169 Axonal Loss in Narcolepsy Type 1 Cross classified multilevel models were implemented for each outcome measure and each ROI for the axon counts and axonal orientation ratios in the reticular formation, pyramidal tract, corpus callosum, anterior cingulate gyrus and vermis. Additional cross classified multilevel models were implemented for each pathological variable of interest (SMI312 percentage of area stained and Bielschowsky, PLP and LFB colour intensity) to combine all ROIs into one analysis. In each measure the pathological variable of interest served as the dependent factor, group as the fixed effect, and ROI location as a random effect. The cross classified multilevel models allowed us to compare groups while accounting for multiple measurements per ROI within donors (i.e., nested data), and for the colour intensity analyses it additionally accounted for intrinsic colour differences of ROIs between donors. In case of significant differences between groups, we correlated the respective pathological outcome measure with remaining hypocretin cell counts in donors with narcolepsy type 1 using Pearson correlation when data were normally distributed and Spearman’s rank correlation when the normality assumption was not met. Subsequent post-hoc comparisons were performed between groups per pathological variable of interest for the individual ROIs. Student’s t-tests were used when data were normally distributed with equal variances, Welch’s t-tests when data were normally distributed with unequal population variances, and Wilcoxon-MannWhitney U tests when the normality assumption was violated. Significant group differences in pathological markers were reported as percentage changes using the mean group values. We also report adjusted q-values (besides the uncorrected p-values) when performing analyses on individual ROIs using the Benjamini–Hochberg procedure to decrease the false discovery rate to 0.05. Correction for multiple comparisons was performed per pathological variable of interest. Hedge’s g statistic was implemented as a measure of effect size as it is most suitable for small sample sizes. Results The four donors with narcolepsy type 1 were comparable to the control groups on the matched parameters as no significant differences were found in sex, age, postmortem delay, cerebrospinal fluid pH, brain weight, Braak AD stage, amyloid stage and Braak Lewy body stage (Table 2). The hypocretin levels in the cerebrospinal fluid of the donors with narcolepsy were undetectably low except for the case with chronic opiate use (200 pg/mL). 6
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