Objective—To create high-quality sequence data for
the generation of an equine gene expression microarray
and evaluate array performance by use of
lipopolysaccharide (LPS) exposure of synoviocytes.
Sample Population—Public nucleotide sequence
database from Equus caballus and synoviocytes from
clinically normal adult horses.
Procedure—Computer procurement of equine gene
sequences, probe design, and manufacture of an
oligomicroarray were performed. Array performance
was evaluated by use of patterns for equine synoviocytes
in response to LPS.
Results—Starting with 18,924 equine gene
sequences, 3,098 equine 3' sequences were annotated
and met the inclusion criteria for an expression
microarray. An equine oligonucleotide expression
microarray was created by use of 68,266 of the 25-oligomer probes to uniquely identify each gene. Most
genes in the array (68%) were expressed in equine
synoviocytes. Repeatability of the array was high (r,
> 0.99), and LPS upregulated (> 5-fold change) 84
genes, many of which were inflammatory mediators,
and downregulated (> 5-fold change) 14 genes. An initial
pattern of gene expression for effects of LPS on
synoviocytes consisted of 102 genes.
Conclusions and Clinical Relevance—Use of a computer
algorithm to curate an equine sequence database
generated high-quality annotated species-specific
gene sequences and probe sets for a gene
expression oligomicroarray, which was used to document
changes in gene expression associated with
LPS exposure of equine synoviocytes. The equine
public database was expanded from 290 annotated
genes to > 3,000 provisionally annotated genes.
Similar curation and annotation of public databases
could be used to create other species-specific
microarrays. (Am J Vet Res 2004;65:1664–1673)
Objective—To evaluate the association between subjective
lameness grades and kinetic gait parameters
and assess the variability in kinetic parameters in
horses with experimentally induced forelimb lameness.
Procedures—Forelimb lameness was induced in
each horse via injection of lipopolysaccharide into 1
metacarpophalangeal joint (40 experimental trials).
Subjective lameness grading and 13 kinetic gait parameters
(force plate analysis) were assessed before
(baseline) and at 12, 18, and 24 hours after
lipopolysaccharide injection. While horses were trotting,
kinetic gait analysis was performed for 8 valid
repetitions at each time point. Repeated-measures
analyses were performed with 8 repetitions for each
kinetic parameter as the outcome, and lameness
grades, time points after lipopolysaccharide injection,
and repetition order as explanatory variables.
Sensitivity and specificity of kinetic parameters for
classification of horses as sound or lame (in relation
to subjective lameness scores) were calculated.
Between- and within-horse variabilities of the 13
kinetic parameters were assessed by calculation of
coefficients of variation.
Results—Subjective lameness grades were significantly
associated with most of the kinetic parameters.
Vertical force peak and impulse had the lowest
between- and within-horse coefficients of variation
and the highest correlations with subjective lameness
grade. Vertical force peak had the highest sensitivity
and specificity for lameness classification. Vertical
force peak and impulse were significantly decreased
even in horses with mild or unobservable lameness.
Conclusions and Clinical Relevance—Among the
kinetic gait parameters, vertical force peak and
impulse had the best potential to reflect lameness
severity and identify subclinical forelimb gait abnormalities.
(Am J Vet Res 2005;66:1805–1815)
OBJECTIVE To investigate effects of hyaluronic acid (HA) or HA combined with chondroitin sulfate (CS) and N-acetyl-d-glucosamine (NAG) by use of a lipopolysaccharide (LPS) in vitro method.
SAMPLE Monolayer cultures of synovial cells from 4 adult horses.
PROCEDURES Synovial cell cultures were untreated or treated with HA alone or HA-CS-NAG for 24 hours, subsequently unchallenged or challenge-exposed with 2 LPS concentrations (20 and 50 ng/mL) for 2 hours, and retreated with HA or HA-CS-NAG for another 24 hours. Cellular morphology of cultures was evaluated at 0, 24 (before LPS), 26 (after LPS), and 50 (24 hours after end of LPS) hours. At 50 hours, cell number and viability and prostaglandin (PG) E2, interleukin (IL)-6, matrix metalloproteinase (MMP)-3, and cyclooxygenase (COX)-2 production were measured.
RESULTS LPS challenge exposure induced a significant loss of characteristic synovial cell morphology, decrease in cell viability, and increases in concentrations of PGE2, IL-6, MMP-3, and COX-2. Cells treated with HA or HA-CS-NAG had significantly better viability and morphology scores and lower concentrations of PGE2, MMP-3, IL-6, and COX-2 than untreated LPS challenge-exposed cells. Cells treated with HA had significantly better morphology scores at 50 hours than cells treated with HA-CS-NAG. Cells treated with HA-CS-NAG had significantly superior suppression of LPS-induced production of PGE2, IL-6, and MMP-3 than cells treated with HA alone.
CONCLUSIONS AND CLINICAL RELEVANCE HA and HA-CS-NAG protected synovial cells from the effects of LPS. Treatment with HA-CS-NAG had the greatest anti-inflammatory effect. These results supported the protective potential of HA and HA-CS-NAG treatments.
Objective—To compare articular cartilage from horses
with naturally developing osteochondrosis (OC)
with normal articular cartilage and healing cartilage
obtained from horses with experimentally induced
Sample Population—109 specimens of articular cartilage
from 78 horses.
Procedure—Morphologic characteristics, proteoglycan
(PG), and type II collagen were analyzed in articular cartilage
of OC specimens (group 1), matched healing cartilage
obtained 40 days after experimentally induced
osteochondral fractures (group 2), and matched normal
cartilage from the same sites (group 3).
Results—79 specimens of OC cartilage were
obtained from horses. Ex vivo PG synthesis was significantly
greater in the femoral cartilage, compared
with synthesis in the tibial cartilage, and significantly
greater for groups 1 and 2, compared with group 3.
For groups 1 and 2, femoral fragments had significantly
greater PG content, compared with PG content
in tibial fragments. Keratan sulfate content was significantly
less in group 3, compared with groups 1 and
2. Cartilage from the OC specimens had loss of structural
architecture. The OC tissue bed stained positive
for chondroitin sulfate and type II collagen, but the
fracture bed did not.
Conclusions and Clinical Relevance—Our analyses
could not distinguish articular cartilage from horses
with OC and a healing fracture. Both resembled an
anabolic, reparative process. Immunohistochemical
analysis suggested a chondromyxoid tissue in the OC
bed that was morphologically similar to fibrous tissue
but phenotypically resembled hyaline cartilage. Thus,
tissue in the OC bed may be degenerative cartilage,
whereas tissue in the fracture bed may be reparative
fibrous callus. (Am J Vet Res 2005;66:1881–1890)
OBJECTIVE To assess efficiency of gravity filtration to enhance recovery of equine bone marrow elements including stem and progenitor cells.
ANIMALS 12 healthy adult horses.
PROCEDURES Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to obtain bone marrow elements. Raw and harvested bone marrow and marrow effluent were evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, mesenchymal stem cell CFUs, cell viability, and differentiation capacity. Isolated cells were analyzed for CD90 and major histocompatibility complex (MHC) class I and II antigens.
RESULTS Mean cell viability of harvested bone marrow was 95.9%. Total WBCs and platelets were efficiently captured on the filter (> 95%), and mean recovery in harvested bone marrow was 30%. Cytologic cell differential counts indicated that the percentage of neutrophils was significantly less and the progenitor cell population was significantly higher and concentrated 1.56-fold in harvested bone marrow, compared with results for raw bone marrow. Flow cytometry and cell culture were used to characterize harvested bone marrow cells as positive for expression of CD90 and negative for MHCI and MHCII, which indicated stem cells with a multipotent phenotype that differentiated into chondrocytes, osteocytes, adipocytes, and tenocytes.
CONCLUSIONS AND CLINICAL RELEVANCE Gravitational filtration of bone marrow efficiently yielded platelets and cells and produced a progenitor-enriched, leukocyte-reduced product, compared with raw bone marrow.
Objective—To determine outcome of Standardbred racehorses with moderate to severe midbody suspensory ligament desmitis (MSD) treated by means of ultrasound-guided intralesional injection of a single dose of platelet-rich plasma (PRP) followed by a program of gradually increased exercise.
Design—Nonrandomized clinical trial.
Animals—9 Standardbred racehorses.
Procedures—Following injection of PRP, horses were allowed a controlled, gradual return to exercise. Race records for the year prior to injury and for 3 consecutive years after horses returned to racing were reviewed. For comparison purposes, race records of 9 Standardbred racehorses with no history of MSD racing at the same time were also reviewed.
Results—All 9 horses with MSD returned to racing after treatment; median time to return to racing was 32 weeks. All 9 horses raced at least once during the first and second years after returning to racing, but only 5 raced during the third year. When number of starts, total earnings, and earnings per start were compared between case and comparison horses, the only significant differences were number of starts during the third year after case horses returned to racing and earnings per start during the first year after case horses returned to racing, with values being significantly lower for case horses than for comparison horses.
Conclusions and Clinical Relevance—Results suggested that horses with moderate to severe MSD treated by means of intralesional injection of a single dose of PRP followed by a program of gradually increased exercise had an excellent prognosis for returning to racing.
Objective—To evaluate the effects of anti-inflammatory
drugs on lipopolysaccharide (LPS)-challenged and
-unchallenged equine synovial membrane in terms of
production of prostaglandin E2 (PGE2) and hyaluronan,
viability, and histomorphologic characteristics.
Sample Population—Synovial membranes were collected
from the carpal, tarsocrural, and femoropatellar
joints of 6 adult horses.
Procedure—Synovial membranes from each horse
were minced and pooled and explants were treated
with one of the following: no drug (control), drug, LPS
alone, or LPS and drug. Treatment drugs were
phenylbutazone (PBZ), flunixin meglumine (FNX),
ketoprofen (KET), carprofen (CRP), meloxicam (MEL),
low-concentration methylprednisolone (METH), highconcentration
METH, dimethyl sulfoxide (DMSO), or
an experimental COX-2 inhibitor (dissolved in DMSO).
Following 48 hours of culture, medium was assayed
for PGE2 and hyaluronan concentration. Synovial
explants were assessed for viability and histomorphologic
Results—For the LPS-challenged explants, PBZ, FNX,
KTP, CRP, MEL, and low-concentration METH suppressed
PGE2 production, compared with LPS challenge
alone. Only MEL suppressed PGE2 production
from LPS-challenged explants, compared with unchallenged
explants. Synovial explants maintained > 90%
viability and there was no significant difference in viability
or hyaluronan production among explants.
Histomorphologic scores were significantly
decreased for explants treated with low-concentration
METH or DMSO.
Conclusions and Clinical Relevance—PBZ, FNX,
KTP, CRP, MEL, and low-concentration METH suppressed
PGE2 production in LPS-challenged explants.
Meloxicam appeared to have more selective suppression
of COX-2 activity. Histomorphologic scores suggest
detrimental effects of METH, DMSO, and the
experimental COX-2 inhibitor. Commonly used nonsteroidal
anti-inflammatory drugs suppress induced
synovial membrane PGE2 production without detrimental
effects on synovial membrane viability and
function. ( Am J Vet Res 2001;62:54–60)
Objective—To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow–derived mesenchymal stem cells (BMD-MSCs).
Sample Population—Articular cartilage, synovium, and bone marrow from 15 adult horses.
Procedures—Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1–deficient adenovirus vector encoding the β-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1–deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection.
Results—Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors.
Conclusion and Clinical Relevance—Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.
Objective—To evaluate the effects of triamcinolone acetonide (TA), sodium hyaluronate (HA), amikacin sulfate (AS), and mepivacaine hydrochloride (MC) on articular cartilage morphology and matrix composition in lipopolysaccharide (LPS)-challenged and unchallenged equine articular cartilage explants.
Sample Population—96 articular cartilage explants from 4 femoropatellar joints of 2 adult horses.
Procedures—Articular cartilage explants were challenged with LPS (100 ng/mL) or unchallenged for 48 hours, then treated with TA, HA, AS, and MC alone or in combination for 96 hours or left untreated. Cartilage extracts were analyzed for glycosaminoglycan (GAG) content by dimethyl-methylene blue assay (ng/mg of dry wt). Histomorphometric quantification of total lacunae, empty lacunae, and lacunae with pyknotic nuclei was recorded for superficial, middle, and deep cartilage zones.
Results—LPS induced a significant increase in pyknotic nuclei and empty lacunae. Treatment with TA or HA significantly decreased empty lacunae (TA and HA), compared with groups without TA or HA, and significantly decreased empty lacunae of LPS-challenged explants, compared with untreated explants. Treatment with AS or MC significantly increased empty lacunae in unchallenged explants, and these effects were attenuated by TA. Treatment with MC significantly increased empty lacunae and pyknotic nuclei and, in combination with LPS, could not be attenuated by TA. Content of GAG did not differ between unchallenged and LPS-challenged explants or among treatments.
Conclusions and Clinical Relevance—Treatment with TA or HA supported chondrocyte morphology in culture and protected chondrocytes from toxic effects exerted by LPS, AS, and MC.