Name: full-circle cauterization of limbal vascular plexus for surgically induced glaucoma in rodents
Uploaded: 2022-02-15T14:00:00
Description: Watch this Scientific Journal Video about Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents at JoVE.com

私たちは、検査技師のホセを認めます。ニルソン・ドス・サントス、ダイアン・マンダリーノ・トーレス、ホセ・フランシスコ・ティブルシオ、ジルド・ブリト・デ・ソウザ、ルチアーノ・カヴァルカンテ・フェレイラ。この研究は、FAPERJ、CNPq、およびCAPESから資金提供を受けました。

すべての手順は、Association for Research in Vision and Ophthalmology(ARVO)の眼科および視覚研究における動物の使用に関する声明に準拠して実施され、リオデジャネイロ連邦大学健康科学センターの科学実験における動物の使用に関する倫理委員会によって承認されました(プロトコル083/17)。本研究では、生後2〜3ヶ月、体重180〜320gのリスターフード付きラットを用いた。しかし、この手順は、さまざま?...

<ol>
	<li>Weinreb, R. N., Aung, T., Medeiros, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Pathophysiology+and+Treatment+of+Glaucoma+A+Review.">The Pathophysiology and Treatment of Glaucoma A Review.</a> JAMA. 311 (8), 1901-1911 (2014).</li><li>Tham, Y. 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=Global+prevalence+of+glaucoma+and+projections+of+glaucoma+burden+through+2040:+A+systematic+review+and+meta-analysis.">Global prevalence of glaucoma and projections of glaucoma burden through 2040: A systematic review and meta-analysis.</a> Ophthalmology. 121 (11), 2081-2090 (2014).</li><li>Quaranta, 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=Quality+of+Life+in+Glaucoma:+A+Review+of+the+Literature.">Quality of Life in Glaucoma: A Review of the Literature.</a> Advances in Therapy. 33 (6), 959-981 (2016).</li><li>Heijl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+intraocular+pressure+and+glaucoma+progression:+results+from+the+Early+Manifest+Glaucoma+Trial.">Reduction of intraocular pressure and glaucoma progression: results from the Early Manifest Glaucoma Trial.</a> Archives of Ophthalmology. 120 (10), 1268-1279 (2002).</li><li>Susanna, R., De Moraes, C. G., Cioffi, G. A., Ritch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+Do+People+(Still)+Go+Blind+from+Glaucoma.">Why Do People (Still) Go Blind from Glaucoma.</a> Translational Vision Science &amp; Technology. 4 (2), 1 (2015).</li><li>Fujikawa, K., 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=VAV2+and+VAV3+as+candidate+disease+genes+for+spontaneous+glaucoma+in+mice+and+humans.">VAV2 and VAV3 as candidate disease genes for spontaneous glaucoma in mice and humans.</a> PLoS One. 5 (2), 9050 (2010).</li><li>Mao, M., Hedberg-Buenz, A., Koehn, D., John, S. W., Anderson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+segment+dysgenesis+and+early-onset+glaucoma+in+nee+mice+with+mutation+of+Sh3pxd2b.">Anterior segment dysgenesis and early-onset glaucoma in nee mice with mutation of Sh3pxd2b.</a> Investigative Ophthalmology &amp; Visual Science. 52 (5), 2679-2688 (2011).</li><li>John, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+iris+atrophy,+pigment+dispersion,+and+glaucoma+in+DBA/2J+mice.">Essential iris atrophy, pigment dispersion, and glaucoma in DBA/2J mice.</a> Investigative Ophthalmology &amp; Visual Science. 39 (6), 951-962 (1998).</li><li>Aihara, M., Lindsey, J. D., Weinreb, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+hypertension+in+mice+with+a+targeted+type+I+collagen+mutation.">Ocular hypertension in mice with a targeted type I collagen mutation.</a> Investigative Ophthalmology &amp; Visual Science. 44 (4), 1581-1585 (2003).</li><li>Chou, T. H., Tomarev, S., Porciatti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+expressing+mutated+Tyr437His+human+myocilin+develop+progressive+loss+of+retinal+ganglion+cell+electrical+responsiveness+and+axonopathy+with+normal+iop.">Transgenic mice expressing mutated Tyr437His human myocilin develop progressive loss of retinal ganglion cell electrical responsiveness and axonopathy with normal iop.</a> Investigative Ophthalmology &amp; Visual Science. 55 (9), 5602-5609 (2014).</li><li>vander Zypen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+morphological+study+on+structure+and+function+of+the+filtration+angel+of+the+rat+eye.">Experimental morphological study on structure and function of the filtration angel of the rat eye.</a> Ophthalmologica. 174 (5), 285-298 (1977).</li><li>Aihara, M., Lindsey, J. D., Weinreb, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+humor+dynamics+in+mice.">Aqueous humor dynamics in mice.</a> Investigative Ophthalmology &amp; Visual Science. 44 (12), 5168-5173 (2003).</li><li>Morrison, J. C., Fraunfelder, F. W., Milne, S. T., Moore, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+microvasculature+of+the+rat+eye.">Limbal microvasculature of the rat eye.</a> Investigative Ophthalmology &amp; Visual Science. 36 (3), 751-756 (1995).</li><li>Morrison, J. 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=A+rat+model+of+chronic+pressure-induced+optic+nerve+damage.">A rat model of chronic pressure-induced optic nerve damage.</a> Experimental Eye Research. 64 (1), 85-96 (1997).</li><li>Sappington, R. M., Carlson, B. J., Crish, S. D., Calkins, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbead+occlusion+model:+A+paradigm+for+induced+ocular+hypertension+in+rats+and+mice.">The microbead occlusion model: A paradigm for induced ocular hypertension in rats and mice.</a> Investigative Ophthalmology &amp; Visual Science. 51 (1), 207-216 (2010).</li><li>Zhu, M. D., Cai, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+experimental+chronic+intraocular+hypertension+in+the+rabbit.">Development of experimental chronic intraocular hypertension in the rabbit.</a> Australian and New Zealand Journal of Ophthalmology. 20 (3), 225-234 (1992).</li><li>Shareef, S. R., Garcia-Valenzuela, E., Salierno, A., Walsh, J., Sharma, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+ocular+hypertension+following+episcleral+venous+occlusion+in+rats.">Chronic ocular hypertension following episcleral venous occlusion in rats.</a> Experimental Eye Research. 61 (3), 379-382 (1995).</li><li>Levkovitch-Verbin, 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=Translimbal+laser+photocoagulation+to+the+trabecular+meshwork+as+a+model+of+glaucoma+in+rats.">Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats.</a> Investigative Ophthalmology &amp; Visual Science. 43 (2), 402-410 (2002).</li><li>Zhao, 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=Characterization+of+the+Circumlimbal+Suture+Model+of+Chronic+IOP+Elevation+in+Mice+and+Assessment+of+Changes+in+Gene+Expression+of+Stretch+Sensitive+Channels.">Characterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels.</a> Frontiers in Neuroscience. 11, (2017).</li><li>Biswas, S., Wan, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+rodent+hypertensive+glaucoma+models.">Review of rodent hypertensive glaucoma models.</a> Acta Ophthalmologica. 97 (3), 331-340 (2019).</li><li>Pitha, I., 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=Sustained+Dorzolamide+Release+Prevents+Axonal+and+Retinal+Ganglion+Cell+Loss+in+a+Rat+Model+of+IOP-Glaucoma.">Sustained Dorzolamide Release Prevents Axonal and Retinal Ganglion Cell Loss in a Rat Model of IOP-Glaucoma.</a> Translational Vision Science &amp; Technology. 7 (2), 13 (2018).</li><li>Grozdanic, S. 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=Temporary+elevation+of+the+intraocular+pressure+by+cauterization+of+vortex+and+episcleral+veins+in+rats+causes+functional+deficits+in+the+retina+and+optic+nerve.">Temporary elevation of the intraocular pressure by cauterization of vortex and episcleral veins in rats causes functional deficits in the retina and optic nerve.</a> Experimental Eye Research. 77 (1), 27-33 (2003).</li><li>Lani, R., 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+subacute+model+of+glaucoma+based+on+limbal+plexus+cautery+in+pigmented+rats.">A subacute model of glaucoma based on limbal plexus cautery in pigmented rats.</a> Scientific Reports. 9 (1), 16286 (2019).</li><li>vander Merwe, E. L., Kidson, 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=The+three-dimensional+organisation+of+the+post-trabecular+aqueous+outflow+pathway+and+limbal+vasculature+in+the+mouse.">The three-dimensional organisation of the post-trabecular aqueous outflow pathway and limbal vasculature in the mouse.</a> Experimental Eye Research. 125, 226-235 (2014).</li><li>Prusky, G. T., Alam, N. M., Beekman, S., Douglas, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+of+adult+and+developing+mouse+spatial+vision+using+a+virtual+optomotor+system.">Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system.</a> Investigative Ophthalmology &amp; Visual Science. 45 (12), 4611-4616 (2004).</li><li>Sasovetz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine+hydrochloride:+an+effective+general+anesthetic+for+use+in+electroretinography.">Ketamine hydrochloride: an effective general anesthetic for use in electroretinography.</a> Annals of Ophthalmology. 10 (11), 1510-1514 (1978).</li><li>Stabio, M. 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辺縁神経叢焼灼術(LPC)は、結膜やほぞの解剖を必要としない、簡単にアクセスできる血管構造を標的にするという利点を備えた新しい海綿後モデルです17,28。脈絡膜静脈ドレナージの外科的障害に基づく有名なOHTモデルである渦静脈焼灼モデルとは異なり、静脈うっ血はLPCモデルでは、静脈静脈が房水流出の上流に位置するため、IOPの上昇に影響を?...

医学文献では、緑内障は、網膜神経節細胞(RGC)、樹状突起、体細胞、および軸索の進行性変性を特徴とする視神経障害の不均一なグループとして理解されており、その結果、視神経の構造的カッピング(発掘)と視神経の機能低下が起こり、眼から脳への視覚情報の伝達を中断することにより、制御されていない症例で黒内障を引き起こします1.緑内障は現在、世界中で不可逆的な失明の最も一般的な原因であり、2040年には約1億1,180万人に達すると予測されており2、患者の生活の質(QOL)に深く影響し、重大な社会経済的懸念を引き起こしています3。
眼圧の上昇(IOP)は、緑内障の発症と進行にとって最も重要で唯一の修正可能な危険因子の1つです。複数のタイプの緑内障のうち、正常緊張緑内障(NTG)を除くすべての緑内障は、疾患の病歴のある時点でIOPの上昇に関連しています。眼圧を標的とし、疾患の進行を遅らせたり止めたりするための臨床的および外科的進歩にもかかわらず、患者は依然として緑内障のために視力を失います4,5。したがって、この疾患の複雑で多因子的な病態生理学を完全に理解することは、より効果的な治療法の開発、特にRGCに神経保護を提供するために不可欠です。
疾患のメカニズムを理解するためのさまざまな実験的アプローチの中で、高眼圧症(OHT)に基づく動物モデルは、ヒトの緑内障に最もよく似ています。げっ歯類モデルは、低コストで、取り扱いが容易で、遺伝子操作が可能で、寿命が短く、房水の産生や排液など、人間に匹敵する眼の解剖学的および生理学的特徴を示すため、特に有用です6,7,8,9,10,11,12,13 .現在使用されているモデルには、強膜上静脈への高張生理食塩水の注入後の線維柱帯の硬化症14、マイクロビーズ15または粘弾性物質のカメラ内注入16、渦静脈の焼灼17、アルゴンレーザー18、辺縁周囲縫合糸19による線維柱帯網膜の光凝固、および加齢性OHT(DBA/2Jマウス)のトランスジェニックモデルの使用が含まれます8.しかし、侵襲性、角膜の術後混濁、前眼部の破壊、広範な学習曲線、高価な機器、および術後IOPの変動は、現在のモデルに関連する報告された落とし穴のいくつかであり、OHTの代替モデルの開発は、これらの問題を克服するための要求となっています20,21,22。
本プロトコルは、げっ歯類の辺縁神経叢焼灼術(LPC)に基づいて、緑内障の代理としてOHTを誘発する新しい外科的処置を形式化します23。これは、簡単で再現性が高く、アクセスしやすく、非侵襲的なモデルであり、高効率でIOP上昇の変動性が低く、完全に臨床的に回復する割合が他に類を見ないため、各実験で使用される動物の数を減らしてin vivo で機能評価を行うことができます。手術技術は亜急性OHTを誘発し、数日でベースラインレベルに徐々に戻り、急性閉塞隅角緑内障で見られる高血圧発作をモデル化します。さらに、モデルにおける眼圧回復に続いて、緑内障性神経変性が持続し、これは、眼圧の適切な制御にもかかわらず、ヒト緑内障のいくつかの症例で発生するRGCの二次変性の将来の機構研究に有用である。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Isofar</td>
			<td>201</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
		</tr>
		<tr>
			<td>Active electrode for electroretinography</td>
			<td>Hansol Medical Co</td>
			<td>-</td>
			<td>Stainless steel needle 0.25 mm &#215; 15 mm</td>
		</tr>
		<tr>
			<td>Anestalcon</td>
			<td>Novartis Bioci&#234;ncias S/A</td>
			<td>MS-1.0068.1087</td>
			<td>Proxymetacaine hydrochloride 0.5%</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Vetec</td>
			<td>560</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
		</tr>
		<tr>
			<td>Cautery Low Temp Fine Tip 10/bx</td>
			<td>Bovie Medical Corporation</td>
			<td>AA00</td>
			<td>Low-temperature ophthalmic cautery</td>
		</tr>
		<tr>
			<td>Cetamin</td>
			<td>Syntec do Brasil Ltda</td>
			<td>000200-3-000003</td>
			<td>Ketamine hydrochloride 10%</td>
		</tr>
		<tr>
			<td>DAKO</td>
			<td>Dako North America</td>
			<td>S3023</td>
			<td>Antifade mounting medium</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>28718-90-3</td>
			<td>diamidino-2-phenylindole; blue fluorescent nuclear counterstain; emission at 452&#177;3 nm</td>
		</tr>
		<tr>
			<td>Ecofilm</td>
			<td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td>
			<td>MS-1.0298.0487</td>
			<td>Carmellose sodium 0.5%</td>
		</tr>
		<tr>
			<td>EPON Resin</td>
			<td>Polysciences, Inc.</td>
			<td>-</td>
			<td>Epoxy resin used for electron microscopy, composed of a mixture of four reagents: Poly/Bed 812 Resin (CAT#08791); DDSA - Dodecenylsuccinic Anhydride (CAT#00563); NMA - Nadic Methyl Anhydride (CAT#00886); DMP-30 - 2,4,6-tris(dimethylaminomethyl)phenol (CAT#00553)</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16110</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
		</tr>
		<tr>
			<td>Hyabak</td>
			<td>Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A</td>
			<td>MS-8042140002</td>
			<td>Sodium hyaluronate 0.15%</td>
		</tr>
		<tr>
			<td>Icare Tonolab</td>
			<td>Icare Finland Oy</td>
			<td>TV02 (model number)</td>
			<td>Rebound handheld tonometer</td>
		</tr>
		<tr>
			<td>IgG donkey anti-mouse antibody + Alexa Fluor 555</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31570</td>
			<td>Secondary antibody solution</td>
		</tr>
		<tr>
			<td>LCD monitor 23 inches</td>
			<td>Samsung Electronics Co. Ltd.</td>
			<td>S23B550</td>
			<td>Model LS23B550, for electroretinogram recording</td>
		</tr>
		<tr>
			<td>LSM 510 Meta</td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<td>Confocal epifluorescence microscope</td>
		</tr>
		<tr>
			<td>Maxiflox</td>
			<td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td>
			<td>MS-1.0298.0489</td>
			<td>Ciprofloxacin 3.5 mg/g</td>
		</tr>
		<tr>
			<td>MEB-9400K</td>
			<td>Nihon Kohden Corporation</td>
			<td>-</td>
			<td>System for electroretinogram recording</td>
		</tr>
		<tr>
			<td>monoclonal IgG1 mouse anti-Brn3a</td>
			<td>MilliporeSigma</td>
			<td>MAB-1585</td>
			<td>Brn3a primary antibody solution</td>
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		<tr>
			<td>Neuropack Manager v08.33</td>
			<td>Nihon Kohden Corporation</td>
			<td>-</td>
			<td>Software for electroretinogram signal processing</td>
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			<td>Optomotry</td>
			<td>CerebralMechanics</td>
			<td>-</td>
			<td>System for optomotor response analysis</td>
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			<td>Osmium tetroxide</td>
			<td>Electron Microscopy Sciences</td>
			<td>19100</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
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			<td>Potassium ferrocyanide</td>
			<td>Electron Microscopy Sciences</td>
			<td>20150</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
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		<tr>
			<td>Reference and ground electrodes for electroretinography</td>
			<td>Chalgren Enterprises</td>
			<td>110-63</td>
			<td>Stainless steel needles 0.4 mm &#215; 37 mm</td>
		</tr>
		<tr>
			<td>Sodium cacodylate buffer</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
		</tr>
		<tr>
			<td>Ster MD</td>
			<td>Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A</td>
			<td>MS-1.0497.1287</td>
			<td>Prednisolone acetate 0.12%</td>
		</tr>
		<tr>
			<td>Terolac</td>
			<td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td>
			<td>MS-1.0497.1286</td>
			<td>Ketorolac trometamol 0.5%</td>
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		<tr>
			<td>Terramicina</td>
			<td>Laborat&#243;rios Pfizer Ltda</td>
			<td>MS-1.0216.0024</td>
			<td>Oxytetracycline hydrochloride 30 mg/g + polymyxin B 10,000 U/g</td>
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			<td>Tono-Pen XL</td>
			<td>Reichert Technologies</td>
			<td>230635</td>
			<td>Digital applanation handheld tonometer</td>
		</tr>
		<tr>
			<td>TO-PRO-3</td>
			<td>Thermo Fisher Scientific</td>
			<td>T3605</td>
			<td>Far red-fluorescent nuclear counterstain; emission at 661 nm</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9036-19-5</td>
			<td>Non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
		</tr>
		<tr>
			<td>Xilazin</td>
			<td>Syntec do Brasil Ltda</td>
			<td>7899</td>
			<td>Xylazine hydrochloride 2%</td>
		</tr>
		<tr>
			<td></td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<td>Stereo microscope for surgery and retinal dissection</td>
		</tr>
	</tbody>
</table>

full-circle cauterization of limbal vascular plexus for surgically induced glaucoma in rodents

緑内障は、世界の失明原因の第2位であり、視神経と網膜神経節細胞(RGC)の変性に構造的損傷があり、眼から脳への視覚情報の伝達が遮断されることにより視覚機能障害を引き起こす不均一な眼疾患群です。眼圧の上昇は最も重要な危険因子です。したがって、高眼圧症のいくつかのモデルは、病気の原因と影響を調査するために、遺伝的または実験的アプローチのいずれかによってげっ歯類で開発されています。それらの中で、外科的侵襲性、不十分な機能評価、広範なトレーニングの必要性、網膜損傷の非常に変動の大きい拡張など、いくつかの制限が報告されています。本研究は、房水ドレナージの主要成分である辺縁血管神経叢の低温全円焼灼に基づく、げっ歯類に高眼圧症を誘発する単純で低コストかつ効率的な方法を特徴付ける。この新しいモデルは、進行性のRGCおよび視神経変性に関連する、技術的に簡単で非侵襲的で再現性のある亜急性高眼圧症と、電気生理学的および行動学的方法の両方による in vivo 機能研究を可能にする独自の術後の臨床回復率を提供します。

量的変数は、平均±平均の標準誤差(SEM)として表されます。OHT群と対照群の眼圧動態の比較(図1F)を除き、統計解析は二元配置ANOVAとそれに続くSidakの多重比較検定を用いて行った。p値<0.05は統計的に有意であると見なされました。
図1 は、全円輪姦焼灼術(LPC)モデルの外科的手順を示しており、360°の熱による辺縁血管の消失や?...

Watch this Scientific Journal Video about Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents at JoVE.com

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

このプロトコルの目的は、緑内障性神経変性の新しいモデルを特徴付けることです 辺縁血管神経叢の360°熱焼灼に基づいて、亜急性高眼圧症を誘発します。

げっ歯類の外科的緑内障に対する辺縁血管神経叢の全周焼灼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

我々は、一時的な眼圧上昇に基づいて、強固で進行性の緑内障変性を誘発する簡単な方法を開発しました。これは、緑内障の病態生理学のキーポイントを研究するまたとない機会です。本モデルは、1回の実験で少ない数で生体内機能解析や短期間の実験計画を可能とする、学習が容易で非侵襲的、低コスト、再現性の高いモデルです。網膜と視神経は中枢神経系の評価可能な部分であるため、これらの構造の進行性の障害をモデル化することで、他の神経変性疾患に関する貴重な洞察を得ることができます。実験的および対側対照眼の両方のベースライン眼圧(IOP)を測定するには、麻酔をかけたラットをベンチトップの腹側褥瘡の位置に置き、角膜表面が眼圧計の先端に簡単にアクセスできるようにします。.眼圧計の先端が角膜中央部に垂直にわずかに接触するように配置します。次に、測定ボタンを押して測定値を取得します。次に、実体顕微鏡下でラットをわずかに側方褥瘡の位置に置き、40倍の倍率で実験眼を検査します。処置中は、カルメロースナトリウムまたはヒアルロン酸ナトリウムを一滴点滴注入して、反対側のコントロールアイを潤滑状態に保ちます。次に、湾曲した鉗子を使用して、実験眼球をそっと前方に押して、辺縁部の360度を取り巻く血管系を露出させます。次に、もう一方の手で、低温の眼科用焼灼を使用して、角膜の周りの血管全体を優しく焼灼します。角膜末梢を焼灼しないように注意してください。強膜辺縁部に焼灼の小さな円形の痕跡が現れ、辺縁血管系が抹消され、手術眼の瞳孔が散大していることを観察して、外科的処置の成功を確認します。手術後、先に示したように、両眼の術後直後の眼圧を確認します。次に、眼科用酢酸プレドニゾロンを実験眼の前面に一滴塗布します。40秒後、眼科用抗生物質軟膏と交換します。非ステロイド性抗炎症薬と抗生物質軟膏で構成される局所薬による実験眼の毎日の臨床フォローアップを適用します。視運動反応解析では、4台のコンピューターモニターを四角形に並べたプラットフォームに、馴化用のラットを置きます。赤い十字カーソルは、仮想円柱の中心を示すため、自由に動くネズミの目の間のビデオフレームに保持します。回転するグレーディングによって誘発されるラットの頭頸部の反射的追跡からなる視運動反応を観察します。最初に、空間周波数の低い刺激を導入し、その後、トラッキングの動きに気づかなくなるまで周波数を徐々に上げます。パターン網膜電図を記録するには、ラットに麻酔をかけた後、角膜の側頭末梢に活性電極を慎重に挿入する。さらに、参照電極を同側側頭骨の皮下組織に挿入し、接地電極を後肢の1つに挿入します。次に、ラットを刺激スクリーンから20cmの位置に配置し、信号ベースラインをモニターし、アクイジションシステムのAnalysisボタンを押してアクイジションを開始します。網膜を単離した後、網膜内を上向きにした状態で、1ミリリットルのPBSを含む24ウェル培養プレートに網膜を移します。PBSで希釈した0.5%非イオン性界面活性剤で洗浄することにより、組織を透過処理します。次に、組織とブロッキング溶液を室温で穏やかに振とうしながら1時間インキュベートします。インキュベーション中に、Brn-3a一次抗体溶液を調製し、摂氏4度で保存します。組織ブロッキング後、網膜を200マイクロリットルの一次抗体溶液中で摂氏4度で72時間、穏やかに振とうしながらインキュベートします。次に、組織をPBSで洗浄します。200マイクロリットルの二次抗体溶液中で組織を室温で2時間インキュベートし、次いで核カウンター染色液で10分間インキュベートして蛍光核染色を行います。PBSを3回洗浄した後、硝子体側を上にしたまま、2つの小さなブラシを使用して網膜をガラス顕微鏡スライドに移します。網膜背側象限を顕微鏡スライド上に配置します。最後に、200マイクロリットルの褪色防止剤をガラスカバースリップに塗布し、平らに取り付けられた網膜に置いて顕微鏡分析を行います。共焦点落射蛍光顕微鏡下で、40倍の拡大対物レンズを使用して、網膜神経節細胞を推定するためにフラットマウントを調べます。眼球摘出術後、眼内部分の一部を含む視神経の眼窩内部分の近位部分を切除する。次に、サンプルを200〜300マイクロリットルの低温固定剤を含むバイアルまたはチューブにすぐに入れます。2時間後、冷たい100マイクロモルのカコジル酸ナトリウム緩衝液で組織を洗浄します。次に、1%四酸化オスミウムと5ナノモルの塩化カルシウムに4°Cで1時間、穏やかに振とうしながら組織を固定します。冷たいカコジル酸ナトリウム緩衝液で3回洗浄した後、視神経断片を冷たい蒸留水で洗浄してから、摂氏4度で一晩、尿中の酢酸1%をインキュベートします。翌日、水で3回洗浄した後、アセトン濃度を上げて組織を徐々に脱水します。次に、組織を純粋なエポキシ樹脂に包埋する前に、包埋とエポキシ樹脂アセトン溶液を3回行い、24時間行います。翌日、試料担体からサンプルを取り出し、60°Cで48時間包埋型に移します。重合後、ウルトラミクロトームを使用して、視神経片の横方向の半薄切片を切断します。次に、切片を採取し、顕微鏡スライドガラスに転写します。切片をトルイジンブルーで染色し、光学顕微鏡で100倍の倍率で画像化します。超微細構造解析では、極薄断面を実行し、銅グリッド上に収集します。次に、便器の酢酸とクエン酸鉛で切片を染色し、透過型電子顕微鏡検査を行います。全周焼灼は、対照眼と比較して、手術直後に眼圧上昇を誘発した。術後眼圧のピークは1日目に観察され、6日目にベースラインレベルまで徐々に減少しました。さらに、網膜機能は、それぞれ視運動反射とパターン網膜電図を使用して行動学的および電気生理学的に評価されました。手術後、両方のパラメータは、3日目の高眼圧急性期と30日目の二次変性期という2つの異なる段階の障害を示しました。対照視神経と比較して、軸索数および半薄の横視神経切片は手術後に徐々に減少した。対照眼からの視神経の透過型電子顕微鏡写真は、薄いグリア細胞突起と明らかな軸索微小管およびニューロフィラメントによって分離された密集した有髄線維を示しました。対照的に、高眼圧症の3日後には、いくつかの変性線維、グリア細胞プロセスにおける細胞質空胞化、およびグリア細胞核における凝縮クロマチンが観察された。7日後、変性軸索線維および高張性グリア細胞突起が増加した。そして14日後、軸索間のグリア細胞突起の侵入に関連する視神経線維のさらなる乱れが観察された。さらに、網膜神経節細胞の密度は、主に背側および側頭網膜象限において経時的に減少した。この手順を実行している間、焼灼の先端で辺縁血管にそっと触れるようにし、臍帯の周辺を温存し、強膜の辺縁全体に継続的な焼灼の跡が観察されるようにします。この手順に続いて、眼構造の機能的生化学的および免疫組織化学的評価、および緑内障の生理病理学を認める可能性のある他の方法など、さまざまな方法を適用できます。

full-circle-cauterization-limbal-vascular-plexus-for-surgically

Research

JoVE Journal

Neuroscience

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

げっ歯類で心的外傷後てんかん原のアンダーカットモデルを準備

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

神経科学

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

齧歯類のために全体の動物灌流固定

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

動作げっ歯類の可動シリコンプローブによるニューロンの大規模なレコーディング

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

げっ歯類における行動聴力検査のための低コストのセットアップ

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

げっ歯類におけるランニングホイール活動の概日リズムの記録および分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

げっ歯類での同時のfMRIと脳深部刺激

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

HIV誘導神経炎症のマウスモデルでは二光子顕微鏡を用いた脳血管のアーキテクチャの定量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

齧歯動物の槽注入カニューレ

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

頭蓋内薬物療法と痛みの試金の齧歯動物

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.