This study was carried out in accordance with the German Animal Welfare Guidelines and was approved by the Ethics Committee of the Lower Saxony State Office for Consumer Protection and Food Safety (Approval Number: 10A071). All owners agreed to their dogs participation in the study and signed a consent form.
Patients
All dogs presented to the Small Animal Hospital of the University of Veterinary Medicine Hannover, Foundation (Germany), between March 2010 and October 2011, for a hind limb amputation were included in the study. In total, 12 patients were enrolled. Two additional patients were not included due to aggressiveness in one case, and presence of metallic orthopedic implants in both stifle joints, making it unadvisable to perform the MR examination, in the other case.
Before surgery a thorough physical examination, including an orthopedic and neurologic examination of all remaining limbs and the spine, was performed to rule out any disease which might obscure the results. This examination was repeated 10, 30, 90 and 120 days after the amputation. It was planned that, in case an abnormality was suspected, all necessary diagnostic examination tools would be used to determine the type and location of such an abnormality and its possible relationship with the amputation.
Surgical procedure
On the amputation day, physical status was determined based on the physical examination, blood work and other diagnostic tests as needed. Based on the American Society of Anesthesiologists (ASA) physical status classification system, all patients were classified as ASA 2 (patients with local or mild systemic disease). The animals were premedicated using a combination of levomethadone (0.6 mg/kg) Footnote 1 and diazepam (0.5 mg/kg) Footnote 2; anesthesia was induced with propofol dosed to effect (1–4 mg/kg) .
Footnote 3 After orotracheal intubation, anesthesia was maintained with isoflurane Footnote 4 in a 1:1 oxygen: air mixture adjusted according to the physical signs of anesthetic depth (end-tidal isoflurane 0.7-1.5 vol %) and a continuous rate infusion (CRI) of fentanyl (0.16 μg/kg/min),Footnote 5 lidocaine (50 μg/kg/min) Footnote 6 and ketamine (10 μg/kg/min).Footnote 7 Additionally, a preoperative epidural anesthesia with bupivacaine (0.5 mg/kg) Footnote 8 and morphine (0.1 mg/kg) Footnote 9 and a intraoperative sciatic nerve block with lidocaine (1 mg/kg) Footnote 10 were performed. For postoperative analgesia the aforementioned CRI of fentanyl, lidocaine and ketamine was used for 24 h, and carprofen (4 mg/kg)Footnote 11 was initiated the day of the surgery and continued for 10 additional days.
The surgical procedure was performed by disarticulation of the hip, as described elsewhere [2]. The dogs remained in the hospital for approximately 5 days.
Kinetic and kinematic gait evaluation
Kinetic (forces) and kinematic (movement) gait analysis was performed one to three days before the amputation, as well as 10, 30, 90 and 120 days after surgery.
Kinetics were measured using a specially designed treadmillFootnote 12 consisting of four separate belts, each of them with an integrated force plate underneath. This design allowed the simultaneous measurement of all limb forces.
Kinematic analysis was performed with the aid of retro-reflective markers (Ø 16 mm reflective markers)Footnote 13 positioned on 24 anatomic landmarks (8 per remaining limb), using double-sided adhesive tape; the location of these markers has been previously described [10, 11] and is illustrated in Fig. 1. Six high-speed infrared camerasFootnote 14 were used to record marker movement in all three remaining limbs simultaneously, as the animals were walking at a controlled speed (measurement frequency: 100 Hz). Before each measurement, static and dynamic camera calibration was performed using an L-shaped calibration device.Footnote 15
On each gait analysis session, patients were gently introduced to the gait on the treadmill; on the first day, a speed at which each individual patient walked comfortably on the treadmill was determined; on each subsequent session the patient was evaluated using the same speed, ranging from 0.5 to 0.8 m/s. During each gait analysis session, two to six trials were recorded, each with a duration of approximately 30 s, until at least one valid trial was obtained. A valid trial was defined as 10 consecutive regular steps, in which the dog walked smoothly, without any external forces from the handler being applied, with all paws landing on the appropriate force plate, without overstepping. Video recording was performed, to ensure that the steps were appropriate for analysis.
Both kinetic and kinematic data were simultaneously recorded using commercially available software.Footnote 16
Ten consecutive steps were afterwards analyzed for the following kinetic parameters: peak vertical force (PFz), mean vertical force (MFz), and vertical impulse (integral [IFz]). All forces were normalized to the individual body weight of each dog and data were expressed as percentage of body weight (%BW). Mean ± standard deviation (SD) was calculated from 10 valid consecutive steps. Afterwards, load redistribution (LR) was calculated for each forelimb and remaining hind limb, for each measured parameter (PFz, MFz, IFz), using the following equation (according to Steiss et al. [12]): % load bearing = Fz of the limb/total Fz of all limbs*100. Other kinetic parameters were not calculated to avoid overloading this section with too much information. The kinetic data were processed using commercial softwareFootnote 17 and exported to a commercially available spreadsheet.Footnote 18
In order to process the kinematic data in Vicon Nexus, all markers were labeled in a trial. Then, 10 valid foot strikes were marked manually to define the gait cycle (stance and swing phases) of each limb. Using a 2-dimensional (2-D) model, projected flexion and extension angles of each remaining joint were calculated: contralateral (with respect to the amputated hind limb) scapulohumeral joint, contralateral cubital joint, contralateral carpal joint, ipsilateral (with respect to the amputated hind limb) scapulohumeral joint, ipsilateral cubital joint, ipsilateral carpal joint, contralateral coxofemoral joint, contralateral femorotibial joint and contralateral tarsal joint. Measured angles are illustrated in Fig. 1. In order to compare the movement pattern of each analyzed joint, the gait cycles were normalized to 100 in all dogs and displayed as percentage of one whole stride. The mean joint angle (±SD) and the range of motion (±SD) of the aforementioned joints were calculated from the mean joint angle progression curves (MJAPC) calculated from the 10 strides per dog. Mean joint angles and range of motion were used since the reader can easily understand their comparison between sessions and to avoid overloading the manuscript with too much data. The kinematic data were processed using commercial softwareFootnote 19 and then exported to a commercially available spreadsheet18.
MR evaluation of the contralateral stifle joint
The MR examination was performed under general anesthesia before and 120 days after amputation. The anesthetic protocol was the same described above, excluding local anesthetics and CRIs. The animals were positioned in lateral recumbency with the limb to be examined in a non-dependent position and the stifle joint at an angle of ~135°. Using a state-of-the-art 3 T MR scan,Footnote 20 images were obtained from the contralateral stifle. Small (11 cm Ø) surface ring coils (Achieva 3.0 T Musculoskeletal SENSE Flex S coil 2 elements) were used as image enhancers; these were positioned parallel to each other, lateral and medial to the examined stifle, with the joint centered between the two coils. The MR protocol used included a 3-D (3-dimensional) PDW (proton-density weighted) acquisition sequence, which was afterwards reconstructed in sagittal, dorsal (parallel to patella ligament) and transversal (parallel to tibial plateau) planes (slices every 2 mm), a PDW HR (high-resolution) TSE (turbo spin echo) SENSE (sensitivity encoding for fast MR) sequence in sagittal plane (slices every 2 mm),, a PDW HR SPAIR (spectrally adiabatic inversion recovery) SENSE in sagittal plane (slices every 2 mm), and a T1-weighted TSE clear (constant level appearance) sequence in sagittal plane (slices every 1.8 mm).
This protocol had been previously standardized and regarded as suitable for use in clinical cases, since diagnostic image quality is optimal and acquisition time is only 22 min (total examination time is about 40 min including positioning, reference scan, survey, and sequence planning).
Using a high-resolution diagnostic screenFootnote 21 the images were assessed by a trained evaluator (VGZ), who looked for changes in the signal intensity of the cranial cruciate ligament (CrCL), the caudal cruciate ligament (CdCL) and the lateral and medial menisci. Possible changes in the cartilage surfaces, as well as evidence of osteoarthritic changes were also evaluated in the lateral and medial femoral condyles, femoral trochlear groove, patella and tibial plateau.
It was expected that, due to a possible underlying metastatic disease, some patients could die or be euthanized before the end of the study; if that was the case, it was planned to ask the owner to authorize the MR examination postmortem.
Owner evaluation of patient comfort
The owner was requested to fill out an evaluation form (modified from Hielm-Björkman et al. [13]) before the amputation and 10, 30, 90 and 120 days after the procedure, in order to gather his/her (subjective) impressions with regard to patient comfort and recovery. At the end of the study (day 120), owners filled out a questionnaire to assess their final impression regarding the degree of activity and life quality of the dog, and their general impression of and satisfaction with the procedure; besides, owners were encouraged to make further comments. It was planned that if the animal died before the end of the study, an appropriate moment would be looked for to ask the owner to fill out the questionnaire. The questions of the questionnaire were adapted from Carberry and Harvey [4], Withrow and Hirsch [5], von Werthern et al. [6] and Kirpensteijn et al. [7]. The owners’ assessment of patient comfort and the final questionnaire were made in German and translated into English as accurately as possible.
Statistical methods
Due to the small sample size and very heterogeneous patient population included in this study, it was decided to use non-parametric statistics. Thus, data were analyzed using a Kruskal-Wallis one-way ANOVA test to compare between sessions; when statistically significant differences were found, a Wilcoxon signed-rank test for paired observations was performed to determine which session was different. All tests were considered statistically significant if p < 0.05 and were performed using standard statistical software.Footnote 22 Descriptive statistics were calculated using a commercially available spreadsheet18, where appropriate.