Design
A randomized, controlled animal experiment.

Time and setting
The experiments were performed at the First Affiliated Hospital of China Medical University in China and Yantai Yuhuangding Hospital Central Laboratory in China from October 2009 to January 2012.

Materials
A total of 24 normal male Sprague-Dawley rats, weighing 250–300 g, aged 10 weeks, and of specific pathogen-free grade, were provided by the Experimental Animal Center of China Medical University, China (license No. SCXK (Liao) 2008-0005).

Penehyclidine hydrochloride (C₂₀H₂₉NO₂•HCl) was provided by Lisite Pharmaceutical Co., Ltd., Chengdu City, Sichuan Province, China (batch No. 060502-1). The molecular formula is as follows:

Penehyclidine hydrochloride is a new anticholinergic drug, which can cross the blood-brain barrier and has both anti-muscarinic (M receptor) and anti-nicotinic (N receptor) activities, and retains potent central and peripheral anticholinergic activities. Penehyclidine hydrochloride has been used widely clinically as an antagonist of organic phosphorus and soman poisoning in China. Penehyclidine hydrochloride has little or no effect on M2 receptor subtypes, which has no significant effect on heart rate.

Methods
Establishment of middle cerebral arterial occlusion models in vivo
Middle cerebral arterial occlusion was induced by extracranial vascular occlusion in Sprague-Dawley rats (body weight 250–300 g), according to previously reported procedures with slight modification[58]. Sprague-Dawley rats were anesthetized using 10% (v/v) chloral hydrate via intraperitoneal injection, and body temperature was maintained at 36.5–37.5°C and physiological parameters were monitored continuously during the anesthesia and surgery. Briefly, a 1.5-cm long incision was made at the midline of the neck, the right carotid bifurcation and common carotid artery were separated from the adjacent tissue, avoiding harm to the vagus nerve. After careful isolation of the external carotid artery branches of the occipital and the superior thyroid arteries, as well as the internal carotid artery branch, all of these arteries were exposed. The right common carotid artery and internal carotid artery were carefully separated from the adjacent vagus nerve and connective tissue, and a 0.265-cm diameter nylon intraluminal suture was ligated into the cervical internal carotid artery, advancing intracranially to block blood flow into the middle cerebral artery, and collateral blood flow was also reduced by interrupting all branches of the external carotid artery and all extracranial branches of the internal carotid artery. The suture was withdrawn after occlusion for 120 minutes, and then reperfusion was achieved^[59].

Determination of infarction volume in the rat brain
Brain tissues were stored at –70°C for 8 minutes and sliced at a thickness of 2 mm. Slices were incubated in 2% (v/v) 2,3,5-triphenyl-tetrazolium chloride solution for 30 minutes at 37°C. Samples were then washed in distilled water for 30 seconds and photographed. OSIRIS soft 4.19 (http://www.seekbio.com/soft/176.html) was used to measure the infarct area in each sample, and the percentage of the hemispheric infarction volume was calculated. To minimize errors in the estimation of infarction volume, three measurements were needed on each slice for calculation of the infarction volume. The infarction volume (V) was calculated using the following equation:
V= t(A1+A2+…+An), where t is the thickness of the brain tissues (mm), A is the area of the infarcted hemisphere slice (mm²), and n is the number of the brain tissues^[60].

Analysis of cell apoptosis in the brain by TUNEL staining
To detect cell apoptosis, TUNEL staining was performed according to the protocol of the TUNEL Detection System (In Site Cell Apoptosis Detection Kit I, POD, MK1020; Boster, Wuhan, Hubei Province, China). Three rats in each group were deeply anesthetized using 10% (v/v) chloral hydrate (300 mg/kg), transcardially perfused with sodium chloride and 4% (w/v) formaldehyde (200 mL), and then decapitated at a given time. Brain samples, including the cortex, hippocampus and striatum, were post-fixed in 4% (w/v) formaldehyde for 2 hours, dehydrated in alcohol, hyalinized by dimethylbenzene, embedded in paraffin, sectioned at a thickness of 4 μm, adhered to superfrost plus slides, and finally stored at room temperature. A standard TUNEL method was employed to detect the fragmented nuclear DNA associated with apoptosis on paraffin sections. After standard deparaffinization, hydration, incubation with proteinase K, and blocking of endogenous peroxidase, tissue sections were incubated with terminal deoxynucleotidyl transferase and digoxigenin-deoxyuridine triphosphate at 37°C for 60 minutes and then with peroxidase converter antibody at 37°C for 30 minutes. For negative controls, some slides were incubated with label solution that did not contain terminal deoxynucleotidyl transferase. Under the optical microscope (Olympus, Tokyo, Japan), the nucleus of apoptotic cells appeared yellow. Positive cells were counted and the mean number in five random views was recorded.

Immunohistochemical staining for Bcl-2 and caspase-3 expression in brain tissues
Formalin-fixed and paraffin-embedded brain sections (5 mm thickness) were first dewaxed in xylene and rehydrated with a graded ethanol series. Endogenous peroxidase was inactivated by incubation in 3% (v/v) H2O2 for 10 minutes at room temperature. Sections were placed in citrate buffer (0.01 M, pH 6.0) for antigen retrieval by microwave heating. Nonspecific binding was blocked by incubation in goat serum (1:10 for 30 minutes at room temperature, and then sections were incubated at 4°C overnight with rabbit anti-Bcl-2 and -caspse-3 polyclonal antibodies (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three washes in PBS for 15 minutes, the slides were incubated with the appropriate secondary antibody at room temperature for 30 minutes. Slides were incubated in horseradish peroxidase-labeled anti-rabbit IgG (1:200; DAKO, Copenhagen, Denmark) at room temperature for 20 minutes and developed with 3,3’-diaminobenzidine (Sigma, St Louis, MO, USA). The sections were finally counterstained with hematoxylin. For negative-control purposes, the same procedure was used on tissue sections in which 1% (v/v) bovine serum albumin in PBS was substituted for the primary antibody^[61-62]. Under the optical microscope (Olympus, Tokyo, Japan), brown particles or patches in the cytoplasm indicated positive staining. Ten fields per section were selected in the cortex and striatum. The numbers of positive cells in the grid were counted and a mean value was recorded.

Detection of excitatory amino acid concentrations in the rat brain
Rats were sacrificed by an overdose of chloral hydrate after 24 hours of reperfusion. Brains were removed from the cranium quickly. The right hemisphere (extracted by professional anatomical staff) of each rat was separated, weighed and immediately mixed with 8% (v/v) sulfosalicylic acid, and then freeze centrifuged at 16 000 r/min for 30 minutes. The supernatant was isolated for analysis. Excitatory amino acid concentrations were measured with a high performance liquid chromatography system (Hitachi, Tokyo, Japan) equipped with a 2.6 × 150.0 mm column using O-phthalaldehyde precolumn derivatization. A mixture of potassium dihydrogen phosphate and 35% (v/v) methanol was run through as mobile phase A, and 90% (v/v) methanol as mobile phase B, at a rate of 1.0 mL/min. Excitatory amino acid concentrations were determined by a calibration curve with known amino acid standards^[63].

Measurement of malondialdehyde content and superoxide dismutase activity in the rat brain
Rats were killed immediately after 24 hours of reperfusion. Both sides of the cerebral cortex and hippocampus were separately weighed and stored at –70°C. The samples were homogenized in 0.9% (w/v) saline solution using a homogenizer. The homogenate was then centrifuged at 3 000 r/min for 10 minutes at 4°C. The supernatant obtained was used for assays of malondialdehyde content and superoxide dismutase activity. Malondialdehyde content was determined by the thiobarbituric acid method^[64], whereas superoxide dismutase activity was evaluated according to the kit instructions (Jiancheng Institute of Biological Products, Nanjing, Jiangsu Province, China).

Primary culture and establishment of oxygen-glucose deprivation and reperfusion models of hippocampal cells in vitro
Primary cultures of the hippocampus were prepared from 1-day-old Wistar rats based on previously reported articles^[65-66]. Briefly, the skin and skull were removed and brain tissue was exposed completely. The bilateral hippocampi were bluntly separated and repeatedly washed with D-Hank’s buffer with 10 μg/mL gentamycin to remove blood vessels and meninges. The dissected pieces were digested in trypsin (0.125% (w/v)) for 20– 25 minutes at 37°C, neutralized with trypsin inhibitor, and washed three times with D-Hank’s buffer. Dissociated cell suspensions of 1 × 10⁶/L density were transferred into Dulbecco’s Modified Eagle Medium buffer with 15% (v/v) serum at 37°C in a humidified atmosphere of 5% (v/v) CO₂ in air. Fresh medium was replaced after overnight incubation to remove dead cells and cytarabine was added into the medium on day 3. Half amount of medium was refreshed after overnight incubation, and medium was completely replaced twice per week thereafter. After 8–12 days, oxygen-glucose deprivation and reperfusion was performed. Neuronal cells was incubated with 100 μL Hank’s buffer without glucose at 37°C in 24-well culture plates for 2.5 hours and maintained in a 95% (v/v) N2 and 5% (v/v) CO₂ incubator. Oxygen levels in the medium of cultured cells was recovered^[67] and the cells were randomly divided into six groups according to treatment with different concentrations of penehyclidine hydrochloride for 20 minutes before oxygen-glucose deprivation/ reperfusion: control group with normal Hank’s buffer, model group, high-dose penehyclidine hydrochloride group (1.6 μM), middle-dose penehyclidine hydrochloride group (0.4 μM), low-dose penehyclidine hydrochloride group (0.1 μM) and MK-801-positive group (0.1 μM), and incubated for 1, 6, 12, 24 hours after reoxygenation.

N-methyl-D-aspartate-NR1 mRNA expression in primary cultured hippocampal cells by reverse transcription-PCR
Total RNA was directly extracted from primary cultured cells using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The absorbance at 260 and 280 nm was measured regularly. The quality of total RNA was examined by running it on a 1% (w/v) agarose/denaturing formaldehyde gel. Samples with a ratio of absorbance at 260 and 280 nm of 1.8–2.0 and a 28s/18s of approximately 2 were qualified for the next round. Three micrograms of total RNA was reverse-transcribed by M-MuLV reverse transcriptase (MBI, Vilmus, Lithuania) with oligo(dT)16 as a primer. Using equal amounts of cDNA, PCR amplification was performed with 20 nM of each primer, 25 mM Mg2+ and 1 U Taq polymerase with the following N-methyl-D- aspartate-NR1 primers (20 mM each) and glyceraldehyde-3-phosphate dehydrogenase primers (20 mM each; Table 1).

All experiments were performed in triplicate. The mRNA level of N-methyl-D-aspartate-NR1 or glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was assessed by measuring the density of the PCR generated amplicon on the gel by densitometric analysis. The final expression level was the average value of the three amplicons. Relative N-methyl-D-aspartate-NR1 expression is given as N-methyl-D-aspartate-NR1/ GAPDH absorbance ratio.

Statistical analysis
Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Data were expressed as mean ± SD. Statistical comparison was performed by one-way analysis of variance followed by least significant difference test. P values of less than 0.05 were considered statistically significant.

Acknowledgments: We are grateful for the excellent pathology assistance from Professor Weidong Yao, Department of Pathology in the Affiliated Yuhuangding Hospital of Medical College of Qingdao University, China.
Author contributions: Junke Wang and Cuicui Yu contributed equally to the study and results analysis. Both authors approved the final version of the paper.
Conflicts of interest: None declared.
Ethical approval: The experimental procedures were approved by the Animal Use and Care Advisory Committee of Experimental Center of China Medical University in China, and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).
Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application disputations.

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