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The study was conducted between May 2011 and February 2013. Nine male patients with genetically defined DMD (mean age 12.1 ± 1.5, age range 10–14 years) and 10 sex- and age-matched healthy control subjects (mean age 11.5 ± 1.3, age range 9–13 years) were included. According to the natural history of the disease all boys with DMD manifested at the age of two to three years. One of the patients did not complete the experimental session, therefore data of 8 patients were analysed. Patients were recruited from the outpatient neuromuscular clinic of the Neuropediatric Department of the University of Duisburg-Essen and control subjects by contacting pupils and their parents of a primary or middle school. The study was approved by the local ethics committee and written informed consent was obtained from the subjects and their legal representatives. To determine muscle weakness and possible cerebellar signs or ataxia symptoms a neurological examination was conducted by an experienced neuropediatrician, U.S. or M.B. None of the control subjects had a history of neurological diseases, they were free from any medication and there were no neurological signs in controls on examination. In DMD patients muscle strength was examined according to the Medical Research Council Scale (MRC) [39]. Because muscle weakness was present in all patients and influenced performance on limb coordination, balance and gait testing, use of ataxia scales was not meaningful to determine the severity of cerebellar signs. Seven patients were not able to walk without support. None of the patients had to use a respirator or was provided with a percutaneous endoscopic gastrostomy (PEG). Five of the patients were currently treated with corticosteroids (Deflazacort or Prednisone 0.45–0.75mg/kg/d). The dosages were lower than current international standards due to individual clinical conditions like development of obesity or loss of ambulatory ability. No other medication was applied. Further clinical and genetic characteristics of the patients are summarized in Table 1.
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0126528.t001 Clinical characteristics and X-chromosomal genetic findings in DMD patients. Examination of limb coordination, balance and gait and the use of ataxia scales to determine the severity of cerebellar signs was not meaningful in case of muscle weakness. Muscle strength was examined according to the graduation of the Medical Research Council (MRC). Abbr. : + = mild, ++ = moderate; A. = Ankle; Dis. = Disorder; n.a. = not applicable in case of muscle weakness. See Methodsfor further details.
At the beginning of the experiment hearing thresholds were determined in each subject using 1 KHz, the frequency of the CS. Thresholds of both ears (dB SPL) were within normal age limits in all participants, there was no significant difference between patients and controls. None of the participants suffered from eye diseases. As reported previously in detail a standard delay eyeblink conditioning paradigm was used [40, 41]. All subjects were investigated by the same investigator (M.B.) in a quiet room, seated assured and comfortably on a chair and watching a silent movie. At the beginning ten CS alone and ten US alone trials were presented in an unpaired and random order, this was followed by 100 paired CS-US trials and then 10 CS alone extinction trials. The US consisted of an air puff (duration 100ms, intensity 400 KPa at source, 110 KPa at nozzle), directed near the outer canthus of the right eye at a distance of about 10 mm. As the CS a tone (1 KHz; 70 dB sound pressure level, SPL; duration 540ms) was presented ipsilaterally and coterminated with the air puff. Surface EMG recordings were taken from orbicularis oculi muscles bilaterally. In paired and extinction trials CRs were semiautomatically identified in the CS-US interval using custom made software [42]. The onset of a CR was defined where EMG activity reached 7.5% of the EMG maximum in each recording with a minimum duration of 20ms and a minimum integral of 1 mV*ms. Trials were visually inspected and implausible identification of CRs was manually corrected. Responses occurring within the 150ms interval after CS onset were considered as reflexive responses to the tone (i.e. alpha responses) and not as CRs [43]. Trials with spontaneous blinks occurring prior to CS onset were excluded from the analysis. As a measure of learning the primary outcome parameter was CR acquisition. The number of CRs was expressed as the percentage of trials containing responses with respect to each block of ten trials (percentage CR incidence) and the total number of trials (total percentage CR incidence). As secondary outcome measures, timing and extinction of CRs were assessed. As outlined above onset and peaktime of CRs in paired trials and URs in unpaired trials were automatically quantified. US onset was set as 0 ms. CR onset and peaktime were expressed as negative values i.e. prior to onset of the US [41]. EMG amplitudes were not analyzed due to methodological limitations in surface EMG recordings. As a measure of extinction the CR incidence within the last block of paired trials (block 10) was compared with the extinction block. All subjects exhibited at least one CR during extinction ensuring a sufficient ability of learning in the paired trials [42]. The frequency of spontaneous blinks was measured in each session within one minute both at the beginning and in the end of the experiment. The rate of alpha-blinks was analysed within the 150 ms interval after CS onset of 100 paired trials.
Analysis of variance with repeated measures (ANOVA) was calculated in paired trials with percentage CR incidence as dependent variable, block (1–10: ten blocks of ten paired trials) as within subject factor and group (controls vs. DMD patients) as between subject factor. Level of significance was set at p < 0.05. For all effects, the degrees of freedom were adjusted, if appropriate, according to Greenhouse-Geisser [44]. In addition, ANOVA was calculated comparing the last block of paired trials and the block of extinction trials. CR and UR timing parameters, spontaneous blink rates and alpha were compared between controls and patients using unpaired t-tests.
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