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 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.
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.
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
2. Initial Incision, Heart Puncture, and Euthanasia
3. Pancreas Extraction
4. Data Collection and Storage
5. Clean Up
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: 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: 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: Anesthesia Chamber. Proper environment for anesthesia via isoflurane. Please click here to view a larger version of this figure.
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: 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: 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: 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: 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: 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: 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 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 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: 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: 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.
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.
The authors have nothing to disclose.
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.
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 |