The overall goal of this procedure is to generate a type one diabetes mellitus zebrafish model, which can be used for discovering the molecular basis of diabetes mellitus complications, and more importantly, determining the genetic basis for their persistence. This is accomplished by first inducing hyperglycemia with a series of injections of the diabetogenic drug STZ, and incubation of the fish at a reduced temperature. Next, after three weeks, fasting blood glucose levels are determined to ensure that the hyperglycemic state was induced and the drug is stopped to initiate beta cell recovery.
Then the caudel fin is amputated and allowed to regenerate to isolate genetically transmissible components of metabolic memory and to remove potentially complicating factors of the previous hyperglycemic environment. Finally, a fin regeneration assay is performed to document a persistent reduction in fin regeneration and identify induced changes in the transected tissue via an assay of interest. The main advantage of the diabetic zebrafish model over existing ones is that metabolic memory can be studied in a true U glycemic environment summer to what would be seen in patients after a pancreas transplant.
It is our expectation that the model will be utilized for the discovery of the epigenetic modifications that underlie metabolic memory and the persistence of diabetic complications To generate zebra fish with diabetes mellitus, prepare a recovery tank with normal fish water and an anesthetic tank of fish water with a one to 1000 dilution of two phenoxyethanol under a fume hood. Prepare a 0.3%solution of STREPTOZOTOCIN or STZ by adding six milligrams of STZ to two milliliters of 0.09%sodium chloride, and immediately placed the solution on ice in a separate tube. Aliquot enough saline for control.
Fish fill a half CC syringe equipped with a 27 and a half gauge needle with the STZ or control solutions, ensuring that no air bubbles are trapped. Anesthetize a single fish by placing it in anesthetic water and waiting until the swimming motion ceases about one to two minutes. Once anesthetized briefly placed the fish on a paper towel to absorb any excess water, then place the fish in a way 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 TZ 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 to collect blood. For determining FBGL, prepare a labeled PCR tube for each blood sample containing five microliters of normal saline.
After anesthetizing the fish blot the excess water, place the fish on a microscope slide and using a scalpel, remove the head at the base of the operculom, collect the blood that is released on the slide and quickly add it to the PCR tube of sterile, normal saline pipetting up and down to make sure that the blood does not clot immediately place the sample on ice. Determine the blood sample volume by measuring the total volume of the liquid in the tube and subtracting out the five microliters of saline. Transfer five microliters of the diluted blood from each PCR tube into a 1.5 milliliter micro fuge tube, and use the quantum chrome glucose assay kit according to the manufacturer's instructions to determine the blood glucose concentration.
After anesthetizing a fish, place it in a Petri dish and under a dissecting scope, use a sterile size 10 scalpel to amputate the coddle fin by cutting a straight line proximal to the first lipid trachea branching point for the regenerative growth phase of the assay. Place the fish in a recovery tank at 33 degrees Celsius to image regenerating fins. After anesthetizing a fish, spread the fin so that it is fully extended and use a dissecting scope equipped with a camera and NIS element software.
Collect images at one x magnification to measure regenerative growth. Print the images and use image J software and a drawing pad to trace around the whole area of new growth for accuracy of the tracing. Ensure that no shadows or water droplets are present and take each measurement five times to calculate the average.
To normalize the measurements, measure the length of the amputation site along the dorsal ventral axis and divide the previously determined area by this measurement. After generating a group of DM FISH and their controls at 21 days, determine the FBGL for a subset of the group and divide the DM fish into two subgroups for the duration of the experiment. Continue weekly STZ injections for one of the groups as DM controls for the second group cease STZ injections and incubate the fish at normal temperature.
Within 14 days, these zebra fish will restore normal blood insulin and glucose control through pancreas regeneration. These fish are now called metabolic memory or MM, fish at day 30 post drug removal amputate the coddle fins of control DM and MM fish as demonstrated earlier in this video to allow the growth of metabolic memory tissue. Return the fish to normal water conditions for fin regeneration for 30 days at day 60.
Perform a second amputation within the tissue that was regenerated in the period from 30 to 60 days and perform a coddle fin regeneration assay, isolate the tissue and perform an assay of interest. Type one diabetic zebrafish not only display the known secondary complications of retinopathy and nephropathy, but also exhibit an additional complication impaired coddle fin regeneration as can be seen here at 72 hours post amputation. This complication persists due to metabolic memory in fish that have restored normal glucose control following a hyperglycemic period as shown here, the mm zebra fish exhibit a deficit in fin regeneration of approximately 40%when compared to control fish, and the impairment has been observed as far out as 150 days post amputation.
After watching this video, you should have a good understanding of how to initiate the hyperglycemic state in the zebra fish, measure fasting blood glucose levels, perform fin regeneration studies, and ultimately generate metabolic memory fish.
