Nerve crush injury model
Male Sprague–Dawley rats weighing 250–300 g (bought from BioLASCO Taiwan Co.) were used and were anesthetized using isoflurane at 4% in the induction period and 1% in maintenance period. The gluteal splitting method was used to expose left sciatic nerve under the microscope, and the nerve was crushed using a vessel clamp 10 mm from the obturator . The animals were randomly allocated into one of six groups as follows: Group I: sham (n = 6); Group II: nerve crush injury as control (n = 6); Group III (HFI): high-frequency (100 Hz) percutaneous electrical stimulation administrated immediately (n = 12); Group IV (HFL): high-frequency (100 Hz) percutaneous electrical stimulation administrated 7 days after nerve crush (n = 12). Group V (LFI): low-frequency (5 Hz) percutaneous electrical stimulation administrated immediately (n = 6); Group VI (LFL): low-frequency (5 Hz) percutaneous electrical stimulation administrated 7 days after nerve crush (n = 6). The electrical stimulation paradigm featured a treatment consisting of stimulation for 30 min per day for 7 consecutive days using 400 ms of biphasic pulses at 200 μs per phase and 100 or 5 Hz frequency and with 6 s of rest (ElePulsHV-F125, Omron, Japan) . Food and water were provided ad libitum before and after the operation. The animal housing environment was kept under the appropriate condition with 2 animals in a single cage, in a temperature-controlled environment at 20 °C and with alternating light and dark cycles with 12-h intervals. After the experiment, all animals were euthanized using CO2. The care and operation of all animals followed the guidelines recommended by Taichung Veterans General Hospital Institutional Animal Care and Use Committee (IACUC) (Permission No.La-1061455).
Analysis of motor function recovery
A technician blindly assessed the SFI in the various groups of animals before operation and weekly after the surgery according to our previous report [20, 21]. Several essential parameters were taken from the footprint and all measurements were taken in the experimental and control groups. An SFI of 0 indicated normal function and − 100 represented total loss of motor function.
Mechanical allodynia was tested blindly by using von Frey hairs (Touch-Test Sensory Evaluator, North Coast Medical, Inc), as previously described by our group [20, 21]. Von Frey hairs were applied in a series of grams to touch the hind paw bilaterally five times for 5-s intervals when the hind paw was placed appropriately on the platform. The withdrawal threshold was considered to be the force (gram) of the hair that caused hind-paw withdrawal in at least four out of the five applications. Thermal hyperalgesia was evaluated via a hot-plate test (Technical& Scientific Equipment GmbH, TSE systems) according to the pervious procedure . The withdrawal latency was recorded as the interval of the time from which the rat touched the 52 °C hotplate to the time of withdrawal of the paw. A maximal cut-off of twenty-seconds was used to prevent paw tissue injury.
Catwalk gait analysis
The CatWalk XT gait analysis has been previously described by our group . Quantitative analysis of the data included the following parameters: step sequence distribution, regularity index (RI), print area, duration of swing and stance phases, and intensity. The data were presented as the ratio of the measurement for the left side divided by that for the right side.
Evoked potential of the somatosensory cortex
Evoked potential measurements have been previously published by our group [20, 21]. In brief, one active electrode was threaded into the dural surface of the somatosensory area (3 mm lateral and 2 mm posterior to the bregma). Another electrode was placed as a reference over the maxillary area at approximately 20 mm from the active electrode. A stimulation intensity of 20 mA with 20–2000 Hz filtration was applied over the sciatic nerve 1 cm proximal to the injury area. The data for conduction latency and evoked potential were presented as the ratio of the measurement for the right side to the left side, to minimize the effects of anesthesia.
Isolation and cultured dorsal root ganglion cells (DRGs) subjected to electrical stimulation
Dorsal root ganglia cells were dissected from embryonic Sprague–Dawley rat at the embryonic days 14–15 according to previous report [20, 22, 23]. The DRGs were incubated with 0.25% trypsin at 37 °C for 15 min and were dissociated, washed and re-suspended with Neurobasal medium containing 2% B27 (sigma, Inc.), 0.3% l-glutamine and 100 ng/ml nerve growth factor. Finally, these cells were cultured in the dish at a density of 1 × 104 cells per ml of medium (16-well array station, ECIS model 800). Next, the cells were maintained and cultured in an incubator at 37 °C and 5% CO2. The cells were recognized by neuronal markers (βIII tubulin) before the experimental process and were subjected to electrical stimulation for duration of 30 min at frequencies of 5 and 100 Hz with 50 mA.
Western blot analysis
The distal end of the nerve, muscles, dorsal root ganglion cells, and brain (hippocampus/cortex) were harvested 4 weeks after the various treatment and proteins were extracted. The cell lysate of dorsal root ganglion cells after electrical stimulation were collected to determine the expression of synaptophysin, TNF-α, and NGF. Proteins (50 μg) were resolved by SDS–polyacrylamide gel electrophoresis and were transferred onto a blotting membrane . After blocking with non-fat milk, the membranes were incubated with antibodies against S-100 (Neomarkers, 1:500 dilution), NF (Cellsignal, 1:1000 dilution), synaptophysin (Abcam, 1:500 dilution), TNF-α (Abcam, 1:1000 dilution), and NGF-R (1:1000, Abbiotec) overnight at 4 °C. The intensity of the protein bands was determined by a computer image analysis system (IS1000, Alpha Innotech Corporation, CA, USA).
Dorsal root ganglion cell culture after electrical stimulation and serial 8-mm-thick section of nerve, muscle, dorsal root ganglion cells, and the brain were cut using a cryostat, and mounted on superfrost/plus slides (Menzel-Glaser, Braunschweig, Germany) were subjected to immunohistochemistry using antibodies against NGF-R (1:1000, Abbiotec), S-100(1:200, Serotec), neurofilament(1:200, Millipore), anti-synaptophysin (Abcam, 1:200 dilution), and anti-TNF-α (Abcam, 1:300 dilution) to detect the inflammatory response associated with nerve regeneration in sciatic nerve, dorsal root ganglion cells, and the brain. The immunoreactive signals were observed using AF 488 donkey anti–mouse IgG and AF594 donkey anti-rabbit (Invitrogen; 1:200 dilutions) and were then viewed using an Olympus BX40 Research Microscope.
Data are expressed as the mean ± SE (standard error). The SFI and CatWalk data were analyzed via repeated-measure ANOVA followed by Bonferroni’s multiple comparison method. The statistical significance of the differences among the groups was determined via one–way analysis of variance (ANOVA) followed by Dunnett’s test. A p value < 0.05 was considered significant.