The authors are very grateful to Adam Shortland and Nicola Fry who shared their algorithms for the 3-dimensional ultrasound in 2004, which were the inspiration for the development of the software used in this study.

The authors have nothing to disclose.

A valid and reliable 3DUS technique is presented that allows for the fast analysis of morphometric variables of skeletal muscles. Different 3DUS approaches for soft tissue imaging have been available for approximately a decade42,43, however the 3DUS approaches are still not used commonly. MRI is a &#39;gold standard&#39; for estimation of in vivo muscle volumes (e.g., references16,17,18,19,20). MRI validity has been tested and confirmed in studies comparing either phantoms or cadaveric organs of known volume to MRI-based volume estimates44,45. However, MRI availability for research is limited and scans are time consuming and costly. In addition, experimental subject postures are limited by the bore seize of the MRI scanners. Typical MR images generate insufficient contrast to perform measurements of variables of muscle geometry (fascicle lengths and angles). However, 3D muscle geometry can be assessed also using MRI by using additional techniques, e.g., DTI technique21. Similar to MRI, US imaging provides adequate distinction at interfaces between different types of tissues (i.e.&#160;visible within US images), providing a valid modality for soft tissues volume assessment1,30,44,46,47,48,49. In contrast to MRI, 3DUS images have sufficient contrast to perform analysis on both volume and muscle geometry from the same measurement.
In addition, the technique presented allows combining images of multiple sweeps into one array, for the study of larger muscles. This new 3DUS method provides a potential tool for clinical assessment of muscle morphology. This method can be used also for imaging soft-tissue structures other than muscle (e.g., tendons, internal organs, arteries).
Modifications to Improve Offline Processing Time:
Modifications of the 3DUS approach were mainly aimed at improving processing time and measuring larger muscles. The offline processing time of a 3DUS image depends on voxel array settings, sampling frequency, size of ROI, duration and speed of the sweep, number of sweeps, and the used workstation. Previously, a reconstruction time of &#8776; 2 h was necessary for reconstructing only one sweep yielding 750 US images (30 s at 25 Hz)15,25,30. With the present 3DUS method, the same sweep takes only 50 s reconstruction time (improving the &#39;offline&#39; processing time by 99%). This improvement can be explained by the enhanced filling algorithm that utilizes large vector operations to fill the voxels frame-by-frame, instead of pixel per pixel and increased random access memory (RAM) of workstations to construct larger voxel arrays. With the new 3DUS approach, a typical reconstruction representing a sweep length of 30 cm at a speed of 1 cm/s, with a target voxel size of 0.2 x 0.2 x 0.2 mm3 and a sampling frequency of 25 Hz, takes the following time to reconstruct:
a. Approximately 10 s to identify the synchronization pulse and select relevant US images. 
b. Approximately 120 s to determine the calibration transformation matrix (PrTIm). 
c. Approximately 10 s for the bin-filling stage. 
d. Approximately 30 s for executing the gap-filling steps.
In total, taking 170 s. Note, step b only needs to be performed once, assuming a rigid connection of the MoCap markers to the probe, leaving 50 s for the reconstruction of a single sweep. Combining two single sweep reconstructed voxel arrays takes approximately 10 s.
Limitations and Critical Steps:
There are several 3DUS imaging aspects that should be taken into account:
i. US image quality: Higher spatial resolution of 2D US images provide more pixels to be placed within the voxel array. This would allow the voxel dimensions to decrease, leading to higher voxel density. Several currently available ultrasound machines use spatial compounding to reduce the noisy granular texture, allowing for better artifact-free distinction of the interfaces of tissues. Another option to reduce speckle is edge enhancement. However, it should be noted that this approach is not desirable, since it deforms the image in an attempt to create distinct interfaces, thereby distorting the true anatomical position of the interfaces.
ii. MoCap accuracy: Pixels can only be accurately placed into a voxel, if the position sensor accurately quantifies the coordinates of the probe. With an increase in image resolution, MoCap accuracy becomes more important. The presented 3DUS setup works best with a voxel dimension of 0.2 x 0.2 x 0.2 mm3, using a MoCap system with an accuracy of 0.1 mm, providing ample accuracy to reconstruct the 3DUS voxel array.
iii. Sample frequency: The lowest temporal resolution of either the US images or MoCap data stream determines the sample frequency. This affects the sweep time or the voxel array settings. For instance, doubling the sample frequency from 25 to 50 Hz allows a sweep to be performed in half the time. Alternatively, not changing the sweep speed, provides more images to fill the voxel array, leaving fewer gaps to be filled and thereby potentially increasing the voxel array resolution. However, increasing the voxel array resolution, without increasing the sampling frequency, requires a slower scan, which will increase the potential of movement artifacts.
iv. Image reconstruction time: Fast reconstructions require a powerful workstation with sufficient available RAM. In addition, reconstruction time varies largely based on the voxel array volume and complexity of the gap-filling process.
v. Experimental protocol: Standardization of the experimental protocol, as exemplified in the present study for the VL and GM, is essential for comparison of morphological measurements (e.g., fascicle length, fascicle angle, muscle belly length, tendon length, aponeurosis length) between subjects and monitoring within subjects in longitudinal studies. However, note that the morphology assessed at rest may alter during muscle activation. For example, for the VL experiment, the knee extensor morphology during maximal contraction may demonstrate a high pennation angle and shorter fascicles in 60&#176; knee flexion, in comparison with morphology at rest50. In certain conditions (e.g., spasticity), electromyography (EMG) may be used to verify resting muscle activity levels during examination.
vi. Probe pressure and tissue deformation: If ample ultrasound gel is applied on the ROI, the amount of pressure to remain for full contact between probe and the skin is limited. As guidance, we advise that scanning a ROI should feel like hovering over the skin, and pressure should only be applied to keep in contact with the gel and thereby the skin. However, slight tissue deformation may be inevitable, even with a generous amount of ultrasound gel. Probe size and a curved ROI affect the required amount of pressure or gel used. Larger probe size and a more curved ROI require more pressure and/or more gel, than smaller probes with a similar curved ROI. Another possible solution is to discard the reverberation (i.e.&#160;non-skin-contact) region of the US images. In addition, tissue deformation is most likely to occur in the first tissue layers, such as skin and subcutaneous adipose tissue layers. Note that subjects with little to no subcutaneous adipose tissue are therefore more prone to adverse effects of pressure. In addition, the tissue deformation occurs most likely at the center of the probe, which is typically not the region of overlap with other sweeps.
vii. Imaging and anatomical knowledge: Another important consideration in using any imaging modality is that the knowledge of both the anatomy and the imaging modality is essential to obtain meaningful interpretation. Anatomical variation between subjects and image artifacts needs to be recognized and taken into account in the identification process of anatomical structures. Even with healthy and/or well developed muscles, clear identification can be difficult because it requires anatomical knowledge to differentiate between different components of one muscle or between muscle groups51. However, in atrophied muscle (i.e.&#160;elderly, in the case of pathology, or a cadaver), the clear identification is even more complicated because of a smaller size and decreased image contrast, and therefore less distinct tissue interfaces (Figure 4). We believe that without prior anatomical knowledge, we would have been limited in making correct judgements in designing this 3DUS approach and in performing the 3DUS measurements. For example, for GM experiments, different footplate angles do not necessarily cause expected changes in muscle tendon complex lengths, due to deformation within the foot7. Also detailed anatomical information on curvature of the distal aponeurosis was essential for an adequate selection of the mid-longitudinal plane in all subjects38.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55943/55943fig4.jpg" /> 
Figure 4: Variation and quality of reconstructed anatomical cross-sectional 3DUS images of the quadriceps muscle halfway along the thigh. (A) Example of a male human cadaver shows an image of an atrophied state at death (death age: 81 years). Identification of the boundaries of individual heads of the quadriceps muscle is difficult. (B) Example of a sedentary male (aged 30 years). (C) Example of a male athlete rower (aged 30 years). The white squares for scale represent 1 cm x 1 cm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55943/55943fig4large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
Future Applications:
The 3DUS approach provides an imaging tool that can be used for various purposes and settings in sports and clinics. In clinical interventions effectiveness is related to the physical fitness level52. Using 3DUS for monitoring patients who are at risk of losing muscle mass is important (e.g., references53,54,55) and potentially allows for adjustment of the treatment. Another potential application of 3DUS lies in monitoring the morphological adaptation of muscle in response to intervention (training) and/or injury.
This protocol described a cost- and time-effective method of measuring soft tissue structure of the human body based on freehand 3DUS sweeps. Moreover, assessment of the meaningful morphological parameters of m. vastus lateralis and m. gastrocnemius medialis proved to be valid and reliable.

In healthcare and sports, information on the morphology of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training1. Ultrasound (US) imaging is a tool commonly used for visualization of soft tissue structures in muscle diseases2, critical illnesses3,4, cardiovascular diseases5, neurological disorders6,7,8, and effects of physical training6,9,10. US imaging enables quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion11,12,13,14,15.
Currently, US imaging measurements are mostly performed in 2D images, with the examiner choosing a presumably, suitable orientation and location of the ultrasound probe. Such 2D methods restrict morphological measurements to one image plane, while the parameter of interest may not be present within this plane. Morphological analysis requires a 3D approach, providing out-of-plane measurements using 3D reference points. Such a 3D morphological representation of soft tissues is known to be provided by Magnetic Resonance Imaging (MRI)16,17,18,19,20. However, MRI is expensive and not always available. Also, visualization of muscle fibers requires special MRI sequences, such as diffusion tensor imaging (DTI)21. A cost-effective alternative to MRI is 3D ultrasound (3DUS) imaging. The 3DUS approach provides several advantages over MRI techniques, e.g., it imposes less space limitations for positioning the subject during an examination. 3DUS imaging is a technique sequentially capturing 2D (B-mode US) images and positioning them into a volume element (voxel) array22,23,24. The process of 3DUS image reconstruction consists of five steps: (1) Capturing a series of freehand 2D US images; (2) Tracking the position of the US probe, using a Motion Capture (MoCap) system; (3) Synchronizing the MoCap position and US images; (4) Calculating the location and orientation of the ultrasound images within the voxel array using a calibrated system of reference; and (5) Placing these images into this voxel array.
The 3DUS approach has been successfully applied for assessment of morphology of skeletal muscle15,25,26,27,28,29. However, previous approaches7,15,25,30 have proved cumbersome, time consuming and technically limited, as only small segments of large muscles could be reconstructed.
To improve the 3DUS approach, a new 3DUS protocol has been developed that allows reconstruction of complete muscles within a short period of time. This protocol article describes the use of 3DUS imaging for morphometry of the m. vastus lateralis (VL) and m. gastrocnemius medialis (GM).

<table>
	<tbody>
		<tr>
			<td>Ultrasound device (Technos MPX)</td>
			<td>Esaote, Italy</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear array probe (12.5 Mhz, 5 cm)</td>
			<td>Esaote, Italy</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Workstation (HP Z440)</td>
			<td>HP, USA</td>
			<td>http://www8.hp.com/us/en/workstations/z440.html</td>
			<td></td>
		</tr>
		<tr>
			<td>Framegrabber (Canopus, ADVC 300)</td>
			<td>Canopus, Japan</td>
			<td>ADVC 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Motion Capture System (Certus)</td>
			<td>NDI, Canada</td>
			<td>http://www.ndigital.com/msci/products/optotrak-certus/</td>
			<td></td>
		</tr>
		<tr>
			<td>Synchronisation device</td>
			<td>VU, NL</td>
			<td>Contact corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Calibration frame</td>
			<td>VU, NL</td>
			<td>Contact corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Greisinger, Germarny</td>
			<td>GTH 175/PT</td>
			<td></td>
		</tr>
		<tr>
			<td>Examination table</td>
			<td>NA</td>
			<td>NA</td>
			<td>Any examination table</td>
		</tr>
		<tr>
			<td>Inclinometer</td>
			<td>Lafayette instrument, USA</td>
			<td>ACU001</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable Footplate</td>
			<td>VU, NL</td>
			<td>Contact corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Torque wrench</td>
			<td>VU, NL</td>
			<td>Contact corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Extendable rod</td>
			<td>VU, NL</td>
			<td>Contact corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Goniometer (Gollehon)</td>
			<td>Lafayette instrument, USA</td>
			<td>1135</td>
			<td></td>
		</tr>
		<tr>
			<td>Triangular shaped beam</td>
			<td>NA</td>
			<td>NA</td>
			<td>Made out a piece of stiff foam</td>
		</tr>
		<tr>
			<td>Lashing straps</td>
			<td>NA</td>
			<td>NA</td>
			<td>Any lashing strap</td>
		</tr>
		<tr>
			<td>Surgical skin marker</td>
			<td>NA</td>
			<td>NA</td>
			<td>Any surgical skin marker</td>
		</tr>
		<tr>
			<td>Ultrasound transmission gel</td>
			<td>Servoson</td>
			<td>NA</td>
			<td>A sticky gel type is recommended</td>
		</tr>
	</tbody>
</table>

3d ultrasound imaging: fast and cost-effective morphometry of musculoskeletal tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

The described 3DUS technique was used to collect morphological data of the GM and VL in four male human cadavers, age at death 76.8 &#177; 7.9 years (mean &#177; SD). The cadavers were obtained via the donation program of the Department of Anatomy and Neuroscience of the Vrije Universiteit Medical Center (VUmc), Amsterdam, the Netherlands. The bodies were preserved using an embalming method aimed at maintaining the morphological features of the tissue39.
Prior to dissection, a 3DUS image was made of the GM and VL according to the methodology described. During dissection, skin, subcutaneous tissue, and fasciae overlaying the GM and VL were removed. A mid-longitudinal section was cut, taking the orientation of the distal aponeurosis into account. Using a caliper, the fascicle length was measured, halfway between the origin and the distal end of the muscle belly. Subsequently, after tenotomy, the muscle belly was dissected and submerged in a calibrated water column. Using ImageJ, the volumes were measured on photographs of the water column with and without the muscle belly, and muscle volume was calculate from the difference40. Fascicle length and volume were measured three times and the mean and standard deviation values were calculated. Criterion validity between the 3DUS method and dissection measurements was tested using a Pearson&#39;s correlation for average fascicle length and muscle volume. Intra-rater reliability of the 3DUS method derived fascicle length and volume measurements was quantified using a two-way mixed model intra-class correlation coefficient (ICC3,3)41, and after logarithmic transformation of the data, the coefficient of variation (CV) was calculated. The validity of fascicle length and muscle volume measurements were confirmed by significant and high correlations (r = 0.998, p &#60;0.01 and r = 0.985, p &#60;0.01, respectively). Intra-rater reliability of the 3DUS method derived measurements of fascicle length and volume was high (ICC3,3 0.983, CV 7.3%, and ICC3,3 0.998, CV 5.4%, respectively). It is concluded that the 3DUS approach presented is a valid and reliable tool for volume and fascicle length assessment of human VL and GM (Table 1).
<img alt="Table 1" src="/files/ftp_upload/55943/55943table1.jpg" /> 
Table 1:&#160;Cadaver Validation Data. C# is Cadaver number, GM is&#160;m. gastrocnemius medialis, VL is&#160;m. vastus lateralis. &#34;Dissection&#34;shows results from the cadaver dissection, and &#34;3DUS&#34; shows the results from the 3DUS image analysis of the cadavers.

Watch this Scientific Journal Video about 3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue at JoVE.com

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

3d-ultrasound-imaging-fast-cost-effective-morphometry-musculoskeletal

Nanyang Technological University

Research

JoVE Journal

Medicine

22.9K Views.  Amsterdam Movement Sciences. 3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

Video: 3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), ...

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse ...

Providing Visual Biofeedback Using Brightness Mode Ultrasound During a Golf Swing

Using ultrasound biofeedback in conjunction with verbal cueing can increase muscle thickness more than verbal cueing alone and may augment traditional rehabilitation techniques in an athletic, physically active population. Brightness mode (B-mode) ultrasound can be applied using frame-by-frame analysis synchronized with video to understand muscle thickness changes during these dynamic tasks. Visual biofeedback with ultrasound has been established in static positions for the muscles of the lateral abdominal wall. However, by securing the transducer to the abdomen using an elastic belt and foam block, biofeedback can be applied during more specific tasks prevalent in lifetime sports, such as golf. To analyze muscle activity during a golf swing, muscle thickness changes can be compared. The thickness must increase throughout the task, indicating that the muscle is more active. This methodology allows clinicians to immediately replay ultrasound videos for patients as a visual tool to instruct proper activity of the muscles of interest. For example, ultrasound can be used to target the external and internal obliques, which play an important role in swinging a golf club or any other rotational sport or activity. This methodology aims to increase oblique muscle thickness during the golf swing. Additionally, the timing of muscle contraction can be targeted by instructing the patient to contract the abdominal muscles at specific time points, such as the beginning of the downswing, with the goal of improving muscle firing patterns during tasks.

Brightness mode ultrasound can be used to provide visual biofeedback of the muscles of the lateral abdominal wall during a golf swing. Post-swing visual and verbal instruction can increase the muscle activation and timing of the external and internal obliques.

Using ultrasound biofeedback in conjunction with verbal cueing can increase muscle thickness more than verbal cueing alone and may augment traditional ...