Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

The overall goal of this procedure is to create a DMM model in mice and to study the onset of osteoarthritis Following the DMM surgery. After preparing the animals, a one centimeter longitudinal medial para patella incision is made to expose the knee joint. Next, the knee joint capsule is dissected and a medial dislocation of the knee is performed.

The final step is to cut through the medial meniscal tibial ligament and suture up the knee. Ultimately, histopathological analyses are used to show destruction of the joint structure as well as loss of protio, glycan, and osteoarthritis. Severity is evaluated using MANKIN scores of saffron and o staining.

Generally, individuals new to this method will struggle unless they have seen the surgery performed in mice. Visual demonstration of this method is critical. As detailed steps are difficult to learn and now performed under the microscope, every structure of the knee joint must be clearly recognized.

To establish this model, Prepare for the destabilization of the medial meniscus or DMM surgery by anesthetizing an eight to 12 week old male C 57 black six mouse weighing approximately 25 grams. Then place ophthalmic ointment on the eyes after shaving the area around the knee. Sterilize the site with Betadine and alcohol three times and surgically drape the area.

Begin the surgery by making a one centimeter longitudinal medial para patella incision to expose the knee joint. Then carefully cut the knee joint capsule longitudinally through the medial para patella incision until the femur is exposed. Next, use forceps to loosen the capsule structure by making the cut wider and deeper.

Grasping the distal part of the hind paw gently with one hand. Use forceps to perform a lateral dislocation of the patella and patella ligament. The dislocation should not require much force if the joint capsule has been severed longitudinally drip sterile saline on the articular cartilage surface throughout the remainder of the surgery to prevent it from drying out.

Now, identify the medial meniscus, which is located between the medial condyle of the femur and the medial plateau of the tibia. If necessary, pull aside the fat pad that covers the meniscus tibial ligament so as to expose the meniscus tibial ligament, which connects the lateral side of the medial meniscus to the intercondylar eminence of the tibia. Holding the hind paw gently with one hand.

Perform the DMM surgery by carefully cutting through the medial meniscal tibial ligament with a number 10 surgical blade. Make sure not to injure the articular cartilage or other ligaments. Next, close the knee joint capsule with a six aut absorbable suture and close the skin with six aut silk suture.

To minimize postoperative pain, give a subcutaneous injection of 10 milligrams per kilogram meloxicam and return the mouse to its cage after the anesthesia has worn off. Several weeks after the surgery, sacrifice the mouse and remove both hind limbs with a number 10 blade. Then carefully dissect the skin and muscles from the hind limb and fix the sample in 4%Paraform aldehyde for three days at room temperature.

After three days, remove the paraform aldehyde and clean the samples in distilled water for three minutes. Afterwards, decalcify the samples in EDTA at four degrees Celsius for two weeks. After two weeks, dehydrate the samples in an ethanol gradient.

After removing the final ethanol, put the samples in three Lene baths for one hour each. Next, embed the samples in a paraffin mold. Use a rotary microtome to make five micron sections and collect the sections on a glass slide.

Continue to cut serial sagittal sections from the center of the lateral condyle to the center of the medial condyle to perform saffron and o staining. First de parize the slides in Celine for three to five minutes. Repeating the Celine bath twice.

Then hydrate the slides in an ethanol gradient ending with distilled water. Then place the slides through Weigert's iron hematin solution, a 0.05%fast green solution, a 1%acetic acid solution, and a 0.1%saffron and O solution. Now mount the slides using a resinous medium score the saffron and O stained slides based on the loss of the red prot glycan color in the cartilage and on the percentage of destruction of the cartilage structure.

Then perform statistical analysis using the O-A-R-S-I histological scoring system. Them These images show knee joints with saffron and O staining in wild type and PG rrn knockout mice eight weeks following either a sham operation or DMM surgery. There is no obvious degeneration of cartilage in either genotype following a sham operation.

However, eight weeks after A DMM operation, there was loss of protio glycan as shown by the loss of red color in the cartilage, and also destruction of the cartilage structure as indicated by the red arrows in both wild type and PGRN knockout mice indicating the successful induction of an osteoarthritic phenotype. Following DMM surgery, a comparison of osteoarthritis scores between wild type and PGRN knockout mice. Following DMM surgery shows a significantly higher score in the knockout mice demonstrating that a loss of PGRN resulted in a more severe osteoarthritis phenotype As master.

This technique can be done in 10 to 15 minutes if it is performed properly. After watching this video, you should have a good understanding of how to perform DMM operation in mass.