Patients
Patients undergoing acute neurosurgery for ICH followed by subsequent treatment at the neurocritical care (NCC) unit, University Hospital, Linköping were prospectively recruited to this observational study.
Patients received dual microdialysis catheters with a molecular weight cut-off of 100 kDa, and membrane length 10 mm (71 High Cut-off Brain MD Catheter, M-dialysis AB, Solna, Sweden) at the time of surgical evacuation of ICH. One catheter was placed within 1 cm of the evacuated hematoma, in the perihemorrhagic zone (PHZ), and the other at a distance of at least 1 cm from the evacuated ICH in a non-eloquent area of seemingly normal brain cortex (SNX) on the ipsilateral side (Figure 1a-c). Microdialysis sampling was initiated directly post-surgery and microdialysis vials were changed every two hours. The catheters were perfused with a commercially available 5% Human Albumin in a water solution containing the excipients sodium chloride, N-acetyl-DL-tryptophan and caprylic acid (Albunorm, Octapharma, Stockholm, Sweden), at a rate of 0.3 µL/min. The use of albumin in the perfusate, to counteract ultrafiltration and subsequent fluid loss to the tissue, has been clinical routine in our department since 2013. Microdialysate was collected at a time corresponding to 72 hours after ICH onset. The results of 2-DE of this microdialysate has been previously published(13).
Patients received an external ventricular drain (EVD) for ICP monitoring from which CSF was collected at a time point corresponding to removal of the microdialysis membranes, towards the end of the patient’s NCC unit treatment period. The 2 first mL of aspired CSF were discarded and the following 2 mL were immediately sent to the laboratory where the sample was centrifuged for 10 min at 1800 x g at 4 °C, and the supernatant was subsequently stored at −86 °C awaiting analysis.
Microdialysis catheter membranes were removed at the end of the patients’ NCC treatment period, cut directly into a polypropylene tube and stored initially at −20 °C, then at −86 °C, awaiting analysis. Three membranes from the perihemorrhagic zone (PHZ1, PHZ2, PHZ3) and two membranes from seemingly normal brain tissue (SNX1, SNX2) were used. The third SNX membrane was accidentally discarded in the NCC unit and thus not available for analysis.
Sample preparation
Preparation of dialysate samples
The proteomic analysis of the microdialysate of the three patients has been previously published(13) along with a detailed description of the sample preparation. Briefly, 40 µL of each sample was applied onto an Albumin & IgG Depletion column (GE Healthcare, Uppsala, Sweden), before being desalted and lyophilized. Protein concentration was determined(14) before applying the sample to 2-DE.
Preparation of CSF samples
The CSF samples were applied to albumin and IgG depletion columns (Albumin & IgG Depletion SpinTrap, GE Healthcare) in order to improve detection of low abundant proteins. Samples were then desalted with Amicon Ultra Centrifugal Filters, 0.5 mL Ultracel 3k (Merck Millipore Ltd, Cork, Ireland), and dried in SAVANT SPD 111V SpeedVac Concentrator. The dried samples were resolved in 150 µL urea buffer solution (8 M Urea, 4% (w/v) CHAPS, 65 mM DTT, 2% (v/v) pharmalyte 3-10, trace of bromophenol blue) and incubated for 1 h at room temperature before protein concentration was determined by 2-D Quant Kit (GE Healthcare).
Preparation of microdialysis membranes
CMD membranes were cut into small pieces and proteins were extracted by adding 100 µL urea buffer solution (8 M Urea, 4% (w/v) CHAPS, 65 mM DTT, 2% (v/v) pharmalyte 3-10, trace of bromophenol blue) and incubating membrane pieces at room temperature with gentle shaking following centrifugation. Protein concentration was determined by 2-D Quant Kit (GE Healthcare) in accordance with standard protocol. An unused 71 High Cut-off Brain MD Catheter (M-Dialysis AB) membrane was used as a blank control.
Two-dimensional gel electrophoresis (2-DE)
50 µg protein from each sample was separated on 2-DE(15) and visualized by silver staining(16). The protein pattern was analyzed as digitized image. The amount of protein in a spot was assessed as background corrected optical density, integrated over all pixels in the spot and expressed as integrated optical density (IOD).
Protein identification
Selected protein spots were excised from the gels using a home-made spot picker and destained. Briefly, after removal of water, 25 µL of solution A (30 mM potassium ferricyanide, MilliQ water) and solution B (100 mM sodium thiosulphate pentahydrate, MilliQ water) were added to the gel pieces at the same time to decolor the gel. Gel pieces were then washed 6 x 5 min, incubated at room temperature for 20 min in 50 µL of solution C (200 mM ammonium bicarbonate, MilliQ water) followed by washing 3 x 5 min.
Gel pieces were then dehydrated using 100 µL of 100% acetonitrile (ACN) applied twice. After removing ACN the samples were dried in SpeedVac for 15 min. 10 µL trypsin (200 µg/mL) was mixed with 90 µL 25 mM ammonium bicarbonate (ABC), and 25 µL of this mixture was added to the gel pieces, incubated on ice for at least 30 min to reduce the autolytic activity of trypsin, and then incubated at 37 °C overnight. The supernatant was transferred to a new tube and dried in SpeedVac. Gel pieces were incubated in 40 µL 50% ACN/5% trifluoroacetic acid (TFA) for 3-4 hours with gentle shaking to enable further extraction of remaining peptides in the gel pieces. The supernatant obtained from extraction with ACN/TFA was then pooled with the dried peptides and completely dried by SpeedVac, and stored at −20 °C until further analysis.
Protein identification was initially done using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and low abundant proteins were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). For MALDI analysis the dried peptides were dissolved in 4 µL of 0.1% TFA and 1 µl was mixed with 1 µl of matrix solution (0.067 g/ml 2, 5-dihydroxybenzoic acid (DHB) in 70% acetonitrile, 0.3% TFA). One µL was applied on the MALDI plate. Peptide calibration standard mixture II (Bruker Daltonics) was mixed with 0.1% TFA in a ratio of 1:50 and 1 µl was mixed with DHB 1:1. One µL of mixed standard was applied on the MALDI plate next to each sample. Peptide analysis was then performed in the range of 300-3500 Da using MALDI-TOF-MS (Voyager-DE PRO, Applied Biosystems).
Protein identification for the low abundant proteins was performed using LC-MS/MS whereby the trypsinated peptides were dissolved in 6 μL of 0.1% formic acid (FA) and applied to a nano-flow HPLC system, EASY-nLC II (Thermo Scientific) in conjugation with the mass spectrometer, LTQ Orbitrap Velos Pro hybrid mass spectrometer (Thermo Scientific) with a nano-electrospray source as previously described(17).
Database searching was performed using MS-Fit search engine and MaxQuant version 1.5 with trypsin as digestion enzyme against a human taxonomy of the SwissProt and the NCBI databases. The following search parameters were used: maximum two missed cleavages; fragment ion mass tolerance 0.5 Da; parent ion mass tolerance 6 ppm; fixed modification- carbamidomethylation of cysteine; variable modifications - N-terminal acetylation and methionine oxidation. Data was filtered at 1% false discovery rate. Identifications were based on a minimum of two unique peptides.
Statistical analysis
Central tendency and dispersion of data are presented as mean and standard deviation when data are summarized; alternatively numbers are presented comprehensively for clarity.