Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

Summary

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

Abstract

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young's elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

Introduction

Most soft-tissues comprising biological organs are mechanically and structurally complex, of low stiffness compared to mineralized bone or engineered materials, and exhibit non-linear and time-dependent deformation. Compared to other tissues in the body, brain tissue is remarkably compliant, with elastic moduli E on the order of 100s of Pa ¹. Brain tissue exhibits structural heterogeneity with distinct and interdigitated gray and white matter regions that also differ functionally. Understanding brain tissue mechanics will aid in the design of materials and computational models to mimic the response of the brain during injury, facilitate prediction ....

Protocol

Ethics Statement: All experimental protocols were approved by the Animal Research Committee of Boston Children's Hospital and comply with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Mouse Brain Tissue Acquisition Procedures (for AFM-enabled indentation and impact indentation)

1. Prepare a ketamine/xylazine mixture to anesthetize the mice. Combine 5 ml ketamine (500 mg/ml), 1 ml xylazine (20 mg/ml) and 7 ml of 0.9% saline solution.
2. Inject mouse (Breed: TSC1; Syn-Cre; plp-eGFP; Age: p21; Sex: Male or Female) with 7 µl per gram bodyweight of the ketamine/xylazine solution.

Results

Figure 4 shows representative indentation and force vs. time responses (Figure 4B,E) for creep compliance and force relaxation experiments, given an applied force or indentation depth (Figure 4A,D), respectively. Using these data and the geometry of the system, the creep compliance J_c(t) and force relaxation moduli G_R(t) can be calculated for different regions of the brain (Figure 4C,F

Discussion

Each technique presented in this paper measures different facets of brain tissue's mechanical properties. Creep compliance and stress relaxation moduli are a measure of time-dependent mechanical properties. The storage and loss moduli represent rate-dependent mechanical properties. Impact indentation also measures rate-dependent mechanical properties, but in the context of energy dissipation. When characterizing tissue mechanical properties, both AFM-enabled indentation and rheology are commonly used methods. AFM-ena.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Xylazine	Lloyd Laboratoried		perscription drug
Ketamine	AnaSed Injections		perscription drug
Vibratome (Vibrating blade microtome)	Leica	VT1200
Hibernate-A Medium	Gibco	A1247501	CO2-independent neural medium for adult tissue
Atomic Force Microscope, MFP-3D-BIO	Asylum Research	-
Petri Dish Heater	Asylum Research	-
AFM Probe, 0.03 N/m, 10 µm radius borosilicate sphere	Novascan	PT.GS
Cell-Tak	Corning	354240	mussel-derived bioadhesive
Sodium Bicarbonate	Sigma-Aldrich	S5761	alternate suppliers can be used
Sodium Hydroxide, 1 N	Sigma-Aldrich	59223C	alternate suppliers can be used
Instrumented Indenter, NanoTest Vantage	Micro Materials Ltd.	-	probe tip needs to be machined (steel flat punch, 1 mm diameter, 4-5 mm length)
NanoTest Liquid Cell	Micro Materials Ltd.	-
Parallel Plate Rheometer MCR501	Anton-Parr	-
PP25	Anton-Parr	-	25 mm diameter flat measurement plate
Adhesive Sandpaper	McMaster-Carr	4184A48	alternate suppliers can be used
Loctite 4013 Instant Adhesive	Henkel	20268	alternate suppliers can be used