Effect of a single intra-articular injection of bupivacaine on synovial fluid PGE2 concentrations in normal canine stifles

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CHAPTER 2 – Effect of a Single Intra-Articular Injection of Bupivacaine on Synovial PGE2 Concentrations in Normal Canine Stifles

The open access journal, BMC Research Notes, published an abbreviated version of this manuscript in April 2018. Giangarra JE, Barry SL, Dahlgren LA, Lanz OI, Benitez ME, Werre SR. Effect of a single intra-articular injection of bupivacaine on synovial fluid prostaglandin E2 concentrations in normal canine stifles. BMC Res Notes. 2018;11:255.

Introduction

Bupivacaine is a local anesthetic that works directly on the neuronal cell membrane, reversibly binding sodium channels and blocking the impulse conduction of nerve fibers. When used intra-articularly, as part of a multimodal analgesic protocol, bupivacaine provides long-lasting local pain relief (1-3). The ease of administration, wide availability, inexpensive nature, familiarity and reliable clinical efficacy of intra-articular bupivacaine make it an attractive option for the management of postoperative pain in the clinical setting.
In recent years, the safety of intra-articular use of bupivacaine has come into question, despite decades of clinical use without immediate side effects (4-8). A retrospective case series along with several systematic reviews of the literature concluded an association between continuous infusion of intra-articular bupivacaine and glenohumeral chondrolysis in people (4, 9-11). Chondrolysis is characterized by severe, diffuse degeneration of the articular cartilage from the bone surface (4, 8) and when associated with bupivacaine is thought to be the results of direct toxicity leading to increased chondrocyte apoptosis and necrosis (4-8, 12, 13) and is time and dose dependent (4, 7, 11, 13-15). The delay of several months between bupivacaine infusion and clinically evident chondrolysis suggests that chondrolysis may be a sequela to an acute process that indirectly leads to chondrocyte toxicity or makes chondrocytes more vulnerable to subsequent insults (16).
In humans and equids, sustained synovitis can indirectly lead to cartilage destruction via the activation and release of enzymes, inflammatory mediators, and cytokines (17-19). It is well established that an insult to the joint induces release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) from synoviocytes (20-23). These cytokines increase the synthesis of other inflammatory mediators from the joint such as prostaglandin (PG) E2 (20, 21). PGE2 is considered a marker of joint inflammation and synovitis
and stimulates expression of matrix metalloproteinases that play a major role in articular cartilage degeneration (24). PGE2 synovial fluid concentrations are increased and sustained in dogs with osteoarthritis induced by experimental transection of the cranial cruciate ligament
In addition, studies also suggest that synovitis may precede cranial cruciate ligament degeneration, stifle instability and subsequent osteoarthritis (26-28).
The idea that bupivacaine may cause acute synovial joint inflammation is supported by studies involving other local anesthetics in multiple species (29-31). Increased nucleated cell counts and protein levels within synovial fluid has been reported 12-24 hours after injection of lidocaine and mepivacaine in equine joints (29), and lidocaine and procaine in bovine joints (30). In rabbit stifles harvested 24 hours, 48 hours and 10 days after bupivacaine injection, scored histologic changes were consistent with synovitis and were significantly worse than histologic scores in the saline controls (31).
There is reason to question whether ex vivo studies reporting bupivacaine’s chondrocyte toxicity accurately model its actual effects in the clinical patient. Isolation of chondrocytes from the surrounding extracellular matrix and the concentration and duration of bupivacaine to which chondrocytes are exposed in these in vitro and ex vivo studies far exceed synovial fluid concentrations found after a single joint injection in vivo (32). Therefore, it is clinically important to know whether a single intra-articular bupivacaine injection, when given at the dose for perioperative analgesia, causes synovial joint inflammation that could lead to cartilage damage in dogs. The objective of this in vivo study was to identify if there is a synovial joint response in the normal canine stifle following a single intra-articular dose of bupivacaine. We hypothesized that a single intra-articular bupivacaine injection would result in a detectable synovial joint response as indicated by a significantly increased synovial fluid PGE2 concentration, compared to intra-articular saline injection in normal canine stifles.

Materials and Methods

Study Population
Eight healthy adult male intact purpose bred Beagle dogs were used. The study was approved by the Institutional Animal Care and Use Committee. Dogs were housed in pairs in approved 1.5×2.1 meter runs and hand walked daily. The mean age of the dogs was 1.59 years (range: 1.57-1.62 years) and the mean weight was 12.1 kg (range: 10.1-13.4 kg). Dogs were included if they were deemed healthy based on normal physical and orthopedic examinations (i.e. no evidence of lameness at a walk, and absence of cranial drawer test or tibial thrust). Exclusion criteria consisted of evidence of orthopedic disease either at the time of orthopedic examination or on radiographic examination of the stifle. Dogs were heavily sedated with hydromorphone (0.05 mg/kg) and dexmedetomidine (5 mcg/kg) intravenously and eyes were lubricated with a sterile petrolatum ophthalmic ointment. Bilateral orthogonal stifle radiographs were obtained and a single board certified radiologist reviewed all radiographic images. Sample Collection
Stifles were randomized using a random number generator1 such that each dog had one stifle allocated to bupivacaine and the contralateral stifle allocated to saline treatments. Immediately prior to sampling, dogs received a subcutaneous injection of a long acting cephalosporin, cefovecin sodium (8 mg/kg), to protect against bacterial contamination as a result of repeated arthrocentesis during this study. Synovial fluid was sampled at five time points: immediately prior to intra-articular injection (T0), thirty minutes following injection (T30), sixty minutes following injection (T60), twenty-four hours following injection (T24) and forty-eight hours following injection (T48).
Following radiographs, under the same sedation, stifles were clipped and aseptically prepared using 70% alcohol and povidone iodine scrub. When necessary, dogs were administered additional sedation (dexmedetomidine 5 mcg/kg, intravenously). The first three time points (T0, T30 and T60) were collected on the first day (Day 0). A standard medial or lateral para-patellar arthrocentesis technique was utilized as described previously, based on dog positioning (i.e. medial arthrocentesis of the right stifle and lateral arthrocentesis of the left stifle) (33). Following collection of the first sample, the syringe (1 mL) and needle (22 gauge) were removed and a new syringe (3 mL) and needle (22 gauge) were used to administer either 0.5% preservative-free bupivacaine2 (0.2 mL/kg) or an equal volume of 0.9% saline. On Day 0, following intra-articular injection, a digital timer was set for 30 minutes and ten passive range of motion cycles were performed. Following the last sample collection, dogs were reversed with atipamezole (equal volume to dexmedetomidine) intra-muscularly. The remaining time points (T24, T48) were collected on Days 1 and 2, respectively. Dogs were sedated; samples collected and then reversed using the above protocol. Following collection, all synovial fluid was transferred into 0.5 mL microfuge tubes and stored at -80C until batch analysis could be performed.
PGE2 Quantification
Samples were thawed on ice and PGE2 quantified by competitive ELISA3 (25) and read by spectrophotometer4. All samples were processed within 6 months of collection. The manufacturer reports that samples can maintain reliability for quantification for up to 2 years with appropriate storage. Each sample was quantified in duplicate according to the
manufacturer’s instructions using the standard curves generated for each ELISA plate (7.8-1,000 pg/mL). Samples were initially diluted 1:2 with ELISA buffer. When necessary additional dilutions were performed to ensure results were within the standard curve of the plate (32/80 samples).
Statistical Analysis
Prior to the study, a power analysis was performed using data from an unpublished pilot study that measured the increase in PGE2 concentration 30 minutes following intra-articular injection of bupivacaine. A sample size of 6 stifles per group was estimated to achieve 83% power to detect a mean of paired differences of 480 pg/ml between groups with a significance level (alpha) of 0.05.
Data were analyzed using commercial software5. T0 concentrations were corrected to account for the inherent dilution created by infusion of either bupivacaine or saline into the joint using the following formula (T0c):   0   =   0×    . Where initial volume (Vi) equals 0.08 mL/kg (32) and final volume (Vf) equals (Vi – Vaspirated) + Vtreatment.
Normal probability plots showed that PGE2 concentrations were skewed and are therefore summarized as median (range). A logarithmic transformation (base e) was applied to the concentrations before any downstream data analysis. Effects of treatment and time were assessed using mixed-model repeated-measures ANOVA followed by Tukey’s procedure for multiple comparisons. The linear model specified treatment and the interaction between treatment and time as fixed effects with Kenward-Roger approximation as the denominator degrees of freedom. G-side variation in the data was modeled by specifying dog identification as a random effect while the R-side variation in the data was modeled by specifying a first order, autoregressive ,covariance matrix. Residuals were inspected to verify that the errors followed a normal distribution with constant variance. A post hoc analysis (mixed-model ANOVA) was performed to assess any association between number of aspiration attempts required to collect an adequate sample and PGE2 concentration. Based on the post-hoc analysis revealing that ≥3 attempts resulted in significantly elevated PGE2 concentrations, a sensitivity re-assessment of the effects of treatment and time using data obtained after 1 or 2 aspiration attempts was performed. Statistical significance was set at p < 0.05.

Results

All dogs were included in the study. No radiographic evidence of osteoarthritis was identified in any stifle in any dog. Most dogs (7/8) required additional sedation administration for Day 0 sample collections. No other complications were noted before, during or after sample collection. No lameness following injection or joint aspiration was observed at any time throughout the study period.
There was no significant difference in PGE2 concentration between the bupivacaine and saline groups (p=0.229-0.898) or over time within each group (p=0.152 for bupivacaine; p=0.343 for saline) when all data were included in the analysis without consideration for number of aspiration attempts (Table 1). The number of aspiration attempts required to collect an adequate sample was 1 in the majority of cases (62/80), but was as high as 6 attempts in a single dog (Table 2) and PGE2 concentrations varied significantly based on number of aspiration attempts (Table 3; p = 0.007). PGE2 concentrations for 1 attempt was not significantly different than for 2 attempts (p=0.984); however, PGE2 concentrations in samples requiring either 1 (p = 0.005) or 2 (p = 0.041) attempts were significantly lower than those requiring 3 attempts.
Based on the analysis of number of attempts, samples requiring ≥3 aspiration attempts were omitted and the data re-analyzed. A total of 11 of 80 samples were omitted (Table 4). Following re-analysis, PGE2 concentrations in the bupivacaine group increased significantly over time (p = 0.008). PGE2 concentrations in samples collected at T24 (p = 0.003) and T48 (p = 0.041) were significantly higher compared to T0 (Table 4). There were no changes in PGE2 concentrations in saline-treated joints over time (p = 0.207) There were no significant differences between saline- and bupivacaine-injected joints at any time point (p=0.261-0.949).
Finally, a second post hoc analysis was performed to evaluate the effect of ≥3 attempts on all subsequent synovial fluid samples. Using a mixed model ANOVA, all samples collected before ≥3 attempt-samples were compared to samples collected after ≥3 attempt-samples, excluding samples that required ≥3 attempts. No evidence was found to suggest that ≥3 aspiration attempts influenced the PGE2 concentration in subsequent samples, regardless of whether all stifles were analyzed together (p=0.221) or whether bupivacaine and saline stifles were analyzed separately (p=0.092 and 0.582, respectively).

TABLE OF CONTENTS
Title Page 
Abstract – Academic 
Abstract – Public 
Dedication 
Acknowledgements
Attributions 
Table of Contents 
List of Abbreviations 
List of Tables 
Thesis Organization 
Chapter 1: Background and Literature Review
Section 1: Normal Joint Anatomy and Physiology
Section 2: Arthritis
Section 3: Prostaglandin E2 Formation & Metabolism
Section 4: Prostaglandin E2 Functions & Role in Joint Inflammation
Section 5: Prostaglandin E2 Competitive ELISA
Section 6: Sensory Innervation of the Joint and the Pain Pathway
Section 7: Pain Management
Section 8: Bupivacaine and Chondrotoxicity
Section 9: Conclusion
References
Chapter 2: Effect of a single intra-articular injection of bupivacaine on synovial fluid PGE2 concentrations in normal canine stifles
Publication
Introduction
Materials and Methods
Results
Discussion
References
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