Studying the cellular sources of the DW-MRI signal with Organotypic cultures

Introduction Diffusion Weighted MRI (DW-MRI) of the brain is based on signal attenuation that originates from multiple morphological or functional sources [1]. Recently it was also suggested that some of these mechanisms can be used to detect neuronal activity directly by DW-MRI [2]. The quantification of the relative contribution of these physical factors to the DW-MRI signal in-vivo (in health and disease) is complicated due to various sources of physiological ‘noise’ such as bulk motion of the tissue, or flow of oxy/non-oxygenated blood. Organotypic neuronal cultures are one of the possible techniques applicable for the study of the sources of the DW-MRI signal. Organotypic cultures are obtained by slicing newborn rats’ brains, and culturing them for several weeks in supportive conditions (temperature, oxygen, nutrients, etc.). During that period the cultures recover the initial trauma and dying cells are cleared by resident microglia. The surviving cells (neurons and glia) establish a functional neuronal network that can be used to study neuronal activity and networking [3]. Organotypic cultures are suitable analogs for the study of neuronal tissues: they mimic brain tissue and its response to perturbations and they survive for long times (weeks). On the other hand, they lack sources of MRI artifacts such as bulk motion and blood flow. Indeed, Shepherd et al. [4, 5] used multiple hippocampal organotypic cultures simultaneously and performed DWI in a 14.1T scanner at room temperature. However, they suffered from the low partial volume, from air bubbles and the need to perform multiple time consuming repetitions. Petridou et al. [6] used organotypic cultures to study an assumed modulation of NMR phase directly by neuronal activity. Their tissues were not perfused but were kept near physiological temperature. Our goal was to design a system for the use of vital, thermally controlled and perfused organotypic cultures in combination with DW-MRI. We aimed to use these cultures as a tool for the study of the biophysical origins of the diffusion-weighted MRI signal. Fig. 1: Two spots of mature hippocampal organotypic cultures on a cover slip. The typical size of each spot is 2x2 mm. Methods Main experimental challenges – the unique challenges that we had to face include the following: (a) bubbles on the slides’ surface, creating susceptibility artifacts; (b) low partial volume; (c) necessity to have the temperature set stably to 35°C; (d) low SNR, due to the effect of imaging gradients, while, (e) avoiding multiple time-consuming averages; (f) sensitivity of the tissue to manipulation, or to drying. These challenges were solves with the following setting. Preparation of cultures – Brain slices were prepared as described in detail in [3]. Cortical and hippocampal coronal slices (400 Qm) were cut from Sprague-Dawley rat brains at postnatal day 1-2 using a vibratome and attached to glass cover-slips [1]. Cultures were then submerged in Dulbecco's Modified Eagle's Medium and placed in a roller incubator for 2-3 weeks at 35°C. During the incubation period cultures flattened to the thickness of 100-200 Qm [1]. MRI tissue chamber – Tissues were scanned in the MRI in a home-designed MRI chamber. The chamber was composed of an Ultem plastic that allows micro-imaging in high field, while preventing susceptibility artifacts close to the chamber’s surface. The cover slips were attached to the chamber, facing one another, such that during the process of transfer of the cultures from the incubator to the MRI camber, no direct manipulation of the tissue (other than attachment of the slide) was required. The MRI chamber was than inserted into a standard 15mm NMR tube (Wilmad, NJ). Perfusion lines and optic fiber thermal probe were inserted through the tube’s cap. Tissue conditions inside the MRI – Throughout the MR scan the tissue was perfused with Artificial Cerebero Spinal Fluid (ACSF) saturated with oxygen (95% O2; 5% CO2). To avoid perfusion-driven flow, the perfusion was stopped before DW-MRI scans, and resumed immediately afterwards. Tissue temperature was kept at 35±0.2oC throughout the entire experiment, (unless intentionally varied). Temperature was monitored by the optic fiber probe and regulated by the flow of hot gas and by warming up the perfusate. MRI protocol – Experiments were performed in a 7T vertical scanner (Bruker, Ettlingen, Germany) equipped with a Micro2.5 gradient coil, and a 15 mm RF coil. Altogether, experiments included multiple DW-MRI scans under various conditions, and altogether took 2-3 hrs (after placement in the MRI), where during the first 40-60 minutes the temperature was stabilized and MRI parameters were prescribed. MRI scans used the following parameters: TR/TE = 2000/70 msec with no averages. / =20/2.5 msec, FOV = 2.6x1.3cm with 64x32 matrix and 150 m slice thickness, Gd=0-315 mT/m (5-8 values) in read and phase directions.