Resistance Training Induced Osteoarthritis of the Knee: Alterations of Gene Expression in Articular Cartilage
Justin A Jacobson,
Student Box 649,
Class of 2003
Kevin P. Black, M.D., Department of Orthopaedics,
Thorsten Kirsch, PhD., Department of Orthopaedics
June 2000 – Aug 2002
- Histological and immunohistochemical analysis of resistance trained rats to evaluate osteoarthritic changes in articular cartilage structure and protein biomarker production, as well as enzyme assays of synovial fluid.
- Evaluation a novel resistance-training rat model for induction of osteoarthritis.
Osteoarthritis (OA) is a major cause of mortality and morbidity within the United States, most commonly affecting the elderly population.1,2 Recent trends in exercise and fitness within the United States have increased physiologic stress and wear on articular cartilage, especially of the knees and hips. Clinical studies evaluating low impact sports and incidence of osteoarthritis are well cited,3,4,5 as well as the correlation of high impact/intensity occupations and sports with OA.6 This is most prominent in athletes who pursue heavy weight training regimens where loads are significantly larger than normal physiologic loads.7
OA is a progressive degeneration of articular cartilage, often localized to weight-bearing cartilage or to sites of trauma. Repetitive mechanical injury is the proposed mechanism for induction of OA, and the chondrocyte is accepted as the target of these biomechanical factors.8,9 In response to this stress (mechanism unknown), chondrocytes express elevated levels of collagenases (also called matrix metalloproteinases or MMPs), decreased levels of tissue inhibitor of metalloproteinase (TIMP) and produce abnormal type X collagen. It is theorized that these enzymes and their imbalance contribute to proteolytic degradation of articular cartilage.10,11,12,13 Loss of glycosaminoglycans and proteoglycans has also been observed in OA, either in response to microtrauma or increased proteolytic activity.14,15,16
Current animal models used in studying osteoarthritis are induced either surgically or chemically and do not use resistance training in its true form for OA induction.17 This study will also evaluate the use of a rat model which solely relies upon resistance training for induction of osteoarthritis. Other previously studied models are meniscal and/or anterior cruciate sectioning, capsaicin injection,18 collagen knockout mice,19 as well as running treadmill20,21 and jumping models used in rats, canines,22,23 hamsters, rabbits24,25 and horses.26
Sprague-Dawley rats, age 3 months and 10 months, will be operantly conditioned to stand on their hind legs and hit an illuminated, elevated lever with their front paws to avoid electrical footshock stimulus. The animals will be exercised 3 days per week for six weeks, each session consisting of 3 sets of 25 repetitions. The experimental group will exercise while wearing a weighted vest that equals their body weight. Each animal will be weighed once per week and the load adjusted accordingly. Exercise controls will undergo the same training regimen without the weighted vest. Non-exercised animals will be used as overall controls.
The action of the exercise simulates what an athlete would do when performing a "leg press" or full extension squat exercise. The overhead bar is adjusted in height to require full extension of the knee joint. This exercise is within tolerable physiologic loads for the knee joints. Age groups are comparable to adolescent and middle aged humans.
The rats will be cared for by the veterinary technicians at the Animal Research Facility at Penn State College of Medicine. They will be kept in a room on a reverse light cycle and trained in the dark with the aid of red lamps.
The rats will be sacrificed one week following completion of the exercise regime. A synovial lavage (0.2-0.4 cc) will be performed on each knee with normal saline (0.9% NaCl). Suprapatellar approach should be used to avoid damaging articular cartilage. Each joint will be labeled with a suture on the medial and superior aspect of each joint to aid proper orientation and identification of anatomic structures. Each joint will be removed intact and stored in 1X PBS and frozen at -20° C.
Harvested joints will be fixed with 4% paraformaldehyde, decalicified in EDTA and embedded in paraffin,27 and each joint sectioned into medial, intermediate and lateral compartments.
Osteoarthritic changes will be evaluated with H&E stain using a standardized grading scale for OA.
1: Fibrillation of superficial layer of cartilage; no loss of cartilage.
2: Fissuring and loss of tissue.
3: Calficied cartilage exposed, tide mark exposed to surface.
4: Deep lesions into bone, 2/3 of cartilage is lost.
This will be done by blind analysis of independent observers. Cartilage matrix loss will be further graded by Safranin-O stain to determine the grade of OA.
Immunohistochemical staining for MMP-13 (a collagenase for type II collagen) will be used to analyze production by chondrocytes. The articular cartilage will be divided into middle and deep sections, using the tide mark to aid in identification of cartilage layer. Each compartment will be analyzed in 5 different areas, counting 20-70 chondrocytes and any immunopositive cells. Both tibia and femur will be analyzed, attempting to analyze weight-bearing surfaces of the cartilage, to determine the percentage of immunopositive cells.
Additional paraffin embedded sections will be saved for possible future evaluation of keratan sulfate, TIMP-1, TIMP-3, MMP-3, and collagen X.
Synovial fluid will be analyzed for MMP enzyme activity using collagenase assay kit. This will be compared with immunohistochemical and histological analysis of tissues.
Statistical analysis will compare mean percent reactive chondrocytes within each compartment of a given exercise group, compare age groups, exercise groups, tibia versus femur, and middle versus deep layers of cartilage.
- Review of literature of animal models of osteoarthritis, protein markers and histological analysis of early stage osteoarthritis.
- Learn operant conditioning techniques and train rats for resistance training study.
- IACUC protocol and approval.
- Tissue harvest, collection and preparation.
- Immunostaining and tissue analysis/grading.
- Statistical analysis of data.
- Prepare and submit a final MSR manuscript.
- Assist in writing abstract and paper for publication submission.
- Provide guidance in project development and implementation.
- Provide training in immunostaining, tissue analysis, and enzyme activity assays.
- Assist in statistical analysis of data.
- Assist in writing of abstract and paper for publication.
- Review draft and final MSR project manuscript.
Kevin P. Black, M.D.
Thorsten Kirsch, Ph.D.
"I give permission for my proposal to possibly be published on the College of Medicine website."
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