All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at our institute and performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, National Academy Press, Washington, DC, USA, revised 1996).
Animal Model of Acute Ischemic Stoke and Corner Test
The protocol and procedure of using a rodent model of acute IS has been described in details in our recent report . Adult male Sprague-Dawley rats, weighing 300-325 g (Charles River Technology, BioLASCO Taiwan Co., Ltd., Taiwan) were utilized in the current study. All animals were anesthetized by chloral hydrate (35 mg/kg i.p.) and placed in a supine position on a warming pad at 37°C. After exposure of the left common carotid artery (LCCA) through a transverse neck incision, a small incision was made on the LCCA through which a nylon filament (0.28 mm in diameter) was carefully advanced into the distal left internal carotid artery for occlusion of left middle cerebral artery (LMCA) to induce brain infarction of its blood-supplying area. The nylon filament was removed three hours after occlusion, followed by closure of the muscle and skin in layers. The rats were then placed in a portable animal intensive care unit (ThermoCare®) for 24 hours. The sensorimotor functional test (Corner test) was done for each rat at baseline and on day 1 (24 h after procedure), 3, 7, 14, and 21 after acute IS induction as we recently described [16, 23]. Briefly, the rat was allowed to walk through a tunnel and then into a corner, the angle of which was 60 degrees. To exit the corner, the rat could turn either to left or right. The results were recorded by a technician who was blind to the study design. This test was repeated 10 to 15 times with at least 30 seconds between each trial. We recorded the number of right and left turns from 10 successful trials for each animal and used the results for statistical analysis.
Ten sham-operated healthy rats served as normal controls (group 1). The other 40 rats with acute IS were equally divided into IS plus intra-peritoneal 1.0 mL physiological saline (at 0.5, 24 and 48 hour after IS) (group 2, n = 10), IS plus CsA (20.0 mg/kg at 0.5 and 24 hour, intra-peritoneal) (group 3, n = 10), IS plus EPO (5,000 IU/kg at 0.5, 24, and 48 hour, subcutaneous) (group 4, n = 10), and combined CsA (20.0 mg/kg at 0.5 and 24 hour, intra-peritoneal) and EPO (5,000 IU/kg at 0.5, 24 and 48 hour, subcutaneous) treatment (group 5, n = 10).
Two rats died in group 2 and one rat died in each other group (i.e. groups 3 to 5) during the procedure. For the purpose of this study, additional rats were added so that 10 animals in each group went through the whole study.
The dosage of EPO and the timing of treatment were based on previous literature and our recent report [16, 24], whereas the dosage of cyclosporine and the treatment protocol were according to a previous report .
Specimen Collection and Preparation for Individual Study
Rats in all groups were euthanized on day 21 after IS induction, and the brain of each rat was promptly removed and immersed in cold saline. For immunohistofluorescent (IHF) study, the brain tissue was rinsed with PBS, embedded in OCT compound (Tissue-Tek, Sakura, Netherlands) and snap-frozen in liquid nitrogen before being stored at -80°C. For immunohistochemical (IHC) staining, the brain tissue was fixed in 4% formaldehyde and embedded in paraffin. Additionally, the brain tissue of infarct area was collected for Western blot, real-time PCR, and oxidative stress analyses.
Measurement of Brain Infarct Area
To evaluate the impact of CsA, EPO, and combined EPO and CsA treatment on brain infarction, coronal sections of the brain were obtained from six extra animals in groups 2 to 5 (n = 6 for each group) as 2 mm slices. Each cross section of brain tissue was then stained with 2% 3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) (Alfa Aesar) for BIA analysis. The methodology has been described in details in our recent studies [16, 23]. Briefly, all brain sections were placed on a tray with a scaled vertical bar to which a digital camera was attached. The sections were photographed from directly above at a fixed height. The images obtained were then analyzed using Image Tool 3 (IT3) image analysis software (University of Texas, Health Science Center, San Antonio, UTHSCSA; Image Tool for Windows, Version 3.0, USA). BIA was identified as either whitish or pale yellowish regions. Infarct region was further confirmed by microscopic examination. The percentages of infarct area were then calculated by dividing the area with total cross-sectional area of the brain.
All measurements (i.e. Corner test and assessment of BIA) were performed by a skillful senior technician blinded to the treatment and non-treatment groups.
TUNEL Assay for Apoptotic Nuclei
For each rat, six sections of BIA were analyzed by an in situ Cell Death Detection Kit, AP (Roche) according to the manufacturer's guidelines. Three randomly chosen high-power fields (HPFs) (×400) were observed for terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL)-positive cells for each section. The mean number of apoptotic nuclei per HPF for each animal was obtained by dividing the total number of cells with 18.
Frozen sections (4 μm thick) were obtained from BIA of each animal. The sections were fixed with 4% paraformaldehyde and permeated with 0.5% Triton X-100 and then incubated with antibodies against NeuN (1:1000, Millipore), GFAP (1:500, DAKO), PGC-1α (1:100, Santa cruz), and AQP4 (1:200, abcam) at 4°C overnight. Alexa Fluor488, Alexa Fluor568, or Alexa Fluor594-conjugated goat anti-mouse or rabbit IgG were used to localize signals. Sections were then counterstained with DAPI and observed with a fluorescent microscope equipped with epifluorescence (Olympus IX-40).
Western Blot Analysis for Bax, Cytochrome C, Caspase 3, NADPH oxidase 1 (NOX-1), NOX-2, Inducible Nitric Oxide Synthase (iNOS), and Endothelial (e)NOS
Equal amounts (50 mg) of protein extracts were loaded and separated by SDS-PAGE using 12% acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific sites were blocked by incubation of the membrane in blocking buffer [5% nonfat dry milk in T-TBS (TBS containing 0.05% Tween 20)] for overnight. The membranes were incubated with the indicated primary antibodies (Bax, 1:1000, abcam; Cytochrome C, 1:2000, BD; Caspase, 1:3000, abcam; NOX-1, 1:1500, Sigma; NOX-2, 1:500, Sigma; iNOS, 1:200, abcam; eNOS, 1:1000, 1:500, abcam; Actin, 1:10000, Chemicon) for 1 hr at room temperature. Horseradish peroxidase -conjugated anti-rabbit or anti-mouse immunoglobulin IgG (1:2000, Cell Signaling) was used as a second antibody for 1 hr at room temperature. The washing procedure was repeated eight times within 1h, and immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) and exposure to Biomax L film (Kodak). For purposes of quantitation, ECL signals were digitized using Labwork software (UVP).
Protocol for RNA Extraction
Lysis/binding buffer (High Pure RNA Tissue Kit, Roche, Germany) 400 μL and an appropriate amount of frozen brain tissues were added to a nuclease-free 1.5 mL microcentrifuge tube, followed by disruption and homogenization of the tissue by using a rotor-stator homogenizer (Roche).
For each isolation, 90 mL DNase incubation buffer was pipetted into a sterile 1.5 mL reaction tube, 10 mL DNase I working solution was then added, mixed and incubated for 15 min at 25°C. Wash buffer I 500 mL was then added to the upper reservoir of the filter tube, which was then centrifuged for 15 seconds at 8,000g. Wash buffer II 300 mL was added to the upper reservoir of the filter tube, which was centrifuged for 2 min full-speed at approximately 13,000g. Elution Buffer 100 mL was then added to the upper reservoir of the filter tube. Finally, the tube assembly was centrifuged for 1 min at 8,000g, resulting in eluted RNA in the microcentrifuge tube.
Real-Time Quantitative PCR Analysis
Real-time polymerase chain reaction was conducted using LighCycler TaqMan Master (Roche, Germany) in a single capillary tube according to the manufacturer's guidelines for individual component concentrations. Forward and reverse primers were each designed based on individual exons of the target gene sequence to avoid amplifying genomic DNA.
During PCR, the probe was hybridized to its complementary single-strand DNA sequence within the PCR target. As amplification occurred, the probe was degraded due to the exonuclease activity of Taq DNA polymerase, thereby separating the quencher from reporter dye during extension. During the entire amplification cycle, light emission increased exponentially. A positive result was determined by identifying the threshold cycle value at which reporter dye emission appeared above background. For normalization, the housekeeping gene Peptidyl-prolyl cis-trans isomerasa (Ppia, Cyclophilin A) was used as the reference gene.
Oxidative Stress Reaction of BIA
The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). The oxyblot procedure was performed according to the previous study . The 2,4-dinitrophenylhydrazine (DNPH) derivatization was carried out on 6 μg of protein for 15 min according to manufacturer's instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1:150) for 2 h, followed by incubation with second antibody solution (1:300) for 1 h at room temperature. The washing procedure was repeated eight times within 40 min. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). On each gel, a standard control sample was loaded.
Data were expressed as mean values (mean ± SD). Statistical analysis was adequately performed by analysis of variance, followed by Scheffe multiple-comparison post hoc test. SAS statistical software for Windows version 8.2 was utilized. (SAS institute, Cary, NC). A probability value < 0.05 was considered statistically significant.