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 ....

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.

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

List of materials used in this article

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