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).
Comprehensive Murine Colitis Assessment
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).
Corresponding Organization : University of Tasmania
Other organizations : Swinburne University of Technology, CSIRO Land and Water
Protocol cited in 9 other protocols
Variable analysis
- DSS induction
- Body weight loss
- Stool consistency
- Blood in stool
- Disease Activity Index (DAI)
- Colon length
- Colon weight/body weight ratio
- Spleen weight
- Histological damage in distal and proximal colon regions
- Body weight measurements
- Fecal sample collection and storage
- Colon dissection and processing
- Swiss roll technique for histological analysis
- Hematoxylin and eosin (H&E) staining
- Positive control: None specified
- Negative control: None specified
Annotations
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