Name: acute and chronic models of hyperglycemia in zebrafish: a method to assess the impact of hyperglycemia on neurogenesis and the biodistribution of radiolabeled molecules
Uploaded: 2017-06-26T12:30:00
Description: Watch this Scientific Journal Video about Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled

We greatly thank Direction des Usages du Num&#233;rique (DUN) from La R&#233;union University for editing the video (in particular, Jean-Fran&#231;ois F&#233;vrier, Eric Esnault, and Sylvain Ducasse), Lynda-Rose Mottagan for the voiceover, Mary Osborne-Pellegrin for proofreading the voice-over, and the CYROI platform. This work was supported by grants from La R&#233;union University (Bonus Qualit&#233; Recherche, Dispositifs incitatifs), Conseil R&#233;gional de La R&#233;union, European Union (CPER/FEDER), and Philancia association. ACD is a recipient of a fellowship grant from the Minist&#232;re de l&#39;Education Nationale, de l&#39;Enseignement Sup&#233;rieur et de la Recherche, La R&#233;union University (Contrat Doctoral).

Adult wildtype zebrafish (Danio rerio) were maintained under standard photoperiod (14/10 h light/dark) and temperature (28 &#176;C) conditions. All experiments were conducted in accordance with the French and European Community Guidelines for the Use of Animals in Research (86/609/EEC and 2010/63/EU) and were approved by the local Ethics Committee for animal experimentation.
1. Establishing a Model of Acute Hyperglycemia in Zebrafish
<ol>
	<li>Prepare a stock solution of tricaine (M...

<ol>
	<li>Ho, N., Sommers, M. S., Lucki, I. <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+diabetes+on+hippocampal+neurogenesis:+links+to+cognition+and+depression.">Effects of diabetes on hippocampal neurogenesis: links to cognition and depression.</a> Neurosci Biobehav Rev. 37 (8), 1346-1362 (2013).</li><li>Cukierman, T., Gerstein, H. C., Williamson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+decline+and+dementia+in+diabetes--systematic+overview+of+prospective+observational+studies.">Cognitive decline and dementia in diabetes--systematic overview of prospective observational studies.</a> Diabetologia. 48 (12), 2460-2469 (2005).</li><li>Gaudieri, P. A., Chen, R., Greer, T. F., Holmes, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+function+in+children+with+type+1+diabetes:+a+meta-analysis.">Cognitive function in children with type 1 diabetes: a meta-analysis.</a> Diabetes Care. 31 (9), 1892-1897 (2008).</li><li>Brismar, T., 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=Predictors+of+cognitive+impairment+in+type+1+diabetes.">Predictors of cognitive impairment in type 1 diabetes.</a> Psychoneuroendocrinology. 32 (8-10), 1041-1051 (2007).</li><li>Ojo, O., Brooke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Association+between+Diabetes,+Cognitive+Decline+and+Dementia.">Evaluating the Association between Diabetes, Cognitive Decline and Dementia.</a> Int J Environ Res Public Health. 12 (7), 8281-8294 (2015).</li><li>Capes, S. E., Hunt, D., Malmberg, K., Pathak, P., Gerstein, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+hyperglycemia+and+prognosis+of+stroke+in+nondiabetic+and+diabetic+patients:+a+systematic+overview.">Stress hyperglycemia and prognosis of stroke in nondiabetic and diabetic patients: a systematic overview.</a> Stroke. 32 (10), 2426-2432 (2001).</li><li>Stead, L. G., 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+as+an+independent+predictor+of+worse+outcome+in+non-diabetic+patients+presenting+with+acute+ischemic+stroke.">Hyperglycemia as an independent predictor of worse outcome in non-diabetic patients presenting with acute ischemic stroke.</a> Neurocrit Care. 10 (2), 181-186 (2009).</li><li>Kagansky, N., Levy, S., Knobler, 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+role+of+hyperglycemia+in+acute+stroke.">The role of hyperglycemia in acute stroke.</a> Arch Neurol. 58 (8), 1209-1212 (2001).</li><li>Gilmore, R. M., Stead, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+hyperglycemia+in+acute+ischemic+stroke.">The role of hyperglycemia in acute ischemic stroke.</a> Neurocrit Care. 5 (2), 153-158 (2006).</li><li>Desilles, J. P., 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=Diabetes+mellitus,+admission+glucose,+and+outcomes+after+stroke+thrombolysis:+a+registry+and+systematic+review.">Diabetes mellitus, admission glucose, and outcomes after stroke thrombolysis: a registry and systematic review.</a> Stroke. 44 (7), 1915-1923 (2013).</li><li>Dorsemans, A. C., 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=Impaired+constitutive+and+regenerative+neurogenesis+in+adult+hyperglycemic+zebrafish.">Impaired constitutive and regenerative neurogenesis in adult hyperglycemic zebrafish.</a> J Comp Neurol. , (2016).</li><li>Schmidt, R., Str&#228;hle, U., Scholpp, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+zebrafish+-+from+embryo+to+adult.">Neurogenesis in zebrafish - from embryo to adult.</a> Neural Dev. 8, 3 (2013).</li><li>Kizil, C., Kaslin, J., Kroehne, V., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+and+brain+regeneration+in+zebrafish.">Adult neurogenesis and brain regeneration in zebrafish.</a> Dev Neurobiol. 72 (3), 429-461 (2012).</li><li>Grandel, H., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+adult+neural+stem+cell+activity+in+vertebrates.">Comparative aspects of adult neural stem cell activity in vertebrates.</a> Dev Genes Evol. 223 (1-2), 131-147 (2013).</li><li>Lindsey, B. W., Tropepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+framework+for+understanding+the+biological+principles+of+adult+neurogenesis.">A comparative framework for understanding the biological principles of adult neurogenesis.</a> Prog Neurobiol. 80 (6), 281-307 (2006).</li><li>M&#228;rz, 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=Heterogeneity+in+progenitor+cell+subtypes+in+the+ventricular+zone+of+the+zebrafish+adult+telencephalon.">Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon.</a> Glia. 58 (7), 870-888 (2010).</li><li>Chapouton, P., Jagasia, R., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+in+non-mammalian+vertebrates.">Adult neurogenesis in non-mammalian vertebrates.</a> Bioessays. 29 (8), 745-757 (2007).</li><li>Lindsey, B. W., Darabie, A., Tropepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+composition+of+neurogenic+periventricular+zones+in+the+adult+zebrafish+forebrain.">The cellular composition of neurogenic periventricular zones in the adult zebrafish forebrain.</a> J Comp Neurol. 520 (10), 2275-2316 (2012).</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> Zebrafish. , 2611-2619 (2012).</li><li>Oka, T., 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=Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity.">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC Physiol. 10, 21 (2010).</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> Comp Biochem Physiol B Biochem Mol Biol. 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+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> Behav Brain Res. 274, 319-325 (2014).</li><li>Schmidt, R., Beil, T., Str&#228;hle, U., Rastegar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stab+wound+injury+of+the+zebrafish+adult+telencephalon:+a+method+to+investigate+vertebrate+brain+neurogenesis+and+regeneration.">Stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration.</a> J Vis Exp. (90), e51753 (2014).</li><li>Diotel, N., 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+estradiol+in+adult+neurogenesis+and+brain+repair+in+zebrafish.">Effects of estradiol in adult neurogenesis and brain repair in zebrafish.</a> Horm Behav. 63 (2), 193-207 (2013).</li><li>Rodriguez Viales, R., 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=The+helix-loop-helix+protein+id1+controls+stem+cell+proliferation+during+regenerative+neurogenesis+in+the+adult+zebrafish+telencephalon.">The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon.</a> Stem Cells. 33 (3), 892-903 (2015).</li><li>Kaslin, J., Ganz, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+neurogenesis+and+regeneration+in+the+non-mammalian+vertebrate+brain.">Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain.</a> Philos Trans R Soc Lond B Biol Sci. 363 (1489), 101-122 (2008).</li><li>Kroehne, V., Freudenreich, D., Hans, S., Kaslin, J., Brand, M. <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+adult+zebrafish+brain+from+neurogenic+radial+glia-type+progenitors.">Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.</a> Development. 138 (22), 4831-4841 (2011).</li><li>Alunni, A., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+view+of+regenerative+neurogenesis+in+vertebrates.">A comparative view of regenerative neurogenesis in vertebrates.</a> Development. 143 (5), 741-753 (2016).</li><li>M&#228;rz, M., Schmidt, R., Rastegar, S., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+response+following+stab+injury+in+the+adult+zebrafish+telencephalon.">Regenerative response following stab injury in the adult zebrafish telencephalon.</a> Dev Dyn. 240 (9), 2221-2231 (2011).</li><li>Wullimann, M., Rupp, B., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomy of the zebrafish brain: A topological atlas. , 1-144 (1996).</li><li>Pellegrini, 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=Identification+of+aromatase-positive+radial+glial+cells+as+progenitor+cells+in+the+ventricular+layer+of+the+forebrain+in+zebrafish.">Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish.</a> J Comp Neurol. 501 (1), 150-167 (2007).</li><li>Zupanc, G. K., Hinsch, K., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+migration,+neuronal+differentiation,+and+long-term+survival+of+new+cells+in+the+adult+zebrafish+brain.">Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain.</a> J Comp Neurol. 488 (3), 290-319 (2005).</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> iConcept Press. , 2611-2619 (2013).</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. , (2016).</li><li>Prasad, S., Sajja, R. K., Naik, P., Cucullo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+Mellitus+and+Blood-Brain+Barrier+Dysfunction:+An+Overview.">Diabetes Mellitus and Blood-Brain Barrier Dysfunction: An Overview.</a> J Pharmacovigil. 2 (2), 125 (2014).</li></ol>

This work describes various methods to establish acute and chronic models of hyperglycemia in zebrafish. The main advantages of these procedures are that: (1) they allow for a reduction in the number of mammals used for research, (2) they are simple to set up and quick to implement, and (3) they are economical. Therefore, such models allow for the investigation of the impact of hyperglycemia on a large number of animals to study its impact on different physiological processes, including atherothrombosis, cardiovascular d...

Hyperglycemia is defined as excessive blood glucose levels. Although it could reflect a situation of acute stress, hyperglycemia is also a condition that often leads to a diagnosis of diabetes, a chronic disorder of insulin secretion and/or resistance. In 2016, the number of adults living with diabetes has reached 422 million worldwide, and each year, 1.5 million people die from this disease, making it a major health problem1. Indeed, uncontrolled diabetes leads to several physiological disorders affecting the cardiovascular system, kidneys, and the peripheral and central nervous systems.
Interestingly, acute and chronic hyperglycemia may alter cognition and contribute to both dementia and depression2,3,4,5,6. In addition, the admission of patients with hyperglycemia has been associated with worse functional, neurological, and survival outcomes after ischemic stroke7,8,9,10,11. It was also shown that hyperglycemia/diabetes affect adult neurogenesis, a process leading to the generation of new neurons, by impacting neural stem cell activity and neuronal differentiation, migration, and survival2,12.
In contrast to mammals, teleost fish, like zebrafish, display intense neurogenic activity throughout the whole brain and exhibit an outstanding capacity for brain repair during adulthood13,14,15,16. Notably, such capacities are possible due to the persistence of neural stem/progenitor cells, including radial glia and neuroblasts17,18,19. In addition, the zebrafish has recently emerged as a model for studying metabolic disorders, including obesity and hyperglycemia/diabetes20,21,22.
Although the zebrafish is a well-recognized model of hyperglycemia and neurogenesis, few studies have investigated the impact of hyperglycemia on brain homeostasis and cognitive function12,23. To determine the impact of hyperglycemia on constitutive and injury-induced brain cell proliferation, a model of acute hyperglycemia was created through the intraperitoneal injection of D-glucose. In addition, a model of chronic hyperglycemia was reproduced through the immersion of fish in water supplemented with D-glucose12. Zebrafish exhibit many advantages in research. They are cheap, easy to raise, and transparent during the first stages of development, and their genome has been sequenced. In the context of this work, they also display several additional advantages: (1) they share similar physiological processes with humans, making them a critical tool for biomedical research; (2) they allow for the quick investigation of the impact of hyperglycemia on brain homeostasis and neurogenesis, given their widespread and strong neurogenic activity; and (3) they are an alternative model, allowing for the reduction of the number of mammals used in research. Finally, zebrafish can be used as a model for testing the biodistribution of radiolabeled molecules and potential therapeutic agents using PET/CT.
The overall goal of the following procedure is to visually document how to set up models of acute and chronic hyperglycemia in zebrafish, use zebrafish to assess brain remodeling in hyperglycemic conditions, and monitor radiolabeled molecules (here, [18F]-FDG) using PET/CT.

<table border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:328px;">1mL Luer-Lok Syringe</td>
			<td style="width:151px;">BD, USA</td>
			<td style="width:93px;">309628</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">4&#39;,6&#39;-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td style="width:93px;">D8417</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7 mL bijou container plain lab</td>
			<td style="width:151px;">Dutscher, France</td>
			<td style="width:93px;">080171</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">D-glucose</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>67021</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Digital camera</td>
			<td>Life Sciences, Japan</td>
			<td>Hamamatsu ORCA-ER</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Disposable base molds&#160;</td>
			<td>Simport, Canada</td>
			<td>M475-2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Donkey anti-rabbit Alexa fluor 488</td>
			<td>Life Technologies, USA</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Embedding center</td>
			<td>Thermo Scientific, USA</td>
			<td>Shandon Histocentre 3</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fluorescence microscope</td>
			<td>Nikon, Japan</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorodeoxyglucose (18F-FDG)</td>
			<td>Cyclotron, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glucometer test strip</td>
			<td>LifeScan, France</td>
			<td>One-Touch 143 Ultra</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Goat anti-mouse Alexa fluor 594</td>
			<td>Life Technologies, USA</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">In-Vivo Imaging System</td>
			<td>TriFoil Imaging, Canada</td>
			<td>Triumph Trimodality&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microtome</td>
			<td>Thermo Scientific, USA</td>
			<td>Microm HM 355 S</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Monoclonal mouse anti-PCNA</td>
			<td>DAKO, USA</td>
			<td>clone PC10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P6148-500G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Polyclonal rabbit anti-GFAP</td>
			<td>DAKO, USA</td>
			<td>Z033429</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Slide drying bench</td>
			<td>Electrothermal, USA</td>
			<td>MH6616</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium chloride</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium citrate trisodium salt dehydrate&#160;</td>
			<td>Prolabo, France</td>
			<td>27833.294</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sterile needle</td>
			<td>BD Microlance 3</td>
			<td>30 G 1/2 ; 0.3 mm x 13 mm</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Student Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Student surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91400-14</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Superfros Plus Gold Slides</td>
			<td>Thermo Scientific, USA</td>
			<td>FT4981GLPLUS</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Surgical microscope</td>
			<td>Leica, France</td>
			<td>M320-F12</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue embedding cassettes</td>
			<td>Simport, Canada</td>
			<td>M490-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue embedding medium</td>
			<td>LeicaBiosystems, USA</td>
			<td>39602004</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Toluene</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:328px;">Tricaine MS-222</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Triton X100</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>X100-500 mL</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vectashield medium&#160;</td>
			<td>Vector Laboratories, USA</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylene</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fish Strain</td>
			<td>AB</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Saline phosphate buffer (10X PBS) pH 7.4 (for 1 liter)</td>
			<td></td>
			<td></td>
			<td>For preparing 10X PBS, add the following&#160; salts and complete to 1 liter with distilled water</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Potassium chloride (MM : 74.55 g/mol): 2.00 g</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>746436</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Potassium phosphate monobasic (MM: 136,09 g/mol): 2.40g</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>795488</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium chloride (MM : 58.44 g/mol): 80.00 g&#160;</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium phosphate dibasic (MM: 141,96 g): 14,40 g</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>795410</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the procedures described in this article, the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) was performed on adult zebrafish and led to a significant increase in blood glucose levels 1.5 h after injection (Figure 1A). 24 h post-injection, the blood glucose levels were similar between D-glucose and PBS-injected fish12. For chronic treatment, zebrafish were immersed in D-glucose water (111 mM) and became hyperglycemi...

Watch this Scientific Journal Video about Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled

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

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 ...

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.

acute-chronic-models-hyperglycemia-zebrafish-method-to-assess-impact

Research

JoVE Journal

Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...