Agradecemos aos muitos estudantes e companheiros que trabalharam sobre este modelo para melhorar a sua eficácia, e compreender seus mecanismos. Agradecimentos especiais são devidos a Dr de. Mary Johnson (Universidade de Maryland-Baltimore), Nitza Goldenberg-Cohen (Schneiderman Childrens Hospital, Petah-Tikva, Israel), Charles Zhang (Einstein Medical College, Bronx, NY), e Valerie Touitou (Hopital Salpetrie, Paris, França). Este estudo foi financiado em parte por RO1 EY015304 para SLB.

Este protocolo foi aprovado pela Universidade de Maryland Animal Care Institucional e Comitê de Utilização (IACUC; Baltimore, MD, EUA) 1. Experimental Set-up <ol><li> Faça um costume projetado lente de contato de um grau claro óptica circular 7 mm de diâmetro Plexiglas, de 3 mm de espessura. Corte as lentes circulares com uma imprensa de broca. Use uma broca padrão para fazer a curva interior, e, finalmente, polir as curvas internas e externas usando um polidor de lentes de contato do grão ultrafino (1000/3000). </li><li> Prepare 2,5 mM Rose Bengal (RB) em pH 7,4 tampão fosfato salino (PBS) com antecedência, filtro de esterilização com um filtro de seringa de 0,45 micron e armazenamento de alíquotas de 1 ml em -20 ° C num recipiente à prova de luz até 6 meses. NOTA: O uso de animais machos exogâmicas albinos, como Sprague Dawley minimiza as diferenças de resposta dependentes de tensão, permite maior facilidade de indução e reduz a variabilidade que pode ocorrer com cycli estrofeminino ng. </li><li> Configurar a frequência duplicou granada de alumínio de neodímio-ítrio (Fd-YAG) laser médico oftalmológico, o que gera uma luz laser de 532 nm. Montar o laser sobre uma lâmpada de fenda oftálmica Haag-Streit usando um adaptador de laser oftalmológico padrão. Este dispositivo disponível comercialmente permite a visualização simultânea de aplicação mancha olho e laser do animal. O laser médico também tem um feixe de mira para a focagem adequada e centragem utilizando mesmo tamanho de ponto como indução laser. parâmetros de potência do laser são os seguintes: <ol><li> Para a indução rNAION rato: use 500 um ponto de tamanho / 50 do laser mW de potência / 1.000 ms duração / 1.000 intervalo ms. </li><li> Para a indução rNAION mouse: alterar o tamanho do ponto a 300 mm para disco óptico menor e deixar outros parâmetros o mesmo que a configuração do rato. </li><li> Rotineiramente usar um medidor de potência do laser para assegurar a potência do laser. </li></ol></li></ol> 2. Procedimento Experimental <ol><li> Ligue a potência do lasere configurar o parâmetro do laser apropriado. Aquecer a laser para, pelo menos, cinco minutos antes da utilização. </li><li> Pesa-se o animal para determinar a dose apropriada para a cetamina / xilazina e corante RB. Anestesiar o animal por injecção intraperitoneal de uma mistura de 1 ml / kg de 80 mg / ml de cetamina e 4 mg / ml de xilazina. </li><li> Deixe o animal em uma gaiola aquecida até que esteja totalmente anestesiado. Verifique se há nenhuma resposta a estímulos aversivos (cauda ou pitada dedo do pé). Verifique animais para a profundidade da anestesia a cada 10 min. </li><li> Dilatar as pupilas do animais com tropicamida a 1% e anestesiar a superfície do olho com 0,5% de proparacaina. Se o uso de animais pigmentados, como Long Evans, um 2,5% gotas oculares neosynephrine vai aumentar a dilatação da pupila. </li><li> Use a tesoura para cortar os bigodes perto do focinho do lado para ser induzido para evitar o bloqueio a vista. </li><li> Coloque uma gota de 1% de metilcelulose ou outra gota de acoplamento oftálmica para o interior da lente de contato feito por encomenda, e depois aplicar a lente em tele olho de rato. </li><li> Colocar o animal sobre uma plataforma ajustada para a altura da lâmpada de fenda. Definir a cabeça do animal em um ângulo de 45 °, de modo que o olho é perpendicular à lâmpada de fenda e feixe de laser. </li><li> Visualize no olho através da lâmpada de fenda oftálmica. Verifique se o feixe de mira é o tamanho certo, bem como focado e centrado diretamente sobre o nervo óptico visualizado. Fotografar o fundo da retina usando uma câmera digital com alta ASA (1.200 - 2.000) Velocidade montado em uma das peças oculares da lâmpada de fenda com um adaptador personalizado feito. NOTA: A capacidade de ver os vasos da coróide revela a transparência da retina. Este é um sinal importante, a fim de ser capaz de detectar a isquemia retiniana mais tarde, que podem confundir a interpretação, se ocorrer. rNAION é a isquemia do nervo óptico, o que resulta na perda de CGR isolado, enquanto isquemia retinal resulta em danos na retina que afecta todas as células das camadas internas da retina. </li><li> Opcionalmente, ainda mais a imagem da retina enervo óptico usando um domínio de óptica espectral coerência tomógrafo (SD-08 de outubro) para avaliar o olho induzida por un. Digitalizar en face (Figura 3B), bem como 7 varreduras em corte transversal através da retina (Figura 3C). O SD-OCT imagem usa a mesma lente de contato usado para indução laser. </li><li> Injectar 1 ml / kg RB por via intravenosa através da veia da cauda, ​​e aguarde 30 segundos, em seguida, ativar o poder do laser. Este atraso de tempo permite que a RB para distribuir uniformemente por toda a circulação. Nós usamos um pulso de laser de 50 mW em um seg / pulso. energias maiores (≥ 60 mW) pode danificar a retina ou causar isquemia vascular da retina. Cuidado: Certifique-se de toda a gente tem um par de óculos de filtragem de segurança do laser do comprimento de onda de bloqueio adequada para evitar a luz do laser de rua entrando olho do investigador. NOTA: A administração de laser deve ser administrado rapidamente após a injecção IV RB desde que o corante é rapidamente eliminado da circulação. Quanto mais tempo a indução de laser, quanto mais grave a isquemia do nervo óptico. Geralmente, os animais são dadas 7 - 12 s impulsos numa sucessão rápida. O contacto da luz laser com o corante circulante dá os vasos do nervo óptico uma bela brilho de ouro que pode ser visto através da lâmpada de fenda (Figura 4B). Isto prova que o corante foi injectada sistemicamente e distribuída para a corrente sanguínea. Se o brilho é fraco, ou nenhum (Figura 4A), o corante não foi injetado por via intravenosa. Neste caso, não dão uma segunda injecção imediatamente, uma vez que o animal vai necessitar de recuperar durante pelo menos dois dias antes da reinjecção. </li><li> Imediatamente após a indução, retire a lente de contato. Cubra os dois olhos com pomada oftálmica antibiótica triplo (Neosporin / polimixina / bacitracina) com dexametasona, e colocar o animal em uma almofada de aquecimento de 37 ° C em uma única gaiola alojados sob observação até a recuperação completa. <ol><li> Limpe a lente de contato com água destilada water e seque com um pano de limpeza não abrasivo para uso futuro. </li><li> Dois dias após a indução avaliar o edema do nervo óptico por ambos cor fotografia fundo e SD-OCT análise 8. NOTA: Comparando o grau de edema do nervo óptico dá uma estimativa da gravidade da isquemia do nervo óptico. Em dois dias, a margem de disco nervo óptico é turva, e as veias da retina são ligeiramente dilatada, em comparação com o olho contralateral (induzida por un). Execute eletrorretinografia (ERG) e flash potencial evocado visual (VEP 11) em duas e quatro semanas após a indução para análise de eletrofisiologia. </li></ol></li></ol>

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Embora haja um certo número de modelos de lesão do nervo óptico (esmagamento do nervo óptico 12, a transecção do nervo óptico 13, e PION 14), o modelo rNAION é humano, adaptável a ambos os ratos e ratinhos. Assemelha-se mais de perto a situação clínica humana de NAION. Esta condição inclui anterior progressiva edema do nervo óptico, uma síndrome do compartimento anterior do nervo óptico, isquemia focal axonal, isolado lesão axonal de células ganglionares da retina e pe...

Anterior não arterítica neuropatia óptica isquêmica (NAION) é uma lesão isquêmica focal da porção anterior do nervo óptico (ON) 1. NAION é a causa mais comum de súbita nervo relacionada perda de visão óptica em indivíduos com idade superior a 50 2. O mecanismo acredita-se ser uma síndrome do compartimento que resulta em edema intraneural, e provoca a compressão dos capilares que abastecem os axónios no interior do nervo óptico 3. Uma vez que o NO é, na verdade, um aparelho do sistema nervoso central (SNC), o modelo de roedor NAION (rNAION) podem ser usadas para estudar os mecanismos e as respostas a derrames do SNC isolada da substância branca. O modelo rNAION podem portanto ser úteis no dissecando muitos problemas associados com o acidente vascular cerebral relacionado danos à substância branca. Ele pode ser usado para avaliar diferentes estratégias neuroprotectoras e agentes de acidente vascular cerebral em matéria branca. Uma das características mais atraentes do modelo é que ele é, Um procedimento não invasivo indolor. A energia laser pode ser ajustado para produzir vários graus de dano isquémico. Outra característica é que ele se baseia em radicais superóxido induzida por laser para danificar o endotélio capilar, produzindo uma disfunção progressiva capilar. É esta disfunção progressiva e edema que acredita-se ser muito semelhante ao mecanismo que faz com que NAION. A pesquisa mostrou que ele não causa a coagulação do capilar directa, mas funciona através de pelo menos dois mecanismos: superóxido induzida por morte e de extracção de algum do capilar de células endoteliais 4, e de NFkB (factor nuclear-cadeia kappa-luz-potenciador de células B activadas ) associado regulação inflamatória-se em permanecer endotélio, com o aumento do transporte de fluidos através das membranas celulares para o interstício 5. O encerramento dos capilares nervo óptico e compressão causados ​​por resultado a acumulação de fluido intersticial em isquemia cabeça do nervo óptico. Um diagrama esquemático é mostrado naFigura 2. O modelo rNAION pode ser usado em ambas as espécies de ratos e camundongos 6,7, e pode ser variada no nível da sua gravidade, a partir de uma lesão ligeira a uma destruição total, mas indolor do nervo óptico e da retina, tais como a oclusão da artéria central da retina (OACR).

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mW 532nm laser</td>
			<td style="width:99px;">Iridex</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Standard Ophthalmic Laser</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0-100mW 532nm laser</td>
			<td style="width:99px;">Laserglow technologies</td>
			<td style="width:119px;"></td>
			<td>Substitute for iridex</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laser slit lamp adapter</td>
			<td style="width:99px;">Iridex</td>
			<td style="width:119px;"></td>
			<td>SMA coupled adapter for laser output</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cpherent Fieldmate laser meter with thermopile sensor</td>
			<td style="width:99px;">Coherent</td>
			<td style="width:119px;"></td>
			<td>others also appropriate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ophthalmic Examing Slit lamp biomicroscope</td>
			<td style="width:99px;">VArious</td>
			<td style="width:119px;"></td>
			<td>Haag-Streit is the best; cheaper versions available on ebay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rose Bengal</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">330000-1G</td>
			<td>Photoinducing agent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fundus Contact lens or glass cover slip</td>
			<td style="width:99px;">custom/Cantor and Nissel (UK)</td>
			<td style="width:119px;"></td>
			<td>Custom designed planoconvex plastic lens for eye exam and induction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tropicamide 1%</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tamiya polishing compound</td>
			<td style="width:99px;">Tamiya, INC</td>
			<td align="right" style="width:119px;">87068</td>
			<td>polishing contact lens</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2.5% Hypromellose (Goniovisc)/1% Methycellulose</td>
			<td style="width:99px;">HUB Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td>contact lens coupling agent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2.5% Neosynephrine Ophthalmic drops</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>pupil dilating agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tropicamide 1%</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>pupil dilating agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5% Proparacaine</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>topical Anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30ga fused needle insulin syringe</td>
			<td style="width:99px;">Various</td>
			<td style="width:119px;">Various</td>
			<td>for intravenous injection of rose bengal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ophthamic Antibiotic ointment with dexamethasone added (Triple antibiotic ointment)</td>
			<td style="width:99px;">Various</td>
			<td style="width:119px;">Various</td>
			<td>Apply after induciton to minimize corneal scarring</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Heidelberg Corporation Spectral domain-Optical Coherence Tomograph</td>
			<td style="width:99px;">Heidelberg Corportion</td>
			<td style="width:119px;"></td>
			<td>For Optical coherence measurements baseline and post-induction; not essential for induction</td>
		</tr>
	</tbody>
</table>

the rodent model of nonarteritic anterior ischemic optic neuropathy (rnaion)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

<html> A visualização da lente de contato habilitado central de retina (Figura 1). O ponto de laser focal ilumina o disco óptico na parte posterior da retina (Figura 2). A retina normal, induzida por un é mostrado fotografada pela lâmpada de fenda bio-microscópio (Figura 3A) e pela SD-OCT (Figura 3B e 3C). Durante a indução do laser, quando não está presente corante está presente na circulação, de luz laser n...

Watch this Scientific Journal Video about The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION at JoVE.com

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

O modelo de roedor de nonarterítica óptica isquêmica anterior Neuropatia (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

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The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

8.8K visualizações.  University of Maryland-Baltimore School of Medicine. The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Vídeo: The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Transferência de genes para insuficiência cardíaca isquêmica em um modelo pré-clínica

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Medicina

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

Uma técnica simplificada para a produção de um modelo de ferida isquêmica

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

Angiogênese no pulmão de rato Isquêmica

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Um modelo murino de isquemia-reperfusão, através da ligadura da artéria Descendente Anterior Esquerda

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Modelo de Rato Induzida fotoquimicamente posterior neuropatia óptica isquémica

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Um Modelo de Leitão Neonatal encefalopatia hipóxico-isquêmica

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

Demonstração da Wound modelo de pele de rato Isquêmico

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

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 ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

Um modelo de roedor isquêmico de pericárdio intacto

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.