Myoclonus

All these patients were treated with TH, targeting a body temperature between 32 and 34°C for 24 h, according to a standardized institutional protocol, including the use of a cold fluid bolus (20 to 30 mL/kg saline or a balanced crystalloid solution over 30 minutes) and of a water-circulating blanket device (Medi-Therm II, Gaymar, Orchard Park, NY, USA). Midazolam (continuous infusion 0.03 to 0.1 mg/kg/h) and morphine (0.1 to 0.3 mg/kg/h) were administered during the hypothermic phase, while neuromuscular blocking agents (NMBAs, cisatracurium as a bolus of 0.15 mg/kg) were given in the induction phase and, if needed, as a continuous infusion thereafter (1 to 3 mcg/kg/min). Re-warming was performed passively at a maximum rate of 0.5°C/h; sedation, analgesia and NMBAs were discontinued when the body temperature exceeded 37°C.
Patients were kept in a 30° semi-recumbent position; ventilation was set to maintain arterial partial pressure of carbon dioxide (PaCO₂) at 35–45 mmHg and peripheral capillary oxygen saturation (SpO2) >94%. Invasive hemodynamic monitoring (PiCCO, Pulsion, Munich, Germany) was placed whenever needed and transesophageal echocardiography was performed within the first 8 to 12 h after ICU admission in all patients. Mean arterial pressure (MAP) was maintained at >65 to 70 mmHg using fluids, dobutamine and/or norepinephrine, whenever needed. Higher levels of MAP were targeted in patients with a history of hypertension and in patients with low (<60%) cerebral oximetry values (Foresight, CasMed, Branford, CT, USA). Intra-aortic balloon counterpulsation (IABP) or extracorporeal membrane oxygenation (ECMO) was initiated in cases of severe cardiogenic shock. A local insulin protocol was applied to keep blood glucose levels between 110 and 150 mg/dL in all patients.
After re-warming the patient and stopping sedative agents, repeated neurologic examination and standard or continuous electroencephalogram (EEG) were performed. Withdrawal of life-support was an interdisciplinary decision based on bilateral absence of the N20 response to somatosensory evoked potentials (SSEPs), persisting deep coma, or presence of status myoclonus or refractory status epilepticus.

Prior to the start of the trial, items were selected through expert consensus as part of a broad clinical description of both MSA and PSP. The dimensions included (i) functional disability (activities of daily living), (ii) mental function (cognition, mood & behavior); (iii) extra-pyramidal motor disability (rigidity, bradykinesia), (iv) tremor, (v) oculomotor function, (vi) cerebellar signs, (vii) pyramidal signs, (viii) dysautonomia, (ix) bulbar/pseudobulbar symptoms, (x) myoclonus, and (xi) dystonia. Items were selected from the following scales available at that time, the UPDRS (all items from Mental, ADL and Motor examination sections) [13] , the PSP-RS (six items from the mental section) [17] , three items from the International Cooperative Ataxia Rating Scale (ICARS) [21] (link), the global ataxia score of the Expanded Disability Status Scale (EDSS ) [22] (link), and four items evaluating orthostatic signs and three for urinary signs from the Autonomic Symptom Profile [23] (link) adapted to interview record instead of self-rating. Additional items were included to assess oculomotor signs, dystonia, myoclonus, pyramidal signs, sitting down and strength of cough.
A preliminary version of 109 items was evaluated in a pilot study to check each item and category wording. Redundant or inappropriate items were eliminated to obtain the first version comprising 85 items to be tested. Severity levels of items ranged from 0 (“normal”) to a maximum of 6 (very severe), with a majority of items (65) scored on a 5-point scale (0–4) (Supporting information S1). Four sections were interview based with patient and/or caregiver (Mental, Activities of Daily Living-ADL, orthostatic and urinary signs), eleven were assessed through examination. Time to complete the scale was 30–45 minutes depending on clinical state of patient. Throughout the study, the scale was completed in all centres using an English version.

Motor activity and behavioral state were assessed visually using video and corroborated with EMG. We used custom-written MATLAB scripts to detect frame-by-frame changes in pixel intensity within user-defined regions of interest (Dooley et al., 2021 (link)). We selected two regions, one encompassing the right forelimb and the other encompassing the entire body, to allow detection of movement periods. Movements were represented as changes in pixel intensity across time. We then imported movement, neurophysiological, and EMG data into Spike2 (Cambridge Electronic Design, Cambridge, UK).
Recording data were separated into periods of AS and wake. AS was defined by the presence of myoclonic twitches occurring against a background of nuchal muscle atonia. Twitches appear as brief, jerky limb movements and as sharp spikes in the EMG record. Wake was defined by the presence of increased nuchal muscle tone relative to AS, most commonly initiated by and containing large-amplitude movements of multiple limbs (Del Rio-Bermudez et al., 2020 (link); Glanz et al., 2021 (link)). Periods of behavioral quiescence that were not defined as AS or wake were examined but not included in the present analyses. Behavioral state was always scored blind to neural activity.
To quantify differences in firing rate across AS and wake, for each unit we calculated the mean firing rate over the duration of each state; we then calculated mean firing rate across units within each brain area for each pup. At P8, we also assessed whether the occurrence of spindle bursts was state dependent. For each area in each animal, we determined the mean rate of spindle bursts during AS and wake, and then calculated the mean rates across pups. Spectrograms of oscillatory activity were generated using the sonogram function in Spike2. (This analysis was not performed at P12 because spindle bursts are not clearly discernable at this age.)
To delineate differences between state- and movement-dependent changes in firing rate in M1, M2, and mPFC, we calculated the mean firing rate in each area during periods of movement. Movement and non-movement periods were extracted using custom MATLAB scripts from whole-body movement data (derived from video as described above). The onset of a movement period occurred when movement data exceeded a threshold value of 3× greater than baseline for at least 250 ms; the offset of a movement period occurred when movement data decreased below threshold. Movement and non-movement periods were categorized as to whether they occurred during AS or wake. Mean firing rates were calculated for each unit during the following conditions: AS with movement, AS with no movement, wake with movement, and wake with no movement. We determined the mean firing rate across units within an area and then the mean rate across pups.