Neurons were obtained from 20 male Sprague Dawley rats (8-12 weeks old; weight, 300-500 g) purchased from the Animal Experimental Center of Hainan Medical University, The Second Affiliated Hospital (Haikou, China). Rats had free access to water and food and they were kept under the standard laboratory conditions (22-25°C; 65±5% humidity; 12 h light/dark cycle). Following sacrifice, the brain was carefully removed from the skull and placed on ice. The meninges were removed under a dissecting light microscope and the cortical tissue was collected, placed in a centrifuge tube, mixed with 0.125% trypsin (Beyotime Institute of Biotechnology, Haimen, China) and digested at 37°C for 30 min. Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 5% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) was added to the digested tissue and the mixture was carefully pipetted. Then, the tube was left to stand at 37°C. Fluid without large particles was transferred into another centrifuge tube and the cell suspension was carefully pipetted several times to obtain a suspension with no visible tissue. Fluid was pipetted in a six-well plate and the supernatant was transferred to another plate containing 5% FBS, 2% B27 (Gibco; Thermo Fisher Scientific, Inc.) and neurobasal medium (Gibco; Thermo Fisher Scientific, Inc.) after 2 h (21,22).

Neuron viability was detected by MTT assay. A total of 10⁴ neurons/well were seeded in 96-well plates containing 200 µl DMEM with 10% FBS to evaluate neuron proliferation at different concentrations of MDZ (Shanghai Guyan Biological Technology Co., Ltd., Shanghai, China) and GLU (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). MTT was added and incubated for 4 h at 37°C (Cytotoxicity Assay kit; Beyotime Institute of Biotechnology). DMSO (Sigma-Aldrich; Merck KGaA) was used to dissolve the formazan crystals. Obtical density was measured at a wavelength of 570 nm. Neurons without MDZ or GLU treatment represented control group 1, while groups 2-7 were simultaneously treated with GLU (final concentration, 100 µM) and MDZ at concentrations of 0, 0.1, 0.3, 0.5, 0.7 or 0.9 mg/l at 37°C for 4 h (23,24)

Neurons were seeded as described in the previous paragraph, and incubated with GLU and MDZ at different concentrations as aforementioned. LDH was measured using an LDH assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s protocol (16,25).

Neurons were seeded as previously described and incubated with 0.7 mM MDZ and GLU (final concentration, 100 µM). In group 2, only GLU was added (final concentration, 100 µM), while the control group lacked any treatment. The medium was removed, neurons were washed with PBS and EGTA-free trypsin was added. When neurons were detached, PBS was added, and neurons were collected in a 15 ml tube and centrifuged at 1,000 x g for 3 min at room temperature. Next, neurons were washed with PBS and centrifuged at 1,000 x g for 3 min at room temperature. Annexin-V from a Fluorescein Isothiocyanate-Annexin V Apoptosis Detection kit (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer’s protocol and apoptosis was measured by MoFlo Astrios EQ flow cytometry and corresponding Summit version 6.2 software was used to analyze the results (Beckman Coulter, Brea, CA, USA) (26,27).

Neurons were cultured in serum-free DMEM medium for 24 h. NP40 cell lysis buffer (Gibco; Thermo Fisher Scientific, Inc.) was used to lyse neurons on ice for 5 min, and sonication (20-24 kHz) was used to further cell cleavage at 0°C (8 times within 2 min, 5 sec each time and pauses of 10 sec). Cell debris was removed by centrifugation at 1,000 x g for 1 min at 0°C and total protein was obtained and detected by Bicinchoninic acid (BCA) method using BCA Protein Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol.

Graded 4-15% polyacrylamide gels were used (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and 50 µg protein were loaded per lane, followed by electrophoresis to separate the target proteins. Then, proteins were transferred onto Immobilin-P membranes (Sigma-Aldrich; Merck KGaA), and membranes were blocked by 5% non-fat milk for 1 h at room temperature. Samples were incubated overnight with antibodies against Bcl-2 (cat. no. ab692; 1:500), Bax (cat. no. ab32503; 1:500), p53 (cat. no. ab26; 1:1,000), caspase-3 (cat. no. ab13585; 1:500) and GAPDH (cat. no. ab8245; 1:500) all from Abcam (Cambridge, MA, USA), diluted in 5% skim milk powder. Following washing twice with PBS and Dulbecco’s PBS, samples were treated with goat anti-mouse immunoglobulin G H&L (cat. no. ab6785; 1:10,000; Sigma-Aldrich; Merck KGaA) and incubated at room temperature for 1 h. Immunoreactive bands were cut and an Odyssey SA infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) was used to observe the results (28,29).

Semi quantitative RT-PCR amplification was used to evaluate the expression of apoptotic genes with the A1250 Access RT-PCR system (Promega Corporation, Madison, WI, USA). Neurons were digested with 5% trypsin (Beyotime Institute of Biotechnology) and divided into single cells under a light microscope. Then, cells were lysed with NP40 cell lysis buffer in PCR tubes for 2 h on ice. MgSO₄ (25 mM), nuclease-free water, dNTP mix, AMV/Tfl 5X Reaction Buffer, SuperScript^® IV reverse transcriptase (all Invitrogen; Thermo Fisher Scientific, Inc.), and specific upstream and downstream primers for GAPDH, p53, caspase-3, Bcl-2 and Bax were added to a 0.5 ml reaction tube. The thermocycling conditions for the PCR were set according to the manufacturer’s protocol of SuperScript™ One-Step RT-PCR System with Platinum™ Taq DNA Polymerase included in the Prime Script TM RT reagent kit (Invitrogen; Thermo Fisher Scientific, Inc.). PCR products were detected by 1.2% agar gel electrophoresis (0.5 µg/ml ethidium bromide) and observed under ultra violet light, as previously described (30,31). The results were analyzed by Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.). The primers used were the following: GAPDH forward, 5′-ACC ACA GTC CAT GCC ATC AC-3′ and reverse, 5′-TCC ACC ACC CTG TTG CTG TA-3′; p53 forward, 5′-ACC TAT GGA AAC TAC TTC CTG AAA-3′ and reverse, 5′-CTG GCA TTC TGG GAG CTT CA-3′; caspase-3 forward, 5′-TGG AAC AAA TGG ACC TGT TGA CC-3′ and reverse; 5′-AGG ACT CAA ATT CTG TTG CCA CC-3′; Bcl-2 forward, 5′-TTC TTT GAG TTC GGT GGG GTC-3′ and reverse, 5′-TGC ATA TTT GTT TGG GGC AGG-3′; and Bax forward, 5′-TCC ACC AAG AAG CTG AGC GAG-3′ and reverse; 5′-GTC CAG CCC ATG ATG GTT CT-3′.

All animal procedures followed the Guide for the Care and Use of Laboratory Animals (33) and were approved by the Institutional Clinical Experiments Committee of the Second Affiliated Hospital of Hainan Medical University (Haikou, China). Sprague Dawley rats (male, 3 months old, 300-500 g) were randomly divided into 4 groups as follows (n=40; n=10 in each group): Sham group, model group, MDZ (2 mg/kg) group, and MDZ (5 mg/kg) group. Rats had free access to water and food and they were housed under stable conditions (22-25°C; 0.03% CO₂; 65±5% humidity; 12 h light/dark cycle). Sodium pentobarbital 2.5% (36 mg/kg; Sigma-Aldrich; Merck KGaA) was used to anesthetize the rats. The common carotid artery, internal carotid artery and external carotid artery were isolated, while the wing palate artery and the cranial branches of the mentioned arteries were separated. Arterial clips were used to clamp the common carotid artery and the internal carotid artery. The distal end of the external carotid artery was ligated and cut. A 5-cm-long nylon filament (diameter, 0.24-0.28 mm) was then inserted into the middle cerebral artery for 2 h. Sham-operated rats were subjected to the same surgical procedure as the rats from the tMCAO groups (34), except for the occlusion of the middle cerebral artery. Rats were treated with a sublingual intravenous injection of MDZ or the same volume of normal saline 10 min following ischemia (34,35).

A total of 24 and 72 h following tMCAO induction, rats were sacrificed under deep anesthesia induced by sodium pentobarbital injection. The number of animals used for each group was 10. Rats randomly selected from each group were used for TTC staining (Beyotime Institute of Biotechnology). Brains were carefully removed and cut into six 2.0-mm-thick coronal sections. Sections were stained with 2% TTC in normal saline for 30 min at 37°C, and fixed in 4% paraformaldehyde solution overnight at 4°C (34).

Brain tissues were stained for 10 min at room temperature with 0.04% w/v toluidine blue in 0.1 M sodium acetate buffer, pH 4.0, to visualize the glycosaminoglycans, followed by 2 min counterstaining in 0.1% w/v fast green FCF at room temperature (37).

A total of 24 and 72 h following tMCAO induction, rats were terminally anesthetized by 1.5% halothane (Shenzhen YunXing Biological Technology Co., Ltd., Shenzhen, China) in air and perfused with saline through the ascending aorta and next with 4% paraformaldehyde. Neurons cultured in vitro were also subjected to immunofluorescence staining. Sections of terminally anesthetized tMCAO rats were cut (10-20 µm thickness) using a cryomicrotome and incubated with 3% goat serum (R&D Systems, Inc., Minneapolis, MN, USA) at 37°C for 40 min. Sections were then incubated with rabbit polyclonal primary antibody against RNA binding fox-1 homolog 3 (1:500; cat. no. ab177487; Abcam) or rat polyclonal primary antibody against neuron filament protein (1:500; cat. no. ab4666; Abcam) and Alzheimer-associated neuronal thread protein (1:500; cat. no. ab49385; Abcam) overnight at 4°C, followed by incubation with secondary antibody (Alexa Fluor 568-donkey anti-mouse immunoglobulin G; 1:1,000; cat. no. ab175700; Abcam) for 2 h at 25°C. Subsequently, nuclei were counterstained using DAPI (Abcam) at room temperature for 20 min. The stained sections were examined with a Leica fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) (38,39). The number of rats used for each group was 10.

The BBB behavior score was used to assess the effect of MDZ on behavior of tMCAO rats (39). Rats were divided into four groups as described above. The assessment was performed at 0, 24 and 72 h following treatment with MDZ. Movement, balance and coordination of limbs, joints, and the whole body of rats was assessed and scored 0-21. Scoring 0-7 points indicated that rats exhibited few or no hindlimb joint movements, 8-14 points indicated ataxic gait and 15-21 points indicated that rats could perform fine movements including toe and tail movements.

Statistical analysis was performed using SPSS 13.0 software package (SPSS, Inc., Chicago, IL, USA). Results are expressed as the mean ± standard deviation, and one-way analysis of variance with Dunnett’s t-test was used to evaluate significance. P<0.05 was considered to indicate a statistically significant difference.

To begin, extracted neurons were identified and neuron purity was tested. Neurons were observed under a microscope and were revealed to have normal morphology and clear nerve fiber growth (Fig. 1A). In addition, immunofluorescent staining revealed the purity of the extracted neurons (Fig. 1B). Thus, they were used for further experiments.

Next, the function of GLU in the induction of cytotoxicity and apoptosis was demonstrated in vitro. The change in staining absorbance with or without GLU treatment was measured. The MTT assay results revealed that the number of cells was reduced to 1/3 following GLU treatment (Fig. 1C). Furthermore, LDH release was increased to 257% compared with the control (Fig. 1D). A significant increase in apoptotic cells following GLU treatment was observed using flow cytometry (Fig. 2A).

Subsequently, the effect of MDZ was evaluated, demonstrating a reduced cytotoxicity and apoptosis in vitro. MDZ reduced neuronal death and LDH release, and the protective effect was concentration-dependent (Fig. 1C and D), reaching an optimal effect at 0.7 mg/ml. At this concentration, neuron density was close to the density of the control group, with LDH release of 134%, and was thus considerably reduced compared with group 2. Furthermore, flow cytometry results revealed that the effect of MDZ on apoptosis (Fig. 2) was also significant, since GLU increased apoptosis from 3.8 to 16.9%, but apoptosis was reduced to ~5.8% following MDZ addition.

Next, the anti-apoptotic mechanism of MDZ treatment was investigated. p53 and caspase3 protein expression were noticeably increased following GLU treatment, which may increase cell apoptosis, and the expression of these proteins was significantly inhibited following MDZ treatment (Fig. 3A). Furthermore, BCL-2 gene expression was inhibited, and the expression of the apoptosis promoter gene Bax was increased following GLU treatment. MDZ also inhibited this effect (Fig. 3B). The PCR results confirmed the western blotting results, and the difference in the expression of apoptosis-associated genes between experimental groups was statistically significant (P<0.05; Fig. 3C). Therefore, GLU induced the expression of p53, caspase-3 and Bax, and inhibited Bcl-2 expression, while MDZ treatment reversed these effects.

Clinical use of MDZ primarily occurs through peripheral intravenous injection, since MDZ passes through the blood-brain barrier to exert its effects relatively quickly. The involvement of MDZ in the reduction of cytotoxicity in peripheral blood vessels or brain tissue was confirmed by animal experiments. LDH concentrations in the peripheral plasma and brain tissue were measured in different tMCAO experimental animal groups. The results revealed that serum LDH concentration in the tMCAO animal model significantly increased from 502±24 to 1,866±71 U/l, and decreased following MDZ treatment in a dose-dependent manner (Fig. 4A). LDH concentration in the brain tissue decreased from 11,073±1,631 to 2,337±276 U/l in the tMCAO animal model, and increased with MDZ treatment in a dose-dependent manner (Fig. 4B).

TTC staining in rat brain tissue revealed that the tMCAO experimental animal model was successfully constructed in the present study, as demonstrated by the ischemic and boundary areas in rat brain tissue, which were consistent with the physiological characteristics of cerebral infarction (Fig. 5A). After 24 and 72 h, the brain of the corresponding treated animals was subjected to morphological analysis and histological staining. The results of toluidine blue staining revealed that the number of neuron in the tMCAO experimental animals was decreased compared with the Sham group, but was increased following MDZ treatment compared with the tMCAO + vehicle group, in a dose-dependent manner (Fig. 5B). Immunofluorescent staining showed similar results: tMCAO produced a remarkable neuronal damage, while MDZ could significantly play a role in protecting neurons. Furthermore, 5 mg/kg achieved improved results compared with 2 mg/kg MDZ (Fig. 5C). Furthermore, the staining results revealed that this effect occurred in the boundary core. The number of neurons in the boundary core also increased under MDZ treatment. At 24 and 72 h, neuron density in the ischemic and boundary areas was statistically analyzed. The results revealed a clear increase in cell density following MDZ treatment compared with the tMCAO, although this density was not considerably different between 24 and 72 h (Fig. 5D and E). In the ischemic core, cell density was decreased from 1.08±0.11×10³ to 0.31±0.03×10³ cells/mm² in tMCAO rats treated with the vehicle, and increased to 0.74±0.08×10³ cells/mm² following 5 mg/kg MDZ treatment within 24 h. Furthermore, in the boundary zone, MDZ achieved a similar effect; increasing cell density from 0.42±0.02×10³ to 0.83±0.08×10³ cells/mm². The behavior of tMCAO rats was also analyzed. The BBB method was used to assess rats’ behavior, including movement, coordination and fine movement of the hind limbs. The rats in the control group and sham group were all given a score of 21. Following tMCAO operation all rats were assessed at 7-10. A total of 3 rats were assessed 12-14 following 24 h of treatment with MDZ (2 mg/kg) and only one of them scored 17 following 72 h of treatment with MDZ (2 mg/kg). In addition, in the MDZ (5 mg/kg) group only two rats scored 16 at 24 h and 17 and 18 at 72 h following treatment with MDZ (data not shown). The results indicate that BBB scores of behaviors improved in certain rats, but not all following treatment with MDZ.