Colonoscopic Guided Murine Wound Model: A Method to Isolate Biopsy Induced Wound Bed from Colon to Understand the Healing Process

프로토콜

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Collection of the wound bed for molecular analysis

1. Euthanize mouse using CO₂ asphyxiation followed by cervical dislocation (or equivalent technique) on the selected day following biopsy.
2. Harvest the distal region of the colon by opening the skin and abdominal muscle to expose the body cavity. Place closed scissors under the colon and gently lift to release it from the underlying mesentery and then cut the colon at its midpoint and at the anus to remove it from the mouse.
3. Flush fecal content using a rat gavage needle attached to a 20 mL syringe filled with ice-cold 1x PBS then lay down the colon onto filter paper.
4. Cut open the colon longitudinally on filter paper ensuring that the mesenteric side is face down against the filter paper. Apply 0.2% methylene blue to the mucosa using a squeeze pipet while tissue is still on the filter paper and then drain off excess methylene blue. View the colon under a dissecting microscope and locate the wound bed (Figure 1A).
5. Using 4 inch micro iris scissors, cut around the edge of the wound bed (Figure 1A, dashed circle) being careful not to cut into the muscle layer (unless the muscle is desired) and transfer the dissected wound bed to a tube using fine point tweezers for snap-freezing or desired storage method.       
   NOTE: The amount of tissue collected in this manner is enough for extracting RNA for RNA-seq or equivalent analysis.

결과

Figure 1: Ex vivo and sectioned images of wound bed. (A) A colon was harvested from a mouse 2 days following biopsy, stained with 0.2% methylene blue and imaged under a dissecting microscope. The dashed circle indicates the edges of the wound bed. The black line indicates the proper location to bisect the wound bed/colon prior to embedding for sectioning. (B) Representative image of an H&E-st...

자료

Name	Company	Catalog Number	Comments
Biopsy forceps, 3 Fr	Karl Storz	61071ZJ
isofluorane	Covetrus	2905
methylene blue	Sigma-Aldrich	M9140
micro iris scissors	Integra	18-1619
scalpal with #10 blade	Hill-Rom	372610