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/stress control. It is important to keep the tanks, airlines/airstones, lids, and cleaning supplies separate for each treatment group</li>
	<li>Use a 2 L tank if the total number of fish used is less than 20. Use a 4 L tank if the total number of fish used is more than 20. 
	NOTE: Use an N of 5-10 per treatment group per sampling time point.</li>
	<li>Keep the tanks in a water bath at 28-29 &#176;C to maintain water temperature.</li>
	<li>On Day 1, place the fish into their respective treatment solutions (glucose, mannitol, water) for 24-hours (&#8216;Water to Treatment&#8217;). On Day 2, transfer the fish from their treatment solutions to water for 24-hours (&#8216;Treatment to Water&#8217;). On Day 3, transfer the fish from water to treatment solutions (&#8216;Water to Treatment&#8217;). This alternating exposure continues for the remainder of the experiment (Figure 1). Transfer water-treated control fish from water to water daily.</li>
	<li>Ensure that the fish are fed and transferred within the same 2-hour window each day throughout the duration of the experiment.</li>
</ol>
2. Preparing the fish
<ol>
	<li>Use adult zebrafish (4 months &#8211; 1 year)5.</li>
	<li>Feed the fish ground Tetramin flakes daily upon arrival to the lab.</li>
	<li>Record the pH and the temperature of all the tanks and record the general condition of the fish.</li>
</ol>
3. Transferring fish
<ol>
	<li>Transfer fish in each treatment group from the housing tank to the corresponding solution tank using a standard fish net.</li>
	<li>Place the tank containing the fish back in the water bath, replace the airstone and tank lid. This tank is now the &#8216;housing tank&#8217; and the tank that previously held the fish is now the &#8216;solution tank&#8217;.</li>
	<li>Discard the old solution and clean the tank, along with the tank lids, airlines, airstones, and nets to prevent buildup of glucose and mannitol. 
	NOTE: Do not wash items with soap. Use water and a dedicated scrub brush/sponge for each treatment condition to properly clean the tanks.</li>
	<li>Dry the newly cleaned &#8216;solution tanks&#8217; with a paper towel. Prepare the solutions for the following day using this tank. Ensure the other items are dried and separated by appropriate treatment groups. 
	NOTE: Keep a log of what solutions the fish are being transferred out of and into each day, as well as the solutions that are prepared for the following day. For example: Fish transferred from glucose to H2O, new 1% glucose solution prepared for tomorrow.</li>
</ol>
4. Post-transfer solution preparation
<ol>
	<li>Preparing sugar solutions
	<ol>
		<li>Fill each solution tank with 2 L (or 4 L) of System Water (system water is defined as water that has been treated with the correct ratio of salt solution and is at the same temperature as stock and treatment tanks).</li>
		<li>Measure the correct amount of glucose and mannitol (see step 5 below) using a top loading scale and separate weigh boats for each chemical.</li>
		<li>Add the weighed glucose or mannitol aliquot to the appropriate, cleaned solution tank, which contains only system water.</li>
		<li>Stir the glucose and mannitol solutions with separate glass stir rods until the sugars are completely dissolved.</li>
		<li>Return solution tanks to the water bath and cover with their corresponding lids.</li>
	</ol>
	</li>
	<li>Preparing water solutions
	<ol>
		<li>Fill experimental tanks (2 L or 4 L) with System Water.</li>
		<li>Return these &#8216;solution tanks&#8217; to the water bath and cover with their corresponding lids.</li>
	</ol>
	</li>
</ol>
5. Changing percentages
<ol>
	<li>Maintain the fish in a 1% solution during the first 2-weeks of treatment: 40 g of glucose or mannitol in a 4 L tank.</li>
	<li>Maintain the fish in a 2% solution during weeks 3 and 4 of treatment: 80 g of glucose or mannitol in a 4 L tank.</li>
	<li>Maintain the fish in a 3% solution for the final 4 weeks of treatment: 120 g of glucose or mannitol in a 4 L tank.</li>
</ol>
6. Measuring blood glucose levels and collecting tissue
<ol>
	<li>Anesthetize fish 2 at a time in a 0.02% Tricaine solution.</li>
	<li>Decapitate the fish directly behind the gills using a razorblade.</li>
	<li>Measure blood sugar value. 
	NOTE: We use a blood glucose meter (e.g., Freestyle Lite) to measure blood glucose and place the test strip directly on the exposed heart (cardiac blood sample).</li>
	<li>Dissect the wanted tissue from the fish (brain, muscle, etc.).</li>
	<li>Store collected tissue by flash freezing on dry ice and storing in a -80 &#176;C freezer, fixing in 4% paraformaldehyde, or placing in a buffer solution for immediate use.</li>
</ol>

<ol><li>Rubinstein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish%3A+from+disease+modeling+to+drug+discovery%5BTitle%5D)+AND+%22Current+Opinion+in+Drug+Discovery+and+Development%22%5BJournal%5D)">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/pubmed?term=((Associative+learning+in+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Think+small%3A+zebrafish+as+a+model+system+of+human+pathology%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">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/pubmed?term=((Regeneration+of+the+pancreas+in+adult+zebrafish%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((Alternate+immersion+in+an+external+glucose+solution+differentially+affects+blood+sugar+values+in+older+versus+younger+zebrafish+adults%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">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/pubmed?term=((Animal+models+for+studying+diabetes+mellitus%5BTitle%5D)+AND+%22Agriculture+and+Biology+Journal+of+North+America%22%5BJournal%5D)">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/pubmed?term=((Retinal+glial+cell+immunoreactivity+and+neuronal+cell+changes+in+rats+with+STZ-induced+diabetes%5BTitle%5D)+AND+%22Current+Eye+Research%22%5BJournal%5D)">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/pubmed?term=((Nitric+oxide+synthase+activity+in+retinas+from+non-insulin-dependent+diabetic+Goto-Kakizaki+rats%3A+correlation+with+blood-retinal+barrier+permeability%5BTitle%5D)+AND+%22Nitric+Oxide%22%5BJournal%5D)">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/pubmed?term=((An+electrophysiological+study+of+retinal+function+in+the+diabetic+female+rat%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">An electrophysiological study of retinal function in the diabetic female rat.</a> Investigative Ophthalmology & 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/pubmed?term=((The+impact+of+diabetes+on+cognition%3A+what+can+be+learned+from+rodent+models%5BTitle%5D)+AND+%22Neurobiology+of+Aging%22%5BJournal%5D)">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/pubmed?term=((A+zebrafish+model+of+diabetes+mellitus+and+metabolic+memory%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Use+of+zebrafish+as+a+disease+model+provides+a+unique+window+for+understanding+the+molecular+basis+of+diabetic+metabolic+memory%5BTitle%5D)+AND+%22Research+on+Diabetes%22%5BJournal%5D)">Use of zebrafish as a disease model provides a unique window for understanding the molecular basis of diabetic metabolic memory.</a> Research on Diabetes. , iConcept Press Ltd. Hong Kong. (2013).</li>
<li>Gleeson, M., Connaughton, V., Arneson, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+hyperglycaemia+in+zebrafish+%28Danio+rerio%29+leads+to+morphological+changes+in+the+retina%5BTitle%5D)+AND+%22Acta+Diabetologica%22%5BJournal%5D)">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/pubmed?term=((Hyperglycemia+induces+memory+impairment+linked+to+increased+acetylcholinesterase+activity+in+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">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/pubmed?term=((Persistent+impaired+glucose+metabolism+in+a+zebrafish+hyperglycemia+model%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+Part+B%3A+Biochemistry+and+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Hyperglycemia+alters+E-NTPDases%2C+ecto-5%27-nucleotidase%2C+and+ectosolic+and+cytosolic+adenosine+deaminase+activities+and+expression+from+encephala+of+adult+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22Purinergic+Signaling%22%5BJournal%5D)">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/pubmed?term=((Photoreceptor+Degeneration+Accompanies+Vascular+Changes+in+a+Zebrafish+Model+of+Diabetic+Retinopathy%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Photoreceptor Degeneration Accompanies Vascular Changes in a Zebrafish Model of Diabetic Retinopathy.</a> Investigative Ophthalmology & Visual Science. 61 (2), 43(2020).</li>
<li>Wiggenhauser, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+Retinal+Angiogenesis+in+Hyperglycemic+pdx1-%2F-+Mutants%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((Involvement+of+HMGB1+mediated+signalling+pathway+in+diabetic+retinopathy%3A+evidence+from+type+2+diabetic+rats+and+ARPE-19+cells+under+diabetic+condition%5BTitle%5D)+AND+%22Journal+of+Ophthalmology%22%5BJournal%5D)">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/pubmed?term=((Effects+of+light+exposure%2C+pH%2C+osmolarity%2C+and+solvent+on+the+retinal+pigment+epithelial+toxicity+of+vital+dyes%5BTitle%5D)+AND+%22American+Journal+of+Ophthalmology%22%5BJournal%5D)">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/pubmed?term=((Predominant+cone+photoreceptor+dysfunction+in+a+hyperglycemic+model+of+non-proliferative+diabetic+retinopathy%5BTitle%5D)+AND+%22Disease+Models+and+Mechanisms%22%5BJournal%5D)">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/pubmed?term=((Dysfunction+of+retinal+neurons+and+glia+during+diabetes%5BTitle%5D)+AND+%22Clinical+and+Experimental+Optometry%22%5BJournal%5D)">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/pubmed?term=((Neuronal+and+glial+abnormality+as+predictors+of+progression+of+diabetic+retinopathy%5BTitle%5D)+AND+%22Current+Pharmaceutical+Design%22%5BJournal%5D)">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/pubmed?term=((Retinal+glial+cell+immunoreactivity+and+neuronal+cell+changes+in+rats+with+STZ-+induced+diabetes%5BTitle%5D)+AND+%22Current+Eye+Research%22%5BJournal%5D)">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/pubmed?term=((Altered+expression+of+retinal+occludin+and+glial+fibrillary+acidic+protein+in+experimental+diabetes%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes.</a> Investigative Ophthalmology & Visual Science. 41, 3561-3568 (2000).</li>
<li>Lieth, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glial+reactivity+and+impaired+glutamate+metabolism+in+short-term+experimental+diabetic+retinopathy%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((Glial+reactivity%2C+an+early+feature+of+diabetic+retinopathy%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Glial reactivity, an early feature of diabetic retinopathy.</a> Investigative Ophthalmology & 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/pubmed?term=((Neuronal+and+microglial+response+in+the+retina+of+streptozotocin-induced+diabetic+rats%5BTitle%5D)+AND+%22Visual+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Muller+cell+changes+in+human+diabetic+retinopathy%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Muller cell changes in human diabetic retinopathy.</a> Diabetes. 47, 445-449 (1998).</li>
<li>Tanvir, Z., Nelson, R., DeCicco-Skinner, K., Connaughton, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One+month+of+hyperglycemia+alters+spectral+responses+of+the+zebrafish+photopic+electroretinogram%5BTitle%5D)+AND+%22Disease+Models+and+Mechanisms%22%5BJournal%5D)">One month of hyperglycemia alters spectral responses of the zebrafish photopic electroretinogram.</a> Disease Models and Mechanisms. 11, (2018).</li>
<li>Hancock, H. A., Kraft, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oscillatory+potential+analysis+and+ERGs+of+normal+and+diabetic+rats%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Oscillatory potential analysis and ERGs of normal and diabetic rats.</a> Investigative Ophthalmology & Visual Science. 45, 1002-1008 (2004).</li>
<li>Layton, C. J., Safa, R., Osborne, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oscillatory+potentials+and+the+b-wave%3A+partial+masking+and+interdependence+in+dark+adaptation+and+diabetes+in+the+rat%5BTitle%5D)+AND+%22Graefe%27s+Archives+for+Clinical+and+Experimental+Ophthalmology%22%5BJournal%5D)">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/pubmed?term=((Early+retinal+damage+in+experimental+diabetes%3A+electroretinographical+and+morphological+observations%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">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. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+inner+retinal+dysfunction+in+streptozotocin-induced+diabetic+rats%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Early inner retinal dysfunction in streptozotocin-induced diabetic rats.</a> Investigative Ophthalmology & Visual Science. 49, 3595-3604 (2008).</li>
<li>Phipps, J. A., Yee, P., Fletcher, E. L., Vingrys, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rod+photoreceptor+dysfunction+in+diabetes%3A+activation%2C+deactivation%2C+and+dark+adaptation%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Rod photoreceptor dysfunction in diabetes: activation, deactivation, and dark adaptation.</a> Investigative Ophthalmology & Visual Science. 47, 3187-3194 (2006).</li>
</ol>

The authors declare no conflicts of interest.

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. Using our noninvasive protocol, we can induce hyperglycemia for up to 8-weeks, therefore simulating prolonged hyperglycemia in otherwise healthy zebrafish.
After blood sugar is measured, tissue (retina and brain) can be collected for subsequent analysis. Physiological differences, such as electroretinogram (ERG) recordings, can be measured directly from retinal eye cups21,30. Behavioral responses (i.e., optomotor responses or cognitive differences (Rowe et al., 2020) can be assessed prior to blood sugar measurement. For Western Blot determination of protein levels, tissue is placed into RIPA buffer or flash frozen and stored at -80 &#176;C. Due to differences in size/protein content, several retinas may have to be pooled prior to analysis. For immunocytochemistry, tissue is fixed in a 4% paraformaldehyde solution for 24 hours and then equilibrated in a 30% sucrose solution prior to until frozen sectioning (20 &#956;m).
To optimize results, carefully weigh out both mannitol and glucose, and make sure that the solutions are thoroughly mixed. It is also very important to keep an active schedule of the transfer days, to make sure sugar and water are alternating daily and at the same time of day. Furthermore, make sure to carefully measure out the water as too little or too much may change the concentration of the solution. Lastly, carefully monitor the pH and the temperature of the solutions, as transferring fish into extreme solutions can shock the fish and cause mortality. There has been no evidence that prolonged glucose exposure or high concentrations of glucose, when exposed gradually, has toxic effects on zebrafish. If the protocol is followed correctly, no extraneous deaths of the zebrafish should be expected.
While this is a proven method of inducing hyperglycemia, a limitation of this protocol is that one cannot ascertain that the fish are hyperglycemic until after they are sacrificed. Another limitation is that we do not assess the pancreas or insulin levels and, therefore, we cannot claim to induce Type 2 Diabetes, only that we induce hyperglycemia. However, this is also what makes this procedure better than competing methods: it is non-invasive. We have observed complications of prolonged hyperglycemia induced by this protocol that are reported in rodent models and diabetic individuals. In the future, it is possible that we can use this procedure to look at therapeutic techniques that help alleviate symptoms of DM, such as retinopathy.
In summary, this noninvasive alternating immersion protocol is an effective way to induce hyperglycemia for up to 8 weeks. This method is a powerful tool that allows for the complications of chronic hyperglycemia to be examined and subsequent determination of therapeutic treatments. It also offers the opportunity to compare biomedical research findings across model organisms.

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.

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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 studies19,20, as it is a 6-carbon sugar like glucose but is not taken up by cells. To be consistent with those studies, and other studies in zebrafish21, we administered mannitol in the same concentrations as glucose to determine if observed effects were due to the high osmolarity resulting from glucose exposure or a glucose-specific effect.
Blood sugar is measured by anesthetizing the fish using 0.02% Tricaine until gill movements have slowed, and then decapitating. Blood glucose levels, measured with a blood glucose meter (Figure 2B), are determined from placing the glucometer test strip directly on the punctured heart (i.e., cardiac blood).
Retinal tissue collected after 4-weeks of hyperglycemia displays an increase in Glial Fibrillary Acidic Protein (GFAP) levels (Figure 3A). GFAP expression is observed in Muller glial cells in the retina, which are altered in diabetic retinopathy22,23. Increased GFAP content and/or immunoreactivity patterns are also observed in STZ-induced diabetic rats24,25,26,27,28, pdx1-/- mutant fish17, and in retinas from diabetic humans29. This increase in GFAP is associated with an increase in nuclear factor Kappa B (NF-kB) levels (Figure 3B)30, suggesting the hyperglycemia induced in zebrafish using the alternate immersion protocol triggers an inflammatory response and reactive gliosis. ERG recordings after 4-weeks of treatment identified a decreased response in glucose-treated retinas compared to mannitol-treated controls (Figure 4A). Amplitudes of both a-wave (photoreceptor) and b-wave (bipolar cells) components are decreased in hyperglycemic fish (Figure 4B). These altered ERG responses are correlated to specific changes in red and/or green cones30,21, which appear particularly sensitive to hyperglycemic insult. Altered ERG responses are also observed in animal models of diabetes31,32,33,34,35 and diabetic humans. Glucose-treated zebrafish also show decreased cognitive performance (see Rowe et al., 2020, in this issue), suggesting prolonged hyperglycemia also leads to cognitive function deficits which is also reported in older diabetic patients.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61935/61935fig01v2.jpg" /> 
Figure 1: Schematic of the alternate immersion protocol.&#160;This is a visual representation of the transfer process. Fish are maintained in 1% solution for 2-weeks, 2% solution for 2 weeks, and then 3% solution for the remaining 4 weeks. Each day, fish are transferred into either sugar or water solution. The control treatments transfer&#160;fish in and out of water (0% glucose - handling control) every 24 hours or into and out of mannitol (osmotic control), with mannitol concentrations paralleling those used for glucose. We have measured blood glucose levels, performed experiments, and collected tissue after 4 and 8 weeks of treatment (boxed). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61935/61935fig01v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61935/61935fig02v2.jpg" /> 
Figure 2: Blood Glucose Levels are elevated after 4 and 8 weeks of treatment.&#160;(A) The glucose-treated fish have more than 3x the amount of blood sugar compared to water- and mannitol-treated control fish, a significant increase (p = 0.029 at 4-weeks; p &#60; 0.001 at 8-weeks). This means that after both 4- and 8-weeks the zebrafish treated with glucose were hyperglycemic. Data were collected from n = 5 mannitol treated fish, n = 8 glucose-treated fish, and n = 3 water control fish at 4 weeks; n = 5 mannitol-treated fish, n = 10 glucose-treated fish, and n = 7 water treated fish at 8 weeks. (B) A visual representation of a zebrafish and the Freestyle Lite Blood Glucose Meter that we use to measure blood glucose levels. Blood sugar levels are measured from a cardiac blood sample after the fish are anesthetized in a 0.02% tricaine solution and decapitated. Values are mean &#177; SE. Asterisks denote significant differences where * = p &#60; 0.05; *** = p &#60; 0.001. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61935/61935fig02v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61935/61935fig03.jpg" /> 
Figure 3: GFAP levels are increased in glucose-treated zebrafish.&#160;Glucose-treated zebrafish have increased levels of (A) glial fibrillary acidic protein (GFAP; 1:1000) and (B) Rel-A (NfK-B; 1:1000) as determined from densitometry analysis of Western blots. &#946;-actin (1:1000) served as the loading control. The increase in Rel-A levels was significant (p &#60; 0.003, asterisks). W = water-treated control, G = glucose-treated, M = mannitol-treated. W2, G2, M2 are replicates for W, G, and M. This suggests that there is insult to the retina in hyperglycemic zebrafish that causes reactive gliosis. (Modified from Figure 7, Tanvir et al., 201830, published originally under the terms of a CC-BY license). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61935/61935fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61935/61935fig04v2.jpg" /> 
Figure 4: ERG response is reduced in glucose-treated fish.&#160;(A) Representative ERG traces from glucose-treated (red), mannitol-treated (green), and water-treated (black) zebrafish retinas evoked with a 570nm stimulus on a red adapting background. The glutamate receptor blocker CNQX (50&#181;M) was present in the bath solution to isolate photoreceptor and ON bipolar cell responses. Units: y-axis = &#181;V, x-axis = time. The square wave pulse reflects the duration of the light pulse. (B) Quantifying changes in response amplitudes. Mean normalized response amplitudes for photoreceptor a-waves (a1 amplitude, right), measured as the peak downward deflection at the start of the square light pulse, and bipolar cell b-waves (b2 amplitude, left), measured from trough to peak during the light pulse. Both values were significantly reduced in glucose- vs. mannitol-treated tissues (p &#60; 0.001 for a1; p &#60; 0.0001 for b2; n = 14 eyes per treatment). Values were normalized to the water-treated control. This is consistent with other diabetic animal models (such as rodents) and in humans with diabetes. (Taken from Figure 4, Tanvir et al. 201830, published originally under the terms of a CC-BY license). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61935/61935fig04v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

alternate-immersion-glucose-to-produce-prolonged-hyperglycemia

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

4.8K Views.  American University. 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.

Video: Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality 

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major challenge in conducting conditioning studies in humans is the ability to strongly manipulate or simulate the environmental contexts that are associated with conditioned emotional behaviors. In this regard, virtual reality (VR) technology is a promising tool. Yet, adapting this technology to meet experimental constraints requires special accommodations. Here we address the methodological issues involved when conducting fear conditioning in a fully immersive 6-sided VR environment and present fear conditioning data.
In the real world, traumatic events occur in complex environments that are made up of many cues, engaging all of our sensory modalities. For example, cues that form the environmental configuration include not only visual elements, but aural, olfactory, and even tactile. In rodent studies of fear conditioning animals are fully immersed in a context that is rich with novel visual, tactile and olfactory cues. However, standard laboratory tests of fear conditioning in humans are typically conducted in a nondescript room in front of a flat or 2D computer screen and do not replicate the complexity of real world experiences. On the other hand, a major limitation of clinical studies aimed at reducing (extinguishing) fear and preventing relapse in anxiety disorders is that treatment occurs after participants have acquired a fear in an uncontrolled and largely unknown context. Thus the experimenters are left without information about the duration of exposure, the true nature of the stimulus, and associated background cues in the environment1. In the absence of this information it can be difficult to truly extinguish a fear that is both cue and context-dependent. Virtual reality environments address these issues by providing the complexity of the real world, and at the same time allowing experimenters to constrain fear conditioning and extinction parameters to yield empirical data that can suggest better treatment options and/or analyze mechanistic hypotheses.
In order to test the hypothesis that fear conditioning may be richly encoded and context specific when conducted in a fully immersive environment, we developed distinct virtual reality 3-D contexts in which participants experienced fear conditioning to virtual snakes or spiders. Auditory cues co-occurred with the CS in order to further evoke orienting responses and a feeling of "presence" in subjects 2 . Skin conductance response served as the dependent measure of fear acquisition, memory retention and extinction.

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

Classical fear conditioning paradigm was adapted for human participants in a fully immersive virtual reality setting. Using a discrimination paradigm, conditioned fear, cue and context memory retention, and extinction was measured with skin conductance response to dynamic virtual snakes and spiders (the conditioned stimuli) in two distinct virtual contexts.

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major ...

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

Developmental Biology

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

Medicine

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

Behavior

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo &#946;-Cell Function

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in &#946;-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, &#946;-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying &#946;-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how &#946;-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate &#946;-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate &#946;-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in &#946;-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect &#946;-cell function.

Here, the study presents a protocol for calcium imaging and glucose stimulation of the pancreatic &#946;-cells of the zebrafish in vivo.

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Biology

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Streptozocin Treatment: A Zebrafish Model of Type 1 Diabetes Mellitus

Source: Intine R. V., et. al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/50232/a-zebrafish-model-of-diabetes-mellitus-and-metabolic-memory" target="_blank">A Zebrafish Model of Diabetes Mellitus and Metabolic Memory</a>. J. Vis. Exp. (2013).
This video describes the technique of creating a zebrafish model for Type 1 diabetes mellitus with the help of the drug Streptozocin, to study the pathophysiology of diabetic complications and metabolic memory.

Source: Intine R. V., et. al. A Zebrafish Model of Diabetes Mellitus and Metabolic Memory. J. Vis. Exp. (2013).
This video describes the technique of ...

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

Summary

Explore More Videos

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo β-Cell Function

Intraperitoneal Injection into Adult Zebrafish

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

High Throughput Danio Rerio Energy Expenditure Assay

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Streptozocin Treatment: A Zebrafish Model of Type 1 Diabetes Mellitus