Alleviation and management of pain and suffering in animals are important aspects of veterinary medicine. Despite this, accurate identification and quantification of pain in animals continue to be an unfulfilled goal, with most clinical research involving subjective and variable outcome measures.
The subjective quantification of pain in the form of visual assessment, such as a numeric pain scale or a visual analog scale, is largely relied upon for pain assessment; however, use of such scales results in considerable inter- and intraobserver variation.1–3 Studies4,5 have been designed to incorporate more objective measures of pain based on changes in physiologic variables such as HR, blood pressure, and plasma catecholamine and serum cortisol concentrations. Unfortunately, these studies1,6,7 have not involved comparison of these measures to a sensitive, objective measure of pain. As a result, findings are based on subjective interpretation of pain. This has led to inconsistent results and hindered progress toward assessment of pain in veterinary patients.
Serum cortisol concentration is believed to increase in response to a painful stimulus through activation of nociceptors and increased activity of the sympathetic nervous system.8,9 Although cortisol concentration has been used in studies of pain in animals, its use has not been validated and thus its validity has been questioned because of the episodic nature of cortisol secretion8 and increased production due to other physiologic stimuli, particularly during times of stress.10
Intra-articular injection of sodium urate has been used to induce acute synovitis, producing a reliable non–weight-bearing lameness of short duration. Use of this model has shown that lameness reliably becomes severe within 1 to 2 hours after injection but ceases to exist within 24 to 36 hours.11–13 This lameness can be measured objectively with force platform gait analysis.11,12,14–20 Given that no other orthopedic or neurologic abnormalities were induced in the lameness model,12 it is reasonable to conclude that the lameness was caused by joint pain; thus, if one can sensitively and objectively measure the lameness, one can effectively estimate joint pain.
The objective of the study reported here was to determine whether a correlation existed between serum cortisol concentration and the severity of lameness resulting from experimentally induced synovitis of the stifle joint measured with force platform gait analysis. The null hypothesis was that there would be no correlation between pain score and serum cortisol concentration.
Materials and Methods
Animals—Ten healthy research-specific hound-type dogs enrolled in a parallel study of acute synovitis were used for this study. To be included in the study, dogs needed to weigh between 25 and 35 kg and have no abnormalities detected during physical examination; musculoskeletal radiographic imaging was not performed prior to inclusion. A CBC, serum biochemical profile, and urinalysis were performed prior to inclusion; results were unremarkable for all dogs. In addition, during a 2-month acclimation period, no dog had any signs of clinical abnormalities. The study was conducted following a protocol approved by the Institutional Animal Care and Use Committee of the University of Minnesota.
Control experiment—Dogs were acclimatized to the study facility, gait laboratory, and handling procedures for 2 months prior to the commencement of the study. Five days prior to induction of synovitis, a blood sample was obtained via jugular venipuncture from each dog for measurement of serum cortisol concentration at 0 (first collection), 2.5, 5, 7.5, 10, and 24 hours, followed by measurement of SAPa and HR. Three measurements of SAP and HR were obtained at each point, and measurements were averaged for analysis. Immediately following blood sample collection and BP and HR measurements, GRFs in the affected limb were measured as described elsewhere2 at 0, 2.5, 5, 7.5, and 10 hours. Dogs were also visually assessed for lameness by a single observer who was not blinded to group assignment at 0, 2.5, 5, 7.5, 10, and 24 hours by use of a visual analog scale.
Synovitis experiment—The protocol was repeated on day 5 with the addition of induction of synovitis via intra-articular injection of sodium urate into the left stifle joint. Synovitis was induced immediately after the 0-hour data collection point, providing 1 measurement point before synovitis, 4 points during which synovitis was in effect (2.5, 5, 7.5, and 10 hours), and 1 point after synovitis had resolved (24 hours). Synovitis was induced while the dogs were lightly sedated with propofol.
Acute synovitis model—Acute synovitis was induced via injection of uric acid into the left stifle joint as follows. Four hundred milligrams of sodium uric acid crystalsb was added to 40 mL of saline (0.9% NaCl) solution to achieve a monosodium urate solution with a concentration of 10 mg/mL. The suspension was heated at low temperature (approx 40°C) on a mechanical stirrer. Prior to transfer to sterile glass vials, the pH was adjusted by adding NaOH or HCl (0.5M) to achieve a pH of 7.0. Next, 40 mL of the suspension was transferred to two 30-mL glass vials (20 mL in each). The vials were autoclaved with venting at 121°C for 15 minutes. After cooling, vials were labeled and stored for use in the study.
Light sedation was induced by administration of propofolc to effect via the right cephalic vein, and dogs were positioned in right lateral recumbency. Hair over the left stifle joint was clipped, and the skin was aseptically prepared for injection. A 20-gauge, 1.5-inch hypodermic needle was aseptically inserted into the stifle joint just medial to the patellar ligament. Following aspiration of joint fluid to confirm needle placement, the sodium urate crystal suspension was vigorously agitated, and 1.0 mL of the suspension was injected into the joint. Digital pressure was then placed on the inoculation site following withdrawal of the needle for approximately 10 seconds in an attempt to minimize leakage into the subcutaneous tissues. The limb was then manipulated through range of motion several times to disperse the fluid within the joint.
Cortisol assay—Blood samples for determination of serum cortisol concentration were collected into serum-separator tubes and allowed to clot for 30 to 60 minutes. The samples were then centrifuged at 894 × g for 15 minutes, and the supernatant serum was frozen at −20°C until transported to a commercial laboratory.d Serum cortisol concentration was determined by use of a chemiluminescent immunoassay.e The assay has been validated in dogs21 and has a test sensitivity of 0.2 μg/dL.
GRFs—Force platform gait analysis was performed by completion of 5 valid trials at a walk, with peak vertical pulse, vertical impulse, and mean falling slope recorded for the left hind limb. To do so, a force platformf measuring 0.5 m2 was mounted in the center of a 10-m runway. Five photoelectric cells mounted 1 m apart and coupled with a triggered timing mechanism were used to measure velocity and acceleration. Data were recorded at rate of 1,000 Hz. Trials were conducted within the velocity range of 1.0 to 1.3 m/s, and an acceleration range of −0.5 to 0.5 m/s2 was used for all trials. Trials were considered valid when the feet of the forelimb and ipsilateral hind limb were isolated on the force platform and gait abnormalities were not detected. The first 5 valid trials for each limb were used for analysis and were normalized for body weight. Data collected and stored on a dedicated personal computerg were used to generate mean force plate values for velocity, acceleration, PVF, vertical impulse, and mean falling slope. A dog was considered to have 5 invalid trials when the dog failed to bear any weight on the limb when walking over the force plate during continual passage for the 30 minutes assigned for gait analysis at each measurement point.
Rescue protocol—Assessment of pain in dogs was performed every 2.5 hours after induction of synovitis by use of a visual analog scale. Dogs were removed from the study and orally administered analgesia in the form of carprofenh (2.2 mg/kg, q 12 h) and tramadol hydrochloridei (2.2 mg/kg, q 8 h) when it was determined the severity of lameness had plateaued for 3 consecutive measurements or at the end of the 24-hour study period.
Statistical analysis—Statistical analyses were performed by use of a commercial software program.j Ground reaction forces are reported as a percentage of body weight, and summary statistics are reported as mean ± SE. To identify lameness (a decrease in GRFs), data collected from all trials when the dogs did not have induced synovitis were considered nonlame data. The maximum variation in PVF from each dog's first trial (0 hours, control experiment) was identified, and the largest of these values (7.1%) was used as a cutoff to differentiate nonlame from abnormal or lame. This effectively classified data from all trials before synovitis was induced as nonlame and only data from trials with a variation > 7.1% after synovitis as lame. Trials from the synovitis study were subsequently classified as lame or nonlame. A receiver-operating characteristic curve was used to measure the ability of serum cortisol concentration to differentiate lame and nonlame dogs, and the area under the curve was used as a measure of accuracy. The sensitivity and specificity were determined by use of the Youden index of the most parsimonious model22 with the highest area under the curve.
A matched pair for time analysis was performed by means of a Wilcoxon signed rank test to calculate statistical differences between dogs with and without synovitis for GRF, cortisol concentration, HR, and SAP. Statistical differences between group means were determined by use of crossed analysis paired t tests. A value of P < 0.05 was considered significant for all analyses.
Results
Animals—All 10 dogs completed the study without a protocol deviation. Dogs had a mean body weight of 28.05 kg (range, 26.2 to 30.3 kg), with equal sex distribution (5 males and 5 females). All dogs in the synovitis experiment were considered lame at 2.5 and 5 hours, 9 dogs were lame at 7.5 hours, and 5 dogs were lame at 10 hours. Statistical analysis revealed that a serum cortisol concentration of ≥ 1.6 μg/dL indicated lameness or pain, with a mean ± 2 SE sensitivity of 0.91 ± 0.1%. That is, the probability of correctly predicting a dog was in pain was 91% if the dog had a cortisol concentration ≥ 1.6 μg/dL. However, the specificity for the same cutoff was only 0.35 ± 0.11%, indicating that 65% of control evaluations yielded a cortisol concentration ≥ 1.6 μg/dL.
During the control study, serum cortisol concentration, SAP, HR, and GRF did not change significantly over time. In addition, no dog developed a visually detectable lameness. During the synovitis study, data collected before (0 hours) and after (24 hours) the period of synovitis were statistically similar to data collected during the control study. Serum cortisol concentration was significantly increased at 2.5 hours, and GRFs (PVF, vertical impulse, and mean falling slope) were significantly decreased at 2.5, 5.0, 7.5, and 10 hours after induction of synovitis, compared with values at 0 hours (Figure 1). No significant difference in serum cortisol concentration was found between male and female dogs at any point during the control and synovitis experiments.
Mean ± SE serum cortisol concentration (A) and PVF (B) of 10 hound-type dogs with (solid lines) and without (dashed lines) intra-articular injection of 10 mg of monosodium urate solution to induce synovitis in the left stifle joint. Control measurements were obtained on day 1 at 0 (first measurement), 2.5, 5, 7.5, and 10 hours. On day 5, the protocol was repeated with the induction of acute synovitis immediately after initial measurement collection at 0 hours. *Value is significantly (P < 0.05) different from value at 0 hours.
Citation: American Journal of Veterinary Research 71, 8; 10.2460/ajvr.71.8.940
Mean ± SE serum cortisol concentration (A) and PVF (B) of 10 hound-type dogs with (solid lines) and without (dashed lines) intra-articular injection of 10 mg of monosodium urate solution to induce synovitis in the left stifle joint. Control measurements were obtained on day 1 at 0 (first measurement), 2.5, 5, 7.5, and 10 hours. On day 5, the protocol was repeated with the induction of acute synovitis immediately after initial measurement collection at 0 hours. *Value is significantly (P < 0.05) different from value at 0 hours.
Citation: American Journal of Veterinary Research 71, 8; 10.2460/ajvr.71.8.940
Mean ± SE serum cortisol concentration (A) and PVF (B) of 10 hound-type dogs with (solid lines) and without (dashed lines) intra-articular injection of 10 mg of monosodium urate solution to induce synovitis in the left stifle joint. Control measurements were obtained on day 1 at 0 (first measurement), 2.5, 5, 7.5, and 10 hours. On day 5, the protocol was repeated with the induction of acute synovitis immediately after initial measurement collection at 0 hours. *Value is significantly (P < 0.05) different from value at 0 hours.
Citation: American Journal of Veterinary Research 71, 8; 10.2460/ajvr.71.8.940
Heart rate was significantly decreased at 10 hours after induction of synovitis (85 beats/min), compared with HR at 0 hours (110 beats/min). Systemic arterial blood pressure did not change over time in either study. Results of visual assessment of lameness indicated that all dogs were nonlame at 0 and 24 hours and lame at all other measurement points.
Discussion
Assessment of pain is a topic of considerable interest in clinical and scientific communities. Researchers often use pain as a measure of morbidity associated with various procedures, techniques, or medications. In addition, clinicians are required to make assessments of the degree of pain with respect to the need for surgery or alterations in analgesic protocols. However, there is no gold standard available for the measurement of pain in animals.
Physiologic variables such as HR or SAP are insensitive predictors of pain in veterinary patients because of considerable variation among animals.23 Accordingly, in our study of dogs before and after induction of experimentally induced synovitis of the stifle joint, results indicated large variation among dogs in physiologic measurements and no significant correlation between HR or SAP and PVF.
Force plate gait analysis has been used as an objective quantitative measure of lameness in veterinary patients with urate-induced synovitis.11–20 Although used at various concentrations and volumes, the urate suspension results in a lameness of predictable duration. Our results were consistent with the following prediction: all dogs were non–weight bearing at 2.5 hours after synovitis induction and clinically normal at 24 hours. Because dogs were weight bearing by 5.0 hours after induction, they did not receive any rescue analgesics.
In this model of acute synovitis, lameness is believed to be induced by pain rather than altered joint mechanics. Injection of the urate suspension results in inflammation and subsequent joint effusion, pain, and lameness. Resolution of lameness with anti-inflammatory medication has been demonstrated, despite continued evidence of effusion suggesting analgesic alleviation of pain as its primary mode of action.12 In addition, lameness reportedly improves when dogs are treated with a pure analgesic that does not reduce joint swelling.12
Our study was designed to assess the validity of serum cortisol concentration as a potential objective measure of pain in dogs with acute synovitis of the stifle joint as a first step to determine whether this method might serve as a useful clinical tool to determine whether pain is present in dogs. Several studies5,24–34 have involved use of serum cortisol concentration in the assessment of pain in postoperative patients; however, its sensitivity for detecting pain has not been assessed because of the lack of an objective comparison.
Many factors influence cortisol secretion. In healthy individuals, daily cortisol secretion assumes a circadian rhythm in many species including humans,35 rhesus monkeys,36 sheep,37 and cattle.38 In dogs, secretion appears to be episodic, with no obvious circadian rhythm; however, considerable inter- and intrasubject variability exists.39–42 Although we did not find any difference between sexes of dogs in this study, sex variation has been reported, with female dogs having significantly greater mean cortisol concentrations as well as a greater frequency and amplitude of concentration peaks than their male counterparts.40 Infrequent sample collection and small sample size may have contributed to the failure to detect differences between dog sexes in our study.
Considerable variability in serum cortisol concentration was evident among the dogs in the present study. As a result, although serum cortisol concentration was increased at 2.5, 5.0, 7.5, and 10.0 hours after synovitis induction, it was significantly increased only at 2.5 hours. At 2.5 hours, when dogs were the most lame and likely having the most pain, serum cortisol concentration was increased in 9 of 10 dogs. Serum cortisol concentration has a half-life of only 50 minutes. Additional statistical differences may have been detected if cortisol concentration had been measured at additional points during maximal lameness or if more dogs were included, thereby increasing the power of the study.
Sleep, activity, and stress (eg, environmental changes, handling, or venipuncture) can influence cortisol secretion. To minimize the stress and environmental factors associated with the procedures of this study, the dogs were acclimatized to the facility, gait laboratory, and handling procedures for several weeks prior to study commencement and the control and synovitis experiments were performed in identical fashion, with the only difference being induction of synovitis. The study protocol required blood sample collection to be performed via venipuncture because of the potential interference of an indwelling catheter with gait analysis and concern regarding catheter maintenance in this group of dogs. The stress of handling and blood sample collection could have led to an artifactual increase in cortisol concentration. Intraindividual variation was in part addressed by having the dogs serve as their own controls. Zero-hour cortisol concentrations for dogs during both the control and synovitis experiments were not significantly different.
Although visual analog scale assessment was performed during the study, the scale was used specifically for assessment of lameness to determine the need for rescue analgesia. It was not used as an outcome measure because the observer was not blinded to synovitis status during this assessment.
A significant difference in serum cortisol concentration was detected following induction of synovitis, suggesting a serum cortisol concentration ≥ 1.6 μg/dL could be used to identify pain in dogs with 91% sensitivity. A high sensitivity ensures pain will be detected in most dogs. On the other hand, the low specificity (35%) would inaccurately classify many dogs as having pain when in fact they do not and therefore limits the use of cortisol values for identifying animals that are not in pain in the experimental setting. Our data does, however, suggest that cortisol concentration can be used as an objective measure of pain in the synovitis-induction model used here and, with proper evaluation, may become a useful tool in clinical settings. If dogs have pain, it is likely their serum cortisol concentration will be high; however, if the cortisol value is < 1.6 μg/ mL, at least in situations similar to those in the present study, it is unlikely the dog has pain.
One limitation of the methods used in our study was the lack of light sedation of dogs during the control experiment; hence, the effect of light sedation with propofol could not be discounted as a cause of the differences detected. With that in mind, it is important to consider that in another study10 in dogs, serum cortisol concentration did not increase significantly with induction and maintenance of anesthesia alone. In addition, cortisol has a mean half-life of 50 minutes in serum,40 whereas 150 minutes elapsed between the time light sedation was administered and the following cortisol measurement was obtained.
Although an attempt was made to ensure the absence of joint disease in study dogs by orthopedic examination and visual observation over the 2-month acclimation period, cytologic evaluation of joint fluid and radiography were not performed. However, because the dogs served as their own controls, we believed that any minor, undetected clinical abnormality would be inconsequential to our findings.
ABBREVIATIONS
| GRF | Ground reaction force |
| HR | Heart rate |
| PVF | Peak vertical force |
| SAP | Systolic arterial blood pressure |
Dinamap, Critikon, Tampa, Fla.
Sigma-Aldrich Inc, St Louis, Mo.
Abbott Laboratories, Abbott Park, Ill.
Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minn.
Immulite 1000, Siemens Healthcare Diagnostics, Deerfield, Ill.
AMTI OR 6-5 force platform, Advanced Mechanical Technology, Watertown, Mass.
Sharon Software Inc, Dewitt, Mich.
Rimadyl, Pfizer Animal Health, Exton, Pa.
Caraco Pharmaceutical Laboratories Ltd, Detroit, Mich.
JMP, version 5.0.01, SAS Institute Inc, Cary, NC.
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