著者はダンFirl、クリス・コルベットとジェシーデンソン、博士に感謝しています。資金は、ニューメキシコ大学のLJに、SRにNIH NINDS R01 NS060765によってLJにP30 CoBREパイロットプログラムと子の健康署名プログラムを用意しました。

両方のボストン小児病院、ニューメキシコ州健康科学センターの大学の制度管理使用委員会は、すべての実験手順を承認しました。 注：前の手順を開始する、シール、すべての手術器具および外科用ドレープを滅菌し、オートクレーブ。さらに、0.125％のbipivucaineおよび0.1mg / kgのブプレノルフィンを含む滅菌バイアルで術後の薬を準備します。また無菌リポポリサッカライド（LPS）溶液を調製：0.04 mg / mlのLPS（0111：B4）エバンスブルー色素を希釈含む滅菌生理食塩水中のを。 1.麻酔<ol><li>胎生18日（E18）3％イソフルランバランス70％の窒素と酸素30％の混合物を妊娠スプラーグドーリーラットに麻酔を誘導します。 </li><li>誘導チャンバーからラットを削除し、37℃に設定したドレープ手術用循環水の毛布の上にラット仰向けに置きます。ノーズコーンに麻酔を移し、isofluraを減らします2％のNEレベル。 </li><li>静かに角膜の乾燥を防ぐために、それぞれの眼に眼軟膏を適用します。手順の間に継​​続的に温度、呼吸数及び動物の心拍数を監視します。母体の生理は手順を通して安定のままであるべきです。 </li></ol> 2.外科準備とスクラブ<ol><li>小動物のバリカンを使用すると、下腹部にすべての毛を削除します。乳首をニッキングまたはライブ生まれた仔の将来の介護のために刺激を与えることができるかみそりの発疹の発生を回避するために注意して矩形のパターンで剃ります。 </li><li>滅菌綿棒でポビドンヨードおよび70％エタノールのスクラブのアプリケーションを交互に腹部の皮膚を準備します。ポビドンヨードおよび70％エタノールを交互に3回、それぞれに適用されるようにスクラブを繰り返します。乾燥してみましょう。 </li><li>つま先ピンチ反射の不在を経て、麻酔の深さを確認してください。痛みに反射し、刺激の非存在下では、1％にイソフルランレベルを下げます。 </li><li>無菌手術用タオルを使用して、動物をドレープ。彼らは子宮角への血流を妨げない一方で吸収洗浄流体の量を最大化するように、適切な角度でドレープを配置するように注意してください。 </li></ol> 3.腹部開腹<ol><li>メスを用いて調製腹部の皮膚の3センチ正中切開を行います。ぶっきらぼうにはさみで腹部の筋膜からスキン層を分析。鉗子および外科はさみを使用して、腹部の筋膜層を昇格し、腹膜腔へのアクセスを得るために、無血管白線の切開を行います。 </li><li>切開部の外側に手術用ガーゼを置き、滅菌生理食塩水で湿らせてから掃き入れます。腹部に鈍い鉗子や外圧を使用して、ゆっくりと腹腔から子宮角を除去し、湿らせたガーゼの上に配置します。 </li><li>慎重にもつれを回避し、腸にお問い合わせください。個々の羊膜嚢の間の唯一の筋肉組織を接触させることにより、ピンセットを用いて胎児を配置します。公開と鈍的切開を使用して、4子宮動脈を分離します。 注：ケアは、子宮動脈を分析するために取られなければなりません。周囲の組織と血管自体は非常にデリケートです。母体の血管への損傷は出血し、重症例では、胎児と母体の死を引き起こす可能性があります。 </li></ol> 4.配置瘤のクリップ<ol><li>各子宮動脈のラット30のG動脈瘤クリップを配置します。個々の胎盤を含む子宮血管の近位および遠位のパルス、および黒ずみなどの血流の停止を確認してください。ガーゼで露光角と全体外科分野をカバーし、滅菌生理食塩水で灌漑。灌漑ごとに約10分で湿ったフィールドを維持するように注意してください。 </li><li> 60分後、ガーゼを除去し、フィールドを灌漑。子宮角と血管が十分に成功したクリップを除去するために湿らせていることを確認します。静かに鉗子を使用して、各動脈瘤クリップを削除します。容器に外傷を起こさないように注意してください、とのmaint除去中AIN組織の整合性。 </li><li>徹底的羊膜嚢からガーゼの任意の浮遊スレッドを削除するように注意しながら、子宮角とフィールドを灌漑。 </li></ol>羊水嚢の中のリポ多糖の5インジェクション<ol><li>各個々の羊膜嚢の基部には、ちょうど、胎盤プレートの前方羊水の中に希釈したエバンスブルー色素とLPS（4μgの/ SAC）の100μLを注入します。注射のための最適な位置に各羊膜嚢を安定させ、回転させる鈍鉗子を使用してください。エバンスブルー色素を希釈し、適切な注射器の配置および注入を確認するのに有用である造影剤です。 注：羊膜内注射のための付属の8ミリメートル31 G針でのみを使用し、超微細0.3ミリリットルのインスリン注射器。大きなゲージの針を使用すると、慢性羊水損失、胎児死亡や妊娠の再吸収になります。注射器の取り外し時に羊水の漏れ少量のmitigすることができます羊膜嚢に直接圧力によってated。一部のラット胎児は、羊水過少の程度を許容することができます。しかし、大きなゲージの針で穿刺から急性羊水損失、または慢性液漏れが生じる偶発穿刺、胎児の損失の深刻なケースでは、結果、近隣の妊娠の喪失。 </li><li>ホーンは、滅菌生理食塩水で3回子宮を灌漑。 </li></ol> 6.開腹を閉じます<ol><li>鉗子を使用して、慎重に腹腔に子宮角を返します。羊膜嚢と正中切開、再おおよその実行3-0絹縫合糸を使用してmusculofascial層エッジ間の十分なスペースを確保。筋肉の切開を閉じながら羊膜嚢の配置に注意してください。嚢にまたはを介して縫合しないように注意してください。 </li><li>スキン層を閉じ、実行中の3-0絹​​縫合糸を使用して、スキン層を再近似します。 注：開腹を可能にするために、連続縫合の2層に閉じる必要があります皮膚や妊娠の増加に伴う筋肉の拡張のため。連続縫合糸は均等に分散創傷テンションを可能にします。多重ノットが刺激性で、簡単に麻酔から回復時にラットによって噛むことができるように結節縫合はあまり望まれています。外科用ステープルは、所望されていません。外科的な結び目の尾が非常に短い（&lt;3 mm）をカットする必要があります。 </li><li> 26 G針を使用して、0.125％ブピバカインを皮下巻き付けエッジの1ミリリットルを注入。首のうなじにブプレノルフィンを皮下/ kgで0.1 mgの1用量を投与します。 </li><li>イソフルランし、必要に応じてタオルドライしたラットの電源をオフにします。きれいなホームケージに置き、麻酔からの回復を監視します。ラットは低体温にならないことを確認してください。 </li></ol> 7.術後の回復とケア<ol><li>子犬が生まれされるまで（約E22またはE23）毎日、その後、ラットを72時間毎に8-12時間を監視し。ブプレノルフィンq8-12時間/ 72時間の追加の用量を投与またはIACUCによって決定されるようprnを。 </li><li>痛み、不快感、膣出血または手術部位からの出血の兆候がラットを監視します。縫合および切開を点検し、ラットが途中で彼らの縫合糸を咀嚼または削除されないように注意してください。非常に珍しいが、その縫合糸を侵害したラットは、創傷裂開のリスクがあります。 注：時々ラットは寝具や非食品の過剰な量を摂取することが、ブプレノルフィン投与の副作用として、パイカと呼ばれます。非常に珍しいが、ラットはパイカと潜在的な後続の腸閉塞のために監視する必要があります。 </li></ol> 8.組織処理および凍結切片化<ol><li>生後脳のヘマトキシリン​​＆エオシン（H＆E）染色のために準備するために、生後2日目（P2）に自分のホームケージから子犬を削除します。手術用ハサミを使用すると、仔ラットを刎ねるとそっと頭蓋骨から脳を削除します。 </li><li>リン酸緩衝生理食塩水で4％パラホルムアルデヒドの7ミリリットルを含む15ミリリットルコニカルチューブに修正脳をドロップ（PBS）。 4℃で脳を置き、72時間固定します。 </li><li> 72時間後、30％スクロースを含む滅菌PBS溶液に脳を転送する（w / v）の、4℃に戻します。 注：脳はショ糖溶液中にドロップすると、彼らはクライオスタットに区分される準備ができています。 </li><li>急速に脳を凍結し、凍結した冠状断面を取得するためのクライオスタット乳棒でマウントします。 20μmの凍結した冠状切片を切断し、スライド上にマウントします。確認のセクションでは、連続的に収集されます。 </li><li>スライドを室温で一晩乾燥することができます。ストアを-20℃でスライドします。 </li></ol> 9.ヘマトキシリン​​＆エオシン染色<ol><li>スライドが凍結切片をマウントし、室温に戻してください。 注：新鮮なすべてのソリューションを準備します。 </li><li> 2時間50°Cまでのスライドウォーマーセットの上に置いてスライドします。 </li><li>転送は、染色ラックにスライドさせます。ディップは、二重蒸留脱イオン水（のddH 2 O）で10倍をスライド。 </li><li> 5分間、100％のヘマトキシリン​​でスライドをインキュベートします。チタン私は、ヘマトキシリン​​で紫色の染色の程度に応じて最適化することができます。 </li><li>ディップは、タップ、H 2 Oで4倍をスライドし、1分間きれいな水道H 2 O中に放置します。 </li><li>ディップ（250 mlの70％エタノール+ 1mlを濃塩酸）酸アルコールで15倍をスライド。 </li><li>ディップは、タップ、H 2 Oで4倍をスライドし、1分間きれいな水道H 2 O中に放置します。 </li><li> 2分間、1％の炭酸リチウムでインキュベートします。 </li><li>ディップは、タップ、H 2 Oで4倍をスライドし、1分間きれいな水道H 2 O中に放置します。 </li><li> 1分間、95％エタノール中でインキュベートします。 </li><li>ディップスライドを100％エオシンで7倍。エオシンでディップの数がピンクの染色の度合いに応じて変更することができます。 </li><li>ディップスライドを95％エタノール中で5倍。 </li><li> 95％のエタノールの新鮮な変化ディップスライド5倍。 </li><li> 1分間100％エタノール中でスライドをインキュベートします。 </li><li> 1分間100％エタノールの新たな変化でスライドをインキュベートします。 </li><li> 15分間、100％キシレンでスライドをインキュベートします。 注：キシレンのステップと封入は、ドラフト内で行う必要があります。 </li><li> 15分間のキシレンの新鮮な変化でスライドをインキュベートします。 </li><li>パーマウントでカバースリップし、ドラフト内で乾燥させてください。 </li><li>画像は、光学顕微鏡でスライドします。 </li></ol>

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L., 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=Developmental+Expression+of+N+Methyl+d+Aspartate+(NMDA)+Receptor+Subunits+in+Human+White+and+Gray+Matter+Potential+Mechanism+of+Increased+Vulnerability+in+the+Immature+Brain.">Developmental Expression of N Methyl d Aspartate (NMDA) Receptor Subunits in Human White and Gray Matter Potential Mechanism of Increased Vulnerability in the Immature Brain.</a> Cereb Cortex. 25, 482-495 (2015).</li><li>Jantzie, L. L., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+Models+of+Encephalopathy+of+Prematurity.">Preclinical Models of Encephalopathy of Prematurity.</a> Devel Neurosci. , (2015).</li><li>Workman, A. D., Charvet, C. J., Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+transformations+of+neurodevelopmental+sequences+across+mammalian+species.">Modeling transformations of neurodevelopmental sequences across mammalian species.</a> J Neurosci. 33, 7368-7383 (2013).</li><li>Herlenius, E., Lagercrantz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+neurotransmitter+systems+during+critical+periods.">Development of neurotransmitter systems during critical periods.</a> Exper Neurol. 190, S8-S21 (2004).</li><li>Kelsom, C., Lu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+specification+of+GABAergic+cortical+interneurons.">Development and specification of GABAergic cortical interneurons.</a> Cell Biosci. 3, 19 (2013).</li><li>Kinney, H., Back, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+oligodendroglial+development+Relationship+to+periventricualr+leukomalacia.">Human oligodendroglial development Relationship to periventricualr leukomalacia.</a> Semin Pediatr Neuro. 5, 180-189 (1998).</li><li>Robinson, S., Li, Q., DeChant, A., Cohen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+amino+butyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma amino butyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg Peds. 104, 396-408 (2006).</li><li>Xu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+development+of+the+GABAergic+system+in+the+human+cerebral+cortex+and+white+matter.">Late development of the GABAergic system in the human cerebral cortex and white matter.</a> J Neuropathol Exp Neurol. 70, 841-858 (2011).</li><li>Segovia, K. N., 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=Arrested+oligodendrocyte+lineage+maturation+in+chronic+perinatal+white+matter+injury.">Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury.</a> Ann Neurol. 63, 520-530 (2008).</li><li>Robinson, S., Li, Q., Dechant, A., Cohen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+aminobutyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma aminobutyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg. 104, 396-408 (2006).</li><li>Boksa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prenatal+infection+on+brain+development+and+behavior+a+review+of+findings+from+animal+models.">Effects of prenatal infection on brain development and behavior a review of findings from animal models.</a> Brain Behav Immun. 24, 881-897 (2010).</li><li>Burd, I., Brown, A., Gonzalez, J. M., Chai, J., Elovitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+term+chorioamnionitis+unraveling+causes+of+adverse+neurological+outcomes.">A mouse model of term chorioamnionitis unraveling causes of adverse neurological outcomes.</a> Repro Sci. 18, 900-907 (2011).</li><li>Bergeron, J. 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=White+matter+injury+and+autistic+like+behavior+predominantly+affecting+male+rat+offspring+exposed+to+group+B+streptococcal+maternal+inflammation.">White matter injury and autistic like behavior predominantly affecting male rat offspring exposed to group B streptococcal maternal inflammation.</a> Devel Neurosci. 35, 504-515 (2013).</li><li>Uchida, 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=Effects+of+Ureaplasma+parvum+lipoprotein+multiple+banded+antigen+on+pregnancy+outcome+in+mice.">Effects of Ureaplasma parvum lipoprotein multiple banded antigen on pregnancy outcome in mice.</a> J Reprod Immunol. 100, 118-127 .</li></ol>

著者らは、開示することは何もありません。

未熟児の脳症が原因傷害のメカニズムを重複病因、神経発達の時間経過、人間の脳のネットワーク形成の複雑さの複雑な相互作用の動物でモデル化することは困難であり、中枢神経系の多様な表現型は、ヒト早産児でマニフェスト侮辱します。 EOPは、特定の細胞型の脆弱性に関連している（ すなわち 、未成熟オリゴデンドロサイト）21だけでなく、多様な発生調節経路（ ?...

37週の推定妊娠期間1前にアメリカで生まれた乳児の12％以上では、未熟児の周産期脳損傷（PBI）は、永久的な障害の重要な原因です。未熟児からPBI、未熟児とも呼ばれる脳症（EOP）は、全体の中枢神経系（CNS）に影響を与えます。 CNS損傷は、多くの場合、 子宮内で開始し、絨毛羊膜炎や、低酸素症および敗血症などの合併症を含む出生後の出産前のプロセスによって悪化します。全身侮辱からPBIは、神経発達を変化させ、感情的な規制、メモリと実行機能1,2に影響を与え 、脳性麻痺、てんかん、認知遅延及び多数の神経精神障害につながります。多くの進歩がなされているが、限られた理解は、早産からCNS損傷の細胞および分子結果が早産で生まれている子供の神経学的後遺症の多くに変換する方法のまま。知識後肢の欠如リアルタイムのCNS損傷の重症度の診断や新興介入の通知投与者。また、この脆弱性の患者集団のための年齢に適切な治療戦略は、とらえどころのないまま。 子宮内炎症は、極端な未熟児では非常に一般的であり、3複雑な胎児母体の胎盤炎症カスケードを伴います。子宮内感染はしばしば無症状です。急性炎症、または組織学的絨毛羊膜炎と一致特定の胎盤所見は、胎児の炎症反応の主要な決定であり、早産3-5に関連した脳損傷と一致しています。実際、胎児の炎症反応は、早産からの長期転帰改善のための明確な臨床的意義を持っています。妊娠期間（SGA）のために小さいまたは感染を経験する人幼児は、神経学的欠損3,4に対して非常に脆弱です。絨毛羊膜炎は、一般的な病理診断以下の早産で<sup&gt; 6,7、および組織学的検査は、非常に早産4児の胎盤の70％に炎症の徴候を明らかにしています。さらに、絨毛羊膜炎は、2年8で認知機能障害と関連しています。非常に早産で生まれた乳児の胎盤における母体の血管underperfusionの証拠はまた、子供の頃9に脳性麻痺と関連しています。絨毛羊膜炎および胎盤灌流欠損の相乗影響はよく年齢10,11の2年間で、この患者集団における異常な神経学的転帰の非常に高いリスクによって示されています。 病原体によって誘発される炎症に関連する人間の全身胎盤灌流欠陥や絨毛羊膜炎を模倣するために、我々はラットで羊膜内リポ多糖（LPS）と組み合わせ出生前過渡全身低酸素虚血（TSHI）のモデルを開発しました。私たちの目標は、子宮内炎症が含まれるように、単独でラット12-16でTSHIの我々のモデルを適応させることでした早産に関連するCNS損傷の前臨床モデル化を容易にします。 TSHIだけではオリゴデンドログリア系細胞、皮質ニューロンの永続的な損失を明らかにした、出生前脳損傷16と一致してけがの段階的なパターンにつながる進行性の虚血性の間隔で、細胞死を増加させ、およびプロ炎症性サイトカインのレベルの上昇。彼らは17-19を老化としてこのモデルの虚血性の構成要素への変更は、メモリの符号化の欠損、短期および長期記憶とラットにおける軽度の筋骨格系の変化を示しました。実際、我々は以前にTSHI + LPSの組み合わせは、オリゴデンドロサイトおよびニューロン喪失、軸索損傷、細胞の炎症や機能異常20を含む EOPの病態生理学的特徴を、再現することが実証されています。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Saline Solution, 0.9%</td>
			<td >Sigma</td>
			<td >S8776</td>
			<td ></td>
		</tr>
		<tr >
			<td >LPS 011B4</td>
			<td>Sigma</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr >
			<td >Evan&#39;s Blue Dye</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical gloves</td>
			<td>Biogel</td>
			<td>40870</td>
			<td></td>
		</tr>
		<tr >
			<td >OR Towels</td>
			<td>Cardinal Health</td>
			<td>287000-008</td>
			<td>Sterile</td>
		</tr>
		<tr >
			<td >PDI Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>06-669-62</td>
			<td></td>
		</tr>
		<tr >
			<td >Mini Arco Rechargeable Clippers</td>
			<td>Kent Scientific Corp.</td>
			<td>CL8787</td>
			<td></td>
		</tr>
		<tr >
			<td >Betadine surgical scrub</td>
			<td>Purdue Products L.P.</td>
			<td>67618-151-17</td>
			<td></td>
		</tr>
		<tr >
			<td >Eye Lubricant</td>
			<td>Refresh Lacri Lube</td>
			<td>00023-0312</td>
			<td></td>
		</tr>
		<tr >
			<td >Blunt Forceps</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td>Roboz</td>
			<td>RS-6808</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Scissors</td>
			<td>Roboz</td>
			<td>RS-5880</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Scissors</td>
			<td>F.S.T.</td>
			<td>14002-16</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td>1 ml</td>
		</tr>
		<tr >
			<td >Needle</td>
			<td>BD</td>
			<td>305122</td>
			<td>25G 5/8</td>
		</tr>
		<tr >
			<td >Needle</td>
			<td>BD</td>
			<td>305128</td>
			<td>30G 1</td>
		</tr>
		<tr >
			<td >Cotton-tipped Applicators</td>
			<td>Fisherbrand</td>
			<td>23-400-114</td>
			<td>Small, 6 inch sterile</td>
		</tr>
		<tr >
			<td >Cotton Gauze Sponge</td>
			<td>Fisherbrand</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr >
			<td >Needle Holders</td>
			<td>Kent Scientific Corp.</td>
			<td>INS600109</td>
			<td>12.5 CM STR</td>
		</tr>
		<tr >
			<td >Vessel Clips</td>
			<td>Kent Scientific Corp.</td>
			<td>INS600120</td>
			<td>30G Pressure</td>
		</tr>
		<tr >
			<td >3-0 Perma Hand Silk Sutures</td>
			<td>Ethicon</td>
			<td>1684G</td>
			<td>Black braided, 3-0 (2 metric), 18&#34;, non-absorbable,&#160; PS-1 24mm needle, 3/8 circle</td>
		</tr>
		<tr >
			<td >Insulin Syringes</td>
			<td>BD</td>
			<td>328438</td>
			<td>0.3cc 3mm 31G</td>
		</tr>
		<tr >
			<td >Pentobarbital</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Buprenorphine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Bupivacaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Lithium Carbonate</td>
			<td>Acros Chemicals</td>
			<td>554-13-2</td>
			<td></td>
		</tr>
		<tr >
			<td >Superfrost Plus Microscope Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr >
			<td >Hematoxylin</td>
			<td>Leica</td>
			<td>3801521</td>
			<td>Surgipath Gill II Hematoxylin</td>
		</tr>
		<tr >
			<td >Eosin</td>
			<td>Leica</td>
			<td>3801601</td>
			<td>Surgipath Eosin</td>
		</tr>
		<tr >
			<td >Xylenes</td>
			<td>Fisherbrand</td>
			<td>X3S-4</td>
			<td>Histological Grade</td>
		</tr>
		<tr >
			<td >Permount</td>
			<td>Fisherbrand</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr >
			<td >Coverglass</td>
			<td>Fisherbrand</td>
			<td>12-548-5P</td>
			<td>Fisher Finest Premium Coverglass</td>
		</tr>
	</tbody>
</table>

modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats

未熟児の脳症（EOP）が最高の事前並進目標に。早産に関連する異常、中枢神経系（CNS）に含まれる用語であり、早産に関連した脳損傷のための新しい治療戦略を発見、EOPの前臨床モデルは、同様のメカニズムが含まれている必要があります出生前のグローバル傷害のヒトで観察され、母体の胎盤胎児システムの複数のコンポーネントを含みます。理想的には、モデルが成熟した動物において機能欠損の類似のスペクトルを生成し、病態生理学の複数の側面を再現する必要があります。人間の全身胎盤灌流欠損、胎盤underperfusionおよび/または早期早産で病原体誘発性の炎症に関連する絨毛羊膜炎を模倣するために、我々は、羊膜内リポ多糖（LPS）と組み合わせ出生前過渡全身低酸素虚血（TSHI）のモデルを開発しました。妊娠のSprague Dawleyラットにおいて、TSHI子宮動脈閉塞oを介してnは胎生18日（E18）は、胎児における増加CNS損傷に関連した段階的な胎盤underperfusion欠陥を誘発します。羊膜内LPS注射と組み合わせると、胎盤の炎症が増加し、CNS損傷は、関連する白質、歩行や画像異常に配合されます。出生前TSHIとTSHI + LPS出生前侮辱は、ニューロン、オリゴデンドロサイトおよび軸索の喪失を引き起こし、子宮内侮辱を反復するなど、EOPモデルの基準のいくつかを満たす、サブプレートの喪失、および非常に生まれた子供で観察されたものを模倣成体動物における機能欠損早産。また、このモデルは、発散損傷の種類によって誘発される炎症の切開を可能にします。

E18でTSHI + LPSの後、ヘマトキシリンおよびエオシン染色は有意な組織病 ​​理学的胎盤両方の異常（ 図1）、脳内（ 図2）を明らかにする。 E19とE21を調べ胎盤は脱落膜と迷路を通してマイクロ出血、および壊死を伴う肉眼的浮腫です。著しい炎症性浸潤及び血管分布の増加も観察されます。 P2に調べた脳を脳室のほか、シャムに比べて白質とサブプレートニューロン?...

Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

ラットにおける羊膜内リポ多糖で出生前低酸素性虚血を使用した未熟児のモデル化脳症

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

modeling-encephalopathy-prematurity-using-prenatal-hypoxia-ischemia

Research

JoVE Journal

Medicine

11.2K ビュー.  University of New Mexico. Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

ビデオ: Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

マウスにおけるモデリングのストローク - フィラメントモデルと中大脳動脈閉塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

医学

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

と陽電子放射断層撮影法を用いた非侵襲的イメージングとリビングラットにおける脳虚血の分析<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

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 ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

と伝達性海綿状脳症種の障壁を評価する<em&gt;インビトロ</em&gt;プリオンタンパク質変換アッセイ

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

マウス脳低酸素性虚血の間に同時PET / MRIイメージング

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

一過性脳低酸素性虚血に基づく血栓性脳卒中モデル

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

光血栓法を用いた外接莢梗塞モデル

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

ラットにおける脱細胞化腎臓足場の調製

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

原発性月経困難症ラットにおける液体クロマトグラフィー-タンデム質量分析法を用いた生および加工されたCyperi根腫サンプルの分析

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

ラットの変形性膝関節症に対する標準化されたTuinaマニピュレーション

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

著者スポットライト:ラットにおけるTuina操作のメカニズムの理解 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

変形性膝関節症ラットの炎症と軟骨喪失を軽減するためのTuina操作

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...