Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
SCHOOL OF VETERINARY MEDICINE
Non Technical Summary
Partial rupture of the suspensory ligament (SL) is an important, but poorly understood condition of horses This project will determine the association between SL partial rupture and expression of HIF-1(alpha) and TRAP which may be an important first step in determining the primary mechanism that leads to SL mechanical degradation during injury
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Hypothesis 1: Partial rupture of the equine SL is associated with a tissue metabolic response to ligament hypoxia. We propose to use histomorphometry and immunohistochemistry to address the following specific questions: 1.Are alterations to ECM collagen fiber diameter and crimp pattern associated with phenotypic transformation of ligament cells in partially ruptured SL? 2.Is SL partial rupture associated with cellular immunoreactivity to the nuclear transcription factor HIF-1alpha? 3.Is SL partial rupture associated with increased apoptosis of ligament cells? Hypothesis 2: Partial rupture of equine SL is associated with expression of tartrate-resistant acid phosphatase. We propose to use immunohistochemistry to determine whether the expression of TRAP in SL tissue is co-localized with CD80 immunoreactivity (CD80-IR), suggesting that the TRAP positive cells are derived from the bone marrow progenitor cells. The following specific questions will be addressed: 1.Is SL
partial rupture associated with expression of TRAP? 2.Is SL partial rupture associated with expression of CD80-IR cells? 3.Is TRAP and CD80 protein expression in partially ruptured SL tissue localized in the same cells?
Project Methods
Specimens will be collected at the time of euthanasia from 10 horses with SL partial rupture, and from 20 clinically normal horses for control tissue. The following methods will provide the framework to assess collagen morphology, cell signaling and collagenolytic enzyme response to ligament injury in the equine suspensory ligament. Samples of SL, 4 cm in length, will be collected from the proximal third of the ligament, the mid-metacarpal or metatarsal region, and from the lateral and medial branches of the SL distally. A section, 1 cm in length will be used for transmission electron microscopy (TEM). The remaining 3 cm length of each ligament specimen will be fixed Zamboni's fixative. One segment, 2 cm in length, will be paraffin-embedded, and 5 micron longitudinal, mediolateral sections will be cut and stained with hematoxylin and eosin. The remaining 1 cm segment will be used for preparation of transverse frozen sections. Metabolic changes: Transverse frozen
sections, 10 microns in diameter, will be cut and mounted on charged slides. HIF-1(alpha), CD80, and TRAP localization will be determined using immunohistochemistry. The HIF levels provide an index of metabolic response to hypoxia. CD80 is a cell surface marker of macrophages. TRAP has been demonstrate to have potent collagenolytic activity and is associated with ligament damage in cruciate ligament injury. The positive control will be ruptured canine cruciate ligament. Apoptosis will be detected using the TUNEL assay, which identifies DNA fragments generated by endonuclease activity in cells. Mayer's hematoxylin will be used as a counter-stain. Small intestine will serve as a positive (+ve) control. The number density and phenotype of HIF-1-alpha-IR, TUNEL, and TRAP positive cells in the central and peripheral ROI will be recorded for each specimen. Histochemical: Staining for TRAP positive cells will be performed using naphthol ASBI phosphate mixed with hexazotized pararosanaline,
in the presence of 50 mmol/l L (+) tartaric acid added to the substrate solution. The counterstain will be Mayer's hematoxylin. Matrix: Mass average diameter of collagen fibrils will be determined from the TEM transverse sections. Tissue sections will be observed in circularly polarized light (605229 RH, 605230 LH circularly polarizing filters, 3M Polaroid). Mean crimp angle and mean crimp period length will be determined by measurements at 50 sites. Ligament fibroblast: Using a standard magnification of x400, images of 5 fields-of-view will be collected from the peripheral and central region-of-interest of the SL sagittal longitudinal paraffin-embedded tissue sections. Using NIH image, the total number density and phenotype (fusiform {aspect ratio >10}, ovoid {aspect ratio of 5-10}, or spheroid {aspect ratio of <5}) of ligament fibroblasts will quantified in each ROI. Chondroid metaplasia may be associated with ligament injury and repair and result in mechanical failure during
repair.