Traumatic arthritis and osteoarthritis may be the most common cause of lameness in equine athletes of all types.1 In the Thoroughbred racehorse industry worldwide, musculoskeletal injury accounts for the highest proportion of days lost from training2–5 and is the most common cause of athletic morbidity.3,4,6–8
Synovitis has been implicated in the initiation of osteoarthritis in racehorses.9–13 Intra-articular analgesics (mepivacaine, lidocaine, and bupivacaine14) and intra-articular therapeutic agents (corticosteroids, polysulfated glycosaminoglycans, and hyaluronic acid derivatives) remain important aids in the diagnosis of acute synovitis and treatment of affected horses and help in the prevention of additional degenerative articular changes.13,15
Current practice is to inject a local anesthetic into an affected joint and follow up with intra-articular administration of a corticosteroid between 1 and 7 days later because reactive synovitis (synovial flare) can be incited after injection of the local anesthetic,16 thus interfering with the absorption and efficacy of the corticosteroid. The prevalence of synovial flare responses in humans treated for arthritis is reportedly 2%.17 In clinical practice, the ability to administer 1 intra-articular injection of a combination of TAa and mepivacaineb would be useful for confirming the site of lameness and treating any synovitis or arthritis that existed there. This single-injection technique would eliminate the need for additional synovial injections, thus reducing potential injury to veterinarians and the risk of synovial sepsis for horses.18,19
Triamcinolone acetonide has recently become popular as an intra-articular medication for high-motion joints because of its purported chondroprotective and anti-inflammatory properties.20–22 In studies of the effects of TA in horses, the analgesic has been injected into the same joint multiple times23 or into multiple joints at 1 time point.24 To our knowledge, the effects of 1 TA injection or TA combined simultaneously with other medications injected into 1 joint of a horse have not been evaluated. Mepivacaine hydrochloride is the local anesthetic of choice for diagnostic intra-articular analgesia because it causes less irritation of the articular environment than lidocaine.25
The purpose of the study reported here was to determine the efficacy, safety, and duration of action of 1 injection of TA or mepivacaine alone, or TA and mepivacaine in combination, on the degree of lameness, joint kinetics, and indicators of synovial inflammation by use of an established protocol for induction of lameness and synovitis in equine MCP joints with Escherichia coli LPS.26,27 In addition, we sought to determine whether local and systemic drug concentrations would be correlated with clinical efficacy scores.
Materials and Methods
Animals—Eighteen skeletally mature, healthy teaching horses were used in the study. Horses were included when they had unremarkable results of clinical examination and routine hematologic and serum biochemical analyses, were not clinically lame, and had mean bilateral forelimb VFP (as a percentage of body weight) values consistent with soundness.26,28–33 All had pain-free range of motion of the MCP joint, and there was no clinical or radiographic (routine lateromedial and dorsoproximal-palmarodistal oblique [15°] views) evidence of pathologic conditions of the articular or periarticular MCP joint. None of the horses had a history of lameness in the previous 12 months, and none had been or were being treated with anti-inflammatory medications. The hooves of all horses were trimmed flat, and hair on the MCP joints was clipped. Horses were acclimated to 4 × 4-m box stalls 1 week prior to the study. Each horse was physically examined to determine rectal temperature, heart rate, and respiratory rate twice daily and was maintained in the box stall for the duration of the study. All experimental procedures were approved by an institutional laboratory animal care and use committee.
Experimental induction of lameness and treatment—The experiment took place in 2 phases, with the control phase preceding the treatment phase. Identification of the MCP joint that would receive 1 of 3 treatments (mepivacaine, TA, or both; designated the treatment joint) was made randomly (blind selection of right or left marker) for each horse; the MCP joint in the contralateral limb was designated the control joint. Lipopolysaccharide (E coli O55:B5c) was thawed from stock, diluted within 30 minutes before injection, and stored in glass vials on ice until injection to minimize the adherence of LPS to the vial and the degradation of LPS. Synovial inflammation and forelimb lameness were induced in the MCP joints of all horses via aseptic intra-articular injection34 (20-gauge needle) of LPS (100 ng/mL in Dulbecco PBS solutiond; 0.0002 μg/kg; range, 0.9 to 1.2 mL/joint). In the control phase of the experiment, lameness was induced in the control joint of each horse on day 0 (LPS1), day 5 (LPS2), and day 10 (LPS3). A 7-day washout period was allowed. Then, in the treatment phase of the study, the treatment joint was injected with LPS on day 18 (LPS1), day 23 (LPS2), and day 28 (LPS3).
In the control phase, control limbs (n = 18) were injected with 9 mL of PBS solutione 12 hours after the LPS1 injection. For the treatment phase, horses were assigned by shuffled charts to receive an intra-articular injection (9-mL total volume) containing 1 of 3 treatments at 12 hours after the LPS1 injection, such that there were 6 horses in each treatment group. Shuffled charts were assigned in order of the 3 treatments consisting of 7.5 mL (150 mg) of mepivacaineb diluted with PBS solution to a total volume of 9 mL, 1.5 mL (9 mg) of TAa diluted to a total volume of 9 mL with PBS solution, and 7.5 mL (150 mg) of mepivacaine and 1.5 mL (9 mg) of TA.
Data collection—Various indicators of synovial inflammation were measured in both forelimbs 3 days prior to the first injection of LPS (day −3) and on day 18 of the experiment (baseline measurements). The same indicators were measured during the control phase of the experiment at the following times: day 0 (at LPS1 injection), 12 hours after LPS1 was injected, and after the control treatment was administered; days 2 and 3; day 5 (prior to LPS2 injection); 12 hours after LPS2 injection on day 5; days 6, 7, and 8; day 10 (prior to LPS3 injection); 12 hours after LPS3 injection on day 10; and day 11. These indicators were also measured during the treatment phase of the experiment at the following times: day 18 prior to LPS1 injection; 12 hours after LPS1 was injected and after 1 of 3 analgesics was administered on day 18; days 19, 20, and 21; day 23 (prior to LPS2 injection); 12 hours after LPS2 injection on day 23; days 24, 25, and 26; day 28 (prior to LPS3 injection); 12 hours after LPS3 injection on day 28; and day 29.
Indicators measured included lameness score,35 mean VFP, joint edema score, pain-free range of motion of the MCP joint,36 and joint circumference. Other indicators included WBC count and total protein concentration in synovial fluid and concentrations of TA and mepivacaine in synovial fluid and serum.
Lameness evaluation and gait analysis—Physical and clinical lameness evaluations were performed by 2 experienced investigators (DMB and ALB), who were unaware of the treatment that each horse had received. Immediately before force plate analysis, each horse was evaluated on the same firm, level surface when walking in a straight line, walking in circles to the right and left, and trotting in a straight line. Observers independently scored the horses and then conferred to produce a consensus score. A modified lameness grade, in increments of 0.5, was assigned in accordance with a grading system that ranged from 0 to 5.37 Specifically, horses without visible lameness when walking but with lameness when trotting were assigned a grade of 2. Horses without visible lameness when walking straight but with lameness when circling were assigned a grade of 2.5. Horses with visible lameness when walking in a straight line were assigned a grade of 3.
Quantitative gait analysis was performed by means of a 3 × 20-m aisle with an inground stationary force platef and computer analysis system.26 The same experienced handler guided each horse to trot over the force plate at a consistent speed (1.8 to 2.2 m/s), which was confirmed via 2 photoelectric switches that were placed 5 m apart and connected to the computer analysis system. The central force plate and aisle were covered by a mat to prevent horses from slipping or from seeing the plate. Data acquisition rate was set at 500 Hz, and filtering stop band frequency was set at 80 Hz. Five valid repetitions were recorded to ensure accuracy.26 A valid repetition was defined as one in which the hoof fully contacted the plate and landed within a 10-cm square boundary from the edges of the force plate within the speed range. The 5 valid measurements were averaged for each horse per limb per time point and reported as a percentage of body weight.
Evaluation of pain-free range of joint motion—The MCP joint was flexed until each horse raised its head or moved the limb in resistance.35 A handheld goniometerg was positioned so that the center of the rotation was over the center of rotation of the MCP joint. The maximal angle of flexion was obtained for 3 consecutive evaluations and averaged.37
Metacarpophalangeal joint swelling (edema score and joint circumference)—Forelimbs were palpated by 2 investigators (DMB and ALB), who were unaware of the treatment horses had received. Joint swelling was graded by means of a scale ranging from 0 to 4 (0 = no swelling; 1 = minimal swelling localized to the injection site; 2 = mild swelling localized to the MCP joint; 3 = moderate swelling extending proximally toward the carpus; and 4 = marked swelling extending to or above the carpus). Joint circumference (mm) was measured with a tape at the height of the mid aspects of the proximal sesamoid bones, 2 cm proximal to the ergot.38,39
Synovial fluid analysis—Two to 6 mL of synovial fluid was collected from MCP joints into EDTA and heparinized tubes.g Samples from the EDTA tubes were immediately evaluated microscopically for total WBC count (cells/μL) and WBC differential (percentage of polymorphonuclear and mononuclear cells), and total protein concentration was measured with a refractometer.h
Measurement of drug concentrations in synovial fluid and plasma—To determine plasma concentrations of TA and mepivacaine (ng/mL), 10-mL blood and synovial fluid samples were collected into heparinized evacuated tubes,g processed on ice, centrifuged at 1,000 × g for 8 minutes, frozen in low-temperature freezer vials,i and stored at −80°C. Blood was collected from a jugular vein from 2 randomly selected horses from each treatment group immediately before the treatment phase of the experiment, from all horses at 12 hours after analgesic treatment, and from the same 2 randomly selected horses 5.5 days (132 hours) after analgesic treatment. A subset of synovial fluid samples was selected from the MCP joints of all horses immediately before LPS injection (day 0 for control joints and day 18 for treatment joints); before treatment (12 hours after LPS1 injection for either joint); and at 36, 60, 120, 132, and 240 hours after treatment. Samples of plasma and selected synovial fluid samples were thawed and shipped to an external laboratorye for determination of concentrations of TA and mepivacaine via quantitative ELISA.j The ELISA for each drug was calibrated with samples containing predetermined concentrations of TA and mepivacaine. Each test was performed in duplicate.
Statistical analysis—All statistical analyses were performed by use of commercially available statistical software.k Overall effects of the treatments on lameness scores, values from vertical force plate analyses, values from assessments of pain-free range of motion of MCP joints, circumferences of MCP joints, WBC counts, and total protein concentrations were analyzed by use of repeated-measures ANOVA. Explanatory variables included horse, MCP joint (right or left), analgesic (TA, mepivacaine, TA and mepivacaine, or control), and measurement time points. Horse was treated as a random variable, and repeated measures (joint and time points) were considered to be nested within horse. The time points included in the analysis were considered in relation to the first LPS injection and were baseline (0 hours), 12, 12.5, 24, 48, 72, and 120 hours.
For the data concerning concentrations of TA and mepivacaine in plasma or joint fluid, multiple pairwise comparisons were made among treatments (ie, TA or mepivacaine alone vs TA and mepivacaine) at the selected time point (12 hours). A crude Bonferroni correction was made to adjust for multiple comparisons.
Lameness scores, values from vertical force plate analyses, values from assessments of pain-free range of motion of MCP joints, circumferences of MCP joints, WBC counts, and total protein concentrations were compared among the 4 treatment groups within each time point by means of the least square means from the repeated-measures ANOVA model. Multiple comparisons were also made among time points within each treatment. Specifically, to assess the long-term anti-inflammatory effects of the analgesics, comparisons were made within each treatment group between values at 12 hours after LPS1 (synovitis prior to drug injection), 12 hours after LPS2, and 12 hours after LPS3. Similarly, to assess possible carryover effects of LPS injection, comparisons were made within each treatment group among values at baseline (before LPS1) and before LPS2 and LPS3.
Edema scores comprised ordinal data and were analyzed descriptively by plotting the median values of each treatment group at each time point over the study period. A value of P < 0.05 was considered significant for all analyses. Values for VFP, pain-free range of motion, synovial WBC count, synovial total protein concentration, and synovial and plasma analgesic concentrations were expressed as mean ± SEM.
Results
Animals—Eighteen adult horses with a mean age of 9.8 years (range, 2 to 19 years) and a mean weight of 548 kg (range, 432 to 732 kg) were included for a total of 36 MCP joints. There were 8 geldings, 1 stallion, 3 fillies, and 6 mares. Six breeds were represented as follows: 10 Quarter Horses, 3 Thoroughbreds, 2 Standardbreds, 1 Hanovarian, 1 Saddlebred, and 1 Appaloosa. All horses met the inclusion criteria and completed the study.
Lameness evaluation and gait analysis—All horses were sound (lameness score of 0) at the start of the experiment and had mean ± SEM VFP readings of 105.2 ± 3.9% (Figures 1 and 2). At 12 hours after the first LPS injection, peak lameness was evident in all limbs. At that time point, the median lameness score was 3.7 (range, 1 to 5) and mean VFP was 53.1 ± 7.8%, which corresponded to a decrease in mean VFP of 52.0 ± 4.0%, compared with the value before LPS was injected. There was no significant difference between gait measurements (clinical lameness scores and VFP values) at baseline, and values obtained before LPS injections were repeated in treatment or control joints.
Mean ± SEM lameness scores at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution (control limbs; black diamonds with solid line), mepivacaine hydrochloride (white circles with dashed line), TA (white diamonds with dashed line), or mepivacaine andTA (white squares with dashed line). *Within the treatment group, value differs significantly (P < 0.05) from that at 12 hours after LPS1. a,bDifferent letters indicate significant (P < 0.05) differences among treatment groups.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM lameness scores at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution (control limbs; black diamonds with solid line), mepivacaine hydrochloride (white circles with dashed line), TA (white diamonds with dashed line), or mepivacaine andTA (white squares with dashed line). *Within the treatment group, value differs significantly (P < 0.05) from that at 12 hours after LPS1. a,bDifferent letters indicate significant (P < 0.05) differences among treatment groups.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM lameness scores at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution (control limbs; black diamonds with solid line), mepivacaine hydrochloride (white circles with dashed line), TA (white diamonds with dashed line), or mepivacaine andTA (white squares with dashed line). *Within the treatment group, value differs significantly (P < 0.05) from that at 12 hours after LPS1. a,bDifferent letters indicate significant (P < 0.05) differences among treatment groups.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM VFPs at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM VFPs at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM VFPs at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Each initial LPS injection (LPS1) induced lameness that was not significantly different in score between control and treated joints. Treatment with mepivacaine hydrochloride alone or in combination with TA improved lameness scores, with a significant (P = 0.002) decrease in lameness detected within 30 minutes after injection of analgesic (median lameness score, 1.3) and a significant (P = 0.003) increase in VFP detected within 45 minutes after injection (mean VFP, 90.8 ± 7.1%), compared with control values.
Subsequent injections of LPS resulted in less lameness at 12 and 24 hours after LPS2 and 12 hours after LPS3 in the joints treated with TA (median lameness score, 1.5; P < 0.001; mean VFP, 89.3% ± 2.7%), compared with lameness scores and VFPs for control joints and joints treated with mepivacaine only and with lameness scores recorded at 12 hours after LPS1 injection for joints treated with TA. There was no difference between lameness values for joints treated with TA alone or TA and mepivacaine. The reduction in LPS-induced lameness persisted for at least 10 days after injection of TA.
Values for VFP were generally consistent (low SEM) among the 5 repetitions during which mean VFP was determined (Figure 3); however, when effects of treatments were evaluated 30 minutes after administration (12.5 hours after LPS1 injection), mepivacaine alone or in combination with TA resulted in a small but significant (P = 0.006) increase in mean VFP as the number of trotting repetitions increased.
Within-horse variation in mean ± SEM VFP for 5 valid repetitions of VFP assessment during trotting before lameness was experimentally induced via LPS injection (black triangles with solid line), after the first LPS injection (white triangles with dashed line), and after subsequent treatment with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. *First and fifth VFP values differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Within-horse variation in mean ± SEM VFP for 5 valid repetitions of VFP assessment during trotting before lameness was experimentally induced via LPS injection (black triangles with solid line), after the first LPS injection (white triangles with dashed line), and after subsequent treatment with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. *First and fifth VFP values differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Within-horse variation in mean ± SEM VFP for 5 valid repetitions of VFP assessment during trotting before lameness was experimentally induced via LPS injection (black triangles with solid line), after the first LPS injection (white triangles with dashed line), and after subsequent treatment with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. *First and fifth VFP values differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Pain-free range of joint motion—Before LPS was injected into MCP joints, mean ± SEM maximal pain-free range of flexion was 130 ± 2° (Figure 4). Peak reduction in MCP flexion was evident at 12 hours after LPS1 injection (mean pain-free range of flexion, 144 ± 2°). Triamcinolone acetonide administered alone (mean, 133 ± 3°) or in combination with mepivacaine (mean, 134 ± 3°) resulted in a significant (P < 0.001) increase in the range of flexion of MCP joints at 12 hours after LPS2 injection, compared with values for control limbs or limbs treated with mepivacaine only.
Mean ± SEM maximal degrees of pain-free MCP joint flexion at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM maximal degrees of pain-free MCP joint flexion at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM maximal degrees of pain-free MCP joint flexion at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Metacarpophalangeal joint swelling (edema score and joint circumference)—At commencement of the experiment and before each replicate LPS injection, horses had no evidence of edema in the region of the MCP joint. A median peak edema score of 3 (range, 2 to 3) was attained at 12 hours after LPS1 injection in all joints before analgesic treatment was administered. Treatment with TA combined with mepivacaine reduced edema scores at 36 hours after drug injection (median score, 0; range, 0 to 3), compared with scores for mepivacaine alone (median score, 3; range, 2 to 3). Treatment with TA, with or without mepivacaine, also reduced peak limb edema scores relative to scores for control joints and joints treated with mepivacaine only and restored scores to baseline values after LPS2 and LPS3 injections (Figure 5). Edema scores for control joints and joints treated with mepivacaine only (median score, 2; range, 0 to 3) did not return to baseline values after LPS2 and LPS3 injections. Circumference of MCP joints did not significantly decrease relative to control values following intra-articular administration of any analgesic.
Median limb edema scores at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Median limb edema scores at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Median limb edema scores at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Synovial fluid measurements—Baseline synovial WBC count was within the reference range (< 1,000 cells/μL) in all MCP joints (mean WBC count, 264 ± 111 cells/μL; Figure 6). In all treatment joints, synovial WBC count peaked at 12 hours after LPS1 injection (mean, 96,759 ± 15,054 cells/μL) and the predominant cell type was nondegenerate neutrophils, which constituted > 91% of WBCs. There was no significant difference in mean neutrophil counts among treatments at 12 hours after LPS1 or LPS3. Triamcinolone acetonide alone significantly (P < 0.001) increased the mean WBC count in synovial fluid, compared with the effects of control or mepivacaine, to a mean peak of 170,717 ± 15,616 cells/μL at 12 hours after LPS2 injection. This increase was not evident after LPS3 injection.
Mean ± SEM WBC counts in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM WBC counts in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM WBC counts in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Baseline synovial fluid concentration of total protein was within the reference range (< 2.5 g/dL) in all limbs at 2.0 g/dL (Figure 7). In all treatment joints, total protein concentration peaked at 12 hours after LPS1 injection (mean concentration, 5.6 ± 0.2 g/dL). Triamcinolone acetonide, with or without mepivacaine, significantly (P < 0.001) reduced the mean total protein concentration (compared with control or mepivacaine values) to 4.3 ± 0.4 g/dL at 12 hours after LPS2 injection, and that reduced value persisted for up to 36 hours, accelerating return to baseline values.
Mean ± SEM concentrations of total protein in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM concentrations of total protein in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM concentrations of total protein in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced via 3 consecutive LPS injections in MCP joints and that were also treated with an intra-articular injection of PBS solution, mepivacaine hydrochloride, TA, or mepivacaine and TA. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Drug concentrations in synovial fluid and plasma—Selected samples of synovial fluid and plasma from horses at baseline contained no TA or mepivacaine prior to administration of the analgesics (Table 1). At 12 hours after drug administration, the concentration of mepivacaine detected in synovial fluid when administered with TA (845.83 ± 202.12 ng/mL) was significantly (P = 0.004) greater than the concentration when mepivacaine was administered alone (209.17 ± 178.34 ng/mL; Figure 8). Mepivacaine was also detected at a significantly (P = 0.004) greater mean concentration in plasma at 12 hours after administration when combined with TA (11.67 ± 4.59 ng/mL), compared with the concentration when mepivacaine was administered alone (0.83 ± 0.8 ng/mL). Mepivacaine was no longer detectable in serum 5.5 days after administration.
Mean ± SEM concentrations (ng/mL) of mepivacaine hydrochloride (MH) and TA at various time points in synovial fluid and plasma obtained from horses in which synovitis was experimentally induced in MCP joints via injection of LPS after joints were injected once with MH, TA, or both (MH+TA).
| Sample | Drug injected | Drug analyzed | Time after drug administration (min) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| 0 | 12 | 36 | 60 | 120 | 132 | 240 | |||
| Synovial fluid | MH | MH | ND | 209.2 ± 178.3 | 0.8 ± 0.8 | — | — | — | — |
| MH+TA | MH | ND | 845.8 ± 202.1 | 75.8 ± 60.3 | — | — | — | — | |
| Plasma | MH | MH | ND | 0.8 ± 0.8 | — | — | — | ND | — |
| MH+TA | MH | ND | 11.7 ± 4.6 | — | — | — | ND | — | |
| Synovial fluid | TA | TA | ND | 10,466.7 ± 1,074.1 | 2,175.0 ± 721.5 | 340.0 ± 105.8 | 109.2 ± 79.3 | — | 0.7 ± 0.5 |
| MH+TA | TA | ND | 10,600.0 ± 951.8 | 2,866.7 ± 834.4 | 466.7 ± 98.9 | 151.7 ± 78.7 | — | 1.8 ± 1.2 | |
| Plasma | TA | TA | ND | 0.6 ± 0.1 | — | — | — | ND | — |
| MH+TA | TA | ND | 0.8 ± 0.1 | — | — | — | ND | — | |
ND = Not detected. — = Sample collected but not analyzed.
Mean ± SEM concentrations of TA and mepivacaine hydrochloride in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced in MCP joints and that were also treated with an intra-articular injection of mepivacaine hydrochloride (MH), TA, or mepivacaine and TA (MH+TA).
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM concentrations of TA and mepivacaine hydrochloride in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced in MCP joints and that were also treated with an intra-articular injection of mepivacaine hydrochloride (MH), TA, or mepivacaine and TA (MH+TA).
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean ± SEM concentrations of TA and mepivacaine hydrochloride in synovial fluid obtained from MCP joints at various time points in horses in which synovitis was experimentally induced in MCP joints and that were also treated with an intra-articular injection of mepivacaine hydrochloride (MH), TA, or mepivacaine and TA (MH+TA).
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1646
Mean concentration of TA in synovial fluid at 12 hours after injection was not significantly different whether mepivacaine was injected simultaneously (10,600.0 ± 951.8 ng/mL) or not (10,466.7 ± 1,074 ng/mL). Triamcinolone acetonide was still detected at 10 days whether TA was administered alone (mean concentration, 1.75 ± 1.2 ng/mL) or simultaneously with mepivacaine (mean concentration, 0.67 ± 0.49 ng/mL). Triamcinolone acetonide was detected at low concentrations in serum at 12 hours after administration with mepivacaine (mean concentration, 0.8 ± 0.1 ng/mL) or without mepivacaine (mean concentration, 0.6 ± 0.1 ng/mL) and was not detected at 5.5 days.
Discussion
The results of the study reported here supported our hypothesis that TA and mepivacaine hydrochloride can be administered together intra-articularly to diagnose acute MCP joint synovitis and treat affected horses without compromising anti-inflammatory or analgesic efficacy or duration of action of either drug. Almost complete joint analgesia was obtained with mepivacaine, and the coadministration of TA did not alter this effect. Triamcinolone acetonide significantly suppressed detectable joint pain and inflammation on the basis of results of evaluations for lameness, pain on joint flexion, and edema formation, and this effect was not influenced by coadministration of mepivacaine. Triamcinolone acetonide alone significantly increased the WBC count in synovial fluid at 12 hours after the second injection of LPS and reduced the synovial fluid concentration of total protein for up to 36 hours after TA was administered, regardless of whether mepivacaine was also injected. These findings may suggest that TA exerts its effects by reducing joint permeability and fluid flux across the synovium rather than by influencing cellular influx.40,41 A similar effect of TA on total protein concentration and reduction in synovial fluid volume in equine middle carpal joints is reported elsewhere.20 Reduced permeability may also explain the high concentrations of mepivacaine in serum and synovial fluid after TA was coadministered.
The protocol for multiple intra-articular injections of MCP joints with LPS used in the study reported here served to effectively evaluate effects on pain and lameness and anti-inflammatory efficacy associated with administration of a local anesthetic (mepivacaine) and a corticosteroid (TA). The 100-μg dose of LPS resulted in a consistent and repeatable lameness without the development of systemic clinical signs of endotoxaemia10,26,27 in horses. As reported elsewhere,26,27,42–45 peak lameness following intra-articular injection of LPS occurs at approximately 12 hours after injection. The timing of induction of peak lameness was useful for critical assessment of the effect of different drug combinations.
Evaluation of control limbs before treated limbs removed the possibility of any potential interference from the influence of a corticosteroid on the development of MCP joint inflammation24 after repeated LPS injections. Such an influence reportedly affects both the ipsilateral (treated) and contralateral (nontreated) MCP joint.23 Similarly, ongoing anti-inflammatory or analgesic effects may not be totally dependent on the detectable existence of analgesic (TA) in synovial fluid or plasma20,24 but rather on the effect created at the cellular level46 and the continued occupation of cell receptors.47,48
To minimize the small but appreciable iatrogenic effects on synovial inflammation, each horse acted as its own control animal and mepivacaine was selected as the analgesic of choice.25,49,50 Baseline values for all variables were obtained immediately prior to commencement of experimental procedures on the contralateral MCP joint to ensure residual lameness would not confound subsequent measurements.
Our study was able to detect TA in synovial fluid samples for at least 10 days following drug administration. Triamcinolone acetonide was detected in serum at low concentrations 12 hours after administration, regardless of whether mepivacaine was coadministered, but TA was not detected in serum 5.5 days after administration. These findings are in accordance with those of another study24 in which TA (6 mg) was injected into each of 3 separate equine joints and detected at low concentrations in those joints for up to 14 days and in plasma for at least 48 hours.
When mepivacaine was administered with TA, significantly greater concentrations of mepivacaine were detected in synovial fluid and serum 12 hours after administration. Low concentrations could still be detected in the MCP joint in TA groups 36 hours after administration of analgesics. These results suggested a potential synergistic effect between mepivacaine and TA, which may alter the rate of diffusion or metabolism of either drug. Values recorded for limbs treated with mepivacaine only correspond to findings reported by other researchers,51 who detected a peak plasma concentration of mepivacaine 4 hours after SC injection (162 mg) and plasma concentrations < 4 ng/mL after 24 hours. Other reports indicate 24- to 48-hour drug clearance times after administration of 200 mg (SC),52 300 mg (intra-articular),53 and 400 mg (SC and intra-muscular)54 of mepivacaine, which supports our inability to detect mepivacaine in plasma 5.5 days after intra-articular injection.
In agreement with results of other studies in apparently clinically sound28–33 and lame horses,29,55–57 all horses in our study had a consistent and repeatable peak lameness at 12 hours after LPS injection, which was restored to baseline values within 2 to 5 days. Mepivacaine at an intra-articular dose of 150 mg with or without TA was effective at reducing the degree of LPS-induced lameness within 30 minutes after administration, which is a protocol commonly used in clinical practice to correspond to maximal analgesic effect.14,58 In our study, as the number of trotting exercise repetitions increased, the horses treated with mepivacaine appeared to become less lame, likely because of the ongoing short-term articular and periarticular effects of local anesthetic diffusion14,58,59 and adaptation of horses to a reduced degree of pain. The severity of lameness recorded was significantly reduced for at least 10 days following intra-articular administration of TA with or without mepivacaine.
Mean range of motion of MCP joints in all horses before LPS was injected is comparable to the reported MCP joint angulation in athletic horses.38 Peak reduction in range of motion, corresponding to increased joint pain, was evident 12 hours after the first LPS injection. This finding agrees with that for Thoroughbred racehorses with moderate MCP effusion and clinical idiopathic synovitis.38 In that study, TA administered alone or in combination with mepivacaine resulted in a significant increase in the range of motion of the MCP joint (ie, less joint pain) 12 hours after the second LPS challenge.
Maximal periarticular edema score was detected 12 to 24 hours after initial LPS injections in all horses, reflecting the commonly reported delay in tissue fluid extravasation and resorption following an inflammatory insult.26,27,42–44 Triamcinolone acetonide in combination with mepivacaine appeared to reduce edema scores the most rapidly (by 36 hours after administration), compared with effects of PBS solution or TA or mepivacaine alone. This finding may indicate the immediate analgesic effect of mepivacaine (still detected at 24 hours after administration) and the medium-term, overlapping analgesic and anti-inflammatory effects of TA. The combination of mepivacaine and TA may promote more rapid weight bearing and use of limbs, despite clinical synovial effusion and increased intra-articular pressure,40,60 thus promoting mechanical removal of tissue fluid via the lymphatic drainage system.
Joint motion and increased synovial microvascular pressures (> 7.3 mm Hg61,62 or > 11 mm Hg40) are required to activate lymphatic drainage and overcome the tissue fluid pressure that results in tissue fluid diffusing toward the joint cavity because of the greater permeability of the synovium and the greater oncotic pressure of the synovial fluid in the presence of inflammation.62,63 In the present study, reduced edema formation was evident for up to 10 days after administration of TA, with or without mepivacaine, compared with results for other treatments. Triamcinolone acetonide reduced limb edema and synovial total protein concentration, reflecting a reduction in permeability of the vasculature. Increased permeability and leakage of proteins out of the vascular space is a standard mechanism of inducing clinical signs of inflammation.64
The finding that numbers of WBCs increased in synovial fluid of MCP joints treated with TA suggests that cells migrated into the joint to a greater degree or less fluid was in the joint, thereby concentrating the cells. The latter explanation appears more likely because edema and permeability of the joint were less in joints treated with TA, compared with results in other joints. Our data may support the hypothesis that the main mechanism of TA in LPS-induced synovitis is an anti-inflammatory one that acts by decreasing permeability of the synovium and fluid flow across the synovium, similar to the action of the known synovial inflammatory mediator, interleukin-1β.40
In the study reported here, mepivacaine effectively eliminated lameness within 30 minutes after intra-articular administration, regardless of whether TA was coadministered. Triamcinolone acetonide reduced values for lameness, limb edema, and synovial fluid concentration of total protein after the second LPS challenge, regardless of whether MCP joints were simultaneously injected with mepivacaine. Triamcinolone acetonide also increased the WBC count and concentration of mepivacaine in synovial fluid, probably via concentrating effects. These findings confirmed that TA is a potent analgesic and anti-inflammatory medication for acute synovitis, and simultaneous administration with mepivacaine does not alter its potency or duration of action.
ABBREVIATIONS
| LPS | Lipopolysaccharide |
| MCP | Metacarpophalangeal |
| TA | Triamcinolone acetonide |
| VFP | Vertical force peak |
Vetalog, Fort Dodge Animal Health, Fort Dodge, Iowa.
Carbocaine-V, Pfizer Inc, New York, NY.
Difco Laboratories, Detroit, Mich.
Sigma Chemical Co, St Louis, Mo.
Truesdail Laboratory Inc, Tustin, Calif.
Model 9281C, Kistler Instrument Corp, Amherst, NY.
Vacutainer, Tyco Healthcare, Mansfield, Mass.
10482 ABBE Mark II Refractometer, Reichert Scientific Instruments, Buffalo, NY.
VWR-International, West Chester, Pa.
Neogen test protocol, Neogen Corp, Lexington, Ky.
SAS, version 9.1, SAS Institute Inc, Cary, NC.
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