Insulitis Grading

One of the hallmarks of type 1 diabetes is the development of insulitis—lymphocytic infiltration of the islets. Grading the severity of the insulitis is a useful immunological parameter to monitor when evaluating immunological therapies or genetic influences.

1. Mice are killed by CO2 inhalation. All four legs are pinned down on a Styrofoam board and the abdomen is cleaned with 70% ethanol.

2. Using forceps, an incision through the fur but not into the peritoneal cavity is made just below the sternum and extended bilaterally to the back. The fur is pulled upward and backward at the incision, exposing the abdominal wall. Any fur is washed away with the ethanol spray bottle.

3. An incision is made just below the sternum, exposing the liver and stomach.

4. The pancreas is just below the stomach with the tail of the pancreas attached to the spleen and the head of the pancreas is attached at the common bile duct near the ligament of Tritz. The spleen is the best landmark for finding the pancreas. Locate the spleen while approaching the abdomen from the left flank just above the kidney.

5. Carefully remove the spleen and you will see the tail of the pancreas attached to the right side of the spleen. The pancreas is somewhat darker yellow than the adipose tissue.

6. The pancreas is removed and fixed in 4% neutral-buffered paraformaldehyde and stained with hematoxylin and eosin and sectioned at three nonoverlapping levels.

7. Six to 10 islets per mouse were individually scored by two independent observers using a semiquantitative scale ranging from 0 to 4: 0, normal islet with no sign of T-cell infiltration; 1, islet associated with perivascular, periductal leukocytic infiltration only; 2, more extensive peri-islet infiltration but with lymphocytes with less than 25% islet destruction; 3, >25% islet destruction; 4, complete islet destruction (6).

8. An insulitis score for each mouse was obtained by dividing the total score for each mouse by the number of islets examined (6).

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