NSI-189, a small molecule with neurogenic properties, exerts behavioral, and neurostructural benefits in stroke rats

Abstract

Enhancing neurogenesis may be a powerful stroke therapy. Here, we tested in a rat model of ischemic stroke the beneficial effects of NSI-189, an orally active, new molecular entity (mol. wt. 366) with enhanced neurogenic activity, and indicated as an anti-depressant drug in a clinical trial (Fava et al., , Molecular Psychiatry, DOI: 10.1038/mp.2015.178) and being tested in a Phase 2 efficacy trial (ClinicalTrials.gov, , ClinicalTrials.gov Identifier: NCT02695472) for treatment of major depression. Oral administration of NSI-189 in adult Sprague-Dawley rats starting at 6 hr after middle cerebral artery occlusion, and daily thereafter over the next 12 weeks resulted in significant amelioration of stroke-induced motor and neurological deficits, which was maintained up to 24 weeks post-stroke. Histopathological assessment of stroke brains from NSI-189-treated animals revealed significant increments in neurite outgrowth as evidenced by MAP2 immunoreactivity that was prominently detected in the hippocampus and partially in the cortex. These results suggest NSI-189 actively stimulated remodeling of the stroke brain. Parallel in vitro studies further probed this remodeling process and demonstrated that oxygen glucose deprivation and reperfusion (OGD/R) initiated typical cell death processes, which were reversed by NSI-189 treatment characterized by significant attenuation of OGD/R-mediated hippocampal cell death and increased Ki67 and MAP2 expression, coupled with upregulation of neurogenic factors such as BDNF and SCF. These findings support the use of oral NSI-189 as a therapeutic agent well beyond the initial 6-hr time window to accelerate and enhance the overall functional improvement in the initial 6 months post stroke.

Keywords: behavioral recovery; cerebral ischemia; neurite outgrowth; neurogenesis; pharmacotherapy; trophic factors.

© 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Figures

Figure 1

NSI‐189 promotes behavioral recovery in stroke. Stroke animals that received NSI‐189 displayed significant amelioration of stroke‐induced motor and neurological deficits as early as day 3 post‐stroke, and continued to improve over the 24 weeks survival compared to vehicle‐treated stroke animals. The observed behavioral recovery in NSI‐189‐treated stroke animals was noted in all monthly test time points, and in both EBST (A) and neurological examination (B)

Figure 2

NSI‐189 enhances cell proliferation in peri‐infarct cortex. Comparison of the Ki67 immuno‐positive cell counts in the SGZ (A and C), SVZ (B and D) and cerebral cortex (G and H) after 12‐week treatment with either vehicle (12 Weeks Vehicle; A, B and G) or NSI‐189 (12 Weeks NSI‐189; C, D and H) and after additional 12 weeks of treatment‐free observational period of vehicle (24 Weeks Vehicle) or NSI‐189 (24 Weeks NSI‐189). Quantifications of Ki67 expression in SGZ (E), SVZ (F), and cortex (I) are shown in bar graphs. Representative images were taken from 12 weeks of survival period. Ki67 expression was significantly increased in the peri‐infarct cortex of NSI‐treated animals at 12 weeks, but did not differ with vehicle‐treated animals in SGZ or SVZ at either survival period

Figure 3

NSI‐189 enhances neurogenesis in stroke. Histopathological examination of secondary cell death in the peri‐infarct cortex (A) and the remote hippocampal area (B) revealed increased MAP2 immunoreactivity in NSI‐189‐treated stroke animals compared to vehicle‐treated stroke animals (*p's < 0.05). These results suggest that NSI‐189 promoted an active remodeling of the stroke brain. Quantification of MAP‐2‐immunopositive staining densities in the hippocampus and the cerebral cortex of NSI‐189‐ and vehicle‐treated animals. Brain sections after MAP‐2‐immuno reactivity, developed by HRP‐DAB reaction, were microscopically scanned and converted into digital images. The regions of interest to quantify were manually lassoed, and individual pixels and their optical densities were automatically captured by a software. (A and B) Representative examples of ipsilateral hippocampal subfields demarcated to quantify the densities within CA1 (deep blue), CA3 (red), and dentate gyrus (DG, light blue), from an animal administered with 12 weeks of vehicle (A) or with 12 weeks of NSI‐189. (C and D) Representative examples of ipsilateral brain section demarcated to quantify the densities within cortex (deep blue), from an animal administered with 12 weeks of vehicle (C) or with 12 weeks of NSI‐189 (D). ST, striatum; V, ventricle; cc, corpus callosum. Bar graphs correspond to quantification of hippocampus (E) and cerebral cortex (F) showing significant increments in MAP2 density, especially at 12 weeks of NSI‐189 treatment

Figure 4

NSI‐189 upregulates neurogenic factors in tandem with neurogenesis. Parallel in vitro studies that exposed primary rat hippocampal neurons to the oxygen glucose deprivation (OGD) insult model revealed reduced cell death viability, Ki67 expression and decreased MAP2 expression under standard media (SM) condition, which were significantly attenuated by NSI‐189 (NSI) treatment (p's < 0.05). Growth factors were upregulated in the CM of NSI‐treated hippocampal cells compared to SM‐grown cells (*p's < 0.05), in particular BDNF and SCF (**p's < 0.05). Combined antibody‐BDNF and antibody‐SCF suppressed the NSI‐189‐mediated rescue of cell viability against OGD (p > 0.05)

Figure 5

NSI‐189's mechanism of action. The entry to the brain of the small molecule, NSI‐189, is prerequisite to realizing therapeutic benefits in stroke (1), likely by upregulating growth factors (2), and increasing neurite outgrowth (3)