我々は、次の資金源を認める: NIH NINDS – K08NS101122 (JB), R01NS040337 (JK), R01NS044370 (JK), バージニア大学医学部 (JB).

すべての動物研究は、バージニア大学の施設動物のケアと使用委員会(IACUC)によって承認されました。
1. 電極建物/ケーブルビルディング
<ol><li>単極絶縁ステンレス鋼線(0.005"裸径0.008"被覆)を使用して、メスソケットコネクタ(メスレセプタクルコネクタ0.079)で接続された電極を作ります。</li>
	<li>特別なカスタムケーブルを使用して、動物をアンプに接続します。<ol>
<li>オスの4ピンコネクタ(オスコネクタ0.079")を4チャンネルユニティゲインインピーダンスマッチングオペアンプ(op-amp)に取り付けます。9 V バッテリに接続するワイヤに 10K の抵抗を接続します。オペアンプに接続されていないアース線は、バッテリーの中点として機能します。</li>
		<li>ケーブルの一方の端(AWG、0.012"OD)をop-ampに接続し、ケーブルのもう一方の端をアンプに接続します。</li>
	</ol>
</li>
</ol>2. 電極注入手術
<ol><li>下向きのフローフードで4-5%のイオブルランで子犬(出生後9日目)を麻酔します。手順の開始前に、ブピバカイン(0.02-0.05 mL、0.25%皮下局所浸潤)で子犬を注入する。</li>
	<li>動物が不動になったら、鼻コーンで立体的な段階に移します。耳をしっかり止めるには柔らかいので、イヤーバーの裏側を使用します。この年齢では、子犬は耳棒の尖った端を使用するために完全に発達した耳を持っていません。</li>
	<li>イオフルランの流れを下げ、2.5~3%に維持します。手術を通して子犬の安定した呼吸に目を光らせる。尾をつまんで痛みの反応を確認し、切開に進みます。</li>
	<li>頭蓋骨の切開領域をベタジンとアルコール(ヨウ素と70%エタノールの3サイクル交互)で殺菌する。切開領域が見えるように周囲の身体部分をドレープする。</li>
	<li>目の上から頭皮前部後部を開き、約0.5cmの皮膚を引き込みます。皮膚が頭蓋骨を露出して外側に引っ張るように、ステレオタックスステージ上でマウスの頭を再配置します。</li>
	<li>綿棒を使用して頭蓋骨に過酸化水素を塗布し、メスの刃を使用して頭蓋骨をきれいに削ります。頭蓋骨は非常に柔らかいです。擦り傷をしながら注意を払ってください。</li>
	<li>接着剤を1滴(約50μL)塗布し、そのアプリケータを使用して露出した頭蓋骨領域の周りに広げます。40sの紫外線に露出して接着剤をセットします。</li>
	<li>参照として、露出したブレグマを使用して座標を測定します。海馬のCA1領域に両側電極を移植する [-3.5 mm側側腹孔(DV),±2mm内側側面(ML)、-1.75mm深部(D)、頭頂皮質内の両側[-1.22mm DV、±0.5mm D]および小脳内の参照電極。32G針を使用して、マークされた領域に穴を開けます。</li>
	<li>頭蓋骨の表面から血液をきれいにします。立体的な腕の助けを借りて脳に女性のソケットコネクタに取り付けられた下の電極と歯科アクリルで所定の位置に固定します。脳に電極を埋め込む。ソケットコネクタヘッドセットは、歯科アクリルで接着頭蓋骨の上に座っています。</li>
	<li>電極が固定されたら、ケトプロフェン(5mg/kg)を皮間領域に皮下に注入します。子犬を母親と一緒に戻します。 注:一度に1つずつ導入するのではなく、一度にヘッドセットでゴミの半分を導入してください。これは、母親が子犬のヘッドセットを損傷するのを防ぎます。</li>
</ol>3. EEGのセットアップと記録(ベースライン/プレ傷害)
<ol><li>電極注入後24時間の回収後、各動物をEEG記録用の加熱(37°C)カスタムメイドのプレキシグラスチャンバーに入れます。このチャンバーは低酸素室としても機能します。</li>
	<li>柔軟なケーブル(カスタムメイドのオペアンプケーブル)を介してビデオ-EEG監視システムにチャンバー内の子犬を接続します。 メモ:ヘッドセットを設置すると、マウスは自由に移動可能で、動作に違いはありません。電極ワイヤーに取り付けられたら、ワイヤーは、子犬がチャンバー全体を自由に移動できるように、適切な量の緩みを提供するためにチャンバーテザー内で調整する必要があります。</li>
	<li>草のアンプを使用して1Kゲインで1000 HzでEEGデータをデジタル化します。ソフトウェア(例えば、LabChart Pro)を使用して、後でEEG信号(3〜70 Hzの間のバンドパスフィルタ)を確認してください。</li>
	<li>頸動脈結紮術のために動物を切断する前に、傷害前のベースラインEEGを30分間記録する。</li>
</ol>4. 左頸動脈結紮
<ol><li>下向きのフローフードで4-5%のイオブルランで子犬(出生後10日目)を麻酔し、ウォーターバスパッドに特別に配置されたセットアップに置きます。動物のスピージンを配置し、紙テープで前肢を固定します。<ol>
<li>イオブルランの流れを2~3%に下げる。痛みの反応のために尾をつまみ、手順全体の呼吸を監視します。</li>
	</ol>
</li>
	<li>ベタジンとアルコール(ヨウ素と70%エタノールの3サイクル)で首の左側の切開領域(下顎骨と鎖骨の間)を殺菌します。</li>
	<li>マイクロシザーを使用して、首の左側に約1cmの長さの切開を行います。解剖顕微鏡を用いて、皮下組織と皮膚を慎重に引き込み、頸動脈を露出させる。迷走神経(動脈に横向きに走る)を特定し、動脈から繊細に分離して引き込むのに注意してください。</li>
	<li>マイクロ鉗子を使用して動脈の下に長さ5cmの無菌シルク縫合糸を通す。動脈の周りに二重結び目の縫合糸を結んで、閉塞流れにします。</li>
	<li>余分な縫合糸をカットし、皮下組織と皮膚を引き戻すことによって露出した動脈を閉じます.切開を密封するために獣医の結合を使用してください。</li>
	<li>動物を温かいマットレスの上に置かれる室温の部屋の連続的なEEGの監視に戻す。チャンバーを開けないように、子犬のコア温度のスポット赤外線温度チェックを取ります。動物が低酸素症の前に1時間回復することを許可します。</li>
</ol>5. EEGと低酸素症
<ol><li>酸素モニターを介してチャンバー内のFiO2 (インスパイアされた酸素の一部)を継続的に監視します。</li>
	<li>100%N2 の60 L/minと100%O2のための0.415 L /分でチャンバーを洗い流す。チャンバーの酸素飽和度が12%に達したら、O2の流れを変えずにN2 の流れを10 L/分に減らします。小さな調整で、45分間8%でFiO2 を維持します。</li>
	<li>低酸素暴露の45分後、21%にFiO2 を戻す。</li>
	<li>子犬はチャンバーで回復し、2時間ポスト低酸素症のEEG上で監視しています。</li>
	<li>記録期間終了後、マウスをEEG記録から切り離し、母親に戻します。</li>
</ol>6. EEG分析
<ol><li>ラボチャートプロのビデオでEEGファイルを分析します。目の不自由な研究者に発作と背景パターンのEEGをマークしてもらいます17。発作は、高周波のリズミカルな鋭波放電(≥3xベースライン)を持つ10秒以上続く電写イベントと定義され、明確な進化17を有する。</li>
	<li>2番目の盲目の研究者レビューは、合意のためにランダムにマークされたイベントを持っています。</li>
	<li>マークされた各電気イベントの関連ビデオをレビューし、新生児げっ歯類行動発作スコア16に従って分析する。簡単に言えば、このスコアは0-6(不動から重度の強壮性クロニック挙動)の範囲です。発作セミオロジーをさらに特徴付けるには、横性(多焦点/二国間運動対焦点/一方的な混合)の行動を分析する。</li>
	<li>パワースペクトログラムを作成します。1024 データ ポイントのサイズを持つコサイン ベル データ ウィンドウを使用して高速フーリエ変換を使用します。87.5%のウィンドウオーバーラップの助けを借りてスペクトログラムに滑らかなX軸を作成します。電源をμV218として表現します。</li>
</ol>

<ol><li>Vasudevan, C., Levene, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+and+aetiology+of+neonatal+seizures%5BTitle%5D)+AND+%22Seminars+in+Fetal+%26+Neonatal+Medicine%22%5BJournal%5D)">Epidemiology and aetiology of neonatal seizures.</a> Seminars in Fetal & Neonatal Medicine. , (2013).</li>
<li>Volpe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+Seizures%5BTitle%5D)+AND+%22Volpe%27s+Neurology+of+the+Newborn%22%5BJournal%5D)">Neonatal Seizures.</a> Volpe's Neurology of the Newborn. , Elsevier. 275-321 (2018).</li>
<li>Shankaran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Network+EKSNNR.+Childhood+outcomes+after+hypothermia+for+neonatal+encephalopathy%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Network EKSNNR. Childhood outcomes after hypothermia for neonatal encephalopathy.</a> New England Journal of Medicine. 366 (22), 2085-2092 (2012).</li>
<li>Pappas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cognitive+outcomes+after+neonatal+encephalopathy%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">Cognitive outcomes after neonatal encephalopathy.</a> Pediatrics. 135 (3), 624-634 (2015).</li>
<li>van Schie, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+motor+and+behavioral+outcome+after+perinatal+hypoxic-ischemic+encephalopathy%5BTitle%5D)+AND+%22European+Journal+of+Paediatric+Neurology%22%5BJournal%5D)">Long-term motor and behavioral outcome after perinatal hypoxic-ischemic encephalopathy.</a> European Journal of Paediatric Neurology. 19 (3), 354-359 (2015).</li>
<li>Rensing, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Longitudinal+analysis+of+developmental+changes+in+electroencephalography+patterns+and+sleep-wake+states+of+the+neonatal+mouse%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Longitudinal analysis of developmental changes in electroencephalography patterns and sleep-wake states of the neonatal mouse.</a> PLoS One. 13 (11), 1-17 (2018).</li>
<li>Rice, J. E., Vannucci, R. C., Brierley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+immaturity+on+hypoxic-ischemic+brain+damage+in+the+rat%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">The influence of immaturity on hypoxic-ischemic brain damage in the rat.</a> Annals of Neurology. 9 (2), 131-141 (1981).</li>
<li>Burnsed, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia-ischemia+and+therapeutic+hypothermia+in+the+neonatal+mouse+brain--a+longitudinal+study%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Hypoxia-ischemia and therapeutic hypothermia in the neonatal mouse brain--a longitudinal study.</a> PLoS One. 10 (3), 0118889(2015).</li>
<li>Semple, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+development+in+rodents+and+humans%3A+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species%5BTitle%5D)+AND+%22Progress+in+Neurobiology%22%5BJournal%5D)">Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.</a> Progress in Neurobiology. , 1-16 (2013).</li>
<li>Comi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gabapentin+neuroprotection+and+seizure+suppression+in+immature+mouse+brain+ischemia%5BTitle%5D)+AND+%22Pediatric+Research%22%5BJournal%5D)">Gabapentin neuroprotection and seizure suppression in immature mouse brain ischemia.</a> Pediatric Research. 64 (1), 81-85 (2008).</li>
<li>Comi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+model+of+stroke+and+ischemic+seizures+in+the+immature+mouse%5BTitle%5D)+AND+%22Pediatric+Neurology%22%5BJournal%5D)">A new model of stroke and ischemic seizures in the immature mouse.</a> Pediatric Neurology. 31 (4), 254-257 (2004).</li>
<li>Kadam, S. D., White, A. M., Staley, K. J., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+Electroencephalographic+Monitoring+with+Radio-Telemetry+in+a+Rat+Model+of+Perinatal+Hypoxia-Ischemia+Reveals+Progressive+Post-Stroke+Epilepsy%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Continuous Electroencephalographic Monitoring with Radio-Telemetry in a Rat Model of Perinatal Hypoxia-Ischemia Reveals Progressive Post-Stroke Epilepsy.</a> Journal of Neuroscience. 30 (1), 404-415 (2010).</li>
<li>Burnsed, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+Circuit+Activity+during+Neonatal+Hypoxic+-+Ischemic+Seizures+in+Mice%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">Neuronal Circuit Activity during Neonatal Hypoxic - Ischemic Seizures in Mice.</a> Annals of Neurology. 86, 927-938 (2019).</li>
<li>Sampath, D., White, A. M., Raol, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+neonatal+seizures+in+an+animal+model+of+hypoxic-ischemic+encephalopathy%5BTitle%5D)+AND+%22Epilepsia%22%5BJournal%5D)">Characterization of neonatal seizures in an animal model of hypoxic-ischemic encephalopathy.</a> Epilepsia. 55 (7), 985-993 (2014).</li>
<li>Sampath, D., Valdez, R., White, A. M., Raol, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anticonvulsant+effect+of+flupirtine+in+an+animal+model+of+neonatal+hypoxic-ischemic+encephalopathy%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">Anticonvulsant effect of flupirtine in an animal model of neonatal hypoxic-ischemic encephalopathy.</a> Neuropharmacology. 123, 126-135 (2017).</li>
<li>Kang, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((and+sex-dependent+susceptibility+to+phenobarbital-resistant+neonatal+seizures%3A+role+of+chloride+co-transporters%5BTitle%5D)+AND+%22Frontiers+in+Cellular+Neuroscience%22%5BJournal%5D)">and sex-dependent susceptibility to phenobarbital-resistant neonatal seizures: role of chloride co-transporters.</a> Frontiers in Cellular Neuroscience. 9, 1-16 (2015).</li>
<li>Zanelli, S., Goodkin, H. P., Kowalski, S., Kapur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+transient+acute+hypoxia+on+the+developing+mouse+EEG%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">Impact of transient acute hypoxia on the developing mouse EEG.</a> Neurobiology of Disease. 68, 37-46 (2014).</li>
<li>Lewczuk, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EEG+and+behavior+patterns+during+experimental+status+epilepticus%5BTitle%5D)+AND+%22Epilepsia%22%5BJournal%5D)">EEG and behavior patterns during experimental status epilepticus.</a> Epilepsia. 59 (2), 369-380 (2017).</li>
<li>Wu, D., Martin, L. J., Northington, F. J., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oscillating+gradient+diffusion+MRI+reveals+unique+microstructural+information+in+normal+and+hypoxia-ischemia+injured+mouse+brains%5BTitle%5D)+AND+%22Magnetic+Resonance+in+Medicine%22%5BJournal%5D)">Oscillating gradient diffusion MRI reveals unique microstructural information in normal and hypoxia-ischemia injured mouse brains.</a> Magnetic Resonance in Medicine. 72 (5), 1366-1374 (2014).</li>
</ol>

我々は、低酸素虚血発作の間に新生児マウスにおける連続的なビデオ-脳細胞モニタリングのためのモデルを提示した。EEGと連動したビデオ分析により、発作セミロジーの特徴付けが可能になります。EEGの分析は、パワースペクトログラムの抽出とバックグラウンド振幅分析を可能にします。
電極の配置中または不正確な配置時の損傷は結果に大きな影響を与える可能性?...

低酸素性虚血性脳症(HIE)は、新生児1000人に1.5人の新生児に影響を及ぼし、新生児発作の最も一般的な原因である1,2である。生き残った乳児は、脳性麻痺、知的障害、てんかん3,4,5などの様々な神経障害の危険にさらされています。
動物モデルは、低酸素虚血および新生児発作の病態生理を理解し、調査する上で重要な役割を果たす6,7。修飾されたヴァンヌッチモデルは、出生後10日目(p10)7,8に低酸素虚血(HI)を誘導するために使用される。この年齢のマウスの子犬は、人間の新生児の完全な用語に神経学的に大まかに変換します 9.
この傷害モデルと組み合わせて使用される連続ビデオ脳波(EEG)モニタリングは、新生児低酸素虚血性発作のさらなる理解と特徴付けを可能にする。これまでの研究では、ビデオ録画、限られたEEG記録、テレメトリEEG記録10、11、12、13、14、15、16など、げっ歯類の新生児発作を分析するための様々な方法を使用してきました。以下の原稿では、低酸素虚血中のマウスの子犬に連続したビデオEEGを記録する過程について詳しく述べる。新生児マウスの子犬における連続的なビデオEEGモニタリングのためのこの技術は、様々な疾患および発作モデルに適用することができる。

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continuous video electroencephalogram during hypoxia-ischemia in neonatal mice

低酸素虚血は新生児発作の最も一般的な原因である。動物モデルは、新生児発作や低酸素虚血の根底にあるメカニズムと生理学を理解するために重要です。本稿では、新生児マウスにおける連続的な脳波(脳波)モニタリングを行い、低酸素虚血中の脳波背景を解析する方法を説明する。ビデオとEEGを併用すると、発作のセミオロジーの記述と発作の確認が可能になります。この方法は、実験期間におけるパワースペクトログラムおよびEEG背景パターンの傾向を分析することも可能である。この低酸素虚血モデルでは、この方法は、損傷前のEEG記録が規範的ベースラインを得ることを可能にし、傷害および回復の間に。総モニタリング時間は、4時間以上母親から子犬を分離できないことによって制限されます。この原稿では低酸素虚血性発作のモデルを用いてきたが、新生児ビデオ脳Gモニタリングのこの方法は、げっ歯類の多様な疾患および発作モデルに適用することができる。

発作セミロジー
新生児低酸素-虚血曝露は、マウスの全身発作と焦点発作の両方をもたらす(図1A-C)。ビデオ脳波録画により、電解調査結果をビデオの動作と相関させます。これらの行動は、以前に公表された新生児げっ歯類行動発作スコア(BSS)16を用いて採点された。BSS に加えて、行動が焦点/一方的、二国間、?...

Watch this Scientific Journal Video about Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice at JoVE.com

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

本稿では、低酸素虚血を起こす新生児マウスにおける複数の深度電極を用いた連続的なビデオEEG記録の方法を説明する。

新生児マウスにおける低酸素虚血期における連続映像脳波

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

continuous-video-electroencephalogram-during-hypoxia-ischemia

Research

JoVE Journal

Neuroscience

3.3K ビュー.  University of Virginia. 本稿では、低酸素虚血を起こす新生児マウスにおける複数の深度電極を用いた連続的なビデオEEG記録の方法を説明する。

ビデオ: Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

の配達<em&gt;インビボ</em&gt;急性間欠性低酸素症新生児げっ歯類で首相脳室下帯由来の神経前駆細胞の培養に

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

発生生物学

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

新生児仮死の新型モデル

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

医学

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

てんかんのマウス ・ モデルにおける Neurocardiac 機能障害を識別するために監視同時ビデオ脳波心電図

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

遺伝学

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

周産期虚血低酸素虚血性脳症モデル

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

生物学

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生児低酸素性虚血性脳症の子豚モデル

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

新生児低酸素性虚血性脳損傷のマウス モデルにおける神経行動学的評価

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

神経科学

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

周産期マウスにおける非侵襲性心電図

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

新生児マウスにおける左心室構造、機能、冠動脈流の心エコー特性評価

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

<meta charset="utf8"></head><body>新生児低酸素性虚血性脳症のラットモデルを用いた脳損傷の早期病理学的および磁気共鳴検出

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

新生児マウスにおける低酸素虚血期における連続映像脳波

要約

さらに動画を探す

の配達

新生児仮死の新型モデル

てんかんのマウス・モデルにおける Neurocardiac 機能障害を識別するために監視同時ビデオ脳波心電図

周産期虚血低酸素虚血性脳症モデル

新生児低酸素性虚血性脳症の子豚モデル

新生児低酸素性虚血性脳損傷のマウスモデルにおける神経行動学的評価

周産期マウスにおける非侵襲性心電図

新生児マウスにおける左心室構造、機能、冠動脈流の心エコー特性評価

新生児低酸素性虚血性脳症のラットモデルを用いた脳損傷の早期病理学的および磁気共鳴検出

新生児低酸素性虚血性脳症のラットモデルを用いた脳損傷の早期病理学的および磁気共鳴検出

新生児マウスにおける低酸素虚血期における連続映像脳波

要約

さらに動画を探す

の配達

新生児仮死の新型モデル

てんかんのマウス ・ モデルにおける Neurocardiac 機能障害を識別するために監視同時ビデオ脳波心電図

周産期虚血低酸素虚血性脳症モデル

新生児低酸素性虚血性脳症の子豚モデル

新生児低酸素性虚血性脳損傷のマウス モデルにおける神経行動学的評価

周産期マウスにおける非侵襲性心電図

新生児マウスにおける左心室構造、機能、冠動脈流の心エコー特性評価

新生児低酸素性虚血性脳症のラットモデルを用いた脳損傷の早期病理学的および磁気共鳴検出

新生児低酸素性虚血性脳症のラットモデルを用いた脳損傷の早期病理学的および磁気共鳴検出

てんかんのマウス・モデルにおける Neurocardiac 機能障害を識別するために監視同時ビデオ脳波心電図

新生児低酸素性虚血性脳損傷のマウスモデルにおける神経行動学的評価