High resolution 13C HR-MAS spectroscopy analysis of different brain regions from rats bearing C6 implanted gliomas.

Cerebral tissue heterogeneity plays a fundamental role in phystological and pathological conditions. Previous 13C NMR approaches in rodents allowed the in vivo investigation of sufficiently large tissue regions (approaching the size of the whole brain) or in vitro analysis of extracts derived from abundant tissue biopsies (> 1g). These limitations precluded a detailed 13C NMR analysis of regional cerebral metabolism in smaller regions. Recently, High Resolution Magic Angle Spinning (HR-MAS) approaches have allowed to obtain high quality 13C spectra from small tissue biopsies (ca. 10 mg), opening the way to investigate cerebral tissue heterogeneity within the microliter range. Here we report, for the first time to our knowledge, a 13C HR MAS study of normal and diseased brain regions of rats bearing implanted C6 gliomas. C6 gliomas were induced Wistar rats (180-250 g) by stereotaxic injection of C6 cells in the left caudate nucleus. Tumor growth was evaluated in vivo using T, and T2 weighted MRI (Bruker Pharmascan 7 Tesla). Three weeks after implantation, rats were anesthetized with isoflurane and infused with [l-13C] glucose (8 mol/100g, 45 minutes). After the infusion cerebral metabolism was arrested using a high-power focused microwaves (5 kW), the fixed brain removed from the skull and five biopsies taken from different brain regions; 1: contralateral brain, 2: normal brain region limiting to the edema limits of the tumor, 3: peripheral (vascularised) tumor zone, 4: tumor central (necrotic) zone and 5; ipsilateral normal hemisphere. Biopsies were analyzed by 1H and 13C HR-MAS (4 KHz spinnng, 4° C, 11.7 Tesla Bruker AVANCE500 WB spectrometer). Figure 2 illustrates the different 1H HR-MAS spectra obtained. It is remarkable the increase in lactate and mobile lipids in the tumour biopsies (regions 3 and 4). Figure 3 shows the 13C labelling patterns obtained by 13C HR-MAS of the same tissue regions. In the tumor biopsies, it is possible to detect significant increases of (3-13C) lactate and decreases of (4-13C) glutamate and (4-13C) glutamine, revealing a marked increase in glycolitic metabolism in the tumor. We quantified these changes determining 1H HR-MAS (LC model analysis) the (3-13C) lactate concentration and used this to quantify the remaining 13C HR-MAS observable metabolites. Ex vivo 13C HR-MAS spectroscopy reveals important metabolic heterogeneity changes in different regions of the brain of rats bearing C6 gliomas, previously not detectabteble in vivo or in vitro 13CNMR