Supplemental Methods 1
METHODS
Automated pupillometry
Automated pupillometry was performed by two operators (SH and LP) using an automated pupillometer (NeuroLightAlgiscan, ID-MED, Marseille, France). A burst of light of fixed intensity (320 lux) and duration (one second) was emitted by the device to each eye and the device then used a period of 3 seconds to determine the minimal pupil size. The measurement was completed in less than one minute for each eye and a minimum duration of one minute was allowed between appraisals of the two pupils to obtain full recovery of baseline pupil diameter after light stimulation. A total of 4 seconds is needed to evaluate baseline pupil size (mm), pupillary constriction rate (i.e., the difference between baseline and post-stimulation pupil size, expressed as % of constriction from the baseline value), CV (mm/sec) and latency (msec). Mean values from both eyes were used for comparison with EEG findings. The initiation of light stimulation and automated pupillometry was noted on the EEG recording.
Anisocoria was defined as a difference of at least 1.0 mm between the two eyes.13
In comatose patients, as part of routine pain assessment in unconscious patients in our unit, an electrical stimulation with variable intensity (increasing from 10 mA to a maximum of 60 mA) was applied on the left and right forearm of each patient, using two electrodes linked to the automated pupillometer. The start of such stimulation was also noted on the EEG recording. Both pupils were assessed during the stimulation of each arm for a total of 4 automated pupillometry recordings; the median values for these analyses were reported for comparison with EEG findings. Baseline pupil size (mm), pupillary dilation to pain (i.e., the difference between post-stimulation and baseline pupil size, expressed as a %) and the
pupillary pain index (PPI) were obtained. This index measures pupil dilation in response to a continuously increasing 1 second electric stimulus varying from 10 to 60 mA in steps of 10 mA. When an increase of 13% in pupil size is detected, the stimulus stops, and PPI is calculated by the device; it assigns a score from 1 (pupillary dilation < 5% to the maximal stimulation intensity) to 10 (pupillary dilation > 13% with 10 mA stimulus).14
EEG recording
Twenty-one EEG electrodes were placed on the scalp, according to the International 10–20 system and EEGs were recorded using a clinical grade system (BrainRT, OSG Inc., Rumst, Belgium). A 20-min epoch around the onset of the automated pupillometry assessment was assessed by three independent neurophysiologists and the EEG background classified into one of four categories of encephalopathy using a modified Synek scale (see below - Appendix 1).
The EEG category was retained when it was attributed by at least two of the three
neurophysiologists, otherwise, the EEG was excluded from the analysis. The presence of EEG changes to external stimulation (i.e. name call for awake and painful stimuli for unconscious patients), defined as “EEG reactivity”, was also assessed, categorizing the EEG background pattern as “reactive”, “non-reactive”, or “unclear”. The presence or absence of EEG reactivity was noted when at least two of the three neurophysiologists gave the same interpretation;
otherwise (or when two raters categorized the trace as “unclear”), EEG reactivity was considered as “unclear” and the recording was excluded from the analysis. We assessed the ability of the automated pupillometry procedure to elicit EEG changes (referred to as
“reactivity to automated pupillometry stimulation”) in a similar fashion.
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