All experimental protocols were approved by our Institutional Committee on Animal Research, and were carried out in accordance with the National Institutes of Health guidelines for animal use and care (National Institutes of Health publication no. 96- 23, revised 1996). Experiments were performed on 36 adult male New Zealand White rabbits (provided by Experimental Animal Center of the Xi'an Jiaotong University) weighing 2.5 to 3.0 kg. The animals were initially anaesthetised with pentobarbital sodium (30 mg/kg IV, sigma, USA, NO: 20030709), followed by a half-dose as required during surgical procedure. No animals received hemodynamic or ventilatory support. The left ear vein was cannulated with a 24-gauge catheter for intravenous drug administration. The right femoral artery was catheterized for blood pressure and heart rate monitoring (Spacelab, USA, model 90206A). Arterial blood was sampled for determination of blood gases (AVL-2, Switzerland) and blood glucose (One Touch II, USA). The rectal body temperature was maintained close to 38°C with the aid of a heating pad during the study.
Experimental groups and Animal models
Rabbits were randomly assigned to 3 groups (n = 12 each). In the TMP group, TMP (30 mg/kg) (Changzhou Pharmacological Co., China, NO: 99091401) was injected via ear vein 30 min before aortic clamping and at the onset of reperfusion. Control animals underwent standard aortic occlusion and intravenous injection of 0.9% sodium chloride under conditions identical to the TMP injection. Sham operated animals subjected to operative dissections without aortic occlusion.
Each group of animals was divided into four experimental subgroups: group A for Biochemical analysis (n = 3), group B for hematoxylin and eosin staining (H&E), Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick End-Labeling (TUNEL) staining and immunohistochemistry (n = 3), group C for electron microscopy (n = 2), group D for Western blot assay (n = 4). The rabbit model of spinal cord I/R injury was established according to Savas'discription [14]. Briefly, after sterile preparation, a 10-cm midline incision was performed. Following anticoagulation with 400 unit's heparin, the abdominal aorta was cross-clamped at the level just inferior to the origin of the left renal artery and at the level of aortic bifurcation for 30 min. Reperfusion was initiated by removal of the occlusion and lasted 48 h. The abdomen was then closed.
Neurologic evaluation
Neurological function was observed at the 24th and 48th hour after reperfusion according to Johnson's score[15].
0: Hind-limb paralysis;
1: Severe paraparesis;
2: Functionalmovement, no hop;
3: Ataxia, disconjugate hop;
4: Minimal ataxia;
5: Normal function.
Two individuals without knowledge of the treatment graded neurological function independently.
Histological study
The animals were euthanized by intravenous administration of a high concentration of pentobarbital at the 48th hour and the spinal cords were quickly removed. The spinal cords were immersed in 4% paraformaldehyde in 0.1 mol/l phosphate buffer and stored at 4°C for 2 weeks. The specimens for microscopy were prepared by obtaining spinal cord cross sections from the L2 or L3 vertebra. The specimens were then embedded in paraffin, cut into sections of 5μm thickness, stained with hematoxylin-eosin (H&E). The specimens were examined under the light microscope by a neuropathologist who was blinded to the study.
Preparation for electron microscopic examination of excised cords
The specimens were fixed in 2.5% glutaraldehyde for 6 h, washed in phosphate buffer (pH 7.4), postfixed in 1% osmium tetroxide in phosphate buffer (pH 7.4), and dehydrated in increasing concentrations of alcohol. Then the tissues were immersed in propylene oxide and embedded in epoxy resin embedding media. Ultrathin sections (thickness 60 nm) were cut and stained with uranyl acetate and lead citrate, and examined with a ZEISS-EM902 transmission electron microscope (Carl Zeiss, Thornwood, NY).
Biochemical analysis
Spinal cord tissues were washed two times with cold saline solution and stored in a deep freeze kept at -30°C until analysis. Tissue MDA levels were determined by the method described by Wasowicz[16]. Briefly, MDA was reacted with thiobarbituric acid by incubating for 1 h at 95–100°C. Following the reaction, fluorescence intensity was measured in the n-butanol phase with a fluorescence spectrophotometry(Hitachi, Model F-4010, Japan), by comparing with a standard solution of 1,1,3,3 tetramethoxypropane. Results were expressed in terms of nmol/g wet tissue. Total (Cu-Zn and Mn) SOD activity was measured by reduction of nitrobluetetrazolium (NBT) by xanthine-xanthine oxidase system. Enzyme activity leading to 50% inhibition was accepted as one unit. Results were expressed as U/mg protein [17]. Protein concentrations were determined according to Lowry's method [18].
TUNEL staining and immunohistochemistry
TUNEL staining was performed on paraffin sections using an in situ cell death detection kit (Rochev, Germany) according to the manufacturer's instructions. Sections were counterstained with hematoxylin. A negative control was similarly performed except for omitting TUNEL reaction mixture. Only cells showing nuclear condensation/fragmentation and apoptotic bodies in the absence of cytoplasmic TUNEL reactivity were considered apoptotic. For immunohistochemistry, sections, blocked using 2% normal goat serum in PBS, were incubated for overnight at 4°C with mouse monoclonal antibody against Bcl-2/Bax at a dilution of 1:50 (Maxim Biotech Inc, China) followed by followed by a biotinylated sheep anti-mouse antibody and avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA.) for 2 h. The slices were colorized with DAB/H2O2 solution, and then cell nucleuses were counterstained with hematoxylin. Each procedure was followed by several rinses in PBS. Blank staining was carried out in the same way as the above, except for eliminating the primary antibodies. Brown color of nuclei was taken as the positive staining of apoptotic neuronal cells and Brown color of cytoplasm was taken as the positive staining of Bcl-2/Bax. For quantitative analysis, 10 microscopic fields were taken, and all neurons, including neurons with TUNEL staining were counted. The mean values of the percentage of neurons with TUNEL positive staining were taken for further processing.
Western blot assay of Bcl-2 and Bax proteins
Spinal cord tissue was placed in lysis buffer containing inhibitors(leupeptin, pepstatin A, and aprotinin), homogenized, and then centrifuged(12,000 × g). After determining concentration of protein in each sample using a protein assay (Bio-Rad, Hercules, CA, USA), Samples were loaded (50 mg of protein/lane), electrophoresed on a 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and blotted to a nylon filter. Blots were probed with mouse monoclonal antibody against Bcl-2/Bax at a dilution of 1:200 (Maxim Biotech Inc, China) and visualized with horseradish peroxidase-conjugated secondary antibodies by enhanced chemiluminescence detection reagents (Amersham). Bcl-2 and Bax proteins were detected as 26 and 21 kDa bands, respectively, using molecular weight marker bands. The filter was scanned by FluorImager 595 (Amersham) and quantified with NIH Image J.
Statistical analysis
Statistical analysis was performed using SPSS 10.0. An unpaired t-test was used for comparisons in physiological parameters, MDA levels, SOD activity, TUNEL-positive rate and Bcl-2/Bax expression between the groups. Neurological scores were analyzed with nonparametric method (Kruskal-Wallis test) followed by the Mann-Whitney U test with Bonferroni correction. Data were expressed as mean ± S.D. and statistical significance was set at P <0.05.
