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This protocol noninvasively induces hyperglycemia in zebrafish for up to 8 weeks. Using this protocol, an in-depth study of the adverse effects of hyperglycemia can be made.
Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This alternate immersion protocol is a noninvasive, step-wise method of inducing hyperglycemia for up to eight weeks. Adult zebrafish are alternately exposed to sugar (glucose) and water for 24 hours each. The zebrafish begin treatment in a 1% glucose solution for 2 weeks, then a 2% solution for 2 weeks, and finally a 3% solution for the remaining 4 weeks. Compared to water-treated (stress) and mannitol-treated (osmotic) controls, glucose-treated zebrafish have significantly higher blood sugar levels. The glucose-treated zebrafish show blood sugar levels of 3-times that of controls, suggesting that after both four and eight weeks hyperglycemia can be achieved. Sustained hyperglycemia was associated with increased Glial Fibrillary Acidic Protein (GFAP) and increased nuclear factor Kappa B (NF-kB) levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.
Zebrafish (Danio rerio) are quickly becoming a widely used animal model to study both disease and cognition1. The ease of genetic manipulation and embryonic transparency through the early developmental stages, make them a prime candidate to study human diseases with a known genetic basis. For example, zebrafish have been used to study Holt-Oram syndrome, cardiomyopathies, glomerulocystic kidney disease, muscular dystrophy, and diabetes mellitus (DM) among other diseases1. In addition, the zebrafish model is ideal because of the species’ small size, ease of maintenance, and high fecundity2,3.
The zebrafish pancreas is both anatomically and functionally similar to the mammalian pancreas4. Thus, the unique characteristics of size, high fecundity, and similar endocrine structures make zebrafish a suitable candidate for studying DM-related complications. In zebrafish, there are two experimental methods used to induce the prolonged hyperglycemia that is characteristic of DM: an influx of glucose (modeling Type 2) and cessation of insulin secretion (modeling Type 1)5,6. Experimentally, to stop insulin secretion, pancreatic β-cells can be chemically destroyed using either Streptozotocin (STZ) or Alloxan injections. STZ has been used successfully in rodents and zebrafish, resulting in complications associated with retinopathy7,8,9, cognitive impairments10, and limb regeneration11. However, in zebrafish, β-cells regenerate after treatment, causing “booster injections” of STZ to be necessary to maintain diabetic conditions12. Alternatively, the pancreas of the zebrafish can be removed6. These are both highly invasive procedures, due to the multiple injections, and extensive recovery time.
Conversely, hyperglycemia can be induced noninvasively through exposure to exogenous glucose. In this protocol, fish are submerged in a highly concentrated glucose solution for 24-hours5,13 or continually for 2-weeks14,15,16. Exogenous glucose is taken up transdermally, by ingestion, and/or across the gills resulting in elevated blood sugar levels. Since this non-invasive technique does not directly manipulate insulin levels, it cannot claim to induce Type 2 DM. However, it can be used to examine complications induced by hyperglycemia, which is one of the main symptoms of Type 2 DM.
Recently, the zebrafish mutant pdx1-/- was developed by manipulating the pancreatic and duodenal homeobox 1 gene, a gene linked to the genetic cause of Type 2 DM in humans. Using this mutant, researchers have been able to replicate pancreatic development disruption, high blood sugar, and study hyperglycemia-induced diabetic retinopathy17,18.
In this paper, we describe a noninvasive hyperglycemia induction method that uses an alternating immersion protocol. This protocol maintains hyperglycemic conditions for up to 8 weeks with subsequent complications observed. In brief, adult zebrafish are placed in a sugar solution for 24 hours and then a water solution for 24 hours. As opposed to continuous immersion in external glucose solutions, alternating days between sugar and water mimics the rise and fall of blood sugar in diabetes. An alternating glucose protocol additionally allows hyperglycemia to be induced for longer periods of time, as the zebrafish are not as able to compensate for the high external glucose conditions. As proof of principle, we provide data showing that hyperglycemia induced using this protocol alters retinal chemistry and physiology.
All procedures were approved by the Institutional Animal Care and Use Committee at American University.
1. Preparing the Solution Tanks
2. Preparing the fish
3. Transferring fish
4. Post-transfer solution preparation
5. Changing percentages
6. Measuring blood glucose levels and collecting tissue
Using this protocol (Figure 1), blood sugar values are significantly elevated after both 4-weeks and 8-weeks of treatment (Figure 2A), with hyperglycemia defined as 3x the control averages from both water-treated and mannitol-treated groups. Water-treated controls are transferred in and out of water daily, providing a stress/handling control. Mannitol serves as an osmotic control in in vitro glucose studies19,...
Diabetes is a nationwide problem. Studies show that by 2030, an estimated 400 million people will have some form of diabetes. In rodent models, Type 2 DM is studied using genetic manipulation. In rats, the Zucker diabetic fatty rats (ZDF), and the Otsuka Long-Evans Tokushima fatty rats (OLETF), are providing more information on the effects of Type 2 DM10. In addition, high fat diets have been used in rodent to induce hyperglycemia. This mirrors the noninvasive procedure proposed in this paper. Usi...
The authors declare no conflicts of interest.
We would like to acknowledge VPC, CJR, and MCP for the development of this protocol. EMM received financial support from the American University College of Arts and Sciences Graduate Student Support to carry out this research. This work was also supported by an American University Faculty Mellon Award and funding through the American University College of Arts and Sciences (both to VPC).
Name | Company | Catalog Number | Comments |
Airline Tubing | petsmart | 5291863 | This can be used in the tank to circulate air |
Airpump | petsmart | 5094984 | This can be used in the tank to circulate air |
Airstones | petsmart | 5149683 | This can be used in the tank to circulate air |
D-glucose | Sigma | G8270-5KG | |
D-mannitol | Acros Organics | AC125340050 | |
Freestyle Lite Meter | Amazon | B01LMOMLTU | |
Freestyle Lite Strips | Amazon | B074ZN3H2Z | |
Net | petsmart | 5175115 | |
Tanks | Amazon | B0002APZO4 |
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