Evaluation of ligament fibroblast viability in ruptured cranial cruciate ligament of dogs.
OBJECTIVE
To determine fibroblast viability, assess development of apoptosis, and evaluate tissue hypoxia via histochemical, in-situ hybridization, or immunohistochemical staining in ruptured and intact cranial cruciate ligaments (CCLs) of dogs.


METHODS
32 dogs with ruptured CCLs, and 8 aged and 19 young dogs with intact CCLs.


METHODS
Markers of cell viability (lactate dehydrogenase [LDH]), apoptosis (terminal deoxynucleatidyl transferase-mediated deoxyuridine triphosphate-nick end labeling [TUNEL] method), and hypoxia (hypoxia-inducible factor-1alpha [HIF-1alpha] monoclonal antibody) were applied to CCL specimens; positive cells were assessed objectively (LDH) and subjectively (TUNEL and HIF-1alpha) in the main axial tissue component (core) and synovial intima and subintima (epiligamentous tissue).


RESULTS
Viable fibroblasts were seen in all intact and ruptured CCLs. More nonviable cells were found in the core regions of ruptured CCLs and intact CCLs of young dogs than in the epiligamentous regions. Number of nonviable cells in the core region of ruptured CCLs was greater than that in intact CCLs of young and aged dogs, whereas the number in the epiligamentous region was similar in all specimens. The TUNEL and HIF-1alpha staining was only found in the epiligamentous region of ruptured CCLs.


CONCLUSIONS
Ruptured CCLs contained a high number of nonviable cells but not a great number of apoptotic cells. Repair processes in the epiligamentous region of the CCL include a metabolic response to hypoxia, suggesting that necrosis of ligament fibroblasts and transformation of surviving cells to a spheroid phenotype may be a response to hypoxia cause by microinjury or inadequate blood flow.
Hayashi
Kei
K
Comparative Orthopaedic Research Laboratory, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Dr, Madison, WI 53706, USA.
Frank
Joseph D
JD
Hao
Zhengling
Z
Schamberger
Gwenn M
GM
Markel
Mark D
MD
Manley
Paul A
PA
Muir
Peter
P
D000375:Aging; D000818:Animals; D016118:Anterior Cruciate Ligament / Q000473:pathology*; D000070598:Anterior Cruciate Ligament Injuries; D002470:Cell Survival; D004285:Dogs / Q000293:injuries*; D005260:Female; D005347:Fibroblasts / Q000473:pathology*; D006614:Hindlimb / Q000293:injuries* / Q000473:pathology*; D008297:Male; D012421:Rupture / Q000473:pathology* / Q000662:veterinary*