Slide 1

Evaluation of Gene Expression in Whole Blood of Osteoarthritic and Normal Horses

Thomas E. Kellerman, David A. Wilson, Aaron M. Stoker

University of Missouri – College of Veterinary Medicine

Clinical Relevance

Introduction

Materials & Methods

Preliminary Data

Acknowledgements

Thoroughbred geldings (n=5) were selected for the study. The horses were separated into experimental and control groups. Horses in the experimental group (n=3) were all diagnosed with OA in the forelimb and horses in the control group (n=2) were not affected with OA. The mean age of the experimental group was 10 years and the mean age of the control group was 15 years. 5ml of whole blood from each horse was collected into two PAXgene Blood RNA tubes (PreAnalytiX). The samples were refrigerated overnight at 4°C. After incubating for 2 hours at room temperature RNA was extracted following the PAXgene Blood RNA Kit protocol. RNA quantity was assessed by measuring absorbance at 260nm and using the RiboGreen RNA test kit (Molecular BioProbes). RNA quality was determined by electrophoresis of total RNA stained with ethidium bromide to assess 28S and 18S ribosomal RNA (rRNA) bands. One total RNA sample per horse was selected for gene expression analysis based on RNA quality and quantity. Both samples were used for horse 5 based on low RNA yield in both samples. Each total RNA sample was amplified and biotin-labeled using the MessageAmp II-Biotin Enhanced Single Round aRNA Amplification Kit (Ambion). Fragmented RNA was submitted to the DNA Core Lab. Each sample was run in duplicate on U133A Microarrays (Affymetrix). GeneChip Operating Software (Affymetrix) will be used to analyze the data and determine significant differences between sample sets. Cross-species homology of genes of interest will be confirmed using RT-PCR.

The University of Missouri Veterinary Research Scholars Program was supported by funds from Merck-Merial, Pfizer and the MU College of Veterinary Medicine. This project was funded by the E. Paige Laurie Endowed Program in Equine Lameness. We thank Robin Hurst-March and Zac March of Out 2 Pasture Thoroughbred Retirement Farm for providing horses for sample collection. We also thank Don Connor and Howard Wilson for their technical advice.

Fig. 2

Lateral radiographic views demonstrating normal (a) and osteoarthritic (b) pastern joints.

Fig. 1

Arthroscopic views of normal (a) and osteoarthritic (b) cartilage.

Lameness is the most frequently occurring medicial condition of the horse. Data suggests that 60% of lamenesses are associated with osteoarthritis. Osteoarthritis (OA) is a progressive and irreversible deterioration of articular cartilage. Any number of insults to the joint can result in OA. The pathogenesis of OA involves the application of abnormal stresses to normal cartilage or the application of normal stresses to abnormal cartilage. The classic manifestation of OA is cartilage degredation characterized by softening, fibrillation and erosion (Fig. 1). Increased volumes of abnormal synovial fluid, synovitis, capsulitis and osteophyte and enthesiophyte formation are also seen. Traditional diagnosis of OA is based upon clinical examination. Lameness is often present as well as joint effusion and heat. Diagnosis is often supported by radiographic findings including, narrowed joint space, osteophyte and enthesiophyte formation and subchondral bone lysis (Fig. 2). Although less frequently used, computerized tomography (CT) and magnetic resonance imaging (MRI) are other imaging modalities that can be utilized in diagnosis of OA. Once an injury progresses to OA it cannot be cured and management is aimed towards minimizing pain, maximizing function and slowing the degredation of the joint.

Following extraction, quantity of RNA per sample was measured using the Ribogreen RNA kit (Fig. 4). Total RNA was amplified to increase yield by approximately 80 fold (Fig. 5). Quality of extracted RNA was evaluated by electrophoresis of total RNA stained with ethidium bromide to visualize 28S and 18S rRNA bands (Fig. 6).

Fig. 6

Electrophoresis gel stained with ethidium bromide showing two characteristic ribosomal RNA bands (18S and 28S).

Fig. 4

Yield from samples 1, 3, 5, and 7 was greater than 2.4 µg.

Fig. 3

Histopathology sections showing normal cartilage (a), mild OA (b), and severe OA (c).

Osteoarthritis is a career ending and possibly life threatening condition. The most effective treatments of OA require early detection, before clinical signs are present. There are no specific and sensitive tests that exist for the early detection of OA. Research has focused on studying the mediators of inflammatory processes as targets for developing diagnostic tests. To date there have been no studies that have evaluated more than a few inflammatory mediators simultaneously. Evaluating genome wide expression rather than just a selected few inflammatory mediators may lend new insight into the inflammatory process. By utilizing microarray technology the expression of thousands of genes can be examined at the same time. It is hoped that this pilot study will identify genes that have not yet been examined for their role in OA. If there is a change in the regulation of a specific gene during inflammatory processes it may be a target for developing novel diagnostic tests and therapeutic agents.

Fig. 5

Yield of amplified RNA.