この作品は、国立衛生研究所[助成金なしでサポートされていました。 R01NS069726]と援助グラントのアメリカ心臓協会グラント【グラントありません。 13GRNT17020004] SDへ。

注：マウス（C57BL / 6J、オス）高齢者 10から12週目は、この研究で使用しました。すべての手順は、実験動物の管理と使用に関するNI​​Hガイドに従って行われ、ミズーリ州制度動物実験委員会（IACUC）の大学によって承認されました。 1.手術前<ol><li>日手術のオートクレーブの前に、すべての手術器具を滅菌します。乾燥の30分（121°Cの 、15 PSI、30/30サイクル）、続いて30分間、15 psiで121°Cので楽器やオートクレーブを包みます。さらに使用するまで清潔で無菌環境で機器を配置します。 </li><li>新鮮なローズベンガル（RB）溶液（滅菌生理食塩水中の20 mg / mlの）手術の前にするたびに準備します。完全にRBを溶解し、ボルテックスチューブをアルミニウム箔19 W.ラップ管の出力電力と50/60 Hzで5分間超音波処理に続いて、さらにシュール中に使用されるまで、光から保護しますGeryの。 </li><li>滅菌生理食塩水にケタミン/キシラジンの混合物を準備します。麻酔薬混合物の最終容量1mlを作るために：（100 mg / mlのストック濃度）および滅菌生理食塩水550μlのとketmaineの325μL：キシラジン125μlの（20 mg / mlのストック濃度）を追加します。 </li><li>恒温加熱パッドを事前に温めます。 </li><li>ランプ電力を安定させるために30分間、メタルハライドランプ（FN1エピ蛍光顕微鏡用の光源）を事前温め。 </li><li> 10倍対物レンズと直立FN1落射蛍光顕微鏡で視野絞りを調整することにより、レチクルを使用して1mmの直径に照明領域のサイズを調整します。 </li></ol> 2.手術手順<ol><li>ケタミンの用量でマウスを麻酔（130ミリグラム/ kgのBの重量。）およびキシラジン（10mg / kgのBとの重量）。マウスのB.重量g当り1.3、4μlのカクテルに基づきます。必要とされています。アルコール綿棒で注射部位を消毒し、内を通って麻酔薬を投与します-peritoneal（IP）ルート。動物の誘導と回復を遅らせることになる。このような血管や筋肉に麻酔薬を注入しないように注意してください。 </li><li>乾燥を防止し、低体温症を防ぐために加熱パッドの上に動物を配置するために、マウスの両眼に人工涙軟膏を適用します。 </li><li>動物の作製<ol><li>つま先のピンチ応答を使用して、適切な外科的レベルの麻酔のために動物を確認してください。 </li><li>動物が麻酔の外科的レベルに達したら、電気ヘアトリマーを使用して、動物の正中線の周りの背側表面の毛をクリップ。ベータダイン液で3回、続いて70％エタノールで手術部位を洗浄。次のステップまで、滅菌外科用ドレープを持つサイトをカバーしています。 </li></ol></li><li>薄い骨の外科的処置は、脊髄を露出させます<ol><li>手術用プラットフォーム（ 図1A）の恒温加熱パッドの上にうつ伏せにマウスを置きます。適切にマウスを確保細長いネック領域（ 図1A、B）を維持するために鼻のクランプを用いて姿勢。 </li><li> T12に胸椎T9から延びる背半ば線に沿って手術用はさみを使用して（約1センチ）の切開を行います。手術領域を露出するために皮膚を脇に移動します。 </li><li>メスを使用して、慎重にT9で背棘を公開するために筋肉をクリア - T12椎骨を。滅菌綿棒で穏やかな圧力を適用することにより、各ステップにおいて出血を止めます。別々のT10 -周辺の筋肉からT12椎骨と安定させ、任意の移動を防止するために椎骨クランプを使用して固定します（ 図1A、B）。 </li><li> T10やT11の椎骨の背側表面（ 図1C）SCの背面に脊椎静脈および他の小血管を可視化するために慎重に、ゆっくりと薄く、ドリルビット研磨骨と高速ドリルを使用します。 </li><li>間引き手順中に発生する熱による熱損傷を防止するために、破片を除去するために一定の吸引と一緒に通常の生理食塩水の穏やかで一定のストリームを適用します。 </li><li>主血管がはっきりと見えるまでメスを使用すると、慎重に骨の表面を滑らかに。このプロセスの間に、脊髄を損傷しないように注意してください。 </li><li>血管が可視化されると、インスリン注射器を用いて眼窩洞を介して経路を30mg / kg（体重）の用量でRBを投与します。 </li><li> RB注射後3分後、レーザードップラー流量計を使用して血流を測定する必要に応じて（ 図2A、B）。全体の手順の間に無菌状態を維持します。 </li></ol></li></ol> PTの3誘導<ol><li>ラボ·ジャックの高さを調整することができる上にXY位置調整可能なステージ上に動物を置きます。 T11脊髄の露出領域を直接FN1エピ蛍光顕微鏡（ 図3A）の10倍の目的の下にあるように、マウスの位置を調整します。 </li><li>李の電力を設定12％でGHTソースと（注：この領域は脊椎静脈およびその他の毛細血管を含む）、薄型化、脊髄の中央に0.75ミリメートルの直径を有するT11領域を照射する緑色光（波長540と - である、580 nmの2分間の10倍の対物レンズを介して顕微鏡でフィルターキューブ）により達成しました。照射（ 図3B、C）の最初と最後に画像を撮影し、この時点では実験の時間を記録します。 </li><li>必要であれば、2.4.8（ 図2A、B）のような脊髄上記と同じ位置に、レーザードップラープローブを配置することにより、10分間、再び血流を測定します。 </li><li>任意の出血のための照射確認後と場合いずれも、動物を縫合に進んで見つかりませんでした。吸収性縫合糸または4-0サイズの絹縫合糸を用いて、脊髄の両側の筋肉と一緒に浅筋膜を縫合。露出されたSCを傷つけないように注意してください。 4-0絹縫合糸で皮膚を縫合。ベタジンまたはヨウ素を適用します。縫合後の皮膚の縁に。 </li></ol> 4.手術後のケア<ol><li>縫合した後、回復のための加熱パッド上に動物を配置します。回収後の両方の後肢の動きを観察することによって神経学的欠損の徴候のために動物を確認してください。それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人動物を放置しないでください。 </li><li>ホームケージに動物を転送します。完全に回復するまで、他の動物の会社に手術を受けた動物を返しません。 </li><li>一定の間隔で動物を確認してください。重度の神経学的欠損の場合には、膀胱の避難、鎮痛薬の投与（ - 0.1ミリグラム/ kgのブプレノルフィン、0.05）のように適切なケアを提供しています。脱水のためにチェックし、重度の場合には、通常の生理食塩水を皮下に投与します。通常、ブプレノルフィン（0.1 mgの/ kg）は、手術部位の痛みを軽減するために縫合した後に投与されるであろう。 </li><li>動物はすぐにされていない場合動物は簡単に食べ物に到達することができますので、手術後に安楽死させ、我々は、ケージの床の上に、高含水量の食事を配置します。 </li></ol> 5. Transcardial灌流、ニッスル染色および免疫染色<ol><li>先に説明したように17-20経動物を灌流。 <ol><li>以前のプロトコルで説明したように、動物を麻酔し、経PBS中の氷冷4％パラホルムアルデヒド（PFA）、続いてリン酸緩衝生理食塩水（PBS、pH7.4）中で灌流。 </li><li>灌流後、脊髄（SC）を除去し、4°CO / NでPBS中4％PFAでそれをポストは、固定してください。 30％のショ糖を含むPBSに固定SCを転送し、2のためにそれを維持する - それはチューブの底に沈むまで3日。 </li><li>クライオスタットを使用することで、厚さ30μmの切片に脊髄を切断し、ゼラチンコートしたスライドガラス上に、または0.01 M PBSで48ウェルプレートにシリアルに配置します。 </li></ol></li><li>ニッスル染色：ニッスルstainiを行うPTによって引き起こされる損傷を検査します以前17-20に記載されているように、脊髄切片にngの。 <ol><li>簡単に説明すると、0.25％クレシルバイオレットで五回ごと脊髄ガラススライド上のスライスや汚れを収集します。染色した切片（ 図4）の画像を撮影します。 </li></ol></li><li>免疫染色：浮動セクション法17,18,20を用いて前述したように。 <ol><li>ウサギ抗のNeuN抗体（1：300）、およびウサギ抗：簡潔には、ウサギ抗グリア線維性酸性タンパク質（GFAP）ポリクローナル抗体（300 1）を用いて4℃でO / Nインキュベートすることにより脊髄切片を染色IBA1抗体（1：500）ロバ抗ウサギアレクサ568コンジュゲートのIgG（1：400）、続いて室温で4時間二次抗体。蛍光顕微鏡（ 図5）を入れて撮影します。 </li></ol></li></ol>

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(76), (2013).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+imaging+of+photothrombotic+stroke+in+conscious+and+freely+moving+rats.">Induction and imaging of photothrombotic stroke in conscious and freely moving rats.</a> J Biomed Opt. 19 (9), 96013 (2014).</li><li>Seto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+ischemic+stroke+in+awake+freely+moving+mice+reveals+that+isoflurane+anesthesia+can+mask+the+benefits+of+a+neuroprotection+therapy.">Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy.</a> Front Neuroenergetics. 6 (1), (2014).</li><li>Bell, M. T., 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=Toll-like+receptor+4-dependent+microglial+activation+mediates+spinal+cord+ischemia-reperfusion+injury.">Toll-like receptor 4-dependent microglial activation mediates spinal cord ischemia-reperfusion injury.</a> Circulation. 128 (11 Suppl 1), S152-S156 (2013).</li><li>Smith, P. 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=The+evolution+of+chemokine+release+supports+a+bimodal+mechanism+of+spinal+cord+ischemia+and+reperfusion+injury.">The evolution of chemokine release supports a bimodal mechanism of spinal cord ischemia and reperfusion injury.</a> Circulation. 126 (11 Suppl 1), S110-S117 (2012).</li><li>Jia, Z., 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=Oxidative+stress+in+spinal+cord+injury+and+antioxidant-based+intervention.">Oxidative stress in spinal cord injury and antioxidant-based intervention.</a> Spinal Cord. 50 (4), 264-274 (2012).</li></ol>

The authors have nothing to disclose.

本研究では、SCの虚血の光血栓モデルを説明しました。遺伝工学の進歩が可能SCにおける虚血性病態生理に関与する特定の遺伝子の影響を研究するために作られた市販のトランスジェニックマウスの急増がありました。研究の目的は、脊髄虚血の再現可能なマウスモデルを開発することでした。ここでは、マウスでSCIを誘導するために皮質PTモデルを適応しました。手術後のT11胸?...

外傷性脊髄損傷（SCI）は、SCの感覚と自律神経機能に影響を与える壊滅的な臨床症状です。 SCIを生き残った患者は、多くの場合、大幅に日常活動や生活1の品質に影響を与える対麻痺を衰弱が残されています。実験SCIモデルはSCIの病態生理と関連する神経修復プロセスを理解するために、科学的な調査で必要不可欠なツールとなっています。これらのモデルはまた、機能回復を目的とする種々の実験の神経保護的介入の前臨床効力を試験するために使用されています。現在、実際にSCIモデルの大部分は、機械的に破壊し、SCを傷つけるために物理的な鈍力の使用を採用しています。これらの方法は、SC 2の挫傷、圧縮、転位や離断を含みます。ことが示唆されている主要な機械的な損傷、負傷したSC内における虚血セットの形での二次損傷後<html> 3,4。二次虚血の原因は、大規模な組織変性、実質内出血、時には組織浮腫5-7による血管の閉塞によって含まれています。 SCの整合性がさらに影響される二次的損傷の結果として、ニューロンとグリア細胞がひどく機能および生存度が損なわれ、虚血性ペナンブラ脳卒中後の成長に類似した損傷の慢性期の間に成長を梗塞につながるアポトーシスを受けます8,9。興奮毒性、フリーラジカル生成、および炎症などのいくつかのメカニズムは、SCI 10,11次の虚血性細胞死の原因であることが報告されています。また、SCの虚血は、多くの場合、患者12,13に対麻痺を引き起こす胸腹部大動脈瘤修復手術の重篤な合併症です。このような高い臨床的影響にもかかわらず、高い再現性脊髄虚血の非常にいくつかのモデルは、現在利用可能です。 NTは&quot;&gt;光血栓（PT）は、技術が高度に再現、かなり非侵襲的である。脳14-20における局所的虚血の誘導のために一般的に使用される方法であり、脳17の露出した領域に正確な焦点虚血性病変を生成します-21。これは、ローズベンガル（RB）16-20,22又はエリスロシンBなどの光活性染料の全身投与により達成されるが、適切な光源を用いて血管の局所照射し23。色素の光活性化は、フリーラジカルの生成を引き起こします滑らかな血管内皮の完全性を破壊し、そして血小板がその後容器24によって供給される領域内の梗塞内血栓結果により血液の流れの閉塞、血栓を形成し、蓄積させる。ためには、制御を容易にします照射の強度および持続時間は、この手順は非常に均一で再現性の梗塞を生じる。また、この方法はinfarcを誘導するために使用することができます虚血の影響の空間（ 例えば 、対白質、灰白質）の理解を可能にする様々な解剖学的位置でT。 現在の研究の目的は、マウスにおけるSC虚血の簡単かつ再現性の高いモデルを開発することです。我々は、マウスにおけるSC虚血のPTモデルの手順を説明しました。組織学および免疫染色の結果から、PTが効果的にSC梗塞、神経細胞脱落と反応性神経膠症を誘発することができることを実証しました。

<table border="0" cellpadding="0" cellspacing="0" width="1050">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
			<td style="width:233px;">Sigma-Aldrich</td>
			<td style="width:180px;">330000</td>
			<td style="width:421px;">20 mg/ml in sterile saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57Bl6/J</td>
			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-996-06</td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-234-11</td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine solution</td>
			<td>Purdue</td>
			<td>NDC-67618-150-01</td>
			<td>10% povidone iodine topical solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal saline</td>
			<td>Abott Laboratories</td>
			<td>04930-04-10</td>
			<td>For diluting RB, anaesthesia and for preventing tissue from drying</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artificial tears ointment&#160;</td>
			<td>Rugby</td>
			<td>NDC-0536-6550-91</td>
			<td>83% white petrolatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal halide lamp</td>
			<td>EXFO, Canada</td>
			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td>Fine Science Tool</td>
			<td>15000-10</td>
			<td>for minor dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors (angled to side)</td>
			<td>Fine Science Tool</td>
			<td>14063-011</td>
			<td>No. 3 handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard scalpel</td>
			<td>Fine Science Tool</td>
			<td>10003-12</td>
			<td>for removing muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
			<td>2976</td>
			<td>No. 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (curved)</td>
			<td>Fine Science Tool</td>
			<td>11150-10</td>
			<td>for holding tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (straight)</td>
			<td>Fine Science Tool</td>
			<td>11151-10</td>
			<td>for holding tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holder&#160;</td>
			<td>Fine Science Tool</td>
			<td>12002-12</td>
			<td>for suturing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue adhesive glue</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>to adhere to edges of the cut skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
			<td>USSC Sutures</td>
			<td>VP-521</td>
			<td>Size = 4-0 (for fascia)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perma-hand silk</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td>Size = 4-0 (for skin)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro drill</td>
			<td>Roboz Surgical Instrument Co. Inc.</td>
			<td>RS-6300</td>
			<td>with bone polishing drill bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser doppler flowmeter</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td>for monitoring change in blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tool</td>
			<td>21052-00</td>
			<td>to prevent hypothermia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Jack</td>
			<td>Fisher scientific&#160;</td>
			<td>14-673-50</td>
			<td>4x4 in plate to adjust the height of the animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-Y gliding stage&#160;</td>
			<td>Amscope</td>
			<td>GT100</td>
			<td>for positioning the animal under microscope&#160;&#160;</td>
		</tr>
	</tbody>
</table>

photothrombosis-induced focal ischemia as a model of spinal cord injury in mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

本研究の目的は、PTモデルを用いて、マウスにおける脊髄虚血を生成することでした。脊髄（T10 - T12）上記骨の所望の領域の後薄くした、ローズベンガルは眼窩洞経路で注入し、虚血はPTによって誘導された、図1A、Bは内に配置マウスを示してカスタムメイドの外科。手術中のプラットフォーム。マウスは、鼻クランプにより所定位置に保持され、2つの調整可能脊椎動物は、脊髄...

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

マウスの脊髄損傷のモデルとしての光血栓誘発性局所虚血

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

Research

JoVE Journal

Medicine

13.1K ビュー.  University of Missouri. Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice. 

ビデオ: Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

ラット脊髄損傷後の急性および慢性の触覚官能検査

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

医学

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

マウスで制御子宮裂傷の傷害

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

ラットにおける重度の脊髄損傷挫傷モデル

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

マウスにおける急性腎障害とポスト傷害線維の虚血再灌流モデル

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

脊髄虚血再灌流のマウスモデルにおいて胸部大動脈閉塞によって再現可能対麻痺

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

外傷性脳損傷のための制御皮質衝撃モデル

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

ラジオ遠隔測定システムは、脊髄離断および胚性神経細胞移植片幹ラットの心臓血管機能を監視する

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

脊髄損傷の実験的挫傷モデルにおける神経幹細胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

脊髄圧縮傷害の校正済み鉗子モデル

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生児マウス脊髄圧縮損傷モデル

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...