Male Sprague-Dawley rats (200–250 g) were obtained from Charles River Laboratories (France) and were housed in an air-conditioned room at 22°C with a 12:12 hours light–dark cycle (light period 07:00–19:00 hours) in a fully equipped animal facility. Rats were kept in holding cages (three rats per cage) for 1 week before the experiment, in order to acclimatize them to the housing conditions. Standard pelleted rodent chow (RM1 diet, SDS, UK) was available ad libitum. Three days before MTX administration, the rats were transferred to metabolism cages (Tecniplast, Lyon, France) where they were fed with the same RM1 diet but in powdered form (SDS). Drinking water was always available ad libitum. Animal care and experimentation were in accordance with guidelines established by the National Institutes of Health, USA and complied with both French and European Community regulations (Official Journal of the European Community L 358, 18/12/1986).

Rats were divided into two groups (n = 12 in each) to achieve similar mean body weight: (i) MTX-treated and (ii) controls. A single dose of MTX (2.5 mg/kg, Teva Pharma, Courbevoie, France) dissolved in phosphate-buffered saline (PBS) was injected subcutaneously (S.C.) each day for three consecutive days (days 1–3). Control rats were injected S.C. with PBS. The dose and duration of MTX administration were selected based on our previous data showing typical response to MTX in rats characterized by anorexia and cachexia and loss of both lean and fat mass (31). Rats were gently handled daily during measurements of their body weight.

Behavioral tests, performed to evaluate the anxious- and depressive-like behaviors in MTX-treated anorectic and in control rats, were carried out between 9.00 and 16.00 during the light period. A habituation to conditions of experimentation was performed the day preceding the experimental procedures. The rat’s spontaneous locomotion was recorded for 120 min, using a Versamax Animal Activity Monitor (AccuScan Instruments, Columbus, OH, USA). The rats were tested for their anxiety in an elevated plus-maze, which is commonly used and pharmacologically validated for anxiety testing in rodents (34). The plus-maze is elevated approximately 1 m from the floor, and the rat is given the choice of spending time in open, unprotected arms or enclosed, protected arms. The rats were tested for their depressive-like behavior in a forced-swim test. Rats were placed into a cylinder filled with tap water (25°C) to 1/3 of its volume. The total time of immobility during 15-min test procedure was registered. A rat was judged to be immobile when it floated in the water in an upright position and made only small adjustments movements to keep its head above water.

Rats were killed by decapitation between 10:00 and 11:00 a.m. at day 6 after beginning of MTX or PBS treatments. Trunk blood was collected into tubes containing EDTA (1 mg/ml), aprotinin (500 U/ml), and 1N HCl (1:10 vol). Plasma was separated by centrifugation at 4°C and stored at −80°C until assayed. Plasma levels of ghrelin and des-acyl ghrelin were measured using enzymatic immunoassay kits from Mitsubishi Chemical Med Corp. (Tokyo, Japan), according to manufacturer’s instructions. The manufacturer specified the sensitivity of both kits at 2.5 fmol/ml and absence of cross-reactivity between the active and des-acylated forms of ghrelin in the corresponding detection assays. For in situ hybridization, the stomach tissue was frozen on dry ice, stored at −80°C, then cut in a cryostat (Leica Microsystems, Nanterre, France) to obtain 14-μm thick transversal sections collected on Superfrost glass slides (Thermo Scientific, Braunschweig, Germany).

All solutions were made using diethylpyrocarbonate (DEPC, Sigma, St. Louis, MO, USA) treated water. Stomach sections were fixed with 4% paraformaldehyde in PBS, pH 7.5. After washing with PBS for 5 min, the sections were incubated with 0.5M HCl in DEPC water for 5 min and then washed in PBS twice for 3 min. Further, the sections were treated with 0.25% acetic anhydride in 0.1M triethanolamine, pH 8.0, for 20 min. The sections were washed in PBS twice for 3 min, and immersed in a graded ethanol series (70, 80, and 99.5%) for 2 min each. Finally, sections were dried for 30 min and then stored at −80°C.

RNA probes specific to preproghrelin mRNA (Accession number: NM_021669.2, National Center for Biotechnology Information, Bethesda, MD, USA) were prepared from rat stomach mRNA. The rat stomach extracted mRNA was reverse-transcribed to generate cDNA using the iScript select cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). The cDNA was amplified using the following specific primers: ghrelin-F: 5′-AGCACCAGAAAGCCCAGCAGAGAA-3′ and ghrelin-R: 5′-TTGCAGAGGAGGCAGAAGCTGGAT-3′ (product size of 335 bp from position 121–455) (Invitrogen, Carlsbad, CA, USA). The PCR fragment was gel purified using QIAquick Gel extraction kit (Qiagen) and subcloned into PCR1II-TOPO vector (Invitrogen, Carlsbad, CA, USA). The sequence of cDNA probe was confirmed by nucleotide sequencing (KIGene, Stockholm, Sweden). The plasmids were linearized using restriction enzyme BAmHI and XbaI (Promega, Madison, WI, USA) and transcribed to generate sense and antisense RNA probes. In vitro transcription and labeling were carried out using SP6/T7 RNA polymerases (Ambion, Austin, TX, USA) and digoxigenin (DIG) RNA labeling mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The transcripts were purified using NucAway Spin Columns (Ambion). Sense probes were used as negative controls.

Sections were prehybridized in a humidified chamber using 50% (v/v) deionized formamide, 0.1M Tris–HCl (pH 7.6), 50 mM EDTA (pH 8.0), 40 mM NaCl, 1.25 mg/ml yeast tRNA (Roche), and 5× Denhardt’s solution for 4–6 h at 55°C followed by hybridization overnight (14–16 h) at 55°C. The labeled probes were diluted to a final concentration 0.8 ng/μl in solution containing 50% (v/v) deionized formamide, 0.3M NaCl, 0.1 M DTT, 10% (vol/vol) dextran sulfate, and 1× Grundmix solution. Grundmix solution is made of 0.2M Tris–HCl (pH 7.6), 5 mg/ml yeast tRNA (Roche), 1 mg/ml poly-A-RNA (Roche), 10× Denhardt’s solution, and 10 mM EDTA (pH 8.0). After hybridization, sections were washed with constant stirring as follows: twice for 30 min in 1× SSC with 0.1% SDS at 55°C, 1 h in 50% (v/v) formamide/0.5× SSC at 55°C, 5 min in 1× SSC with 0.1% SDS at 55°C, 30 min in 36 μg/ml RNase A diluted in RNase A buffer at 37°C, and twice for 10 min in 1× SSC with 0.1% SDS at 55°C. RNase A buffer contains 0.5M NaCl, 10 mM Tris (pH 8.0), and 0.5 mM EDTA. The sections were then incubated three times for 5 min in buffer 1 (100 mM Maleic acid, pH 7.5, 150 mM NaCl, 0.02% tween 20), immersed in 1% blocking reagent (Roche) diluted in buffer 1 for 20 min, and incubated with the alkaline phosphatase-conjugated anti-DIG antibody (Roche) diluted at 1:200 in buffer 1 with 1% blocking reagent at 4°C overnight. The sections were then washed in buffer 1 for 5 min three times, in buffer 2 (100 mM Tris, pH 9.5, 100 mM NaCl, 0.05% tween 20) for 5 min, and in buffer 2 with 5 mM of tetramisole hydrochloride (Sigma) for 5 min. A chromagen solution made of 337 g/ml nitro-blue tetrazolium chloride (NBT) and 175 g/ml 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP) (Roche) in buffer 2 with 5 mM of tetramisole hydrochloride (Sigma) was made, and the sections were incubated for 24 h. The reaction was stopped with PBS. The sections were then washed with distilled water, mounted in a solution of PBS and glycerol (85%), and viewed under a light microscope (Axioskop, Zeiss, Germany). Pictures from the in situ hybridization were taken with an objective 20×. Total number of preproghrelin mRNA-positive cells was counted using Image J 1.45s software (National Institutes of Health, Bethesda, MD, USA) from five representative sections of each mouse. The square area including the gastric mucosa and sub-mucosa were delimited in Image J 1.45s in each picture, and the number of positive cells in the measured surface area was calculated.

Ghrelin-reactive Ig levels were measured in rat plasma using an enzyme-linked immunosorbent assay (ELISA) accordingly to a published protocol (35). Briefly, 96-well plates (Nunc Immunoplate, Rochester, NY, USA) were coated with 2 mg/ml of rat ghrelin (Peptide institute, Inc., Japan) in 0.5M sodium carbonate (Na₂CO₃), 0.5M sodium bicarbonate (NaHCO₃), pH 9.6, and incubated for 72 h at 4°C. Plates were washed using PBS (0.05% Tween20, pH 7.4). Plasma was diluted 1:100 in either normal buffer (PBS, 0.02% sodium azide, pH 7.4) to measure free IgG or in dissociative 3M NaCl, 1.5M glycine (pH 8.9) buffer, to measure total ghrelin IgG levels. After incubation at 4°C overnight, plates were washed and incubated for 3 h with alkaline phosphatase-conjugated secondary antibody (anti-rat IgG; Jackson ImmunoResearch, West Grove, PA, USA). The reaction was developed for 40 min with 150 μl p-nitrophenyl phosphatase substrate solution (Sigma, St Louis, MO, USA) at 1 mg/ml in 0.1M Tris–HCl, 0.1M NaCl, 5 mM magnesium chloride hexahydrate (MgCl₂⋅6H₂0), pH 9.5. About 100 μl sodium hydroxyde (NaOH) (2M) was added to stop the reaction and optical density (OD) was measured at 405 nm in an ELISA plate reader.

Plasma samples were purified using a Melon Gel kit according to the manufacturer instruction (Thermo Fisher Scientific, Rockford, IL, USA). After the purification, IgG was frozen at −30°C and lyophilized overnight. Then, they were suspended in the HBS-EP buffer (GE Healthcare). The IgG concentration of each sample was determined with a Nanodrop^®, and HBS-EP buffer was used as a blank.

Affinity kinetics of mice IgG for ghrelin and des-acyl-ghrelin was determined by surface plasmon resonance (SPR) on a BIAcore 1000 instrument (GE Healthcare) according to a published protocol (36). In brief, rat ghrelin (Peptide institute) was diluted at 0.5 mg/ml in 10 mM sodium acetate buffer (pH 5.0) (GE Healthcare) and was covalently coupled on the sensor chip CM5 (GE Healthcare), using the amine coupling kit (GE Healthcare). All the measurements were done on the same sensor chip. For the affinity kinetic analysis, a multi-cycle method was run with five serial dilutions at 1/2 of each IgG sample: 1680–105 nM plus a duplicate of a blank (buffer only) at the beginning and at the end of the kinetic. During each cycle, the flow speed was 30 μl/min. About 60 μl of each sample dilution were injected correspond to 2-min period followed by 5 min of dissociation. Then, between injections of each dilution, the binding surface was regenerated with 10 mM NaOH resulting in the return of the sensorgram to the same baseline. The affinity kinetic data were analysed using BiaEvaluation 4.1.1 programme (GE Healthcare). For fitting kinetic data, the Langmuir’s 1:1 model was used, and the sample values were corrected by subtracting the blank values resulting from the injection of HBS-EP buffer.

Group differences were statistically analyzed using GraphPad Prism 5.02 (GraphPad software Inc., San Diego, CA, USA). Differences in daily food intake, body weight, and water intake were analyzed by a two-way repeated measurements (RM) ANOVA taking in account effects of time and treatment followed by the Bonferroni post-tests. Individual groups were compared using the Student’s t-test or Mann–Whitney test according to the Kolmogorov–Smirnov (KS) normality test, and p < 0.05 was considered as significant.

Methotrexate-treated rats displayed significant body weight loss (Figure 1A) and decreased food intake (Figure 1B) starting from day 3 after the first MTX injection. Water intake was reduced from day 4 (Figure 1C). Food intake was completely inhibited in MTX-treated rats at days 5 and 6 when they lost 33.7% of body weight as compared to control rats. The ratios of water to food intake were increased in anorectic MTX-treated rats (Figure 1D).

In situ hybridization revealed the presence of preproghrelin mRNA-expressing cells in the mucosal and sub-mucosal layers of the gastric wall of all control and MTX-treated rats (Figures 2A,B). The negative control did not show any positive cells (not shown). The number of preproghrelin mRNA-producing cells was increased by 51.3% in MTX-treated rats as compared to controls (Figure 2C). The gastric mucosa in MTX-treated rats did not show signs of atrophy.

Plasma levels of ghrelin and des-acyl ghrelin were robustly increased in MTX-treated vs. control rats (Figures 3A,B) while their ratios remained similar (Figure 3C).

Methotrexate-treated rats showed decreased plasma levels of both free and total acyl ghrelin-reactive IgG (Figures 4A,B) and free and total des-acyl ghrelin-reactive IgG (Figures 4D,E). The ratios of free to total IgG reactive with both ghrelin and des-acyl-ghrelin were increased in MTX-treated rats (Figures 4C,F).

The analysis of affinity kinetics using SPR revealed increased dissociation rates (small Kd) of IgG for ghrelin in MTX-treated rats (Figure 5B), who also displayed increased dissociation equilibrium constants (big KD) of their IgG for des-acyl-ghrelin (Figure 5F). Other parameters of affinity kinetics were not significantly different among the groups (Figures 5A,C,D,E).

Locomotor activity, including time of movement and crossed distance, was unchanged between MTX and control groups (Table 1). The number of entries in both open and closed arms in the elevated plus-maze was decreased in the MTX-treated rats (Table 1). The time spent in both open and closed areas was not significantly different between the groups, although MTX-treated rats tended to spend less time in open arms. There was no either significant differences between MTX and control groups in the immobility time of the forced-swim test (Table 1).