We acknowledge support of this work by the National Multiple Sclerosis Society and Simons Center for the Social Brain. BQ acknowledges support from the U.S. National Defense Science &amp; Engineering Graduate Fellowship program.

Ethics Statement: All experimental protocols were approved by the Animal Research Committee of Boston Children&#39;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)
<ol>
	<li>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.</li>
	<...

<ol>
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Each technique presented in this paper measures different facets of brain tissue&#39;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...

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 of mechanical damage, and enable engineering of protective strategies. Additionally, such information can be used to consider design targets for tissue regeneration, and to better understand structural changes in brain tissue that are associated with diseases such as multiple sclerosis and autism. Here, we describe and demonstrate several experimental approaches that are available to characterize the viscoelastic properties of mechanically compliant tissues including brain tissue, at the micro-, meso-, and macro-scales.
At the microscale, we conducted creep-compliance and force relaxation experiments using atomic force microscope (AFM)-enabled indentation. Typically, AFM-enabled indentation is used to estimate the elastic modulus (or instantaneous stiffness) of a sample 2-4. However, the same instrument can also be used to measure microscale viscoelastic (time- or rate-dependent) properties 5-10. The principle of these experiments, shown in Figure 1, is to indent an AFM cantilevered probe into the brain tissue, maintain a specified magnitude of force or indentation depth, and measure the corresponding changes in indentation depth and force, respectively, over time. Using these data, we can calculate the creep compliance JC and relaxation modulus GR, respectively.
At the mesoscale, we conducted impact indentation experiments in fluid-immersed conditions that maintain the tissue structure and hydration levels, using a pendulum-based instrumented nanoindenter. The experimental setup is illustrated in Figure 2. As the pendulum swings into contact with the tissue, probe displacement is recorded as a function of time until the oscillating pendulum comes to rest within the tissue. From the resulting damped harmonic oscillatory motion of the probe, we can calculate the maximum penetration depth xmax, energy dissipation capacity K, and dissipation quality factor Q (which relates to the rate of energy dissipation) of the tissue 11,12.
At the macroscale, we used a parallel plate rheometer to quantify the frequency dependent shear elastic moduli, termed the storage modulus G&#39; and loss modulus G&#34;, of the tissue. In this type of rheometry, we apply a harmonic angular strain (and corresponding shear strain) at known amplitudes and frequencies and measure the reactional torque (and corresponding shear stress), as shown in Figure 3. From the resulting amplitude and phase lag of the measured torque and geometric variables of the system, we can calculate G&#39; and G&#34; at applied frequencies of interest 13,14.

<table border="0" cellpadding="0" cellspacing="0" width="1078">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Xylaxine</td>
			<td style="width:161px;">Lloyd Laboratoried</td>
			<td style="width:135px;"></td>
			<td style="width:401px;">perscription drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td>AnaSed Injections</td>
			<td></td>
			<td>perscription drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome (Vibrating blade microtome)</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hibernate-A Medium</td>
			<td>Gibco</td>
			<td>A1247501</td>
			<td>CO2-independent neural medium for adult tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Atomic Force Microscope, MFP-3D-BIO</td>
			<td>Asylum Research</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish Heater</td>
			<td>Asylum Research</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AFM Probe, 0.03 N/m, 10 um radius borosilicate sphere</td>
			<td>Novascan</td>
			<td>PT.GS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>mussel-derived bioadhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>alternate suppliers can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide, 1N</td>
			<td>Sigma-Aldrich</td>
			<td>59223C</td>
			<td>alternate suppliers can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instrumented Indenter, NanoTest Vantage</td>
			<td>Micro Materials Ltd.</td>
			<td>-</td>
			<td>probe tip needs to be machined (steel flat punch, 1mm diameter, 4-5 mm length)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoTest Liquid Cell</td>
			<td>Micro Materials Ltd.</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parallel Plate Rheometer MCR501</td>
			<td>Anton-Parr</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PP25&#160;</td>
			<td>Anton-Parr</td>
			<td>-</td>
			<td>25 mm diameter flat measurement plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive Sandpaper</td>
			<td>McMaster-Carr</td>
			<td>4184A48</td>
			<td>alternate suppliers can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loctite 4013 Instant Adhesive</td>
			<td>Henkel</td>
			<td>20268</td>
			<td>alternate suppliers can be used</td>
		</tr>
	</tbody>
</table>

characterizing multiscale mechanical properties of brain tissue using atomic force microscopy, impact indentation, and rheometry

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&#39;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.

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 Jc(t) and force relaxation moduli GR(t) can be calculated for different regions of the brain (Figure 4C,F</stro...

Watch this Scientific Journal Video about Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry at JoVE.com

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

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

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

The overall goal of these mechanical characterization techniques is to measure the viscoelastic properties of biological tissue at different length scales and loading rates. These methods can be used to answer key questions in biological engineering. For example, how does the brain deform under very high rates of loading, or how do diseases like multiple sclerosis or autism affect the mechanical properties of the brain tissue.The main advantage of these techniques is for materials of very low stiffness and very high hydration, like a biological tissue, you can test over a wide range of loading conditions, and you can also test over a wide range of material volumes, down to the level of a single cell, and up to the level of an entire brain. The implications of these techniques, extends towards modeling of the response of brain during injury, which is important for engineering protective strategies. Though this method can provide insight into brain mechanical properties, it can also be applied to other compliant biological tissues, such as heart and liver.During the mechanical characterization of compliant tissues, establishing appropriate contact between the measurement probe and the tissue is crucial. Carefully load an AFM probe with a nominal spring constant of 0.03 newtons per meter and a 20 micrometer diameter borosilicate bead into the probe holder. Place a brain slice mounted in a petri dish onto an AFM stage-mounted heater that has been pre-warmed to 37 degrees celsius.Then add about two milliliters of pre-warmed medium. Next, carefully add a drop of medium onto the AFM probe to protect it from breaking due to surface tension when it is lowered into the medium surrounding the brain slice. Then reposition the AFM head onto the stage, and begin lowering the head until it is submerged in the medium.Using the optical microscope, move the stage so that the region of interest is below the calibrated AFM probe, then lower the AFM probe to make contact with the surface of the tissue. In order to conduct the creep compliance experiments, construct an applied force function in the software's function editor. The function consists of a 0.1 second ramp to a set point of 5 nanonewton which is held for 20 seconds followed by a one second ramp down to zero nanonewton.The software will record data on the AFM probe's indentation into the tissue during the applied force function. After running the creep compliance experiment, conduct force relaxation experiments by creating an applied indentation function in the software. Run this function while the software collects data on the force experienced by the AFM probe as it indents into the tissue.To begin impact indentation tests, match a spherical probe by sliding it onto the pendulum using tweezers. Then attach the fused quartz sample post onto the plate and screw the plate into the translational stage. To enable dynamic impact experiments on hydrated brain tissues, first perform the liquid cell calibration.Go to the Calibration menu in the software, select Liquid Cell and follow the software prompts to make contact with the fused quartz sample. Next, select Normal for the Indenter Type, and use the default value of 0.05 millinewtons for the Indenter Load. Then click on continue to perform the calibration for the normal indenter configuration.Now move the sample stage back by at least five millimeters and mount the lever arm. Repeat the liquid cell calibration in the new configuration by selecting Liquid Cell for the indenter type. Click Continue to obtain the liquid cell calibration factor.Next, increase the capacitor plate spacing. Increasing the capacitor plate spacing will increase the maximum measurable depth which is necessary when testing highly compliant materials. With a wrench, turn the three nuts that control the capacitor plate spacing clockwise in small increments.After each complete clockwise turn, select Bridge Box Adjustment under the Maintenance menu and obtain a good pendulum test. Continue to slowly adjust the nuts until the Approx. Depth Calibration reads a value of 70, 000 nanometers per volt or higher.Then position a new limit stop at the bottom of the pendulum that can be switched on and off via a power supply. Retract the original limit stop sitting behind the pendulum to remove a potential obstruction of the pendulum motion and allow for higher impact velocities as well as higher penetration depths into compliant samples. Turn on the power supply for the solenoid and set it to 10 volts.Next, go to the Experiment menu and select Impact and Adjust Impulse Displacement. Follow the software instructions to calibrate the swing distance of the pendulum. When the impact indentation setup is fully complete, aspirate the medium and dry the brain slice.Then use a thin layer of cyanoacrylate adhesive to secure the sliced brain to the aluminum sample post. Then slide the liquid cell over the second O-ring on the sample post, and fill the liquid cell with five milliliters of carbon-dioxide-independent medium to fully immerse the tissue. Move the bath in the negative X direction until the tip on the lever arm is properly located above the bath.Next, move in the positive Z direction until the tip is fully submerged in the bath and is in front of the sample. Using the Sample Stage Control window, carefully make contact, and then back the stage away from the sample surface by about 30 micrometers. Under the Experiment menu, click Impact and setup an impact experiment.Choose a specific impulse load that will relate directly to the resulting impact velocity based on the swing distance calibration. And then run the scheduled experiment. When the pendulum swings back and the sample surface continues to move to the measurement plane, turn the bottom limit switch off.The displacement of the probe as a function of time will be recorded by the software. Attach sand paper to the 25 millimeter diameter measurement probe. Next, attach the thermal system and mount the probe.Finally, attach another piece of sand paper to the bottom plate aligned with the top plate. Calibrate the rheometer as per the manufacturers instructions. First, zero the force on the probe.Second, establish contact between the probe and the bottom plate. Then measure the inertia of the probe. Finally, perform a motor adjustment.Then slowly lower the measurement plate. When the plate is within a millimeter of the tissue, lower it in 0.1 millimeter increments until the plate is fully in contact with the top surface of the tissue and the measured normal force is of the desired value. Pipe out a small volume of medium on the edges of the sample to maintain hydration during the procedure.Lower the thermal hood. Next, click File, New, and under the Gel tab select Frequency Sweep. Then click on window measurement one frequency sweep and double click on the oscillation box.Enter the frequency range, the strain, and the number of points. Finally, select OK and click Start to initiate the frequency sweep. Here are representative indentation and force versus time responses for both creep compliance and force relaxation experiments.Using these data and the geometry of the system, the creep compliance and force relaxation moduli can be calculated for different regions of the brain. Impact indentation measures the mechanical properties of the tissue at high rates of spatially and temporally concentrated loading. The resulting impact response parameters can be quantified at different impact velocities which provides a means to study the rate-dependent properties of the tissue.Rheology measures the frequency-dependent viscoelastic properties of bulk tissue in terms of the storage and loss moduli. The storage modulus is nearly an order of magnitude larger than the loss modulus at low frequencies, indicating that elastic properties dominate the behavior of brain tissue. While attempting this procedure, it is important to keep the tissue adequately hydrated or immersed in a fluid that helps the tissue maintain its proper structure.The development of these demonstrated techniques has paved the way for materials researchers to design an optimize synthetic gels that can mimic the mechanical response of the brain. After watching this video, you should have a good understanding of how atomic force microscope enabled indentation, impact indentation, and rheology are used to characterize the viscoelastic mechanical properties of tissue. When interpreting the collected data, remember the underlying assumption that the deformed volume of the tissue is structurally homogeneous and elastically isotropic.This is not necessarily true for all biological tissues. As your questions about mechanics of biological tissues become better defined, you can choose one or more of these mechanical experiments to answer the question at the appropriate length scale or time scale of interest.

characterizing-multiscale-mechanical-properties-brain-tissue-using

Research

JoVE Journal

Neuroscience

12.0K visualizações.  Massachusetts Institute of Technology. The overall goal of these mechanical characterization techniques is to measure the viscoelastic properties of biological tissue at different length scales and loading rates. These methods can be used to answer key questions in biological engineering. For example, how does the brain deform under very high rates of loading, or how do diseases like multiple sclerosis or autism affect the mechanical properties of the brain tissue.The main advantage of these techniques is for materials of v...