The overall experimental protocol is shown in Figure 1. First, we continuously measured the physiological changes, including heart rate (HR), mean arterial pressure (MAP), intracranial pressure (ICP) and cerebral perfusion pressure (CPP) for 120 minutes after TBI. Then, we tested whether TBI effects were associated with nitrosative stress related neuronal apoptosis, cerebral infarction in the ipsilateral injury side of cortex using immunofluorescence and TTC staining. The motor function was examined by incline plane on the 3rd day after TBI. During the same time course, the effects of AMN082 on TBI association parameters were also evaluated. All methods were carried out in accordance with relevant guidelines and regulations.
Ten-week-old adult male Sprague-Dawley rats were used in the experiments. The animals were kept under a 12/12-h light/dark cycle and allowed free access to food and water. The Chi Mei Medical Center’s Animal Care and Use Committee approved all of the experimental procedures, which conformed to the NIH guidelines, including minimizing discomfort to the animals during surgery and during the recovery period. At the end of the experiments, rats were sacrificed with an overdose of urethane.
The study was carried out in compliance with the ARRIVE guidelines.
Traumatic brain injury
Animals were anesthetized with a mixture of ketamine (44mg/kg, i.m.; Nankuang Pharmaceutical, Taiwan), atropine (0.063 mg/kg, i.m.; Sintong Chemical Ind. Co., Taiwan), and xylazine (6.77 mg/kg, i.m.; Bayer, Germany). On a stereotaxic frame (Kopf 1406; Grass Instrument, Quincy, MA), craniectomy was performed with 2 mm in radius, 3 mm from the sagittal sutures and 4 mm from the bregma in the right parietal cortex. After implantation of the injury cannula, the fluid percussion device (VCU Biomedical Engineering, Richmond, VA, USA) was connected to the craniectomy site via Luer-lock fitting, and the brain was injured with 2.2atm, 25 ms percussion as originally described by Mclntosh et al.25 Selection criteria of successful TBI insult of rat were transient hypertensive response, apnea, and seizure observed immediately following fluid percussion injury.
Surgery and physiological parameters monitoring
The right femoral artery of rats was cannulated with polyethylene tubing (PE50) under for blood pressure monitoring. Mean arterial pressure (MAP) and heart rate (HR) were monitored continuously with a pressure transducer after TBI. An intracranial pressure (ICP) microsensor (Codman and Shurtlef, Inc., Rayman, MA, USA) was placed in the parenchyma of the left frontal lobe of each rat. ICP was monitored continuously, and ICP and cerebral perfusion pressure (CPP) values were recorded at 5-minute intervals in the period of 120 minutes after TBI. The CPP value is defined as MAP-ICP.26 Colon temperatures were measured with an analog electronic thermometer (model 43 TE; YSI, Inc, Yellow Springs, OH) and temperature probe (series 400; YSI, Inc).
Using the random number table, the rats were numbered in random order and assigned to three major groups: sham operated (n=6); TBI control treated with vehicle, dimethylsulfoxide (DMSO) (4%, 1ml/kg, injected intraperitoneally, K42088831, vehicle; Merck, Darmstadt, Germany); and TBI +AMN082-treated (10 mg/kg, dissolved with DMSO, injected intraperitoneally; U. S. Pharmacopeia) (n=6). The TBI without treatment group received equal volumes of vehicle via intraperitoneal injection. Vehicle or AMN082 was administered for three consecutive days after TBI. The first injections occurred immediately after TBI, the second injection was administered 24 h later, and the third injection was administered 48 h later. All tests were performed with operators and assessors blinded to the study rats, which were revealed only at the end for the analyses.
Motor function test
An inclined plane was used to measure limb strength for motor function test.27
This test can measure the motor function of rats after neural injury by evaluating the rat’s ability to prevent falls and the endurance strength of the upper and lower limbs in an inclined plane. The animals were initially placed on a 30˚ angle, 20 x 20cm buffer ribbed surface incline plane. To determine the maximal angle at which an animal could remain on the incline plane without drop, the angle was increased in 1˚ increments. Motor deficit were measured with the mean maximal angles of the left upper and lower limbs on the 3rd day after TBI.
Cerebral infarction assay
The triphenyltetrazolium chloride (TTC) staining procedures followed those described elsewhere.28 Briefly, the brain tissue was removed, immersed in cold saline for 5 min, and sliced into 1mm sections. The brain slices were incubated in 2% TTC dissolved in phosphate-buffered saline for 30 min at 37°C and then transferred to 10% formaldehyde solution for fixation. The volume of infarction, as revealed by negative TTC stains indicating dehydrogenase-deficient tissue, was measured in each slice and summed using computerized planimetry (PC-based Image Tools software). The volume of infarction was calculated as 1 mm (thickness of the slice) x (sum of the infarction area in all brain slices [mm2]).29
Immunofluorescence assay was measured on the 3rd day after TBI.30 Adjacent 6-μm sections corresponding to coronal coordinates 0.20 mm to 0.70 mm anterior to bregma were incubated in 2 mol/L HCl for 30 min, rinsed in 0.1 mol/L boric acid (pH 8.5) for 3 min at room temperature, and then incubated with primary antibodies in PBS containing 0.5% normal bovine serum at 4°C overnight. After being washed in PBS, the sections were incubated with secondary antibodies for 1 h at room temperature. The number of immunofluorescent positive cells was calculated using computerized planimetry corresponding to the peri-contusion cortical region (400 x magnification, Image-Pro Plus Media Cybernetics, Inc. Washington Street, Rockville, USA), and the number of positive cells was expressed per mm2 in interested region. The detailed antibodies used were summarized on Table 1.
According to the ANOVA with the level of type I error at 0.05 and the smallest effect size at 0.2 for power calculation, so the sample size available will be at least 6 rats in each groups. This proposed sample size is sufficient to detect a statistical significant of ANOVA at the 0.05 level with power of 80%. All of the data were analyzed using SigmaPlot, version 10.0 for Windows (Systat Software, San Jose, CA) in this study. The results are expressed as the means ± standard deviation of the means for the experiments. For each time points, the mean difference between these three groups was estimated using the analysis of variance (ANOVA).The analysis of variance (ANOVA) with post hoc Scheffe’s test was used to estimate the statistical difference between these three groups. P-values < 0.05 were considered statistically significant.