Anterior Cruciate Ligament Transection and Synovial Fluid Lavage in a Rodent Model to Study Joint Inflammation and Posttraumatic Osteoarthritis

Summary

Herein, we describe a surgical method to transect the anterior cruciate ligament (ACL) as a reliable model to induce knee osteoarthritis in rodents and methodologies for harvesting knee synovial fluid, measuring knee edema, and assessing osteoarthritis severity.

Abstract

Osteoarthritis (OA) is a joint disease characterized by chronic pain and currently has no cure. A subset of OA, posttraumatic osteoarthritis (PTOA), can occur after a joint injury. The knee is one of the most affected joints, and among knee injuries, injuries to the ACL are among the most common. Symptomatic OA, which is accompanied by changes to the joint such as cartilage degradation and osteophyte formation, can develop years after the initial injury, but early joint changes can be detected shortly after. Inflammation is considered one of the contributing factors in PTOA development, and the pro-inflammatory mediators produced in response to an injury can catalyze PTOA development. In animal models, surgically injuring structures in the knee is a widely used method of OA induction, and a common surgical technique is an ACL transection (ACLT). The purpose of this study is to describe our ACLT technique for the induction of OA, as well as methods to monitor joint inflammation and quantify OA severity. To assess inflammation post-surgery, we describe a technique for measuring knee edema as well as a technique for harvesting and analyzing knee synovial fluid. To evaluate the long-term joint changes associated with OA, we present a semi-quantitative scoring system we developed to evaluate OA severity via microCT imaging.

Introduction

The anterior cruciate ligament (ACL) is a critical structure in the knee joint, providing stability and preventing excessive forward movement of the tibia relative to the femur¹. ACL injuries are among the most common knee injuries, particularly in sports that involve sudden stops, jumps, or changes in direction². These injuries can range from partial tears to complete ruptures and often necessitate surgical intervention for optimal recovery³. However, even after surgical treatment, ACL injuries often lead to long-term joint changes, including chondral wear, osteophyte formation, and structural al....

Protocol

All animal studies were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Brigham and Women’s Hospital.

1. Animal preparation

NOTE: The choice of mouse sex should align with the study's objectives, as even though ACLT reliably induces PTOA in both sexes, the severity and progression may differ.

1. Anesthesia preparation: Prepare ketamine-xylazine (KX) anesthetic solution in advance.
   NOTE: Inhalant anesthesia (2% isoflurane) can also be used.
2. Surgical con....

Results

The ACLT procedure is an effective and reproducible surgical method to induce osteoarthritis in the rodent knee. After the ACLT procedure was performed under sterile conditions, the mice showed no signs of distress or infection at the incision site and moved freely without signs of lameness in the operated limb.

Knee size was measured to monitor knee edema following ACLT (Figure 7A). To control for variation in mouse size, the width of the injured knee was normali.......

Discussion

This technique description provides a detailed surgical method for inducing ACL injuries in mice by transecting the ACL, offering a controlled model for studying PTOA. The ACLT surgical model is a widely used preclinical approach for inducing PTOA and effectively replicates key pathological features observed in human ACL injuries, including altered joint biomechanics, synovial inflammation, and cartilage degradation (Figure 7 and Figure 8)

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x phosphate buffered saline (PBS)	Cytiva	SH30256.02
4% paraformaldehyde	Fisher Scientific	AAJ19943K2
70% Ethanol	Fisher Scientific	BP82031GAL
Absorbable 6-0 sutures	Patterson Veterinary Supply	07-893-1916
Blunt-tip forceps	Fine Science Tools	11051-10
Cotton swabs	Fisher Scientific	22-029-685
Dissection microscope	Leica Microsystems	M165 FC
Electric razor	Braintree Scientific	CLP-9961
Ethanol wipes	Fisher Scientific	22-246073
Ethiqa XR (Buprenorphine SR)	Covertus	72117	1.3 mg/mL
Eye lubricant	Patterson Veterinary Supply	NC1701236
Fine scissors	Fine Science Tools	14061-10
Heat pad	Fisher Scientific	NC1061604
Horos	Horos	horosproject.org
Isoflurane	Patterson Veterinary Supply	07-890-8115
Ketamine	Patterson Veterinary Supply	07-890-8598
Meloxicam	Patterson Veterinary Supply	07-893-7565	5 mg/mL
Multiplex assay	MesoScaleDiscovery	K15069M-1
Needleholder	Codman	36-2000
No. 11 scalpel	Fine Science Tools	10011-00
P10 pipette	Gilson	F144055M
P10 pipette tips	Fisher Scientific	02-707-439
Povidone-Iodine swabstick	Medline	MDS093901ZZ
Scanco Medical µCT-35 system	Scanco	https://www.scanco.ch/systems-overview.html	7 µm voxel size, 55 kVp, 0.145 mA, 600 ms
Sterile drapes	McKesson General Medical	NC2032042
Surgical face mask	Fisher Scientific	18-048-010
Surgical gloves	Encore	7823PF
Surgical tape	Patterson Veterinary Supply	07-888-0614
Syringes	BD	309623	1 mL
Vannas spring scissors	World Precision Instruments	501778	3 mm blades
Vernier capiler	Neiko	01407A
Xylazine	Patterson Veterinary Supply	07-893-8424