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

Agradecemos aos nossos técnicos de laboratório José; Nilson dos Santos, Daianne Mandarino Torres, José Francisco Tibúrcio, Gildo Brito de Souza e Luciano Cavalcante Ferreira. Pesquisa financiada pela FAPERJ, CNPq e CAPES.

Todos os procedimentos foram realizados de acordo com a Declaração para o Uso de Animais em Pesquisa Oftalmológica e Visual da Association for Research in Vision and Ophthalmology (ARVO) e aprovada pelo Comitê de Ética no Uso de Animais em Experimentação Científica do Centro de Ciências da Saúde da Universidade Federal do Rio de Janeiro (protocolo 083/17). No presente trabalho, foram utilizados ratos Lister Hooded, de ambos os sexos, com idade entre 2-3 meses e peso de 180-320 g. No entanto, o procedimento pode...

<ol>
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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. 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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. 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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. 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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=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. 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A cauterização do plexo limbal (LPC) é um novo modelo pós-trabecular com a vantagem de ter como alvo estruturas vasculares de fácil acesso, não necessitando de dissecção conjuntival outenonal 17,28. Diferentemente do modelo de cauterização de veias vórtices, um renomado modelo de OHT baseado no comprometimento cirúrgico da drenagem venosa coroide, não se espera que a congestão venosa influencie a elevação da PIO no modelo LPC, uma vez que as veias...

A literatura médica entende o glaucoma como um grupo heterogêneo de neuropatias ópticas caracterizadas por degeneração progressiva das células ganglionares da retina (CGRs), dendritos, soma e axônios, resultando em escavação estrutural (escavação) do disco óptico e deterioração funcional do nervo óptico, levando à amaurose em casos não controlados pela interrupção da transmissão de informações visuais do olho para océrebro1. Atualmente, o glaucoma é a causa mais comum de cegueira irreversível em todo o mundo, com previsão de atingir aproximadamente 111,8 milhões de pessoas em 20402, afetando profundamente a qualidade de vida (QV) dos pacientes e levando a importantes preocupações socioeconômicas3.
A pressão intraocular (PIO) elevada é um dos mais importantes e o único fator de risco modificável para o desenvolvimento e progressão do glaucoma. Dentre os múltiplos tipos de glaucoma, todos, exceto o glaucoma de tensão normal (GNT), estão associados à PIO elevada em algum momento da história clínica da doença. Apesar dos notáveis avanços clínicos e cirúrgicos para atingir a PIO e retardar ou interromper a progressão da doença, os pacientes ainda perdem a visão devido ao glaucoma 4,5. Portanto, o conhecimento aprofundado da fisiopatologia complexa e multifatorial dessa doença é imperativo para o desenvolvimento de tratamentos mais eficazes, especialmente para fornecer neuroproteção aos CGRs.
Entre uma variedade de abordagens experimentais para a compreensão dos mecanismos da doença, os modelos animais baseados em hipertensão ocular (OHT) mais se assemelham ao glaucoma humano. Os modelos de roedores são particularmente úteis por serem de baixo custo, fácil manuseio, manipulação genética, vida útil curta e características anatômicas e fisiológicas oculares comparáveis aos humanos, como produção e drenagem do humor aquoso 6,7,8,9,10,11,12,13 . Os modelos atualmente utilizados incluem esclerose da malha trabecular após injeção de solução salina hipertônica em veias episclerais 14, injeção intracameral de microesferas15 ou substâncias viscoelásticas 16, cauterização de veias vórtices 17, fotocoagulação da tela trabecular com laser de argônio18, sutura circunlimbal 19 e uso de um modelo transgênico de OHT relacionada à idade (camundongos DBA/2J)8. No entanto, invasividade, opacificação pós-operatória da córnea, ruptura do segmento anterior, curvas de aprendizado extensas, equipamentos caros e PIO pós-operatória altamente variável estão entre algumas das armadilhas relatadas associadas aos modelos atuais, tornando o desenvolvimento de um modelo alternativo de ESB uma demanda para superar esses problemas20,21,22.
O presente protocolo formaliza um novo procedimento cirúrgico para induzir OHT como substituto do glaucoma, baseado na cauterização do plexo limbal (LPC) emroedores23. Trata-se de um modelo fácil, reprodutível, acessível e não invasivo, que proporciona alta eficiência e baixa variabilidade de elevação da PIO, associado a uma taxa excepcionalmente alta de recuperação clínica completa, proporcionando avaliação funcional in vivo em um número reduzido de animais utilizados em cada experimento. A técnica cirúrgica induz OHT subaguda com retorno gradual aos níveis basais em poucos dias, o que modela o ataque hipertensivo observado no glaucoma agudo de ângulo fechado. Além disso, a recuperação da PIO no modelo é seguida por neurodegeneração glaucomatosa contínua, o que é útil para futuros estudos mecanísticos da degeneração secundária dos CGRs, que ocorre em vários casos de glaucoma humano apesar do controle adequado da PIO.

<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>
		</tr>
		<tr>
			<td>Neuropack Manager v08.33</td>
			<td>Nihon Kohden Corporation</td>
			<td>-</td>
			<td>Software for electroretinogram signal processing</td>
		</tr>
		<tr>
			<td>Optomotry</td>
			<td>CerebralMechanics</td>
			<td>-</td>
			<td>System for optomotor response analysis</td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Electron Microscopy Sciences</td>
			<td>19100</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide</td>
			<td>Electron Microscopy Sciences</td>
			<td>20150</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr>
			<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

O glaucoma, segunda causa de cegueira no mundo, é um grupo heterogêneo de doenças oculares caracterizadas por danos estruturais ao nervo óptico e degeneração das células ganglionares da retina (CGR), resultando em disfunção visual pela interrupção da transmissão de informações visuais do olho para o cérebro. A pressão intraocular elevada é o fator de risco mais importante; Assim, vários modelos de hipertensão ocular têm sido desenvolvidos em roedores por abordagens genéticas ou experimentais para investigar as causas e efeitos da doença. Dentre essas, algumas limitações têm sido relatadas, como invasividade cirúrgica, avaliação funcional inadequada, exigência de treinamento extensivo e extensão altamente variável do dano retiniano. O presente trabalho caracteriza um método simples, de baixo custo e eficiente para induzir hipertensão ocular em roedores, baseado na cauterização em círculo completo do plexo limbal a baixa temperatura, um dos principais componentes da drenagem aquosa do humor. O novo modelo proporciona uma hipertensão ocular subaguda tecnicamente fácil, não invasiva e reprodutível, associada à degeneração progressiva do CGR e do nervo óptico, e uma taxa de recuperação clínica pós-operatória única que permite estudos funcionais in vivo por métodos eletrofisiológicos e comportamentais.

As variáveis quantitativas foram expressas como média ± erro padrão da média (EPM). Com exceção da comparação da dinâmica da PIO entre os grupos ESB e controle (Figura 1F), a análise estatística foi realizada por meio de ANOVA two-way seguida do teste de comparações múltiplas de Sidak. Um valor de p < 0,05 foi considerado estatisticamente significativo.
A Figura 1 ilustra os passos cirúrgicos do modelo de cauterizaçã...

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

O objetivo deste protocolo é caracterizar um novo modelo de neurodegeneração glaucomatosa baseado na cauterização térmica 360° do plexo vascular limbal, induzindo hipertensão ocular subaguda.

Cauterização em círculo completo do plexo vascular limbal no glaucoma induzido cirurgicamente em roedores

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

Desenvolvemos um método simples para induzir degeneração robusta e progressiva do glaucoma baseado na elevação temporária da pressão intraocular. Isso representa uma oportunidade única para estudar pontos-chave da fisiopatologia do glaucoma. Trata-se de um modelo de fácil aprendizado, não invasivo, de baixo custo e altamente reprodutível, que permite a análise in vivo da função e o planejamento experimental de curto prazo utilizando um número reduzido de animais para cada experimento.A retina e o nervo óptico são partes avaliáveis do sistema nervoso central, de modo que a modelagem do comprometimento progressivo dessas estruturas pode fornecer informações valiosas sobre outras doenças neurodegenerativas. Para medir a pressão intraocular basal, ou PIO, dos olhos controles experimentais e contralaterais, coloque o rato anestesiado em decúbito ventral na bancada, de modo que a superfície corneana seja facilmente acessível à ponta do tonômetro. Posicione a ponta do tonômetro de forma que ela toque levemente a zona corneana central perpendicularmente.Em seguida, adquira a medição pressionando o botão de medição. Em seguida, colocar o rato em leve decúbito lateral sob estereomicroscópio e inspecionar o olho experimental em aumento de 40 vezes. Durante o procedimento, mantenha o olho de controle contralateral lubrificado instilando uma gota de carmelose sódica ou hialuronato de sódio.Em seguida, usando pinça curva, empurre suavemente o globo ocular experimental para frente para expor a vasculatura ao redor de 360 graus do limbo. Em seguida, com a outra mão, usando um cautério oftálmico de baixa temperatura, cauterizar suavemente os vasos ao redor da córnea. Cuidado para não cauterizar a periferia da córnea.Confirmar o sucesso do procedimento cirúrgico observando o surgimento de pequenas marcas circulares de cauterização no limbo escleral, a obliteração da vasculatura limbal e a dilatação pupilar no olho operado. Após a cirurgia, verificar a PIO no pós-operatório imediato em ambos os olhos, conforme demonstrado anteriormente. Em seguida, aplicar uma gota de acetato de prednisolona oftálmica na superfície anterior do olho experimental.Após 40 segundos, substitua-o por uma pomada antibiótica oftálmica. Aplicar um acompanhamento clínico diário do olho experimental com medicamentos tópicos compostos por um anti-inflamatório não esteroidal e pomada antibiótica. Para análise da resposta optomotora, colocar o rato para habituação em uma plataforma construída com quatro monitores de computador em um quadrângulo.Mantenha o cursor de cruz vermelha no quadro de vídeo entre os olhos do rato em movimento livre, pois indica o centro do cilindro virtual. Observar a resposta optomotora que consiste em um rastreamento reflexivo da cabeça e pescoço do rato provocado pelas graduações rotatórias. Inicialmente, introduzir um estímulo com baixa frequência espacial, em seguida, aumentar a frequência progressivamente até que o movimento de rastreamento não seja notado.Para registrar o eletrorretinograma padrão, após anestesiar o rato, insira cuidadosamente o eletrodo ativo na periferia temporal da córnea. Além disso, insira o eletrodo de referência no tecido subcutâneo do canto temporal ipsilateral e o eletrodo terra em um dos membros posteriores. Em seguida, posicione o rato a 20 centímetros da tela de estímulo, monitore a linha de base do sinal e inicie a aquisição pressionando o botão Análise no sistema de aquisição.Após o isolamento das retinas, transferi-las para uma placa de cultura de 24 poços contendo PBS de um mililitro, mantendo a retina interna voltada para cima. Permeabilizar o tecido lavando com um tensoativo não iônico a 0,5% diluído em PBS. Em seguida, incubar o tecido e a solução de bloqueio por uma hora à temperatura ambiente com agitação suave.Durante a incubação, preparar a solução primária de anticorpos Brn-3a e armazenar a quatro graus Celsius. Após o bloqueio tecidual, incubar as retinas em 200 microlitros de solução de anticorpos primários a quatro graus Celsius por 72 horas com agitação suave. Em seguida, lave o lenço de papel com PBS.Incubar o tecido em 200 microlitros da solução de anticorpos secundários por duas horas à temperatura ambiente e, em seguida, com solução de contracoloração nuclear por 10 minutos para coloração de núcleos fluorescentes. Após três lavagens com PBS, transfira a retina para uma lâmina de microscópio de vidro usando duas pequenas escovas mantendo o lado vítreo para cima. Posicione o quadrante dorsal da retina para cima na lâmina do microscópio.Finalmente, aplique 200 microlitros de meio de montagem antifade em uma tampa de vidro e coloque-o na retina plana montada para análise microscópica. Sob um microscópio confocal de epifluorescência, usando uma objetiva de aumento de 40 vezes, examine as montagens planas para estimar as células ganglionares da retina. Após a enucleação do globo ocular, remover o segmento proximal da porção intraorbital do nervo óptico, incluindo parte da porção intraocular.Em seguida, coloque imediatamente as amostras em frascos ou tubos contendo 200 a 300 microlitros de fixador a frio. Após duas horas, lavar o tecido com tampão cacodilato de sódio 100 micromolar frio. Em seguida, pós-fixar o tecido por uma hora em tetróxido de ósmio a 1% e cloreto de cálcio nanomolar a quatro graus Celsius sob agitação suave.Após três lavagens com tampão cacodilato de sódio frio, lavar os fragmentos do nervo óptico com água destilada fria antes de incubar durante a noite e acetato de mictório a 1% a quatro graus Celsius. No dia seguinte, após três lavagens com água, desidratar progressivamente o tecido em concentrações crescentes de acetona. Em seguida, realizar três rodadas de incorporação e soluções de acetona de resina epóxi antes de incorporar o tecido em resina epóxi pura por 24 horas.No dia seguinte, retire as amostras do porta-amostras e transfira-as para moldes de incorporação por 48 horas a 60 graus Celsius. Após a polimerização, use um ultramicrótomo para cortar cortes transversais semifinos dos fragmentos do nervo óptico. Em seguida, colete e transfira as seções para uma lâmina de vidro do microscópio.Coloração dos cortes com azul de toluidina e obtenção de imagens em microscópio óptico com aumento de 100 vezes. Para análises ultra-estruturais, execute seções transversais ultrafinas e colete-as em uma grade de cobre. Em seguida, corar os cortes com acetato de mictório e citrato de chumbo para exame de microscopia eletrônica de transmissão.A cauterização completa induziu elevação da PIO imediatamente após a cirurgia em comparação com os olhos controles. O pico da PIO pós-operatória foi observado no primeiro dia e diminuiu gradualmente até os níveis basais no sexto dia. Além disso, a função retiniana foi avaliada comportamental e eletrofisiologicamente usando reflexo optomotor e eletrorretinograma padrão, respectivamente.Após a cirurgia, ambos os parâmetros mostraram duas fases distintas de comprometimento: hipertensão ocular fase aguda no terceiro dia e fase de degeneração secundária no 30º dia. Em comparação com os nervos ópticos de controle, as contagens axonais e as secções semifinas do nervo óptico transversal diminuíram progressivamente após a cirurgia. Micrografias eletrônicas de transmissão de nervos ópticos do olho controle demonstraram fibras mielinizadas densamente embaladas separadas por processos de células gliais finas e microtúbulos axonais e neurofilamentos evidentes.Em contraste, após três dias de hipertensão ocular, algumas fibras degeneradas, vacuolização citoplasmática em processos de células gliais e cromatina condensada em núcleos de células gliais foram observadas. Após sete dias, houve aumento das fibras axonais degeneradas e dos processos hipertônicos das células gliais. E após 14 dias, observou-se maior desarranjo das fibras do nervo óptico associado à invasão de processos celulares gliais entre os axônios.Além disso, a densidade de células ganglionares da retina diminuiu com o tempo, principalmente nos quadrantes dorsais e temporais da retina. Durante a realização deste procedimento, tente tocar suavemente os vasos límbicos com uma ponta de cautério, poupando a periferia do cordão e garantindo que marcas de cauterização contínua sejam observadas ao redor do limbo escleral. Após esse procedimento, uma variedade de métodos pode ser aplicada, como a avaliação bioquímica funcional e imuno-histoquímica das estruturas oculares e outros métodos que possam ser reconhecidos na fisiopatologia do glaucoma.

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.

Preparando Modelo de rebaixo de epileptogênese Pós-Traumático em Roedores

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

Neurociência

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.

Fixação de Perfusão todo animal para Roedores

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.

Em larga escala de gravação de Neurônios por sondas de silicone móveis em Comportando Roedores

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.

A configuração de Baixo Custo para audiometria comportamental em roedores

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.

Registro e análise dos ritmos circadianos em Running rodas Atividade em Roedores

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.

Estimulação Cerebral Profunda com fMRI simultâneo em Roedores

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.

Quantificação de Vascular Cerebral Arquitetura usando dois fótons Microscopia em um modelo de mouse Neuroinflammation induzida por HIV

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.

Cânula de implantação para a Cisterna Magna de roedores

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.

Farmacoterapia intracraniana e ensaios de dor em roedores

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.