2.1 Experimental design
Male adult Wistar rats (approximately 12 weeks old, obtained from the Animal Facilities at the State University of Campinas, UNICAMP, Brazil) were housed in collective cages under controlled environmental conditions (light and dark 12/12 h; temperature 22±2°C; and humidity 50-60%). Semi-purified diets were prepared following the recommendations of the American Institute of Nutrition (AIN-93 ( 16)) and according to our previous studies (17-19). The control diet contained 18% protein and was composed of 20% casein (protein source), 39.7% corn starch, 13.2% dextrin, and 10% sugar (carbohydrate sources), 7% soy oil (fat source), 5% cellulose micro fiber (fiber source), 3.5% salt mix, 1.0% vitamin mix, 0.3% cysteine, and 0.25% choline. The leucine-rich diet also contained 18% protein and was composed of the same amounts of casein, fat, fiber, salt, vitamin mix, cysteine, and choline as the control diet. The addition of 3% leucine was followed by a 1% reduction in corn starch (38.7%), dextrin (12.2%), and sugar (9%). Whiting those adjustments, both diets, control and leucine, were normoproteic, isocaloric, and normolipidic. The widely used model of cancer cachexia, Walker 256 carcinosarcoma (20) was used in this study. Cell suspension (2.5 × 106 viable cells) of Walker 256 cells were injected subcutaneously into the right flank of the rats. The tumor inoculation and diet administration started on the same day (Figure 1).
The animals were randomly distributed into four experimental groups. Two groups were fed with a control diet: Control (C) and Walker 256 tumor bearing (W), and two other groups were fed with a leucine-rich diet : Leucine Control (L) and Leucine Walker 256 tumor-bearing (LW). The minimal number of animal per group was 6. The animals were monitored daily, weighed 3 times/week and given food and water ad libitum. Food intake was measured once per week, and the functional activities were accessed one week before (to assess the health condition) and after approximately 18 days after tumor and diet administration (pre-agonic moment) (Figure 1).
The final endpoint criteria (pre-agonic moment) utilized in the present study were determined based on the data derived from daily observation of discomfort symptoms such as piloerection, diarrhea or constipation, hunched posture, tremors, closed eyes and red tears (chromodacryorrhea). These symptoms were based on the indicators of quality of life proposed by Betancourt et al. [15]. At the endpoint moment (~ 18 days following tumor inoculation), rats were killed by decapitation, and different body tissues, such as spleen, perirenal fat and skeletal muscle (musculus tibialis anterior) were removed and weighed (Figure 1). The tibia length of each animal was used to normalize their corresponding tissues weights. Muscle samples were frozen directly in liquid nitrogen and stored at −80°C for further gene and protein expression analysis. Also, muscle fragments were also immediately fixed in 2.5 % glutaraldehyde and 2.5% paraformaldehyde in sodium cacodylate buffer (0.1M) at pH 7.4 and CaCl2 (3mM) for 24 h at 4ºC before being processed for electron microscopy analysis. Additional muscle samples were fixed in 4% paraformaldehyde for light microscopic assay. The general guidelines of the United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR), 1998 [10] regarding animal welfare were followed, and the experimental protocol was approved by the Institutional Committee for Ethics in Animal Research (CEEA/IB/UNICAMP, protocol # 4289-1).
2.2 Muscle Functional Analyses
Catwalk walking test
Catwalk walking test (Noldus Inc., Wageningen, Netherlands) consists of an automated tool that quantifies gait parameters. Rats were placed in an illuminated-walkway glass-floor with a video camera (Gevicam GP-3360; Gevicam Inc., CA, USA). The camera positioned under the walkway at a distance of 56 cm recorded the paws prints automatically by using the CatWalkTM XT10.6 software, as the animal crossed the pathway in a calibrated 20 x 10 cm length lane. The maximum intensity measurements were analyzed, which represent the intensity of the complete paw. Besides, the maximum contact area (cm2) was accessed, which corresponds to the max area of a paw that comes into contact within the glass-floor and the print area (cm2). The surface area of the complete paw print of both tumor-bearing groups (W and LW) was compared with the gait patterns at the initial time point (pre-tumor inoculation) and the pre-agonic moment (~18 days after tumor inoculation). All experimental animals used were acclimated to the test one week before the experiment started. The software detects all paws during natural gait, recording all right front (RF); left front (LF); – right hind (RH) and left hind (LH) paws. The average of forepaws (RF and LF) was considered as forelimb, and the average of hind paws (RH and LH) were considered hindlimb.
Behavior test (Video recording system and analysis)
The animal behavior was accessed by night vision cameras placed in front of each individual cage at an adequate height. The cameras recorded all rats activities during their nocturnal behavior. The video recording system commenced one week before the experiment started. Video files were analyzed using the video tracking software EthoVisionXT12 (Noldus Information Technology, Netherlands) to access the total distance covered (cm), the average velocity (cm.s-1) and the time spent in movement (s) of both tumor-bearing groups (W and LW). All data from tumor groups were compared to their behavior at the initial time point (pre-tumor inoculation) and at the pre-agonic moment (~18 days after tumor inoculation) and also compared to control groups.
Grip Strength test
The grip strength test was performed randomly in all experimental groups at the beginning and endpoint moment of the experiment to access force measurement. The equipment use procedures followed the manufactures’ instructions (BIOSEB's Grip Strength Test), and was always operated by the same researcher during the morning period. The animals were placed in the grip strength room 15 minutes before the test to acclimate them to the environment. Briefly, rats were held by the tail and lowered towards the grip strength meter. The animals were allowed to grab the metal grip and were then pulled backwards in the horizontal plane. The force applied to the grid just before it loses grip was recorded as the peak tension. Measurements were repeated 10 times for each animal, and it was recorded in grams and then normalized by the tibia length of each individual animal.
Light Microscopy
Muscle samples were removed from animals and immersed in a fixative solution (4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4) for 24 hours at 4°C. Then, tissues were dehydrated in graded concentrations of alcohol, embedded in historesin (Leica Microsystems, Heidelberg, Germany) and sectioned at a width of 3 μm. The sections were mounted on slides and stained with hematoxylin-eosin-floxin. The sections were then examined for image analysis of cross-sectional area using a Nikon Eclipse E800 light microscope (Nikon Corporation, Tokyo, Japan). The cross-sectional area of myofibers was measured using Image Pro-Plus Premium software (v.3.01, Media Cybernetics, Silver Spring, MD, USA) after capturing the image in a Leica microscope (Leica DMLM, Wetzlar, Germany) using a 20× magnification.
Transmission Electron Microscopy
The transmission electron microscopic was accessed as briefly described: the skeletal muscle tissue was immersed in a fixative solution consisting of 2.5 % glutaraldehyde and 2.5% paraformaldehyde in sodium cacodylate buffer (0.1M) at pH 7.4 and CaCl2 (3mM) for 24 h at 4ºC. Then, tissue samples were rinsed with cacodylate buffer/CaCl2 and were post-fixed in 1% OsO4 in sodium cacodylate buffer (0.1M), CaCl2 (3mM), and potassium ferrocyanide solution (0.8%) for 1 h on ice. Following, tissue samples were washed with milli-Q water and stained with uranyl acetate (2%) overnight at 4 ºC. Then, tissue samples were washed in milli-Q water and dehydrated in an ethanol gradient. The samples were embedded in Epon 812 resin. Resin polymerization was controlled in an incubator (60°C) for 72h. Ultra-thin sections were stained with uranyl acetate, and lead citrate then observed in a transmission electron microscope LEO 906 (Zeiss), operated at 60 kV.
Serum cytokines assay
The serum cytokine (IL-6, TNF, IL-10) profile was measured by Luminex assay using a specific kit (Rat Premixed Multi-Analyte kit) from R&D Systems® (Minneapolis, USA) following the manufacturer’s technical procedures.
Quantitative RT-PCR
Total RNA from the tibialis anterior muscle tissue was extracted with TRIZOL® reagent (Invitrogen) following the manufacturer’s instructions. The quality of the RNA samples was examined at 260/280 nm and 260/230 nm with a UV spectrophotometer (Nanovue Spectrophotometer 28923215 Ge BioSciences, USA). cDNA was produced using a high capacity cDNA reverse transcription kit (Applied Biosystems®, USA) containing Multiscribetm Reverse Transcriptase. cDNA synthesis was performed on 1 μg of RNA at 42 °C. Real-time reactions were performed using standard methods (ABI Prism 7500 Sequence Detection System; Applied Biosystems, Foster City, USA) and qPCR analysis was normalized to GAPDH. The genes evaluated using qPCR were FoxO3 (forward primer 5′- AACTTTGAC TCC CTC ATC TC -3′ and reverse primer 5′- TTT TCT CTG TAG GTC TTC GG - 3′), IL-6 (forward primer 5′- ACT CAT CTT GAA AGC ACT TG -3′ and reverse primer 5′- GTC CAC AAA CTG ATA TGC TTA G -3′), Ubiquitin (forward primer 5′- CAA GCT CAG TCT TTT GCC TCA GA -3′ and reverse primer 5′- GGA TCG GCG GGT AAT GAA G -3′), COX5a (forward primer 5′-TGTTGGCTATGATCTGGTTCC-3′ and reverse primer 5′-TTATGAGGTCCTGCTTTGTCC-3′), CS (forward primer 5′-TATGGCATGACGGAGATGAA-3′ and reverse primer 5′-CATGGACTTGGGCCTTTCTA-3′), and GAPDH forward primer 5′- CCA TGG AGA AGG CTG GG -3′ and reverse primer 5′- CAA AGT TGT CAT GGA TGA CC -3′).
Western blotting
Samples of tibialis anterior muscle biopsies were lysed in RIPA buffer (150 mM NaCl, 25 mM Tris-Cl, pH 7,4, 0,1% SDS, 1% NP-40, 0,5% sodium deoxycholate) and supplemented with protease and phosphatase inhibitors. After protein extraction protocol, protein concentration was measured by the bicinchoninic acid (BCA) method, following the manufacture’s instructions (Pierce™ BCA Protein Assay Kit, Sigma Aldrich, Poole, UK). The proteins (40 μg) were separated by electrophoresis, transferred to nitrocellulose membranes and stained proteins with Ponceau S. After that, the membranes were incubated with primary antibodies against FoxO1 (CellSignaling, 1:1000), MurF (CellSignaling 1:1000), 20S (CellSignaling 1:1000), OXPHOS (Abcam 1:1000), and GAPDH (CellSignaling 1:1000) as a loading control. After that, the membranes were probed with secondary antibodies conjugated with peroxidase, and bands were visualized using a chemiluminescent reagent (ThermoFisher Scientific). The membrane images were captured using an image system (Amersham Imager 600, GE Healthcare), and band volume quantitation was quantified.
Statistical Analysis
Data are expressed as mean ± SEM. Differences between 3 or more groups were analyzed by variance test ANOVA, followed by Tukey post hoc test and by t-test for comparison among 2 groups. For correlation analysis, the Pearson coefficient was calculated. For all statistical analyses, P< 0.05 was considered significant. The statistical analyses were performed using the software Graph Pad Prism 6.0 (Graph-Pad Software, Inc).