1。患者の準備<ol><li>患者は化学療法開始前にテストベースラインの神経興奮性のための医療腫瘍科から呼ばれています。 </li><li>興奮性のテストのために患者適合性を決定する必要があります。彼らは、歴史や末梢神経障害のベースライン生理学証拠を持って前に神経毒性の化学療法治療を受けているか、興奮性テストのために、任意の禁忌がある場合は、患者は除外する必要があります。 </li></ol> 2。軸索の興奮性の手順<ol><li>半自動化されたコンピュータシステムのQTracS（神経学研究所、クイーンスクエア、英国）、隔離された線形バイポーラ定電流刺激（Digitimer、Welwyn Garden Cityの、英国）とアンプ（使用して、正中神経の感覚と運動の興奮性プロトコルを引き受けるサファイアIIA、Medelec、英国）。 </li><li>肌を削減するために研磨ゲルまたはパッドと手首と前腕での皮膚表面を準備します。アルコールワイプのアプリケーションに続いて抵抗。 </li><li>モーターの録音付き短母指外転·ブレビスと化合物のモータ活動電位（活動持続時間を計測）を記録するために遠位基準電極4cmの筋腹上に配置、非分極性電極の記録サイトを準備します。 </li><li>感覚のレコーディングの記録サイトを準備 - 複合感覚の活動電位（CSAPs）を記録するために、それぞれの記録と参照電極の近位および遠位指節間関節に配置されたリング電極を用いた。 </li><li>導電性のゲルで、手のひらに電気中性のアース板を配置します。 </li><li>レコーディングのセットアップで電気的なノイズは、ばかげた50/60 Hzのノイズ除去（クエスト科学機器、ノースバンクーバー、カナダ）を使用して、可能な限り削除する必要があります。 </li><li>手首で正中神経を刺激する。刺激部位は、その後、再配置可能バイポーラ電極と非pを使用して、最も低いしきい値の場所として選択する必要がありますolarizable電極。 </li><li>アノード電極は、骨上の刺激電極から近位10cmに配置する必要があります。 </li><li>テスト全体の刺激部位の温度を監視し、温度が32℃以上であることを確認してください</li></ol> 3。軸索の興奮性プロトコル<ol><li>刺激強度がさらに増加すると応答が最大となると増大しなくなるまで徐々に刺激を増やすことによって、刺激 - 応答曲線を記録します。 </li><li>しきい値を追跡するための目標振幅は、刺激 - 反応曲線の急峻な斜面の面積に対応する30パーセントの最大振幅の40％、自動的に設定されています。 </li><li>ボストックらの方法ごとに、1998のように、ターゲット振幅を達成するために必要なしきい値の変更は、現在はオンラインで追跡されます。 </li><li>閾値電気緊張（TE）、リカバリサイクル（RC）、電流閾値（I / V）の関係を含む複数の興奮性のパラメータを記録としてキールナンら、2000、キールナンら、2001で説明しています。 </li><li>閾値電気緊張は、制御しきい値の±40％（ 図1）に現在のセットを偏光と、100msのサブスレッショルド電流の偏光を用いて評価されています。両方ドおよびHyper-偏光は、次のターゲット応答振幅を維持するために必要なしきい値電流の変化が記録されます。閾値電気緊張が強く、膜電位に関連付けられている偏光パルス（90〜100ミリ秒）の終了時に過分極方向への応答で、結節コンダクタンスと膜電位の評価を提供しています。 </li><li>リカバリサイクルは、テスト刺激（2.5ミリ秒から200ミリ秒まで）することにより、異なる間隔で続いて初期の最大上コンディショニング刺激して、ペアのパルスのパラダイム（ 図2）を用いて評価されています。最大上刺激に続いて、それは電圧-Gの活性化を反映して、 &quot;不応性&quot;と呼ばれる、その後の応答を生成することがより困難であるatedのNa +チャネル。不応期に続いて、superexcitabilityとして知られているファシリテーションの期間が発生します。 </li><li>電流しきい値の関係は50パーセントからしきい値の100％の強度に変化は200 msの偏光電流を用いて評価されています。 </li></ol> 4。患者評価<ol><li>これらの技術を使用して、患者が化学療法を越えて、両方の急性および縦方向に評価されます。急性神経毒性を評価するために、患者が治療を受けてから48時間以内に化学療法後の評価に戻ります。 </li><li>慢性的な神経毒性を調べるために、化学療法注入する前の評価は、治療サイクルの向こう側に長手方向に比較されます。 </li><li>軸索の興奮性のテストに加えて、従来の臨床評価尺度は、国立がん研究所を含む化学療法誘発性神経毒性を評価するために使用されるべき - 有害事象の神経感覚サブスケール、総神経障害スコアとパットのためのコモンクライテリアient報告アウトカム評価。 </li></ol> 5。分析と解釈<ol><li>急性神経毒性を識別するために、結果は前と後の化学療法と比較されます。神経機能の縦の変化の発展を評価するために、結果は処理間で比較されています。評価のための重要なパラメータは、ピーク振幅と遅延時間などの従来のパラメータに加えて、閾値電気緊張のしきい値の変化の耐火性、superexcitability、範囲（90から100ミリ秒を過分極）が含まれています。 </li><li>治療全体の興奮性のパラメータの全体的な変化を評価するために、コンポジット興奮性のスコアが計算されます。 superexcitability、耐火性と閾値電気緊張（90-100ミリ秒を過分極） - - 3つのパラメータの変更は、変更の全体的なマーカーを提供するために、初期から最終的な治療に加算されます。 </li></ol> 6。代表的な結果パットの興奮性の結果の例オキサリプラチンで治療ientが用意されています。すぐにポスト·オキサリプラチン注入、開発、感覚と運動の両方の興奮性の急性変化、機能のNa + channelopathy 10 -13の開発を示唆する。ただし、複数の興奮性のパラメータに有意な変化は、広範囲の感覚軸索損傷と膜電位の変化を反映し、影響を受けないモータの軸索（ 図3）のみで、感覚神経軸索にオキサリプラチン治療を越えて徐々に開発しています。このパターンは、慢性のオキサリプラチン誘発性神経毒性症状の臨床的発現と一致しています。感覚軸索の興奮性の変化は、従来の神経伝導技術を使用して評価されるようにピーク振幅の減少に先行し、その軸索の興奮性の技術が早期にオキサリプラチン誘発性神経毒性のために敏感なアセスメントツールを提供することが示唆された。 <img alt="図1" src="/files/ftp_upload/3439/3439fig1.jpg" /> 図1。 </st現在の偏光長引くサブスレッショルド（100 ms）の応答波形を描いた栄&gt;しきい値の電気緊張は、分極方向と下の象限（青）でプロットし、分極方向は、上部象限（赤）にプロットされます。閾値以下の電気緊張応答を生成するために適用された刺激波形である。 <img alt="図2" src="/files/ftp_upload/3439/3439fig2.jpg" /> 図2。5でピークに増加した興奮性（superexcitability）の期間、続いて最大上刺激後に最大3ミリまで縮小興奮の期間（耐火性）で、インパルス伝導以下の興奮性の変化の特徴的な配列を示す興奮性の回復サイクル、 -7ミリ秒、その後減少し興奮（subexcitability）。ペアのパルスのパラダイム刺激プロトコルははめ込みです。 <img alt="図3" src="/files/ftp_upload/3439/3439fig3.jpg" /> イチジクURE 3。オキサリプラチンの治療の4-6ヶ月後に白で表示黒と後処理のレコーディングに示すように、ベースラインのレコーディングの感覚軸索オキサリプラチン投与患者における興奮性が変化します。これらの変更は広範囲に軸索損傷と膜電位の変化を反映すると考えられている。腫瘍の設定で興奮セットアップの絵が左側に表示されます。 <a href="http://www.jove.com/files/ftp_upload/3439/3439fig3large.jpg" target="_blank">拡大画像を表示するには、ここをクリック</a> 。

<ol>
	<li>Hausheer, F. H., Schilsky, R. L., Bain, S., Berghorn, E. J., Lieberman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+management,+and+evaluation+of+chemotherapy-induced+peripheral+neuropathy.">Diagnosis, management, and evaluation of chemotherapy-induced peripheral neuropathy.</a> Semin. Oncol. 33, 15-49 (2008).</li><li>Park, S. B., Krishnan, A. V., Lin, C. S., Goldstein, D., Friedlander, M., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+chemotherapy-induced+neurotoxicity+and+the+potential+for+neuroprotective+strategies.">Mechanisms underlying chemotherapy-induced neurotoxicity and the potential for neuroprotective strategies.</a> Curr. Med. Chem. 15, 3081-3094 (2008).</li><li>Park, S. B., Lin, C. S., Krishnan, A. V., Goldstein, D., Friedlander, M. L., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Neuropathy+after+Oxaliplatin+Treatment:+Challenging+the+Dictum+of+Reversibility?.">Long-term Neuropathy after Oxaliplatin Treatment: Challenging the Dictum of Reversibility?.</a> The Oncologist. , (2012).</li><li>Postma, T. J., Heimans, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grading+of+chemotherapy-induced+peripheral+neuropathy.">Grading of chemotherapy-induced peripheral neuropathy.</a> Ann. Oncol. 11, 509-513 (2000).</li><li>Cascinu, S., Catalano, V., Cordella, L., Labianca, R., Giordani, P., Baldelli, A. M., Beretta, G. D., Ubiali, E., Catalano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+reduced+gluthathione+on+oxaliplatin-based+chemotherapy+in+advanced+colorectal+cancer:+a+randomized,+double-blind,+placebo-controlled+trial.">Neuroprotective effect of reduced gluthathione on oxaliplatin-based chemotherapy in advanced colorectal cancer: a randomized, double-blind, placebo-controlled trial.</a> J. Clin. Oncol. 20, 3478-3483 (2002).</li><li>Lehky, T. J., Leonard, G. D., Wilson, R. H., Grem, J. L., Floeter, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin-induced+neurotoxicity:+acute+hyperexcitability+and+chronic+neuropathy.">Oxaliplatin-induced neurotoxicity: acute hyperexcitability and chronic neuropathy.</a> Muscle Nerve. 29, 387-392 (2004).</li><li>Bostock, H., Cikurel, K., Burke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+tracking+techniques+in+the+study+of+human+peripheral+nerve.">Threshold tracking techniques in the study of human peripheral nerve.</a> Muscle Nerve. 21, 137-158 (1998).</li><li>Burke, D., Kiernan, M. C., Bostock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+of+human+axons.">Excitability of human axons.</a> Clin. Neurophysiol. 112, 1575-1585 (2001).</li><li>Krishnan, A. K., Lin, C. S., Park, S. B., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+ion+channels+from+bench+to+bedside:+a+translational+neuroscience+perspective.">Axonal ion channels from bench to bedside: a translational neuroscience perspective.</a> Prog. Neurobiol. 89, 288-313 (2009).</li><li>Park, S. B., Lin, C. S., Krishnan, A. V., Goldstein, D., Friedlander, M., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin-induced+neurotoxicity:+changes+in+axonal+excitability+precede+development+of+neuropathy.">Oxaliplatin-induced neurotoxicity: changes in axonal excitability precede development of neuropathy.</a> Brain. 132, 2712-2723 (2009).</li><li>Park, S. B., Goldstein, D., Lin, C. S., Krishnan, A. V., Friedlander, M. L., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+abnormalities+of+sensory+nerve+function+associated+wtih+oxaliplatin-induced+neurotoxicity.">Acute abnormalities of sensory nerve function associated wtih oxaliplatin-induced neurotoxicity.</a> J. Clin. Oncol. 27, 1243-1249 (2009).</li><li>Krishnan, A. V., Goldstein, D., Friedlander, M., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin-induced+neurotoxicity+and+the+development+of+neuropathy.">Oxaliplatin-induced neurotoxicity and the development of neuropathy.</a> Muscle Nerve. 32, 51-60 (2005).</li><li>Krishnan, A. V., Goldstein, D., Friedlander, M., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin+and+axonal+Na++channel+function+in+vivo.">Oxaliplatin and axonal Na+ channel function in vivo.</a> Clin. Cancer Res. 12, 4481-4484 (2006).</li></ol>

我々は、開示することは何もありません。

化学療法誘発性の神経障害は治療過程に影響を与え、長期的な患者の障害を生じることがあり癌治療の重篤な副作用です。特に化学療法を受けた患者の神経機能障害を測定する高感度かつ客観的評価指標の欠如があります。軸索の興奮性技術の臨床開発は、化学療法誘発性神経毒性の評価に有用な予測情報を提供しています。イオンチャネルの機能、静止電位、及び軸索膜機能に関する情報を提供することにより、これらの技術は、化学療法で治療がん患者における軸索の機能障害の根底にある病態生理学的プロセスへの洞察を可能にします。さらに、軸索の興奮性の技術は、臨床腫瘍学の設定で可能であることが実証されており、個々のテストは5-10分で完了することができます。 オキサリプラチン投与患者では、軸索の興奮性技術が提供する重度の神経毒性のリスクがある患者の早期発見を可能に敏感なバイオマーカー。オキサリプラチン誘発性の神経障害を識別し、従来の神経伝導の研究に比較して軸索損失が既に発生した後にのみ、軸索の興奮性の研究は、前軸索消失への神経機能障害の予測マーカーを提供しています。このように、軸索の興奮性の研究は、客観的に神経保護効果を決定するために、潜在的な神経保護戦略の臨床試験で神経機能の評価を提供するために利用されることがあります。

<table border="1"><tbody><tr><td> 機器と材料 </td><td> 会社 </td></tr><tr><td> QTracSプログラム</td><td> Digitimer、神経学研究所、クイーンスクエア、英国</td></tr><tr><td> DS5隔離された線形バイポーラ定電流刺激</td><td> Digitimer </td></tr><tr><td>サファイアIIAアンプ</td><td> Medelec </td></tr><tr><td> 50/60 Hzのノイズ除去をだます</td><td>クエスト科学機器</td></tr><tr><td>非分極性電極</td><td> Unomedical </td></tr><tr><td>電気ニュートラルアース板</td><td> Unomedical </td></tr></tbody></table>

nerve excitability assessment in chemotherapy-induced neurotoxicity

化学療法誘発性神経毒性は、最もよく使用される化学療法のいくつかの1,2で発生する癌治療の重大な結果である。化学療法誘発性の末梢神経障害は、手足のしびれや感覚異常の症状を生成し、微細運動能力と歩行の困難へと進行する可能性があり、機能障害につながる。厄介な症状を生産することに加えて、化学療法誘発性の神経障害は、減量または治療の早期中止に至る治療の成功を制限される場合があります。神経障害の症状は、そうでなければ良好な予後3の患者に永久的な神経損傷を残して、長期的な持続可能性があります。化学療法が予防措置、および生存率が増加するにつれてより頻繁に利用されているように、長期的な、重要な神経毒性の重要性が増加します。 は確立神経保護または治療のオプションと敏感な評価手法の欠如はありません。 Appropr神経毒性のiate評価は予後因子として、神経保護剤の今後の試験に適したエンドポイントとして重要になります。化学療法誘発性の神経障害の重症度を評価する現在の方法は客観性4を変更し、間観察するために不足感度に実証されている臨床医ベースの評価尺度を利用しています。従来の神経伝導の研究は、比較的非具体的な措置であり、イオンチャネルの機能、または静止膜電位への洞察を提供していない複合活動電位の振幅と伝導速度に関する情報を提供しています。したがって、先行研究は、従来の神経伝導の研究は、化学療法誘発性神経毒性4-6の早い変化に敏感ではないことを明らかにした。比較では、神経の興奮性の研究は、大規模な有髄軸索のヒトin vivo でのイオンチャネル、ポンプ、交換の評価を可能にするために開発されたしきい値の追跡技術を利用7-9 神経の興奮性技術は、化学療法誘発性神経毒性10月13日の開発と重症度を調べるためのツールとして確立されている。興奮性パラメータの数を含む、神経興奮性の研究は、点滴や慢性、累積毒性の開発直後に生じる急性神経毒性を評価するために使用することができます。神経の興奮性のテクニックだけで5〜10分で完了し必要な各試験で、臨床の現場で実現可能である。神経の興奮装置は、容易に市販されていると、患者は点滴センターの設定で、その場でテストすることができるようにポータブルシステムが考案されている。さらに、これらの手法は、複数の化学療法での使用に適合させることができます。 主に大腸癌のために利用化学療法、オキサリプラチン、治療を受けた患者で、神経の興奮性のテクニックは、患者を識別する方法を提供慢性的な神経障害の発症前に神経毒性のためにT-リスク。神経の興奮性の研究は、運動感覚軸索10月13日に急性のNa + channelopathyの開発を明らかにした。重要なのは、早期治療に興奮性の変化を示した患者は重篤な神経毒性11〜中等度の開発に続いて多かった。しかし、治療を越えて、印象的な長手方向の変更は、患者だけで10の80％で、臨床神経学的ア ​​ウトカムを予測することができた感覚軸索で同定された。これらの変更は、オキサリプラチン投与後、急性見られるものに異なるパターンを示して、最も可能性が高い重要な軸索 ​​損傷とオキサリプラチン治療10の間に長手方向に開発を感覚神経の膜電位変化の発展を反映しています。前の神経機能の従来の施策の低下への早期治療の間に開発著しい異常は、その元を示唆citabilityパラメータは、敏感なバイオマーカーを提供することがあります。

Watch this Scientific Journal Video about Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity at JoVE.com

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

この抽象は、化学療法の治療を受けている患者では神経の発達を評価する手法を提案する。従来の評価方法は、神経機能の早期の変化を検出する能力が限定されていますが、神経興奮性技術は、病態に重度の神経毒性と洞察力のリスクがある患者の早期発見を提供しています。

化学療法誘発性神経の神経興奮性の評価

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

nerve-excitability-assessment-in-chemotherapy-induced-neurotoxicity

Research

JoVE Journal

Neuroscience

18.0K ビュー.  University of New South Wales. この抽象は、化学療法の治療を受けている患者では神経の発達を評価する手法を提案する。従来の評価方法は、神経機能の早期の変化を検出する能力が限定されていますが、神経興奮性技術は、病態に重度の神経毒性と洞察力のリスクがある患者の早期発見を提供しています。

ビデオ: Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

神経科学

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

経皮的電気神経刺激を使用した神経筋機能の評価

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

ナノ粒子神経毒性を決定するために、代理バイオセンサーとしてミクログリア

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

生体内で軸索興奮性のテストでラットの尺骨神経の電気生理学的測定

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

孤立チェック視覚誘発電位による患者における早期オープンアングル緑内障の評価

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

体外における感覚神経と運動神経からの線維芽細胞およびシュワン細胞の精製

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory 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 ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

神経炎症と神経毒性を評価するためのリポ多糖の仔魚への脳室マイクロインジェクション

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral manipulations, there remain significant gaps in the understanding of the neural processes involved in reach planning, execution, and control. The novel approach described here combines a two-dimensional reaching task with transcranial magnetic stimulation (TMS) and concurrent electromyography (EMG) recording from multiple muscles. This method allows for the noninvasive detection of corticospinal activity at precise time points during the unfolding of reaching movements. The example task code includes a delayed response reaching task with two possible targets displayed &#177; 45&#176; off the midline. Single pulse TMS is delivered on the majority of task trials, either at the onset of the preparatory cue (baseline) or 100 ms prior to the imperative cue (delay). This sample design is suitable for investigating changes in corticospinal excitability during reach preparation. The sample code also includes a visuomotor perturbation (i.e., cursor rotation of &#177; 20&#176;) to investigate the effects of adaptation on corticospinal excitability during reach preparation. The task parameters and TMS delivery can be adjusted to address specific hypotheses about the state of the motor system during reaching behavior. In the initial implementation, motor evoked potentials (MEPs) were successfully elicited on 83% of TMS trials, and reach trajectories were recorded on all trials.

Reaching is a fundamental skill that allows humans to interact with the environment. Several studies have aimed to characterize reaching behavior using a variety of methodologies. This paper offers an open-source application of transcranial magnetic stimulation to assess the state of corticospinal excitability in humans during reaching task performance.

目標指向到達行動中の皮質脊髄興奮性の評価

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral ...