本研究は、博士白-荘Shyu（NSC 99から2320-B-001から016-MY3、NSC 100から2311-B-001-003-MY3、およびNSCに国家科学委員会の助成金によってサポートされていました102-2320- B-001-026-MY3）。この作品は、中央研究院から資金提供を受け医科学研究所、で行いました。

本研究におけるプロトコルは、台湾で中央研究院動物実験からの承認及び利用委員会を受け取りました。 1.動物の準備<ol><li> （ - 400グラム約300）のオスのSprague-Dawleyラットを入手します。食料と水に自由にアクセスして、12時間/ 12時間の明/暗サイクル（午前8:00に点灯）下に - （22°C、湿度50％21）エアコン付きの部屋にラットを維持します。 </li></ol> 2.実験方法<ol><li>ラットのすべてが実験前に1週間のために彼らのホームケージ内の環境に適応できるようにします。適応中に、ベースラインを確立するために、フォン・フレイと足底テストを実行します。 </li><li> フォン・フレイテスト <ol><li>居住のための30分間のアクリルエンクロージャ（30センチ×30センチ×80センチ）にラットを置きます。 </li><li> （ノーフィラメント11から20）のvon Freyフィラメントを得る同じ長さを持っていますが、直径を変化させることの範囲を提供するために、100グラム - 2の力の。 </li><li>ラットの後肢に足引っ込め閾値を評価するために、5分の試行間隔でアクリル板上に網目状のポートを介して後足の中心を刺激するためのvon Freyフィラメントを使用します。最大印加圧力が記録されるまで、ローからハイに、昇順にフィラメントを使用し、最大印加圧力を記録する。10 </li><li>ラットは刺激に対する足引っ込め応答を示す場合には、フィラメント数を記録します。最低の刺激に応じて、この裁判で撤退応答を決定します。 </li><li>同じラットに連続して3回の合計、すぐに同じ手順を繰り返します。 （グラム）の対応力にフィラメント数を変換した値を平均します。 </li></ol></li><li> 足底テスト <ol><li>居住のための30分間（4つのフレームに分かれて80センチ×30センチ×15センチ）透明プレキシガラス箱にラットを置きます。 </li><li> infファイルを使用してくださいraredビームは、ガラス板を介して後肢の中心を刺激します。約10秒の平均足引っ込め応答待ち時間を得るために、赤外光の強度を調整します。赤外線光源をオンにし、デジタルソリッドステートタイマを起動するキーを押すことにより、トライアルを実施しています。赤外光の持続時間を操作します。 </li><li>ラットが足引っ込め応答を示すときに、赤外光の持続時間を記録します。最長の期間は、組織の損傷を避けるために、各試験で20秒を超えてはなりません。連続的な刺激を避けるために、少なくとも5分の試行間間隔を使用してください。 </li><li>左右の後足のための3つの臨床試験でテストを繰り返し、各ラットについて各後足の平均を計算します。 </li></ol></li><li> コラゲナーゼ病変手術 <ol><li>つま先ピンチ応答と発生し、外科的刺激に対する身体的応答の喪失まで4％イソフルランでラットを麻酔。 1.5で麻酔を維持 - 2％isoflur手術の期間ANE。 </li><li> ℃〜37.5から36.5に体温を維持するための簡単​​な加熱パッドと定位装置にラットを置きます。麻酔下ながら乾燥を防ぐために、目にアイクリームを適用します。 </li><li>滅菌電気バリカンで毛を剃る、と頭皮の正中線に沿ってメスでスムーズな切開（約2〜2.5センチメートル）を作ります。摂理のヨウ素と消毒のための75％アルコール溶液を交互に皮膚や頭蓋骨を清掃してください。外科的なフェーズでは、無菌状態を維持するために、すべてのデバイス、ツール、およびワークベンチを滅菌するために75％のアルコールを使用しています。その前に、すべての外科的材料は、蒸気オートクレーブで滅菌する必要があります。 </li><li>滅菌手袋や楽器を使用し、視床の（腹側後内側視床核[VPM]および腹側後外側視床核[VPL]を含む）VB以上の電気ドリルで頭蓋骨に小さな穴（直径3mm）をドリル（3.0 -3.5ミリメートルの後方、3.0〜3.5ミリメートル、横）ブレグマ、および頭蓋表面5.5〜5.8ミリメートルの深さにします。 </li><li>それぞれ通常の生理食塩水または対照群と実験群における4型コラゲナーゼ溶液の0.125 Uの顕微注入する0.5μlを、。 </li><li>薬物拡散を可能にするために追加の5分間の代わりに注射針を保管してください。 </li><li>頭蓋骨に穴を埋めるために歯科用セメントを使用し、切開部を縫合。縫合切開後、創傷に局所鎮痛剤麻酔薬（リドカイン軟膏）を適用し、ホームケージに戻します。 </li><li>彼らは胸骨横臥位を維持し、麻酔から回復するまで暖かく保つまで手術後、単独でプラスチックケージにラットを収容します。 </li></ol></li><li> 外科回復後の行動試験 <ol><li>手術からの回復の7日後、セクション2.2およびテストフェーズ2.3の手順を繰り返します。動物の健康と発育状態を監視しながら、4週間以上の行動試験を行います。 </li><li&gt;手術後フェーズの対照群と実験群との間に、動物の健康状態（ 例えば 、体重、摂食量、および自由な動き）を監視します。 </li></ol></li><li> PE-50チューブでカニューレ注入を行います <ol><li> 4％イソフルランでラットを麻酔し、36.5で体温を維持する - 単純な加熱パッドで37.5°C。 </li><li>メスで、それぞれ、左肩の腹側部分と胸腔の前肢と交差点の背部の正中線に二つの穴（直径2cmずつ）カット。 </li><li>二つの穴の間にはさみで皮膚と筋肉を解離。 </li><li>腹穴から外頸静脈にPE-50チューブ（長さ20cm）の一方の端を接続します。背側の穴に同じPE-50チューブの終端に接続し、皮膚に貼り付けます。 </li><li> PE-50チュービングことを確認するために、頸静脈に生理食塩水を注入するために注射器を使用して、妨害されません。 </li><li>切開部を縫合糸、および6ミリグラム/ kgのゲンタマイシン（腹腔内）でラットを注入します。 </li><li> 20 U / mlのヘパリン生理食塩水の0.1ミリリットル、続いて0.3ミリリットルの0.9％の生理食塩水でチューブを手術後一日おきにフラッシュします。 </li></ol></li><li> 最終的な行動試験 <ol><li>一週間ステップ2.6の後、繰り返しは、手術回復後のステップ2.5に比べて安定している、その動作を確認するために、2.2から2.3を繰り返します。 </li></ol></li><li> 放射性トレーサー注射 <ol><li>適応のための10分 - 5のための休憩ケージにラットを置きます。 </li><li>スプリッタを使用して、2つの1ミリリットルのシリンジにPE-50チューブを接続します。生理食塩水で1注射器を記入し、[14 C] -IAP溶液（0.3容積の125μCiの/キロ- 0.5ミリリットル）で他を記入してください。 </li><li>外頚静脈に放射性トレーサーを注入し、そして3 M塩化カリウムで満たされた別の注射器で注射器を交換してください。 </li><li>放射性トレーサーのINJ後10秒ectionは、イソフルランの過剰摂取の下で3 M塩化カリウムを注入し、台湾の中央研究院動物実験及び利用委員会のガイドラインに基づいて、標準的な安楽死の方法（50分間の気化器からのすなわち 、イソフルラン）によると、動物を生け贄に捧げます。 </li><li> 1分後、頭蓋骨を露出させ、残りの筋肉を切り落とします。鉗子を使用して、脳から頭蓋骨の背側表面をはがします。骨鉗子を用いて、頭蓋骨の側面を切り取ります。次に、スパチュラを用いて、脳の腹側表面に沿って嗅球と神経接続を切断し、脳を取り出します。 </li><li> （約-55℃）ドライアイスとメチルブタンで脳を凍結する最適な切削温度（OCT）化合物を使用してください。冷凍庫に脳組織を保管してください。11、12 </li></ol></li><li> 脳スライス <ol><li>後脳を下に向けて、ミクロトームで組織を向けます。 20＆に脳をスライスするクライオスタットを使用します＃181;メートルの厚さの切片。 </li><li>各スライスの間に240μmの間隔で、-20℃でクライオスタットに顕微鏡スライド上に脳スライスを配置します。 </li><li> -20℃で3日間露光カセットに顕微鏡スライドと段階的な放射能を持つ5つの標準の濾紙を置きます。脳スライスの順序に従って、上から下に顕微鏡スライドを配置します。最後に、カセットの底にろ紙を置きます。12 </li><li>露光カセットから蛍光体スクリーンを外し、脳切片のための[14 C] -IAPの取り込みを示すために、蛍光体スクリーンを読み、画像を生成する可変モードイメージャーを使用しています。 </li></ol></li></ol> 3.データ解析<ol><li>ステップ2.9.4の後、統計的パラメトリックマッピング（SPM）およびImageJソフトウェアを使用して画像を調整します。シリアル冠状切片を用いて、すべての画像を再構成します。スムーズで参照ラット脳モデルに応じた画像を正規化します。12、13 </li><li>定量的評価のために、画素の信号強度を決定するために、ImageJソフトウェアを使用して、脳画像の関心領域（ROI）を測定し、分析のための統計ソフトウェアを使用する。12、13 </li><li>異なる脳核の間の接続を調べるため、地域間の相関マトリックス中の放射能比を表示するMATLAB相関解析ソフトウェアを使用し、カラーマップとして行列を可視化します。最後に、ネットワーク分析のためPajekソフトウェアを使用しています。12、13 </li><li>ベースライン時に偽とCPSPグループに、von Frey試験で足底試験及び機械的な力で耐熱性の持続時間を比較するために、因子のようなグループと数週間で、分散の2×5の双方向混合分析（ANOVA）を使用し、週1 - 5は、グループと脳の領域に応じて放射能比を測定するために、2×31 2ウェイANOVAを使用してください。適切な場合、TukeyのHSD（HSD） 事後試験を行います。カルculateピアソン相関は、偽とCPSPグループ内の選択された脳領域のすべての間で相関を評価するために係数。 </li></ol>

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著者らは、開示することは何もありません。

行動試験では、CPSPグループは、ベースライン時に、von Frey試験における熱痛試験および機械的な力で足引っ込め閾値の削減と1週間展示- 。5.調査結果は先行研究と一致していたが14 [14 C] -IAP方法は、異なる脳切片の定量分析のために脳画像のピクセル強度に依存しています。脳画像のデータを評価するために、画素の信号強度を定義しました。 46.​​979 C...

脳卒中の出血は、神経因性疼痛に苦しむ患者の8％以上で起こることが示されており、中央の脳卒中後疼痛（CPSP）と呼ばれる。1-3 CPSPそれにより過敏症および異痛症を誘発する、感覚機能不全から生じ得る。4が、CPSPにおける体性感覚機能障害の病態生理学的メカニズムは不明のまま。例えば、体性感覚の喪失は出血性脳領域の神経求心路遮断に起因します。痛覚過敏は、中央侵害受容ニューロンまたは中枢脱抑制、5、6しかしCPSP症状に関与している神経基盤は不明のままの過剰興奮によって引き起こされることがあります。いくつかの研究は、背外側前頭前野（DPFC）、吻側前帯状皮質（ACC）、扁桃体、海馬、中脳水道周囲灰白質（PAG）、吻側腹内側髄質、および互いの接続が侵害受容処理を仲介することを示唆している。追加の7LY、内側前頭前皮質（のmPFC）-amygdala回路は、疼痛関連の知覚に関与していることが示された。CPSPの病態生理学的メカニズムの8データは多様であり、かつCPSPの神経基質の活性化がさらに精査が必要です。 [14 C] -Iodoantipyrine（IAP）の取り込みは、脳活動とCBFの関係を仮定して、間接的に局所脳血流量（のrCBF）を観察するために使用されます。 [14 C] -IAPは、機能的磁気共鳴画像法（fMRI）のように、リアルタイムで脳活動を評価することはできないが、それはいくつかの利点を有します。たとえば、[14 C] -IAPは自然に測定する病理学的状態の間に、脳のイベントを発生するのに適している。9また、[14 C] -IAPの取り込みは麻酔なしで測定されます。また、fMRIの陽電子放出断層撮影（PET）を含む他の画像化方法、より少ないコスト。 [14 C] -IAP方法はmeasurinために適切であることが示唆されています視床の腹側基底核（VB）の病変によって誘発されるグラム自発痛（ 例えば 、CPSP）。9 現在のプロトコルは、動物モデルにおける視床のVBの病変によって誘発されるCPSPの神経基盤の関与を評価するための[14 C] -IAP方法を実行する方法について説明します。技術は、行動と神経細胞レベルでCPSP症状の根底にある病態生理学的メカニズムを決定する方法を提供しています。

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon Products Corporation&amp;nbsp;</td><td>NDC 12164-002-25</td><td>4%</td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>homeothermic blanket system</td><td>Harvard Apparatus</td><td>Model 50&amp;ndash;7079</td><td>body temperature were maintained at 36.5 - 37.5 &amp;deg;C.</td></tr><tr><td>10 &amp;micro;l micro syringe</td><td>Hamilton</td><td>80008, Model 1701SN</td><td>injected with collagenase</td></tr><tr><td>polyethylene-50 tubing</td><td>Becton, Dickinson and Company</td><td>427411</td><td>catheterized into external jugular vein</td></tr><tr><td>1 c.c syringe</td><td>Terumo Medical Products</td><td>SS-01T</td><td>injected with &lt;sup&gt;14&lt;/sup&gt;C-IAP and saline.</td></tr><tr><td>saline (Sodium Chloride 0.9 gm)</td><td>Taiwan Biotech Co., LTD.</td><td>100-130-0201</td><td>To flush the tube</td></tr><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>type 4 collagenase</td><td>Sigma</td><td>C5138-500MG</td><td>0.125 U</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1264-250MG</td><td>6 mg/kg</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H9399</td><td>20 U/ml; 0.1 ml/day</td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-iodoantipyrine (IAP)</td><td>PerkinElmer</td><td>NEC712</td><td>125 mCi/kg in 300 ml of 0.9% saline</td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>1.04936.1000</td><td>3 M</td></tr><tr><td>&lt;strong&gt;Behavior system:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>von Frey esthesiometer</td><td>Fabrication Enterprises, Inc.</td><td>Baseline Tactile Monofilaments 12-1666</td><td>mechanical hyperalgesia was assessed by measuring the withdrawal response to a mechanical stimulus</td></tr><tr><td>plantar test apparatus</td><td>IITC Life Science</td><td>IITC 390G Plantar Test</td><td>Thermal hyperalgesia was assessed by measuring the hind paw withdrawal latency in response to radiant heat.</td></tr><tr><td>&lt;strong&gt;Brain slice:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature compound</td><td>Sakura Fintek Inc</td><td>4583</td><td>embedded the brain</td></tr><tr><td>dry ice</td><td></td><td></td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>methylbutane</td><td>Sigma</td><td>M32631-1L</td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>Cryostat</td><td>&amp;nbsp;Leica Biosystems Nussloch GmbH, Germany</td><td>Leica CM1850</td><td>Coronal brain slice were sectioned on this machine.</td></tr><tr><td>&lt;strong&gt;Data analyze&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>exposure cassettes with a phosphor screen</td><td>Amersham Biosciences&amp;nbsp;</td><td>20 cm x 25 cm</td><td>The slices were dried on glass slides and placed alongside five standard filter papers with graded radioactivity. All of the slides were exposed to the cassettes at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>&amp;gamma;-counter</td><td>Beckman Coulter</td><td>Beckman LS 6500 Liquid Scintillation Counter</td><td>To measure the radioactivity count of the filter papers.</td></tr><tr><td>Typhoon 9410 Variable Mode Imager</td><td>&amp;nbsp;GMI, Inc.</td><td>WS-S9410</td><td>To read&amp;nbsp; phosphor screen which was exposed by brain slice</td></tr><tr><td>Statistical Parametric Mapping (SPM)</td><td>Wellcome Centre for Neuroimaging</td><td>version 8</td><td>all of the brains were averaged to create the final brain template. To determine significant differences between the images in these two groups, the images were derived by subtracting the sham group from the CPSP group.</td></tr><tr><td>ImageJ</td><td>http://imagej.nih.gov/ij</td><td>version 1.46</td><td>Adjacent sections were aligned both manually and using Stack- Reg, an automated pixel-based registration algorithm in ImageJ software. All of the original three-dimensionally reconstructed brains were smoothed and normalized to the reference rat brain model.</td></tr><tr><td>Matlab</td><td>MathWorks</td><td>version 2009b</td><td>used Pearson correlation coefficients to examine the relationships between the CPSP and sham groups. An inter-regional correlation matrix was calculated across animals from each group.</td></tr><tr><td>Pajek</td><td>http://Pajek.imfm.si/</td><td>version 3.06</td><td>Graphical theoretical analysis was performed on networks defined by the above correlation matrices using Pajek software.</td></tr></tbody></table>

autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.

図1Aは、実験的なタイムラインを示しています。ラットを行動試験（ すなわち 、フォン・フレイテストと足底試験）のための偽とCPSP群に割り当てました。実験の最初の日は、ベースラインを務め、およびテストは1週間後に繰り返された - 4.ヘパリン（20 U / mlの、0.1ミリリットル/日）中に注入した週に5 PE-50カテーテル法は、外頸静脈に行きまし?...

Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

[オートラジオグラフィー測定<sup&gt; 14</sup&gt; C]中央郵便ストローク痛みに続いてラット脳で-Iodoantipyrine

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to ...

autoradiographic-measurements-14c-iodoantipyrine-rat-brain-following

Research

JoVE Journal

Medicine

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

8.7K ビュー.  Fo Guang University. We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

ビデオ: Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管閉塞/低血圧：グローバル脳虚血のラットモデル

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

医学

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

ラットの脳卒中後うつ病を誘導するための中大脳動脈閉塞技術

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

行動学

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

神経科学

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

生化学

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

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

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

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

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

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

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

頭蓋内薬物療法と痛みの試金の齧歯動物

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

生体内における脳タンパク質合成の局所速度の決定のための定量的自動放射線法

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

虚血性脳卒中のマウス モデルで [11C] DPA-713 Neuroinflammation のイメージングをペットします。

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

シンプルかつ強力な可視化と薬理学的ターゲットの評価法として放射線写真法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

げっ歯類の脳サンプルの単一のセットで脳卒中後脳浮腫、梗塞ゾーンおよび血液脳関門破壊を測定する

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

[オートラジオグラフィー測定

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2血管閉塞/低血圧：グローバル脳虚血のラットモデル

ラットの脳卒中後うつ病を誘導するための中大脳動脈閉塞技術

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

Measurement of Glutamate Uptake using Radiolabeled L-[³H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

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

頭蓋内薬物療法と痛みの試金の齧歯動物

生体内における脳タンパク質合成の局所速度の決定のための定量的自動放射線法

虚血性脳卒中のマウスモデルで [¹¹C] DPA-713 Neuroinflammation のイメージングをペットします。

シンプルかつ強力な可視化と薬理学的ターゲットの評価法として放射線写真法

げっ歯類の脳サンプルの単一のセットで脳卒中後脳浮腫、梗塞ゾーンおよび血液脳関門破壊を測定する

[オートラジオグラフィー測定

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この動画の章

2血管閉塞/低血圧：グローバル脳虚血のラットモデル

ラットの脳卒中後うつ病を誘導するための中大脳動脈閉塞技術

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

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

頭蓋内薬物療法と痛みの試金の齧歯動物

生体内における脳タンパク質合成の局所速度の決定のための定量的自動放射線法

虚血性脳卒中のマウス モデルで [11C] DPA-713 Neuroinflammation のイメージングをペットします。

シンプルかつ強力な可視化と薬理学的ターゲットの評価法として放射線写真法

げっ歯類の脳サンプルの単一のセットで脳卒中後脳浮腫、梗塞ゾーンおよび血液脳関門破壊を測定する

Measurement of Glutamate Uptake using Radiolabeled L-[³H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

虚血性脳卒中のマウスモデルで [¹¹C] DPA-713 Neuroinflammation のイメージングをペットします。