この作品は、北京の自然科学財団 (No.7162124)、北京大学中国語医学新 ao 財団によって支えられました。

すべての動物は、ガイドラインに従って中国の法律倫理的な使用や実験動物の世話によって維持されました。
1 前モデル準備
<ol><li>
研究室の準備
	<ol>
<li>実験前に、少なくとも 30 分間 UV ランプを実行します。</li>
		<li>25 ± 3 ° C、相対湿度 30% 前後で研究室の温度を制御します。</li>
		<li>6:00 光の演習を切り、18:00 12/12 h 明暗サイクルを確立するためにそれをオフにします。</li>
	</ol>
</li>
	<li>
複数のプラットフォーム ボックス構造
	<ol>
<li>110 × 60 × 40 cm3のカバーなしの不透明なプラスチック タンクを構築します。</li>
		<li>15 円形プラットフォームを修正 (h = 8 cm、d = 6.5 cm) タンクの底部には、どの整然と配布 3 行で 5 つの列。スペースを残して十分な各プラットフォーム間約 10 cm の列と行の間の 13 cm。</li>
		<li>タンクの側面の水の出口を設定して蛇口をインストールします。</li>
		<li>それに掛かっているフード ボックスとタンクの鉄金網カバーを作る。</li>
	</ol>
</li>
	<li>
グループ化と住宅ラット 注: 8 週齢, 体重約 200 210 g の Wistar 系雄ラットは実験で使用されます。ラットは、モデリング プロセスの間にグループに住んでいます。<ol>
<li>マーカー ペンとラットの尾根を数します。</li>
		<li>ラットの重量を量る、非常に軽いまたは重いものを除外し、モデルと制御グループにランダムに残りの部分を分けます。</li>
		<li>優しくきれいなケージにラットを入れ、少なくとも 3 日間ラボに順応できるように。十分な水および食糧補給を提供します。</li>
	</ol>
</li>
</ol>2. MMPM でモデリング
注: プロセスは、18:00 から開始、8:00 21 日以上、1 日あたりの 14 時間の合計は、次の日で終わります。干渉の要因を避けるためには、同一人物が同じ白衣を着用しながら、全体の実験を行う必要があります。10 ラットは実験で使用されます。
<ol><li>平らな面、例えば床にタンクを配置します。その後、ぬるま湯 (25 ± 3 ° C)、フラットのプラットフォームの下約 1 cm の約 7 cm でタンクを満たします。</li>
	<li>1 日のタンクのすべてのラットの十分な食べ物や飲み物を準備します。フード ボックスに飼料と水を入れ、表紙にハングアップします。 注: いくつかのスマートのラットはフード ボックスの残りの部分を学ぶ。もしそうなら、タンクに戻ってそれらを運転します。</li>
	<li>ケージのモデル群を取る、尾によってそれらをつかむし、タンクにそっと入れます。水の代わりに、水の恐怖をやる気にさせるプラットフォームのすべてのラットを起動します。すべてのラットが対照群のラットのまま十分な食べ物と水で元檻の中で、上に立つプラットフォームを取得するかどうかを確認します。</li>
	<li>タンクをカバーします。傷害を避けるためにラットを監視します。ラット プラットフォーム上に登って 1 時間以上水に滞在場合、タンクからそれを拾うし、テストから削除します。</li>
	<li>14 時間後のモデルラットのタンクを取り出すし、ドライヤーで髪を乾かします。それはフェードする場合ラットの尾をマークし直す。元檻にラットを戻り、十分な食料と水を提供します。</li>
	<li>タンクの隅々 をフラッシュします。タンクの 1 つの側面を昇格し、下水の流出に蛇口を開きます。</li>
	<li>75% エタノール スプレーでタンクを殺菌、UV ライトにそれを公開します。</li>
</ol>3. モデルの評価: 行動テスト
注: すべてのテストは、行動のラボで実行されます。ノイズと余分な光されていない場合、テスト中に妨害を避けるため。できれば、同じ人を使用して、各テストを実施します。暗いコートと手袋が画像処理でグレー スケール認識するため必要です。OFT を実行最初のラットの行動で最も効果があり。
<ol><li>
しばしば
	<ol>
<li>それはワークステーションに正しく接続されているし、ボックスのすべての角をカバーを確認するオープン フィールド ボックスの上のレコーダーを確認してください。ボックスに影を排除するために照明を調整します。</li>
		<li>元檻の中で行動の実験室にネズミを移動します。テストの前に少なくとも 1 時間を順応させます。</li>
		<li>きれいにし排泄物やにおいが以前の実験から左がないことを確認し、75% エタノールとボックスをサニタイズします。</li>
		<li>その裏でネズミをケージから取り外し、優しく、ボックスの中央の領域にそれを置きます。すぐにシュートをブロックしないようにボックスから腕を撤退します。</li>
		<li>ラットの番号を入力して録音を開始します。カウントし、飼育、登山など、ラットの垂直活動の頻度を記録します。</li>
		<li>5 分後に録画を停止、アウト ボックスのネズミを取る、ケージに戻る。</li>
		<li>3.1.3 - 3.1.6 すべてのラットがテストを完了するまでの手順を繰り返します。</li>
	</ol>
</li>
	<li>
EPM
	<ol>
<li>OFT (ステップ 3.1.1 - 3.1.2) に関しては事前確認と馴化の手順に従います。</li>
		<li>その裏でネズミをケージから取り外し、2 本の腕の接合部に軽くそれを置きます。左の開いている腕に向かってラットを土地し、迅速に残すシュートをブロックしないようにします。</li>
		<li>ラットの番号を入力して録音を開始します。カウントし、異なる腕の入り口の頻度を記録します。ラットをテストで迷路を落とした場合、それを拾うし、迷路にそれを送り返します。データ分析のための詳細な情報を記録します。</li>
		<li>5 分後に録画を停止、ラットを取る、ケージに戻る。</li>
		<li>排泄物を削除し、元ラットの臭いを除去するために 75% エタノールで迷路を拭いてください。</li>
		<li>3.2.2 - 3.2.5 すべてのラットがテストを完了するまでの手順を繰り返します。</li>
	</ol>
</li>
	<li>
ES テスト
	<ol>
<li>暖かい (25 ± 3 ° C) 水の 80 cm で水泳タンク (70 × 30 × 110 cm3) を記入します。 メモ: タンク内にサーモスタットがある場合水の温度には 37 ° C 前後、ラットの体温に似ているが設定する必要があります。ない場合は、部屋の温度を一定に保つ設定。</li>
		<li>ピン各ラットのための負荷房し、その尾ルート優しくそれを結ぶ。負荷は、ラットの体重の 10% の重量を量る。</li>
		<li>尻尾をラットをつかむし、スイミング タンクにそれを投げます。ラットの密談、または壁にしがみつく、一線を画すと水に戻ってそれらを駆動します。</li>
		<li>開始時間時水と停止タイミングが使い果たされるときにネズミを投入する場合、10 以上の水の下に鼻、口と水から苦労する失敗としてことを s。 注: 場合によっては、疲労や溺死突然発生します。必ず十分な実験者の記録し、同時に動物を保存します。</li>
		<li>他の人を中断することがなく水の枯渇ラットを削除します。自分の髪を乾燥、その番号をマークし直すとケージに戻ってそれらを送信します。</li>
		<li>1 つのグループが終了した後は、タンク内の水を変更します。すべてのラットが完了、スイミング タンクを空し、きれいエタノールと紫外線殺菌します。</li>
	</ol>
</li>
</ol>4. モデルの評価: 中央神経伝達物質検出
<ol><li>意識まで 10% 抱水クロラール (3 mL/kg) の腹腔投与でラットを麻酔します。</li>
	<li>ネズミの首をはねます。</li>
	<li>後内側の線に沿って縦切開を行う、両サイドに頭蓋を開き、脳を公開します。頭蓋を裏返し、脳を取り外し、脳アイス袋にします。</li>
	<li>分離し、周囲の組織との明確な境界を持つ脳の基底部にダイヤモンドの形をした領域である視床下部を削除します。生殖不能の管に配置し、液体窒素で凍結します。-80 ° C の冷却装置ですべてのサンプルを格納します。</li>
	<li>高速液体クロマトグラフィー (HPLC)8を使用して視床下部で DA と 5 HT のコンテンツを検出します。</li>
</ol>

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著者が明らかに何もありません。

MMPM もともと睡眠剥奪9です。ラットに水槽に底面固定プラットフォームで起動されます。水の本能的な恐怖によって駆動される、ラットのままプラットフォームの上に立って、睡眠が発生しません。研究は、睡眠不足の別の時間は様々 なラットの行動および認識減損10、否定的な感情の11、中枢性疲労などの気分の変化をもたらすことを?...

疲労は、1人の健康を脅かす主な要因のひとつです。過去数十年で様々 な研究は、疲労は末梢発生しますが、一元的に駆動、常に感情と認知の障害が伴うことを証明しています。イタリアの生理学者 A. Mosso は最初、中枢性疲労2単語を提案しました。それは一般に限られた自主的な活動と中枢神経系 (CNS)3のインパルスの伝達の機能障害による認知障害として定義されます。末梢筋疲労と比べると、中枢性疲労は、中枢神経系だけでなく、結果として感情/行動の障害は、うつ病、不安、認知障害、記憶喪失を含む変更を強調しています。1 つの研究は、多くの要因が、過度の身体活動と精神的ストレスは非常に不可欠な4、中枢性疲労を引き起こすことができることを示しています。病因としてトリプトファン キヌレニン経路仮説5のような理論説明特定の経路の変化しかし、綿密な研究がまだ中枢性疲労の中央周辺の相関関係を明らかにするため必要です。
中枢性疲労のメカニズムはまだ明らかでは、効果的な動物モデルはさらなる研究のためとても重要です。既存の疲労モデルは大抵トレッドミル6と重量ロード スイミング7、精神的な要因に少し関心を持ってのような過度の運動による。中枢性疲労の開発シミュレート、するためには、私たちのグループは、MMPM でラット モデルを開発しました。モデリング プロセス中にラットのまま長時間睡眠時間の一部を含む複数のプラットフォーム ボックスの狭いプラットフォームの上に立って。過度の運動モデルとは異なり、MMPM モデルは、中枢性疲労の複雑な病態を考慮した精神的な要因として部分的な睡眠の剥奪を使用します。
モデル評価不安ムードと自主的な活動を決定するために OFT と EPM のテストを使用します。ES テストを実行して、末梢筋のパフォーマンスを測定します。さらに、我々 はラットの脳を取るし、ドーパミン (DA) を検出/中央神経伝達物質の違いを観察する両方の hypothalamuses のセロトニン (5-HT) のコンテンツ。
以下に示すプロトコル モデル中枢性疲労による繰り返しの物理的な活動と人間の生活の一般的な条件を模倣、睡眠不足です。ただし、モデル期間を調整すると、それすること他の多くの分野でのような観察とストレスの睡眠の研究。将来的に研究、我々 はこのモデルが中枢神経系の変更と中枢性疲労の病態メカニズムを明らかにするため、周辺システムとの接続を発見に役立つことを願っています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>multiple platform sleep deprivation water tank</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>110cm x 60cm x 40cm. There are 15 plastic small platforms at the bottom. The small platform is 6.5cm in diameter and 8cm high</td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Beijing Weitong Lihua Experimental Animal Technology Company</td>
			<td>license number SYXK (Beijing) 2016-0011</td>
			<td>Use 32 Wistar healthy male rats ,8 week old (200-210 g)</td>
		</tr>
		<tr>
			<td>Agilent 1100LC high performance liquid chromatograph&#160;</td>
			<td>Agilent</td>
			<td>&#160;G1379A, G1311A, G1313A , G1316A&#160;</td>
			<td>&#160;G1379A, G1311A type chromatographic pump, G1313A automatic sampler, G1316A column temperature box</td>
		</tr>
		<tr>
			<td>DECADE II SDC electrochemical detector</td>
			<td>Dutch ANTEC company</td>
			<td></td>
			<td>glassy carbon electrode, Ag/AgCl reference electrode, workstations (Clarity CHS)</td>
		</tr>
		<tr>
			<td>Biofuge Stratos high-speed refrigeration centrifuge</td>
			<td>HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VCX130 ultrasonic fracturing instrument</td>
			<td>SONICS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACS-ZEAS electronic scale</td>
			<td>Phos technology development, Beijing.</td>
			<td></td>
			<td>The weight of the weighing rats can be accurate to 0.1g.</td>
		</tr>
		<tr>
			<td>Open Field Box</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>wooden box of open field&#160; 100 cm by 100 cm x 40 cm, inside wall and bottom as the gray.The bottom is divided into 25 equal area squares, each of which is 20cm x 20cm, and the 16 grids along the outer wall are the external ones, and the other 9 grids are central.The camera is mounted above the median.</td>
		</tr>
		<tr>
			<td>Elevated Plus-maze</td>
			<td>Beijing zhongshi dechuang technology development co. LTD.</td>
			<td></td>
			<td>The open arms and close&#160; arms of the cross are composed of 30cm x 5cm x 15cm, and the central area is 5cm x 5cm, with a camera mounted above the center and 45cm high.</td>
		</tr>
		<tr>
			<td>rat swimming bucket.</td>
			<td>Zhenhua biological instrument equipment co., LTD. Anhui,China.</td>
			<td></td>
			<td>The volume of plastic drum is 70cm x 30cm x 110cm, which is used for swimming in rats.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Shiya instrument co., LTD., changzhou,China.</td>
			<td></td>
			<td>Control water temperature</td>
		</tr>
		<tr>
			<td>Small water pump</td>
			<td>Xincheng technology co., LTD., chengdu,China.</td>
			<td></td>
			<td>Used for water tank and swimming behavior.</td>
		</tr>
		<tr>
			<td>Ethovition3.0 behavioral software.</td>
			<td>Nuldus,Netherlands</td>
			<td></td>
			<td>Measurement analysis of rat behavior videos.</td>
		</tr>
	</tbody>
</table>

a rat model of central fatigue using a modified multiple platform method

この記事で紹介した修正中枢性疲労のモデルラット複数プラットフォーム メソッド (MMPM)。[複数のプラットフォーム] は、水タンク下部の狭いプラットフォームとして設計されました。モデルラットしたタンクに入れ、コントラストの設定空のコントロール グループ連続 21 日間、1 日あたり 14 時間 (18:00-8:00) プラットフォームに立っていた。モデリングの最後に、モデル グループのラットは明らかな疲労出現を示した。モデルを評価するためにいくつかの行動試験を行い、オープン フィールドを含むテスト (OFT) 高架プラス迷路 (EPM) テスト、および徹底的なスイミング (ES) テスト。結果、不安、空間認知障害、悪い筋肉のパフォーマンスやモデルラットにおける提示拒否したボランティア活動中枢性疲労の診断を確定します。中央の神経伝達物質の変化では、結果も検証されます。結論としては、正常に再現された中枢性疲労と病気の病態を明らかにするモデルでは、今後の研究が役立つことがあります。

MMPM を使用して中枢性疲労のモデルラットについて述べる。Wistar 系ラットを 24 は、コントロール グループと各グループの 12 のラットをモデル グループにランダムに分かれています。モデル装置は、水タンク下部 (図 1) に狭いプラットフォームとして設計されています。モデルラット 21 日 (図 2)、部分的な睡眠時間を含め...

Watch this Scientific Journal Video about A Rat Model of Central Fatigue Using a Modified Multiple Platform Method at JoVE.com

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

ここでは、変更されたを使用して中枢性疲労のラットのモデルを紹介するプロトコルを提案複数プラットフォーム メソッド (MMPM)。

ラット モデル改良を使用して中枢性疲労の複数プラットフォーム メソッド

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

a-rat-model-central-fatigue-using-modified-multiple-platform

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.9K ビュー.  Beijing University of Chinese Medicine. ここでは、変更されたを使用して中枢性疲労のラットのモデルを紹介するプロトコルを提案複数プラットフォーム メソッド (MMPM)。

ビデオ: A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

ダイナミッククランプ法を用いたラット中脳ドーパミン作動性ニューロンにおけるNMDA受容体のコンダクタンスのアプリケーション

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

神経科学

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

位置選択的微粒子銃は、改変された遺伝子銃を使用した器官型脳切片におけるターゲティング

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

スプラーグ - ドーリーラットのメソッド節状神経節の注入

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

記録と刺激アプリケーションの高接触密度、フラットインターフェイスの神経電極の作製

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

ダイナミック定量的感覚検査は、中枢性疼痛処理を特徴づけるために、

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

多発性硬化症のマウスモデルで進行性の神経障害を測定します

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

くも膜下出血のマウスモデルにおける脳血管攣縮の定量化法の体積

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

レンチ ウイルスのベクトルの直噴ハイライト ラット脊髄における複数の運動経路

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

複数の中枢神経系領域からのマウスグリアの解剖と分離

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

ラット脳組織の疎水性組織除去法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

ラットモデル改良を使用して中枢性疲労の複数プラットフォームメソッド

要約

さらに動画を探す

この動画の章

ダイナミッククランプ法を用いたラット中脳ドーパミン作動性ニューロンにおけるNMDA受容体のコンダクタンスのアプリケーション

位置選択的微粒子銃は、改変された遺伝子銃を使用した器官型脳切片におけるターゲティング

スプラーグ - ドーリーラットのメソッド節状神経節の注入

記録と刺激アプリケーションの高接触密度、フラットインターフェイスの神経電極の作製

ダイナミック定量的感覚検査は、中枢性疼痛処理を特徴づけるために、

多発性硬化症のマウスモデルで進行性の神経障害を測定します

くも膜下出血のマウスモデルにおける脳血管攣縮の定量化法の体積

レンチウイルスのベクトルの直噴ハイライトラット脊髄における複数の運動経路

複数の中枢神経系領域からのマウスグリアの解剖と分離

ラット脳組織の疎水性組織除去法

ラット モデル改良を使用して中枢性疲労の複数プラットフォーム メソッド

要約

さらに動画を探す

この動画の章

ダイナミッククランプ法を用いたラット中脳ドーパミン作動性ニューロンにおけるNMDA受容体のコンダクタンスのアプリケーション

位置選択的微粒子銃は、改変された遺伝子銃を使用した器官型脳切片におけるターゲティング

スプラーグ - ドーリーラットのメソッド節状神経節の注入

記録と刺激アプリケーションの高接触密度、フラットインターフェイスの神経電極の作製

ダイナミック定量的感覚検査は、中枢性疼痛処理を特徴づけるために、

多発性硬化症のマウスモデルで進行性の神経障害を測定します

くも膜下出血のマウスモデルにおける脳血管攣縮の定量化法の体積

レンチ ウイルスのベクトルの直噴ハイライト ラット脊髄における複数の運動経路

複数の中枢神経系領域からのマウスグリアの解剖と分離

ラット脳組織の疎水性組織除去法

ラットモデル改良を使用して中枢性疲労の複数プラットフォームメソッド

レンチウイルスのベクトルの直噴ハイライトラット脊髄における複数の運動経路