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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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><tbody><tr><td>MyLab 70</td><td>Esaote (Genoa, Italy)</td><td></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

24.1K 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

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Developmental Biology

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Neuroscience

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Bioengineering

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Hemodynamic Precision in the Neonatal Intensive Care Unit using Targeted Neonatal Echocardiography

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, reliable, and real-time information on developmental hemodynamics in sick newborns. The comprehensive assessment is based on a multiparametric approach that overcomes the reliability issues of individual measurements, allows for earlier recognition of cardiovascular compromise and promotes enhanced diagnostic precision and timely management. TnECHO-driven research has led to an enhanced understanding of the mechanisms of illness and the development of predictive models to identify at-risk populations. This information may then be used to formulate a diagnostic impression and provide individualized guidance for the selection of cardiovascular therapies. TnECHO is based on the expert consultative model in which a neonatologist, with advanced training in neonatal hemodynamics, performs comprehensive and standardized TnECHO assessments. The distinction from point of care ultrasonography (POCUS), which provides limited and brief one-time assessments, is important. Neonatal hemodynamics training is a 1-year structured program designed to optimize image acquisition, measurement analysis, and hemodynamic knowledge (physiology, pharmacotherapy) to support cardiovascular decision-making. Neonatologists with hemodynamic expertise are trained to recognize deviations from normal anatomy and appropriately refer cases of possible structural abnormalities. We provide an outline of neonatal hemodynamics training, the standardized TnECHO imaging protocol, and an example of representative echo findings in a hemodynamically significant patent ductus arteriosus.

Presented here is a protocol to perform comprehensive neonatal echocardiography by trained neonatologists in the neonatal intensive care unit. The trained individuals provide longitudinal assessments of heart function, systemic and pulmonary hemodynamics in a consultative role. The manuscript also describes the requirements to become a fully trained neonatal hemodynamics specialist.

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, ...

A Modified Sonographic Algorithm for Image Acquisition in Life-Threatening Emergencies in the Critically Ill Newborn

The use of routine point-of-care ultrasound (POCUS) is increasing in neonatal intensive care units (NICUs), with several centers advocating for 24 h equipment availability. In 2018, the sonographic algorithm for life-threatening emergencies (SAFE) protocol was published, which allows the assessment of neonates with sudden decompensation to identify abnormal contractility, tamponade, pneumothorax, and pleural effusion. In the study unit (with a consulting neonatal hemodynamics and POCUS service), the algorithm was adapted by including consolidated core steps to support at-risk newborns, aiding clinicians in managing cardiac arrest, and adding views to verify correct intubation. This paper presents a protocol that can be applied in the NICU and the delivery room (DR) in relation to three scenarios: cardiac arrest, hemodynamic deterioration, or respiratory decompensation. 
This protocol can be performed with a state-of-the-art ultrasound machine or an affordable handheld device; the image acquisition protocol is carefully detailed. This method was designed to be learned as a general competence to obtain the timely diagnosis of life-threatening scenarios; the method aims to save time but does not represent a substitute for comprehensive and standardized hemodynamic and radiological analyses by a multidisciplinary team, which might not universally be on call but needs to be involved in the process. From January 2019 to July 2022, in our center, 1,045 hemodynamic consultation/POCUS consults were performed with 25 patients requiring the modified SAFE protocol (2.3%), and a total of 19 procedures were performed. In five cases, trained fellows on call resolved life-threatening situations. Clinical examples are provided that show the importance of including this technique in the care of critical newborns.

Here, we present a protocol that can be applied in the neonatal intensive care unit and the delivery room in relation to three scenarios: cardiac arrest, hemodynamic deterioration, or respiratory decompensation. This protocol can be performed with a state-of-the-art ultrasound machine or an affordable handheld device; an image acquisition protocol is carefully detailed.

The use of routine point-of-care ultrasound (POCUS) is increasing in neonatal intensive care units (NICUs), with several centers advocating for 24 h ...

State of the Art Cranial Ultrasound Imaging in Neonates

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Chapters in this video

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Murine Fetal Echocardiography

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

Hemodynamic Precision in the Neonatal Intensive Care Unit using Targeted Neonatal Echocardiography

A Modified Sonographic Algorithm for Image Acquisition in Life-Threatening Emergencies in the Critically Ill Newborn