-NR2B9c(ADA)-treated rats (t25.6=2.73, P<0.05; Figure 5A). They were also heavier than controls on subsequent days, including day 56 postinjury (t28=1.67, P=0.05). They had improved daily sensorimotor cumulative deficit scores (F[1,28]=8.30, P=0.008; Figure 5B). Horizontal ladder tests on days 5, 9, and 14 revealed that Tat-NR2B9c(SDV)-treated rats had significantly reduced latencies (ANOVA, F[,28]=12.59, P=0.001) relative to controls and made significantly fewer paw slips (ANOVA; F[1,28]=5.71, P=0.024). No differences between groups were found in number of right rotations or alley swim performance (Supplemental supplemental Table II).
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Emotionality was tested in an open field arena on days 5, 10, and 15. There were no significant differences in total grid crosses, time in locomotion, or right rotations between groups (Figure 5E; Supplemental Table II). However, Tat-NR2B9c(SDV)-treated rats habituated to the open field arena, which occurs in normal rats,21 whereas the controls did not (t[28]=3.68, P=0.001; Figure 5F). Emotionality was also tested in the elevated plus maze on days 6, 13, and 20, and rats treated with Tat-NR2B9c(SDV) showed reduced latencies to enter the closed arm (Supplemental Table II). Thus, the behavior of Tat-NR2B9c(SDV)-treated rats is closer to that of animals without strokes than to those in the groups with strokes and those treated with vehicle or Tat-NR2B9c(ADA). Retention of memory in response to treatment was measured using the Barnes circular maze14 (days 36 to 40) and the Morris water maze15 (days 56 to 61). Shorter mean escape latencies approached significance during the acquisition phase of Barnes maze testing in rats treated with Tat-NR2B9c(SDV) (Figure 5G; F[1,28]=3.80, P=0.061). On retest (to assess memory), Tat-NR2B9c(SDV)-treated rats performed significantly better than the controls (Figure 5H; t[23]=2.77, P<0.01). In the Morris water maze, there were no group differences in the acquisition phase (Figure 5I), but animals injected with Tat-NR2B9c(SDV) had reduced memory deficits as evidenced by increased time in the target quadrant when compared with the combined control groups (Figure 5J; t[28]=2.07, P=0.024).
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Discussion |
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A neuroprotective agent for stroke should have a clearly determined mechanism of action, have an established molecular target, produce long-term histological and neurobehavioral neuroprotection when administered poststroke in a reasonable time window, and have limited side effects. These conditions appear to be met by PSD95 inhibitors, although the full therapeutic window still remains to be characterized, and additional neurobehavioral studies, including further controls such as sham-operated animals, need to be done. However, unlike NMDAR inhibitors, PSD95 inhibition maintains glutamatergic signaling.2,3 In previous studies, genetically disrupting PSD95 in vivo22 or with antisense oligonucleotides2 or PSD95 inhibitors3 has not affected NMDAR function or normal excitatory neurotransmission. Consistent with this, in mutant mice lacking PSD95, long-term potentiation is enhanced, but synaptic NMDA-receptor currents, subunit expression, localization, and synaptic morphology are all unaffected.22 This may be an advantage of PSD95 inhibitors over NMDAR blockers, which cause sedation, psychotropic effects, and potential dose-limiting toxicity1 in the human situation.
PSD95 inhibitors have a clear mechanism based on a specific and restricted set of molecular targets. We recently demonstrated, using a proteomic and biochemical analysis of the interactions of Tat-NR2B9c(SDV) with all known human proteins that it may bind, that this compound has high specificity, impacting excitotoxicity solely through its interactions with PSD95 and with nNOS.4 Although we cannot exclude the possibility that PSD95 inhibitors may also protect neurons through an as-yet uncharacterized off-target effect, all our previous work confirms that when the NMDAR/PSD95 interaction is unaffected, neuroprotection is not achieved.2–4
It is recognized that presently even drugs that were apparently effective in reducing ischemic damage in animals in a posttreatment paradigm have failed in humans. However, past failures enhance, not diminish, the need for future neuroprotection research. To our knowledge, PSD95 inhibitors are the first pharmacological compounds that show efficacy in a posttreatment paradigm even in severe (39°C) hyperthermia. Also, they are the first compounds that, while addressing excitotoxic mechanisms, produce permanent neuroprotection when administered hours after the ischemic insult. This justifies a continuing evaluation of this class of compounds for the treatment of stroke.
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Acknowledgments |
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Sources of Funding
Supported by grants (to MT) from the Canadian Institutes of Health Research (CIHR; MOP 79295), National Institutes of Health RO1 NS048956, and the Canadian Stroke Network, and by funding (to M.T.) from Ontario Research Fund from the Ontario Ministry of Research and Innovation. HSS is a recipient of Postdoctoral Fellowship Awards from Heart and Stroke Foundation of Canada–Focus on Stroke Training Initiative Program.
Disclosures
None.
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Footnotes |
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H.-S.S., T.A.D., and Y.L. contributed equally to this work.
Received October 2, 2007; revision received February 6, 2008; accepted February 13, 2008.
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References |
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1. Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G, Norris J. Selfotel in acute ischemic stroke: possible neurotoxic effects of an NMDA antagonist. Stroke. 2000; 31: 347–354.
2. Sattler R, Xiong Z, Lu WY, Hafner M, MacDonald JF, Tymianski M. Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science. 1999; 284: 1845–1848.
3. Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT, Salter MW, Tymianski M. Treatment of ischemic brain damage by perturbing NMDA receptor- PSD-95 protein interactions. Science. 2002; 298: 846–850.
4. Cui H, Hayashi A, Sun H, Belmares MP, Cobey C, Phan T, Schweizer J, Salter MW, Wang Y, Tasker RA, Garman D, Rabinowitz J, Lu PS, Tymianski M. PDZ protein interactions underlying NMDA-receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors. J Neurosci. 2007; 29: 9901–9915.
5. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 1999; 285: 1569–1572.
6. Gill R, Alanine A, Bourson A, Buttelmann B, Fischer G, Heitz MP, Kew JN, Levet-Trafit B, Lorez HP, Malherbe P, Miss MT, Mutel V, Pinard E, Roever S, Schmitt M, Trube G, Wybrecht R, Wyler R, Kemp JA. Pharmacological characterization of Ro 63–1908 (1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol), a novel subtype-selective N-methyl-D-aspartate antagonist. J Pharmacol Exp Ther. 2002; 302: 940–948.
7. Forder JP, Munzenmaier DH, Greene AS. Angiogenic protection from focal ischemia with angiotensin II type 1 receptor blockade in the rat. Am J Physiol Heart Circ Physiol. 2005; 288: H1989–H1996.
8. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989; 20: 84–91.
9. Bogo V, Hill TA, Young RW. Comparison of accelerod and Rotorod sensitivity in detecting ethanol- and acrylamide-induced performance decrement in rats: review of experimental considerations of rotating rod systems. Neurotoxicology. 1981; 2: 765–787.[Medline] [Order article via Infotrieve]
10. Hughes RN. Intrinsic exploration in animals: motives and measurements. Behav Processes. 1997; 41: 213–226.[CrossRef]
11. Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST. CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology. 2000; 39: 777–787.[CrossRef][Medline] [Order article via Infotrieve]
12. Ryan CL, Doucette TA, Gill DA, Langdon KD, Liu Y, Perry MA, Tasker RA. An improved post-operative care protocol allows detection of long-term functional deficits following MCAO surgery in rats. J Neurosci Methods. 2006; 154: 30–37.[CrossRef][Medline] [Order article via Infotrieve]
13. Markgraf CG, Green EJ, Hurwitz BE, Morikawa E, Dietrich WD, McCabe PM, Ginsberg MD, Schneiderman N. Sensorimotor and cognitive consequences of middle cerebral artery occlusion in rats. Brain Res. 1992; 575: 238–246.[CrossRef][Medline] [Order article via Infotrieve]
14. Barnes CA. Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat. J Comp Physiol Psychol. 1979; 93: 74–104.[CrossRef][Medline] [Order article via Infotrieve]
15. Morris RG, Garrud P, Rawlins JN, O'Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature. 1982; 297: 681–683.[CrossRef][Medline] [Order article via Infotrieve]
16. Joshi CN, Jain SK, Murthy PS. An optimized triphenyltetrazolium chloride method for identification of cerebral infarcts. Brain Res Brain Res Protoc. 2004; 13: 11–17.[CrossRef][Medline] [Order article via Infotrieve]
17. Osborne KA, Shigeno T, Balarsky AM, Ford I, McCulloch J, Teasdale GM, Graham DI. Quantitative assessment of early brain damage in a rat model of focal cerebral ischemia. J Neurol Neurosurg Psychiatry. 1987; 50: 402–410.
18. Abraham H, Somogyvari-Vigh A, Maderdrut JL, Vigh S, Arimura A. Filament size influences temperature changes and brain damage following middle cerebral artery occlusion in rats. Exp Brain Res. 2002; 142: 131–138.[CrossRef][Medline] [Order article via Infotrieve]
19. McCullough LD, Zeng Z, Blizzard KK, Debchoudhury I, Hurn PD. Ischemic nitric oxide and poly (ADP-ribose) polymerase-1 in cerebral ischemia: male toxicity, female protection. J Cereb Blood Flow Metab. 2005; 25: 502–512.[CrossRef][Medline] [Order article via Infotrieve]
20. The Stroke Therapy Academic Industry Round Table (STAIR). Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke. 1999; 30: 2752–2758.
21. Eilam D, Golani I. Home base behavior of rats (Rattus norvegicus) exploring a novel environment. Behav Brain Res. 1989; 34: 199–211.[CrossRef][Medline] [Order article via Infotrieve]
22. Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RGM, Morrison JH, O'Dell TJ, Grant SGN. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature. 1998; 396: 433–439.[CrossRef][Medline] [Order article via Infotrieve]
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