After obtaining approval from the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China), 108 healthy male Sprague-Dawley rats aged 24-month-old and weight 400-600 g, were randomly assigned to three groups, receiving 40% O2 for three hours only, 1.8% isoflurane in 40% O2 for three hours or left nephrectomy under 3-hour 1.8% isoflurane anesthesia, respectively. One week before treatment, all rats were housed in standard cages, in a temperature (23℃) and humidity (45-55%) controlled environment with a 12/12-hour modified dark-light cycle (light on 11:00 PM-11:00 AM). Food and water were available ad libitum.
Left nephrectomy was conducted under general anesthesia with 1.8% isoflurane under aseptic conditions by a professional urologist. Simply, after the surgical site was shaved and sterilized, a local anesthetic bupivacaine (0.375% and 2 ml) was injected into the skin and the subcutaneous tissue of abdominal area, and then a 1-cm incision was made in the upper left quadrant through the skin and muscles. The kidney was mobilized, isolated, and removed, and then the muscle and skin were closed. Finally, EMLA cream (2.5% lidocaine and 2.5% prilocaine) was used every eight hours for the first and second postoperative days.
During surgery and/or anesthesia, the continuous electrocardiogram monitoring, pulse oximetry and noninvasive blood pressure were measured by using a 150B3 monitor (Philips, Suzhou, China). The rectal temperature was measured and maintained at 37-39℃ by using a heating board as needed. After surgery and/or anesthesia, rats were allowed to awaken in an oxygen-enriched environment, and then were transferred to their own cage after completely awakened with free access to food and water.
A well-trained observer who was blinded to group assignment performed behavioral tests of rats daily. 12 rats in each group were used for the behavioral tests.
Y Maze Test
After treatment, the learning and memory ability of rats was assessed daily by using a modified Y-maze apparatus in a dark and quiet room (15). The Y shaped apparatus (50 cm×6 cm×22 cm) consists of three black plastic branches with an illuminant at each end and a joint area. The "floor" of Y-maze is installed with copper rods through which an electric foot shock (30 ± 5 V for 10 seconds) can be applied. Firstly, rats were placed in the joint area of the Y maze, and then one of the arms was randomly illuminated, followed by electric shock applied in 5 seconds in the other two dark arms and the joint area. Normally, rats would run to the other two dark arms for they preferred to avoid an illuminated area. After receiving electric foot shocks, rats would run out, explore and finally enter the illuminated arm. Secondly, rats were manually returned to the joint area of the Y maze and the next episode began. The same episode was repeated for 20 times for each rat every day. If the rat run directly into the illuminated arm before electric shocks were applied, which was defined as active response, this suggested a good learning and memory ability. Learning and memory impairment was presented by a decrease of the number of active response.
Locomotor Activity Test
Locomotor activity of animals was assessed daily in an open field apparatus (160 cm×160 cm×20 cm) which was divided into 36 grids by black lines. Rats were individually placed in the center of the apparatus, and then the number of grid crossings (motor activity) and rearings (exploratory activity) was counted in a five-minute period (16).
In the present study, rats in the three groups were harvested on 6 hours, 1, 3, and 7 days post-treatment for biochemistry studies, and six rats in each group were used for the biochemistry studies at every time point. Blood was collected from retinal vein under 4% isoflurane anesthesia, put in pre-heparinized Eppendorf tubes, and then centrifuged at 5000 rpm for 20 minutes at 4℃, finally the serum was stored at -80℃ until assay. For hippocampal samples, animals were rapidly decapitated with their brain quickly removed. All dissections were performed on an ice-cold frosted glass plate. Hippocampus was disrupted in an immunoprecipitation buffer (10 mmol Tris-HCl, pH 7.4, 150 mmol NaCl, 2 mmol EDTA, 0.5% Nonidet P-40) plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A) (Roche, Indianapolis, IN), and then was homogenized. The homogenate was then centrifuged at 4℃ and 13,000 rpm for 15 minutes. The supernatant was removed and stored at -80℃ until assay. The protein content of each sample was determined by using the bicinchoninic acid protein assay kit (Pierce, Iselin, NJ) according to the manufacturer's protocol.
Determination of Interferon Gamma (IFN-γ) and Interleukin-6 (Il-6) Level in Hippocampus and Serum by Employing A MILLIPLEX MAP Rat Cytokine Panel
Luminex Corporation has created an open source hardware platform combining flow cytometry- and bead-based antibody capture that is able to detect multiple analytes from a single sample and have a greater detection capacity (17). In the present study, IFN-γ and IL-6 levels in hippocampus and serum were quantified on 6 hours, 1, 3, and 7 days post-treatment by using MILLIPLEXTM MAP Rat Cytokine Kits (cat # RCYTO-80K, Millipore, Billerica, MA, USA) in a MILLIPLEX MAP Chemokine Panel which employed Luminex xMAP® technology, according to manufactures' recommended procedures (18). The multiplex assay was sensitive to 4.88 pg/ml IFN-γ and 9.80 pg/ml IL-6, and the inter-assay and intra-assay coefficients of variation were 8%.
Quantification of IDO Level by Using A Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)
IDO levels in hippocampus and serum were determined with a Sandwich ELISA assay by using a Rat IDO ELISA Kit (Cat. No. E1547Ra, Uscn Life Science Inc., Wuhan, China) according to the instructions supplied by the manufacturers. Specifically, 96-well plates were coated with rat monoclonal antibodies specific to IDO. Following blocking with Block Ace, wells were incubated overnight at 4℃ with test samples, and then an anti-IDO antibody conjugated to horseradish peroxidase was added. Plates were then developed with TMB reagent and well absorbance was measured at 450 nm. IDO levels in test samples were determined by comparison with the signal from unconditioned media spiked with known quantities of IDO.
Determination of IDO Activity by Using High Performance Liquid Chromatography (HPLC)
Serum and hippocampal IDO activity were presented as the ratio of level of tryptophan and kynurenine, and kynurenine aminotransferase (KAT) activity was presented as the ratio of level of kynurenic acid and kynurenine. Serum and hippocampal levels of tryptophan, kynurenine and kynurenic acid were analyzed on 6 hours, 1, 3, and 7 days post-treatment by using an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA). HPLC was carried out on a Venusil MP C18 analytical column (5 µm, 250 mm×4.6 mm) operated at 25℃ and a fluorescent detector with a 225 nm wavelength according to classic methods (19). The linear ranges were from 0.3366 to 420.8 μmol/L for tryptophan, from 0.1302 to 1627 μmol/L for kynurenine and from 0.246 to 98.4 μmol/L for kynurenic acid.
An observer who was blinded to the experimental protocol analyzed all data. MLwiN2.25 statistical software and a two-level logistic regression model were used in the comparison of learning curves (based on the number of active responses) among the three groups (control group was defined as reference group). Active responses were divided into two groups, with 1 denoting active response while 0 denoting no active response. Other analyses were performed using the SPSS 13.0 statistical software (SPSS, Chicago, IL, USA). Data are expressed as means ± standard errors of the means (SEM), with (N) indicating the number of subjects. The data were tested for normal distribution or not by the Kolmogorov-Smirnov test. Logarithmic transformation was done if the data did not meet the normal distribution (data not shown). A repeated-measures analysis of variance (ANOVA) was applied to analyze the difference of vital signs, and locomotor activity (based on the number of grid crossings and rearings) among rats in the control group, rats treated with anesthesia and rats treated with nephrectomy under anesthesia, and Bonferroni tests were used for the post-hoc comparisons. Covariates were treatment and time. Finally, basal body weight, the levels of IFN-γ, IL-6 and IDO, and IDO and KAT activity among the three groups were compared using a one-way ANOVA, and Bonferroni tests was used for the post-hoc comparisons. P values less than 0.05 (* or #) and 0.01 (** or ##) were considered statistically significant.