The present protocol was part of a study approved by the Institutional Review Board at the University of Central Florida. Informed consent was received from all human participants for the present study. To be included in the study, participants had to be between 18 years and 75 years of age, play golf at least once per month for the past year or once per week for the past 2 months, have played golf for at least 2 years, and have had at least two episodes of low back pain in the past 12 months. The exclusion criteria were balance disorders, current pregnancy, surgery to the low back or lower extremities in the past year, or an open wound in the abdominal area where the transducer must be placed.
1. Ultrasound setup and data collection
<ol>
	<li>Open and turn on the ultrasound device (see Table of Materials) using the On/Off button.</li>
	<li>Press the Patient button on the keyboard to add a new patient and select New Patient on the left side of the screen.
	<ol>
		<li>Enter the desired patient ID number, ensure MSK is selected as the exam type, and click on Register. Exit to begin the exam and enter B-mode. 
		NOTE: Ultrasound settings in MSK Abdominal Preset, B-mode: B color = Tint Map D, Write Zoom Height = 4, Write Zoom Width = 4, Thermal Index = Tls, ATO Level = Low, Focus Number = 2, Focus Number CrossXBeam = 2, Focus Depth = 50, Depth (cm) = 3, Compression = 1, Focus Width = 1, Focus Width CrossXBeam = 1, Line Density = 3, Line Density CrossXBeam = 3, Suppression = 0, Frame Average = 4, From Average CrossXBeam = 2, CrossXBeam 2, CrossXBeam # = Low, CrossXBeam Type = Mean, Edge Enhance = 3, B Steer = 0, Gray Scale Map = Gray Map C, Gain = 34, Dynamic Range = 69, Rejection = 0, Frequency (MHz) = 12.</li>
	</ol>
	</li>
	<li>Position the linear array transducer in the elastic belt by placing the head of the linear transducer through a horizontal slit in the middle of the belt (see Table of Materials). Next, apply one to two foam blocks to secure the transducer in its place.
	<ol>
		<li>Apply ultrasound gel (see Table of Materials) to the linear transducer. Position the transducer on the lateral abdominal wall, approximately 10 cm lateral to the umbilicus8.</li>
		<li>Secure the belt to the participant using the hook and loop fasteners. Ensure the belt is tight enough so that the transducer is secured perpendicular to the lateral abdominal wall8.</li>
		<li>Adjust the depth and gain (brightness) of the ultrasound if necessary to obtain a clear image of the fascial borders and muscle thickness of the external oblique, internal oblique, and transverse abdominis. Ensure that the fascial border of the transverse abdominis is visible on the edge of the screen in this longitudinal view (Figure 1)8. 
		NOTE: The depth will depend on the size of the individual being measured, but ensure that the deep fascial border of the transverse abdominis is visible and the image is clear.</li>
		<li>Once the image is clear, position the patient in a manner that mirrors the task they will complete. For example, if they perform a golf swing, have them stand in their setup position. Press Freeze, then Store to capture a static image, save it to the patient&#39;s exam, and measure in real time or access it later to measure the resting muscle thickness. 
		NOTE: See Figure 1 for an example of a clear image of the lateral abdominal wall using B-mode ultrasound.</li>
	</ol>
	</li>
	<li>Select Freeze again to return to live imaging. Press Store to begin recording in B-mode video. On the bottom-right corner of the screen, check that a timer begins and is highlighted in bright green, indicating that a video is being captured. Instruct the participant to take a full golf swing once the recording has begun.
	<ol>
		<li>Press Store again to end the video and save it to their exam. 
		NOTE: Saved images and videos will appear below the active image, with up to five visible per row. Select the cursor to scroll through or select previous images.</li>
	</ol>
	</li>
	<li>Repeat step 1.4 as many times as necessary for the study. Adjust the positioning of the ultrasound probe within the foam block as needed if the image becomes blurry. 
	NOTE: Typical protocols comparing biofeedback and non-biofeedback conditions use between 3 and 10 repetitions per condition11,17.
	<ol>
		<li>After each trial, review the video for image clarity. If the image becomes anechoic (entirely black) at any point, it indicates that the ultrasound probe moved during the swing. In such a case, exclude the trial and re-measure.</li>
	</ol>
	</li>
	<li>Following each swing, have the patient positioned where they can view the ultrasound screen. Open the most recent trial.
	<ol>
		<li>Provide a brief overview and description of the muscles of interest (for a rotational task, this will include the external and internal obliques). Describe how the muscles should get thicker during the movement and show them their trial, providing education based on each attempt.</li>
		<li>For example, if the obliques stay relatively the same size throughout the task, instruct them to focus during the next attempt to contract their core muscles forcefully throughout the movement instead of passively rotating the trunk. 
		&#8203;NOTE: Emphasis on trunk rotation as a power source during a golf swing is a common trait of higher-skilled golfers18. During rotational movements, it is generally not feasible to provide biofeedback during the task. Therefore, providing feedback following each trial is thought to be acceptable19.</li>
	</ol>
	</li>
	<li>Repeat the biofeedback trials for as many attempts as desired. Provide the ultrasound images as well as verbal cueing or instruction following each attempt, adjusting the cueing as needed.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64333/64333fig01.jpg" /> 
Figure 1: Image of the right lateral abdominal wall during quiet standing. (A) External oblique. (B) Internal oblique. (C) Transverse abdominis. <a href="https://www.jove.com/files/ftp_upload/64333/64333fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Resting image processing
<ol>
	<li>Open the first resting image to measure. Using the cursor, hover over the desired image from the library below the active image and click on Enter.
	<ol>
		<li>Click on Measure once to open the measurement tool. Click on Enter when the cursor is over the superior fascial border of the muscle of interest. 
		NOTE: For the present study, the external and internal obliques were measured as they are the muscles of interest, but one can also measure the transverse abdominis in this view.</li>
		<li>Click on Enter again once the cursor is over the inferior fascial border. 
		NOTE: The length of the measured segment will appear on the bottom-left portion of the screen, measured in centimeters. Record this length in a spreadsheet organized to include the participant or patient name/number and resting muscle thickness.</li>
	</ol>
	</li>
	<li>Repeat steps 2.1.1-2.1.2 for all the images needed to measure for both the external and internal obliques.</li>
</ol>
3. B-Mode video processing
<ol>
	<li>Once the desired number of videos have been captured, open the first video to be processed. Using the cursor, hover over the desired image from the library below the active image and click on the Enter button.</li>
	<li>Adjust the viewing frame within the B-mode video until the first frame is reached. Determine the desired sampling rate. 
	NOTE: Previous literature has used 10% intervals of the total frames from 0%-100% for analysis, but one may wish to use more or less depending on the aims20.</li>
	<li>Once there is the desired frame open, use the Measure tool. Click the button once to open the tool, place the cursor on the superior fascial border of the muscle of interest, and click on Enter to place the first end of the measurement.</li>
	<li>Drag the measurement line to the inferior fascial border of the muscle of interest and click on Enter again to complete the measurement. 
	NOTE: The distance measured will appear on the bottom-left portion of the screen.</li>
	<li>Press Store to save the measured image.</li>
	<li>Record the measurement in a spreadsheet organized to include participant or patient name/number, frame number, and thickness measurements. Return to the original B-mode video frame (step 3.2).</li>
	<li>Scroll to the next frame to be analyzed. For example, if one is sampling 11 time points (10% intervals from 0%-100%) of a swing that captured 100 frames, one will now use frame 10 (i.e., 10/100 corresponds to frame 10 out of 100 total frames).
	<ol>
		<li>Repeat steps 3.3-3.4 to measure the next frame.</li>
	</ol>
	</li>
	<li>Repeat step 3.7 until all the desired frames are measured.</li>
	<li>Move to the next video and repeat steps 3.1-3.8 until all the videos are measured. 
	&#8203;NOTE: See Figure 2 for a compilation of the processed frames throughout phases of a golf swing.</li>
</ol>
4. Activation ratio calculation
NOTE: An activation ratio is commonly used to determine the degree of muscle thickness change8,9,11. The formula for the activation ratio is contracted thickness (cm)/resting thickness (cm).
<ol>
	<li>To determine the activation ratio at a specific time point, measure the thickness by following step 3. Divide this value by the resting image thickness obtained from step 2.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64333/64333fig02.jpg" /> 
Figure 2: Frame-by-frame analysis of a B-mode video on the trail side (right) lateral abdominal wall of a right-handed golfer. EO = external oblique; IO = internal oblique. <a href="https://www.jove.com/files/ftp_upload/64333/64333fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have no relevant disclosures to report.

Providing ultrasound biofeedback following a rotation-based sports movement such as a golf swing can be used to increase the muscle thickness of the lateral abdominal wall. As shown in the representative results, a single trial of ultrasound biofeedback can lead to short-term increases in oblique muscle activity throughout a golf swing.
Previous research has also used B-mode ultrasound secured with an elastic belt during dynamic tasks20. This was measured while individu...

The muscles of the lateral abdominal wall include the external oblique, internal oblique, and transverse abdominis. The external obliques perform lateral flexion and contralateral&#160;trunk rotation, while the internal obliques perform ipsilateral&#160;trunk rotation. The transverse abdominis is the deepest layer of the abdominal musculature, and it functions to increase intra-abdominal pressure and increase the segmental stability of the spine1. The proper function of these muscles is important to reduce low back pain risk and improve athletic performance as core stability allows for increased strength and power through the extremities2.
During sports with an emphasis on trunk rotation, such as golf, tennis, baseball, or softball, there is a high demand for the core muscles. For example, during a golf swing, the obliques on the trail side of the body peak at 64% of the maximal voluntary isometric contraction (MVIC) when measured using surface electromyography, while the lead obliques peak at 54% MVIC3. Trunk rotation is a key contributor to the distance and accuracy of golf shots4. The stresses of the golf swing and the high demand for core muscle activity may contribute to low back pain, which is the most common injury in golf5. Additionally, in elite golfers with low back pain, the timing of external oblique activity is delayed during the golf swing compared to healthy individuals6. Another study using electromyography found golfers with low back pain have an earlier onset of the erector spinae than golfers without low back pain7, suggesting a focus on anterolateral muscles may be beneficial. Therefore, measuring the extent and timing of abdominal muscle activity during a golf swing is important to improve performance and reduce the risk of low back pain.
Rehabilitative ultrasound is commonly used to assess lateral abdominal wall muscles due to the layered nature of this musculature8,9,10. There is no difference in transverse abdominis activation in college golfers with and without low back pain in the supine position or in a more functional golf swing setup position11. However, transverse abdominis activity is only one component of a golf swing, and rotation may be more important for this population. Previous literature has used an elastic belt and foam block to secure the ultrasound transducer to the abdomen, allowing for ultrasound assessment of the core musculature during dynamic movement such as a single leg squat or gait8. Applying ultrasound during dynamic movements has been shown to have acceptable to excellent reliability12. This technique can be applied to measure thickness changes in the lateral abdominal wall during a golf swing or other sport-specific task. While surface electromyography is commonly used to measure the electrical activity of muscles, this is less feasible in the abdominal region. The layered anatomy leads to cross-talk between the muscles and does not allow for a visual representation of the individual muscle layers of the core13. Ultrasound provides an advantage over alternatives like surface electromyography for the core musculature because it allows for a representation of each individual muscle while also giving an image for feedback14.
Since ultrasound provides an image of the muscles of interest in real time, it can also be used as a tool for visual biofeedback. Ultrasound biofeedback has improved the ability to increase the muscle thickness of the transverse abdominis and lumbar multifidus compared to verbal cueing alone15,16. Additionally, in golfers with and without low back pain, real-time ultrasound biofeedback increases transverse abdominis thickness in supine and in the golf-setup position11. Biofeedback training in supine also translates to upright loaded tasks17. More research is needed to determine the required frequency and duration of biofeedback training, as most studies are single-session or short-term training protocols15. Since ultrasound has been applied during functional tasks and there is evidence that golfers can increase deep muscle pre-activation in the setup position, research should next investigate the use of ultrasound biofeedback to increase oblique muscle thickness during the golf swing.
Therefore, this methodology aims to use ultrasound as a feedback mechanism to improve the activation and timing of the abdominal obliques during the golf swing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquasonic 100</td>
			<td>Parker</td>
			<td>BT-025-0037L</td>
			<td>Ultrasound gel</td>
		</tr>
		<tr>
			<td>GE NextGen Logig e Ultrasound Unit</td>
			<td>GE Healthcare</td>
			<td>HR48382AR</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Array Probe</td>
			<td>GE Healthcare</td>
			<td>H48062AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Velcro straps</td>
			<td>VELCRO</td>
			<td></td>
			<td>Fasteners for the elastic belt used to secure the ultrasound transducer</td>
		</tr>
	</tbody>
</table>

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Non-feedback</td>
			<td></td>
			<td></td>
			<td>Biofeedback</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Swing Duration</td>
			<td>External Oblique Thickness (cm)</td>
			<td>Internal Oblique Thickness (cm)</td>
			<td>Combined Oblique Thickness (cm)</td>
			<td>External Oblique Thickness (cm)</stron...

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Providing Visual Biofeedback Using Brightness Mode Ultrasound During a Golf Swing

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 ...

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1.9K Views.  University of Central Florida. 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.

Video: Providing Visual Biofeedback Using Brightness Mode Ultrasound During a Golf Swing

Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using biofeedback of real-time functional magnetic resonance imaging (rt-fMRI) data. Increased activity of this region is seen in relationship with contamination anxiety both in control subjects1 and in individuals with obsessive-compulsive disorder (OCD),2 a relatively common and often debilitating psychiatric disorder involving contamination anxiety. Although many brain regions have been implicated in OCD, abnormality in the orbitofrontal cortex (OFC) is one of the most consistent findings.3, 4 Furthermore, hyperactivity in the OFC has been found to correlate with OCD symptom severity5 and decreases in hyperactivity in this region have been reported to correlate with decreased symptom severity.6 Therefore, the ability to control this brain area may translate into clinical improvements in obsessive-compulsive symptoms including contamination anxiety. Biofeedback of rt-fMRI data is a new technique in which the temporal pattern of activity in a specific region (or associated with a specific distributed pattern of brain activity) in a subject's brain is provided as a feedback signal to the subject. Recent reports indicate that people are able to develop control over the activity of specific brain areas when provided with rt-fMRI biofeedback.7-12 In particular, several studies using this technique to target brain areas involved in emotion processing have reported success in training subjects to control these regions.13-18 In several cases, rt-fMRI biofeedback training has been reported to induce cognitive, emotional, or clinical changes in subjects.8, 9, 13, 19 Here we illustrate this technique as applied to the treatment of contamination anxiety in healthy subjects. This biofeedback intervention will be a valuable basic research tool: it allows researchers to perturb brain function, measure the resulting changes in brain dynamics and relate those to changes in contamination anxiety or other behavioral measures. In addition, the establishment of this method serves as a first step towards the investigation of fMRI-based biofeedback as a therapeutic intervention for OCD. Given that approximately a quarter of patients with OCD receive little benefit from the currently available forms of treatment,20-22 and that those who do benefit rarely recover completely, new approaches for treating this population are urgently needed.

Here we present a method for training people to control a brain area involved in contamination anxiety and for probing the relationship between contamination anxiety and brain connectivity patterns.

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using ...

Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily life, despite similar visual deficits. We assume this could be due to variable development of compensatory strategies such as altered visual scanning behavior. Scanning compensatory therapy (SCT) is studied as part of the visual training after infarction next to vision restoration therapy. SCT consists of learning to make larger eye movements into the blind field enlarging the visual field of search, which has been proven to be the most useful strategy1, not only in natural search tasks but also in mastering daily life activities2. Nevertheless, in clinical routine it is difficult to identify individual levels and training effects of compensatory behavior, since it requires measurement of eye movements in a head unrestrained condition. Studies demonstrated that unrestrained head movements alter the visual exploratory behavior compared to a head-restrained laboratory condition3. Martin et al.4 and Hayhoe et al.5 showed that behavior demonstrated in a laboratory setting cannot be assigned easily to a natural condition. Hence, our goal was to develop a study set-up which uncovers different compensatory oculomotor strategies quickly in a realistic testing situation: Patients are tested in the clinical environment in a driving simulator. SILAB software (Wuerzburg Institute for Traffic Sciences GmbH (WIVW)) was used to program driving scenarios of varying complexity and recording the driver's performance. The software was combined with a head mounted infrared video pupil tracker, recording head- and eye-movements (EyeSeeCam, University of Munich Hospital, Clinical Neurosciences).
The positioning of the patient in the driving simulator and the positioning, adjustment and calibration of the camera is demonstrated. Typical performances of a patient with and without compensatory strategy and a healthy control are illustrated in this pilot study. Different oculomotor behaviors (frequency and amplitude of eye- and head-movements) are evaluated very quickly during the drive itself by dynamic overlay pictures indicating where the subjects gaze is located on the screen, and by analyzing the data. Compensatory gaze behavior in a patient leads to a driving performance comparable to a healthy control, while the performance of a patient without compensatory behavior is significantly worse. The data of eye- and head-movement-behavior as well as driving performance are discussed with respect to different oculomotor strategies and in a broader context with respect to possible training effects throughout the testing session and implications on rehabilitation potential.

Patients with visual deficits after stroke report about different constraints in daily life most likely due to variable compensatory strategies, which are difficult to differentiate in clinical routine. We present a clinical set-up which allows measurement of different compensatory head- and eye-movement-strategies and evaluating their effects on driving performance.

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

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 ...

Muscle Function Obtained with Motion Mode Ultrasound and Surface Electromyography during Core Endurance Exercise

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured between fascial borders at a given time point during an exercise. This selected time point produces a one-dimensional image resulting in real-time, live observation of anatomy. Ultrasound used during functional movement can be referred to as dynamic ultrasound; this is feasible and reliable with the use of a linear transducer, elastic belt, and foam block to secure consistent transducer placement. The lateral abdominal wall is commonly investigated using ultrasound due to the overlapping nature of the muscles. Surface electromyography (sEMG) can complement M-mode ultrasound imaging because it measures the electrical representation of muscle activation. There is minimal evidence using M-mode ultrasound and sEMG simultaneously during core exercise. Exercises that challenge the core musculature involve both isometric holds (e.g., side plank), as well as oscillatory extremity movements (e.g., dead bug). In this study, both instruments will be used simultaneously to measure core muscle function during exercise. Ultrasound measurements will be obtained using a linear transducer and ultrasound unit, and sEMG measurements will be acquired from a wireless sEMG system. To make comparisons between participants and exercises, normalization methods using static, exercise starting positions for both instruments will be used. An activation ratio will be used for ultrasound and calculated by dividing the contracted thickness (thickness during a time point of exercise) by the rested (starting position) thickness. Muscle thickness will be measured in centimeters from the superior inferior fascial border to inferior superior fascial border. These methods aim to offer an innovative and practical measurement of muscle function with M-mode ultrasound and sEMG during core endurance exercises.

This protocol uses motion mode ultrasound and surface electromyography simultaneously to measure muscle function of the core. Muscle thickness and activation of the local stabilizers (e.g., transverse abdominis, internal oblique) and global movers (e.g., external oblique) is achievable during specific time points of the side plank and dead bug exercises.

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured ...

Continuous Venous-Arterial Doppler Ultrasound During a Preload Challenge

A preload challenge (PC) is a clinical maneuver that, first, increases the cardiac filling (i.e., preload) and, second, calculates the change in cardiac output. Fundamentally, a PC is a bedside approach for testing the Frank-Starling-Sarnoff (i.e., "cardiac function") curve. Normally, this curve has a steep slope such that a small change in the cardiac preload generates a large change in the stroke volume (SV) or cardiac output. However, in various disease states, the slope of this relationship flattens such that increasing the volume into the heart leads to little rise in the SV. In this pathological scenario, additional cardiac preload (e.g., intravenous fluid) is unlikely to be physiologically effective and could lead to harm if organ congestion evolves. Therefore, inferring both the cardiac preload and output is clinically useful as it may guide intravenous (IV) fluid resuscitation. Accordingly, the goal of this protocol is to describe a method for contemporaneously tracking the surrogates of cardiac preload and output using a novel, wireless, wearable ultrasound during a well-validated preload challenge.

The Frank-Starling-Sarnoff curve is clinically important and describes the relationship between cardiac preload and output. This report illustrates a novel method of simultaneous jugular venous and carotid arterial Doppler velocimetry as transient surrogates of cardiac preload and output, respectively; this approach is enabled by wireless, wearable Doppler ultrasound.

A preload challenge (PC) is a clinical maneuver that, first, increases the cardiac filling (i.e., preload) and, second, calculates the change in cardiac ...

Point-of-Care Ultrasound: A Review of Ultrasound Parameters for Predicting Difficult Airways

Airway management remains a crucial part of perioperative care. The conventional approach to assessing potentially difficult airways emphasizes the LEMON method, which looks for and evaluates the Mallampati classification, signs of obstruction, and neck mobility. Clinical findings help predict a higher likelihood of difficult tracheal intubation, but no clinical result reliably excludes difficult intubation. Ultrasound as an adjunct to clinical examination can provide the clinician with a dynamic anatomical airway assessment, which is impossible with clinical examination alone. In the hands of anesthesiologists, ultrasound is becoming more popular in the perioperative period. This method is particularly applicable for identifying proper endotracheal tube positioning in specific patient populations, such as those who are morbidly obese and patients with head and neck cancer or trauma. The focus is on identifying the normal anatomy, correctly positioning the endotracheal tube, and refining the parameters that predict difficult intubation. Several ultrasound measurements are clinical indicators of difficult direct laryngoscopy in the literature. A meta-analysis revealed that the distance from the skin to the epiglottis (DSE) is most associated with a difficult laryngoscopy. An ultrasound of the airway could be applied in routine practice as an adjunct to the clinical examination. A full stomach, rapid sequence intubation, gross visual anatomical abnormalities, and restricted neck flexibility prevent using ultrasound to assess the airway. The airway evaluation is performed with a linear array transducer of 12-4 MHz, with the patient in the supine position, with no pillow, and with the head and neck in a neutral position. The central axis of the neck is where the ultrasound parameters are measured. These image acquisitions guide the standard ultrasound examination of the airway.

A point-of-care ultrasound (POCUS) is a simple, non-invasive, and portable tool that enables dynamic airway assessment. Several studies have attempted to determine the role of ultrasound parameters as an adjunct to clinical examination in predicting difficult laryngoscopies.

Airway management remains a crucial part of perioperative care. The conventional approach to assessing potentially difficult airways emphasizes the LEMON ...

Reduction of Radiation Exposure during Endovascular Treatment of Peripheral Arterial Disease Combining Fiber Optic RealShape Technology and Intravascular Ultrasound

Vascular surgeons and interventional radiologists face chronic exposure to low-dose radiation during endovascular procedures, which may impact their health in the long term due to their stochastic effects. The presented case shows the feasibility and efficacy of combining Fiber Optic RealShape (FORS) technology and intravascular ultrasound (IVUS) to reduce operator exposure during the endovascular treatment of obstructive peripheral arterial disease (PAD). 
FORS technology enables real-time, three-dimensional visualization of the full shape of guidewires and catheters, embedded with optical fibers that use laser light instead of fluoroscopy. Hereby, radiation exposure is reduced, and spatial perception is improved while navigating during endovascular procedures. IVUS has the capacity to optimally define vessel dimensions. Combining FORS and IVUS in a patient with iliac in-stent restenosis, as shown in this case report, enables passage of the stenosis and pre- and post-percutaneous transluminal angioplasty (PTA) plaque assessment (diameter improvement and morphology), with a minimum dose of radiation and zero contrast agent. The aim of this article is to describe the method of combining FORS and IVUS stepwise, to show the potential of merging both techniques in view of reducing radiation exposure and improving navigation tasks and treatment success during the endovascular procedure for the treatment of PAD.

Described here is a stepwise method of combining Fiber Optic RealShape technology and intravascular ultrasound to show the potential of merging both techniques, in view of the reduction of radiation exposure and improvement of navigation tasks and treatment success during an endovascular procedure for the treatment of peripheral arterial disease.

Vascular surgeons and interventional radiologists face chronic exposure to low-dose radiation during endovascular procedures, which may impact their ...

Ultrasound in Evaluating Diaphragm Dysfunction and Monitoring Treatment

Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound

The diaphragm is the main component of the respiratory muscle pump. Diaphragm dysfunction can cause dyspnea and exercise intolerance, and predisposes affected individuals to respiratory failure. In mechanically ventilated patients, the diaphragm is susceptible to atrophy and dysfunction through disuse and other mechanisms. This contributes to failure to wean and poor long-term clinical outcomes. Point-of-care ultrasound provides a valid and reproducible method for evaluating diaphragm thickness and contractile activity (thickening fraction during inspiration) that can be readily employed by clinicians and researchers alike. This article presents best practices for measuring diaphragm thickness and quantifying diaphragm thickening during tidal breathing or maximal inspiration. Once mastered, this technique can be used to diagnose and prognosticate diaphragm dysfunction, and guide and monitor response to treatment over time in both healthy individuals and acute or chronically ill patients.

Author Spotlight: Unraveling the Impact of Mechanical Ventilation on Diaphragm Function and Patient Outcomes 

Diaphragm thickness and function can be assessed in healthy individuals and critically ill patients using point-of-care ultrasound. This technique offers an accurate, reproducible, feasible, and well-tolerated method for evaluating diaphragm structure and function.

The diaphragm is the main component of the respiratory muscle pump. Diaphragm dysfunction can cause dyspnea and exercise intolerance, and predisposes ...