Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

Summary

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.

Abstract

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 (Ptf1a^cre/+;LSL-Kras^G12D/+) 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.

Introduction

The mouse has emerged as an important animal model of human pancreatic cancer^1,2. In the Ptf1a^cre/+;LSL-Kras^G12D/+ 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 cancer^4,5, with the additional advantage that the PanINs emerge within the first five months of life and frequently progress to PDAC within a single year^4,5, whereas pancreatic cancer most frequently occurs in humans 60-70 years of age.

Extraction of the pancreas by dissection from the Ptf1a^cre/+;LSL-Kras^G12D/+ 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 PDAC^3,4,5. 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 tissue^6,7.

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.

Protocol

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

1. 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.
   1. 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.
   2. Establish a postoperative table in the same room and near the main table of operation. Maintain both tables as sterile environments throughout the procedure.
2. 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.
3. 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.
4. Fill one 50 mL and one 15 mL tube to 75% volume with formalin.
5. 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.
6. 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.
7. Pour liquid nitrogen into the shallow wide mouth dewar until the maximum fill line is reached.
8. 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.
9. 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.
   1. If this test fails and the mouse responds to the foot pinch test, repeat step 1.8.

2. Initial Incision, Heart Puncture, and Euthanasia

1. Pin the limbs of the mouse to the surgical foam board and wet the ventral side of the mouse with 70% ethanol.
2. 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.
   1. 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.
   2. 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.
3. Locate the heart and remove the pericardium, which is the sac around the heart, to avoid clogging of the syringe needle.
   1. 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.
   2. 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.
   3. After completing the blood collection, dispel the blood into the centrifuge tube and dispose of the syringe into the sharps container.
   4. 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.
4. 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.

3. Pancreas Extraction

1. 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.
   1. Continue to separate the pancreas from the small intestine jejunum and ileum sections, and lastly from the caecum of the large intestine.
2. 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.
3. 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.
4. Remove the pancreas and spread it out for examination. Leave the spleen attached to the pancreas for identification purposes.
   1. 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 Ptf1a^cre/+;LSL-Kras^G12D/+ 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.
5. If desired, remove other organs at this point.

4. Data Collection and Storage

1. After extraction of the organs, move the samples to the postoperative area for preservation.
2. 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.
3. Once the organs are weighed, place them into the liquid nitrogen for snap freezing.
4. After snap freezing, store the organs at -80 °C for long-term storage.
5. 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 °C for long-term storage.
6. For long term storage, freeze the blood and ear punch at -80 °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 °C.

5. Clean Up

1. 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's animal disposal policy.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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