A micro-silicon chip for in vivo cerebral imprint in monkey

Abstract

Access to cerebral tissue is essential to better understand the molecular mechanisms associated with neurodegenerative diseases. In this study, we present, for the first time, a new tool designed to obtain molecular and cellular cerebral imprints in the striatum of anesthetized monkeys. The imprint is obtained during a spatially controlled interaction of a chemically modified micro-silicon chip with the brain tissue. Scanning electron and immunofluorescence microscopies showed homogeneous capture of cerebral tissue. Nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis of proteins harvested on the chip allowed the identification of 1158 different species of proteins. The gene expression profiles of mRNA extracted from the imprint tool showed great similarity to those obtained via the gold standard approach, which is based on post-mortem sections of the same nucleus. Functional analysis of the harvested molecules confirmed the spatially controlled capture of striatal proteins implicated in dopaminergic regulation. Finally, the behavioral monitoring and histological results establish the safety of obtaining repeated cerebral imprints in striatal regions. These results demonstrate the ability of our imprint tool to explore the molecular content of deep brain regions in vivo. They open the way to the molecular exploration of brain in animal models of neurological diseases and will provide complementary information to current data mainly restricted to post-mortem samples.

Figure 1

Tool design and X-ray scan of a cerebral imprint (A) Schematic representation of the imprint tool. The guide tube prevents the capture of nonspecific tissue during the descent. The exposure of the silicon chip by a 180° rotation of the inner tool allows harvesting of the desired tissue. (B) Picture of the imprint tool. (C) X-ray scan of the monkey skull during the execution of a cerebral imprint in the caudate nucleus. The window of exposure (white arrow) is easily seen.

Figure 2

Motor activities of monkeys after repeated cerebral imprints. Distance moved (A) and velocity of movement (B). Animals were assessed by the video system for 60 min before the first cerebral imprint (PRE, n = 5) and after each imprint (POST, n = 5). Evolution of the distance moved after each cerebral imprint (n = 5) for both monkeys (C). Finger skill tests (D). Animals were assessed by the time required to pick up three peanuts in 1 cm wells before the first (PRE, n = 15) and after each cerebral imprint (POST, n = 15). Data are presented as the means ± SD ** p < 0.01, *** p < 0.001, Mann–Whitney test.

Figure 3

Microscopic observation of a cerebral imprint (A) Scanning electron micrograph showing (left) the homogeneous capture of tissue on the silicon chip, (middle) a variety of cells on the micropillar, and (right) an elongated cell with extensive processes. (B) Immunostaining of glial fibrillary acid protein (GFAP) and neuronal nuclei (NeuN) on the captured cerebral imprint. Astrocytes were identified as GFAP-positive and NeuN-negative cells (top) and neurons showed NeuN-positive nuclei without GFAP labeling (bottom). The chip was stained for GFAP (red), NeuN (green), and Hoechst (blue).

Figure 4

Proteomic analysis of the cerebral imprint. Analysis of proteins extracted from a cerebral imprint by 1D SDS-PAGE (A) and mass spectrometry profiling (B). (C) Functional annotation of the cerebral imprint proteome identified by nano-LC-MS/MS. The bar graph represents the number of identified proteins in each disease or disorder signaling pathways classified as the most significant using the Ingenuity Pathway Analysis software.

Figure 5

Assessment of RNAs from the cerebral imprint in comparison with RNAs from the corresponding post-mortem sections. (A) Venn diagram representing the extent of overlap of probe sets detected from RNA extracted from cerebral imprint and post-mortem sections. (B) Linear regression curve representing the intensity of expression of the common probe sets between cerebral imprints and post-mortem sections.

Figure 6

Brain tissue imprinting in monkey: A combined MRI and histological analysis. (A) A coronal plane of the Rhesus monkey brain atlas with the imprint tool trajectory. (B) Coronal MRI view of an imprint tool track in monkey caudate nucleus. (C) Photomicrograph showing cresyl violet staining around an imprint tool track. Calibration bar = 200 μm. (D) Photomicrograph showing astrocytes (GFAP staining, green) and counterstained cells (PI staining, red) around one imprint tool track (asterisk) following acute implantation in monkey caudate nucleus. Calibration bar = 100 μm.