解剖的小鼠胰腺的组织学分析与代谢谱

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.