Author:

Abstract:

MALDI MS imaging in a mammalian brain plasticity model with new software tools The mammalian neocortex adapts to changing sensory stimulation. The changes occurring in the visual cortex, following retinal lesioning, are a well documented model for brain plasticity, which we study using functional proteomics. To reveal specific protein distribution profiles in the brain, we routinely perform in situ hybridization and immunocytochemistry. Both rely on prior knowledge of the identity of the biomolecules to be visualized. Conversely, MALDI MS imaging (MSI) is a relatively new technique that bypasses the need for a reporter system (stains or labels). Therefore, this method is an ideal complement to our current molecular imaging work. In this study we seek to visualize the differential protein distributions in visual areas affected by the retinal lesion, versus unaffected visual cortex. Prior to sacrifice all animals were placed overnight in a dark room, followed by a normal light environment for one hour. The brains were snap-frozen and a cryostat was used to cut coronal 10 µm sections. Sections were transferred to a stainless steel MALDI target, and subsequently freeze-dried. Following a brief washing procedure (5s in 70% ethanol, 5s in 95% ethanol), deposition of the sinapinic acid matrix was performed using aerosol spray with a coaxial N2 gas flow. Every tenth section was collected on glass slides for Nissl staining. Spectra from 4-25 kDa were acquired on a Bruker Reflex IV MALDI-TOF instrument, operated in the linear mode. Laser energy attenuation was at 50%. Forty shots were accumulated per pixel position. We examined mouse visual cortex located between Bregma levels -4.60 and -2.18 mm in the coronal plane according to the mouse brain atlas of Paxinos and Franklin (2001, Academic Press, Dan Diego). Sections of 10µm yielded the best results. Matrix deposition was carried out using a low flow-rate (30 µl/min) to minimize lateral dispersion due to droplets diffusing laterally over the section. The matrix deposition process was automated using a home-built sprayer based on an XY-plotter. At the time of writing, no MSI software was available for the Bruker Reflex IV. Therefore we developed two software tools to overcome the two major hurdles on this instrument. The first program creates a custom raster pattern to the specifications of the user, according to the dimensions and position of the section. When imported in the MALDI control software, this pattern allows for automated pixel-by-pixel acquisition of spectra using the AutoXecute routines. The second program reads the resulting spectra or a subset, and converts them to a file in the Analyze 7.5 image format. This image is subsequently imported in the BioMap software (M. Rausch, Novartis), allowing image interpretation. The sample preparation method was optimized for reproducibility and resolution using normal mouse brain. The differential protein profiles occurring during brain plasticity were investigated by comparing retinally lesioned mouse brain MS images with normal mouse brain MS images. To enable internal validation of distribution profiles, both hemispheres were imaged. Adjacent Nissl-stained sections were used to provide an anatomical reference view, and were compared with or overlaid on the MALDI MS images in BioMap. The application of MALDI MS imaging to a retinal lesion model represents an important research strategy in our ongoing investigation of mammalian brain plasticity.