私たちは、獣医医療用動物資源スタッフのエモリー大学本部に感謝します。資金源が含まれます：ALS協会、国防総省、そしてNeuralstem、株式会社

1。動物使用本明細書に示さ手続きはエモリー大学機関動物実験委員会（IACUC）によって承認されている。約15〜20 kgの女性ゲッティンゲンのミニブタが使用されます。 2。麻酔動物は、手術の前に約12時間を絶食させる。動物の鎮静および麻酔導入は、筋肉内ケタミン（35 mg / kg）を、アセプロマジン（1.1 mg / kg）であり、アトロピン（0.04 mg / kg）のカクテルで構成されています。動物は、その後挿管し、酸素および1-3％イソフルラン麻酔で維持されます。この時点で、それぞれの動物の背中と頭部を剃毛されています。麻酔の深さは、獣医スタッフによって監視されています。 、すだれ状角膜と眼瞼反射神経だけでなく、心臓や呼吸数、パルスオキシメータ、直接/間接血圧、呼気終末二酸化炭素の測定、および筋緊張/侵害刺激に対する応答の有無は、深さを監視するために使用される<html>麻酔の。 3。ポジショニング動物は、手術室に連れて行かれ、ジャクソン脊椎手術台に患者の位置決めを模倣するように設計されたフレームのカスタムに腹臥位に配置されます。フレームは腹部と胸部とその結果として硬膜外静脈出血（ 図1）に圧力を最小限に抑えるため、腹部は無料垂れ下がらと、動物の胸と骨盤の下に置かれている調節可能なスリングを利用しています。フレームには、手順1の背骨の外部固定化を提供します。 また、動物は体温と体液管理や手術中に必要な薬物送達のためにカテーテルを挿入された辺縁耳静脈を維持するために加熱された再循環パッド上に配置されます。最後に、手術野は、アルコールとChlorhexadineまたはベタジン溶液と整形処理され、外科用ドレープは、手術フィールドに配置されます。 椎弓切除術LE &quot;&gt; 4。 約10〜15 cm商品皮膚切開が行われ、傍脊柱筋組織は、二国間で背骨をオフに解剖されています。次に、背マルチレベルの椎弓切除術が行われます。 3つの椎骨を覆うC3〜C5またはL2-L4セグメントのラミナと棘突起はrongeursと外科用ドリルを使用して除去される。 5。脊髄デリックの配置我々は、脊髄デリック脊髄2-5に（遺伝子細胞）療法の配信のために設計されたデバイスと呼びます。このデバイスの設計と進化に関する詳細な議論は、ライリーら 、2011に記載されています。4 患者へのデバイスの安全性を確保するために、経皮的ポストは主切開上下1センチメートル皮膚切開を介して配置され、主切開上下に薄板に取り付けられている。 次に、2の統合開創は、foに接続されている椎弓切除術を受けている背骨の領域を露出させるために切開部位の上下ウル皮の投稿。 6。硬膜オープニングウッドソン歯科用ツールおよび11ブレードの助けを借りて、2.5 cm商品切開は脊髄を露出し、硬膜を介して行われます。硬膜を4から0 Nurolon縫合糸を使用して軟膜層から反射と深い傍脊柱筋組織に固定されている。 手術のパテは、開口部の吻側および尾側の両極端に配置されます。これらは、脳脊髄液の流れへの部分的なバリアを提供し、また、脊髄に損傷を与えることなく、吸盤を配置する外科医のための安全なターゲットを提供する。ヒトでは、手術用顕微鏡の倍率で、軟膜表面は、この時点で解剖されています。技術的な制限があるため、この手順は必須または動物で現実的ではありません。 7。脊髄注射と追加注射用脊髄デリックの横方向の変位の注射の直前に、メチルプレドニゾロン（125mgを、IV）のボーラスが腫れ脊髄を防止するために与えられている。 この時点では、プラットフォームのレールシステムを装着し、サイドレールは、適切な長さに合わせて調整されます。ゴンドラです 2つのバーの上にトップロードが、Zドライブは、ユニバーサルジョイントにマウントされています。次に、ロードされたカニューレは、マイクロドライブに配置されます。マイクロインジェクションプラットフォーム上でユニバーサルジョイントを使用して、冠状および矢状角は脊髄注射はカニューレの配置をたどるの表面に直交する軌道を確保するために調整されます。針は背根入り口帯（DREZ）に応じて内側に配置されている。 DREZは3.5X外科ルーペの倍率で識別し、ポイントでコードの表面に直交する軌道上に貫通されている&lt;1ミリメートル内側。 ヒトでは、術前のMRIは、ベースラインアセスメントOを提供手術計画のためのf脊髄寸法。また、脊髄の厚さは、前角の目標深度を決定するために測定される。 懸濁液は、軟膜の接触から4ミリメートルの深さに注入される。ウルテムプラスチック製のフランジに、必要以上に深く進んでから針を防ぐために軟膜表面上のストッパーとして機能します。針の先端がターゲットで配置されると、硬い金属外側スリーブは柔軟性のあるチューブが露出したまま、プルアップされています。注入が完了すると、針がカニューレ噴射管アップセルの逆流を防止するためにさらに1分間場所に残されています。 ケアは若干マイクロドライブ側性またはrostro-尾のどちらかを調整することによって、表面の血管系を回避するように注意されています。浸透のサイトからいくつかの出血が発生することがあります。出血などが発生した場合、マイクロパテは出血穿刺部位の上に配置されており、吸引は、カニューレpenetrから血を吸い取るために、それらに適用されるationサイト​​とコードの蓄積を防ぐ。血液が凝固するためにこれが確実にできます。直接の圧力があるよう焼灼は回避される。 針を除去した後、定位固定装置は、脊髄の背側表面に見える血管を避けるために必要に応じて、2または4 mm以上で区切っrostro - 尾軸に沿って次の標的部位に再配置されます。与えられた研究で提案したように、このプロセスが何度も繰り返されます。 8。フローティングカニューレ細径のカスタム注入カニューレ、注射のために使用されます。カニューレは、可変長の30ゲージのフレキシブルシラスティックチューブに接続されている固定長の30ゲージベベル針で構成されています。遠位端がマイクロインジェクターポンプに接続されているハミルトンルアーロックが装備されています。近シラスティックチューブは24番ゲージの剛性アウターカニューレ内ensheathedされている注射針のフランジの近位端上の席。このフランジ両方席外側カニューレ、注射針の深さストップとして機能します。各注射のためには、治療上の懸濁液の適切なボリュームが1分あたり5μlの割合で校正済みM​​INJ-PDマイクロインジェクターポンプ（Tritechリサーチ社、ロサンゼルス、カリフォルニア州）を用いて注入する。 9。閉鎖すべての注射が行われた後、脊髄デリックを穏やかに除去され、切開は4層で閉鎖されています。硬膜防水ファッションで、4.0 Nurolonステッチを使用して閉じられます。 0 VICRYL縫合糸は深い筋層に使用されます。筋膜は、その後防水ファッションでも0 VICRYL縫合糸で閉じられます。真皮層は、最終的にランニングステッチで、2.0 VICRYLで閉じられます。皮膚縫合は3から0ナイロン縫合糸を使用して完了します。 10。回復と疼痛管理動物は2時間以下の麻酔回復のために抜管され、監視されています。次に、動物を個々のCに転送されます年齢や摂餌量、排便、排尿のためにと少なくとも1日1回監視した。 疼痛管理のために、経皮フェンタニルパッチ（75 mcgか）は、術後鎮痛の3日間、動物の背中にステイプルされています。また、ブプレノルフィン（0.05 mg / kgで、BID、IM）はまた、最大3日間、術後のために与えることができます。 11。その結果、代表アウトカム臨床的および行動観察は研究デザインによると終点まで術前に行われると、その後1〜7日目に記録し、毎週しています。行動データは、前述の6として神経学的罹患率を評価するために収集されます。感覚機能は、前面と後肢のつま先への機械的刺激に対する離脱応答の有無により評価される。 - ; 1 -後肢における知覚緊張、わずかメートル麻痺、動かない0：モータ関数はTarlovスコア（ 表1）を次のovement、2 - 後肢での動きが、座ったり立つことができ、3 - スタンドと歩行能力が、運動失調と短い期間、4 - 完全回復、正常な運動機能。 手順の安全性は、手術前のベースラインに戻るには、動物の能力によって決定されます。一過性神経障害は、主に動物の品種や手順（他のパラメータのうち、注射の回数）に応じていくつかのバリエーションで、術後1日目と7の間に解決する必要があります。永久的な罹患率は、動物が動物実験委員会、デフォルトのエンドポイント（ 図2）に達する時間によって解決されない持続的な神経学的欠損によって定義されます。

<ol>
	<li>Usvald, 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=Analysis+of+dosing+regimen+and+reproducibility+of+intraspinal+grafting+of+human+spinal+stem+cells+in+immunosuppressed+minipigs.">Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs.</a> Cell Transplant. 19, 1103-1122 (2010).</li><li>Riley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+spinal+cord+therapeutics+delivery:+stabilized+platform+and+microelectrode+recording+guidance+validation.">Targeted spinal cord therapeutics delivery: stabilized platform and microelectrode recording guidance validation.</a> Stereotact. Funct. Neurosurg. 86, 67-74 (2008).</li><li>Federici, T., Riley, J., Park, J., Bain, M., Boulis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+safety+validation+of+a+stabilized+viral+vector+direct+injection+approach+to+the+cervical+spinal+cord.">Preclinical safety validation of a stabilized viral vector direct injection approach to the cervical spinal cord.</a> Clin. Transl. Sci. 2, 165-167 (2009).</li><li>Riley, J. P., Raore, B., Taub, J. S., Federici, T., Boulis, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platform+and+Cannula+Design+Improvements+for+Spinal+Cord+Therapeutics+Delivery.">Platform and Cannula Design Improvements for Spinal Cord Therapeutics Delivery.</a> Neurosurgery. 69, 147-154 (2011).</li><li>Riley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cervical+spinal+cord+therapeutics+delivery:+preclinical+safety+validation+of+a+stabilized+microinjection+platform.">Cervical spinal cord therapeutics delivery: preclinical safety validation of a stabilized microinjection platform.</a> Neurosurgery. 65, 754-761 (2009).</li><li>Raore, B., 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=Cervical+multilevel+intraspinal+stem+cell+therapy:+assessment+of+surgical+risks+in+Gottingen+minipigs.">Cervical multilevel intraspinal stem cell therapy: assessment of surgical risks in Gottingen minipigs.</a> Spine. 36, 164-171 (2011).</li><li>Boulis, N. M., 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=Translational+stem+cell+therapy+for+amyotrophic+lateral+sclerosis.">Translational stem cell therapy for amyotrophic lateral sclerosis.</a> Nat. Rev. Neurol. , (2011).</li><li>Riley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspinal+Stem+Cell+Transplantation+in+ALS:+A+Phase+I+Safety+Trial,+Technical+Note+&#38;+Interim+Safety+Outcomes.">Intraspinal Stem Cell Transplantation in ALS: A Phase I Safety Trial, Technical Note &#38; Interim Safety Outcomes.</a> Neurosurgery. 71 (2), 405-416 (2012).</li><li>Boulis, N. M., 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=Lumbar+intraspinal+injection+of+neural+stem+cells+in+patients+with+ALS:+results+of+a+Phase+I+trial+in+12+patients.">Lumbar intraspinal injection of neural stem cells in patients with ALS: results of a Phase I trial in 12 patients.</a> Stem Cells. 30 (6), 1144-1151 (2012).</li></ol>

博士Boulisはヒトの脊髄の安全かつ正確な注入を可能にするための装置の発明者です。 Neuralstem社は、この技術への排他的なライセンスを購入した。博士Boulisは、この手数料の棚卸のシェアを受けており、この技術の配布のためのロイヤルティの支払いに対する権利を持っています。他の著者は、開示することは何もありません。

人間7-9の記載された技術を進めるための承認にもかかわらず、重要な問題は、成功するために脊髄療法の順に回答されずに残っている。実質内注入に脊髄寛容の厳格な理解は、退行性脱髄、および外傷性脊髄疾患の治療法を開発する臨床試験の計画と実行を有効にする必要があります。現在、大型哺乳類の脊髄は一過性​​および永久的な罹患せずに耐えることができる注射?...

<table border="1"><tbody><tr><td> 機器の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>脊髄デリック</td><td> Neuralstem株式会社</td><td></td><td> Neuralstem社は、この技術への排他的なライセンスを購入した</td></tr><tr><td> MINJ-PDマイクロインジェクターポンプ</td><td> Tritechリサーチ社</td><td></td><td>この特定のアプリケーション用にカスタマイズ</td></tr><tr><td>フローティングカニューレ</td><td> Neuralstem株式会社</td><td></td><td>この特定のアプリケーションのためのカスタム設計</td></tr></tbody></table>

surgical technique for spinal cord delivery of therapies: demonstration of procedure in gottingen minipigs

これは脊髄に（遺伝子細胞）療法の送達のための手術手技とデバイスの組み合わせのコンパクトな視覚的な説明です。技術が動物で実証されているが、手順がFDA承認されており、現在はALS患者の脊髄に幹細胞移植に使用されている。 FDAは非常に特徴げっ歯類モデルにおける治療効果に関する実証の原則データを認識しているが、大型動物の使用は、外科手術の組み合わせで、デバイス、およびヒトでの使用のための最終的な治療の安全性を検証するために重要とみなされ。大きさ、解剖学、そして豚の背骨と脊髄の一般的な脆弱性は、より良いモデル人間に認識されます。また、露光および操作脊髄ならびに豚に創傷を閉鎖の手術プロセスは、人間とほとんど見分けがつかない。私たちは健康なブタモデルはcritiを表していることを信じている手続き的安全性の研究では、CALの第一歩。

<table border="1"><tbody><tr><td colspan="2"> Tarlovスコア</td></tr><tr><td> 0 </td><td>麻痺、動かない</td></tr><tr><td> 1 </td><td>後肢における知覚緊張、わずかな動き</td></tr><tr><td> 2 </td><td>後肢の動きが、座ったり、立ってすることができません</td></tr><tr><td> 3 </td><td>立って、歩く能力が、運動失調と短期間</td></tr><tr><td> 4 </td><td>完全な回復は、通常の運動機能</td></tr><...

Watch this Scientific Journal Video about Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs at JoVE.com

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

脊髄に（遺伝子細胞）治療​​の送達のために使用手術手技とデバイスの短い視覚的な説明。技術が動物で実証が、完全に翻訳可能であり、現在、人間のアプリケーションに使用されています。

セラピーの脊髄配達のための外科テクニック：ゲッティンゲンミニブタの手順のデモンストレーション

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

surgical-technique-for-spinal-cord-delivery-therapies-demonstration

Research

JoVE Journal

Medicine

15.2K ビュー.  Emory University. 脊髄に（遺伝子細胞）治療​​の送達のために使用手術手技とデバイスの短い視覚的な説明。技術が動物で実証が、完全に翻訳可能であり、現在、人間のアプリケーションに使用されています。

ビデオ: Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

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

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 ...

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 ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

マウスにおける心筋梗塞を誘発する低侵襲手術手順

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

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 ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

実験的脊髄損傷における脊髄血流の評価のための強化された超音波画像コントラスト

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

蝸牛細胞の変性を誘導するための迅速かつ信頼性の高いテクニック：耳毒性剤送達のためのマウスラウンドウィンドウアプローチ

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

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 ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

ラットにおける心筋虚血再灌流後の冠動脈流量の評価

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...