The present study focused on the latter one, which incorporates a natural way of protecting, and possibly repairing the damaged brain and correcting the behavioral impairments. In this study, we tested in a stroke model the therapeutic effects of NSI‐189, a small molecule with enhanced neurogenic activity, which is already in clinical trial for treatment of major depression and prevention against suicide ( ClinicalTrials.gov , 2016 ; Fava et al., 2015 ). While the brain exerts self‐repair acutely, over time the stroke‐induced cascade of cell death events outweighs the endogenous regenerative mechanisms (Acosta et al., 2013 ; Fava et al., 2015 ; Kokaia & Darsalia, 2011 ; Sun et al., 2013 , 2016 ; Zhang et al., 2012 ). Thus, finding a therapeutic strategy to stimulate the brain to mount a prolonged and stable reparative machinery during the secondary cell death progression after stroke will likely afford beneficial effects. Although NSI‐189 has been reported to show neurogenic effects (Fava et al., 2015 ), the exact mechanisms mediating this observed neurogenesis remains not well understood. To this end, we posit that a therapeutic approach against stroke may be achieved by rendering the brain to maintain its reparative capacity via enhancement of host neurogenesis that likely entails growth factor upregulation and neurite outgrowth enhancement. The proposed study elucidated the biological effects of orally administered small molecule NSI‐189, on promoting neurogenesis, and exerting functional benefits in a stroke model.

Impaired neurogenesis (Acosta et al., 2013 ; Esposito, Hayakawa, Maki, Arai, & Lo, 2015 ; Kokaia & Darsalia, 2011 ; Sun et al., 2013 , 2016 ; Xia et al., 2006 ; Zhang et al., 2012 ) accompanies many neurological disorders, including stroke. Enhancement of host neurogenesis may serve as a novel therapy for stroke, which remains a significant unmet clinical need with efficacy of tissue plasminogen activator or tPA limited to 4.5 hr. Until recently, the non‐regenerative capability of the adult damaged brain was an accepted scientific dogma. However, accumulating evidence over the last decade indicates that neurogenesis occur during adulthood, in that neurons and astrocytes can be generated from isolated cells of the adult mammalian CNS (Reynolds & Weiss, 1992 ). Largely based on this phenomenon of neurogenesis, several laboratory studies have examined stem cell therapy for treating various diseases in the CNS, including stroke, and neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease (Borlongan, 2011 ; Borlongan, Glover, Tajiri, Kaneko, & Freeman, 2011 ). Stem cell therapy, however, remains as an experimental treatment. While brain injury has been shown to trigger transient and limited neurogenesis, this endogenous protective mechanism is not capable of reversing the cell death cascade in the CNS. It is, however, recognized that strategies designed to enhance the endogenous neurogenesis are potentially beneficial for treating brain disorders (Borlongan et al., 2011 ; Borlongan, 2011 ; Hess & Borlongan, 2008a ; Yasuhara et al., 2006 ). Regenerative medicine has emerged as a new scientific field advancing stem cell therapy for treating brain disorders, with emphasis on either transplanting exogenous stem cells or amplifying endogenous stem cells via neurogenesis (Hess & Borlongan, 2008b ; Picard‐Riera, Nait‐Oumesmar, & Baron‐Van Evercooren, 2004 ).

2 METHODS

This study was designed to evaluate potential therapeutic value of treatment with a novel neurogenic compound, NSI‐189. Phosphate, hereafter referred to as NSI‐189 (supplied by Neuralstem Inc., Germantown, MD) in an animal model of adult ischemic stroke. A summary of timeline and procedures is provided for clarity of experimental design (Table 1). Drug treatment was initiated at 6 hr after stroke and daily thereafter for 12 weeks, with functional readouts of behavioral and histological deficits conducted during the subsequent 12‐ or 24‐week period post‐treatment. We characterized motor and neurological performance at baseline (prior to stroke), then at 1, 3, and 7 days after stroke and at weekly/monthly intervals thereafter. Following completion of behavioral testing at 12 weeks or 24 weeks post‐treatment, animals were randomly euthanized by transcardial perfusion and brains harvested to histologically characterize the extent of cerebral ischemia and to reveal the host tissue endogenous repair mechanism (i.e., neurogenesis). Investigators who were involved in drug treatments, behavioral testing, and post‐mortem analyzes were blind to the treatment conditions.

Table 1. Treatment conditions Group Size Test article Timing (first dose) Survival time post stroke 1 24 Vehicle 6 hr post‐stroke, daily by oral gavage for 12 wks A: N = 12 @ 12 wks C: N = 12 @ 24 wks 2 24 NSI‐189 6 hr post‐stroke, daily by oral gavage for 12 wks B: N = 12 @ 24 wks D: N = 12 @ 24 wks

2.1 Stroke surgery A total of 60 adult Sprague–Dawley, male rats (weighing around 250 g at beginning of the study) received experimental stroke surgery using the middle cerebral artery occlusion (MCAo) model. All surgical procedures will be conducted under aseptic conditions. The animals were anesthetized with 1–2% isoflurane in nitrous oxide/oxygen (69%/30%) using a face mask and checked for pain reflexes. Under deep anesthesia, animals underwent the MCAo surgery. The MCAo suture technique involved insertion of a filament through the carotid artery to reach the junction of the MCA, thus blocking the blood flow from the common carotid artery, as well as from the circle of Willis. The right common carotid artery was identified and isolated through a ventral midline cervical incision. The suture size was 4–0, made of sterile, non‐absorbable suture (Ethicon, Inc., Somerville, NJ), with the diameter of the suture tip tapered to 24–26‐gauge size using a rubber cement. About 15–17 mm of the filament was inserted from the junction of the external and internal carotid arteries to block the MCA. The right MCA was occluded for 1 hr. A heating pad and a rectal thermometer allowed maintenance of body temperature within normal limits (37 ± 0.3°C). To determine successful occlusion and reperfusion, a laser Doppler probe was placed at the distal end of the MCA and revealed at least 80% reduction in regional cerebral blood flow. To further ensure similar degree of stroke insults, physiological parameters including PaO 2 , PaCO 2 , and plasma pH measurements were monitored, and we found no significant differences in our stroke animals. Based on laser Doppler readouts and behavioral tests after MCAs, a total of 48 animals were enrolled in this study.

2.2 Drug treatment All drug treatments were conducted under aseptic conditions. Test article was NSI‐189 H 3 PO 4 (mol. wt. 464.50), with lot number DAJ‐F‐40(2). Purity was 99.8% and stability was determined as stable under storage condition (−10 to −30°C). The administration dose of 30 mg/kg was based on the weight of the API (active pharmaceutical ingredient) base without the weight of phosphate salt. For vehicle, 0.03N HCl in deionized water was used. Following MCAo, animals were randomly assigned to receive oral NSI‐189 (n = 24) or vehicle (n = 24), starting at 6 hr after stroke, and daily for the next 12 weeks.

2.3 Motor and neurological tests All investigators testing the animals were blinded to the treatment condition. Animals will be subjected to elevated body swing test (EBST) and neurological exam. EBST involved handling the animal by its tail and recording the direction of the swings. The test apparatus consisted of a clear Plexiglas box (40 × 40 × 35.5 cm). The animal was gently picked up at the base of the tail, and elevated by the tail until the animal's nose is at a height of 2 inches (5 cm) above the surface. The direction of the swing, either left or right, was counted once the animals head moves sideways approximately 10° from the midline position of the body. After a single swing, the animal was placed back in the Plexiglas box and allowed to move freely for 30 s prior to retesting. These steps were repeated 20 times for each animal. About 1 hr after the EBST, the neurological exam was conducted. Neurologic score for each rat was obtained using three tests which include (1) forelimb retraction, which measured the ability of the animal to replace the forelimb after it was displaced laterally by 2–3 cm, graded from 0 (immediate replacement) to 3 (replacement after several seconds or no replacement); (2) beam walking ability, graded 0 for a rat that readily traversed a 2.4‐cm‐wide, 80‐cm‐long beam to 3 for a rat unable to stay on the beam for 10 s; and (3) bilateral forepaw grasp, which measured the ability to hold onto a 2‐mm‐diameter steel rod, graded 0 for a rat with normal forepaw grasping behavior to 3 for a rat unable to grasp with the forepaws. The scores from all three tests, which were done over a period of about 15 min on each assessment day, were added to give a mean neurologic deficit score (maximum possible score, 9 points divided by 3 tests = 3). Animals were subjected to both tests at baseline (prior to stroke), then at 1, 3, 7 days after stroke and at weekly intervals post‐treatment.

2.4 Histology All post‐mortem histology was performed by Neurodigitech. At scheduled intervals post‐stroke (either 12 weeks or 24 weeks), rats were randomly euthanized (n = 12 per treatment), perfused by transcardial perfusion with 4% paraformaldehyde. The brains were dissected, post‐fixed for overnight in 4% paraformaldehyde, then subsequently immersed in 30% sucrose until immunohistochemical processing. Brain section preparations were designed to identify stroke‐induced cerebral infarction and NSI‐189‐induced neurogenic effects. All brains were embedded in gelatin blocks and sectioned on a freezing sliding microtome at 40‐μm. The sections were washed in PBS 5 × 10 min to remove the antigen preservative solution. Endogenous peroxidase was blocked using 3% H 2 O 2 for 15 min. The sections were then incubated in 1% Triton‐X100 for 30 min, and then blocked with 5% Normal Horse Serum (NHS) for 1 hr. The sections were incubated with the primary antibody, Ki67 (1:2000, Cat. #: ab16667, Abcam, CA) or MAP2 (1:2000, Cat. #: AB5622, Millipore, MA) overnight. The sections were rinsed 5 × 5 min in PBS prior to pre‐incubation in 5% NHS for 1 hr, then incubated with the secondary antibody which corresponded to the respective host of the primary antibody (Donkey α Rabbit, 1:2000, Cat. #: 711‐066‐152, Jackson ImmunoResearch Laboratories, Inc, PA) for 90 min. After rinsing in PBS (5 × 5 min), the sections were incubated with peroxidase‐conjugated streptavidin (1:5000, Cat. #: 016‐030‐084, Jackson ImmunoResearch Laboratories) with 1% NHS. The sections were washed 5 × 5 min in PBS after the streptavidin, prior to developing with DAB (3,3′‐Diaminobenzidine Tetrahydrochloride Hydrate, Cat #: 1001306853, Sigma–Aldrich, St. Louis, MO) and Nickel chloride. After DAB processing, the sections were rinsed, mounted, and air‐dried overnight. The slides were dehydrated, and coverslipped with DPX Mounting Medium (Cat #: 13512, Electron Microscopy Sciences, Hatfield, PA). The slides were imaged and reviewed under Nikon bright‐field microscope (Nikon, Tokyo, Japan). The regions of interest (ROIs), including the cerebral cortex, the hippocampal subfields (CA1 + CA2, CA3, and DG) were contoured rostro‐caudally and then the immunoreactivity of the MAP2 density was measured by Nikon NIS‐Element software and Ki67+ cells counted rostro‐caudally along the subgranular zone (SGZ) by Image‐Pro Premier (v10.10), respectively. It was noted that all IDs were coded and blinded to the analysts during the course of the study. After completion, all of the quantitative data were extracted and transferred for the statistical analysis (Prism, GraphPad®, La Jolla, CA). The data values between groups were compared using ANOVA with Tukey's post‐hoc tests (p ≤ 0.05).