Male New Zealand white rabbits (n=48, weight, 1.2–1.5 kg) were obtained from the Experimental Animal Center of Cheonan Yonam College (Cheonan, South Korea). They were housed under standard conditions at 22°C and 60% humidity in a 12-h light/dark cycle and with free access to food and water. The handling and care of the animals was in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, 1985, revised 1996) (22). Ethical approval was obtained from the Institutional Animal Care and Use Committee of Seoul National University (Seoul, South Korea; approval no. SNU-141021-2).

Preparation of the Tat expression vector has been described in a previous study (23). Human SUMO-1 was amplified by polymerase chain reaction (PCR) using the following primer sequences: Forward, 5′-CTCGAGATGTCTGACCAGGAGGCA-3′ (containing an XhoI restriction site) and reverse, 5′-GGATCCCTAAACTGTTGAATGACCCC-3′ (containing a BamHI restriction site). The resulting PCR products were ligated into the TA vector and cut with XhoI and BamHI. Fragments were subsequently ligated into the Tat expression vector to generate Tat-SUMO-1. Control SUMO-1 was manufactured without the Tat peptide. Recombinant Tat-SUMO-1 plasmid was transformed into E. coli BL21 (DE3) and cultured in 0.5 mM isopropyl-β-D-thio-galactoside (Duchefa Biochemie, Haarlem, Netherlands) at 18°C for >24 h. Harvested cells were lysed by sonication and the Tat-SUMO-1 protein was purified using Ni²⁺-nitrilotriacetic acid sepharose affinity column and PD-10 column (Qiagen, Inc., Valencia, CA, USA) chromatography to generate a Tat-SUMO-1 protein. Various concentration (0–1,000 mg) of bovine serum albumin (Thermo Fisher Scientific, Waltham, MA, USA) was used as a standard and protein concentration was measured by Bradford assay (24).

To evaluate the effects of Tat-SUMO-1 against ischemic damage, rabbits were divided into the following groups: Sham-operated, vehicle (glycerol)-treated ischemia-operated and Tat-SUMO-1-treated ischemia-operated groups, at 24 h and 72 h following sham operation or ischemia/reperfusion. Tat-SUMO-1 (1 mg/kg) or an equal volume of glycerol was administered to rabbits immediately following reperfusion by intraperitoneal injection. Arterial blood gases [partial pressure (Pa)O₂ and PaCO₂], pH and glucose levels were measured using a GEM Premier 3,000 gas analyzer (Instrumentation Laboratory, Milan, Italy) 10 min after occlusion of the abdominal aorta and 10 min after reperfusion. Additionally, mean arterial pressure (MAP) was recorded from the caudal artery by physiography (Gould Instrument Systems, Cleveland, OH, USA) and Ponemah acquisition software version 3.0 (Gould Instrument Systems).

Rabbits were anesthetized with 2.5% isoflurane (Baxter, Deerfield, IL, USA) in 67% N₂O and 33% O₂. To spare mesenteric flow to the gut, the abdomen was incised at the ventral midline and the abdominal aorta was isolated beneath the left renal artery, following which it was occluded using an aneurysm clip. Spinal cord ischemia was subsequently induced as described by Kiyoshima et al (25). The aneurysm clip was removed after 15 min of occlusion, and reperfusion was observed from the abdominal aorta. This produced delayed paraplegia within several h of ischemia (26). Free-regulating or normothermic (38.7±0.3°C) conditions and a thermometric blanket were used to maintain body temperature, which was monitored with a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA, USA) throughout the surgical procedure. Once the anesthesia had worn off, rabbits were placed in a thermal incubator (Mirae Medical Industry, Seoul, South Korea) prior to sacrifice. Sham-operated animals underwent the same procedure with the exception of occlusion of the abdominal aorta.

To assess neurological function, modified Tarlov criteria were used (27) as follows: 0) no voluntary hind-limb function; i) only perceptible joint movement; ii) active movement but unable to stand; iii) able to stand but unable to walk; or iv) complete normal hind-limb motor function (28,29). Assessments were conducted by two blinded observers for each experiment. Evaluation of control, vehicle-treated, and 1 mg/kg Tat-SUMO-1-treated groups (n=8/group) were conducted under identical conditions. Neurological function was examined 24, 48, and 72 h after reperfusion as it begins to deteriorate 12–24 h after reperfusion, progressing to complete delayed-onset paraplegia by 48 h (30,31).

For histological analysis, animals (n=4/group) were anesthetized with 2 g/kg urethane (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 24 and 72 h after ischemia/reperfusion and perfused transcardially with 0.1 M phosphate-buffered saline (pH 7.4), followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (pH 7.4). The spinal cords were removed and the 5–6th lumbar segments (L₅-L₆) of the spinal cord were fixed in the same fixative for 4 h. The spinal cord tissues were cryoprotected by incubation with 30% sucrose overnight, and 30-µm-thick spinal cord sections were serially cut in the coronal plane using a cryostat (Leica Microsystems GmbH, Wetzlar, Germany). The sections were stained with cresyl violet acetate as previously described (19).

Counting of cresyl violet-positive neurons at the center of the ventral horn was performed using an analysis system equipped with a computer-based CCD camera (OPTIMAS software version 6.5; CyberMetrics^® Corporation, Phoenix, AZ, USA; magnification, ×100). The image was converted to a gray-scale image and cresyl violet-positive neurons were automatically selected according to the intensity of cresyl violet staining. Cell counts were averaged from 25 sections at 300-µm intervals from each rabbit. Values are reported as a percentage of that obtained from the control groups.

The effects of Tat-SUMO-1 on lipid peroxidation in the control, vehicle-, and Tat-SUMO-treated groups (n=4/group) were assessed by measuring MDA formation using a 2-thiobarbituric acid reactive substances assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) 24 h after ischemia/reperfusion. The MDA concentration was determined from a standard curve and expressed in nmol/g of wet tissue. All experiments were conducted in triplicate.

The enzyme activity of SOD1 was measured using superoxide dismutase assay kits (Cayman Chemical Company) to assess the effects of Tat-SUMO-1 in the control, and vehicle- and Tat-SUMO-1-treated groups (n=4/group). Readings were obtained at 550 nm using a Beckman DU-640B spectrophotometer (Beckman Coulter, Inc., Brea, CA, USA). Final SOD1 activity levels were expressed as units of enzymatic activity/mg protein sample (U/mg protein). One unit of SOD was defined as the amount of enzyme necessary to achieve 50% dismutation of the superoxide radical. All assays experiments were conducted in triplicate.

Multiple comparisons were analyzed by one-way or two-way analysis of variance followed by Bonferroni's post hoc test using GraphPad Prism software verson 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as the mean ± standard error. P<0.05 was considered to indicate a statistically significant difference.

In the vehicle-treated and Tat-SUMO-1 treated groups, blood gases (PaCO₂ and PaO₂), pH and glucose levels were not significantly changed 10 min before induction of ischemia or after reperfusion (Table I). The Tat-SUMO-1 and vehicle-treated groups demonstrated similar patterns of MAP; levels significantly decreased 10 min after occlusion (during ischemia) and were restored to baseline levels 10 min after reperfusion (Table I).

In the control group, all animals exhibited completely normal function 72 h after the sham operation, and had a neurological score of 4.00. In the vehicle-treated group, numerous animals had no voluntary or barely perceptible joint movement 24, 48 and 72 h after ischemia/reperfusion, and had a mean neurological score of 0.625. In the Tat-SUMO-1-treated group, animals could stand; however, could not walk, and had an average neurological score of 2.750 at 24 h after ischemia/reperfusion. These animals did not stand or move voluntarily, and had an average neurological score of 1.125 at 72 h after ischemia/reperfusion (Fig. 1).

In the control group, cresyl violet-positive neurons were abundantly detected in the ventral horn of the spinal cord at 24 h (Fig. 2A) and 72 h (Fig. 2B) after the sham operation; however, no significant differences were observed between the two groups. In the vehicle-treated group, cresyl violet-positive neuron counts were decreased in the ventral horn of the spinal cord at 24 h (Fig. 2C) and 72 h (Fig. 2D) after ischemia/reperfusion (28.5 and 12.4% of the control group, respectively, at 24 h). In the Tat-SOD1-treated groups, cresyl violet-positive neurons were abundantly detected in the ventral horn of the spinal cord 24 h after ischemia/reperfusion (Fig. 2E). However, they were markedly reduced in number 72 h after ischemia/reperfusion (Fig 2F). The proportion of cresyl violet-positive neurons 24 h and 72 h after ischemia/reperfusion was 67.5% and 17.2% of the control group, respectively, 24 h after the sham operation (Fig. 2G).

In the control group, MDA levels in L₅-L₆ spinal cord homogenates were 2.278 and 2.325 nmol/g protein at 24 and 72 h after the sham operation, respectively. In the vehicle-treated group, MDA levels were significantly increased compared with the control group to 5.134 nmol/g protein 24 h after ischemia/reperfusion (P<0.05), and were 2.994 nmol/g protein after 72 h. In the Tat-SUMO-1-treated group, MDA levels in L₅-L₆ spinal cord homogenates decreased significantly to 3.244 nmol/g 24 h after ischemia/reperfusion, compared with the vehicle-treated group (P<0.05). However, compared with the control and vehicle-treated groups, the MDA levels in L₅-L₆ spinal cord homogenates increased to 4.264 nmol/g after 72 h in the Tat-SUMO-1-treated group (P<0.05; Fig. 3A).

In the control groups, SOD1 activity in L₅-L₆ spinal cord homogenates was 154.7 and 158.2 U/mg at 24 and 72 h, respectively, after the sham operation, whereas in the vehicle-treated group, SOD1 activity was significantly decreased compare with the control to 53.4 and 41.2 U/mg after 24 and 72 h, respectively (P<0.05). In the Tat-SUMO-1-treated group, SOD1 activity was significantly increased to 127.3 U/mg after 24 h, compared with the vehicle-treated group (P<0.05). However, SOD1 activity in the Tat-SUMO-1-treated group was significantly decreased to 59.3 U/mg at 72 h, compared with Tat-SUMO-1-treated group at 24 h (Fig. 3B).