Name: automated gait analysis in mice with chronic constriction injury
Uploaded: 2017-10-17T14:00:00.000+00:00
Duration: 6 min 49 s
Description: Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

この研究は忠南大学、韓国研究所の東洋医学 (KIOM) と韓国健康技術総合研究所の助成金によって支持された &#38; 開発プロジェクトを通じて、韓国保健産業開発研究所 (KHIDI)、厚生省によって資金を供給&#38; 福祉、大韓民国 (許可番号: HI15C0007)。

すべての実験痛み研究の国際交流協会の倫理指針に従い実施し、忠南大学校 (で機関動物ケアおよび使用委員会によって承認されました。大田広域市, 韓国).
 1 坐骨神経の誘導の CCI <ol><li>家雄 ICR マウスの部屋温度 (20-25 ° C で維持される) と湿度 (40-60%) で 12 h 明暗周期下 20-25 g の重さで無料の食品へのアクセスと。水。手術する前に、少なくとも 1 週間の動物保育室でマウスの順応期間を許可します。
	<ol><li>住宅、外観とマウスの動作を見学し、異常行動を示すマウスを使用しないでください 。
		注: は、CCI 術前 1 日は von Frey フィラメントを適用することによって、後肢の機械的感度を測定し、通常のベースライン値を取得する自動歩行分析を実行します。その後、対照群と実験群にランダムにマウスを割り当てます</li>。
	</ol></li> <li>2,2,2 tribromoethanol (250 mg/kg、腹腔内 (IP)) を注入することによりマウスの麻酔手術の日 。
	注: 手術用ガウン、手袋、マスクなど保護具を着用します。
	<ol><li>は、2,2,2 tribromoethanol の 2.5 グラムの重量を量る、2-メチル-2-buthanol を 5 mL の最終的なボリュームに追加します。光をさえぎってを保つため (例えば、暗いコンテナーを使用して、ホイルで包む).</li>
		<li>40 ° C で加熱と 10-30 分攪拌して完全に溶解する許可する試薬</li>
		<li>200 mL の最終巻に蒸留水を追加し、よく混合されるまでかき混ぜる</li>。
		<li>は、4 ° C で因数を格納し、暗闇の中で保管します。2 週間後、麻酔薬の新しい因数を置き換える必要があります</li>。
		<li>は、1 g の体重ごと 20 μ L のボリュームを挿入します。たとえば、マウスの体重は 25 g、する場合は、500 μ L のソリューションを与えます。この麻酔の典型的な時間は 1 h. 
		注: このソリューションはライトに露出されたとき有毒になるおよび/または熱、ライトおよび熱への暴露を避けるために注意してください。ルートの注入はお勧めします</li>。
	</ol></li> <li>マウスは、深い麻酔に該当、する場合 (すなわち、腹臥位) を背側とオペレーティング テーブルの上に配置し、70% アルコール綿棒で右サイドの外側の半ば太もも領域を消毒します 。
	メモ: は、ピンチや後足は前足や尻尾に刺激の圧力への応答の欠如を確認することによって深い麻酔を確認します</li>。
	<li>誘発マウスの右の坐骨神経に CCI。
	<ol><li>1.0 〜 1.5 cm の長さの半ば太ももの領域の周りの皮膚にメス刃で切開を行います</li>。
		<li>坐骨神経を公開するマイクロ モスキート止血と太ももの筋肉を解剖ぶっきらぼう</li>。
		<li>4-0 クロム腸線縫合糸を使用して 1.0 〜 1.5 mm 間隔で 3 回の露出した坐骨神経の結紮します 。
		注: は、同側の後肢に表示される穏やかな揺れまで坐骨神経を結ぶことによって緩いを作る。マウスの偽のグループ受け取った神経結紮なしを除いて同様の条件で同じ外科的開口します</li>。
	</ol></li> <li>、5-0 絹糸を使用して 3-4 簡単な縫合と外科的開口を閉じて、手術の外側の領域の消毒をポビドン ヨードで消毒します</li>。
	<li>術後では、加熱パッドできれいなケージの中にマウスを置きます。動物が麻酔からリカバリされる場合、ホームのケージに戻ります 。
	注: この研究では、抗生物質使用されませんでした。肯定的な制御として 1 日 1 回 50 mg/kg の用量で CCI + ポンドを受け取ったグループ IP ガバペンチン (GBP).</li>
</ol> 2。機械的アロディニアの測定 (von Frey テスト) 注: 同側の後肢の足底の表面に von Frey フィラメントの 1 g を使用して撤退に対する機械的刺激の頻度を調べます。
<ol><li>それぞれの試験日に行動テスト部屋にマウスを持って来ると、テストする前に、少なくとも 30 分間自分のホーム ケージ内マウスを順応させる 
	注: 手術用ガウン、手袋、マスクなど保護具を着用します</li>。
	<li>金属メッシュの床に透明なアクリル ボックスにマウスを置くし、30 分マウスを順応させる</li>
	<li>フィラメントが曲がるまで優しく、後肢の足底の表面に von Frey のフィラメントの 1 g を適用します</li>。
	<li>適用フィラメント刺激同側の後肢に少なくとも 10 秒間隔で 10 回足し、結果を記録します 。
	注: この研究では、10 試験から足回収回答数は前足撤退周波数 (PWF、%) の割合として表示されます。Von Frey テストは CCI 手術後は 2 日おきに行った</li>。
</ol> 3。歩行分析の自動化を実行する 注: 動物が歩いている、自動的に様々 な歩行パラメーターを分析し、歩行解析システムは各足のプリントを可視化する、あしあと、足強度、歩幅のスタンス相、ステップ シーケンス、スイング、スイング スピード、等 本研究で示した足印刷領域とデータを割合に変換後単一姿勢変更対側の左同右後肢対。したがって、50% の結果は、印刷の足のサイズと前足部左側と右側の間の床との接触期間が同じであることを示します。さらに、下位のデータ値が 0% に近づいている示す対側と比較すると同側の後肢ではサイズと接触時間の両方が減少する ( 図 2 の B のパネルと 図 3).
<ol><li>順化、10 分の歩行分析装置内でマウスに CCI 術前に、5 日から一日一回を維持します 。
	注: 馴化を含む歩行分析テストは暗闇の中で実行する必要があります</li>。
	<li>歩行解析のためのテスト部屋にマウスを持参しテストする前に、少なくとも 30 分間、ホームのケージに順応
	<ol><li>[プログラム] メニューの [設定] タブでは、設定、&#34; 通路の長さ &#34; 30 cm とセットに、&#34; 最大期間を実行 &#34; 5 s と &#34; 最大のバリエーションを実行 &#34; 50%</li>。
		<li>から登録されているカメラを選択、&#39; セットアップ &#39; のタブ、&#39; プログラム &#39; メニュー</li>。
		<li>選択 &#34; 買収を開く &#34; から、&#39; を取得 &#39; のタブ、&#39; プログラム &#39; メニュー</li>。
		<li>次のステータス メッセージをクリックして、&#39; スナップ背景 &#39; 空 (すなわち ブランク) 通路の背景写真を撮る] ボタンをクリックします</li>。
	</ol></li> <li>クリックして、&#39; 集録を開始 &#39; ボタンをクリックし、歩道にマウスを配置、マウスの動きに合わせて自動的に記録を開始 。
	注: マウスが明らかに通路を歩くとき、プログラムは自動的としてこの動きを分類 &#34; 準拠を実行 &#34; 緑のアイコンで。フット プリントを検出するソフトウェアが失敗した場合、実験者は赤いアイコンが表示され、記録を繰り返す必要があります。解析に必要な少なくとも 5 つの成功した準拠実行します</li>。
	<li>テストの後、選択 &#39; の実行を分類する &#39; の取得] タブから、&#39; プログラム &#39; メニュー</li>。
	<li>は、分析とクリックのための実行を選択、&#39; 自動分類 &#39; ボタン 。
	注: 分類、統計パラメーターはすべて自動的に保存され、実験者をクリックして、結果を見つけることができます &#34; 実行統計を表示 &#34; 分析のメニュー</li>。
</ol>

<ol>
	<li>Loeser, J. D., Treede, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kyoto+protocol+of+IASP+Basic+Pain+Terminology.">The Kyoto protocol of IASP Basic Pain Terminology.</a> Pain. 137 (3), 473-477 (2008).</li><li>Colloca, L., 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=Neuropathic+pain.">Neuropathic pain.</a> Nat Rev Dis Primers. 3, 17002 (2017).</li><li>Vranken, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+treatment+of+neuropathic+pain.">Mechanisms and treatment of neuropathic pain.</a> Cent Nerv Syst Agents Med Chem. 9 (1), 71-78 (2009).</li><li>DeLeo, J. 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=Characterization+of+a+neuropathic+pain+model:+sciatic+cryoneurolysis+in+the+rat.">Characterization of a neuropathic pain model: sciatic cryoneurolysis in the rat.</a> Pain. 56 (1), 9-16 (1994).</li><li>Kim, S. H., Chung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+for+peripheral+neuropathy+produced+by+segmental+spinal+nerve+ligation+in+the+rat.">An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat.</a> Pain. 50 (3), 355-363 (1992).</li><li>Seltzer, Z., Dubner, R., Shir, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+behavioral+model+of+neuropathic+pain+disorders+produced+in+rats+by+partial+sciatic+nerve+injury.">A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury.</a> Pain. 43 (2), 205-218 (1990).</li><li>Bennett, G. J., Xie, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peripheral+mononeuropathy+in+rat+that+produces+disorders+of+pain+sensation+like+those+seen+in+man.">A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.</a> Pain. 33 (1), 87-107 (1988).</li><li>Dixon, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+analysis+of+experimental+observations.">Efficient analysis of experimental observations.</a> Annu Rev Pharmacol Toxicol. 20, 441-462 (1980).</li><li>Bonin, R. P., Bories, C., De Koninck, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+up-down+method+(SUDO)+for+measuring+mechanical+nociception+in+rodents+using+von+Frey+filaments.">A simplified up-down method (SUDO) for measuring mechanical nociception in rodents using von Frey filaments.</a> Mol Pain. 10, 26 (2014).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> J Neurosci Methods. 53 (1), 55-63 (1994).</li><li>Chen, H., Du, J., Zhang, Y., Barnes, K., Jia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+Reliable+Gait+Evaluation+Method+for+Rodent+Studies.">Establishing a Reliable Gait Evaluation Method for Rodent Studies.</a> J Neurosci Methods. , (2017).</li><li>Kang, D. W., 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=Antinociceptive+Profile+of+Levo-tetrahydropalmatine+in+Acute+and+Chronic+Pain+Mice+Models:+Role+of+spinal+sigma-1+receptor.">Antinociceptive Profile of Levo-tetrahydropalmatine in Acute and Chronic Pain Mice Models: Role of spinal sigma-1 receptor.</a> Sci Rep. 6, 37850 (2016).</li><li>Huehnchen, P., Boehmerle, W., Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+paclitaxel+induced+sensory+polyneuropathy+with+&amp;#34;Catwalk&amp;#34;+automated+gait+analysis+in+mice.">Assessment of paclitaxel induced sensory polyneuropathy with &amp;#34;Catwalk&amp;#34; automated gait analysis in mice.</a> PLoS One. 8 (10), e76772 (2013).</li><li>Vrinten, D. H., Hamers, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='CatWalk'+automated+quantitative+gait+analysis+as+a+novel+method+to+assess+mechanical+allodynia+in+the+rat;+a+comparison+with+von+Frey+testing.">'CatWalk' automated quantitative gait analysis as a novel method to assess mechanical allodynia in the rat; a comparison with von Frey testing.</a> Pain. 102 (1-2), 203-209 (2003).</li><li>Martinov, T., Mack, M., Sykes, A., Chatterjea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+changes+in+tactile+sensitivity+in+the+hind+paw+of+mice+using+an+electronic+von+Frey+apparatus.">Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus.</a> J Vis Exp. (82), e51212 (2013).</li><li>Ferland, C. E., Laverty, S., Beaudry, F., Vachon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+analysis+and+pain+response+of+two+rodent+models+of+osteoarthritis.">Gait analysis and pain response of two rodent models of osteoarthritis.</a> Pharmacol Biochem Behav. 97 (3), 603-610 (2011).</li><li>Mogil, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypolocomotion,+asymmetrically+directed+behaviors+(licking,+lifting,+flinching,+and+shaking)+and+dynamic+weight+bearing+(gait)+changes+are+not+measures+of+neuropathic+pain+in+mice.">Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice.</a> Mol Pain. 6, 34 (2010).</li><li>Ferreira-Gomes, J., Adaes, S., Castro-Lopes, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+movement-evoked+pain+in+osteoarthritis+by+the+knee-bend+and+CatWalk+tests:+a+clinically+relevant+study.">Assessment of movement-evoked pain in osteoarthritis by the knee-bend and CatWalk tests: a clinically relevant study.</a> J Pain. 9 (10), 945-954 (2008).</li></ol>

著者が明らかに何もありません。

現在、von Frey フィラメントを用いた機械的アロディニアの測定は、最も広く触覚過敏症を示す痛みの動物モデルでメソッドを使用です。神経因性疼痛の動物モデルが開発され続けている、よう改良された8,9,10,15感覚機能の評価の方法論もされています。これらのレポートに、これらの変更は、古?...

外傷、代謝異常、炎症、感染症、虚血による神経系の病理学的変化や病変や疾患の直接的な結果として生じる痛みとして定義されている神経因性疼痛の時折発生する自己免疫疾患1体性感覚系に影響を与えます。神経因性疼痛は通常、我慢、残念なことに、従来の鎮痛薬一般的に生産しない十分な痛み救済2。神経因性疼痛の主要な機能には、自然と刺激に対する反応 (すなわちアロディニア、痛覚過敏) の痛みが含まれています。アロディニアは、軽いタッチや暖かい刺激などの通常非痛み刺激に発生した侵害受容応答です。痛覚過敏は、有害な機械および/または熱刺激3に強化された痛み応答を示します。これらの 2 症状の両方は非常に患者さんの生活の質を損なう、穏やかな触覚刺激により誘発される機械的アロディニアが最も悪化症状ソフト コンタクトは日常生活の中で避けにくいので。
調べるための基本的なメカニズムと神経因性疼痛の治療のための新しい鎮痛薬、痛み反応の精密測定が不可欠です。多数の神経因性疼痛モデル動物が開発した後肢に侵害受容反応その高いアクセシビリティ4,5,6、7のための地域の足。したがって、ほとんど痛み応答評価されてきた、後肢の足底または背側表面に von Frey フィラメントなどの特殊な器具を使用して機械的刺激を適用することによって。最もよく使用される方法の 1 つは、アップ、ダウン ・ ディクソンによって記述方法8と後変更バージョン9,10。しかし、非常に熟練した、経験豊富な実験者は von Frey テストを実行する必要が、結果は主観的な可能性があります。
自動歩行解析システムは、齧歯動物を自由に移動で歩くの様々 なパラメーターを測定することにより、神経疾患、神経筋疾患を調べることができます。神経損傷モデル動物の様々 な侵害受容性といくつかの治療の鎮痛効果は、痛み刺激11,12,13,を追加することがなく評価できます。14. この解析システムなど静的および動的歩行パラメーターの検出することができます: 印刷領域 (床と接触する完全な足印刷の領域) の足、足の強度 (足との接触面積の平均強度)、ストライド長 (同じ足の連続配置間の距離)、姿勢相 (単一の後肢の接地の期間)、ステップ シーケンス (順序で、4 つの足が床に配置されます)、(遊脚相の期間)、スイング、スイング速度 (計算ストライド長期間をスイングと 1 秒あたりのピクセル数で表されます)。本稿では、分析システムの使用方法を示し、絞扼性損傷 (CCI) 神経因性マウスを用いた von Frey テストとデータの簡単な比較を提供しています。

<table><tbody><tr><td>0.9% Saline</td><td>JW Pharmaceutical</td><td>N/A</td><td>Vehicle for drugs</td></tr><tr><td>1ml syringe</td><td>BD Plastipak</td><td>300013</td><td>Injecting device</td></tr><tr><td>2, 2, 2-tribromoethanol (97% purity)</td><td>Sigma</td><td>T48402</td><td>Anesthetic</td></tr><tr><td>2-methyl-2-buthanol (99% purity)</td><td>Sigma</td><td>152463</td><td>Solvent for 2, 2, 2-tribromoethanol</td></tr><tr><td>Catwalk Automated gait analysis system</td><td>Noldus</td><td>N/A</td><td>Automatic analysis software of aniaml gait</td></tr><tr><td>Chromic catgut (4-0 thickness)</td><td>AILEE</td><td>C442</td><td>Ligature to make chronic constriction injury on the sciatic nerve</td></tr><tr><td>Gabapentin</td><td>Sigma</td><td>Y0001280</td><td>Analgeisc, Used as a positive control drug in this study</td></tr><tr><td>Graefe Forceps</td><td>F.S.T</td><td>11051-10</td><td>Surgical instrument</td></tr><tr><td>Heating Pad</td><td>DAESHIN ELECTRONICS</td><td>M-303AT</td><td>Regulation of body temperature</td></tr><tr><td>ICR Mouse</td><td>Samtaco</td><td>N/A</td><td>Experimental animal</td></tr><tr><td>Mersilk (3-0 thickness)</td><td>ETHICON</td><td>W598H</td><td>Suture material for surgical closure of skin</td></tr><tr><td>Micro-Mosquito</td><td>F.S.T</td><td>13010-12</td><td>Surgical instrument</td></tr><tr><td>Micro-scissors</td><td>F.S.T</td><td>14090-09</td><td>Surgical instrument</td></tr><tr><td>Needle holder</td><td>F.S.T</td><td>12002-12</td><td>Surgical instrument</td></tr><tr><td>Povidone Iodine</td><td>Firson</td><td>N/A</td><td>Disinfectant to prevent infection after surgery</td></tr><tr><td>Scalpel blade</td><td>F.S.T</td><td>10010-00 (#10)</td><td>Surgical instrument to make an incision</td></tr><tr><td>Scalpel handle</td><td>F.S.T</td><td>10003-12 (#3)</td><td>Surgical instrument to make an incision</td></tr><tr><td>Von-Frey filaments</td><td>North Coast</td><td>NC12775-99</td><td>Measurement device to test sensory function for mechanical stimulation</td></tr></tbody></table>

automated gait analysis in mice with chronic constriction injury

Von Frey テストは、神経因性疼痛の動物の感覚の機能を調べるため広く使用されている古典的な方法です。ただし、熟練した、経験豊富な実験者の要件と主観的データなどいくつかの欠点があります。さまざまな変更まで、von frey 毛方法の改善が、それはまだいくつかの制限があります。最近の報告では、歩行分析が神経因性動物からより正確で客観的データを生成することを示唆しています。このプロトコルはマウスの神経因性疼痛の程度を判断する自動歩行分析を実行する方法を示します。馴化の数日後、マウスは自由に足跡を照らすためのガラスの床の上を歩くを許されました。足印刷、スイング時間足等の角部などの様々 な歩行パラメーターの自動解析とビデオ クリップを足跡と歩行の定量化を行ったし、
本研究の主な目的は、自動歩行分析の方法論を記述して、簡単に von Frey フィラメントを使用して古典的な感覚のテストからのデータを比較することです。

Von Frey テストを実行し、CCI 術後後 10 日目まで CCI マウスにおける歩行分析を自動化しました。統計解析、双方向分散分析 (ANOVA) の反復測定全体の効果が決まります、実験群 p 値を決定する Dunnett の事後分析を行った。
図 1に示すように示唆された古典的な von Frey テスト データの時間コース。...

Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

Automated Gait Analysis in Mice with Chronic Constriction Injury

動物モデルでは神経因性の痛みの反応の正確な評価は、痛みの病気の病態を調査し、新しい鎮痛薬の開発に不可欠です。後肢足自動歩行解析システムによって齧歯動物の感覚機能は、機密かつ客観的手法を提案します。

絞扼性損傷マウス自動歩行分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

automated-gait-analysis-in-mice-with-chronic-constriction-injury

Research

JoVE Journal

Neuroscience

10.0K ビュー.  Chungnam National University. 動物モデルでは神経因性の痛みの反応の正確な評価は、痛みの病気の病態を調査し、新しい鎮痛薬の開発に不可欠です。後肢足自動歩行解析システムによって齧歯動物の感覚機能は、機密かつ客観的手法を提案します。

ビデオ: Automated Gait Analysis in Mice with Chronic Constriction Injury

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

複数のスケールでデジタル化された神経形態の自動化されたショールの分析

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

神経科学

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

横方向の流体のパーカッション：マウスにおける外傷性脳損傷のモデル

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

げっ歯類の前臨床研究のための3Dキネマティック歩行分析

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

行動学

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

神経変性疾患を持つマウスの年齢依存性運動障害の歩行分析

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

発生生物学

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

神経筋疾患のマウスモデルの行動表現型に関する低コスト歩行分析

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

MOG35 55 を受ける c57bl/6 マウスの矢状面運動歩行解析による実験的自己免疫性脳脊髄炎

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

末梢神経または脊髄損傷を伴うげっ歯類の機能回復を評価する自動歩行分析

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

一方的坐骨神経クラッシュ傷害後のラットにおける運動行動の多因子的評価

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

げっ歯類における不活動誘発歩行変化の包括的理解

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...

MouseWalkerを用いた脊髄損傷マウスの自発運動機能障害の定量化

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

著者スポットライト:MouseWalkerを使用して脊髄損傷のマウスモデルにおける自発運動機能障害の定量化  

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.