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Sandell1 project protocol

Ear wound healing and articular cartilage regeneration in 6 inbred and 9 LGXSM recombinant inbred strains of mice   (2012)

Sandell LJ
With: Rai MF, Hashimoto S, Johnson EE, Janiszak KL, Fitzgerald J, Heber-Katz E, Cheverud JM




Sandell1 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
Data collected
1
 Mice ear-punched at 6 wks of age
-
2
 Ear cartilage healing determined after 30 d
Ear cartilage wound healing
3 Mice anesthetized and subjected to bilateral full-thickness articular cartilage defects in knee joints at 8 wks of age -
4 Mice euthanized at indicated times and knee joints harvested for microscopy Articular cartilage wound healing

Equipment and supplies

  • Surgical kit
  • 27-gauge needle
  • 6-0 absorbable polypropylene suture
  • VetClose skin glue
  • CO2 chamber
  • Paraffin
  • Microtome
  • Polylysine-coated slides (Fisher Scientific)
  • Brightfield microscope (Eclipse E800, Nikon) with mounted camera (2000R Fast1394, Retiga)
  • QCapturePro software (QImaging)
  • Metal ear punch (Fisher Scientific)
  • Grid-etched reticle

Reagents and solutions

  • Ketamine (100 mg/kg body weight)/Xylazine (20 mg/mL)/Acepromazine (10 mg/mL)
  • 10% neutral buffered formalin
  • 10% formic acid in 5% formaldehyde solution
  • 0.1 M EDTA in phosphate buffered saline (PBS)
  • Ethanol
  • Toluidine blue (0.04% solution prepared in 0.1 M sodium acetate (pH 4.0))
  • Xylene

Procedure: Ear wound punch and analysis

    1. A through-and-through hole (2mm diameter) is created bilaterally in the center of the cartilaginous part of the external ear using a metal ear punch.
    2. Holes are measured 30 days after wounding using a grid-etched reticle.
    3. Healing area is calculated by deducting the diameter of the residual hole from 2 mm.

Procedure: Full-thickness cartilage injury and analysis

    1. Mice are anesthetized i.p. with rodent cocktail (ketamine/xylazine/acepromazine).
    2. A small medial parapatellar skin incision is made, the joint capsule opened, and the patella luxated laterally to expose the trochlear groove articulating surface.
    3. A full-thickness lesion is made by creating a circular defect in the cartilage with a sterile needle, using a circular motion until the subchondral bone is reached (confirmed by appearance of a blood droplet following removal of the needle).
    4. The joint capsule is closed with absorbable sutures; subcutaneous tissue is apposed with a single mattress suture, and VetClose skin glue is applied on the skin to bridge over the closed edges.
    5. Mice are euthanized at indicated time points post-wounding and knee joints are dissected, fixed (formalin), and decalcified (formic acid/formaldehyde) for 48 h.
    6. Mouse joints are washed twice with water and incubated in EDTA for 4-6 h at room temperature.
    7. Samples are washed 4 times with water (15 min each time).
    8. Samples are paraffin embedded, mounted in paraffin blocks, and sagittally sectioned at 5µ intervals extending through the entire trochlear groove.
    9. Sections are mounted on microscope slides and dewaxed.
    10. Every third slide is subjected to toluidine blue staining (5 min), washed 3 times with water, twice with ethanol, and twice with xylene before being coverslipped.
    11. Stained sections are viewed using 4X and 10X objectives and images captured by a mounted camera and software.
    12. Three observers, who were blinded with regard to sample identity, each scored 3 selected sections.
    13. Specimens were assigned a score ranging from 0 (no healing) to 14 (full healing).

Data collected by investigator

  • ear cartilage wound healing (30 d post-injury)
  • articular cartilage wound healing (12 wks, 16 wks post-injury)