A Disease Activity Index (DAI) was determined daily in all mice by scoring for body weight, hemocult reactivity or presence of gross blood and stool consistency during the week of DSS induction, as detailed in [25 (link)]. Stool was collected from individual mice and tested for the presence of blood using Hemoccult II slides (Beckman Coulter Inc., Brea, CA, USA). Briefly, the following parameters were used for calculation: (a) body weight loss (score 0 = 0%, score 1 = 1–5%, score 2 = 6–10%, score 3 = 11–15%); (b) stool consistency (score 0 = normal, score 1 = soft but still formed, score 2 = very soft/loose stool, score 3 = diarrhoea/watery stool); and (c) blood in stool (score 0 = negative hemoccult, score 1 = positive hemoccult, score 2 = Blood traces in stool visible, score 3 = rectal bleeding). DAI was determined by combining the scores from these three categories. Body weights were measured for each animal throughout the experiments and expressed as percent weight loss to the weight immediately before DSS treatment. Fecal samples were collected and stored at −80 °C on day 14 for metabolite analysis.
After sacrificing the mice, the colons were removed from the caecum to the anus following the method of Perera et al. [26 (link)]. The length of the colons from the ileocaecal junction to the rectum were recorded. The colon was subsequently opened along its longitudinal axis and the luminal (mucosal) contents were removed using sterilised 200 μL pipette tips prior to weighing the organ. The length and weight of colon and spleen were documented. Spleen weight, colon length, and colon weight/body weight ratio were calculated as macroscopic markers of inflammation. The mucosal and cecal contents were collected for metabolite profiling and stored at −80 °C. The colon was bisected longitudinally, and one half was prepared using the Swiss roll technique [27 (link)] whereas the remaining colonic tissue was dissected out, segregated into proximal colon (PC) and distal colon (DC) and snap-frozen for molecular analyses. Swiss rolls underwent 24 h fixation in 10% (v/v) neutral-buffered formalin. Swiss rolls were subsequently transferred to 70% ethanol prior to progressive dehydration, clearing and infiltration with HistoPrep paraffin wax (Fisher Scientific, Philadelphia, PA, USA). Swiss rolls were then embedded in wax and 5 μm sections were cut using a rotary microtome. Sections were stained with haematoxylin and eosin (H and E; HD Scientific, Sydney, Australia). Slides stained with H and E (n = 8 per group) graded blindly for the severity of tissue damage at distal and proximal regions as described previously [28 ,29 (link)]. Briefly, frequency of distribution of inflammation graded 0-3, crypt architectural distortion and ulceration graded 0–5, tissue damage graded 0-3, inflammatory infiltrate graded 0–3, goblet cell loss graded 0–3, mucosal thickening (oedema) were graded 0–3. All images were captured on a Leica DM500 microscope using a Leica ICC50 W camera (Leica Microsys-tems, Wetzlar, Germany).
After sacrificing the mice, the colons were removed from the caecum to the anus following the method of Perera et al. [26 (link)]. The length of the colons from the ileocaecal junction to the rectum were recorded. The colon was subsequently opened along its longitudinal axis and the luminal (mucosal) contents were removed using sterilised 200 μL pipette tips prior to weighing the organ. The length and weight of colon and spleen were documented. Spleen weight, colon length, and colon weight/body weight ratio were calculated as macroscopic markers of inflammation. The mucosal and cecal contents were collected for metabolite profiling and stored at −80 °C. The colon was bisected longitudinally, and one half was prepared using the Swiss roll technique [27 (link)] whereas the remaining colonic tissue was dissected out, segregated into proximal colon (PC) and distal colon (DC) and snap-frozen for molecular analyses. Swiss rolls underwent 24 h fixation in 10% (v/v) neutral-buffered formalin. Swiss rolls were subsequently transferred to 70% ethanol prior to progressive dehydration, clearing and infiltration with HistoPrep paraffin wax (Fisher Scientific, Philadelphia, PA, USA). Swiss rolls were then embedded in wax and 5 μm sections were cut using a rotary microtome. Sections were stained with haematoxylin and eosin (H and E; HD Scientific, Sydney, Australia). Slides stained with H and E (n = 8 per group) graded blindly for the severity of tissue damage at distal and proximal regions as described previously [28 ,29 (link)]. Briefly, frequency of distribution of inflammation graded 0-3, crypt architectural distortion and ulceration graded 0–5, tissue damage graded 0-3, inflammatory infiltrate graded 0–3, goblet cell loss graded 0–3, mucosal thickening (oedema) were graded 0–3. All images were captured on a Leica DM500 microscope using a Leica ICC50 W camera (Leica Microsys-tems, Wetzlar, Germany).
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