The authors would like to acknowledge Jonathan Chou, MD for his assistance on preparation and editing of the final manuscript and Wenzhong Liu for the OCT data. We would also like to acknowledge support from the Macula Society Research Grant (AAF), support from an unrestricted grant to Northwestern University from Research to Prevent Blindness, Inc., New York, NY, USA, and support from NIH-EY019951.

Todos os animais são tratados de acordo com o Guia de Cuidados e Uso de Animais de Laboratório 2013 Edition, da Associação de Pesquisa em Visão e Oftalmologia (ARVO) Declaração para o uso de animais em Oftalmologia e Vision Research, e conforme aprovado pelo animal Institucional Cuidado e Utilização Comité para a Universidade de Northwestern. Nota: O procedimento que se segue pode ser feito inteiramente com um operador; No entanto, é muito mais eficientemente conduzida com dois operadores com as tarefas dividido em conformidade. 1. Prepare a Estação do laser do laser e pré- <ol><li> Posicione o laser e lâmpada de fenda, onde ele pode ser facilmente acessível. Ligue a laser e definir os parâmetros pré-determinados (por exemplo, 75 mm de tamanho local, poder mW 100, 100 duração ms). CUIDADO: Certifique-se de operador usa todos os equipamentos de protecção individual dos animais e segurança laser e laser de segurança pertinentes sinais são exibidos fora da sala de procedimento. <ol><li> Antes de usar qualquer animais experimentais, encontrar parâmetros ideais usando uma calibração, rato não-experimental. Os parâmetros do laser irá depender do laser utilizado. Parâmetros ideais são definidos pela configuração de energia menor do laser que constantemente provoca a formação de &quot;bolha&quot; quando focada corretamente. Ver vídeo, por exemplo de lesão com adequada &quot;bolha&quot; de formação. </li></ol></li><li> Prepare a estação de pré-laser de modo que a anestesia, animal mais quente, lenços de tecido, e todos os colírios (tropicamida, Tetracaína, e lágrimas artificiais) são facilmente ao nosso alcance para o operador. <ol><li> Assegure-se que animais mais quente é pré-aquecida para corrigir a temperatura (37 ° C) antes de injectar o anestésico em primeiro ratinho, a fim de evitar a hipotermia induzida pela anestesia. </li></ol></li><li> Coloque fase do mouse sobre mais quente para que ele possa reter o calor e permanecer quente uma vez procedimento a laser começa. </li></ol> 2. Rato Anestesia e Preparação Pré-a laser <ol><li> Antes de injectaring anestesia, inspecionar o olho macroscopicamente para garantir que ele não tem deformidades ou anormalidades que diminuem a clareza da córnea (por exemplo, catarata). </li><li> Pesar mouse. </li><li> Peso Record, sexo e número de identificação animal. </li><li> Usando peso, calcular o montante adequado de anestesia a ser usado com base nas orientações dadas pela instituição (por exemplo, 100 mg / kg de cloridrato de cetamina, 10 mg / kg de xilazina OU tribromoetanol 250 mg / kg; para a 20 g rato injectar 0,20 ml de xilazina / cetamina cocktail OU 0,25 ml de tribromoetanol). Tem uma tabela de dosagens pré-calculada por peso em incrementos de 1 g, a fim de reduzir os erros matemáticos. </li><li> Scruff do mouse e injetar anestésico por via intraperitoneal com base em cálculos na etapa 2.4. </li><li> Coloque o mouse sobre os animais mais quente e esperar até que o mouse é completamente anestesiados, verificando o reflexo pedal via pitada dedo do pé (cerca de 3-5 min). </li><li> Arregaçar um lenço de papel e proteger a área nasal do mouse para evitar aspiration de líquido roll-off. Rato do rolo no seu lado e colocar uma gota (aproximadamente 30 ul) de cloridrato de tetracaína em cada olho para a anestesia tópica. Aguarde 2 min para solução tenha efeito. </li><li> Repita o passo 2.7 com uma gota de Tropicamida tópico para dilatação pupilar. Como alternativa, use o cloridrato de fenilefrina (2,5%) para a dilatação. </li><li> Aguarde 2 min de soluções para surtir efeito; manter animais em mais quente durante este tempo. </li><li> Após o tempo adequado tenha decorrido, rapidamente colocar o mouse sobre o palco do mouse e coloque o estágio no queixo resto da lâmpada de fenda. </li><li> Ligue lâmpada de fenda para o menor brilho luz e verificar o grau de dilatação pupilar. Se pupila dilatada não é adequadamente (aproximadamente 2,5-3 mm), retorne mouse para animais mais quente e esperar. Como alternativa, administrar uma gota de Tropicamida. Uma vez que olho é suficientemente dilatado, avance para o procedimento a laser. </li></ol> 3. Laser Procedimento Nota: Certifique-se de outra ps pessoas na sala de usar óculos de proteção quando está longe de lâmpada de fenda olho peça protegida por laser <ol><li> Ajustar o posicionamento do mouse sobre o estágio do mouse, de modo que ele está na posição ideal para a visualização de nervo óptico (ver 3.1.2). <ol><li> Orientar o mouse ao seu titular para que ele fique na horizontal, perpendicular à fenda feixe de luz, com a cabeça de um lado e cauda na outra. Posicionamento ideal do rato fará a visualização do nervo óptico muito mais fácil uma vez tampa de deslizamento é aplicada. </li><li> Ligeiramente vire o mouse por isso é em um ângulo de 170 ° ~ com a cabeça mais perto do operador de laser. </li><li> Certifique-se de que o mouse é o mais próximo possível para lâmpada de fenda, mas ainda em uma posição onde ele é estável e onde a mão do operador terá espaço suficiente para manipulação fina. </li></ol></li><li> Após o ratinho está idealmente posicionado, colocar uma gota da solução de lágrima artificial em 25 mm x 25 mm lamela de vidro. <ol><li> Coloque uma gota de solução de lágrima artificial em opp de ratoolho osite - este olho irá garantir é hidratada e ajuda a formação de atraso de catarata. </li></ol></li><li> Segure esquina da lamela entre o polegar eo ponteiro dedos; posição, de modo que o vidro é apertado entre as pontas de dois dedos. </li><li> Suavemente embrulhar os restantes três dedos ao redor do corpo do animal para o apoio ea estabilização mão. Posição da mão de modo que a lamela de vidro pode ser facilmente colocado no olho do mouse. <ol><li> Certifique-se de que o pulso está estabilizada em uma superfície firme, a fim de reduzir o tremor da mão. </li></ol></li><li> Uma vez que posição estável é obtido, pressione cuidadosamente lamela de vidro (com gota de lágrima artificial ainda aderido) sobre o olho do mouse. <ol><li> Certifique-se a lamela estiver posicionado o mais perpendicular possível para o feixe de laser, a fim de evitar a dispersão do feixe de laser ou reflexão. A lamela age como uma lente de contacto para aplanar a córnea. </li></ol></li><li> Olhe através de lâmpada de fenda e com a mão livre de alternância concentrar untIL retina pode ser visualizado. A retina vai ter uma cor amarelo claro / vermelho, dependendo da localização visualizado, vasos distintos, vermelho será visível. </li><li> Devagar e com cuidado manipular cabeça de rato e / ou lamela até visualizar o nervo óptico. O nervo óptico será de cor amarela com múltiplos vasos que irradiam a partir dele. </li><li> Uma vez operador confirmou visualização do nervo óptico, ligue a laser com foco feixe. </li><li> Uma vez feixe de laser ter sido ligado, manobrar a laser de focagem do feixe para a posição desejada (aproximadamente 1 diâmetro do disco do nervo óptico). </li><li> Concentre-se feixe de laser sobre o RPE do fundo de olho. A focagem adequada é por ter conseguido o feixe de laser mais nítida e clara. Se visando feixe olha oval ou fora de foco, alternar o foco lâmpada de fenda ou re-posição lamela de vidro. </li><li> Uma vez que o feixe de mira está focada em RPE, iniciar administração laser utilizando gatilho pé do laser. <ol><li> Certifique-se de evitar vasos da retina para evitar que ele intraocularmorrhage. </li></ol></li><li> Prestar atenção para o aparecimento de uma bolha imediatamente após a administração do laser. O esboço do tiro de laser deve ser clara e não nebulosa de qualquer forma. <ol><li> Se o tiro de laser não resulta na formação de bolhas, ou área de impacto parece nebuloso (aparência turva com borda circular mal definido vs. fronteira clara, bem definida de um impacto positivo), ou se a hemorragia é visto após a administração de laser, não incluir essas lesões para análise futura. </li></ol></li><li> Repita os passos de 3,10-3,12 para todas as posições desejadas CNV (geralmente aos 3, 6, 9 e 12 posições ao redor do nervo óptico). Se induções a laser são aplicados em aproximadamente mesma distância do nervo óptico, refocus não deve ser necessário. No entanto, devido à forte curvatura do olho do rato e pequenas variações que podem existir na retina, a recentragem do feixe pode ser necessário entre administrações consecutivas de laser. </li><li> Registro em um notebook a localização e resultado de eadministração tiro a laser e ach resultado (sucesso, obscuro, hemorragia, etc.) de cada tiro administrados para o olho. Certifique-se de colocar o laser no modo stand-by quando não estiver em uso. </li><li> 3,1-3,14 Repetir para o outro olho do rato, se necessário, usando a outra mão para a estabilização e uma nova lamela. </li><li> Depois de todos os disparos de laser desejados são administrados, desligue a laser e lâmpada de fenda. </li><li> Descarte lamela e lugar do mouse sobre mais quente para a recuperação da anestesia. Macroscopicamente inspecionar olho por qualquer prejuízo e coloque uma gota de solução de lágrima artificial para manter o olho hidratado e potencialmente prevenir o desenvolvimento de catarata futuro. Uma vez que o mouse se recupera da anestesia, voltar a gaiola. </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td></td><td> AVERTIN </td><td> AVERTIN </td><td> AVERTIN </td><td> XYL / KET </td><td> XYL / KET </td></tr><tr><td> Peso do rato (g) </td><td> Dose (mg / kg) </td><td&gt; Concentração da Solução (mg / ml) </td><td> Anestésico Dose (ml) </td><td> Dose (mg / kg) </td><td> Anestésico Dose (ml) </td></tr><tr><td> 15 </td><td> 250 </td><td> 20 </td><td> 0,1875 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,15 </td></tr><tr><td> 16 </td><td> 250 </td><td> 20 </td><td> 0,2 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,16 </td></tr><tr><td> 17 </td><td> 250 </td><td> 20 </td><td> 0,2125 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,17 </td></tr><tr><td> 18 </td><td> 250 </td><td> 20 </td><td> 0,225 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,18 </td></tr><tr><td> 19 </td><td> 250 </td><td> 20 </td><td> 0,2375 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,19 </td></tr><tr><td> 20 </td><td> 250 </td><td> 20 </td><td> 0,25</td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,2 </td></tr><tr><td> 21 </td><td> 250 </td><td> 20 </td><td> 0,2625 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,21 </td></tr><tr><td> 22 </td><td> 250 </td><td> 20 </td><td> 0,275 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,22 </td></tr><tr><td> 23 </td><td> 250 </td><td> 20 </td><td> 0,2875 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,23 </td></tr><tr><td> 24 </td><td> 250 </td><td> 20 </td><td> 0,3 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,24 </td></tr><tr><td> 25 </td><td> 250 </td><td> 20 </td><td> 0,3125 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,25 </td></tr><tr><td> 26 </td><td> 250 </td><td> 20 </td><td> 0,325 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,26 </td></tr><tr><td> 27 </td><td> 250 </td><td&gt; 20 </td><td> 0,3375 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,27 </td></tr><tr><td> 28 </td><td> 250 </td><td> 20 </td><td> 0,35 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,28 </td></tr><tr><td> 29 </td><td> 250 </td><td> 20 </td><td> 0,3625 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,29 </td></tr><tr><td> 30 </td><td> 250 </td><td> 20 </td><td> 0,375 </td><td> 100 mg / kg de cetamina; 10 mg / kg de xilazina </td><td> 0,3 </td></tr></tbody></table> Tabela 1: Dose XyIKet Chart.

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The authors declare they have no competing financial interests.

Existem vários fatores que podem afetar a entrega do laser e do desenvolvimento resultante da lesão CNV após laser de indução bem-sucedida. Estes factores devem ser controlados e padronizados para a fim de ter os resultados mais fiáveis. O mais pertinente desses fatores são a seleção do mouse (genótipo, idade e sexo), a seleção anestésico, e as configurações de laser. O modelo de mouse específico usado pode ter um efeito significativo sobre o curso do desenvolvimento CNV. O g...

Degeneração macular (AMD) relacionada com a idade é uma das principais causas de cegueira em indivíduos com idade superior a 50 3/1. AMD podem ser classificados em duas formas: atrófica (&quot;secar&quot;) e AMD neovascular (&quot;wet&quot;) AMD. O primeiro é caracterizada por atrofia geográfica do epitélio pigmentado da retina (RPE), coriocapilar, e fotorreceptores, enquanto o último é caracterizada pela invasão de vasos sanguíneos anormais a partir do coróide para as camadas externas retinianas provocando fugas, hemorragia, e fibrose, e, finalmente levando a cegueira 1,2. Das duas formas, DMRI neovascular representa a maioria da perda de visão 1. Felizmente, essa forma tem inúmeras opções eficazes de gestão farmacológicas, ao passo que o seu homólogo atrófica não tem comprovada tratamentos médicos 3. Além disso, porque a forma neovascular foi facilmente re-capituló num modelo animal, que tem sido mais amplamente acessível a um básicaPesquisa MD explorar os mecanismos patológicos subjacentes, a fim de desenvolver novas terapias 4. O primeiro modelo animal experimental de neovascularização coroidal (CNV) foi desenvolvido por Ryan et al. em primatas não humanos 5. Esta ruptura induzida modelo de membrana de Bruch através fotocoagulação por laser, o que provocou uma resposta inflamatória local na angiogénese resultante semelhante ao observado em DMRI neovascular. A progressão histopatológico de angiogênese indução de pós-laser foi encontrado para imitar neovascular AMD, que confirmou a validade do modelo 6. Os primatas não humanos oferecem a anatomia mais semelhantes aos seres humanos, mas, infelizmente, são caros de manter, não pode ser facilmente manipulado geneticamente, e tem um curso lento tempo de progressão da doença 7. Em contraste, os modelos de roedores são muito mais para manter a relação custo-eficácia, pode ser geneticamente manipulados com relativa facilidade, e têm um muito mais rápido course de progressão da doença (experiências pode ser conduzida numa escala de tempo de semanas contra meses). Estas experiências só devem ser realizados em roedores pigmentadas, uma vez que é muito difícil de visualizar em animais albinos. O modelo CNV induzido por laser do mouse, primeiro desenvolvido pelo grupo Campochiaro no final dos anos 90, 10, tem crescido a ser o modelo animal dominante na maioria dos estudos recentes 11-16. Devido à patogénese complexo e ainda não está claro da CNV, o modelo de laser foi aplicado em todos os aspectos da investigação AMD molhado variando de estudar os mecanismos moleculares que conduzem a angiogénese para avaliar novas modalidades de tratamento para futuro uso humano. Por exemplo, Sakurai et al. e Espinosa-Heidmann et ai. utilizado o modelo de laser para investigar o efeito de macrófagos no desenvolvimento de CNV utilizando ratinhos transgénicos e tratamentos farmacológicos exaustão 15, 16. Giani et ai. e Hoerster et ai. usado óptico de tomografia de coerência (OCT) para a imagem da CNV em um esforço para caracterizar a progressão da CNV e comparar os achados histopatológicos com os resultados vistos em outubro imagem 12,17 induzida por laser. Finalmente, estudos envolvendo injeção intravítrea de agentes anti-angiogênicos têm sido usados ​​como pré-requisitos para testes em humanos e foram vitais no desenvolvimento da primeira geração de agentes anti-VEGF utilizados na gestão de DMRI neovascular hoje 10,18,19. Modelos alternativos para CNV experimental utilizam métodos cirúrgicos para induzir a CNV. Este procedimento envolve a injecção de substâncias pró-angiogénicas (por exemplo, vectores virais recombinantes que sobre-expressam o VEGF, a injecção sub-retiniana de células EPR e / ou pérolas de poliestireno) para simular o aumento da expressão de VEGF na DMRI neovascular visto, com o objectivo de causar a angiogénese 8,20. No entanto, este método produz uma incidência drasticamente menor de neovascularização; estes estudos mostraram que na CNVC57 / BL6 ratos ocorre em 31% de injecções de 70% contra a taxa de sucesso ~ visto no método de fotocoagulação com laser na mesma estirpe de ratinhos 8,14. Por estas razões, e tendo em conta as vantagens do uso de roedores contra primatas não-humanos, o modelo de ratinho de CNV induzido por laser tornou-se o modelo animal padrão de CNV para a maioria experiências de estudo AMD neovascular 8. O olho do rato é um tecido minúsculo, delicado para trabalhar. Manobras do olho de visualizar a retina é difícil e requer muito prática até que seja alcançado o domínio. Esta tarefa é complicada pelo facto de que deve ser aprendido com a mão dominante e não-dominante. Além disso, após os movimentos finos necessários para visualizar a retina foram aprendidas, a coordenação entre ambas as mãos ea pedal operar o laser são importantes. Neste trabalho, buscou-se destilar os desafios de aprender todas as manipulações físicas envolvidas no CNV proc induzida por laseredure em um guia que iria ajudar as operadoras a alcançar o sucesso rápido com este modelo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>532 nm (green) argon ophthalmic laser</td>
			<td>IRIDEX</td>
			<td>GLx</td>
			<td>any ophthalmic 532 nm (green) argon laser can be used</td>
		</tr>
		<tr>
			<td>slit lamp</td>
			<td>Carl Zeiss</td>
			<td>30SL-M</td>
			<td>any slit lamp can be used as long as it is compatible with the laser</td>
		</tr>
		<tr>
			<td>tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>tert-amyl alcohol</td>
			<td>Sigma</td>
			<td>152463-1L</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>amber glass vials + septa</td>
			<td>Wheaton</td>
			<td>WH-223696</td>
			<td>tribromoethanol storage</td>
		</tr>
		<tr>
			<td>tissue wipes</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td>miscellaneous&#160;</td>
		</tr>
		<tr>
			<td>1% tropicamide</td>
			<td>Falcon Pharmaceuticals</td>
			<td>RXD2974251</td>
			<td>pupillary dilation</td>
		</tr>
		<tr>
			<td>0.5% tetracaine hydrochloride</td>
			<td>Alcon</td>
			<td>&#160;0065-0741-12</td>
			<td>topical anesthesia</td>
		</tr>
		<tr>
			<td>artificial tears</td>
			<td>Alcon</td>
			<td>&#160;58768-788-25</td>
			<td>hydration</td>
		</tr>
		<tr>
			<td>heat therapy pump (for animal warming)</td>
			<td>Kent Scientific</td>
			<td>HTP-1500</td>
			<td>used to maintain animal body temp</td>
		</tr>
		<tr>
			<td>warming pad</td>
			<td>Kent Scientific</td>
			<td>TPZ-0510EA</td>
			<td>maintains animal body temperature</td>
		</tr>
		<tr>
			<td>30 gauge insulin needles</td>
			<td>BD</td>
			<td>328418</td>
			<td>IP anesthesia injection</td>
		</tr>
		<tr>
			<td>scale</td>
			<td>American Weigh Scale</td>
			<td>AWS-1KG-BLK</td>
			<td>mouse weighing</td>
		</tr>
		<tr>
			<td>cover slip (25 mm x 25 mm)</td>
			<td>VWR</td>
			<td>48366089</td>
			<td>flatten cornea to visualize mouse retina</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>used for volumetric re-construction</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for image analysis</td>
		</tr>
	</tbody>
</table>

a mouse model for laser-induced choroidal neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Quantificação de lesões de NVC pode ser realizado através da análise da coróide planas montadas usando coloração por imunofluorescência para rotular os vasos NVC. Os dois métodos mais utilizados de preparação do tecido são rotulagem FITC-dextrano, feito através de perfusão imediatamente antes do sacrifício animal, ou post-mortem imuno-coloração com um marcador de células endoteliais. Ambos os métodos foram descritos anteriormente em detalhe 13,14,21; figuras 1 e 2<...

Watch this Scientific Journal Video about A Mouse Model for Laser-induced Choroidal Neovascularization at JoVE.com

A Mouse Model for Laser-induced Choroidal Neovascularization

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Um modelo de rato para induzida por laser coróide Neovascularização

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

a-mouse-model-for-laser-induced-choroidal-neovascularization

Research

JoVE Journal

Medicine

17.4K visualizações.  Northwestern University Feinberg School of Medicine. Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Vídeo: A Mouse Model for Laser-induced Choroidal Neovascularization

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Um modelo de rato induzida por laser de crônica Ocular Hypertension para Caracterizar defeitos visuais

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medicina

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Detectar alterações no coróide Vasculature em um modelo do rato de Degeneração Macular relacionada com a Idade de-curso Tempo Indocianina Verde Angiografia

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Um ferimento Modelo Alkali queima de córnea neovascularização no Rato

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Um modelo de pele Rato Fetal de reparação de feridas Scarless

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

A Neonatal do rato Spinal Cord Injury Compression Modelo

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

Induzida por punção neovascularização de Iris como um modelo do rato da Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Uma nova abordagem para o monitoramento de neovascularização de enxerto na gengiva humana

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Um modelo de rato para pancreatite crônica através da infusão TNBS do ducto biliar

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Lesões semi-automatizadas semi-automatizadas controladas por laser para estudar a regeneração da medula espinhal em larvas de zebrafish

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

Methods Article

Um modelo de rato para induzida por laser coróide Neovascularização