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.
Animals—32 dogs with ruptured CCLs, and 8 aged
and 19 young dogs with intact CCLs.
Procedure—Markers of cell viability (lactate dehydrogenase
[LDH]), apoptosis (terminal deoxynucleatidyl
transferase-mediated deoxyuridine triphosphate-nick
end labeling [TUNEL] method), and hypoxia (hypoxiainducible
factor-1α [HIF-1α] monoclonal antibody)
were applied to CCL specimens; positive cells were
assessed objectively (LDH) and subjectively (TUNEL
and HIF-1α) 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-1αstaining was only found in the
epiligamentous region of ruptured CCLs.
Conclusions and Clinical Relevance—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. (Am J Vet Res 2003;64:1010–1016)
Objective—To compare 2 methods of quantitating chondrocyte viability and to determine chondrocyte response to thermal injury over time.
Sample Population—108 stifle joints from 54 adult rats.
Procedures—Cartilage from the distal aspect of the femur was treated ex vivo with radiofrequency energy at a probe setting that would result in immediate partial-thickness chondrocyte death; untreated sections served as controls. Explants were cultured, and cell viability was compared by use of lactate dehydrogenase (LDH) histochemical staining and calcein AM and ethidium homodimer-1 confocal laser microscopy (CLM) cell viability staining. Terminal deoxynucleotidyl transferase–mediated X-dUTP nick end labeling (TUNEL) was used to detect apoptosis. All labeling studies were performed 0, 1, 3, 7, 14, and 21 days after treatment.
Results—In the treated tissues, a greater percentage of viable cells were found with CLM, compared with LDH staining. This result contrasted that of control tissues in which LDH staining indicated a greater percentage of live cells than CLM. The greatest number of TUNEL-positive chondrocytes was present at day 3, declining at later time intervals.
Conclusions and Clinical Relevance—CLM and LDH histochemistry techniques yield different absolute numbers of live and dead cells, resulting in differing percentages of live or dead cells with each technique. These differences may be related to the enzymes responsible for activation in each technique and the susceptibility of these enzymes to thermal injury. Results of TUNEL indicate that apoptosis contributes to chondrocyte death after thermal injury, with a peak signal identified 3 days after insult.
Objective—To compare expression of tartrate-resistant
acid phosphatase (TRAP) and cathepsin K and
histologic changes in canine cranial cruciate ligaments
(CCLs) and human anterior cruciate ligaments (ACLs).
Study Population—Sections of cruciate ligaments
from 15 dogs with ruptured CCLs, 8 aged dogs with
intact CCLs, 14 human beings with ruptured ACLs,
and 11 aged human beings with intact ACLs.
Procedure—The CCLs and ACLs were evaluated histologically,
and cells containing TRAP and cathepsin K
were identified histochemically and immunohistochemically,
Results—The proportion of ruptured CCLs that contained
TRAP+ cells was significantly higher than the
proportion of intact ACLs that did but similar to proportions
of intact CCLs and ruptured ACLs that did.
The proportion of ruptured CCLs that contained
cathepsin K+ cells was significantly increased, compared
with all other groups. Numbers of TRAP+ and
cathepsin K+ cells were significantly increased in ruptured
CCLs, compared with intact ACLs. The presence
of TRAP+ cells was correlated with inflammatory
changes, which were most prominent in ruptured
Conclusion and Clinical Relevance—Results suggest
that synovial macrophage-like cells that produce
TRAP are an important feature of the inflammation
associated with CCL rupture in dogs. Identification of
TRAP and cathepsin K in intact CCLs and ACLs from
aged dogs suggests that these enzymes have a functional
role in cruciate ligament remodeling and repair.
We hypothesize that recruitment and activation of
TRAP+ macrophage-like cells into the stifle joint synovium
and CCL epiligament are critical features of the
inflammatory arthritis that promotes progressive
degradation and eventual rupture of the CCL in dogs.
(Am J Vet Res 2005;66:2073–2080)
Objective—To determine localization of tartrate-resistant
acid phosphatase (TRAP) and cathepsin K in ruptured
and healthy cranial cruciate ligaments (CCL) in
Animals—30 dogs with ruptured CCL, 8 aged dogs
without ruptured CCL, and 9 young dogs without ruptured
Procedure—The CCL was examined histologically
and cells containing TRAP and cathepsin K were identified
histochemically and immunohistochemically,
Results—Cathepsin K and TRAP were detected within
the same cells, principally within the epiligamentous
region and to a lesser extent in the core region
of ruptured CCL. Numbers of cells containing TRAP
and cathepsin K were significantly greater in ruptured
CCL, compared with CCL from young or aged dogs,
and numbers of such cells were greater in CCL from
aged dogs, compared with those of young dogs. In
aged dogs, small numbers of cells containing TRAP
and cathepsin K were seen in intact CCL associated
with ligament fascicles in which there was chondroid
transformation of ligament fibroblasts and disruption
of the extracellular matrix.
Conclusion and Clinical Relevance—Ruptured
CCL contain greater numbers of cells with the proteinases
TRAP and cathepsin K than CCL from
healthy, young, or aged dogs. Results suggest that
cell-signaling pathways that regulate expression of
these proteinases may form part of the mechanism
that leads to upregulation of collagenolytic ligament
remodeling and progressive structural failure of the
CCL over time. (Am J Vet Res 2002;63:1279–1284).