We thank the nurses (appearing on film) for supporting the neonates. 
We thank J. Hagoort, MA, linguist, Department of Pediatric Surgery, Erasmus MC-Sophia Children&#8217;s Hospital, Rotterdam, the Netherlands, for reading and correcting the manuscript.

NOTE: This protocol follows the guidelines of the local human research ethics committee.
1. General Considerations
NOTE: General considerations regarding equipment, data storage and safety are addressed in the Discussion.
<ol>
	<li>Obtain images using a high-resolution, real-time, mobile 2D ultrasound machine with multiple transducers with a band of frequencies (see Discussion). Typically, obtain images of good quality using a probe with a frequency of 7.5 to 8.5 MHz.</li>
</ol>
2. Preparation of the CUS Exam
<ol>
	<li>Schedule the CUS examination so that it does not coincide with other procedures such as blood sampling.</li>
	<li>Ensure that a health care worker or a parent is available to support and/or comfort the neonate during the examination, using strategies such as those according to the principles of Newborn Individualized Developmental Care and Assessment Program6.</li>
</ol>
3. Examination Through Anterior Fontanel
<ol>
	<li>Install the ultrasound machine along the incubator or cot.</li>
	<li>Apply transducer gel to the probe to ensure good contact between the probe and the skin. Consider warming the gel before use.</li>
	<li>Start imaging through the anterior fontanel with a convex probe in B-mode. Place the probe in the middle of the fontanel with the marker on the probe turned to the right side of the neonate. The left side of the brain will then be displayed on the right side of the monitor. 
	NOTE: Imaging through the anterior fontanel can be performed with the neonate in any position3. For research purposes it may be necessary to strive for a standard head position.
	<ol>
		<li>Record images in at least five coronal and five sagittal planes. In the first image adjust depth, gain and time gain compensation settings to produce an image filling the sector, containing the cranial contours, avoiding too bright or dark images and aiming for an equilibrium between reflections from nearby and deeper structures.</li>
		<li>Coronal planes 
		NOTE: Try to obtain perfectly symmetrical images. When lesions near the frontal lobe convexity are suspected, consider recording specific oblique coronal sections, so that one hemisphere is displayed in better detail (Figure 1).
		<ol>
			<li>For the coronal prefrontal image, angle the probe forwards to visualize the frontal lobes, anterior to the frontal horns of the lateral ventricles at the level of the olfactory sulci.</li>
			<li>For the coronal image at the level of Monro, angle the probe to visualize the coronal section anterior to the tela choroidea to depict the frontal horns of the lateral ventricles, cavum septi pellucidi, corpus callosum, sulcus cinguli. Note the echogenicity of parts of the basal ganglia.</li>
			<li>For the coronal image at the level of thalamus, angle the probe backwards to identify the lateral fissures, tela choroidea in the roof of the third ventricle and temporal lobes. Note the echogenicity of the thalamus (especially ventrolateral nuclei) in relation to the basal ganglia. Note that network injury in pulvinar may be visualized in an extra coronal section just in front of the atria.</li>
			<li>For the coronal image at the level of atria, visualize the lateral ventricles at the level of the choroid plexus. Identify the temporal lobes and cerebellar hemispheres. Note the echogenicity of periventricular white matter compared to choroid plexus. Compare optic radiation with the normal hyperechoic areas above and lateral to the atria in preterm neonates.</li>
			<li>For the coronal parieto-occipital image, angle the probe backwards to the level of the parieto-occipital sulcus to identify the parietal and occipital lobes.</li>
		</ol>
		</li>
		<li>Sagittal planes
		<ol>
			<li>Rotate the probe 90&#176; with the marker on the probe facing towards the neonate&#8217;s face. The anterior part of the brain will be displayed on the left side of the monitor. Record images at the level of the following structures (Figure 2).</li>
			<li>For the midsagittal image, visualize the corpus callosum, cavum septi pellucidi (CSP), third and fourth ventricle, vermis, cisterna magna, pons and mesencephalon. Note the presence of cavum Vergae and cavum veli interpositi7.</li>
			<li>For the parasagittal image through one gangliothalamic ovoid (e.g., the right), angle the probe sideways for a parasagittal view through the lateral ventricle. Identify the choroid plexus and note the echogenicity of thalamus and basal ganglia. The scanned side for parasagittal planes should be appropriately indicated with text tools.</li>
			<li>For the parasagittal insular image, angle the probe further lateral through the insula. Identify the lateral fissure and frontal-, temporal-, parietal- and occipital lobes.</li>
			<li>Repeat parasagittal images for the contralateral side (i.e., the left).</li>
		</ol>
		</li>
		<li>Color Doppler
		<ol>
			<li>Continue imaging through the anterior fontanel with a convex probe using color Doppler. Consider evaluating flow velocities in cerebral arteries and veins and obtaining derived indices. 
			NOTE: Resistive index (RI) is defined as peak systolic velocity &#8211; end diastolic velocity / peak systolic velocity. RI is angle-independent, absolute velocity values are not8-10. RI is not similar in arteries of different caliber. Serial measurements are only useful if performed in the exact same location of the same vessel.</li>
		</ol>
		</li>
		<li>Record images in coronal planes of the following vessels (Figure 3):
		<ol>
			<li>Visualize the transverse sinuses at the level of the cerebellum. If only one or none the transverse sinuses is visualized, try lowering the pulse repetition frequency (PRF). If then still only one or none of the transverse sinuses can be identified through the anterior fontanel, use a high frequency linear probe for visualization through the mastoid fontanel (see section 4.4.2).</li>
			<li>Visualize the circle of Willis with internal carotid arteries, middle cerebral arteries and anterior cerebral arteries at the level of the frontal horns of the lateral ventricles. Distinguishing the left and right anterior cerebral arteries is often challenging, but is usually unnecessary. Identify the striatal candelabra of arteries.</li>
			<li>Angle the probe backwards to visualize the basilar artery with adjacent jugular veins.</li>
			<li>Angle even more backwards to visualize the internal cerebral and thalamostriate veins.</li>
		</ol>
		</li>
		<li>Record an image in the sagittal plane of one anterior cerebral artery (Figure 4). Assess flow velocity and RI at a specific part of this vessel (usually below the genu of the corpus callosum). Near the midline velocities in the internal cerebral vein can be readily measured.</li>
		<li>Using a high frequency linear probe in a coronal plane in the anterior fontanel, identify the superior sagittal sinus. If this should fail, reduce the amount of pressure applied with the probe to the fontanel. 
		NOTE: The linear probe can be used for detailed visualization of superficial structures (meninges, arachnoid and subdural space, cortex). Tangential vessels are in the subarachnoid space. Ideally, Doppler imaging as described in the previous steps will be performed during the first CUS examination of the neonate. During follow-up examination some of the steps may be skipped. In case of suspected cerebral sinovenous thrombosis Doppler imaging as described in steps 3.3.5.1, 3.3.7 and 4.4.2 should be performed.</li>
	</ol>
	</li>
</ol>
4. Examination Through Alternative Acoustic Windows
<ol>
	<li>Next, continue examination through alternative acoustic windows.</li>
	<li>Consider recording images through the lambdoid (posterior) fontanel using a convex probe (Figure 5). The posterior fontanel is located at the junction of the sagittal and lambdoid sutures3,11. Image through the posterior fontanel by placing the neonate in the lateral decubitus position. 
	NOTE: In many premature infants satisfactory images may also be obtained through the posterior aspect of the sagittal suture with the infant in a supine position3.
	<ol>
		<li>Position the probe in the middle of the posterior fontanel for a sagittal view. Angle the probe slightly off midline to identify the body of the lateral ventricle and its occipital horn. Turn the probe roughly 90&#176; to obtain a coronal view. Identify the occipital horns of the lateral ventricles.</li>
	</ol>
	</li>
	<li>Consider recording images through the lateral (temporal) window using a convex or linear probe above the ear (Figure 6).
	<ol>
		<li>If necessary, obtain images through the lateral window to allow a detailed view of the brainstem12. Place the probe horizontally above and slightly in front of the ear. Move the probe until the cerebral peduncles are visualized. 
		NOTE: Other structures that can be identified are the third ventricle, aqueduct and temporal lobes. Using color Doppler, the circle of Willis can be visualized.</li>
	</ol>
	</li>
	<li>Record images through the mastoid fontanel (Figure 7). The mastoid fontanel is located behind the ear, at the junction of the temporal, parietal and occipital bones4. Image through the mastoid fontanel by placing the neonate in a lateral decubitus position3. 
	NOTE: In our experience, neonates often show signs of discomfort when images through the mastoid fontanel are obtained. Therefore, it would be best to do this after imaging through the anterior fontanel and other acoustic windows. We hypothesize that this discomfort could be caused by the mechanism of auditory response to pulses of radiofrequency energy13.
	<ol>
		<li>Image through mastoid fontanel using a convex probe. Place the probe parallel to the ear to obtain a coronal view. Sweep the probe back and forth to identify the cerebellar hemispheres, vermis, third and fourth ventricle, pons and cisterna magna. In small preterms the contralateral cerebellar hemisphere may be well depicted.</li>
		<li>Image through mastoid fontanel using a linear probe. If (one of) the transverse sinuses cannot be identified through the anterior fontanel, use a high frequency linear probe for visualization through the mastoid fontanel. Place the probe parallel to the ear lobe to obtain a coronal view.
		<ol>
			<li>Identify the cerebellar hemisphere and fourth ventricle. Using color Doppler, identify the transverse and sigmoid sinus, tentorial sinus and emissary veins.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Consider additional visualization of the posterior fossa through foramen magnum14.</li>
</ol>

<ol>
	<li>Plaisier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+of+routine+early+MRI+in+preterm+infants.">Safety of routine early MRI in preterm infants.</a> Pediatric Radiology. 42 (10), 1205-1211 (2012).</li><li>Raets, M. M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+Cranial+US+for+Detection+of+Cerebral+Sinovenous+Thrombosis+in+Preterm+Infants.">Serial Cranial US for Detection of Cerebral Sinovenous Thrombosis in Preterm Infants.</a> Radiology. 269 (3), 879-886 (2013).</li><li>Di Salvo, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+view+of+the+neonatal+brain:+Clinical+utility+of+supplemental+neurologic+US+imaging+windows.">A new view of the neonatal brain: Clinical utility of supplemental neurologic US imaging windows.</a> Radiographics. 21 (4), 943-955 (2001).</li><li>Enriquez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mastoid+fontanelle+approach+for+sonographic+imaging+of+the+neonatal+brain.">Mastoid fontanelle approach for sonographic imaging of the neonatal brain.</a> Pediatric Radiology. 36 (6), 532-540 (2006).</li><li>Luna, J. A., Goldstein, R. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Atlas of Neonatal Brain Sonography. , (2010).</li><li>Gray, P. H., Griffin, E. A., Drumm, J. E., Fitzgerald, D. E., Duignan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Wave+Doppler+Ultrasound+in+Evaluation+of+Cerebral+Blood-Flow+in+Neonates.">Continuous Wave Doppler Ultrasound in Evaluation of Cerebral Blood-Flow in Neonates.</a> Archives of Disease in Childhood. 58 (9), 677-681 (1983).</li><li>Seibert, J. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+white+matter+diseases+of+prematurity.">Magnetic resonance imaging of white matter diseases of prematurity.</a> Neuroradiology. 52 (6), 505-521 (2010).</li><li>Wezel-Meijler, G., Steggerda, S. J., Leijser, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+Ultrasonography+in+Neonates:+Role+and+Limitations.">Cranial Ultrasonography in Neonates: Role and Limitations.</a> Seminars in Perinatology. 34 (1), 28-38 (2010).</li><li>Leijser, L. M., de Vries, L. S., Cowan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+cerebral+ultrasound+effectively+in+the+newborn+infant.">Using cerebral ultrasound effectively in the newborn infant.</a> Early Human Development. 82 (12), 827-835 (2006).</li><li>Houston, L. E., Odibo, A. O., Macones, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+safety+of+obstetrical+ultrasound:+a+review.">The safety of obstetrical ultrasound: a review.</a> Prenatal Diagnosis. 29 (13), 1204-1212 (2009).</li><li>Torloni, M. 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The authors have nothing to disclose.

We describe and demonstrate a state-of-the-art approach for neonatal Doppler CUS. In experienced hands, this is an excellent tool for safe, serial bedside neonatal brain imaging. In many NICUs the described possibilities are not optimally used. Adding Doppler studies allows for screening of patency of intracranial arteries and veins. Flow velocities can be evaluated and indices obtained. Doppler CUS permits detection of cerebral sinovenous thrombosis at an early stage in the fragile transverse to sigmoid sinus angle, all...

Since its clinical introduction in the late 1970&#8217;s cranial ultrasound (CUS) has been widely used for detecting congenital anomalies and acquired brain lesions during the neonatal period. In many neonatal intensive care units (NICUs), CUS has become indispensable in the care for critically ill neonates. Major advantages are its relatively low cost and the fact that it can be performed at bedside, even when a patient is unstable. In addition it is radiation-free and allows for serial imaging. Another technique often used for neuroimaging in critically ill neonates is magnetic resonance imaging (MRI). MRI provides excellent image quality, but its clinical use in NICU&#8217;s is currently limited because of logistic and safety issues1. 
Over time, quality of CUS has drastically improved, with advancing technique leading to higher resolution, faster image processing and digital display and back-up. Important brain structures can be adequately visualized using optimal settings. Traditionally, images are obtained through the anterior fontanel. This approach is less suitable for evaluation of infratentorial structures because they are located far away from the transducer and the highly echoic tentorium impedes their assessment. Use of high-frequency linear transducers through alternative acoustic windows and adapted settings also provides access to these brain regions. Examples of these supplemental acoustic windows are the lambdoid (posterior), mastoid and lateral (temporal) fontanels. So far, however, only few NICUs use these additional acoustic windows routinely2-5. Doppler techniques can be used for screening patency of intracranial vessels. Flow velocities and indices in cerebral arteries can also be obtained. Some manufacturers now provide hardware to visualize flow around 2 cm/sec (Raets, et al., unpublished data). Small vessels are well displayed: medullary trunks and channels, subependymal veins tributing to the thalamostriate veins, and perforator arteries. 
We present our approach of neonatal CUS, focusing on the use of different transducers, multiple acoustic windows and Doppler techniques. Neonatologists and radiologists use this approach in daily clinical practice but is also suitable for research purposes. In the practical part of the video we demonstrate bedside use in the NICU.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MyLab 70</td>
			<td style="width:191px;">Esaote (Genoa, Italy)</td>
			<td style="width:119px;"></td>
			<td>Ultrasound system</td>
		</tr>
	</tbody>
</table>

state of the art cranial ultrasound imaging in neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Examples of imaging made according to the described protocol are presented in Figures 1-7. Images should be carefully interpreted by an experienced observer. Symmetrical imaging is necessary for adequate interpretation of coronal images made through the anterior fontanel (Figure 1). Any suspected lesion should be visualized in both a coronal and (mid)sagittal plane or by visualization through an acoustic window other than the anterior fontanel. Use Color Doppler for visu...

Watch this Scientific Journal Video about State of the Art Cranial Ultrasound Imaging in Neonates at JoVE.com

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

state-of-the-art-cranial-ultrasound-imaging-in-neonates

Research

JoVE Journal

Medicine

24.0K Views.  Erasmus MC-Sophia Children's Hospital. Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique. 

Video: State of the Art Cranial Ultrasound Imaging in Neonates

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

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  ...

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

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 ...

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of prion disease transmission and pathogenesis. Vertical transmission is an area of interest among researchers studying infectious diseases, including prion disease, and these investigations require efficient methods for evaluating the effects of maternal infection on reproductive performance. Ultrasonographic examination is a well-established tool for diagnosing pregnancy and assessing fetal health in many animal species1-7, including several species of farmed cervids8-19, however this technique has not been described in Reeves' muntjac deer. Here we describe the application of transabdominal ultrasound to detect pregnancy in muntjac does and to evaluate fetal growth and development throughout the gestational period. Using this procedure, pregnant animals were identified as early as 35 days following doe-buck pairing and this was an effective means to safely monitor the pregnancy at regular intervals. Future goals of this work will include establishing normal fetal measurement references for estimation of gestational age, determining sensitivity and specificity of the technique for diagnosing pregnancy at various stages of gestation, and identifying variations in fetal growth and development under different experimental conditions.

Transabdominal ultrasound is described as an effective, noninvasive means for assessing reproductive status in Reeves' muntjac deer. These methods can be used to achieve early pregnancy diagnosis and to evaluate fetal viability. Future applications of this technique include estimation of gestational age and effects of maternal disease on fetal development.

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of ...

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 ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

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.