This paper is dedicated to my beloved wife Maren and our little Karl.

ウシ目の調製<ol><li>動物が屠殺された直後に牛の目を入手します。 </li><li> 120のNaCl、2のKCl、0.1のMgCl 2、0.15のCaCl 2、1.5 mMののNaH 2 PO 4、13.5のNa 2 HPO 4および5 mMグルコースでを含む特別な媒体」Sickel-ソリューション」で保護された目を輸送RT。 </li><li>暗赤色灯と暗順応条件下での網膜の準備を行います。 </li><li>眼の前の部分を削除します。赤道切開CAを実行します角膜輪部に4ミリメートルの後部。その後、1枚で角膜、虹彩、毛様体およびレンズを取り外します。 Sickel-溶液中の網膜にしてください。 </li><li>機械的に網膜表面にガラス質の添付ファイルを緩め、オープンアイカップから硝子体を除去する。 </li><li>その後4つの象限に目を分割し、約ラウンド領域をパンチアウトトレフィンを使用して直径7mm。 </li><li>静かに網膜を分離色素上皮から、光から保護ボックス内部の記録装置の上に置きます。記録装置は、中央にメッシュプラスチックメンテナで構成されています。メッシュ上に、網膜を配置し、電極上に直接プラスチックリングで固定します。 NOTE：プラスチックメンテ媒体の一定の流れを可能にするために二つのチャンネルを有している。 </li></ol> 2.録音電図（ERG） <ol><li> 、電位図を記録する網膜の両側にある銀/塩化銀電極を使用し、約一定の灌流速度で網膜を灌流するために1ml /分、37℃の一定温度。酸素で飽和した「Sickel-ソリューション」を使用してください。 </li><li>測定を開始する前に、暗い網膜を適応させる（すべての測定中に光から保護する）と、5分の刺激間隔を使用しています。網膜表面で6.3 MLXに設定された強度で刺激のための1 Hzの単一の白色キセノンフラッシュを使用してください。 </LI&gt; <li>使用は、最適な応答を得るために、減光フィルタ、タイマによって制御さ10マイクロ秒の光刺激をキャリブレート。 </li><li>グラスRPS312RMアンプを使用して（100Hzのハイパスフィルタ、50 Hzのノッチフィルタ100,000のx増幅）を測定し、処理したデータを、ERGをフィルタリングして増幅した。信号を乱す可能性のある妨害の周波数をフィルタリングしてみてください。データを処理するために、デスクトップコンピュータ（PC互換性のある）のアナログ·デジタルデータ収集ボードを使用する。 </li><li>安定b波振幅が記録されるまで一定の灌流下暗順応期間の後、電気信号​​の振幅を測定する。 注：5つの測定値が平均値に達し、10％未満を逸脱した場合には、振幅が安定しているとみなされる。単一の測定値の良い例は、 図1に示されている。 </li><li>テストを開始するには、純粋な窒素で純酸素を交換する（単一の実験の数、n = 5）の低酸素状態をテストするまたは250μmのグルタミン酸（n = 5）であった。 </li><li>電気的応答45分間5分毎に記録します。 </li><li>テスト期間の後、75分間酸素で飽和した標準培地で網膜を灌流し、b波振幅の変化を見てください。これはウォッシュアウトフェーズです。 b波のピークまでのA波の谷からb波の振幅を測定します。 </li><li>暗順応条件下で、感光体電位に対する低酸素またはグルタミン酸の効果を調べるために、栄養液に1mMのを追加することによってb波を抑制する。 </li><li> 30分間の安定した感光体の電位を記録した後、1mMのアスパラギン異なる洗浄溶液に網膜45分​​間さらす前と同様の手順を行う。前述のように同じ休薬期間（ステップ2.8）を使用します。 </li></ol> 3.データ分析<ol><li>統計的データを評価するために、 例えば、コルモゴロフ-スミルノフを使用して、すべてのデータのための正規分布を確実にするテスト15。 </li><li>博覧会の前に最後の測定と比較して露光段階後の割合でA及びB波の振幅の減少を計算します。 45分後にERG-振幅の減少を比較する - 展示期間の終わり - ERGへの適用前に測定した。 </li><li>可能な回復を調べるために博覧会前に、対応する振幅にウォッシュアウトフェーズの終了時にA-とB-波の比較。 </li><li>統計分析のために、ソフトウェアJMP統計ソフトウェアまたはSPSSソフトウェアを使用しています。平均±標準偏差として全体のデータを計算します。適切な統計的検定によって有意性を推定します。 注：これらのテストは、実験的な範囲に応じて異なる場合があります。この設定では、スチューデントのt検定を使用しています。 </li></ol>

<ol>
	<li>Varma, D. D., Cugati, S., Lee, A. W., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+central+retinal+artery+occlusion:+clinical+presentation+and+management.">A review of central retinal artery occlusion: clinical presentation and management.</a> Eye (Lond). 27, 688-697 (2013).</li><li>Resch, M., Suveges, I., Nemeth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension-related+eye+disorders).">Hypertension-related eye disorders).</a> Orv Hetil. 154, 1773-1780 (2013).</li><li>Feltgen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+study+of+the+European+Assessment+Group+for+Lysis+in+the+Eye+(EAGLE)+for+the+treatment+of+central+retinal+artery+occlusion:+design+issues+and+implications.+EAGLE+Study+report+no.+1+:+EAGLE+Study+report+no.+1.">Multicenter study of the European Assessment Group for Lysis in the Eye (EAGLE) for the treatment of central retinal artery occlusion: design issues and implications. EAGLE Study report no. 1 : EAGLE Study report no. 1.</a> Graefes Arch Clin Exp Ophthalmol. 244, 950-956 (2006).</li><li>Dreyer, E. B., Zurakowski, D., Schumer, R. A., Podos, S. M., Lipton, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+glutamate+levels+in+the+vitreous+body+of+humans+and+monkeys+with+glaucoma.">Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma.</a> Arch Ophthalmol. 114, 299-305 (1996).</li><li>Bertram, K. 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=Amino-acid+levels+in+subretinal+and+vitreous+fluid+of+patients+with+retinal+detachment.">Amino-acid levels in subretinal and vitreous fluid of patients with retinal detachment.</a> Eye (Lond). 22, 582-589 (2008).</li><li>Diederen, R. 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=Increased+glutamate+levels+in+the+vitreous+of+patients+with+retinal+detachment).">Increased glutamate levels in the vitreous of patients with retinal detachment).</a> Exp Eye Res. 83, 45-50 (2006).</li><li>Ientile, R., 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=Apoptosis+and+necrosis+occurring+in+excitotoxic+cell+death+in+isolated+chick+embryo+retina.">Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina.</a> J Neurochem. 79, 71-78 (2001).</li><li>Mali, R. S., Cheng, M., Chintala, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activators+promote+excitotoxicity-induced+retinal+damage.">Plasminogen activators promote excitotoxicity-induced retinal damage.</a> FASEB J. 19, 1280-1289 (2005).</li><li>Ambati, 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=Elevated+gamma-aminobutyric+acid,+glutamate,+and+vascular+endothelial+growth+factor+levels+in+the+vitreous+of+patients+with+proliferative+diabetic+retinopathy.">Elevated gamma-aminobutyric acid, glutamate, and vascular endothelial growth factor levels in the vitreous of patients with proliferative diabetic retinopathy.</a> Arch Ophthalmol. 115, 1161-1166 (1997).</li><li>Vorwerk, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depression+of+retinal+glutamate+transporter+function+leads+to+elevated+intravitreal+glutamate+levels+and+ganglion+cell+death.">Depression of retinal glutamate transporter function leads to elevated intravitreal glutamate levels and ganglion cell death.</a> Invest Ophthalmol Vis Sci. 41, 3615-3621 (2000).</li><li>Schultheiss, 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=Dulbecco's+Modified+Eagle+Medium+is+neuroprotective+when+compared+to+standard+vitrectomy+irrigation+solution.">Dulbecco's Modified Eagle Medium is neuroprotective when compared to standard vitrectomy irrigation solution.</a> Graefes Arch Clin Exp Ophthalmol. 251, 1613-1619 (2013).</li><li>Januschowski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+effects+of+two+different+irrigation+solutions+on+an+isolated+perfused+vertebrate+retina.">Comparing the effects of two different irrigation solutions on an isolated perfused vertebrate retina.</a> Ophthalmic Res. 48, 59-66 (2012).</li><li>Sickel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+and+Electrical+Responses+to+Light+Simulation+in+the+Retina+of+the+Frog.">Respiratory and Electrical Responses to Light Simulation in the Retina of the Frog.</a> Science. 148, 648-651 (1965).</li><li>Luke, 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=The+isolated+perfused+bovine+retina--a+sensitive+tool+for+pharmacological+research+on+retinal+function.+Brain+research.">The isolated perfused bovine retina--a sensitive tool for pharmacological research on retinal function. Brain research.</a> Brain research protocols. 16, 27-36 (2005).</li><li>Henderson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+experimental+data+for+univariate+normality.">Testing experimental data for univariate normality.</a> Clinica chimica acta; international journal of clinical chemistry. 366, 112-129 (2006).</li><li>Alt, 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=The+neuroprotective+potential+of+Rho-kinase+inhibition+in+promoting+cell+survival+and+reducing+reactive+gliosis+in+response+to+hypoxia+in+isolated+bovine+retina.">The neuroprotective potential of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in response to hypoxia in isolated bovine retina.</a> Cell Physiol Biochem. 32, 218-234 (2013).</li><li>Green, D. G., Kapousta-Bruneau, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+properties+of+a+new+isolated+rat+retina+preparation.">Electrophysiological properties of a new isolated rat retina preparation.</a> Vision research. 39, 2165-2177 (1999).</li><li>Richter, S. H., Garner, J. P., Wurbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+standardization:+cure+or+cause+of+poor+reproducibility+in+animal+experiments.">Environmental standardization: cure or cause of poor reproducibility in animal experiments.</a> Nat Methods. 6, 257-261 (2009).</li><li>Dutescu, R. 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=Multifocal+ERG+recordings+under+visual+control+of+the+stimulated+fundus+in+mice.">Multifocal ERG recordings under visual control of the stimulated fundus in mice.</a> Investigative ophthalmology &amp; visual science. 54, 2582-2589 (2013).</li><li>Perlman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+retinal+toxicity+of+drugs+in+animal+models+using+electrophysiological+and+morphological+techniques.">Testing retinal toxicity of drugs in animal models using electrophysiological and morphological techniques.</a> Doc Ophthalmol. 118, 3-28 (2009).</li><li>Mukhopadhyay, A., Gupta, A., Mukherjee, S., Chaudhuri, K., Ray, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Did+myocilin+evolve+from+two+different+primordial+proteins.">Did myocilin evolve from two different primordial proteins.</a> Mol Vis. 8, 271-279 (2002).</li><li>Januschowski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+retinal+toxicity+of+a+new+heavy+intraocular+dye,+using+a+model+of+perfused+and+isolated+retinal+cultures+of+bovine+and+human+origin.">Evaluating retinal toxicity of a new heavy intraocular dye, using a model of perfused and isolated retinal cultures of bovine and human origin.</a> Graefes Arch Clin Exp Ophthalmol. 250, 1013-1022 (2012).</li><li>Luke, 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=The+isolated+perfused+bovine+retina--a+sensitive+tool+for+pharmacological+research+on+retinal+function.">The isolated perfused bovine retina--a sensitive tool for pharmacological research on retinal function.</a> Brain Res Brain Res Protoc. 16, 27-36 (2005).</li></ol>

The authors have nothing to disclose.

本研究では、低酸素症の45分後にb波の振幅に有意な影響が見られた。この減少は、依然としてウォッシュアウト段階の後に有意であった。感光性に関する同様の効果が観察された。 結果は、他の公開されたデータ16でサポートされているし、私たちに低酸素症の後の可能な神経保護効果を研究する機会を与えている。 250μMのグルタミン酸の4...

神経保護は、過去数十年で眼科研究の調査の強い分野となっている。網膜は、酸素化に大きく依存し、その周囲の細胞の代謝によって強く影響され、高感度の神経回路網である。神経細胞の損傷に関連する主な眼の病状は、網膜血管閉塞、緑内障、および網膜剥離である。 網膜動脈閉塞は網膜血管閉塞のための例として、内側網膜1の低酸素状態に視力の突然の損失につながる。それは、多くの場合、一般的な血管の病理2に関連付けられており、患者のわずか8％を大幅に1視力を回復し、永続的な視覚喪失1につながる。動脈の線維素溶解が治療選択肢として提案されているが、利益は、無作為化臨床試験3に示すことができなかった。 緑内障と網膜剥離両方のグルタミン酸濃度4-6の増加を持っている。生理学的条件下でグルタミン酸は、全体の中枢神経系全体に興奮トランスミッタと内側の網膜7,8として検出された。上昇グルタミン酸レベルは、緑内障及び網膜剥離5,6においても、増殖性糖尿病性網膜9のみならず発見されている。グルタミン酸の増加は、おそらく、したがって、神経細胞の損傷10の興奮毒性につながり。網膜剥離の網膜（毛様体輪の硝子体切除）上の増殖性糖尿病網膜症の手術のいくつかのケースでは、ほとんどの場合必要です。扁平部の硝子体切除機械的操作時には、光ファイバーや長い操作中に灌漑ソリューションの高流量によって発揮されるせん断応力の明るい光が網膜11,12に追加のストレスを発揮する。 すべての疾患は、病理がレチンに局在している点で共通している単独および感覚神経系として網膜を保護する方法を見つけるという課題に眼科コミュニティをもたらす。 電図（ERG）は、インビボで光受容体機能（波）と内網膜（b波）の機能を評価するための標準的な方法である。 ERGは、角膜内に導入銀電極で測定され、眼は、ロッドまたは円錐または内側の網膜の欠陥を検出するための光のレベルの増加によって刺激されている。網膜における異なる欠陥は、振幅（応答の強さ）またはERGの待ち時間（時間 - 応答間隔）の変化によって検出することができる。異なるERGプロトコルと測定方法（パターン-ERG、多焦点-ERGまたは明視野ERG）は、欠陥のさらなる差別化を可能にする。単離された網膜の技術により、試験動物の例からの干渉なしで、網膜への影響を評価すること、最近導入された一般的な反応は13,14。 評価および灌流孤立網膜上の低酸素症およびグルタミン酸ストレスのために定義され、標準化された応力モデルを紹介するこの研究の目的だった。したがって、私たちは、特定の薬剤または眼内灌漑ソリューションの神経保護効果に関する今後の研究のための基礎を築くことを望んでいます。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">120 mM NaCl&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,064,041,000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 mM KCl,&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,050,010,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.1 mM MgCl2,&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">58,330,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.15 mM CaCl2</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td style="width:119px;">111 TA106282</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1.5 mM NaH2PO4/13.5 mM Na2HPO4&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,065,860,500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5 mM glucose</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">40,741,000</td>
			<td></td>
		</tr>
	</tbody>
</table>

glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina

神経保護は、過去数十年で眼科研究の調査の強い分野きや、緑内障、網膜血管閉塞、網膜剥離、および糖尿病性網膜症などの疾患に影響を与えています。それは、将来の前臨床治療試験のための標準化された応力モデルを紹介する本研究の目的であった。 ウシ網膜を調製し、灌流酸素飽和標準溶液とし、ERGを記録した。安定したB波を記録した後、低酸素（純粋N 2）またはグルタミン酸応力（250μmのグルタミン酸）が45分間に発揮した。単独の光受容体機能に対する効果を調べるために、1mMのアスパラギン酸は、波を得た。 ERG-回復は75分間モニターした。 低酸素症の場合、87.0％の波振幅の減少は45分（ウォッシュアウトP = 0.03の終了後に36.5％の減少）の露出時間の後に（P &lt;0.01）が認められた。さらに、初期のDECR87.23パーセントのb波振幅の容易さは、統計的有意に（p &lt;0.01、ウォッシュアウトの終了時の25.5％の減少はp = 0.03）に達したことを記録した。 250μmのグルタミン酸、1.9％た（p&gt; 0.05）の減少が続く波振幅た（p&gt; 0.05）の最初の7.8％の減少である。 b波振幅た（p &lt;0.01）の83.7％の減少が認められた。 75分のウォッシュアウト後に減少が2.3％（P = 0.62）であった。本研究では、標準化されたストレスモデルは、将来の可能性神経保護効果を同定するために有用であり得ることが示されている。

酸素飽和標準溶液（ 図1AおよびB）と網膜製剤の灌流の1時間後にERG-振幅が安定化し、単一の測定値との間の振幅のより少ない変動を示した。 pHは、浸透圧、温度、及び（低酸素症試験を除く）のpO 2は、すべてのテストのために一定に維持した。 内網膜の信号から光受容体の信号を分離するために、1mMのアスパラギン酸は、b波（ 図1A）を抑制する?...

Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

本研究では、我々は将来の前臨床治療試験のための孤立した灌流ウシ網膜のための標準化された応力モデルをご紹介。 A及びB波振幅によって表さ網膜機能上のいずれかの低酸素（純粋なN 2）またはグルタミン酸ストレス（250μMグルタミン酸）の効果を評価した。

単離された灌流脊椎動物の網膜のためのストレスモデルとしてグルタミン酸および低酸素症

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

glutamate-hypoxia-as-stress-model-for-isolated-perfused-vertebrate

Research

JoVE Journal

Medicine

7.8K ビュー.  University Eye Hospital Tübingen. 本研究では、我々は将来の前臨床治療試験のための孤立した灌流ウシ網膜のための標準化された応力モデルをご紹介。 A及びB波振幅によって表さ網膜機能上のいずれかの低酸素（純粋なN 2）またはグルタミン酸ストレス（250μMグルタミン酸）の効果を評価した。 

ビデオ: Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

パーキンソン病の単方向、病変6-OHDAモデルマウスの開発

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

医学

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

心理的ストレスを誘導するためのトリーアの社会ストレステストプロトコル

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

ザ<em&gt;卵で</em肉腫のためXenograftmodelとして&gt; CAMアッセイ

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

ヒアルロン酸ナトリウムの網膜下注入によるげっ歯類における網膜剥離モデル

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

ブドウ膜黒色腫の肝転移の治療として単離した肝灌流

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

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

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

一過性脳低酸素性虚血に基づく血栓性脳卒中モデル

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

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

モデルは、ヒトにおける臨床的に関連する低酸素症をシミュレートします

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...