In these series of experiments, 6 female adult cats (Felis catus; table ) were studied. The experimental and surgical procedures were consistent with US Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Georgia Institute of Technology.
Characteristics of animals and walking cycles used for mechanical analysis
Prior to surgeries and data collection, each cat was trained using methods of operant conditioning with food awards to walk along a custom-made Plexiglas-enclosed wooden walkway (3.0 × 0.4 m) with up to 3 force plates (2 plates, 16 × 11 cm; 1 plate, 11 × 7 cm; Bertec Corporation, Columbus, Ohio, USA) embedded in the walkway floor. The floor and force plates were covered separately with rubberized material to prevent slippage. The walkway was set up at 3 walking conditions: level (0%), downslope (−50% or −27°) and upslope (50% or 27°).
After completion of locomotor training (lasting 3–6 weeks) and initial locomotion data collection (necessary for evaluating effects of electrode implantations on walking mechanics, see below), each animal underwent a first survival surgery under general anesthesia and aseptic conditions to implant EMG electrodes in selected ankle extensor muscles. The cat was anesthetized using ketamine (10 mg/kg, s.c.), atropine (0.05 mg/kg, s.c.) and isoflurane (inhalation, 5%). Subsequently, the cat was intubated and anesthesia was maintained with isoflurane (1–3%). The animal was continuously monitored for temperature, respiration, heart rate and blood pressure throughout the surgery. Before making incisions, the skin overlying dorsal aspects of the right hindlimb and the skull was shaved and cleaned with a surgical disinfectant. Teflon-insulated multi-stranded stainless steel wires (100 μm diameter; Conner Wire, Chatsworth, Calif., USA) were passed subcutaneously along the back from a multi-pin amphenol connector mounted on the skull (with four stainless steel screws and dental cement) to a skin incision overlying muscles of interest in the right hindlimb: SO, LG, MG (in cats No. 3–6) and SO, LG, MG and PL (in cats No. 1 and 2). A portion of each pair of wires with a small strip of insulation (approximately 2 mm) removed was inserted with a hypodermic needle in the mid-belly of each muscle and fixed at an inter-electrode distance of 3–4 mm. After implantation of EMG electrodes and their validation using mild electrical stimulation, skin incisions were closed, pain medication administered (fentanyl transdermal patch, 2–25 μg/h and/or buprenorphine, s.c., 0.01 mg/kg, or ketoprofen, 2 mg/kg, s.c.) and anesthesia stopped. The animal recovered for 1–2 weeks with the pain medication administered for at least 3 days and antibiotics (cefovecin, 8 mg/kg, s.c., or ceftiofur, 4 mg/kg, s.c.) for 10 days. Locomotion experiments followed the EMG electrode implantation and lasted typically for up to 3 weeks (see below). No signs of pain or lameness during walking were detected after implantation of EMG electrodes with locomotor patterns recorded after implantation indistinguishable from those collected before surgery.
Once collection of baseline locomotion and EMG data was completed, the second survival surgery was conducted to cut and reconnect the branch of the tibial nerve innervating SO and LG. The aseptic conditions, anesthesia and animal monitoring and postsurgery pain management were the same as in the first surgery. A longitudinal skin incision was made in the popliteal region of the right hindlimb and the exposed fat pad reflected aside by blunt dissection. The LG-SO nerve was identified and a small portion (2–4 mm) cleared of surrounding tissue. The nerve was cut with sharp scissors between the branch point of the tibial nerve and the entry point to LG. Completeness of SO and LG denervation was tested with mild electrical stimulation of the proximal nerve stump. Subsequently, proximal and distal nerve stumps were aligned to their original position and secured in place using fibrin glue (equal parts of thrombin and a 1:1 mixture of fibrin and fibronectin; Sigma-Aldrich, St. Louis, Mo., USA) [English et al., 2005
]. The cat was allowed to recover for 3–5 days before locomotion experiments resumed.
The repair of the cut peripheral nerve using fibrin glue (or 10-0 suture as used by us previously) allows for reinnervation of cat ankle extensor muscles – first signs of detectable EMG emerge in 4–5 weeks [Gregor et al., 2003
; Prilutsky et al., 2006
; unpubl. data]. In this manuscript, we report data obtained during the first 3 weeks after nerve cut and repair when SO and LG were still in a state of paralysis.
Each cat was tested in 3 series of experiments, i.e. (1) before EMG electrode implantations, (2) after implantations and before denervation, and (3) after denervation, and in 3 locomotion conditions, i.e. level, downslope and upslope walking. Before first testing in each of the 3 series of experiments, the cats were sedated (dexmedetomidine, 40–60 μg/kg, s.c.) and the right hindlimb was shaved. The length of each hindlimb segment (pelvis, thigh, shank, tarsals, digits) was measured using an anthropometer (table ). Following shaving and segment measurements, antipamezole (same dose as dexmedetomidine) was administered to reverse sedation.
Prior to recordings, small (6 or 9 mm) reflective markers were placed on the anatomical landmarks of the right hindlimb (the iliac spine, greater trochanter, lateral femoral epicondyle, lateral malleolus, 5th metatarsophalangeal (MTP) joint and the distal end of the 5th digit; see cat hindlimb diagram in fig. ) using a double-sided adhesive tape. Marker positions during locomotion were recorded in 3D by a 6-camera motion capture system Vicon (Vicon Motion Systems, UK) at a sampling rate of 120 Hz. Positional data were recorded synchronously with the 3 components of the ground reaction force vector and coordinates of its point of application (360 Hz; Bertec Corporation, Columbus, Ohio, USA) and with EMG signals sampled at 3,000 Hz from SO, LG, MG and PL. EMG signals were collected using a 16-conductor shielded flexible cable that was attached to the head mounted connector. The recorded signals were band-pass filtered (30–1,000 Hz) and amplified. During each session, 30–60 trials of walking were recorded. Only trials during which the cat moved with a steady speed and hit at least one force plate with the right hindlimb were used for analysis. Walking cycles that were too fast (stance duration <300 ms) or too slow (>650 ms) were not analyzed. Postdenervation locomotor testing was conducted for 3–5 days each week starting from day 4 or 5 (when possible; week 1) and continued for 12 weeks or longer. In this report, we present results obtained in weeks 1 and 2 after denervation (cats No. 1, 3, 5, 6; table ). Data obtained in week 3 were also included into the dataset for 2 animals (cats No. 2 and 4) because their gait in weeks 1 and 2 was unsteady or too slow, so an insufficient number of good walking cycles were collected. Since no SO and LG EMG activity could be detected in the analyzed postdeneravation walking cycles, the postdenervation data were treated as a single dataset representing short-term effects of SO and LG paralysis.
Fig. 1 Schematic representations of the cat hindlimb. a Definitions of joint angles and joint moment directions. Letters a, k and h and the corresponding arcs denote ankle, knee and hip joint angles; symbols Ma, Mk and Mh and the corresponding arrows indicate (more ...)
Marker coordinates in the sagittal plane were low-pass filtered (4th-order, zero-lag Butterworth filter, with a cutoff frequency of 5–8 Hz). Knee marker position was recalculated from hip and ankle coordinates and length of thigh and shank segments to reduce influence of skin movement around the knee. Linear and angular segment displacements, joint angles as well as their first (velocity) and second (acceleration) time derivatives were computed using the method of finite differences; for details of kinematic analysis and definitions of body segments and joint angles, see Prilutsky et al. 
, Gregor et al. 
and Maas et al. 
Mass and moment of inertia with respect to the frontal axis through the center of mass of each hindlimb segment were obtained from the cat mass and the measured segment length (table ) by means of the regression equations [Hoy and Zernicke, 1985
]. The resultant moments of force at hindlimb joints in the sagittal plane were computed using a standard inverse dynamics analysis described previously [Manter, 1938
; Fowler et al., 1993
; McFadyen et al., 1999
; Prilutsky et al., 2005
; Gregor et al., 2006
]. Briefly, each hindlimb segment was considered in isolation with external forces and moments applied at the distal and proximal joints. The Newton-Euler dynamic equations of motion written for the most distal segment (digits), known segment mass-inertia parameters and the measured ground reaction forces (the normal and tangential components to walking surface), as well as linear and angular segment accelerations enabled calculations of the joint forces and joint moment at the proximal (MTP) joint. Analogous calculations were conducted to compute the forces and moment applied at the proximal joint (ankle) of the next tarsal segment; in these calculations, the role of external moment and forces at the distal joint played the MTP joint moment and force components multiplied by −1. The calculations were repeated for the next two segments (shank and thigh), and joint moments of force at the knee and hip were calculated. According to selected sign convention, positive joint moments and velocities corresponded to joint extension (fig. ).
Joint power was computed as the product of the joint moment and joint angular velocity. Positive values of joint power indicate generation of mechanical energy at the joint, negative – energy absorption.
Measured ground reaction forces (tangential and normal components) and computed joint moments and power during each walking cycle were time normalized separately for the stance and swing phases. These kinetic variables were averaged for each 0.5% of stance and 1% of swing across walking cycles of each cat and then across all cats for each walking condition. Kinetic data were checked for outliers – a variable in a particular cycle was removed from the data set if its values were outside of ±2 standard deviation (SD) of the mean. Such outliers were likely resulted from unsteady gait or measurement artifacts.
Values of the ground reaction force, joint moments and power were normalized by the animal mass, and maximum and minimum values of these variables were determined for statistical analysis.
EMG analysis was described previously [Gregor et al., 2006
; Maas et al., 2010
] and is only briefly outlined here. EMG band-pass filtered signals (30–1,000 Hz, 3 dB) were full-wave rectified (fig. ). EMG burst onset and offset times were determined using a criterion of 2 SD; when the EMG magnitude exceeded 2 SD of the mean EMG during muscle silent periods (most of the swing phase) for at least 50 ms, the muscle was considered active. The onset and offset times enabled identification of EMG bursts. The mean magnitude and duration of the bursts were computed and the mean magnitude normalized to the maximum of the mean EMG magnitudes found for each cat across all trials and walking conditions.
Fig. 2 Examples of rectified EMG activity of ankle extensor muscles before and 1 week after denervation of SO and LG muscles during downslope (−50%), level (0%) and upslope (50%) walking of cat No. 5. Only 3 ankle extensors were recorded in cat No. 5: (more ...)
Mean and SD of minimum and maximum values of ground reaction forces, joint moments and powers during the stance phase as well as the EMG burst duration and mean magnitude were computed for all walking conditions before and after muscle denervation. Pre- and postdenervation values of the above characteristics were tested for statistically significant differences within each cat and across all cats using the Student t test for independent variables. Significance level was set at 0.05. In the statistical analysis, stance duration (indicating walking speed) was not used as a covariate because the stance duration increased after denervation only by about 10% in a small number of cases (6 out of 18; table ). Also, the observed increase in EMG activity of the intact ankle extensors and in the ankle joint moment and power magnitude were opposite to changes that are expected from longer stance times (slower walking speed; see Results and Discussion).