Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.
Part 1: Injury-Repair Colitis Model
Part 2: Necropsy and Harvesting of Colon
Part 3: Processing as “Sausages” for Macroscopic Analysis of the Entire Colon
Part 4: Making Swiss Rolls for the Histological Analysis of Acute Colitis
Part 5: Representative Results
The DSS model for acute colitis allows the researcher to obtain, fix, and analyze a colon that models acute colitis. When the Swiss roll is cut and mounted, it should form a representative slice of the entire colon if rolled properly (Figure 1). The mounted roll can be stained with H&E in order to determine the extent of damage to the colon, from the distal (inside) end to the proximal (outside) end (Figure 2). Immunohistochemistry can also be performed on the Swiss roll to identify and quantify inflammatory infiltrates. If the sausage method has been performed correctly, the fixed colon will be dilated and the entire mucosal surface can be easily manipulated and viewed under the dissecting microscope (Figure 3).
Figure 1. The properly-executed “Swiss roll”. (A) The colon is rolled from the distal to proximal end, transected with a needle and secured by bending the end of the needle. It is then placed in a tissue cassette for fixation. (B) H&E stained 5 μm section of a Swiss roll made from the colon of a mouse treated with DSS (d= distal colon p= proximal colon).
Figure 2. DSS treated colons show signs of acute colitis. Inflammation and crypt damage are apparent in the DSS-treated colon compared to a water treated control.
Figure 3. An example of a “sausage”. The sausage infused with formalin and completely expanded. A slight angle will be present secondary to the natural curvature of the colon. The opened sausage should lie flat.
This protocol can be modified to model acute injury, injury-repair, or chronic colonic injury processes.
Acute Injury Modification:
DSS treatment ad lib for 4-6 days followed by immediate sacrifice
Injury-Repair Modification:
Injury with 4-6 days of DSS treatment followed by recovery period of 3-4 days and sacrifice (as described in above protocol).
Chronic Colitis Modification:
Mice are placed on three five-day cycles of 3% DSS with sixteen days of recovery between each cycle. Mice are sacrificed after the final 16-day rest period.
There are several issues that researchers need to be aware of with this model:
Biochemical analysis of the colon can be performed by taking samples from the proximal or distal margins. Depending on the severity of injury this may impact your ability to generate a histologic injury score. In vivo BrdU labeling to measure proliferation can be achieved by intraperitoneal injection of 16.7 μg/kg BrdU 2 hrs prior to sacrifice followed by α-BrdU IHC processing.
As an alternative to the “sausage”, the colon can be fixed completely flat. Line the bottom of a Tupperware container with Whatman paper and soak the paper with 10% buffered formalin phosphate. Dissect the colon as described in “Part 2: Necropsy and Harvesting of Colon”. Again, keep the distal end of the colon closest to the operator. Using fine-tipped scissors, incise longitudinally from distal to proximal end of the colon. With fine tipped forceps grasp either edge of the incision and open laterally working, thus displaying the colon as a flat sheet. Transfer this flat sheet to the pre-soaked Whatman paper. Cover the flattened colon with another piece of Whatman and wet with 10% buffered formalin phosphate. The Whatman should be completely covered in formalin but not so much that it lifts off the colon. Seal the Tupperware and fix for 24 hours. Remove the colon from the Tupperware and place it in 10 ml 70% ethanol in a 15 ml conical tube. (Colons can be stored in 70% EtOH indefinitely at room temperature.)
There are several methods for quantifying colonic injury 5,6,7. One method, for example, uses a multi-parameter scale including: inflammation, extent involvement, regeneration, crypt damage, and percent involvement 8.
The authors have nothing to disclose.
Funding provided by NIH (1 K08 DK080221-01) and Vanderbilt institutional development funds.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Dextran Sulfate Sodium Salt | USB Corp | 9011-18-1 | mol wt 40,000-50,000 | |
10% Buffered Formalin Phosphate | Fisher Scientific | SF100-4 | ||
Isoflurane, USP | Phoenix Pharmaceuticals | NDC 57319-474-06 | ||
TISSUE PATH Macrosette Cassettes | Fisher Healthcare | 15182706 | ||
BD 10 ml Syringe | B-D | 309604 | ||
Ethyl Alcohol | Pharmco-Aaper | E190 | Dilute to 70% with distilled water | |
20G Straight Feeding Needle | VWR | 20068-612 | ||
27G1/2 PrecisionGlide Needle | B-D | 305109 | ||
Dissecting Scissors Sharp/Blunt Tip | VWR | 82027-588 | ||
Waugh Forceps | VWR | 82027-428 | ||
Non-absorbable Suture | LOOK Surgical Specialties | SP116 | ||
Whatman Blotting Paper | VWR | 28298-020 | Cut as needed | |
Nalgene Surfactant-Free Cellulose Acetate (SFCA) Filter | Cole-Parmer | EW-06731-2 | ||
Carbon fiber composites digital caliper | Fisher Scientific | 15-007-958 |