Study design
The study protocol was approved by the Comité d’éthique de l’utilisation des animaux of the Faculty of Veterinary Medicine, Université de Montréal (18-Rech-1979). This study is reported according to the Consolidated Standards of Reporting Trials (CONSORT) guidelines for randomized clinical trials [20]. This was a prospective, randomized, masked clinical trial.
Animals
Twenty-six female dogs from local animal shelters were admitted to the veterinary teaching hospital (Centre Hospitalier Universitaire Vétérinaire) of the Faculty of Veterinary Medicine, Université de Montréal for elective ovariohysterectomy between May and July 2019. Written consent for participation in the study was obtained for each patient. Dogs were included if they were considered healthy based on medical history, physical examination, hematology and biochemical panel. They were up to date on vaccination and parasite control. Dogs of any breed with age between 6 months and 8 years and weighting between 5 and 30 kg were included. Patients with aggressive or fearful behaviors, pregnancy or any sign of disease were excluded. Dogs were admitted the day before surgery and were housed individually in a dog ward. Each run was equipped with blankets, water and food bowls. Food but not water was withheld for 8–12 h. At the end of the study, dogs returned to their local shelters for adoption.
Anesthesia, surgery and treatments
Randomization of treatments was performed by an individual not involved with pain assessment using a randomization plan generator (www.randomization.com). Upon arrival, each dog was sequentially assigned a number (1–24). According to this number, the patient was allocated to one of the three treatment groups (n = 8/group). If any dog from 1 to 24 was excluded, additional dogs were recruited and given the same treatment of the excluded dog on the order of arrival.
After physical examination and baseline pain assessment, an IV catheter was placed aseptically in each cephalic vein. Dogs were then randomly premedicated with 0.02 mg/kg of buprenorphine (Simbadol) via intravenous (group IV), intramuscular (group IM) or subcutaneous (group SC) route of administration. The IM administration was performed into the epaxial muscles. The SC administration was performed between the scapulae. The two catheters were color-coded: the ones wrapped in blue bandage were used for drug and fluid administration (including premedication in the IV group) and were removed 6 h after surgery. The catheters wrapped in red bandage were used exclusively for blood sampling and removed following collection of the last blood sample.
Thirty minutes after premedication with buprenorphine, anesthetic induction was accomplished with intravenous propofol (PropoFlo 28, Zoetis, Canada) until endotracheal intubation was possible using an appropriately sized cuffed endotracheal tube. General anesthesia was maintained with isoflurane (Isoflurane USP, Fresenius Kabi, Canada) in oxygen. Following induction of anesthesia, carprofen was administered (4.4 mg/kg SC; 50 mg/mL, Rimadyl, Zoetis, Kirkland, QC, Canada) and dogs were positioned in dorsal recumbency over a circulating warm-water blanket. A balanced crystalloid solution (Lactated Ringer’s solution USP, Baxter, Canada) was administered (10 mL/kg/h) throughout surgery. Anesthetic monitoring was performed using a multi-parametric monitor (Lifewindow 6000 V, Digicare Animal Health, USA) with electrocardiography, non-invasive blood pressure via the oscillometric method, capnography, inspired and expired concentrations of isoflurane, pulse oximetry and esophageal temperature. Ovariohysterectomy was performed by the same veterinarian using a midline approach and a two-clamp modified technique. The abdominal wall and subcutaneous tissues were closed with a simple continuous pattern of absorbable suture material whereas the skin was closed using an intradermal suture pattern. Surgery time (time elapsed from the first incision until placement of the last suture), anesthesia time (time elapsed from injection of propofol to turning off the vaporizer dial) and time to extubation (time elapsed from turning off the vaporizer dial until extubation) were recorded. The length of incision was recorded after skin closure. A 2-cm green line tattoo was applied laterally to the ventral midline incision for visual identification of a neutered dog. Dogs recovered from anesthesia in a warm and quiet environment. An additional dose of carprofen (4.4 mg/kg orally) was administered 24 h after the first dose.
Pain and sedation assessments
Assessments were performed by an observer with previous experience in pain assessment who was blinded to the routes of administration. Pain was evaluated using the short-form Glasgow composite pain scale for dogs (SF-GCPS) [21]. This scale includes 30 descriptor options within six behavioral categories. Within each category, the descriptors are ranked numerically according to their associated pain. In this study, section “B” of the SF-GCPS (“put lead on dog and lead out of kennel”) was not performed since dogs were unlikely to stand up and walk shortly after anesthesia. Thus, the maximum total score of the SF-GCPS was 20 [21]. The SF-GCPS evaluations were performed 1 h before anesthetic induction (time 0 h, baseline) and at 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h after extubation. Sedation was evaluated using a dynamic and interactive visual analog scale (DIVAS) where 0 was considered as no sedation and 100 mm as the maximum sedation where dogs would be in lateral recumbency and not respond to stimulation [22]. Rescue analgesia was performed if SF-GCPS scores were ≥ 5/20 using morphine which was administered over 1 min (0.25 mg/kg IV 10 mg/mL, Morphine Sulfate Injection, Sandoz Canada Inc., Boucherville, QC, Canada). Data collected after the administration of rescue analgesia were not included in the statistical analysis; however, dogs were continuously monitored for additional need of analgesics. The number of dogs, timing of rescue administration and number (frequency) of rescue treatments were recorded.
Blood collection
This was performed just before and at 2, 5, 10, 15, 30, 60, 120, 240, 360, 540 and 720 min after the administration of buprenorphine [5, 7]. One individual was responsible for blood collection while another observer was responsible for pain assessment. The volume of blood collected was adjusted for each individual so that less than 10% of the dog’s total blood volume was removed over the study period (2 mL maximum per sample). For each sample, a total of 0.3–0.5 mL of blood was first collected and discarded to avoid contamination from a previous time point. A second syringe was then used to collect the blood sample for analysis, which was immediately transferred to an EDTA K3 tube and stored in ice. The catheter was then flushed with 1 mL of heparinized saline solution. The blood sample was then centrifuged at 2000 g for 10 min. Plasma was separated and stored at − 80 °C until buprenorphine assay was performed.
Buprenorphine assay
The concentrations of buprenorphine and norbuprenorphine were determined in plasma using a validated liquid chromatography with tandem mass spectrometry method (HPLC-MS/MS). Detailed methodology, and test precision and accuracy are described in Additional file 1.
Pharmacokinetic analysis
Buprenorphine plasma concentrations versus time data for each patient were plotted and visually inspected. The pharmacokinetic analysis of buprenorphine was performed using a standard software (PK Solver 2.0). Non-compartmental analysis was performed for the three routes of administration. The area under the time-concentration curve from time zero to infinity (AUC 0-∞) and area under the curve to the last measurable concentration (AUC 0-t) were calculated by use of a log-linear trapezoidal model. Standard equations were used to calculate other estimates including elimination half-life [t (1/2)], plasma clearance (Cl), apparent volume of distribution at steady state [Vz (ss)], and mean residence time (MRT). Time to maximal concentration [T (max)] and maximal serum concentration [C (max)] were taken directly from the data [5, 7].
Statistical analysis
The study would require eight dogs per group to detect a difference of 3 points in pain scores between two means (IV and IM) using the SF-GCPS and considering an alpha value of 5%, a power of 80% and a standard deviation within group of 2 points. These values were based on clinical experience where changes in 3 points in SF-GCPS would likely reflect clinically relevant changes in patient comfort. Statistical analyses were performed with SAS (version 9.3; SAS Institute, USA) and figures were plotted with GraphPad Prism 8 (version 8.0.2; GraphPad Software Inc., USA). Demographic data between groups were analyzed using linear models with group as fixed factor. The prevalence of rescue analgesia was analysed using Fisher’s exact test for each comparison between groups. The number (frequency) of rescue analgesia was analysed using the Mantel-Haenszel chi-square test for ordinal variables. Pain and sedation scores were analysed using a linear mixed model with group as between-subject fixed factor, time as a within-subject fixed factor and dog as the random effect. A series of a priori contrasts were performed to compare scores within- and between-groups. The Benjamini-Hochberg sequential procedure was used for adjustment after multiple comparisons. Significance was set at α < 0.05.