Methods
Electromyography recordings
Surface EMG activity was recorded from the lateral head of the right gastrocnemius muscle,
located by manual muscle testing, using bipolar Ag-AgCl electrodes. The electrodes were
placed 2 cm apart over over the belly of the muscle, with the third reference electrode
(ground electrode) placed over the bony prominence at the fibula head. The area of
electrode placement was prepared by shaving (if required) and cleaned with 70% isopropyl
alcohol. EMG signals were amplified (2 kHz-10 kHz), bandpass filtered (13 kHz-1000 kHz),
digitized (2 kHz), recorded for 500 ms and analysed off-line (Nihon-Koden, Japan).
M-wave and H-reflex recordings
M-wave and H-reflex testing followed the methodological considerations discussed by
Palmeri et al (2004). A 2 cm bipolar stimulating electrode (Nihon Koden, Japan) with the
cathode and anode was placed over the posterior tibial nerve in the popliteal fossa whilst
the participant remained relaxed. H-reflex/M-wave recruitment curves were completed in
order to identify stimulation intensities to illicit maximal amplitude H-reflex and M-wave
(Palmieri et al. 2004). Stimuli of 1 ms pulse width were delivered at a frequency of 0.1 Hz,
starting at 0.2 mA. Once maximal amplitude H-reflex was identified, stimulation intensity
increased until M-wave saturation. After a brief pause, 10 stimuli were delivered at the
identified amplitude for maximal H-reflex, and a further 10 stimuli delivered at 20% above
the stimulation intensity identified for maximal M-wave amplitude to ensure M-wave
maximum (M-max) was achieved.
Transcranial magnetic stimulation
Maximal voluntary contraction (MVC) EMG was recorded to determine background muscle
activity during the TMS protocol. The participant was instructed to dorsiflex the ball of the
foot against the investigator as forcefully as possible for 3 s. The standard criteria for
measurement of MVCs were fulfilled and included a period of familiarisation and verbal
encouragement provided by the investigators and the rejection of the trial in the case the
participant felt it was not a maximal effort (Gandevia 2001).
TMS testing followed the established protocols of Byrnes et al. (1999), Pearce et al. (2000)
and Wilson et al. (1993). MEPs were evoked by TMS of the contralateral motor cortical area
projecting to the right gastrocnemium using a Magstim 2002 stimulator (Magstim Co, UK),
with a 90 mm circular coil placed tangential to the skull in an antero-posterior direction. For
reliability of coil placement participants wore a snugly fitting cap, positioned with reference
to the nasion-inion and interaural lines (Wilson et al. 1993; Byrnes et al. 1999; Pearce et al.
2000; Hortobágyi et al. 2008). The cap was marked with sites at 1 cm spacing in a latitudelongitude matrix to ensure reliable coil position throughout the testing protocol and for
repeated testing sessions over the period of the study. The cap was checked constantly to
ensure that no changes in cap position occurred. Sites near the estimated centre of the
right gastrocnemius area (2-4 cm anterior to the vertex) were explored to determine the
site at which the largest MEP amplitude was observed. This site was defined as the
“optimal” site. At the optimal site, active motor threshold (AMT) was determined by
delivering one set of four stimuli at intensities (5% of stimulator output steps) from a level
below the participant’s AMT until the MEP amplitude became saturated (i.e. until the
amplitude did not increase with increased stimulation). AMT was defined as the intensity at
which an MEP could be obtained in 50% of MEP sweeps during 10% of MVC EMG (Wilson et
al, 1993). For main studies, 10 stimuli were delivered during a controlled, low level voluntary
contraction of the gastrocnemius muscle at 10% (± 3%) of MVC rmsEMG (Pearce et al. 2000;
Wilson et al. 1993). Each stimulus was delivered in random intervals every 5 s to avoid
stimulus anticipation and 30 s rest was provided between each set of 5 stimuli to reduce the
possibility of muscular fatigue.
Data and Statistical analyses
All waveforms collected (H-reflex, M-wave, and MEPs; n=10 each; see Figures 1 and 2 for
examples) were displayed and averaged online for visual inspection as well as stored off-line
for further analysis. H-reflex, M-wave and MEP waveforms were quantified by measuring
the peak-to-trough amplitude on the EMG (Pearce et al. 2000; Palmieri et al. 2004). MEP
latency and SP duration were quantified by measuring the stimulus artefact to onset of MEP
and from the onset of MEP to the return of EMG respectively (Wilson et al. 1993; Pearce
and Kidgell 2009). To allow for intra-participant group comparisons, data for H-reflex and
MEP amplitudes were normalised by expressing the H-reflex and MEP as a percentage of the
M-max amplitude.
All data were first screened to ensure they were normally distributed. No variable’s z-score
of skew or kurtosis was excessive. Further, Shapiro-Wilk tests suggested all variables
Latency, MEP/M-max percentage, and SP duration for pre and post manipulation and
control conditions were normally distributed. H-reflex/M-max percentage for pre control
and post manipulation were also normally distributed, however H-reflex/M-max for post
control (SW=0.84, df = 14, p = 0.02) and pre manipulation (SW=0.80, df = 14, p = 0.06) were
apparently non-normal. Following visual examination of frequency histograms and
detrended Q-Q plots, for these two conditions which did not show excessive skewness, it
was not considered sufficient to warrant a more conservative analytic strategy.
Consequently, it was decided to treat all data as essentially normal in distribution.
To test the hypothesis that HVLA manipulation alters corticospinal measures, single-group
two-condition paired-samples t-tests for each parameter were employed. Data is presented
as means ( SD) effect size conventions were used for trivial (<0.2), small (0.21-0.5), medium
(0.51-0.79) and large (>0.8) comparative effects were t-tests showed significance (Cohen
1988). Level of significants for statistical tests was set at p<0.05
Results
Descriptive data are presented in Table 1. No differences were observed in MEP latency pre
versus post time points for both the manipulation (p=0.41) and control (p=0.09) conditions.
Similarly, no difference in M-wave latency was observed pre versus post time points for
both manipulation (p=0.27) and control (p=0.52) conditions. Similarly, SP duration did not
change between either condition (manipulation p=0.62; control p=0.73). MEP/M-wave
percentage did not change in the control condition (p=0.39) however, MEP/M-wave
percentage did significantly reduce by 2.8% (p=0.02; ES=0.25). Similarly, H-reflex/M-wave
percentage showed a significant reduction of 10.5% (p=0.004; ES=0.84) following HVLA
manipulation. No change in H-reflex/M-wave percentage was observed in the control
condition (p=0.66).
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