Comparative Analysis of Brain Stiffness Among Amniotes Using Glyoxal Fixation and Atomic Force Microscopy

Abstract

Brain structures are diverse among species despite the essential molecular machinery of neurogenesis being common. Recent studies have indicated that differences in the mechanical properties of tissue may result in the dynamic deformation of brain structure, such as folding. However, little is known about the correlation between mechanical properties and species-specific brain structures. To address this point, a comparative analysis of mechanical properties using several animals is required. For a systematic measurement of the brain stiffness of remotely maintained animals, we developed a novel strategy of tissue-stiffness measurement using glyoxal as a fixative combined with atomic force microscopy. A comparison of embryonic and juvenile mouse and songbird brain tissue revealed that glyoxal fixation can maintain brain structure as well as paraformaldehyde (PFA) fixation. Notably, brain tissue fixed by glyoxal remained much softer than PFA-fixed brains, and it can maintain the relative stiffness profiles of various brain regions. Based on this method, we found that the homologous brain regions between mice and songbirds exhibited different stiffness patterns. We also measured brain stiffness in other amniotes (chick, turtle, and ferret) following glyoxal fixation. We found stage-dependent and species-specific stiffness in pallia among amniotes. The embryonic chick and matured turtle pallia showed gradually increasing stiffness along the apico-basal tissue axis, the lowest region at the most apical region, while the ferret pallium exhibited a catenary pattern, that is, higher in the ventricular zone, the inner subventricular zone, and the cortical plate and the lowest in the outer subventricular zone. These results indicate that species-specific microenvironments with distinct mechanical properties emerging during development might contribute to the formation of brain structures with unique morphology.

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

“…Our results pertaining to cortical and hippocampal tissue stiffness are in a comparable range to other studies that used SWEI in adult rodents (within 10 ± 7 kPa; Mace et al, 2011;Lay et al, 2019). However, these results are not of the same order of magnitude as the Young's modulus acquired by AFM-based indentation assessment for the same tissue (ranged within 1 ± 0.5 kPa), which are in agreement with the values of previous reports on post-natal mouse brains (Elkin et al, 2010;Iwashita et al, 2020;Antonovaite et al, 2021). A possible way to account for this difference is that amputation of stiffer tissue components, such as vessels and axons, by slice preparation for AFM measurement may result in decreased stiffness compared to the intact brain.…”

“…Acute slices were incubated in slicing aCSF for 45 min on ice, then transferred to measuring-aCSF buffer (glucose 11 mM, NaCl 120 mM, KCl 3.5 mM, NaH 2 PO 4 1.25 mM, NaHCO 3 26 mM, MgCl 2 1.3 mM, and CaCl 2 2 mM) on a 35-mm poly-D-lysine-coated dish prior to indentation. The measurement of brain tissue stiffness was performed as previously described (Iwashita et al, 2020). Briefly, we used an AFM (Bioscope Resolve, NanoScope 9.4, Bruker, Billerica, MA, USA) mounted on an inverted microscope (ECLIPSE Ti2, Nikon, Japan).…”

Separation and single-cell analysis for free gastric cancer cells in ascites and peritoneal lavages based on microfluidic chips

“… 59 Thus, we fixed our cells with paraformaldehyde to enhance our success rate in cell sorting and trapping for samples from the biobank. Paraformaldehyde fixation can increase tissue stiffness 60 and preserve call shape 61 , 62 by inducing covalent cross-linking between molecules, effectively gluing them together into an insoluble meshwork. 63 After paraformaldehyde, almost no cells showed cell lysis in our chips.…”

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

“…We assume that the effects of temperature on the mechanical properties of soft tissue will be small; however, this might be one of the reasons for differences between the reported in vivo values of mechanical properties of liver tissues and the values in this study. modulus from liver sections, which may be achieved by a different method for fixation of liver tissue 64 .…”