陸軍医科大学第二附属病院脳神経外科の現場サポートに感謝します。資金提供:この研究は、中国自然科学基金会(82104696)の支援を受けました。

この研究の実験は、動物倫理の「3R」の原則に準拠しており、陸軍医科大学(AMUWEC20234543)の実験動物福祉および民族委員会によって承認されています。
1.げっ歯類の体固定装置の準備
<ol><li>中国の実用新案特許9に基づいて小動物用ホルダーを設計します。</li>
	<li>3D印刷技術でデバイスを製造します。</li>
	<li>長さ2mのロープを用意し、6本のロープに分けます。この6本のロープを固定具の穴に通します。バネ仕掛けのバックルを利用してロープの端を固定し、ループサイズの調整を容易にします。 注:固定具は、設計図に基づいて3D印刷技術を備えた任意の会社で製造できます。デバイスの設計図を 図1に示します。本研究で用いた固定具は、溶融堆積モデリング(FDM)、溶融フィラメント製造(FFF)とも呼ばれる印刷法により、3Dプリンターで製造された10。この積層造形法は、溶融した形状の材料を所定の経路に選択的に堆積させることにより、設計の部品を層ごとに印刷します。このプロセスでは、フィラメントに入った熱可塑性ポリマーを使用して、最終的な物理的構造を作成します。本研究で用いた固定装置は、3匹の動物を同時に固定することができ、6本のロープが必要で、各動物には首用と胴体用のロープが1本ずつ必要になります。</li>
</ol>2.げっ歯類の準備
<ol><li>3匹のSprague-Dawleyラット(10-40 g、7-45日齢)と3匹のC57 BL / Cマウス(5-40 g)を購入します。</li>
	<li>昼夜サイクルが12時間、周囲温度が21〜23°C、湿度が45%〜60%の部屋で動物を飼育します。定期的に給餌環境の消毒と換気を行い、動物が自由に食べたり飲んだりできるようにしてください。</li>
</ol>3.げっ歯類を固定具に固定する(腹臥位)
<ol><li>げっ歯類の頭を固定具の前のU字型の穴に置き、げっ歯類をうつ伏せの位置にします。げっ歯類の体を2つのロープループに通します。</li>
	<li>げっ歯類の首と腹部にロープを固定し、逃げられないようにしますが、呼吸困難や死を引き起こすほどきつく締め付けないでください。<ol><li>重要なステップは、首を所定の位置に固定することです。げっ歯類の首とロープの間に2〜5mmの隙間を残します。ロープがきつすぎて動物が首を絞めたり、緩すぎて逃げられなかったりしないようにしてください。</li>
	</ol></li>
	<li>他のげっ歯類をデバイス内で同じ方法で固定します。</li>
	<li>上記の固定手順(3.1-3.2)を3日間繰り返して、固定手順によって引き起こされる潜在的なストレス反応を最小限に抑えます。固定手順を3日間連続して繰り返し、各セッションは1日あたり5分間続きます。 注:固定手順中にげっ歯類に過度のストレスがかからないようにするために、手順を3日間連続して繰り返しました。各セッションは1日あたり5分間続き、マウスまたはラットが手順に慣れ、潜在的な不快感を最小限に抑えるのに十分な時間を提供しました。</li>
	<li>4日目からのストラトEA。</li>
	<li>病気の蔓延を防ぐために、滅菌綿棒またはボールを75%アルコールに浸し、ホルダーを完全に拭いて、使用する前後に清掃および消毒してください。げっ歯類を取り扱う際の潜在的な健康上のリスクを回避するために、手袋やその他の保護具を使用してください。</li>
</ol>4. マウスまたは若齢ラットでのEAの実施(MS6)
<ol><li>0.25 mm x 13 mmの使い捨て滅菌鍼をお選びください。</li>
	<li>解剖学的位置からツボを見つけ、ツボの挿入深さを確認します。 注意: MS6はヘッドの外側にあります。頭部のこの領域は、前頭頂葉(Du21)としても知られる深空(Ex-hn1)と玄里(GB6)の間の接続を通過します。MS6は、頭皮の大脳皮質の中心前回の発現に対応します。Ex-hn1は、頭部の前後正中線の中点から0.5cm後方に位置しています。GB6は、耳の前部と目の端の角度の中間点にあります。上顎上神経、眼窩上神経、後頭神経、および耳介側頭神経が分布しています。MS6は、主に脳卒中や運動障害などの脳症の治療に使用されます。</li>
	<li>3匹のげっ歯類の皮下に沿って、前深中(Exhn1)から玄里(GB6)まで、それぞれ3/5の深さでMS6に滅菌針をゆっくりとねじり、挿入します。</li>
	<li>接続ケーブルの片側をEA機器に接続します。</li>
	<li>プラスとマイナスのEAクリップを含む接続ケーブルの反対側を、左右のMS6の鍼灸シャンクに接続します。</li>
	<li>EAインストゥルメントをオンにします。<ol><li>ボタンを回して(機能/スイッチを選択)、周波数と機能モードを設定します。</li>
		<li>[周波数] を 10 Hz に設定し、[連続波] モードを選択します。</li>
		<li>対応するボタン(強度調整)を回して強度を設定します。各強度調整ボタンは、その下に接続されたワイヤのげっ歯類の現在の強度を制御します。電流強度をげっ歯類の許容範囲(通常は1〜2 mA、5分間持続)に調整します。 注意: 処置中のげっ歯類の呼吸と行動を監視して、苦痛や不快感を感じていないことを確認することが重要です。ラットが苦しんでいる、または痛みを感じているように見える場合は、手順を直ちに中止する必要があります。.</li>
	</ol></li>
</ol>5.げっ歯類を固定具に固定する(仰臥位)
<ol><li>げっ歯類の頭を固定具の前のU字型の穴に入れ、げっ歯類をうつ伏せの位置にします。げっ歯類の体を2つのロープループに通します。</li>
	<li>げっ歯類の首と腹部にロープを固定し、逃げられないようにしますが、呼吸困難や死を引き起こすほどきつく締め付けないでください。 注:固定中の注意点は、手順3.2と同じです。</li>
	<li>他のげっ歯類も同じように修正します。</li>
	<li>ステップ3.4と同様に、げっ歯類のストレス反応を減らすために、上記の固定ステップを3日間繰り返します。</li>
	<li>4日目からのストラトEA。 注意: 消毒は、手順3.6で説明したように実行されます。</li>
</ol>6. EAの実行(ST25)
<ol><li>0.25 mm x 13 mmの使い捨て滅菌鍼をお選びください。</li>
	<li>解剖学的位置からツボを見つけ、ツボの挿入深さを確認します。 注:ST25は臍の高さにあります。げっ歯類にこの点を配置するには、まずげっ歯類の足の長さを測定する必要があります。ST25は、おへその隣の足の長さの2/3にあります。例えば、げっ歯類の測定された足が0.6 cmの場合、ST25は臍の左右0.4 cmに位置します。</li>
	<li>3匹のげっ歯類で、滅菌針をST25両側にゆっくりとねじり、4〜8mmの深さで貫通させます。</li>
	<li>接続ケーブルの片側をEA機器に接続します。</li>
	<li>プラスとマイナスのEAクリップを含む接続ケーブルの反対側を、左右のST25の鍼灸シャンクに接続します。</li>
	<li>EAインストゥルメントをオンにします。<ol><li>ボタンを回して(機能/スイッチを選択)、周波数と機能モードを設定します。</li>
		<li>「周波数」を「10 Hz」に設定し、「連続波モード」を選択します。</li>
		<li>対応するボタン(強度調整)を回して強度を設定します。各強度調整ボタンは、その下に接続されたワイヤのラットの現在の強度を制御します。電流強度をげっ歯類の許容範囲(通常は1〜2 mA、5分間持続)に調整します。 注:観察結果はステップ4.6と同じでした。</li>
	</ol></li>
</ol>

3Dプリントされたホルダーを使用した小動物のバルク電気鍼治療

<ol>
	<li>Lee, S., Kim, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+acupuncture+on+modulating+inflammatory+cytokines+in+rodent+animal+models+of+respiratory+disease:+A+systematic+review+and+meta-analysis.">The effect of acupuncture on modulating inflammatory cytokines in rodent animal models of respiratory disease: A systematic review and meta-analysis.</a> Frontiers in Immunology. 13, 878463 (2022).</li><li>Xin, Y. Y., Wang, J. X., Xu, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroacupuncture+ameliorates+neuroinflammation+in+animal+models.">Electroacupuncture ameliorates neuroinflammation in animal models.</a> Acupuncture in Medicine: Journal of the British Medical Acupuncture Society. 40 (5), 474-483 (2022).</li><li>Mengzhu, 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=Electroacupuncture+at+Tianshu+(ST25)+and+Zusanli+(ST36)+alleviates+stress-induced+irritable+bowel+syndrome+in+mice+by+modulating+gut+microbiota+and+corticotropin-releasing+factor.">Electroacupuncture at Tianshu (ST25) and Zusanli (ST36) alleviates stress-induced irritable bowel syndrome in mice by modulating gut microbiota and corticotropin-releasing factor.</a> Journal of traditional Chinese medicine. 42 (5), 732-740 (2022).</li><li>Xu, D. 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=A+new+attempt+of+re-mapping+acupoint+atlas+in+the+rat.">A new attempt of re-mapping acupoint atlas in the rat.</a> Acupuncture Research. 44 (1), 62-65 (2019).</li><li>Yin, C. 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=A+proposed+transpositional+acupoint+system+in+a+mouse+and+rat+model.">A proposed transpositional acupoint system in a mouse and rat model.</a> Research in Veterinary Science. 84 (2), 159-165 (2008).</li><li>Cao, 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=The+effectiveness+of+acupuncture+for+Parkinson's+disease:+An+overview+of+systematic+reviews.">The effectiveness of acupuncture for Parkinson's disease: An overview of systematic reviews.</a> Complementary Therapies in Medicine. 50, 102383 (2020).</li><li>Yang, 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=Effect+of+warming+moxibustion+Tianshu+(ST+25,+bilateral)+and+Qihai+(CV+6)+for+the+treatment+of+diarrhea-dominant+irritable+bowel+syndrome:+a+patient-blinded+pilot+trial+with+orthogonal+design.">Effect of warming moxibustion Tianshu (ST 25, bilateral) and Qihai (CV 6) for the treatment of diarrhea-dominant irritable bowel syndrome: a patient-blinded pilot trial with orthogonal design.</a> Journal of Traditional Chinese Medicine. 37 (4), 538-545 (2017).</li><li>Delcour, M., 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=Early+movement+restriction+leads+to+maladaptive+plasticity+in+the+sensorimotor+cortex+and+to+movement+disorders.">Early movement restriction leads to maladaptive plasticity in the sensorimotor cortex and to movement disorders.</a> Scientific Reports. 8 (1), 16328 (2018).</li><li>Zhifeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fixing+device+for+mice+body.">A fixing device for mice body.</a> China Patent. , (2023).</li><li>Ismail, K. I., Yap, T. C., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-printed+fiber-reinforced+polymer+composites+by+fused+deposition+modelling+(FDM):+Fiber+length+and+fiber+implementation+techniques.">3D-printed fiber-reinforced polymer composites by fused deposition modelling (FDM): Fiber length and fiber implementation techniques.</a> Polymers. 14 (21), 4659 (2022).</li><li>Lu, 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=Efficacy+of+electroacupuncture+with+different+frequencies+in+the+treatment+of+chemotherapy-induced+peripheral+neuropathy:+A+study+protocol+for+a+randomized+controlled+trial.">Efficacy of electroacupuncture with different frequencies in the treatment of chemotherapy-induced peripheral neuropathy: A study protocol for a randomized controlled trial.</a> Frontiers in Neurology. 13, 843886 (2022).</li><li>Zhang, 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=Modulation+of+colonic+function+in+irritable+bowel+syndrome+rats+by+electroacupuncture+at+ST25+and+the+neurobiological+links+between+ST25+and+the+colon.">Modulation of colonic function in irritable bowel syndrome rats by electroacupuncture at ST25 and the neurobiological links between ST25 and the colon.</a> Frontiers in Neuroscience. 16, 930489 (2022).</li><li>Wang, J. 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=Anti-inflammation+mechanism+of+electro-scalp+acupuncture+in+treatment+of+ischemic+stroke+based+on+IL-12+mediated+JAK/STAT+signaling+pathway.">Anti-inflammation mechanism of electro-scalp acupuncture in treatment of ischemic stroke based on IL-12 mediated JAK/STAT signaling pathway.</a> Chinese Acupuncture &amp; Moxibustion. 42 (10), 1137-1144 (2022).</li><li>Ren, 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=A+novel+design+of+a+plate+for+posterolateral+tibial+plateau+fractures+through+traditional+anterolateral+approach.">A novel design of a plate for posterolateral tibial plateau fractures through traditional anterolateral approach.</a> Scientific Reports. 8 (1), 16418 (2018).</li><li>Wu, L., Dong, Y., Zhu, C., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+and+mechanism+of+acupuncture+on+Alzheimer's+disease:+A+review.">Effect and mechanism of acupuncture on Alzheimer's disease: A review.</a> Frontiers in Aging Neuroscience. 15, 1035376 (2023).</li><li>Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroacupuncture+for+spinal+cord+injury:+A+scientific+study+of+traditional+medicine.">Electroacupuncture for spinal cord injury: A scientific study of traditional medicine.</a> Neurospine. 19 (3), 770-772 (2022).</li><li>Gozzi, A., Zerbi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+brain+dysconnectivity+in+rodents.">Modeling brain dysconnectivity in rodents.</a> Biological Psychiatry. 93 (5), 419-429 (2023).</li><li>Denic, A., Macura, S. I., Mishra, P., Gamez, J. D., Rodriguez, M., Pirko, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+in+rodent+models+of+brain+disorders.">MRI in rodent models of brain disorders.</a> Neurotherapeutics. 8 (1), 3-18 (2011).</li><li>Cong-hui, Q., 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=Efficacy+observation+of+long-retaining+scalp+acupuncture+plus+interactive+training+for+upper-extremity+dysfunction+after+cerebral+stroke.">Efficacy observation of long-retaining scalp acupuncture plus interactive training for upper-extremity dysfunction after cerebral stroke.</a> Journal of Acupuncture and Tuina Science. 19, 43-48 (2021).</li><li>Kvist, L. J., Hall-Lord, M. L., Rydhstroem, H., Larsson, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomised-controlled+trial+in+Sweden+of+acupuncture+and+care+interventions+for+the+relief+of+inflammatory+symptoms+of+the+breast+during+lactation.">A randomised-controlled trial in Sweden of acupuncture and care interventions for the relief of inflammatory symptoms of the breast during lactation.</a> Midwifery. 23 (2), 184-195 (2007).</li><li>Boram, L., Chan-Young, K., Sun Haeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+and+safety+of+auriculotherapy+for+breastfeeding:+a+systematic+review.">Effectiveness and safety of auriculotherapy for breastfeeding: a systematic review.</a> Journal of Traditional Chinese Medicine. 40 (5), 721-737 (2020).</li></ol>

執筆者らは、競合する利益相反は存在しないと宣言した。

電気鍼治療(EA)は、鍼に電気刺激を与える鍼治療の一種である11。この技術では、特定の強度と周波数のマイクロパルス電流を使用して経穴を刺激し、治療効果を高めます3。しかし、EAの治癒メカニズムは限られており、1,2,3を証明するには広範な基礎研究が必要です。げっ歯類モデルは?...

<a href="https://app.jove.com/t/6632">This corrects</a> the article <a href="https://dx.doi.org/10.3791/65648">10.3791/65648</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65648">Bulk Electroacupuncture Operation for Mice or Young Rats</a>. Figure 3 was updated.

Erratum: Bulk Electroacupuncture Operation for Mice or Young Rats

電気鍼治療(EA)は、鍼を頭皮に挿入し、電気機械に接続して特定のツボを刺激するユニークな方法です1。手動刺激とは異なり、EAは特定の周波数と波形を安定させることで刺激をより適切に制御し、最適な治療効果を得ることができます2。調査によると、中国の医療機関の81.2%が、脳性麻痺、神経痛、顔面神経麻痺などの治療にEAまたは手鍼治療を使用しています。その人気にもかかわらず、EAの有効性の特定のメカニズムはまだ不明であり、神経疾患のリハビリテーション治療におけるその促進と応用を妨げています1,2,3。EAの有効性メカニズムを十分に理解し、神経疾患のリハビリテーション治療への応用を促進するためには、さらなる研究が必要です。
鍼治療の影響が世界的に拡大するにつれて、ラットやマウスのモデルなどのげっ歯類モデルで実施されるEAのメカニズムはすでに多くの研究で調査されています1,2,3。EAのげっ歯類の実験では、いくつかの問題がしばしば発生します。1つ目は、覚醒している被験者に行われる鍼治療が臨床診療をより反映しているため、麻酔なしでげっ歯類を固定する方法です。さらに、一部の実験では、治療効果を観察するために動物を目覚めさせる必要があります2,3。また、マウスやラットのツボをヒトのツボと正確に一致させることも課題です。実験的なげっ歯類における経穴の正確な局在は、現在多くの学者によって研究されています4,5。このプロトコルでは、MS6とST25が選択され、ヒトの解剖学的局在化の変換によってげっ歯類で明確に定義されています。MS6は、パーキンソン病6などの脳疾患の治療に一般的に使用されています。ST25は通常、下痢などの胃腸の問題の治療に使用されます7。これらの2つのツボは、主にげっ歯類を仰臥位と腹臥位の両方で効果的に固定する方法を示すために選択されました。さらに、これらのツボは広く研究されており、研究目的に重要な洞察を提供しています6,7。
実験のために1匹のラットを固定する従来の方法は、時間がかかるだけでなく、1人で扱うのも困難でした8。さらに、動物が非協力的であるため、実際の成功率は比較的低いです。そのため、実験の効率化のためには、安定な特性を持つ簡便なモデル動物を作製することが急務となっています。この記事では、複数のげっ歯類を簡単に固定でき、運動制限につながる小動物用の3Dプリントホルダーを紹介しました。この論文の目的は、マウスのバルク制限、MS6およびST25経穴の同定、および刺激の戦略に焦点を当てて、若いラットまたはマウスのバッチにEA治療を投与することです。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D printing batch mice fixator</td>
			<td>MESH INVENT</td>
			<td>Custom made</td>
			<td>The fixator produced by 3D printer. The printing method is called fused deposition modeling (FDM), also known as fused filament fabrication (FFF).&#160;&#160;</td>
		</tr>
		<tr>
			<td>Electroacupuncture instrument</td>
			<td>Hwato, Suzhou Medical Appliance Factory</td>
			<td>SDZ-III</td>
			<td>www.Hwato-med.com</td>
		</tr>
		<tr>
			<td>Disposable sterile acupuncture needle</td>
			<td>Suzhou Medical Appliance Factory</td>
			<td>N/A</td>
			<td>0.25 mm x 13 mm</td>
		</tr>
	</tbody>
</table>

bulk electroacupuncture operation for mice or young rats

電気鍼治療(EA)は、さまざまな健康状態の治療に広く使用されています。しかし、EA治療の根底にあるメカニズムは不明のままであり、その推進を妨げています。メカニズム研究では、この問題に対処するためにマウスまたはラットモデルが必要です。しかし、これらの動物は実験プロセスに従わないため、時間がかかります。これらの問題を解決するために、EAの動物実験の効率を向上させるために、3Dプリントされた小動物の体バルク固定器を設計しました。このビデオでは、固定具を使用してマウスまたは若いラットにバルクEAを実行する方法を詳しく説明します。ツボの選定にあたっては、側頭頂点の前斜線(MS6頭部)と天書点(ST25腹部)を選定し、伏臥位と仰臥位による固定装置の効果を検証した。3Dプリントされた小動物ホルダーを使用すると、複数のげっ歯類を同時に固定して処理できるため、実験に必要な時間とリソースを削減できます。この技術は、さまざまなサイズの3Dプリントによって他の動物モデルに適用でき、さまざまな固定条件に使用できる可能性があります。このデバイスは、EAでの実験的科学研究の促進に役立ちます。

私たちが設計した動物の体固定具の間取り図を図1に示します。さらに、この固定具の3Dグラフィックモデルを提出し、設計の包括的なビューを提供しました(補足ファイル1)。3匹のげっ歯類を固定し、同時にEAを行うことができる装置です。ラットとマウスは覚醒状態で固定具に制限され、うつ伏せと仰臥位の両方をげっ歯類に損傷を与えることなくしっか?...

Watch this Scientific Journal Video about Bulk Electroacupuncture Operation for Mice or Young Rats at JoVE.com

著者スポットライト:複数の電気鍼治療を同時に行うための3Dプリントホルダーの使用

ここでは、3次元(3D)プリントホルダーを使用して、マウスまたは若いラットに効率的かつ迅速な電気鍼治療(EA)を行うためのプロトコルを紹介します。この技術により、複数の動物を同時に操作できるため、時間が節約され、実験効率が向上します。

マウスまたは若齢ラットに対するバルク電気鍼治療手術

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering ...

このビデオでは、複数の覚醒マウスまたはラットに電気鍼治療を使用する効率的な方法を紹介します。膀胱研究では、動物は排尿していますが、協力的ではなく、鍼治療のみに時間がかかります。もう一つの問題は麻酔についてで、これは鍼治療の臨床的適用ではありません。これらの問題に対処するために、3Dプリントされたホルダーを使用して、複数の覚醒マウスまたはラットに電気鍼治療を行う効率的な方法を実証します。私たちのプロトコルは、複数の動物を同時に保持するためのより効率的で安全な方法を示しており、固定を意味する他の方法とは異なり、この方法は時間を節約するだけでなく、実験の成功率も向上させます。さらに、デバイスは取り扱いが簡単で、1人で操作できるように設計されています。ProTechとデバイスは、電気鍼治療における実験的科学研究の促進に有益です。

bulk-electroacupuncture-operation-for-mice-or-young-rats

Research

JoVE Journal

Biology

Bulk Electroacupuncture Operation for Mice or Young Rats

537 ビュー.  Army Medical University. このビデオでは、複数の覚醒マウスまたはラットに電気鍼治療を使用する効率的な方法を紹介します。膀胱研究では、動物は排尿していますが、協力的ではなく、鍼治療のみに時間がかかります。もう一つの問題は麻酔についてで、これは鍼治療の臨床的適用ではありません。これらの問題に対処するために、3Dプリントされたホルダーを使用して、複数の覚醒マ?...

ビデオ: 著者スポットライト:複数の電気鍼治療を同時に行うための3Dプリントホルダーの使用

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

マイクロ流体プローブ：局在表面処理のための操作と使用方法

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

生物学

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

接着細胞培養用の酸素挿入の作製と動作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

マウスとラットの拘束マニュアルと共通の複合投与経路

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

診断剖検及びラット及びマウスで選択された組織とサンプル集

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

ラットとマウスにおける卵巣摘出および17β-エストラジオールの交換：ビジュアルデモ

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

デジタル核酸定量アッセイの簡単な一括読み出し

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

隔離の乳酸脱水素酵素試金-哺乳類細胞における一括 Autophagic 隔離活動を決定するシンプルで信頼性の高い方法

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

動物性食品中の サルモネラ菌 をスクリーニングし、培養分離から サルモネラ菌 を確認するためのループ媒介等温増幅

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...