この作品は、2017 年に gist 補助金を通じて「生物医学統合研究」プロジェクトによって支えられました。

1. 注射針の絶縁
注: 直径 720 μ m, 長さ 32 mm は、注射針の電気絶縁用透明熱収縮チューブ (HST) を採用します。ハッブル宇宙望遠鏡は、ポリエチレンテレフタ レート (PET)、ほとんどの酸に対する耐薬品性を示し、拠点を機械的耐久性と生体適合性製です。初期の内径とハッブル宇宙望遠鏡の壁の厚さは、それぞれ 840 μ m、25 μ m。高温でさらに大きな削減を 100 ° C の温度で 50% 以上減少する傾向にあるハッブル宇宙望遠鏡の直径 190 ° C までペット HST が完全に堅く、堅い硬化時になるプロパティを持つ熱硬化性材料であることに注意してください。注射針、収縮チューブのサイズは、研究の目的とアプリケーションに応じて調整できます。全体の製造プロセスは図 1にグラフィカルに集約されます。
<ol><li>ハッブル宇宙望遠鏡の 3 cm。 調整の長さに注射針の浸透深さによって管の長さをカットします。</li>
	<li>カット ハッブル宇宙望遠鏡に注射針を挿入します。</li>
	<li>脱水は 105 ° c (手順 1.6) でクリーニング プロセスで実施する場合、不要な追加の収縮を防ぐために設定されている 150 の ° C の温度で熱銃を使用してチューブを縮小します。</li>
	<li>そのハブから注射針を分離します。</li>
	<li>30 kHz、出力 350 W の超音波攪拌を伴う脱イオン (DI) 水浴 (20 ° C) のハッブル宇宙望遠鏡による絶縁注射針をきれい。</li>
	<li>105 ° c 10 分ホット プレートにハッブル宇宙望遠鏡によって絶縁されて注射針を退避します。</li>
</ol>2. Au 蒸着スパッタリングによる
注: この研究では、使用可能なスパッタ リング プロセスが使用されて電極の Au 層を沈殿物電子ビーム蒸発のプロセスは、別の方法をすることができます。それは、スパッタ リング プロセスのほとんどによる温度上昇がハッブル宇宙望遠鏡の追加の収縮を引き起こすことが確認されています。しかし、数分以上続くプロセスは、初期収縮温度上 HST を熱かもしれない。先端から加工マージンの増加の結果、ハッブル宇宙望遠鏡の追加の収縮があります。
<ol><li>ハッブル宇宙望遠鏡は Cr/Au 蒸着用両面テープを使用してスライド グラス上に並んでによって絶縁されて洗浄の皮下注射針を配置します。</li>
	<li>
スパッタリング装置を使用して、ハッブル宇宙望遠鏡によって絶縁されて洗浄の皮下注射針の Cr/Au を入金します。 注: この場合、Cr と Au の厚さが 10 nm と 100 nm、それぞれ (ハッブル宇宙望遠鏡と Au 層間の接着層は、Cr が使用された)。<ol>
<li>生産コストと生産時間を短縮するために可能な限り多くの針を配置します。預金 10 以下スパッタ条件を使用する Cr nm と 100 nm Au。</li>
		<li>Cr スパッタ、設定クロム ターゲット直径: 4 インチ、高周波電力: 300 W、アルゴン圧力: 5 mTorr とシャッターが開く時間: 20 s (10 nm)。</li>
		<li>Au スパッタリング、使用 Au ターゲット直径: 4 インチ、DC 電源: 300 W、アルゴン圧力: 10 mTorr とシャッターが開く時間: 80 s (100 nm)。</li>
	</ol>
</li>
</ol>3. スプレー塗装
注: 低粘度 (14 cp) フォトレジストは噴霧効率を高めるためにスプレー コーティング プロセスであります。フォトレジストは Au スパッタ針の針が加熱されるときにのみ簡単にコーティングすることができます。
<ol><li>両面テープを使用してスライド ガラスに Au スパッタ皮下注射針の一つを修正します。</li>
	<li>100 ° C に加熱されてスプレーコーターのチャックにスライド グラスを置く2-3 分待ってから針が十分に加熱します。</li>
	<li>100 ° C で針を加熱しながら Au スパッタ針のフォトレジストをスプレーします。次の条件を使用してスプレー塗装プロセスを実行します。セット ノズル径: 400 μ m、ノズル移動速度: 70 mm/s、噴霧圧力: 500 kPa とチャックとノズル間の距離: 13.5 cm。</li>
	<li>スプレー塗装が完了したら、ソフト焼成プロセスを実行する 3 分間 100 ° C でチャックにスライド ガラスを残します。</li>
	<li>フォトレジストは Au スパッタ針に均一に塗布したかどうかを決定する顕微鏡の 100 倍の倍率に設定を使用して結果を確認します。</li>
</ol>4. 紫外線暴露と開発
注: 一般的に、紫外線照射前に柔軟なフィルム フォトマスク プレートに取り付け、フラット透明フォトマスクと紫外線にさらされるサンプルの間のエアー ギャップを削除します。ただし、この研究では、フォトマスクは、フラット透明プレートなし直接金属の湾曲した注射針の表面パターン形成を実現するために使用されます。フォトマスクが地域において最高の接触のアライナと実現可能な解像度のパターン化を達成するために注射針の曲線に沿って曲がっています。曲げフォトマスクと注射針の湾曲した表面の接触面積をできるだけ大きく保つために柔軟なフォトマスクをことができます。ぬれたエッチングの金属を考慮したパターン形成のプロセス (リフトオフ プロセスではなく) を取って、ポジ型フォトレジストの使用は、ネガ型フォトレジストの使用よりも有利です。これは、電極パターンを除く全域が透明、針の中心に電極パターンを容易に合わせて広い視野を提供するためです。
<ol><li>
ウェッジ エラーを最小化、それは完全に固定のフォトマスク固定プレートが接触するまでゆっくりと可動サンプル保持プレートを持ち上げます。空気圧のポンプを使用してサンプル保持板を固定します。
	<ol>
<li>おそらく空気ギャップの紫外線の散乱によって形成され、サンプルとフォトマスクの不完全な接触によって引き起こされる可能性のある好ましくないパターンを避けるためにこのプロセスを実行します。 注: また、ウェッジ エラーの最小化により、フォトレジスト コーティング注射針が動かない次の配置手順でフィルム フォトマスクに接続するときにもかかわらず、注射針の接触面は円形。</li>
	</ol>
</li>
	<li>アライナのサンプル保持板にフォトレジスト コーティング注射針を配置します。</li>
	<li>
映画フォトマスクの配置パターンとレジスト コーティング注射針の投影画像を合わせます。 注: この場合、フィルム フォトマスクの配置パターンは、ハッブル宇宙望遠鏡とフォトレジスト コーティングの厚さを考慮した、800 μ m の距離で 2 本の平行線として設計されました。<ol>
<li>フォトマスク (図 1e); 2 つの平行ラインに投影されたイメージの 2 つの境界線を配置します。したがって、フォトレジスト コーティング皮下注射針は、10 μ m 以下の配置エラーで 2 つの平行ラインの中央に配置できます。</li>
		<li>配置プロセスを監視、電荷結合素子 (CCD) カメラと顕微鏡に接続してディスプレイ モニターを通じてリアルタイムで。</li>
	</ol>
</li>
	<li>フォトマスクに向かって針をゆっくりと持ち上げてフォトレジスト コーティング注射針固定の柔軟なフォトマスクとの接触をもたらします。</li>
	<li>30 の紫外線露出を行う s (UV 強度: 15 mJ/cm2) 3 分の開発プロセスによってこれに従うと。</li>
	<li>純水を使用してサンプルの開発者をすすいでください。</li>
	<li>フォトレジストは Au スパッタ注射針に明確にパターンかどうかを決定するために 200 倍の倍率設定顕微鏡を通して結果を検査します。露出したフォトレジストが開発プロセスの後に完全に削除されていない場合は、30 秒間隔で開発プロセスを繰り返します。</li>
</ol>5. Cr/Au ウェット エッチング
注意: 皮膚/眼を避ける Cr と Au の湿潤腐食液との接触。
<ol><li>スライド ガラスに固定されたサンプル (フォトレジスト パターン注射針) をデタッチするのにピンセットを使用します。</li>
	<li>サンプルを 1 分 Au ウェット エッチング液に浸します。</li>
	<li>純水を使用してサンプルのうち Au エッチング液を洗い流してください。</li>
	<li>200 X 倍率に顕微鏡を使って結果を検査します。まだ削除されてに金に残ります、ウェット 10 秒間隔でエッチングを繰り返します。過度に長いウェット エッチング時間は櫛型電極 (IDE) を薄く作る。</li>
	<li>30 Cr エッチング液に試料を浸す s。</li>
	<li>純水を使用してサンプルから Cr エッチング液を洗い流してください。</li>
</ol>6. 残留フォトレジストとパッシベーションの除去
<ol><li>サンプル (注射針の金属パターン) を 1 分のアセトン溶液に浸漬します。</li>
	<li>DI 水でサンプルを洗い、105 ° C 10 分のホット プレートに退避。</li>
	<li>接続線の電気パッシベーションのカット シュリンク チューブ、2-3 mm (20 mm、最大の深さに浸透する) 電極より長いハッブル宇宙望遠鏡は縮小後は、ハッブル宇宙望遠鏡の長さが差し引かれますので、図 2に示すよう。</li>
	<li>ハッブル宇宙望遠鏡は、IDE の端から可能な限りの位置後、150 ° C で熱銃を使用するしっかりと針に HST の温度を上げます。</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Electrical+Impedance+Spectroscopy-on-a-Needle+as+a+Novel+Tool+to+Determine+Optimal+Surgical+Margin+in+Partial+Nephrectomy.">Evaluation of Electrical Impedance Spectroscopy-on-a-Needle as a Novel Tool to Determine Optimal Surgical Margin in Partial Nephrectomy.</a> Adv Healthc. , (2017).</li><li>Wu, H., 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=Conformal+Pad-Printing+Electrically+Conductive+Composites+onto+Thermoplastic+Hemispheres:+Toward+Sustainable+Fabrication+of+3-Cents+Volumetric+Electrically+Small+Antennas.">Conformal Pad-Printing Electrically Conductive Composites onto Thermoplastic Hemispheres: Toward Sustainable Fabrication of 3-Cents Volumetric Electrically Small Antennas.</a> PLoS One. 10 (8), e0136939 (2015).</li><li>Ahn, C., 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=Direct+fabrication+of+thin+film+gold+resistance+temperature+detection+sensors+on+a+curved+surface+using+a+flexible+dry+film+photoresist+and+their+calibration+up+to+450&#176;+C.">Direct fabrication of thin film gold resistance temperature detection sensors on a curved surface using a flexible dry film photoresist and their calibration up to 450&#176; C.</a> C. J Micromech Microeng. 23 (6), 065031 (2013).</li><li>Goluch, E. D., 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=Microfluidic+method+for+in-situ+deposition+and+precision+patterning+of+thin-film+metals+on+curved+surfaces.">Microfluidic method for in-situ deposition and precision patterning of thin-film metals on curved surfaces.</a> Appl Phys Lett. 85 (16), 3629-3631 (2004).</li><li>Hu, X., 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=A+degradable+polycyclic+cross-linker+for+UV-curing+nanoimprint+lithography.">A degradable polycyclic cross-linker for UV-curing nanoimprint lithography.</a> J Mater Chem C. 2 (10), 1836-1843 (2014).</li><li>Wu, J. -. T., Lai, H. -. C., Yang, S. -. Y., Huang, T. -. C., Wu, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dip+coating+cooperated+with+stepped+rotating+lithography+to+fabricate+rigid+microstructures+onto+a+metal+roller.">Dip coating cooperated with stepped rotating lithography to fabricate rigid microstructures onto a metal roller.</a> Microelectron Eng. 87 (11), 2091-2096 (2010).</li></ol>

著者が明らかに何もありません。

スプレー コーティングとフィルム フォトマスクを使用してそれの写真平版は 1 mm 以下の細径注射針の曲面に微細 Ide を作製する実行可能な方法を行った。幅と Ide のギャップの低として 20 μ m、先端から加工余白が 680 μ m ほど。、プロトコルの中でくさびエラー除去などの配置プロセスが重要です。イーオンは、厳格な配置プロセスを個別に製造された時、生産量は 90% であった以上。これ?...

皮下注射針は、安価で使いやすいので広く生検と薬剤投与のための病院に利用されます。また、薄型の直径と侵略に適した鋭利構造にもかかわらず優れた機械的特性があります。生検中に超音波ガイダンス1皮下注射針の中空に標的組織をサンプリングします。超音波検査は放射線の胎児や妊娠中の女性にとって安全な無料リアルタイム イメージングを提供します、それは超音波が空気を突き通すことができないので、肥満患者の場合は特に、体の奥深くにある臓器を参照してくださいすることは困難または脂肪組織2。さらに、外科医は医師のスキルを欠いている場合、生検の必要性の結果、病院の大半に従来利用されている二次元超音波検査からの奥行き情報を取得できませんまたはが発生します。、脊髄くも膜下麻酔の薬剤では、医師は、脳脊髄液 (CSF)、慎重に患者さんの背中に針を挿入する際、注射器に逆流する場合針が脊髄の領域に達したことを決定します。CSF の逆流を確認した後、麻酔薬は脊髄領域3に注入されます。しかし、医師を危険にさらす貫通または脊髄領域における神経線維を切断患者とも麻痺4,5に激しい痛みを引き起こします。したがって、この手順には、熟練した医師も必要です。克服し、上記の問題を軽減する 1 つのソリューションは、針の位置について客観的な情報を提供することができますので、皮下注射針にナビゲーション機能を追加することです。これは容易に彼らの経験的判断のみに頼ることがなく生検、薬剤配達およびも手術を実行医師に役立つでしょう。
電体、電気インピー ダンス分光法を取り入れて皮下注射針のターゲット組織をローカライズするために (EIS) センサーは、針上の EIS (永劫)6として導入されています。EIS センサーは DNA 検出7,8,9, 細菌/ウイルス検出10、11,12 などの医用工学の分野で利用されています。、および細胞/組織13,14,15,16,17,18,19,20の分析,21,22.、イーオンを異種材料の電気伝導度、誘電率に基づいた周波数領域で区別します。リン酸緩衝生理食塩水 (PBS)23、豚脂肪/筋肉組織6、23、およびもヒト腎正常/癌組織24 の様々 な濃度にイーオンの弁別能力を検証しました ,25。イーオンのこの機能は、かなり標的病変組織と正常組織との間の電気インピー ダンスの違いによる標的組織を配置することによって生検の精度を向上させると予想されます。同様に、薬剤注入間の電気インピー ダンスの調査の違いスペース (脊椎または硬膜外スペース) と正確なターゲットの場所に麻酔薬を提供医師を助けることができる周囲の組織。さらに、イーオンは電気などとして多く正常組織として維持するために、腎部分切除術、腫瘍の切除を伴う手術中に最適な外科的マージンを確認するに同様に脳・筋を刺激するために利用できます。可能。
イーオンの実現における最大の課題の 1 つは、小さな曲率半径を有する注射針の曲面上の電極の作製です。フォトリソグラフィー プロセスを使用して直接金属パターン形成は、数ミリメートルの直径を持つ湾曲した基板上の微小電極の作製には不向き、または以下と見なされています。等角印刷26、柔軟性など、さまざまな方法が乾燥膜フォトレジスト27、マイクロ流体法28、ナノイン プリント ・ リソグラフィ ・29と基板回転露光30、されているところ、曲面上の金属/樹脂パターンを作製を紹介しました。しかし、直径 1 mm 未満、20 mm 以上の合計電極長さ必要な基板など、イーオンの要件に起因する制限、幅、数十マイクロメートル、および大量生産に至るまでの電極のギャップはまだ。
本研究で注射針の湾曲した表面に微小電極を実現するフォトレジスト スプレー コーティングと柔軟なフィルム フォトマスクを用いて直接金属パターンを提案する.針の直径は 720 μ m (22-ゲージ)、生検と病院で薬物送達のため広く用いられているほどです。本加工法の生産量も手頃な価格で大量生産の可能性を決定する評価されます。

<table>
	<tbody>
		<tr>
			<td>Heat shrink tube</td>
			<td>VENTION MEDICAL, Inc.</td>
			<td>103-0655</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needle (22G)</td>
			<td>HWAJIN MEDICAL co. ltd</td>
			<td>-</td>
			<td>http://www.hwajinmedical.com</td>
		</tr>
		<tr>
			<td>Heat gun</td>
			<td>Weller</td>
			<td>WHA600</td>
			<td>http://www.weller-tools.com/en/Home.html</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>HWASHIN INSTRUMENT CO, LTD.</td>
			<td>POWERSONIC 620-</td>
			<td>http://www.hwashin.net</td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>AS ONE Corporation</td>
			<td>006560</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputtering</td>
			<td>A-Tech System. Ltd.</td>
			<td>ATS/SPT/0208F</td>
			<td>http://www.atechsystem.co.kr</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>1000412</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray coater</td>
			<td>LITHOTEK</td>
			<td>LSC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist</td>
			<td>AZ electronic materials</td>
			<td>GXR 601</td>
			<td>http://www.merck-performance-materials.com/en/index.html</td>
		</tr>
		<tr>
			<td>Developer (solution)</td>
			<td>AZ electronic materials</td>
			<td>MIF 300</td>
			<td>http://www.merck-performance-materials.com/en/index.html</td>
		</tr>
		<tr>
			<td>Aligner</td>
			<td>MIDAS SYSTEM CO.,Ltd.</td>
			<td>MDA-400M</td>
			<td>http://www.midas-system.com</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON Corporation</td>
			<td>L200</td>
			<td>http://www.nikonmetrology.com</td>
		</tr>
		<tr>
			<td>Au wet etchant</td>
			<td>TRANSENE COMPANY, Inc.</td>
			<td>Au etchant type TFA</td>
			<td>http://transene.com</td>
		</tr>
		<tr>
			<td>Cr wet etchant</td>
			<td>KMG Electronic. Chemicals, Inc.</td>
			<td>CR-7</td>
			<td>http://kmgchemicals.com</td>
		</tr>
		<tr>
			<td>Au target</td>
			<td>Thin films and Fine Materials</td>
			<td>-</td>
			<td>http://www.thifine.co.kr</td>
		</tr>
		<tr>
			<td>Cr target</td>
			<td>Thin films and Fine Materials</td>
			<td>-</td>
			<td>http://www.thifine.co.kr</td>
		</tr>
		<tr>
			<td>Argon gas (99.999%)</td>
			<td>SINIL Gas Co.Ltd</td>
			<td>-</td>
			<td>http://www.sigas.kr</td>
		</tr>
		<tr>
			<td>Acetone solution</td>
			<td>OCI Company Ltd</td>
			<td>-</td>
			<td>http://www.ocicorp.co.kr/company/index.asp</td>
		</tr>
		<tr>
			<td>Impedance analyzer</td>
			<td>Gamry Instruments Inc</td>
			<td>Reference 600</td>
			<td>https://www.gamry.com</td>
		</tr>
		<tr>
			<td>Height Controller</td>
			<td>Mitutoyo Corporation</td>
			<td>192-613</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies Corporation</td>
			<td>10010023</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of fine electrodes on the tip of hypodermic needle using photoresist spray coating and flexible photomask for biomedical applications

上の電気インピー ダンス分光 (EIS) の作製方法を紹介して、-針 (EoN: EIS 上針) 計測・異種による生体組織の電気的インピー ダンスの違いを分析し体内の標的組織を検索します。フォトリソグラフィ工程でのフォトレジスト スプレー コーティングと柔軟なフィルム フォトマスク使用注射針の先端に細かい櫛型電極 (Ide) を作製する方法について述べる。ポリエチレンのポリエチレンテレフタ レート (PET) の熱収縮チューブ (ハッブル宇宙望遠鏡) が壁厚 25 μ m では断熱材とパッシベーション層として用いられます。ハッブル宇宙望遠鏡はペットは、誘電体コーティング材として広く使用されている poly(p-xylylene) のポリマーと比較して高い機械的耐久性を示しています。さらに、HST はイーオンに化学的損傷を制限するために有利であるほとんどの酸と塩基、耐薬品性を示します。イーオンの使用は、特に化学物質/材料の酸/塩基の化学物質を使用して作製評価に適しています。作製したギャップと Ide の幅が 20 μ m ほどと全体の幅と、Ide の長さは 400 μ m と 860 μ m それぞれ。注射針の先端 (注射針の先端と Ide の開始点の間の距離) から製造マージンは 680 μ m ほど中を示す尖頭のの不要な侵入を避けることができる、電気インピー ダンス測定。イーオンは、臨床使用の可能性が高いように甲状腺生検と脊髄領域の麻酔薬物送達。さらに、腫瘍の切除を含む手術でもイーオン用いることができる通常の外科的マージン (通常組織腫瘍の外科的切除で除去) を検出することによりできるだけ多く正常な組織として維持するために、病変組織。

櫛型電極 (Ide)、図 2で示すように、電極の他の図形と比較して限られた表面に大きい効果的な検出エリアにあります。Ide の全体の長さは 860 μ m 検出、生検や薬物の配信手順で高位置決め精度を提供する尖頭で 1 mm 間隔以下でインピー ダンス変化を分析して設計されています。Ide の幅の合計は、提案のフォトリソグラフィ プロセスを使用して、...

Watch this Scientific Journal Video about Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications at JoVE.com

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

高級櫛型電極の作製法 (ギャップと幅: 20 μ m) 注射針の先端に (直径: 720 μ m) フォトリソグラフィ工程でスプレー塗装と柔軟なフィルム フォトマスクを使用して示されて。

医用フォトレジスト スプレー塗装と柔軟なフォトマスクを使用して皮下注射針の先端の微細電極の作製

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

fabrication-fine-electrodes-on-tip-hypodermic-needle-using

Research

JoVE Journal

Neuroscience

9.5K ビュー.  Gwangju Institute of Science and Technology (GIST). 高級櫛型電極の作製法 (ギャップと幅: 20 μ m) 注射針の先端に (直径: 720 μ m) フォトリソグラフィ工程でスプレー塗装と柔軟なフィルム フォトマスクを使用して示されて。

ビデオ: Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

老化とアミロイド病理の間にシナプス変化の研究のためのラットおよびトランスジェニックマウスからの急性海馬スライスの準備

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

神経科学

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

拡散うつ病を使用してエピソードと慢性片頭痛の神経免疫シグナルのモデリングインビトロ</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments in vision research require displays with more than 12 bits of luminance resolution. Several solutions are available. Bit++ 1 and DataPixx 2 use the Digital Visual Interface (DVI) output from graphics cards and high resolution (14 or 16-bit) digital-to-analog converters to drive analog display devices. The VideoSwitcher 3 described here combines analog video signals from the red and blue channels of graphics cards with different weights using a passive resister network 4 and an active circuit to deliver identical video signals to the three channels of color monitors. The method provides an inexpensive way to enable high-resolution monochromatic displays using conventional graphics cards and analog monitors. It can also provide trigger signals that can be used to mark stimulus onsets, making it easy to synchronize visual displays with physiological recordings or response time measurements.
Although computer keyboards and mice are frequently used in measuring response times (RT), the accuracy of these measurements is quite low. The RTbox is a specialized hardware and software solution for accurate RT measurements. Connected to the host computer through a USB connection, the driver of the RTbox is compatible with all conventional operating systems. It uses a microprocessor and high-resolution clock to record the identities and timing of button events, which are buffered until the host computer retrieves them. The recorded button events are not affected by potential timing uncertainties or biases associated with data transmission and processing in the host computer. The asynchronous storage greatly simplifies the design of user programs. Several methods are available to synchronize the clocks of the RTbox and the host computer. The RTbox can also receive external triggers and be used to measure RT with respect to external events. 
Both VideoSwitcher and RTbox are available for users to purchase. The relevant information and many demonstration programs can be found at <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a>.

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

有効にする高解像度を表示し、従来のコンピュータ上で正確な応答時間の測定値をグレースケール

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

単一ユニット活動とElectrocorticographic信号を記録するための慢性神経電極の外科的移植

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function.
Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron&#x2019;s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

用ピギーバックMultibarrel電極の製造と使用<emインビボ&gt;</em神経反応の&gt;薬理マニピュレーション

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

嗅覚検索用自律型ロボットに昆虫Electroantennogramセンサを用いた

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

マウス海馬にオリゴマーアミロイドベータの定位注入

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

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 ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

神経系発達中の軸の成長と挙動を解析する3Dコラーゲンベースのヒドロゲルの生成

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

医用フォトレジストスプレー塗装と柔軟なフォトマスクを使用して皮下注射針の先端の微細電極の作製

要約

さらに動画を探す

この動画の章

老化とアミロイド病理の間にシナプス変化の研究のためのラットおよびトランスジェニックマウスからの急性海馬スライスの準備

拡散うつ病を使用してエピソードと慢性片頭痛の神経免疫シグナルのモデリングインビトロ

有効にする高解像度を表示し、従来のコンピュータ上で正確な応答時間の測定値をグレースケール

単一ユニット活動とElectrocorticographic信号を記録するための慢性神経電極の外科的移植

用ピギーバックMultibarrel電極の製造と使用

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

嗅覚検索用自律型ロボットに昆虫Electroantennogramセンサを用いた

マウス海馬にオリゴマーアミロイドベータの定位注入

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

神経系発達中の軸の成長と挙動を解析する3Dコラーゲンベースのヒドロゲルの生成

医用フォトレジスト スプレー塗装と柔軟なフォトマスクを使用して皮下注射針の先端の微細電極の作製

要約

さらに動画を探す

この動画の章

老化とアミロイド病理の間にシナプス変化の研究のためのラットおよびトランスジェニックマウスからの急性海馬スライスの準備

拡散うつ病を使用してエピソードと慢性片頭痛の神経免疫シグナルのモデリングインビトロ

有効にする高解像度を表示し、従来のコンピュータ上で正確な応答時間の測定値をグレースケール

単一ユニット活動とElectrocorticographic信号を記録するための慢性神経電極の外科的移植

用ピギーバックMultibarrel電極の製造と使用

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

嗅覚検索用自律型ロボットに昆虫Electroantennogramセンサを用いた

マウス海馬にオリゴマーアミロイドベータの定位注入

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

神経系発達中の軸の成長と挙動を解析する3Dコラーゲンベースのヒドロゲルの生成

医用フォトレジストスプレー塗装と柔軟なフォトマスクを使用して皮下注射針の先端の微細電極の作製