The Two Way Anova Test Biology Essay


Thirty-six male Wistar Rats, weighing 180-200 g, from the National animal center, Salaya campus, Mahidol University, Bangkok, Thailand were used for this study. All experiments were conducted in accordance with approved protocol from the Faculty of Medicine, Chiang Mai University Institutional Animal Care and Use Committee, in compliance with NIH guidelines. All animals were housed in environmentally controlled conditions (25 ± 0.5 °C, 12 hour light/dark cycle) and allowed to acclimate for one week. Rats were then divided into 2 groups (n = 18/group). Each group was fed either the normal diet (ND) or the high-fat diet (HFD) for 12 weeks. Animals in the ND group were fed with a standard laboratory pelleted diet (Mouse Feed Food No. 082, C.P. Company, Bangkok, Thailand), containing 19.77% total energy from fat, whereas animals in the HFD group were fed a diet containing 59.28% total energy from fat, as described in our previous study (Pratchayasakul et al., 2011). All animals were given free access to drink water and their respective diets. At the end of the 12th week, rats in each group were further divided into 3 subgroups (n=6/subgroup) to receive vehicle (normal saline solution; 2 ml/kg/day), vildagliptin (Gulvus, Novartis, Bangkok, Thailand; 3 mg/kg/day), or sitagliptin (Januvia, MSD, Bangkok, Thailand; 30 mg/kg/day) (Chen et al., 2011a) via gavage feeding for 21 days. It has been shown that either 3 mg/kg/day of vildagliptin or 30 mg/kg/day of sitagliptin reduces peripheral insulin resistance in insulin resistant and diabetic rat models (Burkey et al., 2005).

Experimental Protocols

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The open field activity level test was performed at the end of week 12 (before pharmacological treatment) to measure the locomotive function of each animal. All animals with positive open field activities were tested for cognitive behaviors. Cognitive behaviors were determined via the Morris Water Maze test at the end of week 12 and week 15 (after pharmacological treatment). The body weight of all animals was recorded weekly. Blood samples were collected from a tail vein at week 0 (before dietary feeding), week 12, and week 15 for further plasma analysis. At the end of treatment, animals were deeply anesthetized with isoflurane and sacrificed via decapitation. Each brain was rapidly removed and used to determine brain mitochondrial function and oxidative stress levels.

Plasma analysis

Plasma glucose and cholesterol levels were determined via the colorimetric assay (Biotech, Bangkok, Thailand). Plasma HDL and LDL/VLDL levels were determined via a commercial colorimetric assay kit (Biovision, California, USA). Plasma insulin levels were determined via the Sandwich ELISA kit (LINCO Research, MO, USA). Peripheral insulin resistance was assessed via the Homeostasis Model Assessment (HOMA) as described in previous studies (Appleton et al., 2005; Haffner et al., 1997). Plasma malondialdehyde levels (MDA) were determined using the high performance liquid chromatography (HPLC)-based assay (Grotto et al., 2007).

Open- field test

The open-field test was used to measure locomotive activity and was modified from the methods of Arakawa (Arakawa, 2005). The open field consisted of a black box with a floor (75 cm -75 cm) and walls with a height of 40 cm. The box floor was painted with white lines (6 mm wide) to form 25 equal squares. During a 2-min observation period, the animal was placed in the middle-square of the apparatus. The total number of lines crossed was recorded and used to determine locomotive activity.

Morris Water Maze test

The Morris Water Maze (MWM) test was modified from the methods of Vorhees and Williams (Vorhees and Williams, 2006) and used for the purpose of cognitive, learning, and memory behaviors assessment. The test was performed in a 170-cm diameter water pool virtually divided into 4 quadrants. The pool was filled with water (26±1-C) and made opaque with flour. A clear platform of 10 cm in diameter was submerged in a designated quadrant approximately 1 cm beneath the surface of the water. The MWM test was performed for each rat at the end of week 12 and week 15. Each test included 2 different assessments; the acquisition test (platform present) and the probe test (no platform present). The acquisition test was performed for 5 consecutive days of training with 4 trials per day. Animals were given 120 sec to locate the hidden platform. Any animals that could not find the platform within the 120 sec period were guided to the platform. After the platform was found, the animal was allowed to remain on the platform for 15 sec before the next test began by placing the animal at a starting point within the other 3 remaining quadrants. The acquisition time began at the moment the animal entered the water and ended at the moment the animal reached the submerged platform. In the probe test, animals were tested on the 6th day of training with only one starting point. The probe time was the amount of time the animals spent in the target quadrant during the 90 sec testing period.

Preparation of brain mitochondria

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Brain mitochondria were isolated as described in our previous study (Pipatpiboon et al., 2012). Shortly after decapitation, the brain was removed and placed in 5 ml ice-cold MSE solution, transferred to 10 ml ice-cold MSE-nagarse solution (0.05% nagarse in MSE solution), and homogenized at 600 rpm using a homogenizer. Next, the brain homogenate was centrifuged at 2,000 g for 4 min. The supernatant was collected and further centrifuged at 12,000 g for 11 min. Mitochondrial pellets were collected and resuspended in 10-ml ice-cold MSE-digitonin solution (0.02% digitonin in MSE solution). Finally, the mitochondrial pellets were resuspended in respiration buffer (150 mM KCl, 5 mM HEPES, 5 mM K2HPO4.3H2O, 2 mM L-glutamate, 5 mM pyruvate sodium salt). Mitochondrial protein concentration was measured via the BCA assay (Thummasorn et al., 2011).

Brain mitochondrial reactive oxygen species (ROS) assay

Brain mitochondrial reactive oxygen species (ROS) levels were measured via the use of dichloro-hydrofluoresceindiacetate (DCFHDA). Brain mitochondria (0.4 mg/ml) were incubated with 2-μM DCFHDA at 25°C for 20 minutes. The fluorescent intensity was measured via the use of a fluorescent microplate reader at the excitation wavelength of 485 nm and emission wavelength of 530 nm (Pipatpiboon et al., 2012; Thummasorn et al., 2011).

Brain mitochondrial membrane potential (m) assay

The change in mitochondrial membrane potential (m) within isolated brain mitochondria was measured via the fluorescent dye 5, 5ï‚¢, 6, 6ï‚¢-tetrachloro-1, 1ï‚¢, 3, 3ï‚¢-tetraethyl benzimidazolcarbocyanine iodide (JC-1). JC-1 monomer form (green) fluorescence was excited at a wavelength of 485 nm and detected at the emission wavelength of 590 nm. JC-1 aggregate form (red) fluorescence was excited at a wavelength of 485 nm and detected at the emission wavelength of 530 nm. Brain mitochondria (0.4 mg/ml) were incubated with JC-1 dye at 37 °C for 15 minutes. Mitochondrial membrane potential was determined via fluorescent intensity measured via a fluorescent microplate reader. The change in mitochondrial membrane potential was calculated as the ratio of red to green fluorescent intensity (Pipatpiboon et al., 2012; Thummasorn et al., 2011).

Brain mitochondrial swelling assay

Brain mitochondrial swelling was determined via the change in the absorbance of the brain mitochondrial suspension. Brain mitochondria (0.4 mg/ml) were incubated in 2 ml respiration buffer. The suspension was assessed for absorbance at a wavelength of 540 nm via the use of a microplate reader. Mitochondrial swelling was indicated by a decrease in absorbance (Pipatpiboon et al., 2012; Thummasorn et al., 2011).

Determination of plasma and brain malondialdehyde (MDA) levels

The HPLC method was used to evaluate the concentration of plasma and brain melondialdehyde (MDA) which acts as an indicator of oxidative stress (Candan and Tuzmen, 2008). To briefly summarize, the brain tissue was homogenized in a phosphate buffer (pH 2.8). The plasma or brain homogenate was mixed with 10% trichloroacetic acid (TCA) containing butylated hydroxytoluene (BHT), incubated at 90 oC for 30 min, and centrifuged at 6,000 rpm for 10 min. The supernatant was mixed with H3PO4 and thiobabituric acid solution (TBA) and incubated at 90oC for 30 min to produce thiobarbituric acid reactive substances (TBARS). TBARS levels were measured via the absorbance which was assessed at a wavelength of 532 nm by the HPLC system. Absorbance was determined directly from the standard curve and reported as the equivalent concentration of MDA.

Statistical analysis

Data were expressed as mean ± SE. Comparison between the two groups prior to the treatment was performed using an independent t-test. Comparison among groups after pharmacological treatment was performed using the two-way ANOVA test followed by the Fisher's test as the post hoc analysis. P < 0.05 was considered statistically significant.