Mice are given 3%DSS at libido for four days. While weight and presence of diarrhea and fecal blood is recorded daily TSS is replaced by autoclave water after day four. After three days on water, mice are euthanized and their colons are removed.
Colons are measured and weighed. The colons are then either formed into Swiss rolls or sausages and examined under a microscope. This video article will demonstrate the harvesting and processing of colons in a murn model of colitis.
This procedure is used to study colitis in the laboratory of Dr.Christopher Williams of the Department of Cancer Biology at Vanderbilt University prior to beginning dextrin sulfate sodium treatment. Baseline weights are obtained throughout the course of the experiment. Daily weights are taken after the mice have been weighed, make a 3%weight per volume, dextrin sulfate sodium salt solution, or DSS in water.
The DSS must be of a low molecular weight for this model to be effective, make enough DSS solution to fill a water bottle for each cage of mice and filter the solution with a 0.45 micrometer cellulose acetate filter. Replace the drinking water bottle in the mouse cage with a bottle containing 3%DSS solution. If your institution uses an automated watering system, place exclusion tips on watering spouts to ensure that the mice do not have access to any other source of water.
After four days, the mice are weighed again to quantify systemic consequences of colitis. Weight loss is common with severe injury, however, may not be apparent for several more days on the same day. Day four, replace the DSS solution with water for an additional three days to allow some colonic epithelial recovery.
Three days later, day seven, the mice are weighed again. The colon can then be harvested from the euthanized mice as shown in the next section. To harvest the colon, expose the ventral side of the euthanized mouse to ensure unobstructed access to the abdomen.
Wet the abdomen completely with 70%ethanol. Grasp the midline of the abdomen with tissue forceps and elevate. Thus tenting the skin using fine tipped scissors.
Incise the abdomen to expose the abdominal contents. Extend the incision to the tip of the xiphoid process at the midline, and then extend along the inferior aspect of the costal margins bilaterally. Identify the small intestine cecum and colon.
Carefully dissect slash tease the colon from the surrounding mesentery. After that, transect the colon deepen the pelvis to free. The distal colon from the rectum.
Care should be taken so that no arteries or veins are cut. Also, be aware that the colon can tear easily. So gently remove the colon from the mesentery.
Transect the colon at the Colona sequel margin to free the proximal colon. Remove the colon and total using a 20 gauge feeding needle and 10 milliliter syringe intubate and flush the colon with ice cold PBS until the EIT is completely clear of stool. It is important to maintain the correct orientation of the colon by keeping the distal end of the colon closest to you.
Start by measuring the colon length using a digital caliper. Colon length is another indicator of the severity of injury. Colitis increases edema and shortens the overall colon length.
At this point, the harvested colons can either be processed as sausages for macroscopic analysis or made into Swiss roll for histological analysis of acute colitis. We will demonstrate these techniques in the following sections to process the colon as a sausage For macroscopic analysis of the entire colon, it is important to maintain the correct orientation of the colon. Therefore, keep the distal end of the colon closest to you.
Cut two pieces of non-absorbable suture, one approximately one inch in length and the other two inches in length. Use the two inch piece of suture to tie off the distal end of the colon as close to the margin as possible while still maintaining a good seal. Then place a 20 gauge feeding needle containing five milliliters of 10%buffered formalin phosphate into the proximal end of the colon.
Loosely tie the remaining piece of suture immediately proximal to the bulb of the feeding needle. Now grasp the colon firmly at the bulb of the feeding needle and infuse sufficient amount of formalin ensuring the colon is expanded. Tighten the knot in the suture as you withdraw the feeding needle, thus leaving the infused expanded colon.
As a sausage, fill a 15 milliliter conical tube with 10%buffered, formal and phosphate, and label the tube. Place the colon in the tube and fix for 24 hours. After 24 hours, pour off the formin into an appropriate waste container and replace it with 70%ethanol for an additional 24 hours.
Colons can be stored in 70%ethanol indefinitely at room temperature. When you are ready to analyze the colon, remove it from the conical tube. Cut the strings on each end with a scalpel being careful not to damage the colon.
Remember that the distal end has the longest piece of string. Cut the colon longitudinally from the distal to proximal end so that it forms a long sheet. The colon can now be viewed under a dissecting microscope.
Cut the colon longitudinally and lay out the colon as a flat sheet. Rolling the colon requires two pairs of forceps and a two handed technique. Grasp both lateral edges of the distal colon margin with forceps proceed to sequentially roll the colon by rotating and releasing the forceps proceeding to the proximal margin, thus generating a spiral with a third dimension or Swiss roll.
If the colon adheres to the watman paper, wet the paper and colon with a small amount of PBS to maintain the form. Grasp the roll firmly with forceps and transect with a 27 and a half gauge needle. Secure the roll by bending the needle as it exits the roll.
Now, place the roll in a labeled tissue cassette and place the cassette in a jar of 10%buffered formalin phosphate. Leave it room temperature for 24 hours to fix the tissue. After 24 hours, pour off the formalin and replace with 70%ethanol for an additional to four hours.
Colons can be stored in 70%ethanol indefinitely at room temperature. This model allows the researcher to obtain, fix and analyze colons, thus allowing for analysis of severity of colitis. For macroscopic analysis of the entire colon, the colon can be processed as a sausage that is infused with formalin and completely expanded.
The open sausage should lie flat. 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. For histological analysis, the colon can be processed as a Swiss roll.
The roll is secured with a needle and placed in a tissue cassette for fixation. When the roll is cut and mounted, it should form a representative slice of the entire colon if rolled properly, sections of the Swiss roll can be stained with hematin and ASIN in order to determine the extent of colonic injury. Remembering the distal end of the colon is in the center of the roll on the slide.
In this example, inflammation and crypt damage are apparent in the DSS treated colon compared to a water treated control. We have just shown you how to harvest and process colons from a murine model of acute colitis. When performing this procedure, it's important that you are well organized prior to sacrificing.
The mice, for example, have all tubes and cassettes labeled for each mouse and all of the tools and reagents readily available. In addition, maintain detailed records of mouse weight and colon length. Finally, make sure that you have allotted enough time for processing.
It usually takes 15 minutes per mouse for a novice and six to eight minutes for someone more experienced in this technique.
