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.

注：このプロトコルは、地元の人間研究倫理委員会のガイドラインに従っています。 1.一般的な考慮事項注：機器、データストレージおよび安全性に関する一般的な考慮事項は議論に対処されている。 <ol><li> （説明を参照）の周波数帯域に複数の変換器と、高解像度、リアルタイム、モバイル2D超音波装置を用いて画像を取得する。一般的に、7.5から8.5までの周波数でプローブを使用して良質の画像を得る。 </li></ol> CUS試験の調製<ol><li>それはそのような血液採取などの他の手順と一致しないように、CUSの検査をスケジュールします。 </li><li>そのような新生児個別発達ケアと評価プログラム6の原理に基づくもののような戦略を使用して、検査中に新生児を医療従事者や親がサポートする使用可能であることを確認し、および/ ​​または快適さ&lt;/ SUP&gt;。 </li></ol>大泉門を通して3.審査<ol><li>インキュベーターまたはベビーベッドに沿って超音波装置を取り付けます。 </li><li>プローブと皮膚との間の良好な接触を確実にするために、プローブにトランスデューサゲルを適用する。使用前にゲルを暖め考えてみましょう。 </li><li> Bモードで凸プローブと大泉門を通してイメージングを開始します。新生児の右側になってプローブ上のマーカーと泉門の真ん中にプローブを配置します。脳の左側は、モニタの右側に表示される。 NOTE：大泉門を通してイメージングは、任意の位置3で ​​新生児を行うことができる。研究目的のためには、標準的なヘッド位置のために努力する必要があるかもしれない。 <ol><li>少なくとも5つの冠状および5矢状面におけるレコードのイメージ。最初の画像に頭蓋の輪郭を含むセクタを充填する画像を生成する深さ、ゲイン、時間利得補償の設定を調整し、回避明るすぎたり暗いイメージをると近く、より深い構造物からの反射との間の平衡を目指して。 </li><li>冠状面 注：完全に対称画像を得るようにしてください。前頭葉の凸部の近くに病変が疑われる場合には1半球が（ 図1）より良い詳細に表示されるように、特定の斜めの冠状切片を記録することを検討してください。 <ol><li>冠状前頭前野画像の場合、角度プローブは前方には、嗅覚脳溝のレベルで、側脳室の正面の角に前方に前頭葉を可視化した。 </li><li>モンローのレベルでのコロナル像については、角度側脳室の正面の角を描写するためにテラの脈絡膜への冠状断面の前方を可視化するプローブ、耳甲隔pellucidi、脳梁、溝のcinguli。大脳基底核の部分のエコー源性に注意してください。 </li><li>視床のレベルでのコロナル像の場合、角度は、プローブは後方後半を識別するためにRAL亀裂、第三脳室と側頭葉の屋根でテラ脈絡膜。大脳基底核に関連する視床（特に腹外側核）のエコー源性に注意してください。視床枕でネットワーク障害がちょうど心房の前に余分な冠状断面で可視化することができることに注意してください。 </li><li>心房のレベルでのコロナル画像について、脈絡叢のレベルで側脳を視覚化する。頭葉と小脳半球を特定します。脈絡叢に比べて脳室周囲白質のエコー源性に注意してください。早産新生児における心房に上記と横通常の高エコー領域と光学的放射を比較してください。 </li><li>冠状頭頂後頭の画像の場合、頭頂後頭溝のレベルにプローブ後方に角度が頭頂後頭ローブを識別します。 </li></ol></li><li>サジタルプレーン<ol><li>新生児の顔の方を向いて、プローブ上のマーカーでプローブ90°回転します。目脳の電子の前方部分は、モニタの左側に表示されます。以下の構造（ 図2）のレベルで画像を記録する。 </li><li>正中矢状画像の場合は、脳梁、耳甲隔pellucidi（CSP）、第3および第4脳室、虫部、大槽、橋と中脳を視覚化する。腔Vergaeと耳甲ヴェリinterpositi 7の存在に注意してください。 </li><li> 1 gangliothalamic卵形を通して矢状画像（ 例えば 、右）のために、側脳室を通じて矢ビュー用のプローブ横向きの角度。脈絡叢を特定し、視床や大脳基底核のエコー源性に注意してください。矢状面のためのスキャンされた側が適切にテキストツールで示されるべきである。 </li><li>矢島画像、島を経て、さらに横方向の角度プローブ用。横方向の亀裂とfrontal-、temporal-、parietal-と後頭葉を識別します。 </li><li> Tを繰り返し矢のイメージ彼の反対側（ すなわち 、左）。 </li></ol></li><li>カラードップラー<ol><li>カラードップラーを使用して凸状のプローブで大泉門を通してイメージングを続行します。大脳動脈と静脈の流速を評価し、派生インデックスを取得することを検討してください。 注： - 拡張末期速度/ピーク収縮期速度抵抗指数（RI）は、ピーク収縮期速度として定義されている。 RIは角度に依存しない、絶対速度の値は8-10ではありません。 RIは異なる口径の動脈で類似していない。同じ容器の正確な同じ場所で行われた場合に一連の測定にのみ有用である。 </li></ol></li><li>以下の血管の冠状面での記録画像（ 図3）： <ol><li>小脳のレベルで横洞を視覚化。 1つまたは全く横洞が可視化されている場合、パルス繰り返し周波数（PRF）を下げてください。当時はまだ一つだけまたは横洞の。ない場合大泉門を介して同定することができ、乳様突起泉門を通して可視化するための高周波リニアプローブを使用します（セクション4.4.2を参照）。 </li><li>内頸動脈、中大脳動脈とウィリス動脈輪を視覚化し、側脳室の正面の角のレベルで脳動脈を前方。左右の前大脳動脈を区別することは、しばしば困難であるが、通常は不要です。動脈の線条体燭台を特定します。 </li><li>角度後方に隣接する頸静脈と脳底動脈を可視化するプローブ。 </li><li>さらに後方に角度が内部脳と視床線条体静脈を可視化した。 </li></ol></li><li> 1前大脳動脈（ 図4）の矢状面に画像を記録します。 （通常は脳梁の膝の下）この容器の特定の部分での流速とRIを評価する。近く内部大脳静脈における正中線速度が可能容易に測定。 </li><li>大泉門に前頭面の高周波リニアプローブを用いて、上矢状静脈洞を識別する。これは、失敗泉門のプローブを用いて適用される圧力の量を減らす必要がある場合。 NOTE：リニアプローブは、表面構造（髄膜、くも膜及び硬膜下腔、皮質）の詳細な視覚化のために使用することができる。タンジェンシャル船がくも膜下腔にある。理想的には、前の手順で説明したようにドップラーイメージングは​​、新生児の最初のCUSの検査中に実行されます。フォローアップ検査中のステップの一部は省略してもよい。ステップ3.3.5.1に記載されているように疑われる脳sinovenous血栓症ドップラーイメージングの場合には、3.3.7と4.4.2を実行する必要があります。 </li></ol></li></ol>オルタナティブアコースティック窓から4.審査<ol><li>次に、別の音響窓から審査を継続する。 </li><li> （POSラムダを通して画像を記録考えてみましょう凸プローブ（ 図5）を使用して、内部を塗っ）泉門。後部泉門は、矢状及びラムダ縫合3,11の接合部に位置しています。側臥位で新生児を配置することによって事後泉門を介して画像。 注記：良好な画像も仰臥位3に幼児と矢状縫合の後方側面によって得ることができる多くの未熟児で。 <ol><li>矢状ビューの事後泉門の中央にプローブを配置します。角度側脳室とその後頭部ホーンの本体を識別するために、わずかに正中線オフプローブ。冠状ビューを取得するためにおよそ90°プローブを回します。側脳室の後頭の角を特定します。 </li></ol></li><li>耳（ 図6）上記凸状または直線状のプローブを用いて、横方向（時間）窓から画像を記録することを検討してください。 <ol><li>必要であれば、許可する横窓を通して画像を得る脳幹12の詳細図。耳の前に水平に上記と少しプローブを配置します。脳花梗が可視化されるまでプローブを移動します。 注：他の構造を同定することができる第三脳室、水道橋と頭葉である。カラードップラーを使用して、ウィリス動脈輪を可視化することができる。 </li></ol></li><li>乳様突起泉門を通して録音画像（ 図7）。乳様突起泉門は、時間、頭頂と後頭部の骨4の接合部に、耳の後ろに位置しています。側臥位3で新生児を配置することによって乳様突起泉門スルー画像。 注：乳様突起の泉門を経て画像が得られた場合、当社の経験では、新生児は、多くの場合、不快感の兆候を示している。したがって、大泉門と他の音響窓から撮影した後、これを行うのがベストでしょう。我々は、この不快感がPULSに聴覚反応のメカニズムによって引き起こされる可能性があることを仮定した高周波エネルギー13のES。 <ol><li>凸状のプローブを使用した乳様突起の泉門を介して画像。冠状ビューを得るために、耳にプローブを平行に配置してください。小脳半球、虫部、第3および第4脳室、脳橋と大槽を識別するために、前後にプローブをスイープ。小さなpretermsでは対側小脳半球はうまく描くことができる。 </li><li>リニアプローブを用いた乳様突起の泉門を介して画像。 （一）横洞が大泉門を介して同定することができない場合には、乳様突起泉門を通って可視化するための高周波線形プローブを使用する。冠状ビューを得るために、耳たぶにプローブを平行に配置してください。 <ol><li>小脳半球と第四脳室を識別します。カラードップラーを使用して、横方向およびS状静脈洞、tentorial洞と使者静脈を識別する。 </li></ol></li></ol></li><li>大後頭孔14を介して後頭蓋窩の追加の可視化を考えてみましょう。 </li></ol>

<ol><li>Plaisier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety+of+routine+early+MRI+in+preterm+infants%5BTitle%5D)+AND+%22Pediatric+Radiology%22%5BJournal%5D)">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/pubmed?term=((Serial+Cranial+US+for+Detection+of+Cerebral+Sinovenous+Thrombosis+in+Preterm+Infants%5BTitle%5D)+AND+%22Radiology%22%5BJournal%5D)">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/pubmed?term=((A+new+view+of+the+neonatal+brain%3A+Clinical+utility+of+supplemental+neurologic+US+imaging+windows%5BTitle%5D)+AND+%22Radiographics%22%5BJournal%5D)">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/pubmed?term=((Mastoid+fontanelle+approach+for+sonographic+imaging+of+the+neonatal+brain%5BTitle%5D)+AND+%22Pediatric+Radiology%22%5BJournal%5D)">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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sonographic+visualization+of+neonatal+posterior+fossa+abnormalities+through+the+posterolateral+fontanelle%5BTitle%5D)+AND+%22AJR+Am+J+Roentgenol%22%5BJournal%5D)">Sonographic visualization of neonatal posterior fossa abnormalities through the posterolateral fontanelle.</a> AJR Am J Roentgenol. 174 (2), 561-567 (2000).</li>
<li>Als, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?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%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">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>Govaert, P., de Vries, L. S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGovaert%2C+P+%22An+Atlas+of+Neonatal+Brain+Sonography%22">An Atlas of Neonatal Brain Sonography</a>. , 2nd, Mac Keith Press. London, UK. (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/pubmed?term=((Continuous+Wave+Doppler+Ultrasound+in+Evaluation+of+Cerebral+Blood-Flow+in+Neonates%5BTitle%5D)+AND+%22Archives+of+Disease+in+Childhood%22%5BJournal%5D)">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. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Duplex+Pulsed+Doppler+Ultrasonography+Versus+Intracranial-Pressure+in+the+Neonate+-+Clinical+and+Experimental+Studies%5BTitle%5D)+AND+%22Radiology%22%5BJournal%5D)">Duplex Pulsed Doppler Ultrasonography Versus Intracranial-Pressure in the Neonate - Clinical and Experimental Studies.</a> Radiology. 171 (1), 155-159 (1989).</li>
<li>Volpe, J. J., Perlman, J. M., Hill, A., Mcmenamin, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebral+Blood-Flow+Velocity+in+the+Human+Newborn+-+the+Value+of+Its+Determination%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">Cerebral Blood-Flow Velocity in the Human Newborn - the Value of Its Determination.</a> Pediatrics. 70 (1), 147-152 (1982).</li>
<li>Correa, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Posterior+fontanelle+sonography%3A+An+acoustic+window+into+the+neonatal+brain%5BTitle%5D)+AND+%22American+Journal+of+Neuroradiology%22%5BJournal%5D)">Posterior fontanelle sonography: An acoustic window into the neonatal brain.</a> American Journal of Neuroradiology. 25 (7), 1274-1282 (2004).</li>
<li>Helmke, K., Winkler, P., Kock, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sonographic+examination+of+the+brain+stem+area+in+infants.+An+echographic+and+anatomic+analysis%5BTitle%5D)+AND+%22Pediatric+Radiology%22%5BJournal%5D)">Sonographic examination of the brain stem area in infants. An echographic and anatomic analysis.</a> Pediatric Radiology. 17 (1), 1-6 (1987).</li>
<li>Elder, J. A., Chou, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auditory+response+to+pulsed+radiofrequency+energy%5BTitle%5D)+AND+%22Bioelectromagnetics%22%5BJournal%5D)">Auditory response to pulsed radiofrequency energy.</a> Bioelectromagnetics. , Suppl 6. S162-S173 (2003).</li>
<li>Brennan, C. M., Taylor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sonographic+imaging+of+the+posterior+fossa+utilizing+the+foramen+magnum%5BTitle%5D)+AND+%22Pediatric+Radiology%22%5BJournal%5D)">Sonographic imaging of the posterior fossa utilizing the foramen magnum.</a> Pediatric Radiology. 40 (8), 1411-1416 (2010).</li>
<li>Rutherford, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetic+resonance+imaging+of+white+matter+diseases+of+prematurity%5BTitle%5D)+AND+%22Neuroradiology%22%5BJournal%5D)">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/pubmed?term=((Cranial+Ultrasonography+in+Neonates%3A+Role+and+Limitations%5BTitle%5D)+AND+%22Seminars+in+Perinatology%22%5BJournal%5D)">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/pubmed?term=((Using+cerebral+ultrasound+effectively+in+the+newborn+infant%5BTitle%5D)+AND+%22Early+Human+Development%22%5BJournal%5D)">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/pubmed?term=((The+safety+of+obstetrical+ultrasound%3A+a+review%5BTitle%5D)+AND+%22Prenatal+Diagnosis%22%5BJournal%5D)">The safety of obstetrical ultrasound: a review.</a> Prenatal Diagnosis. 29 (13), 1204-1212 (2009).</li>
<li>Torloni, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety+of+ultrasonography+in+pregnancy%3A+WHO+systematic+review+of+the+literature+and+meta-analysis%5BTitle%5D)+AND+%22Ultrasound+in+Obstetrics+%26+Gynecology%22%5BJournal%5D)">Safety of ultrasonography in pregnancy: WHO systematic review of the literature and meta-analysis.</a> Ultrasound in Obstetrics & Gynecology. 33 (5), 599-608 (2009).</li>
</ol>

The authors have nothing to disclose.

私たちは、記述し、新生児のドップラーCUSのための最先端のアプローチを実証する。経験豊富な手では、これは安全で、シリアルベッドサイド新生児の脳イメージングのための優れたツールです。多くのNICUsに記載されて可能性が最適に使用されていません。ドップラー研究を追加すると、頭蓋内動脈と静脈の開通のスクリーニングが可能になります。流速が評価指標を得ることができる。?...

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.

7 -記載されたプロトコールに従って作製イメージングの例は、 図1に示されている。画像は慎重に経験豊富な観察者によって解釈されるべきである。対称的なイメージングは、大泉門（ 図1）を介して行わコロナル画像の適切な解釈のために必要である。疑わしい病変は大泉門以外の音響窓を通して冠状及び（ミッド）矢状面の両方で、または可視化に?...

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.

新生児におけるアート頭蓋超音波画像の状態

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

24.2K ビュー.  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. 

ビデオ: 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.

高周波超音波を用いて、マウス胚におけるコントラストイメージング

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

発生生物学

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.

ネズミの胎児心エコー検査

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

医学

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.

心血管イメージング (30/45 MHZ) の高周波超音波を用いた胎児マウス

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.

体内の胎児と胎盤の開発の分析のための高周波超音波

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.

非侵襲的実質細胞、血管および代謝高周波超音波および光音響ラット脳深部イメージング

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

神経科学

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.

リアルタイムの生体内多光子顕微鏡で、血液脳関門透過性を高める、集中超音波治療とマイクロバブル治療を可視化

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 ...

生物工学

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.

生体内で新生仔マウスの大脳皮質ニューロンの 2 光子イメージング

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.

げっ歯類における血液脳関門開口のためのハイスループット画像誘導定型神経ナビゲーションと集中超音波システム

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.

標的新生児心エコー検査を用いた新生児集中治療室における血行動態の精度

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.

重症新生児の生命を脅かす緊急事態における画像取得のための修正超音波アルゴリズム

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