To establish a model of cancer bone pain in mice, for testing experimental analgesic therapeutics.
Materials:
NCTC 2472 murine fibrosarcoma cells will be obtained from the ATCC and propagated in tissue culture for implantation in medullary space of the femur.
Animals:
20 female C3H/HeJ normal mice, 5-6 weeks old, 20-25 grams in weight, will be obtained and acclimated for 1 week prior to initiation of bone tumor growth.
Design:
The NCTC 2472 tumor cell line will be propagated in cell culture and harvested while in logarithmic growth and implanted directly in the medullary cavity of the distal femur. Mice will be anesthetized with isofluorane, and a right knee arthrotomy performed and 105 tumor cells injected (20ul) directly into the medullary cavity of the distal femur using a 29 gauge needle attached to an insulin syringe. The entry hole in the bone is then filled with bone wax to prevent tumor cell extravasation into the knee joint space. The groups will consist of: 10 sham implanted mice injected with PBS, and 10 mice implanted with tumor cells. Animals will be assessed for pain response using the von Frey filament test on days 7, 10, 14 and 21, post tumor cell implantation. On day 22 animals will be euthanized under isofluorane anesthesia, perfused intracardially with 4% paraformaldehyde in PBS. The brain, spinal cord and both left and right femurs will be harvested and placed in 10% formalin for further processing. Radiographs of femurs will be prepared to determine the extent of bone density loss. The loss of bone density will be quantified on a scale of 0-3: (0) normal bone; (1) minor loss of bone in medullary canal; (2) substantial loss of bone in medullary canal with some destruction of the distal femur; (3) substantial loss of bone in the medullary canal with major structural destruction of the distal femur. After completion of radiographic imaging, femurs will be decalcified and H&E stained histology performed to determine the extent of tumor growth. Immunohistochemistry and special stains of spinal cords and brain sections will be performed at sponsor’s request.
Treatment Groups:
Group
N
QD (24 hr intervals)
1
10
Sham surgery/ PBS injection into femur
2
10
Surgery/ tumor cell injection into femur
Tentative Calendar:
2/27/2018
Day -7
Receive mice
3/05/2018
Day 0
Implant tumor cells, sham injections
3/12/2018
Day 7
von Frey monofilament assessmen
3/15/2018
Day 10
von Frey monofilament assessment
3/19/2018
Day 14
von Frey monofilament assessment
3/22/2018
Day 17
von Frey monofilament assessment
3/26/2018
Day 21
von Frey monofilament assessment
3/27/2018
Day 22
Terminate, perfusion fixation, harvest tissue for radiographic and histological assessment.
