In horses, detection and suppression of inflammation during the early stages of synovitis may substantially improve prognosis and future athletic function.1 Traditionally, synovitis has been treated by means of parenteral administration of NSAIDs, parenteral or local administration of corticosteroids, topical or local administration of analgesics, and parenteral administration of opioids. Whereas the immunomodulatory and analgesic effects of NSAIDs have been well described,2 the possible immunomodulatory effects and potential clinical applications of locally administered opioids have been an area of research in human medicine for only the past 2 decades.3
Previous investigators have reported the anti-inflammatory4 and analgesic effects of IA morphine administration in human patients,5–8 with all but 1 review concluding that IA morphine administration is associated with substantial analgesic effects.5,6,8 The recent discovery of opioid receptors in the equine synovial membrane9 makes it plausible that IA morphine administration may have similar analgesic and anti-inflammatory effects in horses. On the basis of data extrapolated from human patients, morphine has been administered IA to equine patients and dose recommendations have been published.10 To our knowledge, however, the immunomodulatory and analgesic properties of IA morphine administration have not been studied in horses. Therefore, the objective of the study reported here was to compare the effects of IA versus IV morphine administration on local and systemic inflammatory responses in horses with acute LPS-induced aseptic synovitis. Our hypothesis was that IA administration of morphine would be superior to IV administration for attenuation of the local and systemic inflammatory responses in horses with experimentally induced acute synovitis.
Materials and Methods
Study design—The study was performed as a randomized, controlled, observer-blinded, crossover trial. Each horse received the following 2 treatments in randomized order, with a washout period of 3 weeks between treatments: IA administration of morphine with IV administration of saline (0.9% NaCl) solution, and IA administration of saline solution with IV administration of morphine. Before each treatment, aseptic synovitis was induced by injection of LPS in a radiocarpal joint. The experimental protocol was approved by the Danish Animal Experimentation Board, and all procedures were carried out in compliance with the Danish Animal Testing Act.
Horses—Eight adult riding horses (5 geldings and 3 mares [age, 4 to 9 years]) were included in the study. Prior to enrollment, horses were considered healthy and free from evidence of forelimb joint disease on the basis of results of a thorough clinical examination, including flexion tests, and routine hematologic, serum biochemical, and synovial fluid analyses. Horses with minimal lameness (a score of 1 on a scale from 1 to 511) in a single hind limb were eligible for inclusion in the study. Throughout the study, horses were housed in 3 × 4-m stalls in a barn maintained at a temperature of 13 ± 1°C. Horses were fed a commercial grain mixture twice daily, with access to water and hay ad libitum. To avoid chemical or physical restraint during the study, all horses were trained by means of positive reinforcement techniques to accept handling and all experimental procedures, including radiocarpal arthrocentesis.
Induction of synovitis—Synovitis was induced by means of aseptic IA injection (21-gauge needle) of 3 μg of LPS (Escherichia coli O55:B5a). Lipopolysaccharide was diluted in sterile saline to a final concentration of 1.5 μg/mL and divided into aliquots containing 2 mL of this solution. Aliquots were stored in siliconized glass tubes at −20°C and thawed within 15 minutes of use. To avoid micelle formation and prevent LPS from adhering to the glass, tubes were vortexed for a minimum of 10 minutes after thawing and until the suspension was transferred to the syringe immediately prior to injection. For the first treatment, synovitis was induced in 1 randomly selected radiocarpal joint. For the second treatment, synovitis was induced in the contralateral radiocarpal joint. The time of induction was referred to as PIH 0, and all other time points refer to the number of hours after induction of synovitis.
Experimental treatments—During each of the 2 treatment periods, experimental treatments were administered 4 hours after synovitis was induced. Immediately prior to treatment, synovitis was confirmed on the basis of the presence of joint effusion, lameness, high synovial fluid WBC count, and high synovial fluid TP and SAA concentrations. For all treatments, sterile, preservative-free morphine in individual-use vialsb with an initial concentration of 20 mg/mL was diluted in saline solution to a final concentration of 5 mg/mL. Thereafter, both morphine and saline were administered at the same volume of 1 mL/100 kg of body weight, regardless of route of administration. All morphine and saline treatments were prepared by a person not otherwise involved in the study. Experimental treatments were delivered to the study participants in similar sterile vials marked with horse number, treatment period, and route of administration. Study participants were therefore blinded to the treatments that the horses were receiving. All injections were performed by use of standard aseptic techniques.
Treatment IA consisted of IA administration of morphine (0.05 mg/kg) into the affected radiocarpal joint and IV administration of saline solution (1 mL/100 kg). Treatment IV consisted of IV administration of morphine (0.05 mg/kg) and IA administration of saline solution (1 mL/100 kg). The order of treatments was randomized, and observers were unaware of the treatment administered.
Experimental procedures—During each of the 2 treatment periods, blood and synovial fluid samples were obtained and clinical examinations performed before and 4, 6, 8, 12, 16, 20, 24, 28, 32, 48, 96, and 168 hours after the induction of synovitis. Clinical examinations consisted of examination of general demeanor, mucous membrane color, and capillary refill time; abdominal auscultation; and measurement of rectal temperature and skin temperature over the inflamed joint, heart rate,c and respiratory rate. Blood samples were collected through an indwelling jugular venous catheter and transferred to plain tubesd and tubes containing EDTA.d Approximately 3 mL of synovial fluid was obtained at each sample time by means of repeated arthrocentesis, with a 21-gauge, 40-mm needle. Standard aseptic technique was used. Samples were immediately transferred into tubes containing EDTA.c None of the horses were sedated for any procedures, but 1 horse was physically restrained with a nose twitch.
Evaluation of inflammation—At each examination time point, skin temperature over the dorsal aspect of the radiocarpal joint was measured with an infrared thermometer.e The emissivity of the infrared thermometer was set at 0.98, and each measurement was obtained at the manufacturer's recommended distance of approximately 20 cm from the skin's surface. A 15-second measurement was obtained, and mean skin temperature was calculated. Joint swelling (effusion and periarticular edema) was assessed by measuring the circumference of the carpus at the level of the accessory carpal bone with a tape measure.
Clinicopathologic analysis—Blood samples were stored at 5°C and analyzed within 18 hours after collection. Total and differential WBC counts were obtained from samples anticoagulated with EDTA, by use of an automated cell counter.f Serum SAA concentration was determined with an immunoturbidimetric method as described,12,g and serum cortisol concentration was measured with a chemiluminescence method.h
Synovial fluid TP concentration was determined by use of refractometryi immediately after sample collection. Synovial fluid samples were then stored at 5°C, and WBC counts were determined by use of a hemacytometer within 2 hours after collection. The remaining synovial fluid was then centrifuged at 3,000 × g at 5°C for 10 minutes, and the supernatant was stored at −32°C, until analyzed for synovial fluid SAA concentration, as described for serum.g
Statistical analysis—Statistical analyses were performed with a commercially available software package.j Data were analyzed by calculating the differences between treatments for each horse at each time point. Differences were then analyzed by means of repeatedmeasures models containing factors for time and the difference between baseline (PIH 4) measurements (calculated for each horse). Several correlation structures were examined, and the best-fitting structure was chosen on the basis of the Akaike criterion13 corrected for small samples, accounting for repeated measurements. For all analyses, values of P < 0.05 were considered significant.
Results
All horses developed clinical signs of local and systemic inflammation following IA injection of LPS, including signs of depression and high rectal temperatures, heart rates, and respiratory rates. Lameness became apparent within 4 hours after administration of LPS and persisted for 48 to 96 hours. Degree of inflammation did not differ significantly between treatment groups immediately before treatment (PIH 4).
Clinical signs of inflammation—Joint circumference increased slowly, but significantly, after injection of LPS and peaked in individual horses at 32 to 48 hours after induction of synovitis. Treatment with morphine administered IA resulted in significantly (P = 0.001) lower circumference of the carpal joint at PIHs 28, 32, 96, and 168, compared with treatment with morphine administered IV (Figure 1). Skin temperature did not differ significantly between treatments at any examination time (Figure 2).
Circumference of the radiocarpal joint measured at the level of the accessory carpal bone after IA (circles) and IV (triangles) administration of morphine to 8 adult horses. Synovitis was induced at time 0 by means of IA injection of LPS; treatments were administered 4 hours later. Values are mean ± SEM. *Values were significantly (P < 0.05) different between treatments.
Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.69
Skin temperature (°C) measured over the radiocarpal joint after IA (circles) and IV (triangles) administration of morphine to the same horses as in Figure 1. Values are mean ± SEM.
Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.69
Synovial fluid analysis—Synovial fluid TP concentration ranged from 0 to 5 g/L prior to LPS injection, with peak values of 55 to 60 g/L between 12 and 20 hours later. For the study period as a whole, treatment with IA morphine resulted in significantly (P = 0.004) lower synovial fluid TP concentration. However, because of variation in the data, we were unable to reject the hypothesis of no treatment-by-time interaction (Figure 3).
Synovial fluid TP concentration after IA (circles) and IV (triangles) administration of morphine to the same horses as in Figure 1. Values are mean ± SEM.
Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.69
In all horses, synovial fluid SAA concentration prior to LPS injection was between 0 and 4 mg/L. Peak values of approximately 100 mg/L (IA morphine treatment) and approximately 160 mg/L (IV morphine treatment) were seen at PIH 48. Treatment with morphine IA resulted in significantly (P = 0.038) lower synovial fluid SAA concentrations at PIH 96, compared with concentrations for morphine administered IV. However, significant differences were not detected at any other time (Figure 4).
Synovial fluid SAA concentration after IA (circles) and IV (triangles) administration of morphine to the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.69
Synovial fluid WBC counts peaked at PIHs 8 to 12, but did not differ significantly between treatments at any time (Figure 5).
Synovial fluid WBC count after IA (circles) and IV (triangles) administration of morphine to the same horses as in Figure 1. Values are mean ± SEM.
Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.69
Clinicopathologic analysis—Serum SAA concentrations peaked at PIH 48, and significant (P = 0.018) differences were detected between treatments at PIHs 24, 28, 32, 48, and 96 (Figure 6).
Serum SAA concentration after IA (circles) and IV (triangles) administration of morphine to the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.69
Blood WBC counts peaked at PIH 16. A significant (P = 0.049) difference between treatments was identified only at PIH 32 (Figure 7).
Blood WBC counts after IA (circles) and IV (triangles) administration of morphine to the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.69
Serum cortisol concentrations peaked at PIH 4. Cortisol concentrations did not differ significantly between treatments (Figure 8).
Serum cortisol concentrations after IA (circles) and IV (triangles) administration of morphine to the same horses as in Figure 1. Values are mean ± SEM.
Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.69
Discussion
In the study reported here, all horses developed signs of systemic and local inflammation in response to IA LPS injections, including increases in joint circumference, synovial fluid TP and SAA concentrations, blood WBC count, and serum SAA concentration. These are classic signs of inflammation. Intra-articular morphine administration resulted in significantly less inflammation than did IV administration of the same dose of morphine. Both joint circumference and synovial fluid TP concentration were significantly lower after IA morphine administration than after IV morphine administration. Similarly, several studies14–16 reported that systemically administered morphine reduces edema in rats with experimentally induced inflammation of the hind paws. In one of these studies, the effects were reversed by specific antagonists and were predominantly mediated by peripheral opioid receptors.15 Furthermore, IA administration of the synthetic M-receptor agonist tramadol has also been shown to reduce joint effusion in rats with carrageenan-induced synovitis of the knee joint.17
Serum amyloid A is a major acute-phase protein in several species, including humans and horses.18,19 In the present study, IA morphine administration resulted in significantly lower serum and synovial fluid SAA concentrations, compared with IV morphine administration. Inflammatory stimuli lead to the release of proinflammatory cytokines from monocytes and macrophages, resulting in the hepatic synthesis and release of large quantities of SAA into the bloodstream.20 Serum amyloid A is also produced in peripheral tissues, including synovium, of humans18 and horses.19 Serum amyloid A concentration reflected the severity of joint inflammation in a small study21 in horses, in which synovial fluid SAA concentrations decreased with successful treatment of inflammatory joint conditions.
The significantly lower joint circumference and serum SAA, synovial fluid SAA, and synovial fluid TP concentrations after IA versus IV morphine administration in the study reported here indicate that IA morphine administration induced local and systemic anti-inflammatory effects. However, neither gross nor histologic examinations of the synovial membrane or articular cartilage were performed in our study. Hence, the clinical relevance of the anti-inflammatory effects of IA morphine administration observed in the present study remains undefined. Nevertheless, a beneficial effect is supported by several factors. Joint circumference has been shown to be positively correlated with concentrations of several inflammatory cytokines and chemokines in rats with adjuvant-induced arthritis, including tumor necrosis factor-α, interleukin-1β, interleukin-6, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α.22 In addition, the release of substance P, which is a strong proinflammatory signal in inflammatory joint disease23 and acts to increase vascular permeability and plasma extravasation,24 is attenuated by both μ- and κ-opioid receptor agonists in several studies25,26 of rodents with inflammatory joint disease. Furthermore, synthesis of SAA is induced by proinflammatory cytokines, such as interleukin-6, interleukin-1, and tumor necrosis factor-α, which are all released from monocytes and macrophages during inflammation.20 Serum amyloid A may also induce the release of matrix metalloproteinases, potentially playing a role in cartilage destruction in arthritis.27,28
Morphine as well as other μ- and κ-opioid agonists has previously been shown to decrease the accumulation of leukocytes during inflammation. In humans with chronic arthritis, IA administration of morphine reduced synovial fluid WBC counts significantly more than did IA administration of dexamethasone or saline solution.4 In rats with carrageenan-induced inflammation of the paw, morphine reduced neutrophil accumulation in a dose-dependent manner.15 In the present study, IA morphine administration caused significantly lower synovial fluid TP concentrations, whereas synovial fluid WBC counts did not differ between treatments. This difference may be related to differences in kinetics of these 2 inflammatory markers or to the fact that one is caused by capillary fenestration (TP concentration) and the other by chemotaxis (synovial fluid WBC count). Lipopolysaccharide injection rapidly increased synovial fluid WBC counts to peak values at PIHs 8 to 12, returning to preinjection values by PIH 96. In contrast, synovial fluid TP concentration peaked more slowly after induction of synovitis, peaking at PIHs 12 to 20 and remaining high for the duration of the study. Other inflammatory markers measured in the study reported here (joint circumference and serum and synovial fluid SAA concentrations) also peaked comparatively slowly and were significantly altered by IA administration of morphine. Previous research suggests that the effect of locally administered opioids may be dependent on inflammation-induced increases in the number of functionally active opioid receptors or on increased accessibility of opioid agonists to these receptors.29 In laboratory animals, inflammation increases the number of peripheral opioid receptors,30–32 suggesting that opioid analgesics may be more effective in inflammatory conditions. However, in a study9 examining synovial membrane samples collected from horses, there was no significant correlation between the degree of inflammation and the number of opioid receptors. It has been suggested that activation of opioid receptors occurs as a result of inflammation,4 potentially related to enhanced coupling with G proteins, and that disruption of the perineural barrier may enhance opioid access.33 Taken together, these factors may help explain why IA administration of morphine seemed to have a more substantial effect on inflammatory variables that peaked later in the present study, compared with those that peaked earlier. However, evaluation of these factors was beyond the scope of our study.
In the present study, SAA concentrations in serum and, to a lesser degree, synovial fluid were significantly reduced by morphine administered IA, compared with morphine administered IV, whereas serum cortisol concentration did not differ between treatments. Cortisol is not a specific marker for inflammation because it is also secreted in response to noninflammatory stressful stimuli, such as transportation and exercise.34–36 Although the effect of opioids on the acute-phase response in horses has not been reported previously, studies in other animals and humans have investigated and confirmed this relationship. Results of previous studies of dogs37 and humans38,39 suggest that central administration of a combination of local analgesics and morphine affects the neuroendocrine stress response without affecting the acute-phase response, whereas peripheral administration of morphine or local analgesics decreases acute-phase protein concentrations without affecting cortisol concentrations. This implies that the observed anti-inflammatory effects of morphine administered IA are mediated via peripheral opioid receptors and agrees with findings of the present study.
The present study was conducted as a crossover design to reduce experimental variation. Intra-articular administration of LPS reliably induces a dose-dependent acute synovitis that resolves within approximately 1 week, without any long-term clinical effects on the joint.19,28 The severity of the induced synovitis is considered to be intermediate between that of traumatically induced and infectious synovitis. Thus, we believe that our results are pertinent to the clinical use of morphine in horses. The IA dose used in previous studies5–8 of morphine in human patients ranges from 1 to 5 mg/joint (0.014 to 0.071 mg/kg in a person weighing 70 kg). This is equivalent to less than half of the IV dose required to result in adequate postoperative pain relief in humans.40 For horses, an IA morphine dose of 0.1 mg/kg has been recommended previously.10 Although the basis for this recommendation is unclear, it equals the lowest systemic dose of morphine reported to induce analgesia in horses10,27,41 Based on the knowledge obtained from studies in humans and horses and to avoid adverse systemic effects, we decided to administer half of the lowest recommended IV morphine dose; hence, a morphine dose of 0.05 mg/kg was chosen.
Experimental treatments (IA vs IV administration of morphine) were chosen to allow the effects of morphine on central versus peripheral opioid receptors to be considered. Ideally, a placebo treatment involving no morphine administration should have been included in the present study, but this was not considered acceptable for humane reasons because of the painful nature of the synovitis technique.
Our results suggest that IA morphine administration has anti-inflammatory properties in horses with experimentally induced acute synovitis and that the anti-inflammatory effects are mediated peripherally. However, the mechanism of action was not determined. Intra-articular morphine administration tended to exert maximal effect on inflammatory variables that changed slowly or peaked late in the course of the induced synovitis. Further investigation of the anti-inflammatory and analgesic properties of IA morphine administration in equine patients with clinical synovitis is indicated.
ABBREVIATIONS
| IA | Intra-articular |
| LPS | Lipopolysaccharide |
| PIH | Postinduction hour |
| SAA | Serum amyloid A |
| TP | Total protein |
Sigma-Aldrich, St Louis, Mo.
Morfin DAK, Nycomed, Roskilde, Denmark.
Polar Equine Heart Rate Monitor S810i, Polar Electro Danmark Aps, Holte, Denmark.
BD Vacutainer, BD, Franklin Lakes, NJ.
Raytek Raynger MX4, Raytek, Santa Cruz, Calif.
ADVIA 120, Bayer A/S, Lyngby, Denmark.
LZ test SAA, EIKEN Chemical Co, Tokyo, Japan.
Immulite 2000 Advanced Immunoassay System, Siemens Healthcare Diagnostics, Tarrytown, NY.
Clinical refractometer, Atago Co, Tokyo, Japan.
SAS, version 9.1, SAS Institute Inc, Cary, NC.
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