This work was supported by a research grant from the Iacocca Family Foundation, Rosalind Franklin University start-up funds, and National Institutes of Health Grant DK092721 (to R.V.I.). The authors wish to thank Nikki Intine for aid in manuscript preparation.

All procedures are performed following the guidelines described in "Principles of Laboratory Animal Care" (National Institutes of Health publication no. 85-23, revised 1985) and the approved Rosalind Franklin University Institutional Animal Care and Use Committee animal protocol 08-19.
There are 2 important abbreviations that are used in this manuscript. 1) DM: refers to fish that are in an acute (300 mg/dl) hyperglycemic state and have been for at least 3 weeks. 2) MM: refers to fish that were 21 day (see protocol) DM fish and allowed to restore glycemic control through pancreatic regeneration. This is achieved within (14) days of drug removal. The fish are considered MM fish from this point onward. It is also important to note that fish that are referred to as control are treated identically as DM or MM fish in terms of the number of injections (saline only), the incubation times at the various temperatures and the number and timing of caudal fin amputations.
1. Generation of Zebrafish with Diabetes mellitus, DM Fish
<ol>
	<li>Prepare both recovery and anesthetic water tanks. Recovery water is normal fish water. For anesthetic water add sufficient 2-phenoxyethanol so that a 1:1,000 dilution in normal fish water is achieved.</li>
	<li>Prepare a 0.3% solution (in a fume hood) of streptozocin (STZ) by adding 6 mg of STZ to 2 ml of 0.09% sodium chloride and immediately place the solution on ice . This will provide enough injection solution for the injection of approximately 20 fish in 20 min. If you exceed the 20 minute mark, stop, and make a fresh STZ solution before proceeding. In a separate tube aliquot enough saline solution for control fish. Once the STZ is solubilized all subsequent steps do not require fume hood use.</li>
	<li>Fill a &frac12; cc syringe equipped with a 27 1/2 gauge needle with the STZ or control solutions ensuring that no air bubbles are trapped.</li>
	<li>Anesthetize each fish individually by placing the fish in anesthetic water, and wait until their swimming motion ceases (1-2 min).</li>
	<li>Once anesthetized briefly place the fish on a paper towel to absorb any excess water, place the fish in weigh boat and measure the mass of the fish.</li>
	<li>Place the fish on a firm surface (Petri dish lid) for injection.</li>
	<li>Inject the STZ or control solution into the peritoneal cavity of the fish by inserting the needle past the bevel into the posterior aspect of the ventral peritoneum.</li>
	<li>0.35 mg/g (350 mg/kg) of STZ should be delivered to each fish and the volume of the 0.3% solution required can be calculated in the following manner.
	<ol class="lalpha">
		<li>Multiply mass of fish (g) by 0.35 to yield the amount of STZ in mg required.</li>
		<li>divide the product generated above by 3 to yield the volume of the 0.3% solution required for injection in &mu;l. 
		Sample: For a 0.5 g fish : a) 0.5 x 0.35 = 0.175 b) .0175/3= 0.058 ml = 58 &mu;l. 
		The same volume without the STZ would be injected for a control fish. A sheet for volumes to inject per fish mass should be generated and used for quick reference.</li>
	</ol>
	</li>
	<li>Following injection place the fish in the recovery water tank and monitor them for normal swimming activity. Once this has been achieved the fish are transferred to a normal living tank that is maintained at the reduced temperature range of 22 &deg;C - 24 &deg;C. This reduced temperature is critical for efficient induction of hyperglycemia (diabetes mellitus, DM).</li>
	<li>Although hyperglycemia is detected within 24 hr of the first injection, in order to induce a prolonged state of very high hyperglycemia the zebrafish require a frequent injection induction phase followed by weekly maintenance injections as shown below.</li>
</ol>
Week 1: 3 injections (Day 1, 3, 5), Week 2: 1 injection (Day 12), Week 3: 1 injection (Day 19), 
Week 4: (Day 21) Perform assay of interest.
At this point the zebrafish are considered to have been in a prolonged state of hyperglycemia and exhibit the diabetic complications of retinopathy, nephropathy and also impaired fin regeneration. These are referred to as DM fish. Additionally if desired the fish can be maintained in the hyperglycemic state with weekly maintenance injections. Approximately 5% death during this process should be expected.
2. Blood Collection and Fasting Blood Glucose Level (FBGL) Determination
<ol>
	<li>Each group of DM and control fish must include enough fish to accurately determine the average FBGL of the group as this assay requires these fish to be sacrificed.</li>
	<li>Prepare a labeled PCR tube for each blood sample containing 5 &mu;l of sterile normal saline.</li>
	<li>For blood collection, anesthetize fish as above, remove all water, place the fish on a microscope slide and using a scalpel, remove the head of the fish at the base of the operculum.</li>
	<li>Collect the blood (up to 2 &mu;l) that is released from the fish onto the slide and quickly add it to the 5 &mu;l of sterile normal saline pipetting up and down to make sure that the blood does not clog. Immediately place the sample on ice.</li>
	<li>Determine the blood sample volume by measuring the total volume of the liquid (saline + blood) and then subtracting out the 5 &mu;l of saline.</li>
	<li>Transfer 5 &mu;l of the diluted blood from each PCR tube into a 1.5 ml microfuge tube and determine the blood glucose concentration using the QuantiChrome Glucose Assay Kit. This is performed by following the manufacturer's protocol without exception. Expected results: normal/control fish 60 mg/dl and DM fish 310 mg/dl.</li>
</ol>
3. Caudal Fin Regeneration Studies
<ol>
	<li>Anesthetize fish as described in 1.0-1.4.</li>
	<li>Place fish on a Petri dish lid and amputate the caudal fin in a straight line using a sterile size 10 scalpel proximal to the first lepidotrichia branching point while viewing the fin through a dissecting microscope.</li>
	<li>Allow the fish to recover as in 1.10 but place the fish at 33 &deg;C for the regenerative growth phase of the assay. This is an established temperature for accelerated fin regeneration analysis.</li>
	<li>The fins can be imaged at any time post amputation however we routinely examine the regenerative growth at 24, 28 and 72 hr following trans-section.</li>
	<li>Anesthetize fish as before (see 1.0-1.4), place the fish under a dissecting microscope equipped with a camera (we use a Nikon SMZ-1500 equipped with a Q-imaging Camera) and collect all fin images at 1X magnification with NIS elements software. The fin should be spread for imaging so that it is fully extended without stretching or damaging the tissue and should be free of any water droplets. For consistency always place the dorsal side to the right.</li>
	<li>Print images and measure the regenerative growth area using Image J software and the use of a drawing pad. Trace around the whole area of new growth and determine the area. Each measurement should be made five times and averaged to ensure accuracy of tracing. It is important that the images used do not have shadows or water droplets as these contribute to errors in measuring the outgrowth area.</li>
	<li>Measure the length of the amputation site along the dorsal-ventral axis and divide the area determined in 3.6 by this measurement. This allows fish of different sizes to be compared directly.</li>
</ol>
4. Generation of Metabolic Memory (MM) Zebrafish
<ol>
	<li>Initiate a group of DM fish and their appropriate controls as described in section 1.</li>
	<li>At 21 days determine the FBGL for a subset of the group and divide the DM fish into 2 subgroups. Continue weekly STZ injections for one of the groups as DM controls for the duration of the experiment. Cease STZ injection for the second group and incubate the fish at normal temperature. Within 14 days these zebrafish will restore normal blood insulin and glucose control through pancreas regeneration. These fish are now called MM (metabolic memory fish).</li>
	<li>At day 30 post drug removal amputate the caudal fins of control, DM and MM fish via the method described in 3.2.</li>
	<li>Return fish to normal water conditions for fin regeneration for 30 days. This allows the growth of what we term, metabolic memory tissue.</li>
	<li>At day 60 perform a second amputation (as described in 3.2) within the tissue that was regenerated in the period from 30-60 days and perform the caudal fin regeneration assay (3.1-3.7) Figure 1.</li>
	<li>Isolate tissue and perform assay of interest. See Table 1 for a protocol summary.</li>
</ol>

No conflicts of interest declared.

Diabetes mellitus is a disease of metabolic dysregulation, initially diagnosed as hyperglycemia, that ultimately results in blood vessel damage leading to many complications which all persist even after euglycemia is achieved though pharmaceutical intervention. This persistence of complications is referred to as metabolic memory and several recent studies have examined the role that epigenetic mechanisms play in this phenomenon. Here we have detailed a protocol that allows for the generation of both acute diabetic and me...

Diabetes mellitus (DM) is a serious and growing health problem that results in reduced life expectancy due to disease specific microvascular (retinopathy, nephropathy, neuropathy, impaired wound healing) and macrovascular (heart disease and stroke) complications 1. Once initiated, diabetic complications continue to progress uninterrupted even when glycemic control is achieved 2,3 and this phenomenon has been termed metabolic memory or the legacy effect. The presence of this phenomenon was recognized clinically during the early 1990s as the "The Diabetes Control and Complications Trial (DCCT)" progressed and since has been supported by multiple additional clinical trials 4,5,6,7,8,9,10,11,12,13,14. Animal models of DM have been critical for discoveries related to the patho-physiology of diabetic complications and metabolic memory. In fact, the persistence of diabetic complications was first documented in a canine model of diabetic retinopathy which has since been supported by several lines of experimental evidence using a variety of in vitro culture systems and animal models 15,16,17,18,19,20,21. These studies clearly show that an initial hyperglycemic period results in permanent abnormalities (including aberrant gene expression) of target organs/cells and mechanistically suggests the involvement of the epigenome.
Epigenomes consist of all the chromatin modifications for a given cell type and are responsible for a cell's unique gene expression profile. The chromosome modifications are dynamic during development, support cell differentiation, are responsive to external stimuli, are mitotically stably inherited 22,23 and can be altered in disease 24,25,26. These epigenetic mechanisms include: post translational histone modifications, non-canonical histone variant inclusion in octomers, chromatin access changes through DNA methylation, and gene expression control through non-coding micro RNAs 27,28,29,30. Altogether, epigenetic processes allow cells/organisms to quickly respond to changing environmental stimuli 31,32,33 , they also confer the ability for the cell to "memorize" these encounters once the stimulus is removed 23,22. Therefore, as altered gene expression profiles resulting from epigenetic processes are stable in the absence of the signal(s) that initiated them and are heritable through cell division, they have gained great interest as underlying molecular mechanisms of human pathologies including metabolic memory. The results that are emerging in the context of DM and epigenetics parallel advancements in other diseases in that a plethora of epigenetic changes induced by hyperglycemia cause remarkable persistent changes in transcriptional networks of cells (reviewed in 34,35,36,37,38).
The zebrafish has long been a premier model organism to study vertebrate development however the last 15 years has seen an exponential growth in utilizing this organism for study of human disease. 39. Zebrafish models of human disease have been established spanning a wide range of human pathologies including genetic disorders and acquired disease 40,41,42. The many advantages of the zebrafish over other vertebrate model organisms include high fecundity, short generation time, transparency through early adulthood, reduced housing costs and an array of tools for gene manipulation. Moreover, due to the extensive conservation of genetic pathways and cellular physiology among the vertebrates and the capacity to perform high throughput drug screenings, the zebrafish has been successfully used for pharmaceutical discovery.
We have developed an adult zebrafish model of type I diabetes mellitus using the diabetogenic drug, streptozocin. We have characterized this model to show that diabetic zebrafish not only display the known human secondary complications but in addition, exhibit impaired limb regeneration (caudal fin regeneration) as a consequence of the hyperglycemic environment. In addition, we have reported that hyperglycemic zebrafish revert back to normal glycemia within 2 weeks of drug removal due to regeneration of endogenous pancreatic beta cells resulting in a physiologically normal glycemic state. However, in contrast, limb regeneration in these fish remains impaired to the same extent as in the acute diabetic state indicating this complication persists and is susceptible to metabolic memory. The main impetus for generating this model was to provide a system to study the mitotically stable epigenetic components that support the metabolic memory phenomenon in the absence of the background noise of the previous hyperglycemic environment. At the conclusion of the protocol provided here the zebrafish and or selective tissues can be processed by any assay suitable to the researchers needs. We have successfully used this procedure to identify the genome-wide persistent changes in DNA methylation induced by hyperglycemia that are maintained in the metabolic memory state 21.
We feel that this zebrafish model of type I diabetes mellitus has several innovative advantages over other model systems for examining metabolic memory. 1) All of our studies can be conducted in vivo and as the previous hyperglycemic fish return to euglycemia through regeneration of endogenous insulin production, they do not require exogenous insulin injections. Therefore, this avoids the complicating spikes and valleys in glycemic control that may occur in animals requiring exogenous insulin. 2) As described above, the background stimulation from the previous diabetic state (i.e. the continued presence of advanced glycation end-products and reactive oxygen species markers) are eliminated and therefore one can examine the purely epigenetic factors of metabolic memory. 3) The experiments can be performed rapidly as it takes approximately 80 days from diabetes induction until metabolic memory examination. 4) Caudal fin regeneration is experimentally very approachable and allows for easy genetic and experimental manipulation for which there are a vast array of tools. 5) Caudal fin regeneration provides a very simple and quantifiable method to assess metabolic memory and therefore will allow for future drug discovery.

<table border="1">
	 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Streptozocin</td>
 <td>Sigma Aldrich</td>
 <td>S0130</td>
 <td></td>
 </tr>
 <tr>
 <td>2 phenoxyethanol</td>
 <td>Sigma Aldrich</td>
 <td>P1126</td>
 <td></td>
 </tr>
 <tr>
 <td>Scalpel (size 10)</td>
 <td>Fisher Scientific</td>
 <td>089275A </td>
 <td></td>
 </tr>
 <tr>
 <td>Petri Dishes</td>
 <td>Fisher Scientific</td>
 <td>08-757-13 </td>
 <td></td>
 </tr>
 <tr>
 <td>&frac12; cc syringe, with 27 1/2 gauge needle</td>
 <td>Fisher Scientific</td>
 <td>305620</td>
 <td></td>
 </tr>
 <tr>
 <td>QuantiChrome glucose assay kit.</td>
 <td>Bioassay Systems</td>
 <td>DIGL-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Sigma Aldrich</td>
 <td>S3014</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting Microscope</td>
 <td>Nikon</td>
 <td>TMZ-1500</td>
 <td>Any dissecting microscope is fine.</td>
 </tr>
 <tr>
 <td>Camera for Imaging</td>
 <td>Nikon</td>
 <td>Q imaging</td>
 <td>Any camera is suitable.</td>
 </tr>
 <tr>
 <td>Image J software</td>
 <td>National Institutes of Health</td>
 <td>NIH Image</td>
 <td></td>
 </tr>
 <tr>
 <td>NIS Elements</td>
 <td>Nikon</td>
 <td></td>
 <td>Any imaging software is suitable.</td>
 </tr>
	 </tbody>
 </table>

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.

Type I diabetic zebrafish not only display the known secondary complications of retinopathy and nephropathy, but also, exhibit an additional complication: impaired caudal fin regeneration. This later complication persists due to metabolic memory in fish that have restored normal glucose control following a hyperglycemic period. In Figure 2A (control) and Figure 2B (metabolic memory) representative images of regenerating fins that were captured at 72 hr post-amputation are presented. The ...

Watch this Scientific Journal Video about A Zebrafish Model of Diabetes Mellitus and Metabolic Memory at JoVE.com

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

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 ...

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25.5K Views.  Rosalind Franklin University of Medicine and Science. 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.

Video: A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

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 ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.