Jun 17, 2008 - Shannon Kelly Reed for the degree of Master of Science in Veterinary Science ...... were performed by the
AN ABSTRACT OF THE THESIS OF Shannon Kelly Reed for the degree of Master of Science in Veterinary Science presented on June 17, 2008. Title: A Molecular and Morphologic Study of Idiopathic Fetlock Hyperextension and Suspensory Apparatus Breakdown in the Llama Abstract approved: ____________________________________________ Stacy A. Semevolos
Suspensory apparatus breakdown and hyperextension of the metacarpophalangeal and metatarsophalangeal (fetlock) joints is a common condition in the llama and has been observed in llamas of all ages. Llama breeders refer to the condition as “down in the pasterns” or “down in the fetlocks.” The condition can result in debilitating lameness, most likely due to mineralization of soft tissues including the tendons and ligaments and/or osteoarthritis of the metacarpo/metatarsophalangeal, proximal interphalangeal and distal interphalangeal joints. Two forms exist, an induced form occurring from abnormal weight bearing such as a severe lameness in a contralateral limb, and an idiopathic form that affects multiple limbs. The idiopathic form is poorly characterized in the llama and has not been reported in the literature prior to the studies performed as part of this thesis. The specific aims of this thesis were to characterize the nature of suspensory apparatus breakdown in the llamas by use of ultrasonographic and radiographic evaluation, histologic evaluation, biochemical assessment of collagen, copper concentrations and lysyl oxidase activity, and molecular techniques to identify specific gene expression alterations and matrix connective tissue changes. The specific hypotheses were: 1) affected llamas would have ultrasonographic and histologic
evidence of disruption of fibers in the suspensory ligament 2) affected llamas would have decreased copper concentrations and lysyl oxidase activity 3) affected llamas would have decreased gene expression of collagen type I and lysyl oxidase, and increased gene expression of collagen type III and matrix metalloproteinases as a result of ongoing repair of tendons and ligaments. High serum zinc concentration coupled with low liver copper concentration were found in llamas having metacarpo(tarso)phalangeal hyperextension. However, lysyl oxidase activity was no different between the affected and controls, even though the copper levels were lower in affected animals. In addition, other expected changes on radiographs and ultrasound were not as prevalent as hypothesized. No significant difference was appreciated between affected and control animals in expression levels of collagen types I or III, LOX or MMP-13, although there was a trend towards decreased expression of MMP-13 in affected animals. Mild proteoglycan accumulation was appreciated in the suspensory ligament of two of the six affected animals. No difference in distribution of collagen types I or III was appreciated on histologic section, and elastic fiber appearance was similar between the affected and control animals. This thesis suggests a different etiology to fetlock hyperextension than initially hypothesized.The lack of radiographic, ultrasonographic, histologic, and biochemical differences between affected and control animals supports a nondegenerative etiology for this condition.
© Copyright by Shannon Kelly Reed June 17, 2008 All Rights Reserved
A Molecular and Morphologic Study of Idiopathic Fetlock Hyperextension and Suspensory Apparatus Breakdown in the Llama by Shannon Kelly Reed
A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented June 17, 2008 Commencement June 2009
Master of Science thesis of Shannon Kelly Reed presented on June 17, 2008. APPROVED: ______________________________________________________________ Major Professor, representing Veterinary Science _______________________________________________________________ Dean of the College of Veterinary Medicine _______________________________________________________________ Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. _______________________________________________________________ Shannon Kelly Reed, Author
ACKNOWLEDGEMENTS I would like to thank the Willamette Valley Llama Foundation for funding of this project. Dr. Stacy Semevolos has been an amazing mentor with just the right amount of support and high expectations. Several technicians were instrumental in the project: Kari Gamble in the lab, Becki Francis in radiology, and Shawn Davis, Sharon Ward, Betsy Snyder, Garland Burdock, and Shannon Casserly in the large animal hospital. I thank my family for their never ending support and my husband Jeremy Reed for being everything to me. I also thank the animals who made the ultimate sacrifice in the name of research, you could find no human with this degree of dignity in sacrifice.
CONTRIBUTING AUTHORS Dr. Stacy Semevolos designed the study and was involved in the research of Chapters 1, 2 & 3. Drs. Gheorghe and Ileana Constantinescu assisted in the writing of Chapter 1 with dissections of specimens and naming of structures. Dr. G. Constantinescu illustrated the dissections of the anatomy specimens. Dr. Paul Rist performed and interpreted the ultrasound examinations in chapter one. Dr. Beth Valentine assisted in the design and interpretations of tissue histology.
TABLE OF CONTENTS Page Introduction……………………………………………
2
Suspensory apparatus........................................
2
Tendon structure and function…………………
3
Ligament structure and function………………
5
Comparative anatomy…………………………
6
Superficial digital flexor tendon………
7
Deep digital flexor tendon…………….
9
Suspensory ligament…………………..
13
Sesamoidean ligaments……………….
16
Lumbricalis muscle…………………...
21
Suspensory apparatus breakdown…………….
25
Morphological and biochemical characterization of metacarpophalangeal and metatarsophalangeal hyperextension in llamas…………………………….
32
Introduction…………………………………..
33
Materials and methods………………………..
34
Lysyl oxidase activity………………...
35
Immunohistochemistry……………….
37
Results………………………………………...
38
Discussion…………………………………….
44
Idiopathic hyperextension of the metacarpophalangeal and metatarsophalangeal joints in llamas: molecular and histological characterization…………………………….
53
TABLE OF CONTENTS (Continued) Introduction……………………………………..
Page 54
Materials and methods………………………….
55
Sample preparation……………………..
55
cDNA cloning………………………….
55
Real-time quantitative reverse transcription polymerase chain reaction………………
56
Histologic Evaluation……………………
59
Statistical Analysis………………………
60
Results…………………………………………...
60
Discussion……………………………………….
65
Conclusion……………………………………………….
73
Bibliography…………………………………………….
78
Footnotes………………………………………………..
81
LIST OF FIGURES Figure
Page
1. The palmar aspect of the carpal canal and metacarpus in the llama…………………………………………………………….
11
2. Left pelvic limb with palmar view of metatarsal bones, dorsal view of proximal sesamoid bones and the suspensory ligament/ interosseous muscle………………………………………………....
18
3. Left pelvic limb, plantar aspect of the fetlock joints with the SDFT and DDFT removed ………………………………………….
19
4. Photograph of the distal limb of the llama with the skin and superficial digital flexor tendon removed………………………
23
5. The distal limb of the llama with the superficial flexor tendon reflected to reveal the emergence of the lumbricalis muscle at the splitting of the deep digital flexor tendon……………………
24
6. Distal forelimb in llama with normal fetlock and normal pastern to ground axis ……………………………………………..
27
7. Distal forelimb in llama with fetlock hyperextension and normal pastern to ground axis………………………………………
28
8. Lateral radiographs of the metacarpophalangeal joint….
40
9. Mean liver copper levels comparing llamas affected with fetlock hyperextension and age-matched controls………………….
42
10. Mean serum zinc levels comparing llamas affected with fetlock hyperextension and age-matched controls………………….
43
11. Photomicrographs following immunohistochemical localization with antibodies directed against collagen type I ………
46
12. Photomicrographs following immunohistochemical localization with antibodies directed against collagen type III………
47
13. Hematoxylin and eosin stained sections from normal llamas……………………………………………………………….
48
14. mRNA expression of collagen type I quantified via real-time PCR using suspensory ligaments from affected and control llamas…………… 61
LIST OF FIGURES (Continued) Figure
Page
15. mRNA expression of collagen type III quantified via real-time PCR using suspensory ligaments from affected and control llamas …………
62
16. mRNA expression of lysyl oxidase quantified via real-time PCR using suspensory ligaments from affected and control llamas ………
63
17. mRNA expression of MMP-13 quantified via real-time PCR using suspensory ligaments from affected and control llamas ……
64
18. Alcian blue stained suspensory ligament in affected animal with proteoglycan stained blue……………………………………….
66
19. Longitudinal section of suspensory ligament form and affected llama. ……………………………………………………….….......
67
20. Trichrome stain of the cross section of the suspensory ligament of an affected llama with collagen stained blue, and the cytoplasm, keratin, and muscle fibers stained red (bar=100μm)……
68
LIST OF TABLES Table
Page
1. Species comparisons of origins and insertions of superficial digital flexor tendon in the forelimb………………………………….
10
2. Species comparisons of origins and insertions of deep digital flexor tendon in the forelimb………………………………….
14
3. Species comparisons of origins and insertions of suspensory ligament in the forelimb……………………………………………...
17
4. Species comparisons of origins and insertions of sesamoidean ligaments in the forelimb…………………………………………………………
22
5. Six adult animals having at least 2 metacarpophalangeal joints with hyperextension (affected) and six normal age-and sex-matched controls evaluated for body weight, body condition, serum zinc and copper levels
39
6. Immunohistochemistry mean total scores of the SDF, DDF, and suspensory ligament compared between affected and control llamas for collagen type I and type III protein expression……………………………………
45
7. Oligonucleotides primers for polymerase chain reaction (PCR) assays designed on the basis of concensus sequences for LOX, COLI, COLIII, MMP-13, and ß-actin…………………………………………………….
57
8. Llama specific primer and probe sequences for real-time PCR quantification of gene expression in suspensory ligament from affected and control llamas……………………………………………………….
58
A molecular and morphologic study of idiopathic fetlock hyperextension and suspensory apparatus breakdown in the llama
2
Chapter One Introduction
Suspensory apparatus The suspensory apparatus consists of the superficial digital flexor tendon, the deep digital flexor tendon, the suspensory ligament (interosseous muscle), and the proximal sesamoid bones and associated ligaments. The suspensory apparatus supports the distal limb, allowing the joints to maintain relatively upright anatomical positions and preventing hyperextension of the metacarpophalangeal and metatarsophalangeal joints.
Tendon structure and function The function of tendon is to join muscle to bone and transmit forces between them. Tendons arise from muscles and insert on the bone, with the gradation from tendon to muscle referred to as the myotendinous junction and the junction of tendon to bone referred to as the osteotendinous junction or insertion. The attachment of muscle to bone is referred to as the origin of the muscle.1 Tendons in general show great resistance to mechanical loads, owing most of this resistance to the collagen matrix that constitutes the majority of the tendon. The flexor tendons are predominantly type I collagen, with smaller amounts of collagen types III, V, XII, and XIV.2 The large, highly cross-linked collagen type I fibrils create the high tensile strength of tendons. Tendons have several support structures associated with them including fibrous sheaths and retinacula, reflection pulleys, synovial sheaths,
3
peritendinous sheaths, and tendon bursae.1 These associated structures are responsible for keeping the tendons orientated correctly and allow the tendons to glide smoothly over bony prominences and through changes in direction. Tendons have a surrounding connective tissue structure referred to as the paratenon. The paratenon contains both collagen types I and III, as well as elastic fibrils. It is lined by synovial cells on its inner surfaces, as the paratenon permits free movement of the tendons against the surrounding structures in the absence of a true two layer tendon sheath.1 The epitenon is contiguous with the paratenon on its outer aspect and the endotenon on the inner surface. The tendon is penetrated by the endotenon, which invests each tendon fiber and binds both individual fibers and larger units together. Between the endotenon and the tendon, a well hydrolated layer of proteoglycans exists to improve binding. The endotenon also brings in blood vessels, nerve fibers and lymphatics. Collagen fiber orientation within tendons varies greatly between different tendons and even within a single tendon.1 Collagen fibers are cross-linked together to form primary, secondary and tertiary fiber bundles. Collagen fibers constitute 65-80% of the dry mass of the tendon and the majority of the collagen in healthy tendon is type I. Beyond the collagen component of the tendon, there is an extensive extracellular tendon matrix composed of collagen fibers, elastic fibers (1-2% of dry mass of tendon), ground substance containing proteoglycans glycosaminoglycans (GAG) (0.23.5% of dry mass of tendon), structural glycoproteins, and inorganic components (
