A new look at glutamate and ischemia: NMDA agonist improves long-term functional outcome in a rat model of stroke

Abstract

Ischemic stroke triggers a massive, although transient, glutamate efflux and excessive activation of NMDA receptors (NMDARs), possibly leading to neuronal death. However, multiple clinical trials with NMDA antagonists failed to improve, or even worsened, stroke outcome. Recent findings of a persistent post-stroke decline in NMDAR density, which plays a pivotal role in plasticity and memory formation, suggest that NMDAR stimulation, rather than inhibition, may prove beneficial in the subacute period after stroke. AIM: This study aims to examine the effect of the NMDAR partial agonist d-cycloserine (DCS) on long-term structural, functional and behavioral outcomes in rats subjected to transient middle cerebral artery occlusion, an animal model of ischemic stroke. MATERIALS #ENTITYSTARTX00026; METHODS: Rats (n = 36) that were subjected to 90 min of middle cerebral artery occlusion were given a single injection of DCS (10 mg/kg) or vehicle (phosphate-buffered saline) 24 h after occlusion and followed up for 30 days. MRI (structural and functional) was used to measure infarction, atrophy and cortical activation due to electrical forepaw stimulation. Memory function was assessed on days 7, 21 and 30 postocclusion using the novel object recognition test. A total of 20 nonischemic controls were included for comparison. RESULTS: DCS treatment resulted in significant improvement of somatosensory and cognitive function relative to vehicle treatment. By day 30, cognitive performance of the DCS-treated animals was indistinguishable from nonischemic controls, while vehicle-treated animals demonstrated a stable memory deficit. DCS had no significant effect on infarction or atrophy. CONCLUSION: These results support a beneficial role for NMDAR stimulation during the recovery period after stroke, most likely due to enhanced neuroplasticity rather than neuroprotection.

Figure 1. MRI confirmation of infarction and edema 24 h after middle cerebral artery occlusion

(A) Pretreatment infarction and edema in two animals later randomized to PBS or DCS treatment. Note the high signal intensity in these T₂-weighted images, delineating infarction in the striatum and overlying cortex, and the midline shift and swelling of the infarcted hemisphere characteristic of edema. (B) Bars represent means and standard errors of infarction and edema expressed as percentage of the contralateral hemisphere. The area of infarction and cross-sectional area of both hemispheres were measured using NIH Image routines from serial MRI slices covering the whole forebrain, summed and multiplied by the slice thickness and gap to provide a volume estimate. Percentage infarction was calculated as 100 × (infarct volume/volume of contralateral hemisphere). Percentage edema was calculated as 100 × ([ipsilateral hemisphere volume − contralateral hemisphere volume]/contralateral hemisphere volume). There was no statistically significant difference between the animals randomized to receive vehicle (n = 7) or DCS (n = 7) in percentage infarction or percentage edema. DCS: d-cycloserine; PBS: Phosphate-buffered saline.

Figure 2. Effect of ischemia and d-cycloserine on blood oxygen level-dependent activation maps 30 days after middle cerebral artery occlusion

Structural T₂-weighted coronal images at the level of maximal infarction are shown in the leftmost column, with a nonischemic control in the top row (intact), middle cerebral artery occlusion (MCAO) plus PBS (small and large infarct) in the middle rows and MCAO plus DCS (small and large infarct) in the bottom rows. The color bar corresponds to a positive blood oxygen level-dependent signal increase from 1% (red) to 10% (yellow). Serial coronal echo planar imaging images from the ipsilateral and contralateral hemisphere are shown to the left and right of the color bar, respectively. Note the symmetrical blood oxygen level-dependent signal intensity change produced in the nonischemic control, the absence of ipsilateral activation in the MCAO plus PBS animals and the presence of activation in the somatosensory cortex in a DCS-treated animal with a striatal infarct. Activation shifted towards the midline can be seen in the MCAO plus DCS animals with a large cortical infarct (arrow). DCS: d-cycloserine; PBS: Phosphate-buffered saline; T₂W: T₂-weighted.

Figure 3. Effect of ischemia and d-cycloserine on percentage change in blood oxygen level-dependent signal amplitude 30 days after middle cerebral artery occlusion

Bars represent means and standard errors of the percentage change in BOLD signal amplitude following right and left forepaw stimulation in nonischemic controls (n = 13, red bars), middle cerebral artery occlusion (MCAO) plus PBS (n = 7, blue bars) and MCAO plus DCS (n = 7, green bars). A three-way analysis of variance of percentage change in the amplitude of the BOLD signal revealed significant effects of surgery, side and drug treatment (p < 0.05). Post hoc ana lysis of the differences between individual groups and sides revealed significance for MCAO plus PBS compared with nonischemic controls in the ipsilateral hemisphere (***p < 0.001) and a trend for MCAO plus PBS compared with MCAO plus DCS in the ipsilateral hemisphere (^#p = 0.08). The MCAO plus DCS and nonischemic controls were not significantly different from each other (p > 0.2) in the ipsilateral hemisphere. There were no effects of ischemia or drug treatment on the BOLD signal intensity in the contralateral hemisphere. BOLD: Blood oxygen level-dependent; DCS: d-cycloserine; PBS: Phosphate-buffered saline.

Figure 4. Effect of d-cycloserine on novel object recognition task performance after middle cerebral artery occlusion

Bars represent means and standard errors of the percentage of time spent by rats investigating a new object in the presence of a familiar one during a 5-min testing period. Rats were tested on day 0 (presurgery), and at 7, 21 and 30 days post-middle cerebral artery occlusion (MCAO) with new pairs of objects on each day. Note the initial (presurgical) adequate performance (preference of novel object) in all three groups, the consistent performance of the intact/sham group, the persistent deficit in the MCAO plus PBS rats and the gradual improvement to presurgical performance levels of the MCAO plus DCS group. Two-way analysis of variance with repeated measures revealed significant main effects for surgery and drug and a significant drug by time (repeated measure) interaction. Post hoc comparison of the individual groups and days revealed significance for MCAO plus PBS compared with nonischemic controls (***p < 0.0001), significance for MCAO plus PBS compared with nonischemic controls (**p < 0.001) and significance for MCAO plus PBS compared with MCAO plus DCS (*p < 0.05). The DCS and intact groups were not significantly different from each other at any time point. DCS: d-cycloserine; PBS: Phosphate-buffered saline.

Figure 5. No effect of d-cycloserine on long-term infarction and atrophy after middle cerebral artery occlusion

(A) Long-term tissue loss and infarction. From left, photograph (dorsal view) of a middle cerebral artery occlusion (MCAO) plus PBS brain showing shrinkage of the ipsilateral hemisphere, followed by MRI images of coronal slices and matching histological sections showing infarction, ipsilateral ventricular enlargement and tissue loss in the ipsilateral hemisphere in a MCAO plus PBS- and MCAO plus DCS-treated rat. (B) Results of a morphometric ana lysis of infarct volume and tissue loss perfomed on T₂-weighted MRI images from six MCAO plus PBS and six MCAO plus DCS rats, with percentage infarction = 100 × (infarct volume/contralateral hemisphere volume) and percentage atrophy = 100 × ([contralateral hemisphere − ipsilateral hemisphere]/contralateral hemisphere). Bars represent means and standard errors of the infarction and atrophy expressed as percentage of contralateral hemisphere. DCS: d-cycloserine; PBS: Phosphate-buffered saline.