Microdialysis is an elegant and minimally-invasive approach for the investigations of brain physio-pathology. Utilizing a microdialysis probe to overcome the blood-brain-barrier (BBB) allows for both (i) the local delivery of therapeutic compounds and (ii) the quantification of extracellular brain metabolites and small proteins associated with the disease. In this work, we report an online MR Spectroscopy measurement of dialysate composition in gliobastoma-bearing rats using a MRS micro-coil.

In vitro experiment: Human glioblastoma U87-MG cells were suspended in 300 μL of PBS and 150mM of 3-¹³C sodium pyruvate at 37°C. A microdialysis probe was placed into a cell suspension and infused with a buffer solution (PBS in D₂O with the addition of Gd-DOTA 3mM) under a flux of 500 nL/min. The microdialysis outlet tube was passed through a custom-made microsolenoid coil (750 µm inner diameter and 1 µL total volume) for ¹H detection inside a 7T Bruker Biospec MRI system. The total acquisition time for each MRS spectrum was 3.50 minutes, and a series of MRS spectra was acquired to record the metabolic evolution of the dialysate.

In vivo experiment: Microdialysis catheters with a cutoff of 6kDa were implanted stereotaxically in the cortex region of female healthy and in C6 glioblastoma-bearing Wistar rats (250 ± 10 g). Animals were positioned prone inside a 7 T Bruker Biospec MRI scanner. The microdialysis probe was infused with a buffer solution (PBS in D₂O) with the addition of Gd_DOTA 3mM under a flux of 500 nL/min. The outlet tube of the microdialysis probe was passed through the ¹H microcoil for MRS and an 8 mm surface dual channel-coil was placed on the rat head for brain MRI (Fig.2a). MRI and MRS scans were acquired before and after an intravenous injection of a solution of glucose into the animal. High resolution NMR spectra of the dialysate were further acquired using a Bruker DPX 500 MHz system.

Representative ¹H spectra of the dialysate obtained within the MRI 7 T scanner from the cell suspension are displayed in Fig 1a. Changes in pyruvate, lactate, alanine and glutamine peak intensities can be readily monitored every 3.5 minutes. The presence of ¹³C labeled lactate, glutamine, alanine and pyruvate in the dialysate was further confirmed by ¹³C, ¹H and 2D ¹H/¹³C HSQC experiments in a high field spectrometer at 500 MHz (Fig. 1b). The positioning of the microdialysis probe in the brain and the integrity of the semi-permeable membrane was accurately visualized using T_1-w images in the three orthogonal planes (Fig. 2b). The presence of endogenous lactate was measured around 1mM concentration in healthy and tumor-bearing rats. Following the i.v. injection of glucose, the presence in the MRS spectra of glucose peaks and increasing lactate peaks was observed in the brain dialysate (Fig.2c).The high sensitivity per unit volume of the microsolenoids developed and used in this study is a key factor for obtaining online MR Spectroscopy spectra from very small volume (1 μl) of dialysate. The limit of detection was measured equal to 2.4 mM (2.4 nmol) for a single proton and 0.8 mM (800 pmol) for a methyl group. The application of the microdialysis procedure associated with in vivo MRI and MRS techniques offers important synergetic potential. First of all, with the combined use of MRI and microdialysis, extracted brain metabolites can be correlated with anatomical changes or pathological structures, such as tumors, visualized in brain images. More importantly, MRS spectra of dialysate metabolites can be acquired in the vicinity of the micro-dialysis probe. As a result, online analysis of dialysate can be performed real-time to monitor the variations of metabolites concentration. The use of Gd_DOTA is effective to precisely localize, and monitor the integrity of the semi-permeable membrane inside the rat brain (Fig. 2b). Additional molecules within the perfusate such as meglumine (methyl group at 2.8 pm) can be used as an internal standard for quantifying the metabolites detected in the dialysate (Fig. 2c). In this work, cells and brain metabolites below 1mM concentration were detected in the 1 μl dialysate with a S/N of 23 (using a lb of 4Hz)We demonstrated that the use of a ¹H microsolenoid operating at 7 T within a pre-clinical MRI scanner connected to a microdialysis probe is an excellent tool for visualizing on-line extracellular metabolites concentration from cells suspension and rat brain. The MRI/MRS protocol presented here can be potentially associated with a chemiotherapeutic treatment, monitoring in real time the metabolites evolution and the tumor responsivity to chemotherapeutic drugs. It may be applied as well for probing neuronal and glial cells metabolism in heathy brain. Furthermore, it could have a relevant role in the development of personalized medicine protocols considering the availability of similar instrumentation (micro-coils, microdialysis) for patients.