We thank J. Hagoort for reading and correcting the manuscript.

Dieses Protokoll folgt den Richtlinien des örtlichen menschlichen Forschungsethikkommission. 1. Vorbereitung <ol><li> Zeitplan der Mikrozirkulationsmessung, so dass es nicht mit anderen Verfahren wie Blutentnahme überein. In Neugeborenen ist es am besten nach der Fütterung durchgeführt. Dies verhindert, dass Bewegung und die Messung zu erleichtern. </li><li> Stellen Sie sicher, dass eine Krankenschwester oder ein Elternteil betreut Unterstützung und Komfort das Neugeborene während der Untersuchung unter Verwendung der Prinzipien der Neugeborenen Individualisierte Entwicklungspflege und Assessment Program 12. Hinweis: Obwohl Messungen können von einer einzigen Person durchgeführt werden, ist es dringend empfohlen zu haben, eine zweite Person helfen. Eine Halterung für die Kamera und ist an das Neugeborene konzentriert, während die andere arbeitet, den Computer und Software. Nach unserer Erfahrung ergibt sich eine höhere Bildqualität und eine kürzere Dauer des Verfahrens. </li><li> Wenn der klinische Zustand des Neugeborenen Genehmigungen, legen Sie dieNeugeborene in Rückenlage. Mikrozirkulation Bildgebung kann in Bauchlage durchgeführt werden, aber dies erfordert mehr Geschick und Geduld. </li><li> Sicherstellen, dass die Körpertemperatur des Frühgeborenen innerhalb geeigneten Bereich (36,5-37,5 Grad Celsius). </li></ol> 2. Verfahren <ol><li> Installieren Sie das Gerät auf dem Inkubator. Sicherzustellen, dass der Inkubator in der richtigen Höhe. </li><li> Legen Sie die Einweg-Schutzkappe an der Kamera. </li><li> Gelten Gel, Öl oder Salzlösung über die Spitze der Sonde; dies wird Ihnen helfen zu glätten den Kontakt zwischen Sonde und die Haut. </li><li> Legen Sie die Kamera auf dem ventromedialen Seite der Säuglinge Oberarm. Um den Fokus-Artefakte zu vermeiden, stellen Sie sicher, dass die Sonde senkrecht zur Haut. Dies kann Neupositionierung der Säuglinge Arm erfordern. Anmerkung: Die ventromedialen Seite des oberen Arms ist der primäre Standort, um die Mikrozirkulation der Haut zu messen. Dieser Ort hat wenig Lanugohaare und ist daher weniger anfällig für Artefakte. Es ist sehr leicht zu erreichenwenn der Patient in Rückenlage positioniert. </li><li> Um die Gesamtlänge des Verfahrens zu minimieren, Zeit zu gewinnen, indem du die optimale Tiefenschärfe (Abbildung 3), während die Suche die Position mit den wenigsten Artefakte. Hinweis: Tiefenschärfe hängt in erster Linie auf die postnatale Alter eher als Schwangerschaftsalter. Die durchschnittliche Tiefe des Fokus in der ersten Woche des Lebens ist von 0 bis 80 &amp; mgr; m. Jenseits aufgrund Reifung der Haut, die Fokustiefe steigt schnell mit Durchschnittswerten von 80 bis 200 &amp; mgr; m zwischen 1-4 Wochen postnatalen Alter (Abbildung 2). In Termin geboren Neugeborene die durchschnittliche Tiefe des Fokus ist von 80 bis 160 &amp; mgr; m bei der Geburt. </li><li> Stabilisieren Sie die Sonde, um Bewegungsartefakte zu vermeiden. Um dies zu tun, seien Sie die Ellbogen auf den Inkubator-Fenster und das Handgelenk neben dem Neugeborenen. Alternativ zu positionieren Sie die Sonde neben dem Neugeborenen auf einem Kissen. </li><li> Vermeiden Sie Druckartefakten, indem Sie die Kamera nur die geringste Berührung mit der Haut. Druckartefakten können rwährend die Bildaufnahme ecognized wenn es hin- und hergehende Strömung in Gefäßen oder wenn große Behälter nicht perfundierten während es gute Fließ in kleinen Gefäßen. Auch wenn die Strömungsmuster in der gesamten Bildschirm identisch, passen sie von Druckartefakten. </li><li> Nehmen Sie Videos mit einer Mindestdauer von 5 Sekunden. </li><li> Nach einer erfolgreichen Erfassung, bewegen Sie die Kamera an eine andere Stelle auf dem Oberarm. Hinweis: Es wird empfohlen, insgesamt 5 erfassen - 10 Videos in 3 - 5 verschiedenen Standorten, wie einige Artefakte sind nur bei Offline-Analyse, was bedeutet, dass das Video in Frage ist nicht zur Analyse nutzbaren anerkannt. </li><li> Von der Haut mit einem kleinen Gaze Entfernen Sie das Gel, Öl oder Kochsalzlösung. </li></ol> 3. Offline-Analyse <ol><li> Schneiden Sie das Video, wenn es eine deutliche Bewegung, die Analyse erschwert. Gehen Sie zum Abschnitt &quot;Tools&quot; und verwenden Sie die Schaltfläche &#39;Editor&#39;. Wählen Sie den Bildintervall die für Analyse und klicken Sie auf den &quot;Crop Video &#39;button. Hinweis: Videos sind akzeptabel, wenn Bewegung innerhalb ½ des Feldes Ansicht 13. </li><li> Wählen Sie das zugeschnittene Video-und stabilisieren. Gehen Sie zum Abschnitt &quot;Tools&quot; und verwenden Sie die Schaltfläche &quot;Analysieren&quot;. Klicken Sie auf die &#39;stabilisieren&#39; button. Hinweis: Alle Filme müssen stabilisiert, bevor die automatische Analyse durchgeführt werden kann. </li><li> Wählen Sie das stabilisierte Video. Gehen Sie zum Abschnitt &quot;Analyse&quot; und klicken Sie auf &#39;Erkennen&#39; Button. Achten Sie darauf, die Optionen &#39;Kapillaren &quot;und&quot; Schiffe &quot;werden hervorgehoben. </li><li> Nach der Erkennung (Abbildung 4), klicken Sie auf den &quot;CNA&quot; oder &quot;De Backer-Button für eine vollständige Mikrozirkulations Bericht. Dieser Bericht enthält die am häufigsten verwendeten Outcome-Parameter wie die Gesamtgefäßdichte (TVD), perfundierten Gefäßdichte (PVD) und der Anteil der durchströmten Gefäßen (PPV). Anmerkung: Alternativ können Videos exportiert offline manuell analysiert werden kann. Diese Option finden Sie im Abschnitt &quot;Tools&quot; finden. SeSELECT die Option &quot;Exportieren&quot; und klicken Sie auf &#39;AVA Export &quot;-Taste. </li></ol>

<ol>
	<li>Groner, W., 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=Orthogonal+polarization+spectral+imaging:+a+new+method+for+study+of+the+microcirculation.">Orthogonal polarization spectral imaging: a new method for study of the microcirculation.</a> Nat Med. 5 (10), 1209-1212 (1999).</li><li>Goedhart, P. T., Khalilzada, M., Bezemer, R., Merza, J., Ince, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sidestream+Dark+Field+(SDF)+imaging:+a+novel+stroboscopic+LED+ring-based+imaging+modality+for+clinical+assessment+of+the+microcirculation.">Sidestream Dark Field (SDF) imaging: a novel stroboscopic LED ring-based imaging modality for clinical assessment of the microcirculation.</a> Opt Express. 15 (23), 15101-15114 (2007).</li><li>Sherman, H., Klausner, S., Cook, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incident+dark-field+illumination:+a+new+method+for+microcirculatory+study.">Incident dark-field illumination: a new method for microcirculatory study.</a> Angiology. 22 (5), 295-303 (1971).</li><li>Trzeciak, S., 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=Early+microcirculatory+perfusion+derangements+in+patients+with+severe+sepsis+and+septic+shock:+relationship+to+hemodynamics,+oxygen+transport,+and+survival.">Early microcirculatory perfusion derangements in patients with severe sepsis and septic shock: relationship to hemodynamics, oxygen transport, and survival.</a> Ann Emerg Med. 49 (1), 88-98 (2007).</li><li>Sakr, Y., Dubois, M. J., De Backer, D., Creteur, J., Vincent, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+microcirculatory+alterations+are+associated+with+organ+failure+and+death+in+patients+with+septic+shock.">Persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock.</a> Crit Care Med. 32 (9), 1825-1831 (2004).</li><li>De Backer, D., 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=Microcirculatory+alterations+in+patients+with+severe+sepsis:+impact+of+time+of+assessment+and+relationship+with+outcome.">Microcirculatory alterations in patients with severe sepsis: impact of time of assessment and relationship with outcome.</a> Crit Care Med. 41 (3), 791-799 (2013).</li><li>Buijs, E. 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=Early+microcirculatory+impairment+during+therapeutic+hypothermia+is+associated+with+poor+outcome+in+post-cardiac+arrest+children:+A+prospective+observational+cohort+study.">Early microcirculatory impairment during therapeutic hypothermia is associated with poor outcome in post-cardiac arrest children: A prospective observational cohort study.</a> Resuscitation. , (2013).</li><li>Genzel-Boroviczeny, O., Christ, F., Glas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+transfusion+increases+functional+capillary+density+in+the+skin+of+anemic+preterm+infants.">Blood transfusion increases functional capillary density in the skin of anemic preterm infants.</a> Pediatr Res. 56 (5), 751-755 (2004).</li><li>Ergenekon, E., 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=Peripheral+microcirculation+is+affected+during+therapeutic+hypothermia+in+newborns.">Peripheral microcirculation is affected during therapeutic hypothermia in newborns.</a> Arch Dis Child Fetal Neonatal Ed. 98 (2), F155-F157 (2013).</li><li>Schwepcke, A., Weber, F. D., Mormanova, Z., Cepissak, B., Genzel-Boroviczeny, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+mechanisms+in+postnatal+hypotension+affecting+premature+infants.">Microcirculatory mechanisms in postnatal hypotension affecting premature infants.</a> Pediatr Res. , (2013).</li><li>van Elteren, H. A., Ince, C., Tibboel, D., Reiss, I. K., de Jonge, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+microcirculation+in+preterm+neonates:+comparison+between+sidestream+dark+field+(SDF)+and+incident+dark+field+(IDF)+imaging.">Cutaneous microcirculation in preterm neonates: comparison between sidestream dark field (SDF) and incident dark field (IDF) imaging.</a> J Clin Monit Comput. , (2015).</li><li>Als, H., 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=Individualized+Behavioral+and+Environmental+Care+for+the+Very-Low-Birth-Weight+Preterm+Infant+at+High-Risk+for+Bronchopulmonary+Dysplasia+-+Neonatal+Intensive-Care+Unit+and+Developmental+Outcome.">Individualized Behavioral and Environmental Care for the Very-Low-Birth-Weight Preterm Infant at High-Risk for Bronchopulmonary Dysplasia - Neonatal Intensive-Care Unit and Developmental Outcome.</a> Pediatrics. 78 (6), 1123-1132 (1986).</li><li>Massey, M. J., 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=The+microcirculation+image+quality+score:+development+and+preliminary+evaluation+of+a+proposed+approach+to+grading+quality+of+image+acquisition+for+bedside+videomicroscopy.">The microcirculation image quality score: development and preliminary evaluation of a proposed approach to grading quality of image acquisition for bedside videomicroscopy.</a> J Crit Care. 28 (6), 913-917 (2013).</li><li>Weidlich, K., 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=Changes+in+microcirculation+as+early+markers+for+infection+in+preterm+infants--an+observational+prospective+study.">Changes in microcirculation as early markers for infection in preterm infants--an observational prospective study.</a> Pediatr Res. 66 (4), 461-465 (2009).</li><li>De Backer, D., 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=How+to+evaluate+the+microcirculation:+report+of+a+round+table+conference.">How to evaluate the microcirculation: report of a round table conference.</a> Crit Care. 11 (5), R101 (2007).</li><li>Sallisalmi, M., Oksala, N., Pettila, V., Tenhunen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+sublingual+microcirculatory+blood+flow+in+the+critically+ill.">Evaluation of sublingual microcirculatory blood flow in the critically ill.</a> Acta Anaesthesiol Scand. 56 (3), 298-306 (2012).</li><li>van den Berg, V. J., 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=Reproducibility+of+microvascular+vessel+density+analysis+in+Sidestream+dark-field-derived+images+of+healthy+term+newborns.">Reproducibility of microvascular vessel density analysis in Sidestream dark-field-derived images of healthy term newborns.</a> Microcirculation. 22 (1), 37-43 (2015).</li></ol>

Die Autoren haben nichts zu offenbaren.

In diesem Manuskript wir beschreiben und zeigen die Vorgehensweise für die transkutane mikrozirkulatorischen Bildgebung in der Frühgeborenen. Visualisieren Sie diese Methode hilft Forschern zu überwinden zwei der größten Herausforderungen in der Forschung: die Reproduzierbarkeit und die zeit- und arbeitsintensiv Natur der das Erlernen neuer Techniken. Diese Technik kann nützlich sein, Informationen von peripheren Mikrozirkulation bei Frühgeborenen in einer nicht-invasiven Art und Weise bereitzustellen. Serienmess...

Hemodynamic diagnostics in critically ill preterm neonates has always been difficult. Most diagnostic tools used in adults cannot be applied in these tiny preterm infants; and then there is a problem of the sensitivity of the outcome parameters. But most of all, these infants are so vulnerable, that the risks of diagnostic procedures do not outweigh the benefits. As a result, in the field of neonatology, hemodynamics has been neglected and therefore there is a lack of knowledge on this topic.
An interesting option for handling these problems might be visualizing the microcirculation. The introduction of handheld microscopes in the late 1990s made it possible to visualize the microcirculation in a non-invasive manner. Three generations of devices have been introduced: Orthogonal Polarization Spectral (OPS) imaging1, Sidestream Dark Field (SDF) imaging2, and Incident Dark Field (IDF) imaging3. They all use more or less the same technique in which green light with a specific wavelength (548nm) stroboscopic illuminates the microcirculation. The green light is absorbed by oxy- and deoxyhemoglobin and mostly reflected by the surrounding tissue. This property of green light therefore creates visible contrast. The reflected light passes a magnification lens and is projected on a camera sensor. Hereby it is possible to visualize the flowing red blood cells at a depth of approximately one millimeter of mucosal tissue or directly at solid organs. 
Over the past 15 years, the microcirculation has been mainly studied in adults, especially in patients with septic shock4-6. These observational studies found that persistent microcirculatory alterations were associated with organ failure and mortality. This observation cannot be extrapolated directly to (preterm) infants however, as in the adults the microcirculation was measured sublingually. High quality images of the sublingual microcirculation cannot be obtained in preterm infants because they are unable to cooperate. In term infants the buccal microcirculation has been the area of interest7. Fortunately, in preterm infants the thin skin allows transcutaneous microcirculatory imaging. This approach has been applied in neonatal studies focusing on blood transfusion8, therapeutic hypothermia9 and hypotension10.
In this manuscript we present our protocol for transcutaneous microcirculatory imaging using Incident Dark Field imaging in preterm neonates. We will focus on different strategies to acquire the highest quality images. Technical details and differences between the SDF and IDF devices can be found elsewhere11.

<table>
	<tbody>
		<tr>
			<td>Cytocam</td>
			<td>Braedius</td>
			<td>http://www.braedius.com/magnoliaPublic/braedius/products.html</td>
			<td>Other well known handheld microscopes to visualize the microcirculation are MicroScan (Microvision Medical) using SDF technique or the CytoScan (CytoMetrics) using OPS technique</td>
		</tr>
		<tr>
			<td>Disposable Lens Cover</td>
			<td>Glycocheck</td>
			<td>http://www.glycocheck.com/lenscovers.php</td>
			<td></td>
		</tr>
		<tr>
			<td>CCTools</td>
			<td>Braedius</td>
			<td>http://www.braedius.com/magnoliaPublic/braedius/products.html</td>
			<td>Another well known offline analysis programme is AVA (Microvision medical).&#160;</td>
		</tr>
	</tbody>
</table>

transcutaneous microcirculatory imaging in preterm neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Abbildungen 1 und 2 zeigen repräsentative Standbilder aus hochwertigem MI Videos. Diese Beispiele zeigen den Unterschied in der Dicke der Haut in gleichem Säuglingen zwischen Tag 1 (Abbildung 1) und am Tag 28 (Figur 2) der postnatalen Alter. Am Tag 1, gibt es eine helle Beleuchtung, ausreichende Konzentration auf die Mikrogefäße und minimale Präsenz von Artefakten. Am 28. Tag ist es schwierig, das richtige Gleichgewicht zwischen den...

Watch this Scientific Journal Video about Transcutaneous Microcirculatory Imaging in Preterm Neonates at JoVE.com

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Transkutane Mikrozirkulatorische Imaging in Frühgeborenen

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

transcutaneous-microcirculatory-imaging-in-preterm-neonates

Research

JoVE Journal

Medicine

7.6K Aufrufe.  Erasmus MC-Sophia Children's Hospital. Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Serie: Transcutaneous Microcirculatory Imaging in Preterm Neonates

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Die Verwendung von pharmakologischen fMRT-Herausforderung in der präklinischen Forschung: Anwendung auf die 5-HT-System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Forschung

Medizin

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Automatisierte Messung der Mikrozirkulation Blutflußgeschwindigkeit in Lungenmetastasen von Ratten

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

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.

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.

State of the Art Hirn Ultraschall-Bildgebung bei Neugeborenen

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 ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Transkutane Beurteilung der Nierenfunktion in Conscious Nagetiere

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Quantitative Visualisierung von Leukozyten-Infiltrat in einem Murin-Modell der Fulminant-Myokarditis durch Licht-Blatt-Mikroskopie

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Application of an Amplitude-integrated EEG Monitor (Cerebral Function Monitor) to Neonates

Amplitude-integrated EEG (aEEG) is an easily accessible technique to monitor the electrocortical activity in preterm and term infants in neonatal intensive care units (NICUs). This method was first used to monitor newborns after asphyxia, providing information about future neurological outcomes. The aEEG is also helpful to select newborns who benefit from cooling. The aEEG monitoring of preterm infants is becoming more widespread, as various studies have shown that neurodevelopmental outcome is related to early aEEG tracings. Here, we demonstrate the application of the aEEG monitoring system and present typical patterns that depend upon gestational age and pathophysiological conditions. Furthermore, we mention pitfalls in the interpretation of the aEEG, as this method requires accurate fixation and localization of the electrodes. Additionally, the raw EEG can be used to detect neonatal seizures or to identify aEEG application problems. In conclusion, aEEG is a safe and generally well-tolerated method for the bedside monitoring of neonatal cerebral function; it can even provide information about long-term outcome.

Application of an Amplitude-integrated EEG Monitor Cerebral Function Monitor to Neonates

Here, we show how to apply amplitude-integrated electroencephalography to monitor cerebral function in neonates.

Anwendung eine Amplitude integriert-EEG-Monitor (zerebrale Funktion Monitor) auf Neugeborene

Amplitude-integrated EEG (aEEG) is an easily accessible technique to monitor the electrocortical activity in preterm and term infants in neonatal ...

Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. Those tools are subjective or determine secondary physiological indicators which also have certain limitations particularly when exploring the effectiveness of analgesia. As cortical processing is essential for pain perception, brain activity measures may provide a useful approach to assess pain in infants. Here we present a method to assess nociception with electrophysiological brain activity recordings optimized for the use in newborn infants. To produce highly standardized and reproducible noxious stimuli we applied mechanical stimulation with a flat-tip probe, e.g., PinPrick, which is not skin-breaking and does not cause behavioral distress. The noxious-evoked potential allows the objective measurement of nociception in non-verbal patients. This method can be used in newborn infants as early as 34 weeks of gestational age. Moreover, it could be applied in different situations such as measuring the efficacy of analgesic or anesthetic drugs.

Measuring pain in non-verbal patients is a challenge. In this study we combine EEG recording with stimulation using a flat-tip probe to detect noxious-evoked brain activity in an objective manner.

Elektrophysiologische Messungen der schädlichen-evozierten Hirnaktivität beim Neugeborenen mit einem Flat-Tip Sonde gekoppelt, Elektroenzephalographie

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase Kontrast Magnetresonanz-Bildgebung in der Ratte gemeinsame Halsschlagader

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Quantitative [18F]-Naf-PET-MRI-Analyse zur Bewertung des dynamischen Knochenumsatzes bei Patienten mit facetogenen Rückenschmerzen

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Clinical Practice Protocol of Creative Music Therapy for Preterm Infants and Their Parents in the Neonatal Intensive Care Unit

Creative music therapy for preterm infants and their parents (CMT) has emerged as a promising family-integrated early intervention involving communicative musicality to improve infant development, parental well-being, and bonding. It aims at relaxing and nurturing the infant as well as promoting safety and social interaction for the parent-infant dyad. A music therapist specially trained in CMT hums or sings in an infant-directed, improvised, lullaby style continually adjusting to the individual needs, expressions, and breathing pattern of the preterm infant. Based on the principles of family-integrated care, the family is incorporated individually in the therapeutic process, namely by delivering CMT during kangaroo care (KC) and by motivating and facilitating parental vocal interaction with their infant to strengthen the parent-infant bonding. CMT aims at relaxing, stimulating, and coregulating premature infants at a time when many other interventions are still risky and can overwhelm the vulnerable patient group. CMT may be advantageous not by educating and teaching parents, but rather by uncovering the intuitive capacities of parenting that are often overshadowed by the traumatic experience of preterm birth. However, CMT can only be provided when the infants are clinically stable. CMT with parental integration is feasible when parents are available and receptive to participate. This paper presents a detailed protocol on how to use CMT to empower preterm infants and their families.

Creative music therapy for preterm infants and their parents has emerged as a promising family-integrated early intervention. We present a detailed protocol on how to use vocal interaction, humming, or singing to empower preterm infants and their families.

Creative music therapy for preterm infants and their parents (CMT) has emerged as a promising family-integrated early intervention involving communicative ...