Fig 1.
Types and occurrence frequencies of spastic behaviors during the swimming test.
(A) Configuration of the EMG recording device during the swimming test. (B) Sequential captured images and corresponding recorded muscle activity of the left TA (blue) and the left MG (orange) muscle during the swimming test showing each type of spasticity (the clonus phase and spastic phase). The black box represents a magnified image of the recorded EMG. The red bar on the recorded EMG of the spastic phase indicates co-contraction of the TA and MG muscles. The green bar on the bottom image corresponds to the normal reciprocal stroke phase. White arrows indicate the location of the hind paw of the rat during the clonus phase. Note that all EMG figures were normalized to the peak amplitudes of TA muscle activity. (C) Mean amplitude of muscle activity during spastic behaviors (SCI spasticity; black bar) compared with the mean amplitude of muscle activity during normal reciprocal strokes (SCI normal stroke; white bar) of SCI rats and uninjured rats (Uninjured; hatched bar) at 4 weeks after SCI. (D) The number of the SCI rats with each occurrence frequency of spastic behaviors during swimming test at 4weeks after the injury. (E) Percentage of the spasticity-strong rats from 3 to 6 weeks after SCI. Injured rats were classified as “spasticity-strong” if they showed the occurrence frequency over 40% during the swimming test. (F) Average percentage of the spared area of the epicenter of the injury measured by the GFAP (+) area after SCI in the spasticity-strong group and -weak group. **: p < 0.01 Abbreviations: WT, water temperature; TA, tibialis anterior; MG, medial gastrocnemius; n.s, not significant.
Fig 2.
The occurrence frequencies of spastic behaviors and corresponding amplitudes of muscle activity during the test period.
(A) The occurrence frequency and percentage of each type of spasticity (clonus, black bars; spastic, grey bars; total, white bars) observed in the spasticity-strong group during the swimming tests from 4 to 6 weeks after SCI. (B) The mean amplitude of muscle activity of spastic behaviors in the spasticity-strong group during the swimming test from 4 to 6 weeks after SCI recorded from the left TA muscle (blue) and the left MG muscle (orange). Mean amplitudes of muscle activity at 4 weeks were considered to be 100%. Abbreviations: TA, tibialis anterior; MG, medial gastrocnemius; n.s, not significant.
Fig 3.
Rate-dependent depression (RDD) of the H-wave in the SCI groups, reflecting excitability of the spinal reflex.
(A) Representative Hoffmann (H) reflex waveforms of uninjured, spasticity-strong, and -weak groups were shown. Different colors indicate different stimulation frequency: 0.2 Hz (blue), 0.5 Hz (Green), 1 Hz (Black), 2 Hz (orange), and 5 Hz (red). (B) RDD of the M-wave. The mean amplitude at 0.2-Hz stimulation was set to the baseline amplitude as 100%. Uninjured (pink), spasticity-strong group (solid line), -weak group (dashed line). (C) RDD of H-wave in the uninjured (pink) and total SCI group (black; includes spasticity-strong and -weak rats). (D) Comparison of the RDD values of the H-wave between groups. The RDD of the spasticity-strong group (solid black line), -weak group (dashed line), and uninjured rats (pink line). **: p < 0.01, ***: p ≤ 0.001.
Fig 4.
Immunohistochemistry and quantification of 5-HT and its receptor in SCI groups and uninjured rats.
(A) Robust up-regulation of the 5-HT2A receptor at the lumbar motor neurons of the spasticity-strong group compared with the -weak group and uninjured group (scale bars: 50 μm). (B) Quantification of the 5-HT2A receptor-positive (+) area within spinal motor neurons labeled as ChAT positive (+) at both the cervical region and the lumbar region. (C) The 5-HT fibers were almost completely lost after 250-kd contusive SCI, but were more reduced at the spasticity-strong group (scale bars: 50 μm). (D) Quantification of the area of 5-HT fibers at both the cervical and the lumbar ventral horn region. Values were normalized considering the value for uninjured rats as 100%. (E) Plots of the quantification of 5-HT2A receptor expression and 5-HT fibers at the cervical and lumbar region, respectively in uninjured (yellow), spasticity-strong group (purple), and -weak group (pink). **: p < 0.01, ***: p < 0.001 Abbreviations: 5-HT, 5-Hydroxytryptophan; ChAT, Choline acetyltransferase; n.s, not significant.
Fig 5.
EMG results and occurrence frequencies of spastic behaviors during the 20-hour cage observation.
(A) Recorded EMG results for the clonus and spasm in SCI rat during 20-hour cage observations obtained from the left TA muscle (blue) and the left MG muscle (orange). The red bar indicates co-contraction of the TA and MG muscle activities. Note that all EMG figures were normalized to the peak amplitudes of TA muscle activity. (B) Mean amplitude of muscle activity during spastic behaviors compared with the mean amplitude of muscle activity during 10 gait cycles on BWST 4 weeks after SCI. (C) The occurrence of each type of spastic behaviors during 20-hour cage observations in the spasticity-strong group (solid bars) and -weak group (open bars) at 4 weeks after SCI. (D) The occurrence of spastic behaviors in the spasticity-strong group (solid line) and -weak group (dashed line) shown by each recorded hour. The shaded area shows the default dark time (18:00 to 6:00) of the housing room. The yellow bars indicate the peak time points for the counted spastic behaviors. *: p < 0.05, **: p < 0.01 Abbreviations: TA, tibialis anterior; MG, medial gastrocnemius; BWST, body weight supported Treadmill.