orally treated by gavage with euphol (30 mg/kg, twice a day) or the corresponding vehicle (5% Tween 80 in saline 0.9% NaCl), starting 24 h after TNBS instillation (day 1) during 3 consecutive days (day 1–3). Vehicle solution (5% Tween 80 in 0.9% NaCl solution) was used in control experiments. The dose of each drug was chosen based on preliminary studies. Body weight change and disease activity index Body weight was measured daily, starting on the day preceding DSS administration and up to 7 days thereafter in the DSS model. Body weight was also measured daily in the TNBS model, starting on the day preceding colitis induction (i.e., before fasting) and then again just prior to intracolonic TNBS or vehicle administration (after fasting) and up to 72 h after treatment. In the DSS model only, the clinical disease activity index (DAI) was measured daily using the protocol previously described [68], which ranged from 0 to 4 and was the sum of scores given for body weight loss (scored as: 0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, over 20%), stool consistency (scored as: 0, well formed pellets; 2, loose stools; 4, diarrhea) and presence or absence of fecal blood (scored as: 0, negative hemoccult test; 2, positive hemoccult test; 4, gross bleeding). Macroscopic and microscopic colonic damage In the DSS model at the end of the 7-day period, colons were removed and examined for: weight, the consistency of the stool found within as well as gross macroscopic appearance and length, which was measured from 1 cm above the anus to the top of the cecum. Macroscopic damage was assessed according previously [69], as the sum of scores attributed to stool condition (0, normal well-formed fecal pellets; 1, loosely shaped moist pellets; 2, amorphous, moist, sticky pellets; 3, diarrhea; plus 1 for presence of blood in stool), colon damage (0, no inflammation; 1, reddening, mild inflammation; 2, moderate or more widely distributed inflammation; 3, severe and/or extensively distributed inflamma- tion), colon weight loss (0, for ,5%; 1, for 5–14%; 2, for 15–24%; 3, for 25–35%; 4, for .35%) and colon length shortening (0, for ,5%; 1, for 5–14%; 2, for 15–24%; 3, for 25–35%; 4, for .35%), with up to a maximum total score of 15. To evaluate microscopic colon damage by light microscopy, samples of the distal colon were fixed immediately in 10% formaldehyde solution, embedded in paraffin, cut into 5 mm thick transversal sections, mounted on glass slides, deparaffinized and stained with hematoxylin and eosin stain (H&E). In each specimen, six random fields of view were analyzed by two double-blind observers, using Sight DS-5M-L1 digital camera connected to an Eclipse 50i light microscope (both from Nikon, Melville, NY, USA). The intensity of microscopic colonic damage was assessed according to the scoring system described by Rath et al. [70] and modified by Van der Sluis et al. [71]. In the TNBS model, at 3 days after TNBS administration, mice were sacrificed and their colons removed and rinsed with saline, and macroscopic colonic damage was evaluated using the following scoring system: 0, no damage; 1, hyperemia without ulcers; 2, hyperemia with bowel wall thickening but no ulcers; 3, one site of ulceration without bowel wall thickening; 4, two or more sites of ulceration or inflammation; 5, 0.5 cm of inflammation and major damage; 6, at least 1 cm of major damage (for every additional 0.5 cm of damage, the score was increased by one to a maximum of 10); plus 1 for presence of diarrhea or stricture; plus 1 or 2 for presence of mild or severe adhesions, respectively [72]. Scanning electron microscopy (SEM) SEM was used to visualize topographical alterations of the luminal surface of the colon from control healthy, untreated and treated mice. Fragments of 2 mm thickness from the mid-colon Euphol and Experimental Colitis were pre-fixed in the field in 2.5% glutaraldehyde in 0.1M sodium phosphate buffer (pH 7.4) and kept on ice. The specimens were then rinsed in the same buffer, post-fixed in 2% osmium tetroxide (OsO4) for 4 h, dehydrated in a graded ethanol series (30% to 100%) and dried in hexamethyldisilazane (HMDS). The samples were glued onto stubs, covered with gold particles (Bio-Rad SC502, Hertfordshire, UK) and observed under a scanning electron microscope (JEOL JSM-6390LV, Tokyo, Japan). Myeloperoxidase assay Neutrophil infiltration into the colon was assessed indirectly by measuring myeloperoxidase (MPO) activity. Mid-colon segments were homogenized in ethylenediaminetetraacetic acid (EDTA)/ NaCl buffer (pH 4.7) and centrifuged at 10,000 rpm for 15 min at 4uC. The pellet was ressuspended in 0.5% hexadecyltrimethyl ammonium bromide buffer (pH 5.4) and frozen in liquid nitrogen and thawed repeatedly three times. Samples were then centrifuged again (10,000 rpm, 15 min, 4uC) and 25 ml of the supernatant was used for the MPO assay. The enzymatic reaction was assessed by the addition of 25 ml of 1.6 mM tetramethylbenzidine (TMB) in 80 mM NaPO4, plus 100 ml of 0.3 mM H2O2. MPO activity was measured spectrophotometrically at 650 nm and the results are expressed as optical density (OD) per milligram of tissue. Determination of cytokine levels Mid-colon segments were homogenized in phosphate buffer containing 0.05% Tween 20, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 IU aprotinin A. The homogenates were centrifuged at 3,0006g for 10 min and the supernatants stored at 280uC until assays for the determination of levels of the cytokines IL-1b, keratinocyte- derived chemokine (CXCL1/KC), monocyte chemoattractant protein-1 (MCP-1) and macrophage-inflammatory protein-2 (MIP-2) were carried out. The amount of protein in each sample was measured using the Bradford method [73], using bovine serum albumin as a standard. The levels of each cytokine were evaluated using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s recommendations (R&D systems, Minneapolis, MN, USA) and the results are expressed in pg/mg of protein in each sample. Real-time quantitative PCR Total RNA was extracted from mid-colon samples taken from anesthetized mice treated with DSS using the Trizol protocol (Invitrogen, Carlsbad, CA, USA) and its concentration was determined by NanoDropTM 1100 (NanoDrop Technologies, Wilmington, DE, USA). A reverse transcription assay was performed as described in the M-MLV Reverse Transcriptase protocol according to the manufacturer’s instructions. cDNA (300 ng) was amplified in triplicate using TaqManH Universal PCR Master Mix Kit with specific TaqMan Gene Expression target genes, the 3’ quencher MGB and FAM-labeled probes for mouse TNF-a (Mm00443258_m1), IL-1b (Mm01336189_m1), CXCL1/ KC (Mm00433859_m1), IL-6 (Mm99999064_m1), ICAM-1 (Mm005616024_g1), VCAM-1 (Mm01320970_m1), lymphocyte function- associated antigen 1 (LFA-1, Mm01278854_m1) and GAPDH (NM_008084.2) (which was used as an endogenous control for normalization). The PCR reactions were performed in a 96-well Optical Reaction Plate (Applied Biosystems, Foster City, CA, USA). The thermocycler parameters were as follows: 50uC for 2 min, 95uC for 10 min, 50 cycles of 95uC for 15 sec and 60uC for 1 min. Expression of the target genes was calibrated against conditions found in control non-colitic animals, i.e., those that received vehicle (5% Tween 80 in saline 0.9% NaCl). PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27122