Early proteomic markers of vasospasm can be identified in cerebral microdialysates of patients with subarachnoid hemorrhage

Introduction

A major complication of aneurysmal subarachnoid hemorrhage (SAH) is delayed cerebral ischemia (delayed ischemic neurological deficit [DIND], also called symptomatic vasospasm) several days after SAH, affecting about 30% of the patients. It is unknown why symptomatic vasospasm develops in some patients after SAH and does not in others. In this study, we hypothesized that one or more proteins might be detected in the brain parenchyma of patients before they develop vasospasm and that these proteins do not show up in SAH patients without vasospasm. Therefore, we searched for protein markers in human brain microdialysates which might be related to developing vasospasm.

Patients and Methods

We investigated 10 patients with SAH, 5 developing vasospasm and 5 others without vasospasm. Informed consent was obtained from the patient or legal representative. After the initial surgery, flexible microdialysis probes (CMA 70 custom probes, CMA Microdialysis, Solna, Sweden) were inserted into fronto-temporal cortex for later-on metabolic monitoring in neuro-intensive care. The probes were perfused at 0.3 μL/min with sterile Ringer's solution. We analyzed proteomic profiles of the microdialysate 24 hours and 5–6 days after insertion of the probes by two-dimensional gel electrophoresis. Proteins were separated in the first dimension according to their isoelectric point, and in the second dimension according to their molecular weight. Polyacrylamide electrophoresis gels were silver-stained and analysed by the Phoretix 2D Elite software (Nonlinear Dynamics, Newcastle-upon-Tyne, UK). Proteins were identified by mass spectrometry (Center for Molecular Biology, University of Heidelberg). We compared protein expression profiles by hierarchical clustering using the EMBL online tool EPCLUST, version 0.9.23 beta, at http://ep.ebi.ac.uk.

Results

We found an average of 57+/−22 protein spots in the individual gels, ranging from 37 to 149. Thirty-three spots consistently appeared in at least 50% of the gels analyzed. of these, 20 protein spots were differentially expressed (P < 0.05), of which we could identify 15. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 4 isoforms) was increased by a factor of 3.7 ± 1.1 (n=33 vasospastic; n=47 non-vasospastic, means ± sd) in the vasospasm group, whereas the concentration of Heat shock protein 73 (HSP73, 9 isoforms) was decreased to 0.47 ± 0.2 (n=88 vasospastic; n=101 non-vasospastic). These protein changes occurred 2.4 ± 2.1 days before vasospasm developed. Cluster analysis of the proteome data revealed a close relationship of GAPDH and HSP73 in the context of cell death and apoptosis.

Conclusions

Patients who develop cerebral vasospasm after SAH show a pattern of proteins in their brain microdialysate which is different from that of SAH patients who do not develop vasospasm. Since these differences in the protein pattern are evident as soon as 2 days before the development of vasospasm, they may potentially be used as early prognostic markers for the development of vasospasm after SAH, enabling selective early therapeutic intervention in this high risk group of patients.