A Shift in Tissue Stiffness During Hippocampal Maturation Correlates to the Pattern of Neurogenesis and Composition of the Extracellular Matrix.

Youngjae Ryu,Kenji Uchimura,Wonyoung Lee,Misato Iwashita,Yoichi Kosodo

doi:10.3389/fnagi.2021.709620

Abstract

Aging changes the mechanical properties of brain tissue, such as stiffness. It has been proposed that the maintenance and differentiation of neural stem cells (NSCs) are regulated in accordance with extracellular stiffness. Neurogenesis is observed in restricted niches, including the dentate gyrus (DG) of the hippocampus, throughout mammalian lifetimes. However, profiles of tissue stiffness in the DG in comparison with the activity of NSCs from the neonatal to the matured brain have rarely been addressed so far. Here, we first applied ultrasound-based shear-wave elasticity imaging (SWEI) in living animals to assess shear modulus as in vivo brain stiffness. To complement the assay, atomic force microscopy (AFM) was utilized to determine the Young’s modulus in the hippocampus as region-specific stiffness in the brain slice. The results revealed that stiffness in the granule cell layer (GCL) and the hilus, including the subgranular zone (SGZ), increased during hippocampal maturation. We then quantified NSCs and immature neural cells in the DG with differentiation markers, and verified an overall decrease of NSCs and proliferative/immature neural cells along stages, showing that a specific profile is dependent on the subregion. Subsequently, we evaluated the amount of chondroitin sulfate proteoglycans (CSPGs), the major extracellular matrix (ECM) components in the premature brain by CS-56 immunoreactivity. We observed differential signal levels of CSPGs by hippocampal subregions, which became weaker during maturation. To address the contribution of the ECM in determining tissue stiffness, we manipulated the function of CSPGs by enzymatic digestion or supplementation with chondroitin sulfate, which resulted in an increase or decrease of stiffness in the DG, respectively. Our results illustrate that stiffness in the hippocampus shifts due to the composition of ECM, which may affect postnatal neurogenesis by altering the mechanical environment of the NSC niche.

Highlights

Mechanical properties in the brain, including stiffness, are defined by various heterogeneous components, such as neurons, glial cells, vessels, the extracellular matrix (ECM), and interstitial fluids (Franze et al, 2013; Javier-Torrent et al, 2021)
We explored the transition and function of chondroitin sulfate proteoglycans (CSPGs) in the hippocampus to find out their relevance for maturation-dependent changes in brain-tissue stiffness
The shear wave propagation generated by the acoustic radiation force (ARF) using a focused US beam shows reliable and consistent results to quantify the shear modulus as in vivo stiffness of the brain tissue, including humans and rodents (Mace et al, 2011; Tzschatzsch et al, 2018)

Summary

Introduction

Mechanical properties in the brain, including stiffness, are defined by various heterogeneous components, such as neurons, glial cells, vessels, the extracellular matrix (ECM), and interstitial fluids (Franze et al, 2013; Javier-Torrent et al, 2021). Several in vitro studies have shown that the proliferation, differentiation, and migration of neural stem cells (NSCs), progenitor cells, and post-mitotic neurons can be regulated depending on the stiffness of the surrounding environment (Saha et al, 2008; Leipzig and Shoichet, 2009; Rammensee et al, 2017). Previous studies have reported on heterogeneous tissue stiffness in the rodent hippocampus at the neonatal, juvenile, and adult stages (Elkin et al, 2010; Antonovaite et al, 2021) Based on such findings, it is deduced that the stiffness may be related to the niche of NSCs and their activity, including the maintenance and regulation of NSCs (Urban et al, 2019; Kobayashi and Kageyama, 2021). The following major questions remain to be addressed: (1) Does a shift in hippocampal stiffness correlate to the differentiation status of NSCs in vivo?; and (2) What is the principal component that determines tissue stiffness in the hippocampus?

Methods

Results

Conclusion