5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate and probenecid were obtained from Life Technologies Corporation (Carlsbad, CA). Dulbecco's modified Eagle medium (DMEM), minimum essential media (MEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Laminin and Monocyte Chemoattractant Protein-1 (MCP-1) were purchased from BD biosciences (San Jose, CA). EBSS and HEPES buffer solution were obtained from Welgene (Daegu, South Korea). 2, 2'-azobis-(2-methylpropionamide)-dihydrochloride (AAPH), 1, 1-diphenyl-2-picrylhydrazyl (DPPH), Poly-D-Lysine (PDL), Harris hemotatoxylin, and all other drugs were purchased from Sigma-Aldrich co (St. Louis, MO). LMT497 (Seco-Cl-IB-MECA) was synthesized by Dr. Yongseok Choi at the Department of Biotechnology, School of Life Sciences and Biotechnology, Korea University (For details of chemical synthesis, see the supplementary Data).

Male Sprague-Dawley rats (260 to 300 g; Charles River Laboratories, Seoul, South Korea) were acclimated to their environments. All animal experiments were performed in accordance with the NIH guideline and approved by the Institutional Animal Care & Use Committee. The animal IRB number used for in vitro was KUIACUC-2014-29 and the IRB number used for in vivo was KUIACUC-2014-14.

Rats were initially anesthetized with 3% isoflurane in a 70% N₂O and 30% O₂ mixture via facemask and maintained with 2% isoflurane. Focal cerebral ischemia was induced by endovascular middle cerebral artery occlusion (MCAO) on the right side of the brain as previously described [6]. Briefly, a 4-0 monofilament nylon suture (Ethicon Johnson & Johnson, Brussels, Belgium) with a heat-blunted end was inserted into the right external carotid artery and advanced into the internal carotid artery. After 90 minutes of MCAO, rats were reperfused by removing the suture from the vessel. During the surgery, body temperature was maintained at 37℃ with a homeothermic blanket (Panlab, Barcelona, Spain) and checked with a rectal temperature probe. LMT497 (2 mg/kg) was initially dissolved in dimethyl sulfoxide 5%, Cremophor EL 10% (poluoxyl-35 hydrogenated castor oil; Merck KGaA, Darmstadt, Germany), and saline 85% respectively. Drugs were injected intravenously twice per experiment at 3 h/8 h or 10 h/18 h after the onset of MCAO.

After 24 h of MCAO, rats were anesthetized with chloral hydrate. Brains were extracted from rats and cut into 2 mm coronal sections and stained with 2% triphenyltetrazoliumchloride (TTC; Alfa Aesar A Johnson Matthey Company, Ward Hill, USA) at 37℃ for 10 minutes. The total infarction volume was calculated by integrating 6 sections, manually measuring the infarct area and compensating for brain edema as described previously [7, 8]. The percentage of edema was calculated by measuring the difference in volume between ipsilateral and contralateral hemispheres [% Edema = (Vi - Vc)/Vc; Vi, ipsilateral volume; Vc, contralateral volume].

Neurological scores were evaluated after 24 h of initial MCAO, as described previously [9]. Scores are divided as follows: 0, no observable deficit of motor function; 1, contralateral forelimb flexion when rat was lifted by tail; 2, decreased resistance to lateral push (and forelimb flexion) without circling; 3, same behavior as grade 2, with circling; 4, no spontaneous motor activity.

Primary mixed cortical neuronal/glial co-cultures were prepared from embryonic (E16-E17 day-old) Sprague-Dawley rats. Meninges-free cortices were dissociated by triturating with Pasteur pipette. Dissociated cerebrocortical cells (1.35 × 10³cells/mm) were added to pre-coated culture plates with poly-D-lysine (10 µg/mL) and laminin (4 µg/mL). Cells were then maintained in DMEM containing 10% fetal bovine serum and B27-supplement in a 37℃ in a humidified incubator containing 95% air and 5% CO₂. Experiments were performed 12-14 days after initial plating of cultures. Cultures consisted of 50-60% neurons and 40-50% glial cells (> 95% astrocytes), assessed by immunostaining with neuronal marker NeuN and astroglial marker GFAP.

Pure microglial cells were prepared from primary mixed glial cell cultures. Cerebral cortices from neonatal Sprague-Dawley rats (1 to 2 days old) were triturated to single cells. They were then plated onto poly-D-lysine (1 µg/mL) coated 75-cm² T-flasks and maintained in modified Eagle's medium (MEM) containing 10% fetal bovine serum. Seven to nine days after plating, microglia was detached from the flasks by mild shaking (37℃, 2 min at 200 r.p.m.) and was set for experimental use.

For an in vitro model of hypoxic/ischemic insult, cells were placed in an anaerobic chamber (partial pressure of oxygen <2mm Hg; 95% N2, 5% CO₂), at 37℃ for 1 h, while the culture medium was replaced with a glucose-free DMEM. OGD was terminated by replacing culture medium with DMEM supplemented with 25 mM glucose, and by returning cells to normoxic conditions. Cells were treated with LMT497 (10 µM), during the entire period of OGD/R.

Cell injury or death was assessed by morphological examination with a phase-contrast microscope (Leica, Solms, Germany), and by measuring the amount of LDH released into the culture medium using a diagnostic kit (Sigma-Aldrich, Co., St. Louis, MO). The degree of cell injury was expressed as a percentage of total LDH release, which was defined as the amount of LDH released from the cells after repeated freeze/thaw cycles. Dose-response experiments of LMT497 in the OGD/R model were performed between the ranges of 0.1 and 100 µM.

The intracellular reactive oxygen species (ROS) level was measured with CM-H₂DCF-DA, which diffuses through cell membranes and hydrolyzed by intracellular esterase to its nonfluorescent form, CM-DCF-H. CM-DCF-H then reacts with free radicals to form highly fluorescent CM-DCF. After 3 h reoxygenation, cells were loaded with 1 µM CM-H₂DCF-DA in EBSS containing 2.5 mM probenecid for 10 min. After removing the loading medium, the DCF fluorescence in three non-overlapping optical regions (825 × 625 µm²) per sample was measured at Ex488 nm/Em525 nm with a fluorescence microscope (Leica, Solms, Germany) equipped with a digital camera. The fluorescence intensity was quantified using an image analyzer (TOMORO ScopeEYE 3.5) by a treatment-blinded examiner.

For oxygen radical absorbance-capacity (ORAC) assays, different concentrations of LMT497 or Trolox were allowed to react with peroxyl radicals generated from 2,2'-azobis-(2-methylpropionamide)-dihydrochloride (AAPH; 60 mM), and ROS scavenging activity was determined by monitoring the loss of fluorescein (50 nM) fluorescence caused by peroxyl radicals. Fluorescence decay was measured every 5 min for 3 h at 37℃ at (Ex485nm/ Em530nm), respectively, using a fluorescence microplate reader. Peroxyl radical scavenging capacity was quantified by calculating the area under the curve (AUC) on the basis of kinetic curves using the following equation: AUC = (0.5 + f₁/f₀ + f₂/f₀ + f₃/f₀ + … + f_n-2/f₀ + f_n-1/f₀ + f_n/f₀) × 5, where fi is the fluorescence reading at time i (minutes). The net AUC = AUC_sample - AUC_blank. The concentration of LMT497 producing the same net AUC, when compared to 50 mM Trolox, was presented as its Trolox Equivalents (TE).

For the DPPH reduction assay, LMT497 or Vitamin C (0.1, 1, 10, 25, 50, 100 µM) were mixed with 23.6 µg/mL of DPPH, an organic nitrogen radical generator. After 30 min of incubation at 37℃, the absorbance was measured at 517 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The scavenging activity of free radicals was expressed as the percentage of maximum inhibition [(Abs_maximum - Abs_sample)/(Abs_maximum - Abs_minimum) × 100], obtained from a standard curve generated using vitamin C.

For the measurement of SOD activity, cells were suspended in cold HEPES buffer (20 mM HEPES pH 7.2, 1 mM EGTA, 210 mM mannitol, 70 mM sucrose), and the resulting cell lysates were centrifuged at 1500' g for 5 min at 4℃. SOD activity was measured using a SOD assay kit (Cayman Chemical Company, Ann Arbor, MI). In this assay, tetrazolium salt reacts with superoxide anion to produce formazan, which is detected at 450 nm with an ELISA microplate reader (SpectraMax190; Molecular Devices, Sunnyvale, CA).

For the measurement of catalase activity, cells were homogenized with cold phosphate buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA), and the resulting cell lysates were centrifuged at 10,000'g for 15 min at 4℃. Catalase activity was determined in supernatants by the reaction of catalase with methanol in the presence of an optimal concentration of H₂O₂. Formaldehyde production was determined using a catalase assay kit (Cayman Chemical Company) and was quantified by measuring absorbance at 540 nm using an ELISA microplate reader (SpectraMax190, Molecular Devices).

The intracellular levels of superoxide were determined by measuring the red fluorescence of hydroethidium (HE) produced by oxidation of dihydroethidium (DHE) [10].Cells were loaded with 5 µM DHE for 20 min before the termination of OGD/R (1 h/3 h), after which the medium was replaced with fresh medium. HE fluorescence in three non-overlapping optical sections was examined using a fluorescence microscope (DM IL HC Fluo; Leica Microsystems GmbH, Wetzlar, Germany) equipped with a digital camera (DFC420C, Leica Microsystems GmbH, Wetzlar, Germany). The intensity of the fluorescence was quantified using an image analyzer (TOMORO ScopeEye 3.5; Saramsoft Co.).

Monocyte Chemoattractant Protein 1(MCP-1) was placed in the bottom chamber of a chemotaxis chamber (Neuro Probe, Cabin John, MD) and microglia (5 × 10⁴ cells/mL in serum-free MEM) in the upper chamber was allowed to migrate to the bottom section for 2 hours, through membrane pores (8 mm² filter area; Neuro Probe, Gaithersburg, MD). Migrated cells on the bottom-side filter were stained for nuclei with Harris's hematoxylin and then counted.

Data were expressed as mean±standard error of the mean (SEM) and analyzed for statistical significance by employing an analysis of variance (ANOVA) and followed by the post hoc Tukey's test for multiple comparisons. Otherwise, the data were expressed as median and interquartile range (IQR: Q1-Q3) and analyzed by Kruskal-Wallis test followed by Mann-Whitney U test. p values < 0.05 were considered significant after Bonferroni correction.

To investigate the neuroprotective effects of LMT497 during ischemic conditions in vitro, cortical cells were placed in an anaerobic chamber for 1 h and were re-oxygenated for 8 h in normoxic conditions. LMT497 (10 µM) reduced the bursting and swelling of the neuronal cell bodies induced by OGD/R in microscope phase-contrast observation (Fig. 2A). Using the LDH assay to evaluate the viability of neuronal cells, LMT497 dose-dependently attenuated OGD/R-evoked LDH release significantly compared to the untreated OGD/R group (Fig. 2B).

It is known that oxidative stress occurs in cerebral ischemic stroke due to the imbalance of NO and ROS [11]. Therefore, to assess the anti-oxidative effect of LMT497 on OGD/R-induced ROS generation in cortical neurons, the DCF-DA assay was performed. LMT497-treated cells showed significantly decreased fluorescence in ischemic conditions, compared to the non-treated control (Fig. 3A, B). To further examine the ROS scavenging activities of LMT497 in a cell-free system, ORAC (hydrogen atom transfer) and DPPH (electron transfer) assays were done [12]. In the ORAC assay, LMT497 did not significantly slow the rapid AAPH-induced decay in fluorescein fluorescence (Fig. 3C, D). LMT497 did not react significantly with DPPH radicals (Fig. 3F).

In regards to anti-oxidative defense mechanisms, enzymes such as SOD and catalase have the abilities to alleviate oxidative damage caused by ischemic stroke [13]. To further investigate the anti-oxidative capabilities of LMT497, the levels of SOD and catalase were tested as representative anti-oxidant enzymes. LMT497 significantly increased SOD activity in cortical cells in non-ischemic conditions, but failed to show significance when tested for catalase activity (Fig. 4A, B). Since SOD catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen [13], it was necessary to examine whether LMT497 could decrease the level of superoxide under ischemic conditions. Similar to Trolox, LMT497 significantly decreased OGD/R-induced HE fluorescence, which indicates the decline of superoxide levels. (Fig. 4C, D)

The inflammatory process plays a vital role during the ischemic cascade. The resident immune cells, primarily microglia, are activated once ischemic stroke disables the endogenous inhibitory signals and triggers. Microglia then migrates towards the damaged penumbra area to release anti- and pro-inflammatory cells [14]. To examine whether LMT497 has anti-inflammatory properties, a microglial migration chemotaxis experiment was done. LMT497 significantly inhibited MCP-1 (100 ng/mL)-induced migration of microglia compared to the untreated control (Fig. 5).

In randomized experiments in rats, post-ischemic treatment of LMT497 (2 mg/kg, i.v., twice) significantly reduced ischemic infarction volume, edema and neurological score in two different time windows (3 h/8 h; 10 h/18 h) (Fig. 6). Results obtained from the initial i.v. administration (3 h/8 h) showed that LMT497 significantly diminished infarct volume (58.6%), edema (75.2%), and neurological score compared to the control group (Fig. 6A-D). To further expand the therapeutic time window, we administered LMT497 at 10 h/18 h post-ischemia. At this time point, LMT497 showed dimidiate infarct volume, edema, and neurological score compared to the control group (Fig. 6E-H).