This research was supported by funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R15HD093024 and R01HD104910A and NSF CAREER Award Number 1752513.

All procedures described were approved by the Drexel University Institutional Animal Care and Use Committee (IACUC). The neonatal piglet was acquired from a United States Department of Agriculture (USDA)-approved farm located in Pennsylvania, USA.
1. Stereo-imaging system setup
<ol>
	<li>Attach a stereo-imaging camera system that captures up to 100 frames/s (FPS) to a utility stand. The stereo-imaging camera system used in this study is a passive stereo camera with two horiz...

Biomechanical Analysis of Peripheral Nerves Using DLT-Calibrated Stereo-Imaging

<ol>
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Studies reporting biomechanical properties of peripheral nerves (PNs) because of stretch injury vary, and that variation can be attributed to testing methodologies such as testing equipment and elongation analysis5,6,7,8,9,10,11,12,<sup class="xr...

Peripheral nerves (PNs) undergo stretch during development, growth, normal joint movement, injury, and surgery1. PNs display viscoelastic properties to protect the nerve during regular movements2,3 and maintain the structural health of its nerve fibers2. Because PN response to mechanical stretch has been shown to depend on the type of nerve fiber damage4, injuries to adjacent connective tissues2,4, and testing approaches (i.e., loading rate or direction)5,6,7,8,9,10,11,12,13,14, it is essential to distinguish the biomechanical responses of PNs during normal range of motion versus non-physiological range at both slow- and rapid-stretch rates. This can further the understanding of the PN injury mechanism in response to stretch and aid in timely and optimized intervention1,4,15,16. There has been a growing trend in physical therapy to evaluate and intervene based on the relationship between nerve physiology and biomechanics17. By understanding the differences in PN biomechanics at various applied loads, physical therapists can be better prepared to modify current interventions17.
Available biomechanical data of PNs in response to stretch remains variable and can be attributed to testing equipment and procedures and differences in elongation data analysis5,6,7,8,9,10,11,12,13,14,16. Furthermore, measuring three-dimensional (3D) in-situ nerve displacement remains poorly described in the currently available literature. Previous studies have used stereo-imaging techniques to maximize the accuracy of 3D reconstruction of tissue displacement of facet joint capsules18,19. The direct linear transformation (DLT) technique enables the conversion of two or more two-dimensional (2D) views to 3D real-world coordinates (i.e., in mm)20,21,22. DLT provides a high-accuracy calibration method for stereo-imaging camera systems because it enables precise reconstruction of 3D positions, accounting for lens distortion, camera parameters, and image coordinates, and permits flexibility in stereo-imaging camera setup20,21,22. Studies using DLT-calibrated stereo-imaging camera systems are typically used to study locomotion and gait analysis22,23. This protocol aims to offer a detailed methodology to determine the in-situ strain of PNs at varying degrees of stretch using a DLT-calibrated stereo-imaging camera system and an open-source tracking software22.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Clear Acrylic Plexiglass Square Sheet</td>
			<td>W W Grainger Inc</td>
			<td>BULKPSACR9</td>
			<td>Construct three-dimensional control volume</td>
		</tr>
		<tr>
			<td>Stereo-imaging camera system - ZED Mini Stereo Camera</td>
			<td>StereoLabs Inc.</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Imaging Software - ZED SDK</td>
			<td>StereoLabs Inc.</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Maintence Software - CUDA 12</td>
			<td>StereoLabs Inc.</td>
			<td>N/A</td>
			<td>Download to run ZED SDK&#160;</td>
		</tr>
		<tr>
			<td>Camera stand - Cast Iron Triangular Support Stand with Rod</td>
			<td>Telrose VWR Choice</td>
			<td>76293-346</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>MicroSribe G2 Digitizer with Immersion Foot Pedal</td>
			<td>SUMMIT Technology Group</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Proramming Software - MATLAB&#160;</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>version 2019A or newer</td>
		</tr>
		<tr>
			<td>DLTcal5.m</td>
			<td>Hedrick lab</td>
			<td>N/A</td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>DLTdv7.m</td>
			<td>Hedrick lab</td>
			<td>N/A</td>
			<td>Open Source</td>
		</tr>
	</tbody>
</table>

direct linear transformation for the measurement of in-situ peripheral nerve strain during stretching

Peripheral nerves undergo physiological and non-physiological stretch during development, normal joint movement, injury, and more recently while undergoing surgical repair. Understanding the biomechanical response of peripheral nerves to stretch is critical to the understanding of their response to different loading conditions and thus, to optimizing treatment strategies and surgical interventions. This protocol describes in detail the calibration process of the stereo-imaging camera system via direct linear transformation and the tracking of the three-dimensional in-situ tissue displacement of peripheral nerves during stretch, obtained from three-dimensional coordinates of the video files captured by the calibrated stereo-imaging camera system.
From the obtained three-dimensional coordinates, the nerve length, change in the nerve length, and percent strain with respect to time can be calculated for a stretched peripheral nerve. Using a stereo-imaging camera system provides a non-invasive method for capturing three-dimensional displacements of peripheral nerves when stretched. Direct linear transformation enables three-dimensional reconstructions of peripheral nerve length during stretch to measure strain. Currently, no methodology exists to study the in-situ strain of stretched peripheral nerves using a stereo-imaging camera system calibrated via direct linear transformation. Capturing the in-situ strain of peripheral nerves when stretched can not only aid clinicians in understanding underlying injury mechanisms of nerve damage when overstretched but also help optimize treatment strategies that rely on stretch-induced interventions. The methodology described in the paper has the potential to enhance our understanding of peripheral nerve biomechanics in response to stretch to improve patient outcomes in the field of nerve injury management and rehabilitation.

Stereo-Imaging System DLT Calibration to Capture 3D In Situ Displacements of Stretched Peripheral Nerves

Direct Linear Transformation for the Measurement of In-Situ Peripheral Nerve Strain During Stretching

To begin, acquire three clear acrylic plexiglass square sheets. On each sheet, create a grid and mark at least 10 points, ensuring a minimum of 30 points across the 3D control volume on the x, y, and z-coordinate planes. Stack the sheets at different heights to capture the maximum height of the intended recording.Using a digitizer equipped with a foot pedal, digitize all points on the 3D control volume. Establish the origin of the 3D control cube and define the positive x and y directions to obtain x, y, and z-coordinates. On the digitizer software interface, set the origin and positive x and y-axes respectively on the reference frame.Open a new Excel worksheet to save the x, y, and z-coordinates of the control volume. Then on the digitizer software interface, set the format strings to Excel to store the coordinates on the 3D control volume. As the x, y, and z-coordinates are digitized using the digitizer and foot pedal, they're recorded onto an open Excel sheet.To document them, open an Excel sheet and save the coordinates as a csv file.

Using the described methodology, various output files are obtained. The DLTdv7.m *_xyzpts.csv (Supplemental File 12) contains the (x, y, z) coordinates in millimeters of each tracked point at each time frame that is further used to calculate the length, change in length, and strain of the stretched PN. Representative length-time, change in length-time, and strain-time plots of a stretched PN are shown in Figure 10. The stretched PN had an insertion marker, four markers along...

Read this Scientific Article Protocol about Direct Linear Transformation for the Measurement of In-Situ Peripheral Nerve Strain During Stretching at JoVE.com

This protocol implements a stereo-imaging camera system calibrated using direct linear transformation to capture three-dimensional in-situ displacements of stretched peripheral nerves. By capturing these displacements, strain induced at varying degrees of stretch can be determined informing the stretch injury thresholds that can advance the science of stretch-dependent nerve repair.

Peripheral nerves undergo physiological and non-physiological stretch during development, normal joint movement, injury, and more recently while ...

direct-linear-transformation-for-measurement-situ-peripheral-nerve