Quantification of the Extracellular Matrix Molecule Thrombospondin 1 and Its Pericellular Association in the Brain Using a Semiautomated Computerized Approach

Abstract

The structure and functions of the extracellular matrix (ECM), its spatial distribution and pericellular association of ECM molecules remain poorly understood. Colocalization of ECM molecules with cell phenotypes through immunohistochemistry can provide crucial insights into their juxtacrine signaling role as well as their structural relevance to tissue architecture. As manual quantification of images introduces intra- and inter-user bias and is cumbersome for high-throughput approaches, we implemented an automated high-throughput method to quantify the spatial distribution and cellular association of one ECM molecule, thrombospondin 1 (TSP1) with two major cell phenotypes, neurons, and astrocytes. The distribution of TSP1 was homogeneous throughout the striatum and cortex along the anterior–posterior axis. TSP1 occupied 8.85% of the striatum and 7.40% in the cortex. TSP1 also associated with 94.58% and 88.45% of neurons in the striatum and cortex. The association with astrocytes was significantly lower at 47.55% and 28.09%. These findings highlight the key role that TSP1 plays in neuron physiology in a healthy brain, but also highlights key regional difference in astrocytes secreting ECM molecules. The semiautomated approach implemented here will improve the throughput and reliability of measuring the distribution and cellular colocalization of ECM molecules.