Experimental and Basic Research Studies
Expression of inﬂammatory and structural matrix genes in synovial
ﬂuid following intra-articular administration of isoﬂupredone
acetate to exercised horses
H. K. KNYCH
* , L. HARRISON
, N. CHOUICHA
and P. H. KASS
K.L. Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, California, USA
Department of Veterinary Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA
Willow Oak Equine, Woodland, California, USA
Department of Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA.
*Correspondence email: firstname.lastname@example.org; Received: 19.02.17; Accepted: 10.10.17
Background: Intra-articular use of corticosteroids is commonplace in performance horses. Isoﬂupredone acetate (IPA) is one of four Food and Drug
Administration approved corticosteroids for intra-articular use in horses. The lack of published reports describing the efﬁcacy and duration of effects of
this drug warrant further study.
Objectives: To assess the effects of intra-articular administration of IPA on the expression of selected anti- and pro-inﬂammatory and structural matrix
genes following intra-articular administration to exercised Thoroughbred horses and to correlate these effects with drug concentrations.
Study design: Block design in vivo experiment.
Methods: Twelve exercised horses received either a single intra-articular administration of 8 mg of IPA or 0.9% saline solution. Synovial ﬂuid samples
were collected prior to and up to 42 days post drug administration from the treated joints. Microarray and qRT-PCR analysis were used to assess
changes in expression levels of various inﬂammatory and structural genes post drug administration.
Results: On microarray analysis, 855, 23,358 and 26,411 genes had a measurable fold change (increase or decrease in expression levels) when
comparing baseline samples to 24 h, baseline samples to day 7 and 24 h samples to day 7, respectively. Of the genes selected for further study by qRT-
PCR analysis, expression of ANXA-1 (lipocortin) was signiﬁcantly increased and IL23A and MMP1 and MMP9 signiﬁcantly decreased following IPA
administration. Expression levels of collagen genes were not signiﬁcantly different from baseline.
Main limitations: Limitations include the use of a noninﬂammatory model as results may differ in the presence of an acute inﬂammatory insult and the
inability to measure protein concentrations of inﬂammatory mediators due to limited synovial ﬂuid sample volume.
Conclusions: Expression relative to baseline, for both inﬂammatory and matrix genes for up to 42 days post IPA administration, suggests a prolonged
effect relative to detection time in both plasma and synovial ﬂuid.
Keywords: horse; corticosteroid; isoﬂupredone; joint; synovial ﬂuid; gene expression; inﬂammatory
Corticosteroids are potent anti-inﬂammatory agents that increase
expression levels of genes encoding anti-inﬂammatory proteins and
decrease expression of genes encoding pro-inﬂammatory mediators and
degradative enzymes . By virtue of their anti-inﬂammatory effects, the
use of corticosteroids is widespread in equine medicine, especially in
performance horses. Isoﬂupredone acetate (IPA) is one of four
corticosteroids approved by the Food and Drug Administration for intra-
articular administration in horses.
The pharmacokinetics of IPA in horses has been reported previously
[2,3]. However, while there are numerous published reports describing the
effects of intra-articular administration of the corticosteroids
methylprednisolone acetate [4–9] and triamcinolone acetonide (TCA) in
horses [10–14], to the authors’ knowledge no such studies exist for IPA.
By virtue of their ability to affect gene expression, one can speculate
that corticosteroids likely have a prolonged effect at the molecular level,
relative to the time of drug detection. Our laboratory has previously
described a method by which to assess the effectiveness and duration of
effect of intra-articular TCA in horses by measuring expression levels of
genes involved in the inﬂammatory process in synovial ﬂuid, both prior to
and for several weeks post drug administration . This approach
demonstrated that TCA had a long duration of action, which was
prolonged relative to the detection time of drug in blood and synovial ﬂuid.
In the current study, we use this same gene expression approach to assess
the effects and duration of action of IPA on the expression levels of genes
encoding pro- and anti-inﬂammatory genes and structural proteins in an
exercised horse model.
Materials and methods
Twelve university-owned exercised adult Thoroughbred horses including
six geldings and six mares (age: 4–8 years; weight: 492–600 kg) were
studied. Prior to and throughout the course of the study, horses were
exercised 5 days a week using a protocol that was meant to simulate the
strenuous exercise of race training. The exercise regimen for these horses
consists of 2 days per week on a high-speed treadmill (Mustang 2200)
(Day 1: 5 min @1.6 m/s; 5 min @ 4m/s;5min@ 7m/s;5min@ 1.6 m/s
all at 6% incline. Day 2: 3 min @ 1.6 m/s; 4 min @ 4.0 m/s; 2 min @ 7.0 m/
s; 2 min @ 11.0 m/s and 5 min @1.6 m/s all at 3% incline) and 3 days per
week on an Equineciser
(5 min at walk; 30 min trot; 5 min walk). All
horses were subject to regular ﬁtness testing, including weekly heart rate
measurements and calculation of V
(running velocity that elicits a heart
rate 200 bpm) and monthly measurements of end run plasma lactate
concentrations, as a means by which to ensure that the ﬁtness levels of the
horses used in this study were comparable to the average racehorse.
Horses were not exercised on the day of or the day after synovial ﬂuid
Equine Veterinary Journal 50 (2018) 504–512 © 2017 EVJ Ltd
Equine Veterinary Journal ISSN 0425-1644