Name: alternate immersion in glucose to produce prolonged hyperglycemia in zebrafish
Uploaded: 2021-05-05T15:00:00
Description: Watch this Scientific Journal Video about Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish at JoVE.com

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.&#160;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).

All procedures were approved by the Institutional Animal Care and Use Committee at American University.
1. Preparing the Solution Tanks
<ol>
	<li>Obtain six tanks, two for each experimental group (glucose, mannitol, and water). Label one of the two tanks &#8216;housing tank&#8217; (it will house the fish) and label the other &#8216;solution tank&#8217; (it will hold the solution). 
	NOTE: The mannitol treatment group is the osmotic control, and the water treatment group is a handling/st...

<ol>
	<li>Rubinstein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+from+disease+modeling+to+drug+discovery.">Zebrafish: from disease modeling to drug discovery.</a> Current Opinion in Drug Discovery and Development. 6 (2), 218-223 (2003).</li><li>Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+learning+in+zebrafish+(Danio+rerio).">Associative learning in zebrafish (Danio rerio).</a> Methods in Cell Biology. 101, 249-270 (2011).</li><li>Goldsmith, J. R., Jobin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Think+small:+zebrafish+as+a+model+system+of+human+pathology.">Think small: zebrafish as a model system of human pathology.</a> BioMed Research International. , (2012).</li><li>Moss, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+pancreas+in+adult+zebrafish.">Regeneration of the pancreas in adult zebrafish.</a> Diabetes. 58 (8), 1844-1851 (2009).</li><li>Connaughton, V. P., Baker, C., Fonde, L., Gerardi, E., Slack, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternate+immersion+in+an+external+glucose+solution+differentially+affects+blood+sugar+values+in+older+versus+younger+zebrafish+adults.">Alternate immersion in an external glucose solution differentially affects blood sugar values in older versus younger zebrafish adults.</a> Zebrafish. 13 (2), 87-94 (2016).</li><li>Etuk, E. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+studying+diabetes+mellitus.">Animal models for studying diabetes mellitus.</a> Agriculture and Biology Journal of North America. 1 (2), 130-134 (2010).</li><li>Agardh, E., Bruun, A., Agardh, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+glial+cell+immunoreactivity+and+neuronal+cell+changes+in+rats+with+STZ-induced+diabetes.">Retinal glial cell immunoreactivity and neuronal cell changes in rats with STZ-induced diabetes.</a> Current Eye Research. 23 (4), 276-284 (2001).</li><li>Carmo, A., Cunha-Vaz, J. G., Carvalho, A. P., Lopes, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthase+activity+in+retinas+from+non-insulin-dependent+diabetic+Goto-Kakizaki+rats:+correlation+with+blood-retinal+barrier+permeability.">Nitric oxide synthase activity in retinas from non-insulin-dependent diabetic Goto-Kakizaki rats: correlation with blood-retinal barrier permeability.</a> Nitric Oxide. 4 (6), 590-596 (2000).</li><li>Ramsey, D. J., Ripps, H., Qian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electrophysiological+study+of+retinal+function+in+the+diabetic+female+rat.">An electrophysiological study of retinal function in the diabetic female rat.</a> Investigative Ophthalmology &amp; Visual Science. 47 (11), 5116-5124 (2006).</li><li>Biessels, G. J., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+diabetes+on+cognition:+what+can+be+learned+from+rodent+models.">The impact of diabetes on cognition: what can be learned from rodent models.</a> Neurobiology of Aging. 26 (1), 36-41 (2005).</li><li>Intine, R. V., Olsen, A. S., Sarras, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zebrafish+model+of+diabetes+mellitus+and+metabolic+memory.">A zebrafish model of diabetes mellitus and metabolic memory.</a> Journal of Visualized Experiments. (72), e50232 (2013).</li><li>Sarras, M. P., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+zebrafish+as+a+disease+model+provides+a+unique+window+for+understanding+the+molecular+basis+of+diabetic+metabolic+memory.">Use of zebrafish as a disease model provides a unique window for understanding the molecular basis of diabetic metabolic memory.</a> Research on Diabetes. , (2013).</li><li>Gleeson, M., Connaughton, V., Arneson, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+hyperglycaemia+in+zebrafish+(Danio+rerio)+leads+to+morphological+changes+in+the+retina.">Induction of hyperglycaemia in zebrafish (Danio rerio) leads to morphological changes in the retina.</a> Acta Diabetologica. 44 (3), 157-163 (2007).</li><li>Capiotti, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+induces+memory+impairment+linked+to+increased+acetylcholinesterase+activity+in+zebrafish+(Danio+rerio).">Hyperglycemia induces memory impairment linked to increased acetylcholinesterase activity in zebrafish (Danio rerio).</a> Behavioural Brain Research. 274, 319-325 (2014).</li><li>Capiotti, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+impaired+glucose+metabolism+in+a+zebrafish+hyperglycemia+model.">Persistent impaired glucose metabolism in a zebrafish hyperglycemia model.</a> Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 171, 58-65 (2014).</li><li>Capiotti, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+alters+E-NTPDases,+ecto-5'-nucleotidase,+and+ectosolic+and+cytosolic+adenosine+deaminase+activities+and+expression+from+encephala+of+adult+zebrafish+(Danio+rerio).">Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).</a> Purinergic Signaling. 12 (2), 211-220 (2016).</li><li>Ali, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+Degeneration+Accompanies+Vascular+Changes+in+a+Zebrafish+Model+of+Diabetic+Retinopathy.">Photoreceptor Degeneration Accompanies Vascular Changes in a Zebrafish Model of Diabetic Retinopathy.</a> Investigative Ophthalmology &amp; Visual Science. 61 (2), 43 (2020).</li><li>Wiggenhauser, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Retinal+Angiogenesis+in+Hyperglycemic+pdx1-/-+Mutants.">Activation of Retinal Angiogenesis in Hyperglycemic pdx1-/- Mutants.</a> Diabetes. 69 (5), 1020-1031 (2020).</li><li>Chen, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+HMGB1+mediated+signalling+pathway+in+diabetic+retinopathy:+evidence+from+type+2+diabetic+rats+and+ARPE-19+cells+under+diabetic+condition.">Involvement of HMGB1 mediated signalling pathway in diabetic retinopathy: evidence from type 2 diabetic rats and ARPE-19 cells under diabetic condition.</a> Journal of Ophthalmology. 97, 1598-1603 (2013).</li><li>Costa, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+light+exposure,+pH,+osmolarity,+and+solvent+on+the+retinal+pigment+epithelial+toxicity+of+vital+dyes.">Effects of light exposure, pH, osmolarity, and solvent on the retinal pigment epithelial toxicity of vital dyes.</a> American Journal of Ophthalmology. 155, 705-712 (2013).</li><li>Alvarez, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+cone+photoreceptor+dysfunction+in+a+hyperglycemic+model+of+non-proliferative+diabetic+retinopathy.">Predominant cone photoreceptor dysfunction in a hyperglycemic model of non-proliferative diabetic retinopathy.</a> Disease Models and Mechanisms. 3, 236-245 (2010).</li><li>Fletcher, E. L., Phipps, J. A., Wilkinson-Berka, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunction+of+retinal+neurons+and+glia+during+diabetes.">Dysfunction of retinal neurons and glia during diabetes.</a> Clinical and Experimental Optometry. 88, 132-145 (2005).</li><li>Fletcher, E. L., Phipps, J. A., Ward, M. M., Puthussery, T., Wilkinson-Berka, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+glial+abnormality+as+predictors+of+progression+of+diabetic+retinopathy.">Neuronal and glial abnormality as predictors of progression of diabetic retinopathy.</a> Current Pharmaceutical Design. 13, 2699-2712 (2007).</li><li>Agardh, E., Bruun, A., Agardh, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+glial+cell+immunoreactivity+and+neuronal+cell+changes+in+rats+with+STZ-+induced+diabetes.">Retinal glial cell immunoreactivity and neuronal cell changes in rats with STZ- induced diabetes.</a> Current Eye Research. 23, 276-284 (2001).</li><li>Barber, A. J., Antonetti, D. A., Gardner, T. W., Group, T. P. S. R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+expression+of+retinal+occludin+and+glial+fibrillary+acidic+protein+in+experimental+diabetes.">Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes.</a> Investigative Ophthalmology &amp; Visual Science. 41, 3561-3568 (2000).</li><li>Lieth, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+reactivity+and+impaired+glutamate+metabolism+in+short-term+experimental+diabetic+retinopathy.">Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy.</a> Diabetes. 47, 815-820 (1998).</li><li>Rungger-Brandle, E., Dosso, A. A., Leuenberger, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+reactivity,+an+early+feature+of+diabetic+retinopathy.">Glial reactivity, an early feature of diabetic retinopathy.</a> Investigative Ophthalmology &amp; Visual Science. 41, 1971-1980 (2000).</li><li>Zeng, X. X., Ng, Y. K., Ling, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+microglial+response+in+the+retina+of+streptozotocin-induced+diabetic+rats.">Neuronal and microglial response in the retina of streptozotocin-induced diabetic rats.</a> Visual Neuroscience. 17, 463-471 (2000).</li><li>Mizutani, M., Gerhardinger, C., Lorenzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muller+cell+changes+in+human+diabetic+retinopathy.">Muller cell changes in human diabetic retinopathy.</a> Diabetes. 47, 445-449 (1998).</li><li>Tanvir, Z., Nelson, R., DeCicco-Skinner, K., Connaughton, V. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potentials+and+the+b-wave:+partial+masking+and+interdependence+in+dark+adaptation+and+diabetes+in+the+rat.">Oscillatory potentials and the b-wave: partial masking and interdependence in dark adaptation and diabetes in the rat.</a> Graefe's Archives for Clinical and Experimental Ophthalmology. 245, 1335-1345 (2007).</li><li>Li, Q., Zemel, E., Miller, B., Perlman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+retinal+damage+in+experimental+diabetes:+electroretinographical+and+morphological+observations.">Early retinal damage in experimental diabetes: electroretinographical and morphological observations.</a> Experimental Eye Research. 74, 615-625 (2002).</li><li>Kohzaki, K., Vingrys, A. J., Bui, B. 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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...

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&#8217; 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 &#946;-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, &#946;-cells regenerate after treatment, causing &#8220;booster injections&#8221; 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.

<table>
	<tbody>
		<tr>
			<td>Airline Tubing</td>
			<td>petsmart</td>
			<td>5291863</td>
			<td>This can be used in the tank to circulate air</td>
		</tr>
		<tr>
			<td>Airpump</td>
			<td>petsmart</td>
			<td>5094984</td>
			<td>This can be used in the tank to circulate air</td>
		</tr>
		<tr>
			<td>Airstones</td>
			<td>petsmart</td>
			<td>5149683</td>
			<td>This can be used in the tank to circulate air</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8270-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Acros Organics</td>
			<td>AC125340050</td>
			<td></td>
		</tr>
		<tr>
			<td>Freestyle Lite Meter</td>
			<td>Amazon</td>
			<td>B01LMOMLTU</td>
			<td></td>
		</tr>
		<tr>
			<td>Freestyle Lite Strips</td>
			<td>Amazon</td>
			<td>B074ZN3H2Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Net</td>
			<td>petsmart</td>
			<td>5175115</td>
			<td></td>
		</tr>
		<tr>
			<td>Tanks</td>
			<td>Amazon</td>
			<td>B0002APZO4</td>
			<td></td>
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alternate immersion in glucose to produce prolonged hyperglycemia in zebrafish

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)&#160;levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.

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,...

Watch this Scientific Journal Video about Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish at JoVE.com

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

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 ...

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