The overall goal of this procedure is to evaluate the intestinal immune response induced by dextron sulfate sodium by determining the quantities of myelo peroxidase activity and pro-inflammatory cytokines produced in response to the inflammation. First DSS colitis is induced in mice through the introduction of DSS SALT solution into the drinking water. Next, the disease severity of the colitis is assessed by macroscopic scoring and the colon is removed for further analysis.
The dissected colon is then sectioned into three pieces for histology, cytokine quantification, and NPO activity. Finally, the colon tissue is homogenized and the supernatant is used to determine MPO activity. Ultimately, changes in the MPO activity can be determined through the absorbance data collected using a spectrophotometer.
This method can provide insight into chronic information of various models of inflammatory bowel disease, such as DSS and TNBS induced colitis. In addition, you can use this method in other organs such as the lung and liver Before beginning the procedure. Replace the drinking water in each mouse cage with DSS solution to induce the inflammation.
Give control mice autoclave drinking water without DSS. Then on the day of the experiment, dissect out the colon as previously demonstrated in the JoVE article by Whitham Williams and Williams. Carefully squeeze the feces outta the entire colon using a pair of bent tweezers, collecting it into a weighing paper by rinsing the tissue with sterile PBS.
Assess the stool by using a pair of forceps to press down on the feces to determine its consistency to determine a score for blood in the feces. Note the color of the feces and further validate the evaluation using a hemoccult test, which involves smearing stool onto hemoccult paper, adding developer, and then making a note of the color blue denotes a positive stool test for blood. Then use this table to determine a value for each of the conditions.
Next, while maintaining the proximal to distal orientation of the colon, cut the colon into three equal sections. Then take sample fragments from the proximal and mid colon sections of the colon, and place the samples in individual 1.5 milliliter eph tubes. To evaluate the histological damage of the colitis severity, use the most distal tissue section of the colon and cut a small half to one centimeter length fragment.
Place the fragment in a tissue cassette and submerge the cassette in buffered 10%formalin solution. Then after preparing paraffin embedded cross-sections of the tissue fragments, stain the sections with hematin eoin for scoring by a blinded observer. Finally, freeze the samples from the proximal and mid collon sections in liquid nitrogen and store them until use at minus 70 degrees Celsius.
After removing the colon samples from minus 70 degrees Celsius storage, place them on ice. Then using bent forceps to remove any visible feces or fat, and place them into individual two milliliter tubes suitable for use in a homogenizer. Then determine and record the weight of each sample.
Next, add homogenizer beads to each sample tube, and then add the appropriate amount of H tab buffer to the tube according to the weight of the tissue as just determined, dissociate the fragments with a tissue homogenizer for four minutes at 30 hertz. Finally, centrifuge the cell solution for six minutes at 13, 400 times G and four degrees Celsius. Collect the supernatant into a fresh tube, and then making sure to keep the homogenization bead discard the resulting pellet.
The supernatant can then be stored at minus 70 until use. Begin this step by adding seven microliters of the previously prepared tissue homogenate in triplicate into a 96 well plate. Then add 50 microliters of diluted hydrogen peroxide to freshly prepared OD Dion iodine.
Using a multi-channel pipette, add 200 microliters of the hydrogen peroxide ity mixture to each of the wells. Now, measure the absorbance of 550 nanometers using a spectrophotometer, taking three readings at 32nd intervals. Then using the absorbance data, tissue weight, and buffer volume, calculate the NPO activity in the homogenates as demonstrated in this sample calculation.
During the duration of the DSS treatment, the disease activity index can be used to assess and evaluate the clinical progression of the disease. Animals treated with DSS show significant weight loss compared to their initial weights, loose stools and fecal bleeding. The more substantial than macroscopic systems, the higher the disease activity index.
Upon sacrifice and examination of the colons of mice that were given 5%DSS in drinking water for five days, the severity of colitis based on macroscopic scoring of the shortening of colon length, colonic bleeding, fecal bleeding, loosening of stool consistency, and signs of rectal bleeding for this representative experiment was determined to be about five times worse than in controls treated with water only as demonstrated by these data cross-sections of DSS treated colonic tissue samples, same with h and e, have higher histological scores than water treated controls. Here, the normal architecture and lack of cellular infiltrate could be noted in the area indicated by the asterisk in this h and e stained cross section collected from a control animal that received water only. Now compare this h and e staining of a sample from A DSS treated animal to the previous figure.
The colon from this animal exhibits more histological damage as evidenced by more cellular infiltration as indicated by the pound sign and by more goblet cell depletion and greater distortion. Damage to K crypt architecture as indicated by the asterisk. To further characterize the extent of the disease severity in DSS treated mice, NPO activity, a surrogate marker for inflammation can be assessed from homogenized colon tissue samples.
As expected DSS treated colons have a higher NPO activity than do controls. In addition, the severity of DSS induced colitis is associated with increased levels of pro-inflammatory cytokines, such as IL one beta IL six, and TNF alpha. While attempting this procedure, it's important to remember that MPO activity can decrease over time.
Therefore, the MPO assay should be one of the first assays performed when determining the severity of intestinal inflammation.
