Microdialysis technique
Brain microdialysis is used routinely in our Neurotraumatology Intensive Care Unit in most severe or moderate TBI patients who require ICP monitoring. The protocol used for implanting probes and the analytes routinely monitored at the bedside has been published elsewhere22. In four patients, three of them included in the study group (moderate or severe TBI patients), a high cut-off (100 kDa) cerebral microdialysis catheter (CMA-71, CMA Microdialysis Stockholm, Sweden) was inserted in the less damaged hemisphere in a structurally normal area identified by CT scan. The exact situation of the probe and the absence of any hemorrhagic or hypodense lesion around it was confirmed by a control CT scan showing the location of the probe’s gold tip. Sterile isotonic fluid containing 147 mmol/l Na+, 1.2 mmol/l CaCl2, 2.7 mmol/l KCl, and 0.85 mmol/l MgCl2 was perfused at a flow rate of 0.30 l/min using a CMA-106 pump (CMA Microdialysis, Stockholm, Sweden). Brain microdialysates were collected hourly and routine online analysis for glucose, lactate and pyruvate was performed at the bedside using the CMA-600 analyser (CMA microdialysis, Stockholm, Sweden). After this analysis, the leftover microdialysates were frozen at -80ºC until MMP levels were assayed. All samples collected during the first hour after probe implantation were discarded.
Zymographic measurement of gelatinase activity
In both plasma and microdialysate samples, MMP-2 and MMP-9 were measured by gelatin-substrate zymography as previously described23. We used zymography because this techique is more sensitive than ELISAs and reveal both active and inactive forms of MMPs6. The total protein content of plasma samples was determined by BCA assay (Pierce,Rockford, IL, USA) and samples were adjusted to equal protein content (20 µ). Using this standard method, we can be sure that differences detected in MMP expression (gelatin degradation) are due to true differences and not to variations in the protein content among samples. Moreover, in each gel we included a positive control both for MMP-2 and MMP-9 as an internal control that permits the comparison of results among all zymograms. A total volume of 10 µl of microdialysate was loaded in each lane. Enzymatic activity bands were visualized after staining the gels in Amido Black 1% for a total of one hour and then destaining for a total of 100 minutes in a solution containing 30% methanol and 10% glacial acetic acid.
The gelatinolytic zones were quantified using the Kodak 440 imaging system and analyzed using Kodak 1D image analysis software (Kodak, New Haven, USA). The intensity of the bands (measured in arbitrary units) was normalized to that of recombinant human MMP-9 proform control to allow comparisons between gels. The results of each MMP band were given as ratios. This assay is able to detect all gelatinase MMP isoforms and consequently detects the proforms for MMP-2 and MMP-9 (72 kDa and 92 kDa respectively) and also their cleaved forms (66 kDa and 83 kDa respectively). However, cleaved forms were found only in zymographies of peripheral blood for MMP-9 and in some cases for MMP-2, but never in microdialysates.