Name: Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling (Video) | JoVE Journal
Uploaded: 2017-08-19T14:00:00.000+00:00
Duration: 8 min 21 s
Description: Learn mouse pancreas dissection for histological analysis and metabolic profiling using surgical techniques. This method enables researchers to study pancreatic diseases through accurate tissue harvesting and terminal blood collection.

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5 minutes

MAK acknowledges support for this work from the National Institutes of Health / National Cancer Institute grant number - 1R15CA152985-01A1. This project has also been supported by the Miami University Undergraduate Research Award Program, the Miami University Doctorate-Undergraduate Opportunities for Scholarship Program and the Miami University Summer Scholars Program.

The procedures carried out in the video and described below have been approved by the Institutional Animal Care and Use Committee (IACUC) at Miami University.
1. Preparation and Stimulus Test
<ol>
	<li>Establish two distinct areas for the surgical procedure, the operating table and the post-operation table. Stage both areas with all materials and utensils necessary.
	<ol>
		<li>Stage the operating table under a vented hood. Arrange the table with the equipment in a manner that allows the continuous and unimpeded performance of the procedure.</li>
		<li>Establish a postoperative table in the same room and near the main table of operation. Maintain both tables as sterile environments throughout the procedure.</li>
	</ol>
	</li>
	<li>Place the following supplies on the operating table: one glass jar with lid, one 15 mL tube, one pair of surgical scissors, one squeeze bottle of 70% ethanol, two foam boards, two forceps, two 1 mL 21 gauge syringes, two 50 mL tubes, one centrifuge tube, one cryogenic vial, four surgical pads, ten pins, a dispenser of sterilizing wipes, and a sharps container.</li>
	<li>Place the following supplies on the post-operation table: one analytical balance, one 4 L dewar of liquid nitrogen, a shallow wide mouth dewar, a floating microtube rack, one pair of surgical scissors, two forceps, four cryogenic vials, and a dispenser of sterile wipes.</li>
	<li>Fill one 50 mL and one 15 mL tube to 75% volume with formalin.</li>
	<li>Using a pin in each corner, affix one surgical pad to an approximate 30 cm x 30 cm foam board to serve as the dissection board. Use the remaining four pins during the operation. Prepare a smaller foam board with a surgical pad to transfer the organs to the post-op table. 
	CAUTION: Isoflurane (99.9%) is a toxic chemical, and should be used in a vent hood to ensure the maximum level of safety from the scavenging of waste anesthetic gas8. Additional information regarding the risks to researchers associated with the use of the open-drop method using isoflurane can be found in an article by Taylor and Mook8.</li>
	<li>Place one surgical pad into the glass jar and soak with a few drops of isoflurane (99.9%) and place a paper towel over the top to prevent direct contact of the mouse with the isoflurane. Similarly, use a surgical pad to line the remaining tube and soak with a few drops of isoflurane and place an additional pad over the top to prevent direct contact between the mouse and the isoflurane.</li>
	<li>Pour liquid nitrogen into the shallow wide mouth dewar until the maximum fill line is reached.</li>
	<li>Place the mouse selected for dissection into the anesthesia chamber, i.e. the glass jar with a pad soaked with a few drops of isoflurane (99.9%) covered with the lid, for ~1 min. 
	NOTE: This time varies from mouse to mouse. Once the mouse is unconscious, remove it from the chamber and place it onto the operating board.</li>
	<li>Orient the mouse so that it is lying ventral side up and with its head pointed away from the scientist. Place the head inside the tube lined with a surgical pad soaked with a few drops of isoflurane (99.9%), and perform a stimulus test by a foot pinch to ensure that the mouse is unresponsive to stimuli.
	<ol>
		<li>If this test fails and the mouse responds to the foot pinch test, repeat step 1.8.</li>
	</ol>
	</li>
</ol>
2. Initial Incision, Heart Puncture, and Euthanasia
<ol>
	<li>Pin the limbs of the mouse to the surgical foam board and wet the ventral side of the mouse with 70% ethanol.</li>
	<li>Pinch the fur/skin near the urethral opening with forceps and pull slightly upwards. Make an incision with the surgical scissors through the abdominal cavity starting from the urethral opening, up the midline and ending at the chin.
	<ol>
		<li>Near the starting point of the initial incision, grab one side of the fur/skin with the forceps and make another incision with the scissors downward and diagonally towards the back paw.</li>
		<li>Repeat this in the same manner on the opposite side. 
		NOTE: The fur/skin may be pinned down to create a wider opening, but is not necessary.</li>
	</ol>
	</li>
	<li>Locate the heart and remove the pericardium, which is the sac around the heart, to avoid clogging of the syringe needle.
	<ol>
		<li>Grasp the pericardium with the forceps and cut it with the scissors. Perform the heart puncture by carefully inserting the syringe needle into the beating heart and slowly start to retract the plunger.</li>
		<li>For optimal blood collection, use the plunger of the needle to mimic the pumping action of the heart and avoid drawing too quickly. 
		NOTE: Typically about 1 mL of blood can be collected.</li>
		<li>After completing the blood collection, dispel the blood into the centrifuge tube and dispose of the syringe into the sharps container.</li>
		<li>After the heart puncture is performed, carry out euthanasia by removing the attachments connecting the heart. 
		NOTE: Heparin, an anti-coagulant, was not added to the syringe in this procedure prior to the heart puncture to allow the blood to coagulate for serum collection in this specific study. However, if the researcher wanted to prevent blood coagulation to collect plasma, heparin could be added to the syringe prior to the heart puncture.</li>
	</ol>
	</li>
	<li>If the study involves genotyping of the mouse, snip a portion of the ear with the scissors and place into a centrifuge tube for a genotype verification.</li>
</ol>
3. Pancreas Extraction
<ol>
	<li>Locate the stomach on the left side of the mouse. Begin gently (so as to avoid tearing) separating the pancreas from the stomach and duodenum by using two forceps. 
	NOTE: When detaching the pancreas from the stomach and intestines, it is very important that the forceps are used gently to guide the pancreas tissue away from the organs and to not crush or tear the pancreas with the forceps.
	<ol>
		<li>Continue to separate the pancreas from the small intestine jejunum and ileum sections, and lastly from the caecum of the large intestine.</li>
	</ol>
	</li>
	<li>At the caecum, reposition the forceps and continue separation of the pancreas along the remaining colon towards the rectum. 
	NOTE: At this point, it is convenient to cut and remove the portion from the stomach to the region of the colon immediately preceding the rectum.</li>
	<li>Locate the pancreas and attached spleen. Slide the pancreas towards the right side of the mouse. Separate the remaining connections between the pancreas and thoracic cavity with the forceps to fully detach the pancreas and adjoined spleen.</li>
	<li>Remove the pancreas and spread it out for examination. Leave the spleen attached to the pancreas for identification purposes.
	<ol>
		<li>Remove all connective tissue, fat and mesenteric tissue from the pancreas. 
		NOTE: This tissue is whiter in color and thus can be easily distinguished from the pancreas tissue that is pinker in color. This is particularly important if the whole pancreas needs to be removed. For example, if the pancreas needs to be weighed and compared to body weight or between groups of animals. In the Ptf1acre/+;LSL-KrasG12D/+ mouse model, specifically in the older months, hard fibrous pancreatic tissue could be present. In this case, careful removal of the pancreas must be conducted as the intestines could be interlaced in tumor tissue. In advanced cases, abnormal spleen and liver tissue may also be present.</li>
	</ol>
	</li>
	<li>If desired, remove other organs at this point.</li>
</ol>
4. Data Collection and Storage
<ol>
	<li>After extraction of the organs, move the samples to the postoperative area for preservation.</li>
	<li>Weigh each organ and place them into their respective cryogenic vial. 
	NOTE: Along with the mass of each sample, any irregularities should be recorded for future reference.</li>
	<li>Once the organs are weighed, place them into the liquid nitrogen for snap freezing.</li>
	<li>After snap freezing, store the organs at -80 &#176;C for long-term storage.</li>
	<li>Place the formalin stored samples on the bench top overnight, and the next morning change their solution from formalin to 70% ethanol. 
	NOTE: These samples should be stored at 4 &#176;C for long-term storage.</li>
	<li>For long term storage, freeze the blood and ear punch at -80 &#176;C.. For serum collection from the blood, allow the blood to coagulate for 30 min then centrifuge it. Remove the serum portion using a pipette and then store at -80 &#176;C.</li>
</ol>
5. Clean Up
<ol>
	<li>Sanitize all the dissection tools with the sterilizing wipes. Cap the tube lined with the isoflurane soaked surgical pad. Replace the surgical pad on the foam board with a fresh surgical pad. Dispose the portions of the mouse that were not collected per the facility&#39;s animal disposal policy.</li>
</ol>

<ol><li>Fesinmeyer, M. D., Austin, M. A., Li, C. I., De Roos, A. J., Bowen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differences+in+Survival+by+Histologic+Type+of+Pancreatic+Cancer%5BTitle%5D)+AND+%22Cancer+Epidemiol+Biomarkers+Prev%22%5BJournal%5D)">Differences in Survival by Histologic Type of Pancreatic Cancer.</a> Cancer Epidemiol Biomarkers Prev. 14 (7), 1766-1773 (2005).</li>
<li>Jackson-Grusby, L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/12154411">Modeling cancer in mice.</a> Oncogene. 21 (35), 5504-5514 (2002).</li>
<li>Heid, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+requirement+of+Rac1+in+a+mouse+model+of+pancreatic+cancer%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Early requirement of Rac1 in a mouse model of pancreatic cancer.</a> Gastroenterology. 141 (2), 719-730 (2011).</li>
<li>Hingorani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preinvasive+and+invasive+ductal+pancreatic+cancer+and+its+early+detection+in+the+mouse%5BTitle%5D)+AND+%22Cancer+cell%22%5BJournal%5D)">Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.</a> Cancer cell. 4 (6), 437-450 (2003).</li>
<li>Shi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((KRAS2+Mutations+in+Human+Pancreatic+Acinar-Ductal+Metaplastic+Lesions+Are+Limited+to+Those+with+PanIN+Implications+for+the+Human+Pancreatic+Cancer+Cell+of+Origin%5BTitle%5D)+AND+%22Mol+Cancer+Res%22%5BJournal%5D)">KRAS2 Mutations in Human Pancreatic Acinar-Ductal Metaplastic Lesions Are Limited to Those with PanIN Implications for the Human Pancreatic Cancer Cell of Origin.</a> Mol Cancer Res. 7 (2), 230-236 (2009).</li>
<li>LaConti, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+serum+metabolomics+profiles+associated+with+malignant+progression+in+the+KrasG12D+mouse+model+of+pancreatic+ductal+adenocarcinoma%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Distinct serum metabolomics profiles associated with malignant progression in the KrasG12D mouse model of pancreatic ductal adenocarcinoma.</a> BMC Genomics. 16 (1), 1-10 (2015).</li>
<li>Ludwig, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surveying+the+serologic+proteome+in+a+tissue-specific+kras%28G12D%29+knockin+mouse+model+of+pancreatic+cancer%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Surveying the serologic proteome in a tissue-specific kras(G12D) knockin mouse model of pancreatic cancer.</a> Proteomics. 16 (3), 516-531 (2016).</li>
<li>Taylor, D. K., Mook, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isoflurane+Waste+Anesthetic+Gas+Concentrations+Associated+with+the+Open-Drop+Method%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Isoflurane Waste Anesthetic Gas Concentrations Associated with the Open-Drop Method.</a> J Am Assoc Lab Anim Sci. 48 (1), 61-64 (2009).</li>
<li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+sample+collection+in+small+laboratory+animals%5BTitle%5D)+AND+%22J+Pharmacol+Pharmacother%22%5BJournal%5D)">Blood sample collection in small laboratory animals.</a> J Pharmacol Pharmacother. 1 (2), 87-93 (2010).</li>
<li>Li, D. S., Yuan, Y. H., Tu, H. J., Liang, Q. L., Dai, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+protocol+for+islet+isolation+from+mouse+pancreas%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">A protocol for islet isolation from mouse pancreas.</a> Nature Protocols. 4 (11), 1649-1652 (2009).</li>
<li>Auer, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+frozen+tissue+storage+conditions+on+the+integrity+of+RNA+and+protein%5BTitle%5D)+AND+%22Biotech+Histochem%22%5BJournal%5D)">The effects of frozen tissue storage conditions on the integrity of RNA and protein.</a> Biotech Histochem. 89 (7), 518-528 (2014).</li>
<li>Ma, J., Leung, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limbic+System+Participates+in+Mediating+the+Effects+of+General+Anesthetics%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">Limbic System Participates in Mediating the Effects of General Anesthetics.</a> Neuropsychopharmacology. 31, 1177-1192 (2006).</li>
<li>Ijichi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aggressive+pancreatic+ductal+adenocarcinoma+in+mice+caused+by+pancreas-specific+blockade+of+transforming+growth+factor-%CE%B2+signaling+in+cooperation+with+active+Kras+expression%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-β signaling in cooperation with active Kras expression.</a> Genes Dev. 20 (22), 3147-3160 (2006).</li>
<li>Abiatari, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Moesin-dependent+cytoskeleton+remodelling+is+associated+with+an+anaplastic+phenotype+of+pancreatic+cancer%5BTitle%5D)+AND+%22J+Cell+Mol+Med%22%5BJournal%5D)">Moesin-dependent cytoskeleton remodelling is associated with an anaplastic phenotype of pancreatic cancer.</a> J Cell Mol Med. 14 (5), 1166-1179 (2010).</li>
<li>Goodpaster, A. M., Romick-Rosendale, L. E., Kennedy, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((a+Statistical+significance+analysis+of+nuclear+magnetic+resonance-based+metabonomics+data%5BTitle%5D)+AND+%22Analytical+biochemistry%22%5BJournal%5D)">a Statistical significance analysis of nuclear magnetic resonance-based metabonomics data.</a> Analytical biochemistry. 401, 134-143 (2010).</li>
<li>Yadav, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Idiopathic+Tumefactive+Chronic+Pancreatitis%3A+Clinical+Profile%2C+Histology%2C+and+Natural+History+After+Resection%5BTitle%5D)+AND+%22Clin+Gastroenterol+Hepatol%22%5BJournal%5D)">Idiopathic Tumefactive Chronic Pancreatitis: Clinical Profile, Histology, and Natural History After Resection.</a> Clin Gastroenterol Hepatol. 1 (2), 129-135 (2003).</li>
</ol>

The authors have nothing to disclose.

Significance with Respect to Existing Methods 
While other informal videos of mouse dissections exist, this video article provides the first professional quality, peer reviewed, visual demonstration of all of the detailed steps required for extraction and harvesting of the mouse pancreas by dissection10. With the pancreas being a main organ for metabolic activity and insulin production, dissection and harvesting of the pancreas allows for the preservation of the physiological...

The mouse has emerged as an important animal model of human pancreatic cancer1,2. In the Ptf1acre/+;LSL-KrasG12D/+ mouse model, the Kristen rat sarcoma (K-Ras) oncogene is activated exclusively in the pancreas, resulting in initiation of precancerous lesions in the pancreas, known as pancreatic intraepithelial neoplasias (PanINs), that progress to pancreatic ductal adenocarcinomas, commonly referred to as PDACs3. This mouse model system provides one of the best available animal models for human pancreatic cancer4,5, with the additional advantage that the PanINs emerge within the first five months of life and frequently progress to PDAC within a single year4,5, whereas pancreatic cancer most frequently occurs in humans 60-70 years of age.
Extraction of the pancreas by dissection from the Ptf1acre/+;LSL-KrasG12D/+ mice at various ages allows for detailed longitudinal histological examination of cancer development in the pancreas, ranging from the earliest PanIN stages through the progression to PDAC3,4,5. Harvesting the pancreas at ages ranging from five to fifteen months can also be used to prepare tissue extracts to characterize global changes in pancreas4 metabolism that occur during the transition from healthy to diseased tissue6,7.
This article presents a complete visual guide of the steps required to perform a mouse pancreas extraction and provides guidelines for storage of a pancreas for further analysis. This guide will be equally valuable for individuals conducting research on other pancreatic diseases, including type I diabetes, and should be especially useful to students and investigators new to research involving harvesting of the mouse pancreas using dissection for metabolic profiling or histological analyses.

<table><tbody><tr><td>Glass Jar&amp;nbsp;</td><td>Corning</td><td>3140-150</td><td>The glass jar used in the video has been discontinued.&amp;nbsp; This is its replacement.</td></tr><tr><td>Lid of Glass Jar</td><td>Corning</td><td>9985-150</td><td>The glass jar lid used in the video has been discontinued.&amp;nbsp; This is its replacement.</td></tr><tr><td>15 mL Falcon Tubes</td><td>Fisher Scientific</td><td>339650</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fisher Scientific</td><td>9201</td><td></td></tr><tr><td>Squeeze bottle&amp;nbsp;</td><td>Fisher Scientific</td><td>03-409-10DD</td><td></td></tr><tr><td>100% Ethanol</td><td>Fisher Scientific</td><td>22-032-103</td><td></td></tr><tr><td>Formalin</td><td>Fisher Scientific</td><td>245-684</td><td></td></tr><tr><td>Foam Boards</td><td>Therapak</td><td>562908</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>200205SHN</td><td></td></tr><tr><td>1 mL 21G Syringes</td><td>BD Biosciences</td><td>309624</td><td></td></tr><tr><td>50 mL Falcon Tubes</td><td>Fisher Scientific</td><td>339652</td><td></td></tr><tr><td>2.0 mL Microcentrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific</td><td>02-681-258</td><td></td></tr><tr><td>Surgical Pads</td><td>Fisher Scientific</td><td>S67011</td><td></td></tr><tr><td>T-Pins Length:&amp;nbsp; 2&quot;&amp;nbsp;</td><td>Advance Store Products</td><td>X32T-05</td><td></td></tr><tr><td>Sterilizing Wipes</td><td>Professional Disposables International Inc.</td><td>Q85084</td><td></td></tr><tr><td>Sharps Container</td><td>Fisher Scientific</td><td>14-827-122</td><td></td></tr><tr><td>Analytical Balance</td><td>Marshall Scientific</td><td>ME-AE200</td><td></td></tr><tr><td>4L Dewar</td><td>Taylor-Wharton</td><td>4LD</td><td></td></tr><tr><td>Shallow Wide Mouth Dewar</td><td>Fisher Scientific</td><td>F3087-V</td><td></td></tr><tr><td>Floating Microtube Rack</td><td>VWR</td><td>60986-100</td><td></td></tr><tr><td>Cryogenic Vial 1.2 mL, Sterile</td><td>Fisher Scientific</td><td>10-500-25</td><td></td></tr><tr><td>Isothesia (Isoflurane)&amp;nbsp;</td><td>Henry Schein Animal Health</td><td>050033</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>Wright Brothers</td><td>NIT-60-XX</td><td></td></tr><tr><td>Mouse Kras Strain</td><td>The Jackson Laboratory</td><td>OO8179</td><td></td></tr><tr><td>Mouse Cre Strain</td><td>MMRRC</td><td>OOO435-UNC</td><td></td></tr></tbody></table>

dissection of the mouse pancreas for histological analysis and metabolic profiling

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.

Figure 1 shows an overview of the operating environment area and Figure 2 shows the post operation area. While this setting provides the minimal amount of equipment and staging, individuals may choose to alter this to best suit individual needs. The protocol should be optimized according to the specific needs of the experiment. This procedure is conducted in a manner that terminates the life of a mouse, requiring proper euthaniza...

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 ...

The primary goal of this video article is to provide detailed visual instructions outlining the dissection of a mouse pancreas. The instructions should be useful to researchers who study mouse models of human pancreatic diseases and who need to harvest the mouse pancreas by dissection for histological analysis or metabolic profiling. The video article also provides detailed instructions to perform a terminal blood collection using a heart puncture procedure.Displayed is a operating table with the needed supplies. These include one glass jar with lid, one 15 mL falcon tube, one pair of surgical scissors, one squeeze bottle of 70%ethanol, two foam boards, two forceps, two one mL 21 gauge syringes, two 50 mL falcon tubes, one centrifuge tube, one cryo-vial, four surgical pads, ten pins, a dispenser of sterilizing wipes and a sharps container. Displayed is the post-operating table with the needed supplies.These include one analytical balance, one four liter Dewar of liquid nitrogen, a shallow wide mouth Dewar, a floating microtube rack, one pair of surgical scissors, two forceps, five cryo-vials and a dispenser of sterile wipes. To perform euthanasia, the mouse is first placed in the glass jar containing a gauze pad soaked with a few drops of isoflurane, and then covered with the lid until the mouse becomes unconscious, which typically takes about one minute. The mouse is then removed from the glass jar and a foot pinch test is performed to ensure that the mouse cannot experience discomfort.If the foot pinch test fails, the mouse is returned to the glass jar and the lid replaced until the foot pinch test can be performed successfully. Place the mouse on its back and pin the limbs of the mouse to the surgical foam board and wet the ventral side of the mouse with 70%ethanol. Pinch the fur near the urethral opening with the forceps and slightly pull upward.Make an incision through the abdominal cavity, starting from the urethral opening, up the midline and ending at the chin. Near the starting point of the initial incision, grab one side of the fur and make another incision downward and diagonal toward the back paw. Repeat this in the same manner on the opposite side.Locate the heart to perform the terminal heart puncture. To do this, grasp the sternum with the forceps and cut into the diaphragm. Quickly cut upward through the ribcage and remove the cut portion to expose the beating heart.Once the heart is located, remove the pericardium and perform the terminal heart puncture with the syringe. For optimal blood collection, use the plunger of the needle to mimic the pumping action of the heart and avoid drawing too quickly. Dispel the blood into the centrifuge tube and dispose of the syringe into the sharps container.After the heart puncture is performed, euthanasia is carried out by removing the attachments connecting the heart. Locate the stomach on the left side of the mouse. The pancreas has the appearance of a diffuse lighter color tissue that is attached to the stomach and duodenum and all along the small and large intestines and is attached to the spleen.Begin to gently separate the pancreas from the stomach and duodenum by using the forceps. Continue to separate the pancreas from the small intestine continuing from the jejunum and ileum sections and finally to the cecum and large intestine. At the cecum, reposition the forceps and continue separation of the pancreas along the remaining colon towards the rectum.At this point, it is convenient to cut and remove the section from the stomach to the region of the colon immediately preceding the rectum. Locate the pancreas and attached spleen. Slide the pancreas towards the right side of the mouse.Cut the remaining connections between the pancreas and thoracic cavity to fully detach the pancreas and adjoined spleen. Remove the pancreas and spread it out to examine. If desired, other organs may be removed at this point.After extraction, the samples are moved to the post operative area. Each organ will be weighed and then placed into their respective cryo-vial. Record any observed irregularities.Once the organs are in cryo-vials, place them into the floating microtube rack and then into the Dewar with liquid nitrogen. After snap freezing, the organ should be stored at minus 80 degrees Celsius for long-term storage. All of the dissection tools used should be sanitized using the sterilizing wipes.The falcon tube lined with the isoflurane soaked surgical pad should be capped. The surgical pad on the foam board should be replaced with a fresh surgical pad. Portions of the mouse that were not collected should be disposed of according to the facility's animal disposal policy.The following items should be handled accordingly. Once these items are properly stored, a multitude of experiments can be conducted and include, but are not limited to, fluorescence microscopy, hematoxylin and eosin histology, immunohistochemistry, mass spectrometry, and nuclear magnetic resonance spectroscopy. With the pancreas being a primary organ for metabolic activity and insulin production, this procedure allows for the preservation of the physiological characteristics.By isolating the pancreas, future analysis may be conducted on the sample. This procedure allows for the comparison and study of interactions from other tissues within the same organism, within the same timeframe.

dissection-mouse-pancreas-for-histological-analysis-metabolic

Research

JoVE Journal

Medicine

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

This video describes the technique of differentiating pancreatic acinar cells to ductal cells in ex vivo 3D culture. This helps to understand the mechanisms regulating the acinar-to-ductal metaplasia (ADM), a key process in the early development of pancreatic cancer.

Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture

Assaying in vitro β-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining β-cell function.

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

Pancreatic Tissue Dissection to Isolate Viable Single Cells

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

Isolation of Mouse Pancreatic Endothelial Cells

Learn mouse pancreas dissection for histological analysis and metabolic profiling using surgical techniques. This method enables researchers to study pancreatic diseases through accurate tissue harvesting and terminal blood collection.

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

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