Streptozocin Treatment: A Zebrafish Model of Type 1 Diabetes Mellitus
Transcript
– Type 1 diabetes mellitus is a human disease in which the immune system destroys insulin making beta cells in the pancreas. Streptozocin can induce type 1 diabetes mellitus in zebrafish. First, anesthetize a fish in a solution of 2-phenoxyethanol in fish water. Take the fish and place it on a Petri dish, ventral side facing upwards. Calculate the dose of streptozocin based on 0.35 milligrams per gram of the fish's weight and dilute it in saline solution.
Administer the streptozocin by intraperitoneal injection below the pelvic girdle on the ventral side of the zebrafish. Streptozocin is structurally similar to N-acetylglucosamine, a ligand that binds the GLUT2 receptor on pancreatic beta cells, leading to secretion of insulin. Thus, streptozocin binds to GLUT2 receptors present on pancreatic beta cells which transport it inside the cells.
Upon entering the beta cells, streptozocin triggers an immune response leading to the release of autoantigens. The autoantigens cause the immune system to recognize beta cells as foreign and destroy them leading to reduced levels of insulin. Administer repeated doses of streptozocin on days three and five, followed by weekly doses, one every seven days to maintain the immune response.
In the following example, we will generate a zebrafish model of type 1 diabetes mellitus. To generate zebrafish with diabetes mellitus, prepare a recovery tank with normal fish water and an anesthetic tank of fish water with a 1 to 1,000 dilution of 2-phenoxyethanol. Under a fume hood, prepare a 0.3% solution of streptozocin or STZ by adding 6 milligrams of STZ to 2 milliliters of 0.09% sodium chloride and immediately place the solution on ice.
In a separate tube, aliquot enough saline for control fish. Fill a half CC syringe equipped with a 27 and 1/2 gauge needle with the STZ, or control solutions, ensuring that no air bubbles are trapped. Anaesthetize a single fish by placing it in anesthetic water and waiting until the swimming motion ceases, about one to two minutes.
Once anaesthetized, briefly place the fish on a paper towel to absorb any excess water. Then, place the fish in a weigh boat and weigh it. Next, place the fish on a firm surface. Then insert the needle past the bevel into the posterior aspect of the ventral peritoneum and inject either 0.35 milligrams per gram of STZ or an equivalent volume of control solution into the peritoneal cavity of the fish.
Following injection, place the fish in the recovery water tank and monitor it for normal swimming activity. After injecting enough fish for the experiment, transfer them to normal living tanks and maintain at a temperature of 22 to 24 degrees Celsius. The reduced temperature is critical for efficient induction of hyperglycemia. To induce a prolonged state of very high hyperglycemia, follow a schedule of frequent injections during the induction phase followed by weekly maintenance injections as shown here.
