Animals
This prospective clinical study was evaluated and approved by the National Ethics Committee and written informed client consent was obtained before the dogs were included in the study. All procedures complied with the relevant Slovenian (Animal Protection Act UL RS, 43/2007) and European regulations. Client-owned dogs receiving no medication one month prior to anaesthesia were recruited from the University of Ljubljana Veterinary Faculty subject pool. Their health status was evaluated based on history, physical examination and blood tests including complete blood count with white cell differential count and serum biochemical analyses (glucose, urea, creatinine, sodium, potassium, chloride, calcium, total protein, albumin, alanine aminotransferase, alkaline phosphatase, serum total cholesterol and triglycerides). Cardiovascular disease was confirmed by history, clinical examination, standard electrocardiogram and echocardiography using two-dimensional, M-mode, and colour and spectral Doppler modes (VIVID E9, General Electric Healthcare, Milwaukee, Wisconsin, USA).
All eligible dogs that were presented between June 2016 and January 2017 were included. The final sample size was n = 30. The dogs were diagnosed with periodontal disease and scheduled for a dental procedure under general anaesthesia. Eighteen dogs were diagnosed with MMVD class B1 or B2 according to the American College of Veterinary Internal Medicine (ACVIM) classification [42]. They were randomly assigned (by tossing a coin) to anaesthesia with propofol (MMVD/P, n = 10) or sevoflurane (MMVD/PS, n = 8). The control group consisted of 12 dogs with no evidence of MMVD.
Study protocol
Dogs were premedicated with morphine 0.3 mg/kg administered subcutaneously (SC) 20 minutes before induction to anaesthesia. An intravenous catheter was placed into the cephalic vein, and after 5 minutes of preoxygenation (flow by, 2 L/min), anaesthesia was induced with propofol (3–6 mg/kg) intravenously (IV) and titrated to effect. The dogs were intubated endotracheally within 30 to 60 seconds, connected to a circle breathing system and allowed to breathe oxygen spontaneously. In dogs with MMVD, anaesthesia was maintained with propofol at 0.3–0.6 mg/kg/min IV (MMVD/P group) or with sevoflurane at an end-tidal sevoflurane concentration of 2 to 3% (MMVD/PS group). Dogs with no evidence of MMVD (PS group) were anaesthetised the same way as the MMVD/PS group. Hartmann's solution was infused at 5 mL/kg/h IV during anaesthesia. Perioperative analgesia was supported with oral regional nerve blocks with levobupivacaine 1 to 2 mg/kg and/or IV boluses of ketamine 0.5 mg/kg. Ketamin was administered when the heart rate increased by 20% or if signs of nociceptive responses occurred during anaesthesia. Perioperative antibiotic management was carried out with cefazolin 20 mg/kg IV if clinically indicated.
The dogs were placed in dorsal recumbency and warmed with bags filled with warm water. End-tidal sevoflurane concentration, end-tidal CO2 tension, respiratory rate, ECG, blood pressure (non-invasively) and rectal temperature were monitored during anaesthesia.
Dogs with the majority of teeth present were included in the study, and a detailed oral examination (probing, charting and full-mouth dental radiographs) was performed prior to periodontal treatment. Dogs were divided into two groups based on their oral/dental disease for statistical analysis: ≤ 25% of the teeth affected with periodontitis and/or dental fractures and > 25% of the teeth affected with periodontitis and/or dental fractures.
During recovery, the dogs were administered Hartmann's solution at 2 mL/kg/h IV and morphine 0.3 mg/kg SC every three hours, depending on the invasiveness of the procedure. Carprofen 4 mg/kg IV was administered after the last blood sampling. All dogs were released to home care the same day, with analgesics and antibiotics prescribed as clinically indicated. They were prescribed carprofen 2 mg/kg orally twice daily for 4 to 6 days. In case of severe acute pain, a transdermal fentanyl patch (3 to 4 µg/kg/h) was placed on the skin of the lateral thoracic region at the end of anaesthesia.
Blood sample collection, processing, and analyses
Venous blood samples were collected from the jugular vein before premedication (basal values) and 5 minutes (immediate post induction period), 60 minutes (intraoperative period) and 6 hours after induction to anaesthesia (postoperative period).
Blood samples for determination of ACL and ACW concentrations (total volume 2 mL) were collected in lithium heparin containing tubes (Vacuette, Greiner Bio-One, Kremsmunster, Austria). Samples were immediately centrifuged at 1500 × g for 15 minutes at 4 °C and plasma immediately frozen at – 80 °C until analysis.
Determination of antioxidant capacity of water- and lipid-soluble antioxidants
The method is based on the chemiluminometric detection of photochemically generated superoxide anion radicals from a photosensitizer (luminol), which are partially eliminated from the sample by reaction with antioxidants present in the sample. The remaining radicals react with luminol to produce luminescence, which is measured with a PHOTOCHEM analyser (Analytik Jena, Jena, Germany) using supplied reagent kits. After defrosting and vortexing, 100 μL of plasma sample and 100 μL of methanol were pipetted in a 1.5 mL plastic container with a cap. Samples were then vortexed for 10 minutes and centrifuged at 25,000 rpm for 10 minutes at 4 °C. Sample preparation steps were performed in a dark place. Until analysis (usually within 30 minutes), prepared samples were held in the dark at a temperature below 4 °C. Plasma ACL and ACW were measured in accordance with the manufacturer instructions, using reagent kits (Analytik Jena, Jena, Germany) with the PHOTOCHEM analyser. The results of the ACW measurements are expressed in nmol equivalents of ascorbic acid in mL of the sample, and the results of the ACL measurements in nmol equivalents of trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) in mL of the sample.
To assess whether the propofol formulation used in the study has antioxidative properties, we also measured ACL in a sample of propofol formulation, using the same method as described above.
Statistical analysis
An a priori sample size calculation was not performed as no comparable data from the literature regarding ACW and ACL were available to enable calculation. Post-hoc sample size calculation indicated that with an effect size of 0.5 and significance level p = 0.05, 30 dogs would be enough to achieve more than 80% power of the study. Statistical analysis was supported by the R statistical software program (version 3.2.2) with the nlme package [43]. The Kruskal-Wallis test was used to evaluate the differences in the baseline characteristics of the dogs, age and duration of anaesthesia. Fisher’s exact test was used to evaluate the differences between the MMVD/PS, MMVD/P and PS groups with respect to the baseline characteristics of periodontal/dental disease status and the use of antibiotics, ketamine and nerve blocks. A linear mixed-effect analysis was used to examine the treatment effect and trends over time within groups (MMVD/PS, MMVD/P and PS) and between groups (the MMVD/PS group was compared to the MMVD/P and PS groups). The models included a random intercept for each dog and three fixed effects: time, group and interaction term. In case of significant fixed effects , multiple comparisons were performed using Holm-Bonferroni correction. A value of p < 0.05 was considered significant.