Examples

Resistance Training Induced Osteoarthritis of the Knee: Alterations of Gene Expression in Articular Cartilage

 

Student

Justin A Jacobson,
Student Box 649,
Class of 2003

Faculty Advisor

Kevin P. Black, M.D., Department of Orthopaedics,
Thorsten Kirsch, PhD., Department of Orthopaedics

Research Period

June 2000 – Aug 2002

Objectives

  1. 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.
  2. Evaluation a novel resistance-training rat model for induction of osteoarthritis.

 

Background

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

Methods

Animal Protocol

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.

Tissue Protocol

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.

Tissue Analysis

Osteoarthritic changes will be evaluated with H&E stain using a standardized grading scale for OA.

0: Normal.
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.

Student's Responsibilities

  1. Review of literature of animal models of osteoarthritis, protein markers and histological analysis of early stage osteoarthritis.
  2. Learn operant conditioning techniques and train rats for resistance training study.
  3. IACUC protocol and approval.
  4. Tissue harvest, collection and preparation.
  5. Immunostaining and tissue analysis/grading.
  6. Statistical analysis of data.
  7. Prepare and submit a final MSR manuscript.
  8. Assist in writing abstract and paper for publication submission.

 

Sponsor's Responsibilities

  1. Provide guidance in project development and implementation.
  2. Provide training in immunostaining, tissue analysis, and enzyme activity assays.
  3. Assist in statistical analysis of data.
  4. Assist in writing of abstract and paper for publication.
  5. Review draft and final MSR project manuscript.

 

Signatures

_________________
Kevin P. Black, M.D.

__________________
Thorsten Kirsch, Ph.D.

__________________
Justin Jacobson

"I give permission for my proposal to possibly be published on the College of Medicine website."

References

1.  Strange, C., 1996. Coping with arthritis in its many forms.  FDA Consumer. 30 (2)

2.  CDC. 1999.  Impact of Arthritis and Other Rheumatic Conditions on the Health-Care system – United States 1997.  MMWR  48 (17): 349-359.

3.  Sandmark, H. and Vingard E., 1999. Sports and risk for severe osteoarthritis of the knee.  Scandinavian Journal of Medicine and Science in Sports. 9: (5) 279-284

4.  Saxon L., C. Finch, and S. Bass.,  1999.  Sports participation, sports injuries and osteoarthritis:  implications for prevention.  Sports Medicine. 28: (2) 123-135.

5.  Lane, N.E. and Buckwalter, J.A.  Sep 1999.  Exercise and osteoarthritis.  Current Opinion in Rheumatology. 11(5): 413-416.

6.  Cooper, C. and Coggon, D.  June 1999.  Physical activity and knee osteoarthritis.  The Lancet, 353 (9171): 2177-2178.

7.  Fitzgerald B. and McLatchie, G.R.  Aug 1980.  Degenerative joint disease in weight lifters, fact or fiction?  British Journal of Sports Medicine. 14 (2,3): 97-101. 

8.  Goldring, M., 2000. The role of the chondrocyte in osteoarthritis. Arthritis and Rheumatism,  43 (9): 1916-1926.

9.  Leyes, M. 1998. Articular Cartilage Review.

10.  Yasunori, O. et al.  1992. Localization of MMP-3 (stromelysin) in osteoarthritic cartilage and synovium.  Laboratory Investigation. 66 (6): 680-689.

11.  Dean, D. et al. Aug 1989. Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage.  Journal of Clinical Investigation. 84:678-685.

12.  Hembry, R.M. et al.  1995.  Immunolocalisation studies on six matrix metalloproteinases and their inhibitors, TIMP-1 and TIMP-2, in synovia from patients with osteo-and rheumatoid arthritis. Annals of Rheumatic Diseases. 54: 25-32

13.  Martel-Pelletier, J. et al.  1994. Excess of metalloproteases over tissue inhibitor of metalloprotease may contribute to cartilage degradation in osteoarthritis and rheumatoid arthritis. Laboratory Investigation. 70 (6): 807-815.

14.  Lindhorst, E. et al.  2000. Longitudinal characterization of synovial fluid biomarkers in the canine meniscectomy model of osteoarthritis.  Journal of Orthopaedic Research. 18:269-280.

15.  Lark, M. et al. 1997. Aggrecan degradation in human cartilage: evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic and rheumatoid joints.  The Journal of Clinical Investigation.  100(1): 93-106.

16.  Hazell, P. et al.  1995.  Changes in glycosaminoglycan epitope levels in knee joint fluid following injury.  Arthritis and Rheumatism. 38: 953-959.

17.  Bendele, A., et al, Jan-Feb 1999. Animal models of arthritis: Relevance to human disease.  Toxicologic Pathology, 27: (1) 134-142.

18.  Machner, A. et al,  1999. Deterioration in sensible joint innervation as a possible cause for the development of osteoarthritis. Zeitschrift fuer Rheumatologie.  58: (3) 148-154.19.

19.  Lapvetelainen, T. et al. 1995.  Lifelong moderate running training increases the incidence and severity of osteoarthritis in the knee joint of C57BL mice.   The Anatomical Record. 242: 159-165.

20.  Machner, A., et al.  Gelenkbelastung als eine moegliche Ursache der Arthrose Entstehung.  Zentralblatt fuer Chirurgie 125, 2000, 536-542.

21.  Pap, G., et al 1998. Development of Osteoarthritis in the knee joints of Wistar rats after strenuous running exercise in a running wheel by intracranial self-stimulation.  Pathology Research and Practice, 194: (1) 41-47.

22.  Newton, P. et al.  1997. The effect of lifelong exercise on canine articular cartilage.  The American Journal of Sports Medicine. 25 (3)282-287.

23.  Kiviranta, I. et al.  Oct 1992.  Articular Cartilage Thickness and Glycosaminoglycan distribution in the canine knee joints after strenuous running exercise.  Clinical Orthopaedics and Related Research. 283: 302-308.)

24.  Markku, T. et al.  1983. Proteoglycan alterations in rabbit knee articular cartilage following physical exercise and immobilization.  Connective Tissue Research. 11: 44-55.

25.  Dekel, S. and Weissman S.L., 1978. Joint changes after overuse and peak overloading of rabbit knees in vivoActa Orthopaedic Scandinavia, 49: 519-528.

26.  Palmer, J. et al. 1995. Site-specific proteoglycan characteristics of third carpal areticular cartilage in exercised and nonexercised horses.  American Journal of Vetrinary Research, 56 (12): 1570-1576.

27.  Bancrock, .D. and Stevens, A. Theory and Practice of Histological Techniques.  Churchill Livingston, 1993.

References

1.  Carey, Timothy S., Joanne Garrett, Anne Jackman, Curtis McLaughlin, John Fryer, Douglas R. Smucker, The North Carolina Back Pain Project.  1995.  The Outcomes and Costs of Care for Acute Low Back Pain among Patients Seen by Primary Care Practitioners, Chiropractors, and Orthopedic Surgeons.  The New England Journal of Medicine.  333(14):913-917.

2.  Croft, Peter R., Gary J. Macfarlane, Ann C. Papageorgiou, Elaine Thomas, Alan J. Silman.  1998.  Outcome of low back pain in general practice: a prospective study.  British Journal of Medicine. 316(7141):1356-1359.

3.  Deyo, Richard A.  1996.  Acute low back pain: a new paradigm for management: Limited imaging and an early return to normal activities.  British Medical Journal. 313(7069):1343-1344.

4.  Henley, Eric.  2000.  Understanding and Treating Low Back Pain in Family Practice.  The Journal of Family Practice.  49(9):793-795.

5.  Little, Paul, Lisa Smith, Ted Cantrell, Judith Chapman, John Langridge, Ruth Pickering.  1996.  General practitioner's management of acute back pain: a survey of reported practice compared with clinical guidelines.  British Medical Journal. 312(7029):485-488.

6.  Lurie, Jon D., Paul D. Gerber, Harold C. Sox.  2000.  A Pain in the Back.  The New England Journal of Medicine.  343(10):723-726.

7.  Malanga, Gerard A. MD, Scoot F. Nadler, DO.  1999.  Nonoperative Treatment of Low Back Pain.  Mayo Clinic Proceedings.  74(11):1135-1148.

8.  Reis, Shmuel MD, Doron Hermoni, MD, Jeffrey M. Borkan, MD, Aya Biderman, MD, Chava Tabenkin, MD, Avi Porat, MD.  1999.  A New Look at Low Back Complaints in Primary Care; A RAMBAM Israeli Family Practice Research Network Study.  The Journal of Family Practice.  48(4):299-303.

9.  Suarez-Almazor, Maria E. MD, MSc, PhD, Elaine Belseck, BSCN, Anthony S. Russell, MB, BCH, FRCPC, John V. Mackel, MB, CCFP, FCFP.  1997.  Use of Lumbar Radiographs for the Early Diagnosis of Low Back Pain: Proposed Guidelines Would Increase Utilization.  The Journal of the American Medical Association.  277(22):1782-1786.