Perioperative management
All experimental protocols of this study were approved by the Institutional Ethics Committee of Linyi People’s Hospital. All experimental animals were provided with adequate food and water and housed individually in metal cages at a temperature of 25–26 °C. The handling and use of laboratory animals conformed to the “Guidelines for animal experiments at Linyi People’s Hospital” and “Medical Laboratory Animal Management Regulations” and other relevant laws and regulations.
Study design and management
Thirty-three New Zealand white rabbits, both male and female, weighing 3.0–3.5 kg were randomly selected for this experiment. The rabbits underwent different levels of serial lumbar artery ligation in a craniocaudal direction between the renal artery and the aortic bifurcation. The animals were divided into six experimental groups as follows: sham group, no ligation, n = 8; group 1, ligation of bilateral lumbar arteries at 1 level, n = 5; group 2, ligation of bilateral lumbar arteries at 2 level, n = 5; group 3, ligation of bilateral lumbar arteries at 3 level, n = 5; group 4, ligation of bilateral lumbar arteries at 4 level, n = 5; and group 5, ligation of bilateral lumbar arteries at 5 level, n = 5.
Anaesthesia management
Intravenous access was established in the marginal ear vein. Anaesthesia (3% sodium pentobarbital) was infused through the marginal ear vein at 1 ml/kg and was maintained using 1/3 to 1/2 of the initial dose according to the response of animals during the experiment. Rabbits were intubated and connected to a respirator to control their breathing and provided with nitrous oxide and oxygen at a 2:1 ratio. Intravenous lactate ringer solution was infused according to the amount of bleeding. Body temperature was monitored continuously with a rectal thermometer during the experiment and was maintained between 38 °C and 39 °C with an electric blanket.
Surgical technique
The experimental animals were put in the supine position. After sterile surgical preparation, additional local anaesthesia (0.5% lidocaine hydrochloride) was applied to the abdominal wall. A midline abdominal incision was made, and the bowels were taken out by turning them over to the left and covering them with wet, warm, and sterile gauze to reduce fluid and heat loss. The retroperitoneal cavity was opened and probed, the abdominal aorta between the renal artery and the aortic bifurcation was exposed, and the 5 lumbar arteries between the renal artery and the aortic bifurcation were exposed. The superior and inferior mesenteric arteries were untouched during the surgery.
The experimental groups and the control group
In the experimental groups, a baseline Tes-MEP recording was recorded while anaesthesia conditions were stable. The lumbar arteries between the renal artery and the aortic bifurcation were ligated in a craniocaudal direction. Tes-MEPs were recorded 30 min and 2 days after ligation. The animals’ neurologic functions were assessed after awakening and 2 days after ligation. Spinal cords were quickly harvested 2 days after ligation for histopathologic observation. In the control group, stimulation with different intensities was used to induce Tes-MEPs to determine the most appropriate stimulation intensity. MEPs were recorded before and after the operation and every 30 min for 3 h.
Monitoring technique for transcranial electrical stimulation motor-evoked potentials
The rabbits were placed in the prone position, and the skull was placed in a stereotaxic instrument. The scalp was infiltrated with 1% lidocaine. A 3-cm-long incision was cut in the scalp, exposing the skull. The sagittal and coronal sutures of the calvarium were exposed after the periosteum was removed. Stimulating electrodes were fixed on the skull, with the cathode placed in the C4 position and the anode in the C2 position [4]. The stimulating electrodes were connected to an EpochXP-2000 electrical stimulator (purchased from Axon system of America). Recording electrodes were made using silver acupuncture needles, placed in the subcutaneous area near the gastrocnemius muscle in the hind leg. Stimulation parameters included a stimulation train (three pulses, 120–130 V, 100 ms duration, and 2 ms interstimulus interval) that was used to elicit Tes-MEPs. Tes-MEPs were also recorded from the upper extremities to control for the procedure. Recording parameters were as follows: time base 100 ms, bandpass filter 30–3000 Hz, and amplified 5000 times. Tes-MEPs were recorded before the ligation, after 30 min, and 2 days after ligation. The baseline value was determined just prior to the start of lumbar artery ligation. The amplitude of Tes-MEPs was defined as the voltage from the most positive to the most negative component. After the baseline value of Tes-MEPs was recorded, prepared lumbar arteries were ligated in a craniocaudal direction at different levels.
Evaluation of neurologic outcome
The motor function of the hind limbs of all rabbits was assessed after surgery and at 2 days after ligation. Tarlov’s score includes the following: 1, spastic paraplegia, cannot move; 2, paraparesis, slight movements; 3, paraparesis, powerful movements in the hind limbs but unable to stand; 4, able to stand but unable to walk; and 5, full recovery, normal walking function. Neurological examination was carried out at the same time by two investigators who were blinded to the groupings, who independently assessed the animals’ neurologic functions.
Evaluation of pathologic outcome
All rabbits were killed with deep intravenously administered sodium pentobarbital anaesthesia (100 mg/kg) 2 days after surgery. The spinal cord between L2 and L4 was taken out and soaked in 10% paraformaldehyde/0.1 mol/L phosphate-buffered saline solution at 4 °C for 48 h. Sections were cut before being embedded in paraffin. The experimental slices were stained with haematoxylin-eosin and examined using light microscopy by neuropathologists blinded to the experimental groups. Destruction of spinal cord motor neurons in the anterior horn was quantified.
Statistical analysis
Data are expressed as the means ± standard deviation. Multigroup variables were compared using one-way variance analysis. The paired t test was used for the analysis of Tes-MEP latency and amplitude before and after ligation in each experimental group. The paired t test was used for the analysis of motor function before and after ligation in each group. Analyses were implemented with SPSS 17.0 software. Difference with P values less than 0.05 was considered statistically significant.