Adult patients >18 years old and requiring emergent surgery for ICH using an open craniotomy, at the Department of Neurosurgery, University Hospital Linköping, Sweden, during the period 2016-2018 were prospectively recruited to the study. Exclusion criteria included severe coagulation disorders and a known source of bleeding, such as an aneurysm or an arteriovenous malformation. The ICH was evacuated via a standard craniotomy and by microneurosurgical evacuation. Intracranial pressure (ICP)-monitoring was achieved using either Neurovent-P parenchymal pressure monitoring device (Raumedic AG, Helmbrechts, Germany) or Bactiseal external ventricular drainage (EVD) catheter (DePuy Synthes, Raynham, USA). One microdialysis (MD) catheter (CMA-71 Brain Catheter, M-Dialysis, Solna, Sweden) was inserted via the craniotomy into the perihemorhagic zone (PHZ) aiming for within 1 cm of the evacuated ICH, and one catheter was inserted ipsilateral to the hemorrhage in seemingly normal and non-eloquent cortex (SNX). MD catheters were inserted at a 45 degree angle aiming for the microdialysis membrane to include the cortex-white matter junction. As it is essential for the interpretation of MD sampling to know the location of the membrane(43, 44) a post-operative CT-scan was performed to verify MD catheter placement.
Three of the patients were included in a previous publication from our group evaluating energy metabolic disturbances using MD following ICH surgery.(29)
Patients were treated according to a standardized neurocritical care protocol to avoid secondary insults. This included intubation and mechanical ventilation if the patient was unconscious (Glasgow Coma Scale motor score (GCS-M) ≤5), aiming for normoventilation, normovolemia, and normothermia. ICP was maintained at ≤ 20 mmHg and cerebral perfusion pressure (CPP) > 60 mmHg with the use of volume substitution and inotropic drugs such as norepinephrine or dobutamine, when needed.
To determine outcome according to modified Rankin Scale (mRS) patients or their closest relative were asked to complete a validated questionnaire at 3-6 months post ICH-onset.(29, 45, 46)
Microdialysis catheters of 10 mm length with a molecular weight cut-off (MWCO) of 100 kDa (CMA-71, M-dialysis AB, Solna, Sweden) were used. The catheters were perfused per institutional routine with 5% human albumin in a water solution containing the excipients sodium chloride, N-acetyl-DL-tryptophan and caprylic acid (Albunorm, 50g/l, Octapharma AB, Stockholm, Sweden), at a rate of 0.3 µL/min using the CMA 106 perfusion pump (M-Dialysis AB, Solna, Sweden).(31) The first 2 hours (0-2) of sampling were discarded according to consensus praxis.(44, 47) Samples were collected every 2 hours for routine analysis of small molecular metabolites (glucose, lactate, pyruvate, glycerol and glutamate).(44, 47-49) Following this analysis, the remaining MD sample (approximately 30 µL/vial) was frozen and stored at - 20°C, and typically within 2-8 weeks transferred to Eppendorf vials and stored at -86 °C until further analysis.
Interstitial levels of glucose, lactate, pyruvate, glycerol and glutamate in the MD samples were analysed bedside using an ISCUS Flex® analyser (M Dialysis AB, Solna, Sweden). The lower limit of detection (LLOD) was 1.0 μmol/L for glutamate, 0.1 mmol/L for glucose and lactate, 10 μmol/L for pyruvate and 0.22 mg/mL for glycerol. No sample preparation was needed. Metabolite concentrations in the MD samples were analysed by the enzymatic methods using the ISCUS Flex® analyser, immediately after sample collection. The sample volume required for the different metabolites was 0.5 µL for glucose, 0.2 µL for lactate, 0.5 µL for pyruvate, 0.5 µL for glycerol, 1 µL for glutamate and 0.5 µL for urea. Following the analysis of these metabolites, approximately 30 µL of dialysate remained that were used in the Multiplex immunoanalysis.
MD samples from three different time-periods were analyzed for inflammatory mediators using the MSD (Meso Scale Discovery, Rockville, USA) MULTI-SPOT Assay System V-PLEX Human Proinflammatory Panel 1, Cytokine Panel 1 and Chemokine Panel 1(cat #K15210D). The proteins in each panel were detected by immunoassays. Antibodies with electrochemiluminescent labels bind the proteins, the MSD instrument applies a voltage to emit light from these labels and measures the intensity of the light, providing a quantitative measure of each protein concentration.
Pooling of three MD vials, after analysis of the routine analytes, was needed to achieve a sufficient sample volume of 25 µl per well enabling the analysis and thus a 6-hour time resolution was achieved. The three chosen time points were; early (4-10 hours), intermediate (20-26 hours) and late (44-50 hours after surgery). The pooled samples were diluted to ⅕ of the original concentration by adding 11 μL sample to 44 μL diluent.
All samples were analyzed with the same protocol. Pooling of samples was done 24 h before the analysis of the plates, pooled samples was stored at -20 °C before analysis.
V-PLEX Proinflammatory Panel 1: 67 μL of SULFO-TAG Anti-human IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13 and TNF-α were added to 2400 μL of diluent.
V-PLEX Cytokine Panel 1: 67 μL of SULFO-TAG Anti-human IL-1α, IL-5, IL-7, IL-12/IL-23p40, IL-15, IL-16, IL-17A, TNF-β and VEGF-A were added to 2400 μL of diluent.
V-PLEX Chemokine Panel 1: 67 μL of SULFO-TAG Anti-human Eotaxin, MIP-1β, Eotaxin-3, TARC, IP-10, MIP-1α, MCP-1, MDC and MCP-4 were added to 2400 μL of diluent.
After blocking using the blocker H for one hour with shaking for one hour, plates were washed three times with 150 μL Wash Buffer (PBS + 0.05% Tween 20). Thereafter, 25 μL sample or calibrator were added. The plates were sealed and incubated at room temperature with shaking for two hours. The plates were washed three times with 150 μL Wash Buffer, after which 25 μL of antibody solution was added to each well. The plates were sealed and incubated at room temperature with shaking for two hours, and subsequently washed three times with 150 μL Wash Buffer. To each well, 150 μL of 2X Read Buffer T was added, and the plates were analyzed with a MESO QuickPlex SQ 120 instrument.
Statistical analyses were performed in SIMCA 15.0.2 (Umetrics, Sweden) or in IBM SPSS 27.0 (IBM, Kista, Sweden).
Data distribution was assessed using Shapiro-Wilks’ normality test. Normally distributed data are presented as mean and standard deviation (SD). Non-normally distributed data are presented as median and range, median and interquartile range or median and individual values.
Low-molecular weight metabolite data were analysed using linear mixed model (MML) method using patient number as subject level and catheter location as fixed effect.
Multivariate analysis (MVA) was performed by overviewing the data using principal component analysis (PCA) and thereafter fitting orthogonal projections to latent structures discriminant analysis (OPLS-DA) model to the data. Critical outliers were investigated with Hotelling’s T2, a multivariate generalization of confidence interval, in the PCA. Any data point outside the resulting >99% confidence interval was removed from analysis. Moderate outliers were investigated using distance to model X (DModX)(50). Model validity was investigated with a cross-validated ANOVA (CV-ANOVA) and a p-value < 0.05 considered significant. R2 and Q2 are presented for the OPLS-DA model along with number of latent variables. Scaling to unit variance and mean centering was employed. Variables with a │p(corr)│> 0.4 and VIP > 1 were considered significant.(50)
Univariate analysis was performed using paired t-test for normally distributed data and paired Wilcoxon signed rank test for non-normally distributed data.
In the cytokine data analysis, values below lower limit of detection (LLOD) of the assay were substituted with the exact value given for LLOD by the manufacturer.