Conantokin-G attenuates detrimental effects of NMDAR hyperactivity in an ischemic rat model of stroke

Abstract

The neuroprotective activity of conantokin-G (con-G), a naturally occurring antagonist of N-methyl-D-aspartate receptors (NMDAR), was neurologically and histologically compared in the core and peri-infarct regions after ischemia/reperfusion brain injury in male Sprague-Dawley rats. The contralateral regions served as robust internal controls. Intrathecal injection of con-G, post-middle carotid artery occlusion (MCAO), caused a dramatic decrease in brain infarct size and swelling at 4 hr, compared to 26 hr, and significant recovery of neurological deficits was observed at 26 hr. Administration of con-G facilitated neuronal recovery in the peri-infarct regions as observed by decreased neurodegeneration and diminished calcium microdeposits at 4 hr and 26 hr. Intact Microtubule Associated Protein (MAP2) staining and neuronal cytoarchitecture was observed in the peri-infarct regions of con-G treated rats at both timepoints. Con-G restored localization of GluN1 and GluN2B subunits in the neuronal soma, but not that of GluN2A, which was perinuclear in the peri-infarct regions at 4 hr and 26 hr. This suggests that molecular targeting of the GluN2B subunit has potential for reducing detrimental consequences of ischemia. Overall, the data demonstrated that stroke-induced NMDAR excitoxicity is ameliorated by con-G-mediated repair of neurological and neuroarchitectural deficits, as well as by reconstituting neuronal localization of GluN1 and GluN2B subunits in the peri-infarct region of the stroked brain.

Fig 1. Treatment with con-G showed improved neurological scoring and decreased peri-infarct Ca²⁺ microdeposits.

(A) Rats that underwent MCAO were either non-treated (S) or treated with 2 μM con-G (S/con-G) 30 min after stroke induction. Neurological assessments were performed 26 hr after ischemia induction Data are represented as mean ± SEM. *p = 0.037 between non-treated and con-G-treated stroked rats. (B) Intrathecal administration of con-G reduced swelling at 4 hr post-MCAO by ∼34%. (C) An approximate 50% reduction in infarct size was observed in the ischemic core in rats after 4 hr of con-G treatment. Changes in swelling and infarct sizes have been calculated as % of non-treated rats. (D) Coronal sections were processed with von Kossa stain for Ca²⁺ deposits. Ca²⁺ deposits were averaged from 3–5 peri-infarct fields of non-treated stroked rat brains (S) and con-G-treated stroked rat brains (S/con-G) at 4 hr and (E) 26 hr. Representative images of brain sections of the peri-infarct region stained for Ca²⁺ deposits by von Kossa staining from non-treated rats at 4 hr (F) and 26 hr (H) and con-G-treated brain rats at 4 hr (G) and 26 hr (I). Bars represent mean ± SEM. *p < 0.05 between non-treated and con-G treated group of rats. N = 12 for S and n = 7 for S/con-G at 26 hr, and n = 4 for both S and S/con-G at 4 hr.

Fig 2. Effects of ischemia on cerebral vasculature.

Stroke-mediated compromised vasculature was observed for non-treated (A-C) and for con-G treated rat brains (D-F) at 4 hr postischemia. Decreased vascular staining (red arrows) was observed in the ipsilateral regions of both non-treated (A) and con-G treated (D) stroked rat brains at 4 hr postischemia, and this observation is exacerbated in the ipsilateral region at 26 hr for both non-treated (G) and con-G-treated rat brains (J). The contralateral region shows intact vasculature at 4 hr (B, E) and at 26 hr (H, K) postischemia, while the peri-infarct regions showed vasculature that was between ipsilateral and contralateral at 4 hr in non-treated (C) and con-G-treated rat brains (F), and at 26 hr (I, L).

Fig 3. Effects of ischemia on numbers of cerebral vessels.

Quantitation of the average vascular counts from 3–5 different fields of PAS-stained vasculature per 0.13 mm² area at 4 hr (A) and 26 hr (B). * p<0.05 between stroked rats in the different regions of the brain, and # p<0.05 between con-G-treated rats in the different parts of the brain. Quantitatively, the peri-infarct regions showed average number of vasculature bodies that was between ipsilateral and contralateral regions in non-treated (black bars) and con-G-treated rat brains (white bars) at 4 hr and 26 hr. N = 12 for S and n = 7 for S/con-G at 26 hr, and n = 4 for both S and S/con-G at 4 hr.

Fig 4. Con-G repairs MCAO-induced morphological changes.

(A-L) Representative images of H & E stained coronal brain sections showing perturbed brain architecture of the ipsilateral side at 4 hr and 26 hr for non-con-G treated (A, 4 hr; G, 26 hr) and con-G treated rats (D, 4 hr; J, 26 hr), characterized by perineuronal vacuolation (black arrows) and shrunken neurons (block arrows), which is less dramatic at 4 hr (A) than at 26 hr (G). MCAO-induced pathology in the stroke area is similar in con-G treated brains at 4 hr (D) and 26 hr (J) compared to non-con-G treated brains. (B, E, H, K) show the normal brain architecture of the contralateral region of non-conG treated and con-G-treated rats at 4 hr and 26 hr after stroke induction. (C) The peri-infarct region at 4 hr and (I) 26 hr of con-G-treated and non-treated stroked rats with perineuronal vacuolation and degenerating neurons (<) observed. However, the peri-infarct region of con-G-treated rat brains shows repair of the brain cytoarchitecture at 4 hr (F). The perineuronal vacuolation, while present, is not as pronounced (arrows), with fewer degenerated neurons (<). (L) 26 hr con-G-treated rat brains showed enhanced cytorepair with unpronounced perineuronal vacuolation (black arrow) and diminished eosinophilic neurons (black circle). Total magnification, 200X. Bar scale, 60 μm. N = 12 for S and n = 7 for S/con-G at 26 hr, and n = 4 for both S and S/con-G at 4 hr.

Fig 5. Con-G mitigates ischemic neurodegeneration and restores neuronal integrity.

(A) Representative Fluoro Jade B stained sections of the peri-infarct regions of non-con-G treated rat brains (A) and con-G-treated rat brains (B) at 4 hr, and nontreated (C) and con-G-treated stroked rat brains (D) at 26 hr. Degenerated neurons are indicated by arrows. (E) Quantification of degenerated neurons averaged from 3–5 fields at 4 hr and 26 hr. *p<0.05 between non-treated and con-G-treated groups. N = 12 for S and n = 7 for S/con-G at 26 hr, and n = 4 for both S and S/con-G at 4 hr.

Fig 6. Anti-MAP2 staining in non-treated and con-G-treated stroked rat brains.

At 4 hr postischemia, a lack of MAP2 immunoreactivity in the ipsilateral core of non-treated (A) and con-G-treated rats (D) was observed as compared to the contralateral regions (red staining), which exhibited robust MAP2 staining with distinct soma and dendritic structures in non-treated (B), and also in con-G-treated stroked rats (H). (C) The peri-infarct regions of non-treated stroked rats showed attenuated MAP2 immunoreactivity at 4 hr, but the peri-infarct regions of rats treated with 2 μM con-G demonstrated MAP2 staining in the soma and dendritic regions of the neurons at 4 hr (F). Similarly, at 26 hr postischemia, MAP2 staining was compromised in non-treated (G) and con-G-treated rat brains (J), compared to the contralateral side that showed intact MAP2 staining in the soma and dendrites (H, K) for both groups of animals. (I) The peri-infarct of non-treated rat brains did not show any recovery of MAP2 staining. (L) Con-G was able to sustain reinstitution of neuronal integrity at 26 hr as observed by MAP2 staining in the soma and dendrites. Bar scale, 100 μm for all images. N = 12 for S and n = 7 for S/con-G at 26 hr, and n = 4 for both S and S/con-G at 4 hr.

Fig 7. GluN1 localization pattern in non-treated and con-G-treated stroked rats at 4 hr and 26 hr.

Random punctate staining of GluN1 (red) observed in the ipsilateral regions of stroked rat brain sections at 4 hr (A, D). In the contralateral regions of non-treated (B, 4 hr; H, 26 hr) and-con-G treated stroked rat brains (E, 4 hr; K, 26 hr), distinct GluN1 staining around the soma (blue) is observed (arrows). At 4 hr, the peri-infarct regions of non-treated brains showed similar random punctate staining of GluN1 (C) as the ipsilateral regions; however, the conG-treated stroked rats showed GluN1 staining around the soma (arrows) (F). At 26 hr, the ipsilateral regions of non-treated and con-G-treated-stroked rat brains showed loss of GluN1 staining around the soma (G, J), compared to the contralateral regions (H, K). At 26 hr, the peri-infarct region of non-treated stroked rat brains showed punctate GluN1 staining in the soma (arrow) (I), but the con-G-treated rat brains showed concentrated clusters of somal GluN1 (arrows) (L). Magnification, 100X. Bar scale, 100 μm for all images.

Fig 8. Effect of con-G on GluN2A localization patterns.

Distinct GluN2A localization around the soma was disrupted by MCAO in the ipsilateral region (A, D) compared to the contralateral region (arrows) (B, E). Clusters of GluN2A were observed in the peri-infarct region of non-treated stroked rat brains (arrows) (C), but this was not observed in con-G-treated brains at 4 hr (F). At 26 hr post-MCAO, perinuclear staining of GluN2A was observed (arrows) (G, J) in the ipsilateral region compared to GluN2A staining observed in the soma of the contralateral region (arrows) (H, K). The peri-infarct of non-treated stroked rat brains showed no specific GluN2A staining (I), but the con-G-treated rat brains showed increased perinuclear presence of GluN2A (arrow) (L). Magnification, 100X. Bar scale, 100 μm for all images.

Fig 9. Con-G restores GluN2B localization at 26 hr.

Loss of GluN2B was observed in the ipsilateral regions at 4 hr (A, D) and 26 hr (G, J) compared to the contralateral regions, where polar (B; arrows) or somal (blue) localization (E, H, K; arrows) of GluN2B was observed. The peri-infarct region of untreated rat brains lacked specific GluN2B staining at 4 hr (C) and 26 hr (I), but a distinct GluN2B presence (red) was observed in the peri-infarct area of con-G-treated rats at 4 hr (F) and at 26 hr (L). Magnification, 100X. Bar scale, 100 μm for all images.