Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

Published 8/19/2017
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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.

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Veite-Schmahl, M. J., Regan, D. P., Rivers, A. C., Nowatzke, J. F., Kennedy, M. A. Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling. J. Vis. Exp. (126), e55647, doi:10.3791/55647 (2017).

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 (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.

Introduction

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.

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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 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.
  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.

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Representative 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 euthanization9. When the researcher is ready, the mouse is placed into the anesthesia chamber with the isoflurane-soaked pads (Figure 3).

Once the mouse is unconscious, remove the mouse and place it dorsal side on the board. A toe-pinch procedure should be performed to ensure that the mouse is unresponsive to pain (Figure 4). Apply 70% ethanol to sterilize the initial incision area. The terminal blood draw must be conducted first, prior to the pancreas removal, to ensure adequate blood retrieval. Prior to blood removal, the pericardium should be removed to prevent clogging of the 21 G needle opening. After completing the terminal blood draw, the heart is detached as a secondary method of euthanasia and the pancreas is then removed.

Begin by locating the stomach, which provides a good starting point for pancreas removal (Figure 5). Note: Extreme care should be exercised during removal of the pancreas, which is a delicate and fragile tissue, and therefore all operations should be performed with gentle force. Using forceps, begin the dissection by starting to gently pull the pancreas away from the stomach and continue to separate the pancreatic tissue from the outer lining of the gastrointestinal (GI) tract working from the stomach to the duodenum, jejunum and ileum (Figure 6). Once the caecum is reached, easier removal of the pancreas is achieved by repositioning the forceps so that one forceps is holding the caecum and the other forceps is used to continue to separate the pancreas from the large intestine (Figure 7). After removal from the large intestine, the pancreas is placed on the right side of the mouse and any remaining attachments are severed(Figure 8).

The pancreas should be fanned out for inspection and any abnormalities should be recorded (Figure 9). In the Ptf1acre/+;LSL-KrasG12D/+ mouse strain, the pancreas could potentially contain a hardened tumor (Figure 10). Other organs should also be examined for potential metastasis. Once the pancreas has been removed, it should be weighed and the weight recorded. A portion of the pancreas should be snap-frozen in liquid nitrogen for future metabolic profiling analysis or other testing and a portion of the pancreas should be placed in formalin for future histological analysis. Figure 11 shows the initial storage of the various organs collected from the dissection for use in later analysis. The organs collected by dissection and stored for further study will depend on the goals of the individual researcher.

Tissue and blood samples can be used for histological analyses and for metabolic profiling. An example of the histological analysis of the pancreas tissue is shown in Figure 12. Metabolic profiling can be conducted on the snap-frozen tissue samples and blood sample. Representative nuclear magnetic resonance spectroscopy (NMR) spectra of the hydrophilic and hydrophobic components of pancreas tissue extracts are shown in Figure 13A and 13B, respectively. A representative NMR spectrum collected on a serum sample prepared from blood collected at the time of a terminal blood draw procedure is shown in Figure 14.

Figure 1
Figure 1: Staging of Operating Area. General layout of correct tools and operating conditions for the dissection. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Staging of Post-Operation Area. General layout of correct tools and operating conditions for the postoperative procedures. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Anesthesia Chamber. Proper environment for anesthesia via isoflurane. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Stimulus Examination. The stimulus test conducted on the mouse prior to the initial incision to ensure any pain or discomfort is not being endured. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Beginning Removal of Pancreas. The orientation of the mouse indicating the initial extraction of the pancreas, location indicated by the forceps. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Pancreas Extraction Along the Intestines. Process of isolating the pancreas from the gastrointestinal tract. Please click here to view a larger version of this figure.

Figure 7
Figure 7: Pancreas Removal at the Caecum. Repositioning of the forceps once the caecum is reached. Please click here to view a larger version of this figure.

Figure 8
Figure 8: Pancreas Removal. Place the pancreas on the right side of the mouse. Any remaining attachments should be cut to fully remove the pancreas. Please click here to view a larger version of this figure.

Figure 9
Figure 9: Pancreas Examination. The pancreas with attached spleen being examined after removal from the mouse. Spleen is indicated by the vertical arrow, and the pancreas is indicated by the horizontal arrow. Please click here to view a larger version of this figure.

Figure 10
Figure 10: Pancreas Examination. The pancreas with attached spleen displaying a pancreatic tumor being examined after removal from the mouse. Spleen is indicated by the vertical arrow, and the pancreas is indicated by the horizontal arrow. Please click here to view a larger version of this figure.

Figure 12

Figure 11: Storage of Organs Removed. Appropriate storage of organs and samples collected, prepared for long-term storage and future analysis. Please click here to view a larger version of this figure.

Figure 11
Figure 12: Histological Analysis of Pancreas Tissue. Hematoxylin and eosin stained images from pancreas tissue. A) Normal pancreas tissue from a Ptf1acre/-;LSL-KrasG12D/- control mouse. B) PanIN tissue from the pancreas of a Ptf1acre/+;LSL-KrasG12D/+ study mouse. Please click here to view a larger version of this figure.

Figure 13
Figure 13: Metabolic Profiling Analysis. One-dimensional proton nuclear magnetic resonance spectroscopy (NMR) spectra of A) hydrophilic and B) hydrophobic phase components of pancreas tissue extracts following tissue homogenization and subjected to chloroform/methanol extraction. The NMR spectra were acquired at 850 MHz and are suitable for use in metabolic profiling analyses. Please click here to view a larger version of this figure.

Figure 14
Figure 14: Representative NMR Spectrum of Serum. The blood collected by the terminal blood draw procedure can be used for metabolic profiling analysis. This spectrum shows a typical one-dimensional proton 850 MHz NMR spectrum collected on the serum obtained from a terminal blood draw sample. Please click here to view a larger version of this figure.

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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 characteristics11. 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 time frame.

Limitations of the Technique
The greatest limitation of this procedure is termination of the mouse's life, thereby preventing longitudinal collection and sampling of multiple tissue samples from the same mouse. In order to analyze trends related to age, sex, or other quantifiers, a cross-sectional population must be implemented, as we have done for our study of metabolic biomarkers of pancreatic cancer. Another limitation of this protocol is the inability to pause the procedure. Once euthanization is initiated, the procedure must be carried out in its entirety.

Critical Steps within the Protocol
Execution of the stimulus test by pinching the hind paw of the mouse is critical to ensure that the mouse receives humane treatment. If the mouse does not react to this stimulus, then the procedure may be carried out as planned. However, should the mouse display a distressed response as a result of the stimulus test, the mouse should be returned to the anesthesia chamber for an additional period of time and the test repeated until a reaction to the stimulus test is not observed12.

Similarly, the terminal heart puncture followed by the removal of the connections to the heart immediately after the terminal blood sample is collected as a secondary method of euthanasia ensures the humane sacrifice of the mouse. To ensure an effective blood draw, the scientist should use a pumping motion with the syringe that is similar to the heartbeat of the mouse, allowing for maximum collection of blood for analysis.

Modifications and Troubleshooting
Switching the organs from formalin to 70% ethanol solutions prepares the organs for the embedding process required for histological analysis. Different storage solutions may be required should the scientist choose to perform other experiments with the organs. Before analysis, it is important to limit any potential thawing of the organs stored in the -80 °C freezer to preserve the organ's integrity.

Use of the Ptf1acre/+;LSL-KrasG12D/+ mouse model minimizes the occurrence of non-pancreatic primary tumors and diseases13. Thus, it is important to note any irregularities that are apparent to the pancreas or other organs during dissection and collection of the tissue samples for analysis.

Future Applications
Harvesting of the mouse pancreas by dissection allows for multiple types of analysis to be conducted on the same sample. The most popular of these include, but are not limited to, fluorescence microscopy, hematoxylin and eosin histology, immunohistochemistry, mass spectrometry, and nuclear magnetic resonance spectroscopy6,7,14,15. Diseases like diabetes, pancreatitis, and pancreatic cancer can be studied using the techniques mentioned above16.

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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References

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

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