Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection

Summary

Dunkin-Hartley guinea pigs are an established animal model for osteoarthritis research. Such studies may benefit from intra-articular injections for various reasons, including investigating novel agents or treating disease. We describe a methodology for intra-articular knee injections in Guinea pigs and subsequent micro-computed tomography analysis assessing arthritis-associated knee changes.

Abstract

The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and assessment using micro-computed tomography. Dunkin-Hartley guinea pigs are robust models for osteoarthritis research as they spontaneously develop osteoarthritis in their knees. Intra-articular drug delivery is a common method to study the effects of an investigational drug in vivo. In humans, therapeutic agents administered via intra-articular injection can offer pain relief and delay further progression of osteoarthritis. As with any species, the introduction of a needle into a joint space has the potential to cause injury, which can result in pain, lameness, or infection. Such adverse events can compromise animal welfare, confound study results, and necessitate additional animals to achieve study objectives. As such, it is imperative to develop proper injection techniques to prevent complications, especially in longitudinal studies that require multiple, repeated intra-articular injections. Using the presented methodology, five guinea pigs received bilateral knee injections under general anesthesia. Seven days after injection, animals were humanely euthanized for analysis of osteoarthritis severity. No adverse events occurred following anesthesia or knee injections, including limping, pain, or infection. X-ray micro-computed tomography analysis of the knee can detect pathologic changes associated with osteoarthritis. Micro-computed tomography data indicates osteoarthritis is more severe in older animals, as indicated by increased bone mineral density and trabecular thickness with age. These results are consistent with histologic changes and Modified Mankin scores, an established and widely used scoring system to assess arthritis severity in these same animals. This protocol can be utilized to refine intra-articular injections in guinea pigs.

Introduction

Osteoarthritis (OA) impacts 32.5 million US adults. It is caused by progressive loss of articular cartilage, mild inflammation of the tissues in and around the joints, and formation of osteophytes and bone cysts^1,2. Symptoms typically manifest in the later stages of the disease, with current treatments providing only palliative relief as well as having systemic side effects. The lack of disease-modifying drugs stems from a poor understanding of the underlying mechanisms of the disease³. As a result, there is a critical and ongoing medical need for improved agents to treat OA.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. The study followed the principle of 3R.

1. Intra-articular injection preparations

1. Allow Dunkin-Hartley guinea pigs to acclimate to facility for at least one week prior to starting the experiment.
   NOTE: 5- (n=2), 9- (n=1), and 12- (n=2) month-old male guinea pigs were used. Males display an accelerated development and more severe phenotype of OA.
2. Shave the knee area with an electric razor.
   NOTE: Be careful of the nipples medially.
3. Anestheti....

Results

Before performing intra-articular injections on live animals, the above protocol was practiced on three rat cadavers to ensure correct injection location. During the practice sessions, 50 µL of 70% new methylene blue dye was injected into both knee joints using the methodology described above. This equates to six practice injections. After injections, the knee joint was dissected by incising through the cranial aspect of the joint space, distal to the patella and through the patellar ligament, to visualize the joint spac.......

Discussion

Despite recent advancements in symptomatic treatment of OA, there is a complete lack of therapeutic agents that prevent onset or delay progression of OA²⁴. Currently, the only cure for severe OA is joint replacement, which is costly, invasive, and can result in patient morbidity and mortality²⁵. As a result, there is a dire need for continued research with animal models of OA and the sustained development of novel therapeutics. Several animal models are available to study d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
200 Proof Ethanol	Decon Laboratories	2701	sterilizing agent
3D.SUITE software	Bruker		μ-CT analyzing software
Betadine Surgical Scrub	Avrio Health	67618-151-16	sterilizing agent
Insulin syringe with needle	Ulticare	91008	to perform injections
Isoflurane	Piramal	803249	anesthesize animal
Neutral Buffered Formalin	Fisher Scientific	23-427098	Fix tissue
Nrecon Software	Bruker		μ-CT reconstruction software
Phosphate Buffered Saline	Cytiva	SH30258.01	control and diluting agent
SkyScan 1176	Bruker		to scan samples