Streptozocin Treatment: A Zebrafish Model of Type 1 Diabetes Mellitus

1. Generation of Zebrafish with Diabetes mellitus, DM Fish

1. Prepare both recovery and anesthetic water tanks. Recovery water is normal fish water. For anesthetic water add sufficient 2-phenoxyethanol so that a 1:1,000 dilution in normal fish water is achieved.
2. Prepare a 0.3% solution (in a fume hood) of streptozocin (STZ) by adding 6 mg of STZ to 2 ml of 0.09% sodium chloride and immediately place the solution on ice . This will provide enough injection solution for the injection of approximately 20 fish in 20 min. If you exceed the 20 minute mark, stop, and make a fresh STZ solution before proceeding. In a separate tube aliquot enough saline solution for control fish. Once the STZ is solubilized all subsequent steps do not require fume hood use.
3. Fill a ½ cc syringe equipped with a 27 1/2 gauge needle with the STZ or control solutions ensuring that no air bubbles are trapped.
4. Anesthetize each fish individually by placing the fish in anesthetic water, and wait until their swimming motion ceases (1-2 min).
5. Once anesthetized briefly place the fish on a paper towel to absorb any excess water, place the fish in weigh boat and measure the mass of the fish.
6. Place the fish on a firm surface (Petri dish lid) for injection.
7. Inject the STZ or control solution into the peritoneal cavity of the fish by inserting the needle past the bevel into the posterior aspect of the ventral peritoneum.
8. 0.35 mg/g (350 mg/kg) of STZ should be delivered to each fish and the volume of the 0.3% solution required can be calculated in the following manner.
   1. Multiply mass of fish (g) by 0.35 to yield the amount of STZ in mg required.
   2. divide the product generated above by 3 to yield the volume of the 0.3% solution required for injection in μl.
      Sample: For a 0.5 g fish : a) 0.5 x 0.35 = 0.175 b) .0175/3= 0.058 ml = 58 μl.
      The same volume without the STZ would be injected for a control fish. A sheet for volumes to inject per fish mass should be generated and used for quick reference.
9. Following injection place the fish in the recovery water tank and monitor them for normal swimming activity. Once this has been achieved the fish are transferred to a normal living tank that is maintained at the reduced temperature range of 22 °C – 24 °C. This reduced temperature is critical for efficient induction of hyperglycemia (diabetes mellitus, DM).
10. Although hyperglycemia is detected within 24 hr of the first injection, in order to induce a prolonged state of very high hyperglycemia the zebrafish require a frequent injection induction phase followed by weekly maintenance injections as shown below.

Week 1: 3 injections (Day 1, 3, 5), Week 2: 1 injection (Day 12), Week 3: 1 injection (Day 19), Week 4: (Day 21) Perform assay of interest.

At this point the zebrafish are considered to have been in a prolonged state of hyperglycemia and exhibit the diabetic complications of retinopathy, nephropathy and also impaired fin regeneration. These are referred to as DM fish. Additionally if desired the fish can be maintained in the hyperglycemic state with weekly maintenance injections. Approximately 5% death during this process should be expected.