Cerebral Microdialysis and Analysis of Brain Extracellular MMP-9

Additional Methods

Multimodal neuromonitoring including electrocorticography (EcoG) was approved by the institutional review board of the Innsbruck Medial University (Protocol Nr.: UN3898 285/4.8 and UN4089 292/4.4). Written informed consent was obtained according to federal regulations. Hematoma volume was calculated using the ABC/2 method. PHE volume was calculated by total defect volume minus hematoma volume. Asix platinum contacts subdural EcoG-strip (AD-TECH, Medical Instrument Corporation, WI, USA) was connected to six sequential bipolar channels (A[1-2], B[2-3],C[3-4],D[4-5], E[5-6] and F[6-1]) modified by a bio-signal amplifier (g.BSamp, g.tec Medical Engineering Gmbh, Austria) and recorded using PowerLab (ADInstruments, New South Wales, Australia) and LabChart Software (Version 7.2; ADInstruments; sampling rate: 200 Hz). An electrocardiogram patch at the patients shoulder and bed served as grounding, a surface reference electrode (gold-plated silver) was glued with adhesive paste (SLE Colloidion Adhesive, South Croydon, UK) on the mastoid. Cortical spreading depolarizations (CSDs) were considered as sequential onset of propagating, polyphasic slow potential changes in adjacent channels, accompanied by an EcoG-amplitude loss (EcoG depression) of at least 50%.1,2 The duration of the EcoG-depression was defined as the time (minutes) between depression onset and start of EcoG recovery in the integral of power calculations of the band-pass filtered signal (0.5-45Hz; 60 seconds time constant decay)3. SPC propagation velocity was calculated from the monopolarEcoG channels as dividing 10 (separation in mm between adjacent contacts on the EcoG strip) by the time interval between the onsets of the SPC in adjacent channels.

Cerebral microdialysis and analysis of brain extracellular MMP-9:

Cerebral microdialysis (CMD) was performed using a CMA-71 catheter (100kD cut-off; membrane length 10mm; μDialysis, Stockholm, Sweden) and isotonic perfusion fluid (Perfusion Fluid CNS; μDialysis, Stockholm, Sweden) at a perfusion rate of 0.3 μl /min. The microdialysate was collected hourly and analyzed at the bedside using ISCUS Flex (μDialysis, Stockholm, Sweden). CMD-MMP-9 levels were measured using an enzyme linked immunosorbent assay (ELISA; SearchLight®, AushonBioSystems) according to the manufacturer’s instructions. MMP-9 detection limit was 98 pg/ml.

References:

1.Strong AJ, Fabricius M, Boutelle MG, et al. Spreading and synchronous depressions of cortical activity in acutely injured human brain. Stroke; a journal of cerebral circulation 2002;33:2738-43.

2.Fabricius M, Fuhr S, Bhatia R, et al. Cortical spreading depression and peri-infarct depolarization in acutely injured human cerebral cortex. Brain : a journal of neurology 2006;129:778-90.

3.Dreier JP, Woitzik J, Fabricius M, et al. Delayed ischaemic neurological deficits after subarachnoid haemorrhage are associated with clusters of spreading depolarizations. Brain : a journal of neurology 2006;129:3224-37.