著者らは、げっ歯類の監視、取り扱いや実験で夫人シャロンSpanaの支援を感謝しています。博士課程の資金援助は、ビジョンに優秀シドニー大学とオーストラリア研究センターにより提供されています。

このプロトコルは、シドニー大学の動物ケアのガイドラインに従っています。 1.メイキング電極<ol><li>正極白金線直径1〜2ミリの短い（5 cm）の長さから（角膜に接触することになる1）を構築します。ループ直径が数mmにファッションを。あなたのアンプの入力段に到達するために十分な長さ、従来の鉛にこのループを接続します（ 図4を参照）。 </li><li>銀/塩化銀はまた、大会リードに接続直径1〜2ミリ、ペレット使用して（動物の口の中に移動します）負極を構成する（ 図4参照）。 </li><li> （動物の臀部になります）を参照電極として、清潔な注射針（23 G）を使用し、また、適切な長さのリードに接続された（ 図4参照）。 </li><li>理想的には、（正のU 3つの電極を接続し、機器の製造業者によって提供される3つのリードケーブルを使用94;角膜、負→口、アンプへの臀部→参照）。 </li></ol>光刺激とERGセットアップの2の接続とキャリブレーション<ol><li>ダークにすることができる小型の記録実験室、作成（または見つけ）。赤製オーバーベンチ光または赤ヘッドランプのいずれかまたは両方を装備。 </li><li>セットアップ時に、ラットの目に到達する赤色光照度が1ルクスを超えていないことを確認するルクスメーターを使用してください。 注：中性濃度フィルタは、ランプの明るさを減少させるために使用することができ、ランプ光源は、具体的に、赤色光を放出しなければなりません。光源は低い（可視）波長を放射する場合ダーク適応性が損なわれます。 </li><li>あなたが持っているであろう記録研究室に入るすべての迷光を封鎖（これは多くの場合、不透明なテープで永続性を必要とする）と、オーバーフィットするのに十分な大きさ、など薄暗い減光フィルタ（これはシートに購入することができます）、任意のコンピュータの画面を準備ラボ。 注：迷光と画面の光がラットの眼の暗順応を害するのに十分です。 </li><li>データ集録ハードウェアにアンプを接続します。アンプに正、負の基準リード線を接続します。コンピュータとLED全体野電源ユニットは確実に接地源に接続されていることを確認してください。 注：一部のラボでは、建物のグランドに接続され、接地点を専門にしています。水道管は、効果的な代替手段です。 </li><li>研究品質の放射計とLED光源を調整します。動物の眼は実験中に配置される位置にあるメーターのセンサーを修正しました。 </li><li>フラッシュエネルギー、フラッシュ期間、フラッシュ間のフラッシュの繰り返しや時間の段階的な増加にフルフィールドERGプロトコルを実行するプログラム全体野のLEDは、設定をinterstimuls間隔（ISI）と呼ばれます。例えば、フルフィールドプロトコルについては、表1を参照してください。 注：フルフィールドERGは、Bに繰り返し薄暗いが点滅から増加を点滅段階的な様式で右が点滅します。ツインフラッシュプログラムは、フルフィールドのプロトコルからの続き、ロッドとコーンの応答の分離を可能にします。 </li></ol> ERGの実験に3日前<ol><li>ダーク記録する前に12時間のSDラットを適応させます。これは、迷光が除去された後、記録研究室でこれを行うことが便利です。 </li></ol> ERG実験の4日目<ol><li>録音中、穏やかに加熱される動物のためにアレンジ。私たちは、動物の頭部が全体野へのエントリで正しいポイントで休むことができるように構築された軽金属プラットフォームを使用しています。プラットフォームは、我々は水浴中で40℃に予熱した水をポンプこれを通してチューブを内蔵しています。 注：経験は、これは37℃での動物のコア温度を維持することを示しています。 </li><li>暗条件下でラットを計量。レコードの重みと正しいケタミン（60 mgの/ kg）およびキシラジンを構成する（5ミリグラム/ kg）の用量。ラットGENを抑えますTLYと腹腔内に麻酔薬を注入します。 </li><li>注入時に注意してください。動物が無意識になると（通常は5分以内）軽く反射応答が存在するかどうかを確認するために、片足パッドをつまんで麻酔の深さを確認してください。なお、この反射は先に進む前に、存在しないか、または弱いまで待つのがベストです。 </li><li>角膜へのアトロピンとproxmethacaineの別の一滴を適用します。 </li><li>黒糸の10cmの長さをカットします。簡単な結び目でループ状にして、目の赤道上にループを滑ります。少し締め。影響は最小限の圧力で、やや前方眼球を描画することです。これは、まぶたから透明な角膜を保持します。 </li><li>カルボマー眼は角膜の表面に低下適用されます。カルボマーは、角膜表面上に残り、まぶたや顔の上にこぼれないようにします。 </li><li>加熱されたプラットフォーム上に吸収性寝具を置きます。 </li><li>全体野の開口部に推奨代わりに頭をベッド上の位置ラット、。 </li><li>挿入int型直腸にernal温度プローブ。尾へのプローブコードをテーピングにより所定の位置に温度プローブを固定します。 </li><li>皮下後脚に参照電極（23 G針）を挿入し、アンプに接続します。 </li><li>口の中でしっかりと負極（銀/塩化銀ペレット）を配置します。これは口を滑らないようにするには、安定した面に接続するリード線を取り付けます。 </li><li>角膜の中心の上に正極を配置します。マイクロマニピュレーターを用いて、電極を静かに角膜に触れることを確認してください。 </li><li> 37.5°C - チェック体温は37.0です。 </li><li>動物が適切に配置され、電極が所定の位置にあるされたら、（暗順応を維持するために）、不透明な材料で、全体のセットアップ（全体野および動物）を羽織ります。我々は、柔らかい黒い布を使用しています。 </li><li>取得ソフトウェアで事前収集サンプリングの5ミリ秒で100〜1000ミリ秒の収集時間で2 kHzのサンプリング·レートで設定します。 1〜1000のバンドパスフィルタを設定しますヘルツとそのサンプリングがフラッシュ以下〜250ミリ秒の期間をサンプリングするようにトリガされていることを確認します。 </li><li>記録ベースラインを確認してください。これは、外来ノイズを含まないことが、いくつかのアンプのノイズや呼吸振動を表示する必要があります。 </li><li>ベースラインは外来ノイズを示している場合は、トラブルシューティングを開始します。ほとんどの問題は、電極の位置、またはアースに滑りに関連しています。録音は外来ノイズの自由であることを確認するためにファラデーケージを使用します。 </li><li>テストフラッシュ、0.4ログスコットcd.sm -2を実行します。 図2Aと同様のERG波形が表示されます。 （：-474±39μVとb波：1,512±160μV、N = 11波）当研究室では0.4ログスコットcd.sm -2フラッシュのための典型的な応答があります。 </li><li>ダーク10分間の再適応に動物を可能にします。これは、ベースラインを再確認するために、これらの10分を使用すると便利です。 </li><li>安定した信号の次の確認は、録音を開始します。 </li><li>記録セッションの終了時に、そのボディtemperatをチェックUREを維持しました。電極を取り外します。角膜へのカルボマーポリマーを再適用します。それは、動物のハウジングに戻る前に、完全なモバイルとアクティブになるまで動物が熱パッド上に回復することができます。 </li></ol> 5.リモート虚血<ol><li>覚醒または麻酔げっ歯類のいずれかでリモート虚血を実行します。 </li><li>動物が麻酔されている場合は、膝の明確な、加熱されたプラットフォーム上に置く（上）と後肢の上部にわたって血圧計用カフをスリップ。 </li><li>動物は処理さに慣れている場合には、麻酔なしでこの手順を実行することが可能です。これは、2人が必要です。一人は静かに動物を拘束し、第二は、血圧計用カフを適用し、血圧計を運営しています。 </li><li>覚醒動物の場合、1後肢無料で、静かに動物をラップするために~15センチ×30〜50センチメートルタオルの一部を使用しています。その頭をホルダの腕や胴体、および場所の間に隠れてと、左前腕（例えば）に背に動物を置きますカフは、今説明しました。 </li><li>カフを収縮し、空気圧バルブが閉じていることを確認します。麻酔動物で160 mmHgのにカフをポンプと、覚醒動物における180 mmHgです。これは、収縮期血圧（通常は140 mmHgで160 mmHgのそれぞれ）を超えています。 </li><li>必要に応じて手持ちのポンプを使用して、これらの圧力を維持します。 </li><li>虚血のために計画された時間（私たちは、5分間の再灌流によって分離された5分の2ピリオドを使用）した後、空気圧バルブを緩めてカフ圧を収縮させます。 </li><li>フットパッドに接続されている皮膚の温度プローブを使用して、リモート虚血の効果を確認してください。皮膚温度は、典型的には、5分間かけて、32-30°Cから落下し、再灌流に回復します。 </li></ol> 6.光損傷<ol><li>光損傷の手順の前に、ラットが暗順応一晩であることを確認してください。 </li><li> （遅滞なく我々の実験で）肢虚血以下の適切な時間に、各動物をプレキシガラス箱の中に単独で配置され、WI床式の容器に目の水と食料。 注：光誘起損傷は、アルビノ動物において行うことができます。 </li><li>標準時間（通常は午前9時）に予め較正千ルクスの白色光に切り替え、24時間この状態を維持します。 </li></ol> 7. ERGデータ抽出と分析<ol><li> ERGの平均波形を取得します。減算によって、非ゼロのベースラインの、正しい必要であれば。 </li><li>ベースラインと第一の電圧差として、（高刺激強度半ばで発表）は、A波の振幅を測定（&lt;30ミリ秒の待ち時間）トラフ（ 図1）。 </li><li>典型的には80〜100ミリ秒（ 図1）の待ち時間で発生する、次の波のa波と正のピークとの間の電圧差としてb波の振幅を測定します。 </li><li> 90 Hzの遷移帯域で、60-235ヘルツからデータをフィルタリングするために、フーリエ変換を用いて、振動電位を分離します<sup&gt; 7。必要に応じて、単離された振動電位信号は、A波の谷の身元を確認するためにフィルタリングされていない波形から減算することができます。 </li><li> A及びB波のピークの暗黙の時間（待ち時間）は、有用な測定（ 図1）であることができます。ロッド応答を分離するために双子の点滅を使用します。混合応答（フラッシュ1）ロッド応答を分離する（ 図2）からの錐体応答（フラッシュ2）を減算します。 </li><li>個々の光強度を波およびb波の振幅を正規化（後処理/後処理ベースライン）、または治療群について平均。強度応答曲線は、フラッシュエネルギーに対してグループ振幅と誤差をプロットします。 </li></ol>

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+Mydriatics+Affect+Light-Evoked+Retinal+Responses+in+Anesthetized+Mice).">Topical Mydriatics Affect Light-Evoked Retinal Responses in Anesthetized Mice).</a> Investigative Ophthalmology &amp; Visual Science. 51 (1), 567-576 (2010).</li><li>Fraunfel, F. t., Burns, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACUTE+REVERSIBLE+LENS+OPACITY+-+CAUSED+BY+DRUGS,+COLD,+ANOXIA,+ASPHYXIA,+STRESS,+DEATH+AND+DEHYDRATION.">ACUTE REVERSIBLE LENS OPACITY - CAUSED BY DRUGS, COLD, ANOXIA, ASPHYXIA, STRESS, DEATH AND DEHYDRATION.</a> Experimental Eye Research. 10 (1), 19 (1970).</li><li>Calderone, L., Grimes, P., Shalev, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACUTE+REVERSIBLE+CATARACT+INDUCED+BY+XYLAZINE+AND+BY+KETAMINE-XYLAZINE+ANESTHESIA+IN+RATS+AND+MICE.">ACUTE REVERSIBLE CATARACT INDUCED BY XYLAZINE AND BY KETAMINE-XYLAZINE ANESTHESIA IN RATS AND MICE.</a> Experimental Eye Research. 42 (4), 331-337 (1986).</li><li>Behn, 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=Dark+adaptation+is+faster+in+pigmented+than+albino+rats.">Dark adaptation is faster in pigmented than albino rats.</a> Documenta Ophthalmologica. 106 (2), 153-159 (2003).</li><li>Sugawara, T., Sieving, P. A., Bush, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+relationship+of+the+scotopic+and+photopic+ERG+to+photoreceptor+cell+loss+in+light+damaged+rats.">Quantitative relationship of the scotopic and photopic ERG to photoreceptor cell loss in light damaged rats.</a> Experimental Eye Research. 70 (5), 693-705 (2000).</li><li>Machida, S., 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=P23H+rhodopsin+transgenic+rat:+Correlation+of+retinal+function+with+histopathology.">P23H rhodopsin transgenic rat: Correlation of retinal function with histopathology.</a> Investigative Ophthalmology &amp; Visual Science. 41 (10), 3200-3209 (2000).</li><li>Brandli, A., Stone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+Ischemia+Influences+the+Responsiveness+of+the+Retina.">Remote Ischemia Influences the Responsiveness of the Retina.</a> Observations in the Rat. Investigative Ophthalmology &amp; Visual Science. 55 (4), 2088-2096 (2014).</li><li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina.">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology &amp; Visual Science. 49 (3), 1254-1261 (2008).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+abnormal+rod+photoreceptor+activity+with+the+a-wave+of+the+electroretinogram:+Applications+and+methods.">Assessing abnormal rod photoreceptor activity with the a-wave of the electroretinogram: Applications and methods.</a> Documenta Ophthalmologica. 92 (4), 253-267 (1996).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rod-driven+a-wave+of+the+dark-adapted+mammalian+electroretinogram.">The rod-driven a-wave of the dark-adapted mammalian electroretinogram.</a> Progress in Retinal and Eye Research. 39, 1-22 (2014).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+COMPUTATIONAL+MODEL+OF+THE+AMPLITUDE+AND+IMPLICIT+TIME+OF+THE+B-WAVE+OF+THE+HUMAN+ERG.">A COMPUTATIONAL MODEL OF THE AMPLITUDE AND IMPLICIT TIME OF THE B-WAVE OF THE HUMAN ERG.</a> Visual Neuroscience. 8 (2), 107-126 (1992).</li><li>Wachtmeister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potentials+in+the+retina:+what+do+they+reveal.">Oscillatory potentials in the retina: what do they reveal.</a> Progress in Retinal and Eye Research. 17 (4), 485-521 (1998).</li></ol>

ジョナサン·ストーンは、CSCM Pty Ltdのディレクターであります

上記の暗順応フラッシュERG方法は、ラットの網膜機能を評価するための信頼できる方法です。 A波とB波の両方は、光損傷によ​​って減少しました。リモート虚血プレコンディショニングは、A波とB波の光損傷誘導減少を軽減します。網膜機能のこの保存は遠隔虚血プレコンディショニングは、低酸素症、虚血および運動8-10などの保護事前調整の他の形態に似ている、神経保護を誘?...

ERGは、光に応答して、網膜によって生成され、眼の角膜表面から記録された電位です。記録条件は、慎重に管理されている場合、ERGは、網膜機能を評価するための種々の方法で使用することができます。ここでは、「フラッシュERG」、網膜が全体野の背景に提示簡単な、明るいフラッシュにさらされたときに発生する電位を記録する方法を説明しました。全体野が均一に光を分散し、光のフラッシュがほぼ均一に全体の網膜に到達します。網膜は、暗い記録前適応、及び動物の記録のために準備されるように暗順応を維持し、ERGが得られた場合、ロッドと錐体光受容体の両方によって生成されます。 暗順応フラッシュERGは、次の2つの方法で分析された特徴的な波形を有しています。まず、ERG波形の初期および後期成分を区別し、神経細胞の配列に関連しています網膜中のAlの活性化。最古のコンポーネントは、短い待ち時間負に向かう可能性のある、A波（ 図1）です。これは、b波と呼ばれ、正方向電位が続きます。 b波の立ち上がり位相が別の構成要素（振動電位またはOPS）と考えられている振動を示しています。 A波は、内顆粒層の細胞、およびアマクリン細胞による1のOPによってb波、光受容体によって発生すると考えられます。 刺激強度に基づいて、非常に薄暗いフラッシュへの対応は、暗順応閾値応答が可能であると呼ばれます。暗順応の閾値応答は、網膜神経節細胞2-4から生成されていると理解されます。第二に、フラッシュERGは明順応により分離することができる、または2つのフラッシュプロトコルによって棒状と円錐駆動コンポーネントに、以下に説明します。明所視条件下では、円錐集団が低いため、A波は、ラットでは検出可能ではなく、のOP及びb波であります5明確。その網膜高いコーン集団を持つ霊長類では、ロッド状と円錐経路の両方は、検出波6を発生させます。 多くの場合、フラッシュERGから抽出された2つの有用な尺度は、図2に示される典型的なフラッシュの応答と、 図1のように測定し、A-の振幅とb波である。感光体集団はdamagingly明るいへの曝露によって、例えば、低減された場合光、ERGのすべてのコンポーネントが低減されます。このような遠隔虚血プレコンディショニング（RIP）などの神経保護の介入は、A及びB波（ 図3）の振幅を維持することによって検証することができます。要約すると、ERGの分析は、健康、光損傷を受けたとneuroprotected網膜の間の比較を可能にします。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PC computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab, 4 channel acquistion hardware</td>
			<td>AD Instruments</td>
			<td>PL 35044</td>
			<td>Acquistion of ERG</td>
		</tr>
		<tr>
			<td>Animal Bio Amp</td>
			<td>AD Instruments</td>
			<td>FE 136</td>
			<td>Amplifier for ERG</td>
		</tr>
		<tr>
			<td>Lab chart</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Signal collection software</td>
		</tr>
		<tr>
			<td>Ganzfield</td>
			<td>Photometric solutions</td>
			<td>FS-250A</td>
			<td>Light stimulus</td>
		</tr>
		<tr>
			<td>Ganzfield operating system</td>
			<td>Photometric solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Research Radiometer</td>
			<td>International light technologies</td>
			<td>ILT-1700</td>
			<td>calibrate light series</td>
		</tr>
		<tr>
			<td>Lux meter</td>
			<td></td>
			<td>LX-1010B&#160;</td>
			<td>check red light illumanation</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lead wires</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Connect postive, negative ground electrodes to amplifier</td>
		</tr>
		<tr>
			<td>Lead wires -aligator</td>
			<td>AD Instruments</td>
			<td></td>
			<td>ground ganzfield and acquistion hardware to computer</td>
		</tr>
		<tr>
			<td>Platinum wire 95%</td>
			<td>A&#38;E metals</td>
			<td></td>
			<td>postive electrode</td>
		</tr>
		<tr>
			<td>Mouth electrode Ag/AgCl Pellet</td>
			<td>SDR</td>
			<td>E205</td>
			<td>negative electode</td>
		</tr>
		<tr>
			<td>26 gauge needle</td>
			<td>BD</td>
			<td></td>
			<td>ground electode</td>
		</tr>
		<tr>
			<td>Water pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic blanket system with flexible probe</td>
			<td>Harvard Appartus</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine 1% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical mydriasis</td>
		</tr>
		<tr>
			<td>Proxmethycaine 0.5% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical anaesthetic</td>
		</tr>
		<tr>
			<td>Visco tears eye drops</td>
			<td>Novartis</td>
			<td></td>
			<td>carbomer polymer</td>
		</tr>
		<tr>
			<td>Thread</td>
			<td></td>
			<td></td>
			<td>retract eye lid</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable adhesive</td>
			<td>Blu tac</td>
			<td></td>
			<td>Dim red headlamp. Affix electrodes</td>
		</tr>
		<tr>
			<td>Absorbent bedding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamil - ketamine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>dissociative</td>
		</tr>
		<tr>
			<td>Xylium - Xylazine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>muscle relaxant</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

using the electroretinogram to assess function in the rodent retina and the protective effects of remote limb ischemic preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

プロトコルは、 インビボでげっ歯類の網膜の視覚機能を測定することができます。 A波、光受容体の機能の測定値、及びb波、内網膜の機能の測定値は、 図1に注釈されています。 増加した光刺激を有するロッド支配ERG信号が増加すると、 図2（a）に示すように。 A波は〜0.4ログスコットのcd.sm -2および2.5ログスコットcd.sm -2（</s...

Watch this Scientific Journal Video about Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning at JoVE.com

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

齧歯類網膜とリモート四肢虚血プレコンディショニングの保護効果に機能を評価するために電図を用いて、

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

using-electroretinogram-to-assess-function-rodent-retina-protective

Research

JoVE Journal

Neuroscience

16.0K ビュー.  University of Sydney. The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

ビデオ: Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

コー​​ルド - プレコンディショニングによる神経保護の研究のための戦略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

神経科学

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

スキンと四肢骨の神経支配マウス感覚神経線維の組織の調製と免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

齧歯中枢神経系への薬物の直接脳室内デリバリー

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

マウス網膜を使用してCNSにおける血管再生の評価

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

ゼブラフィッシュ幼虫における視覚反応の電位図解析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

CLARITY / CUBIC技術を用いたマウスの脊髄にセロトニン繊維のイメージング

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

エレクトロポレーションによる齧歯類坐骨神経内のシュワン細胞へのインビボ遺伝子導入で

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

加圧齧歯動物に及ぼす影響の爆発による頭部外傷の中間の大脳動脈

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神経クラッシュ損傷のラットモデルにおける機能的評価のための3D運動学的解析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

行動とEEGを組み合わせて、エピソード記憶に対するマインドフルネス瞑想の効果を研究する

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...