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

Summary

We describe a protocol for the destabilization of the medial meniscus (DMM) model in mice, an effective tool for osteoarthritis (OA) research. In addition, we have demonstrated that deficiency of progranulincan exaggerate OA development and progression by using this model, indicating that progranulin plays a protective role in the pathogenesis of OA.

Abstract

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules in the pathogenesis of osteoarthritis (OA) in vivo. However, the detailed, especially the visualized protocol for establishing this complicated model in mice, is not available. Herein we took advantage of wildtype and progranulin (PGRN)-/- mice as examples to introduce a protocol for inducing DMM model in mice, and compared the onset of OA following establishment of this surgically induced model. The operations performed on mice were either sham operation, which just opened joint capsule, or DMM operation, which cut the menisco-tibial ligament and caused destabilization of medial meniscus. Osteoarthritis severity was evaluated using histological assay (e.g. Safranin O staining), expressions of OA-associated genes, degradation of cartilage extracellular matrix molecules, and osteophyte formation. DMM operation successfully induced OA initiation and progression in both wildtype and PGRN-/- mice, and loss of PGNR growth factor led to a more severe OA phenotype in this surgically induced model.

Introduction

Osteoarthritis (OA), also known as degenerative arthritis, affects 15% of the world's population and over 46 million people within the United States, and is characterized by synovitis, cartilage degeneration, and osteophyte formation¹. It can be a result of a complex interplay of genetic, metabolic, biomechanical and biochemical factors. The underlying mechanisms of OA continue to evade the scientific community. There are presently numerous animal models which can mimic the pathogenesis of OA^2,3. It is important to establish animal models in mice because of both the availability of various genetically modified mice and the cost effectiveness of experimentation. Among the different kinds of experimental OA models, the surgically induced destabilization of medial meniscus (DMM) model is a well-accepted OA model because of its good reproducibility and a relatively slower progression during OA development. Both of these attributes have been key for the evaluation of OA progression in different treatments or transgenes^3-8. However, the consistency of surgical OA model is affected by various factors during the surgery and as a result, the application of surgical mouse model is limited.

Progranulin (PGRN) is a multi-functional growth factor expressed in various cells. It is known that PGRN plays a critical role in various physiological and disease processes such as wound healing ⁹, tumorigenesis¹⁰, and inflammation^11-15. Studies also shown that insufficiency of PGRN can cause degenerative diseases of nervous system in both humans and mice^16-18. It is known that PGRN is expressed in human articular cartilage, and its level is significantly elevated in cartilage of patients with OA and rheumatoid arthritis¹⁹. In addition, PGRN also plays a crucial role in chondrocyte proliferation ²⁰, differentiation and endochondral ossification of growth plate during development ^21,22. Recently, we reported that PGRN antagonized TNF-α through binding to TNF receptors and exhibited an anti-inflammatory function in inflammatory arthritis models^13,14,23,24. However, the role of PGRN in OA, especially in vivo, remains to be an enigma. Herein, we present the procedure to induce a surgical DMM model, and investigate the role of PGRN in OA development through establishing DMM model in WT and PGRN-/- mice.

Protocol

All of the surgical procedures relating to the animals should be approved by local Institution's Animal Care and Ethics Committee, with an effort made to minimize pain and discomfort caused by the surgery.

1. Preparation

1. Select 8-12 weeks old male C57BL/6 mice with a body weight of approximately 25 g for surgery.
2. Anesthetize the animals through intraperitoneal injection of a cocktail containing both xylazine (5 mg/kg) and ketamine (40 mg/kg).
3. Shave the knee with razors, then surgically drape the animal and sterilize the surgical site with betadine and alcohol (3x) and cover the mouse eyes with ointment.

2. Surgical Process

1. Make a 1 cm longitudinal medial para-patellar incision to expose knee joint.
2. Open the knee joint gently through lateral dislocation of the patella and patellar ligament.
   1. Cut the knee joint capsule longitudinally through the medial para-patellar incision in step 2.1.
   2. Dissect knee joint capsule with forceps.
   3. Grab the distal part of hind paw with left hand, and perform lateral dislocation of the patella and patellar ligament with forceps. Hold the hind paw gently and make sure to avoid trauma in the paw. The better the joint capsule is dissected, the less force will be required to make the dislocation.
3. Drip sterile saline on the surface of articular cartilage during operation to avoid drying out of cartilage surface.
4. Cut through the medial meniscotibial ligament which anchors medial meniscus to the tibial plateau. Avoid injuring the cartilage beneath the medial meniscus.
   1. Identify the medial meniscus which locates between medial condyle of femur and medial plateau of tibia.
   2. Identify the meniscotibial ligament which connects lateral side of medial meniscus with intercondylar eminence of tibia.
   3. Hold the hind paw gently with hand, and cut through the medial meniscotibial ligament carefully by using a No. 10 surgical blade. Make sure not to cause injure to articular cartilage and other ligaments. In many cases, the covering fat pad needs to be pulled to the side but not to be removed in order to identify the ligament.
5. Close the knee joint capsule with a 6-0 absorbable suture.
6. Close the skin with 6-0 silk suture.

3. Post-operative Care

1. Apply one drop of 0.25% bupivacaine in the surgical site of each mouse to minimize post-operative pain.
2. Leave the operated mice free to get water and food.

4. Histological Scoring of Surgical DMM Model

1. Sacrifice the mice at indicated time points (e.g. 4 weeks, 8 weeks, and 12 weeks. Here we showed results of 8 weeks as representative) after the surgery.
2. The entire knee joints are fixed, decalcified, embedded by paraffin and then sectioned serially at 5 µm interval.
   1. Cut the whole hind limbs with No. 10 blades.
   2. Dissect the skin and muscles of the hind limbs carefully, and fix the samples with 4% PFA for 3 days in RT.
   3. Remove the PFA, and clean the samples with water. Afterwards, decalcify the samples in EDTA at 4 °C for 2 weeks.
   4. Dehydrate the samples in an ethanol gradient. In detail, keep the samples in 70% ethanol for 1 hr, then change the ethanol and keep the samples in a new set of 70% ethanol for O/N. Afterwards, put the samples in 80% and 90% ethanol consequently, and keep them for 1 hr, respectively, followed by 100% ethanol for O/N.
   5. Remove the ethanol. Keep the samples in oxylene for 1 hr, and change to a new set of oxylene. Repeat this step 3x.
   6. Embed the samples into a paraffin mold using the Leica embedding center. Afterwards, the samples are sectioned at 5 μm using a rotary microtome (Leica RM2255, Germany) and then collected onto glass slides.
   7. Serial sagittal sections are cut for each sample spanning a region from the center of the lateral condyle to the center of the medial condyle.
3. Safranin O staining is performed, followed by scoring and statistical analysis through OARSI scoring system as described previously²⁵.
   1. First, deparaffinize the slides in oxylene, and hydrate them in an ethanol gradient to distilled water. Thereafter, the slides are stained through Weigert's Iron Hematoxylin Solution, 0.05% Fast Green (FCF) Solution, 1% Acetic Acid Solution, and 0.1% Safranin O Solution. Mount the slides using resinous medium.
   2. Score the Safranin O stained slides based on loss of proteoglycan (red color) in cartilage and percentage of destruction in cartilage structure, do statistical analysis for the histological score.

Results

DMM model was successfully established in mice, and deficiency of PGRN exaggerated surgically-induced OA development.

Sham and DMM operations (Figure 1) were performed in WT and PGRN-/- mice. 8 weeks after operation, the mice were sacrificed, and Safranin O staining was performed on the sections from knee joints, followed by statistical analysis of arthritis score based on histology. As shown in Figure 2A, there was no obvious degeneration of ...

Discussion

It is reported that the strain of mice is very important for DMM model induction, as different strains of mice have varying severity of OA after DMM, with highest level in the 129/SvEv strain, followed by C57BL6, 129/SvInJ and then FVB/n²⁶. A large part of transgenes are established in C57BL6 mice, such as PGRN-/- mice we used in the present study, which are relatively susceptible to DMM. However, if the transgene is based on insensitive strain such as FVB/n mice, it is necessary to backcross these mice with s...

Materials

Name	Company	Catalog Number	Comments
No. 10 Surgical blades	Feather	25-2976#10
6-0 suture	Applied Dental	WG-N53133