The overall goal of the following procedure is to assess brain remodeling in hyperglycemic conditions, and to follow radio-labeled molecules in zebrafish. Hyperglycemia is characterized by an excessive concentration of blood glucose. Chronic hyperglycemia may be due to diabetic conditions, and causes different vascular dysfunctions, that may in turn trigger clinical complications, such as myocardial infarction, stroke, or diabetic foot ulcers.
Also, acute hyperglycemia due to stress may promote hemorrhagic complications in ischemic stroke. Zebrafish is an interesting model for understanding human diseases, and their impact on the central nervous system. It is particularly true, given that the brain of adult fish displays an intense neurogenic capacity, and an outstanding capability for brain repair mechanisms, compared to mammals, therefore, zebrafish represent good model for investigating brain remodeling, following stress under acute and hyperglycemic conditions.
It also allows investigating the biodistribution of radio-labeled molecules, and potential therapeutic agents. All experiments were conducted in accordance with the French and European community guidelines for the use of animals in research, and approved by the local ethics committee for animal experimentation. Catch a fish gently with a fish net.
Put the fish in a small volume of tricaine anesthetic, diluted at a final concentration of 0.02%in the water. Wait until the fish stops moving. Remove the fish, using a cut pipette, and place it gently on absorbent tissue paper to remove a maximum of water.
Weigh the fish in order to prepare a syringe of d-glucose for injection, of 50 microliters of 2.5 grams per kilo of body weight. For instance, a 0.6 gram fish will receive 50 microliters of a 3%d-glucose PBS solution. Put the fish on its back, and maintain it with one hand.
With the other hand, insert the needle of the syringe in the intraperitoneal cavity, and slowly inject the d-glucose PBS solution. Remove the needle, and put the fish back into the fish water. Check the fish until it recovers completely.
Their blood glucose levels are usually measured after 1 1/2 hours. In order to mimic chronic hyperglycemia, prepare a two liter tank of d-glucose, at a final concentration of 111 millimolar. For this, weigh 40 grams of d-glucose, and put it in an empty tank.
Fill the tank with two liters of fish water, and place up to seven fish in it. Change the glucose solution every two days, in order to avoid growth of bacteria, or other microorganisms. Chronic hyperglycemia is obtained by a 14 day treatment with d-glucose.
Catch a fish with a fish net, and put it on ice. Cover the fish with ice for a rapid euthanasia. Take the fish and wipe it, in order to remove any drops of water that could dilute blood samples.
Remove an eye with dissecting forceps, and wait until the eye cavity is filled with blood. Put a test strip on the glucometer, and insert the strip into the eye cavity. Note the value of the blood glucose concentration.
For acute hyperglycemia, an intraperitoneal injection of d-glucose induces a significant increase in blood glucose levels, as shown, 1 1/2 hours after injection. For chronic hyperglycemia, immersion of fish in a d-glucose solution results in a significant increase in blood glucose levels, as shown after 14 days of treatment. At the end of the procedure, separate the body from the head with dissecting scissors, and put the head in 4%of paraformaldehyde, diluted in PBS for immunohistochemistry experiments.
Incubate overnight at four degrees celsius. Alternatively, the brain can be directly extracted, and frozen for other experiments, such as mRNA extraction. The next day, fixed heads are rinsed with PBS, and maintained with a needle under a dissecting microscope.
The remaining eye is removed, and the top of the skull is gently opened with forceps. The brain is then carefully extracted, and placed in 1x PBS. Fixed brains are dehydrated stepwise, in a series of increasing ethanol concentrations, followed by two 30 minute baths of toluene.
Brains are then placed in an embedding cassette, and the lid is carefully closed. Put the cassette in melted paraffin, at 58 to 60 degrees celsius. During paraffin embedding of the brain, turn on the warming inclusion forceps.
Put the cassette into a clean paraffin bath. At the end of the paraffin baths, take out the cassette. Pour some liquid paraffin into a mold.
Open the cassette, and place the brain within melted paraffin in the mold. Orientate the brain with the warming inclusion forceps. Fill up the mold with melted paraffin, and let it harden on the cooling part of the embedding machine.
For technical reasons, brain should be positioned according to an anterior-posterior axis. After unmolding the paraffin block, crop it, and fix it on a cassette with melted paraffin, in the right orientation to allow transversal sectioning. Insert the paraffin block in the arm of a microtome, and cut 50 micrometer sections, until reaching the brain sample level.
Then, trim the paraffin block to obtain a trapeze, and adjust the sectioning thickness to seven micrometers. Collect the paraffin ribbons with a fine paint brush, and put it on a black paper. Cut the paraffin ribbons every three to four sections.
Put a slide on a warming plate, and cover it with distilled water. Position gently the sections on the water on the slide, and warm up to 40 degrees celsius. When paraffin ribbons are sufficiently spread, remove the water with absorbent tissue.
The last drops are mechanically removed, as shown. Let the slide dry at least two hours before performing the immunohistochemistry procedure described in the protocol. After immunohistochemistry, the slides are analyzed under an epifluorescence, or a confocal, microscope, for studying the impact of hyperglycemia on brain cell proliferation.
Brain cell quantification is made on at least three successive sections, or a region of interest. Here, proliferative cells corresponding to PCNA positive cells appear in green. As you can see, chronic hyperglycemia induces a decrease in the number of proliferative cells, labeled in green with PCNA antibody, in the ventral telencephalon, the dorsal telencephalon, and the caudal hypothalamus, around the lateral and posterior recess.
Alternatively, for studying the effects of hyperglycemia on injury induced neurogenesis, a stab wound injury of the telencephalon can be performed during chronic hyperglycemic treatment. For this, anesthetize a seven day treated fish, and put it under a dissecting microscope. Push a 30 gauge syringe vertically through the skull, into the center of the right telencephalic hemisphere.
After this procedure, the fish should be returned to its tank for the required time. Here, the fish was allowed to survive seven days in d-glucose water, before being sacrificed. Stab wound injury of the telencephalon strongly upregulates proliferation in the injured hemisphere, seven days post-lesion.
Our data show that chronic hyperglycemia induces a significant decrease in brain healing processes, by decreasing cell proliferation after stab wound injury of the telencephalon. Zebrafish can also be used for studying the biodistribution of radio-labeled molecules. Here, we decided to study glucose metabolism in the zebrafish organism.
The fish should be first anesthetized, and placed on a tissue soaked with anesthetics. The fish is then placed behind a radio protection window. The syringe containing 50 microliters of fluorodeoxyglucose is prepared.
The fish is positioned on its back, and injected intraperitoneally with 50 microliters of FDG at 20 megabecquerel. The fish is then placed on another tissue soaked with anesthetics. It is gently wrapped, and the positioned on the arm of a PET scan imager.
The arm is inserted into the PET scan, and the imaging procedure is performed. Here, you can note that FDG is not only detected at the injection site, but also in the head of the fish, notably, in the brain, and all along the spinal cord. In this study, we provide two different methods for establishing acute and chronic hyperglycemia in zebrafish, and we show that hyperglycemia impairs brain cell proliferation under normal and injury induced conditions.
In conclusion, zebrafish can be used as a relevant model to investigate the deleterious effects of hyperglycemia in the central nervous system. It also allows assessing the biodistribution of radio-labeled molecules by PET scan.
