This research was supported by the Australian Research Council through the ARC Centre of Excellence in Vision Science (CE0561903), the National Health and Medical Research Council (1049990), and Progetto regionale speciale multiasse 'Reti per l'Alta Formazione' - PO FSE Abruzzo 2007-2013 - Azione 4.II.iii.

Todos os experimentos realizados foram de acordo com a Declaração de ARVO para o Uso de Animais em Oftálmica e Vision Research, e com a aprovação do Comitê de Ética da Universidade de L&#39;Aquila animal e ao Comité do Australian National University Ética em Experimentação Animal. Ratos adultos (P100-200) foram utilizados ao longo deste estudo. 1. Prepare os grânulos de resina de copolímero <ol><li> Colocar 20 pastilhas Elvax 40W em um pequeno copo de vidro na câmara de exaustão. </li><li> Encher o balão com 100% de etanol garantindo-se a cobrir todas as pelotas. </li><li> Cobrir a proveta com Parafilm e embeber os granulados em etanol a 100% à temperatura ambiente, durante 7-10 dias. NOTA: A partir desta etapa em diante, não use metal ou plástico para manipular ou armazenar a resina. </li></ol> 2 Preparar a solução de Drogas para carregar <ol><li> Dissolve-se o fármaco de escolha em 0,1% de dimetil-sulfóxido (DMSO). Adicione a solução de fármaco até 40 mL de volume.Carregar a resina com três vezes a concentração do medicamento que seria dado numa dose única. </li></ol> 3. Preparar e carregar o complexo de droga-resina <ol><li> Transferir os 20 pelotas lavadas com outra pequena proveta de vidro. Dissolve-se as pelotas em 4 ml de diclorometano durante aproximadamente 45 min. Cobrir o copo com Parafilm enquanto as pelotas estão se dissolvendo. </li><li> Prepara-se uma solução de Fast Green FCF, dissolvendo-o em 0,1% de DMSO a uma concentração de 0,001 mg / ml. </li><li> Tome uma pipeta, e elaborar 40 mL da solução da droga preparada anteriormente. Coloque outra pipeta, com 40 ml de solução de Fast Green. </li><li> Adicionar as duas soluções em simultâneo para o copo. Mistura-se rapidamente com uma vareta de vidro, até que o corante verde é uniformemente distribuída por toda a mistura. </li><li> Imediatamente transferir a taça sobre gelo seco durante 10 min a congelação rápida da resina. </li><li> Configurar uma &quot;câmara de evaporação&quot; para evaporar o solvente. Encha umamaior recipiente até um terço de pedrinhas de sulfato de cálcio. Criar um poço nas pedras, e coloque cuidadosamente o copo no poço para que as pedrinhas chegar até a metade do copo, para estabilizar o copo na câmara. </li><li> Cubra o recipiente fora maior com Parafilm. </li><li> Transferir a câmara de evaporação para o congelador. Manter a -20 ° C durante 2-3 semanas. </li></ol> 4 Prepare o complexo de droga-resina para implantação cirúrgica <ol><li> Retire a câmara de evaporação do congelador. Transferir o bloco de droga-resina sólido para um prato de vidro mantidos em gelo. </li><li> Com a ajuda de um microscópio ou lupa, cortar um pedaço de aproximadamente 0,05 mm de diâmetro e 0,1 mm de comprimento do bloco, utilizando uma ferramenta ou punção trefina. </li><li> Envolver o remanescente do bloco de material à prova de congelação (por exemplo, uma folha de alumínio) e manter a -20 ° C até ser necessário. </li></ol> 5. Cirurgicamente Implant o complexo de droga-resina para a Rat Eye </p&gt; <ol><li> Prepare o animal por cirurgia. Anestesiar utilizando uma injecção intraperitoneal de uma mistura de cetamina (100 mg / kg de peso corporal) e xilazina (12 mg / kg de peso corporal). Observar o animal até anestesia completa seja alcançada. NOTA: O animal deve ter uma perda de movimentos (exceto a respiração), e uma perda de reflexos pitada da córnea e do dedo do pé. </li><li> Transferir o animal completamente anestesiados para a mesa de cirurgia. Use mydriaticum (por exemplo, sulfato de atropina) e anestesia local (por exemplo, cloridrato de tetracaína) cai no olho (s) submetidos a cirurgia. Aplicar lágrimas artificiais para manter a córnea úmida durante a cirurgia. Utilize agulhas descartáveis ​​e instrumentos esterilizados durante toda a cirurgia. </li><li> Estabilize o olho usando um par de pinças curvas cegas após o aluno está totalmente dilatado. Usar um microscópio de operação para fazer uma perfuração profundidade máxima para alcançar o vítreo, cerca de 2 mm do limbo usando uma agulha de 25 G. </li><li> Insira o pedaço preparado de tele complexo de droga-resina para dentro da ferida punção usando um par de fórceps finos, pontiagudas (por exemplo, uma pinça), que são capazes de penetrar no local da punção. </li><li> Como profilaxia da infecção bacteriana, aplicar uma pomada antibiótica ao olho, para prevenir a inflamação, bem como prevenir a secura da córnea, enquanto o animal está a recuperar. </li><li> Observar o animal até a recuperação completa. Não deixe o animal sozinho até que ele recuperou a consciência. Não devolver o animal para a companhia de outros animais até que esteja totalmente recuperado. Se o animal apresenta sinais de stress ou desconforto, analgésicos devem ser utilizados de acordo com o protocolo de ética aprovado. </li><li> Siga os procedimentos experimentais habituais para coleta e análise do tecido. </li><li> Após a conclusão de experiências, sacrificar o animal com uma injecção intraperitoneal de uma pentobarbital (60 mg / kg de peso corporal) de solução de cloridrato de lidocaína a 2%. </li></ol> NOTA: A lidocaína cloridrato minimizes desconforto local.

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Os autores não têm nada a divulgação.

Este trabalho demonstrou a utilização de um complexo de droga-resina para a entrega de libertação lenta de drogas para a retina. Nosso objetivo foi apresentar um método que é relativamente barato e fácil de aplicar em um modelo animal pequeno. Tendo em conta que a função do APB é actuar como um análogo do glutamato, que vai bloquear a resposta da retina no olho. Os resultados mostram que o bloqueio provocado APB da resposta da retina de uma semana após a implantação. Isto indic...

O tratamento de doenças crônicas, como diabetes e pressão arterial elevada apresenta muitos desafios, como essas doenças geralmente necessitam de tratamento por longos períodos de tempo, muitas vezes por toda a vida. Este apelou para o desenvolvimento de sistemas de liberação de liberação lenta, o que reduz a necessidade de doses freqüentes. A eficácia destes métodos de libertação lenta tem sido demonstrada através de desenvolvimento de bombas de insulina, para reduzir o número de injecções de insulina necessários para o tratamento da diabetes. As doenças crônicas do olho, particularmente aquelas que afetam suas camadas internas, requerem a administração freqüente de drogas através de procedimentos invasivos. Uma doença crônica, que afeta o olho humano é a idade degeneração macular relacionada à (AMD). Ela afecta a retina central, que é uma camada de tecido neural situado na parte de trás do olho responsável por iniciar a visão. AMD é a principal causa de cegueira no mundo ocidental 1. Um desafio particular com o tratamento de redoenças tinal é que a droga é necessária para atingir esta camada profunda no olho, o que muitas vezes requer a entrega de métodos invasivos. As drogas são geralmente administradas a câmara vítrea e retina utilizando injecções intravítreas. No entanto, com cada injecção há o risco de complicações após a injecção, incluindo endoftalmite, descolamento da retina, catarata e hemorragia vítrea 2. Este risco é multiplicado com cada reinjecção do fármaco. Reduzindo a necessidade de múltiplas injeções seria um grande benefício no tratamento da AMD. Para tratar a forma úmida de DMRI, onde crescimento de novos vasos é uma característica marcante, a estratégia terapêutica estabelecida é atingir os factores de crescimento endotelial (VEGF) que usam inibidores de VEGF 3. No momento, estes são entregues através de injeções intravítreas repetidas. De igual modo, no tratamento de edema macular, uma complicação comum da retinopatia diabética, os corticosteróides são fornecidos através de injecções repetidas <saté&gt; 4. A entrega de drogas através destes métodos de libertação lenta pode, de facto reduzir o risco de complicações após a injecção. A noção de um sistema de entrega de droga de libertação lenta foi descrita pela primeira vez utilizando um veículo de borracha de silicone para proporcionar moléculas pequenas em tecidos animais 5. Desde então, outros métodos de libertação lenta foram desenvolvidos para entregar moléculas grandes, algumas das quais foram testadas no olho. Transportadoras de partículas, tais como microesferas biodegradáveis, poli-láctido-co-glicólido (PLGA) nanopartículas e vesículas de fosfolípidos (lipossomas) podem ser úteis como veículos de distribuição de 6,7. Nanopartículas e lipossomas PLGA foram comparados num ambiente in vitro quanto à sua capacidade para entregar agentes anti-cancerígenos através da esclera 7 ao longo do tempo. Ambos os veículos foram eficazes na lentamente liberando as drogas. No entanto, o estudo foi realizado apenas num ambiente in vitro. Bochot et al. (2002) 8 testadosa eficácia de lipossomas para entregar moléculas para a retina in vivo. Eles demonstraram que os lipossomas entregam com sucesso oligonucleótidos pequenos para a retina do coelho. Os autores sugeriram que os lipossomas podem ser benéficos no tratamento de doenças da retina 8. No entanto, a natureza destas vesículas para flutuar no vítreo significa que eles provavelmente borrar ou prejudicar a visão 9. Okabe et ai. (2003) 10 não utilizados discos de polímero biodegradável constituída por 33% de etileno-acetato de vinilo para aplicar beta-dexametasona em coelhos. Eles discos implantados no bolso escleral e demonstraram a libertação eficaz da droga no vítreo e retina de até um mês 10. No entanto, este protocolo em particular, o implante foi relativamente grande e rígida, e é necessária uma cirurgia mais complexa, incluindo uma grande incisão escleral e sutura. Um estudo anterior investigou a tissue resposta a vários veículos de polímero por implantá-las para a córnea do olho de coelho, e verificou que os copolímeros de etileno-acetato de vinila, lavados em álcool não causou inflamação ou irritação. Estes complexos foram mostrados para sustentar a entrega de compostos de maior dimensão em tecidos animais, por períodos de tempo alargados, excedendo alguns até 100 dias dependentes da droga 11. Um tal tipo de resina de copolímero foi desenvolvido industrialmente, sob a forma de Elvax 40W (40% em peso de etileno-vinil-acetato com conteúdo de comonómero de «W» amida aditivo para melhorar o tratamento da pelota). Esta resina de copolímero é uma substância inerte, que é estável a ambos os quarto e a temperatura do corpo. Não tem sido demonstrado causar alergias ou toxicidade no tecido biológico. Esta resina tem efectivamente fornecida uma grande variedade de drogas, em diferentes modelos experimentais que investigam as funções de vários sistemas, tais como o sistema ductal de mama 12, o córtex auditivo primário 13,14 &lt;/ Sup&gt;, eo sistema visual 15 sapo. Esta resina tem também sido utilizada no olho para entregar drogas para o desenvolvimento de tartaruga 16,17, 18,19 embrião de galinha, e de adultos furão retinas 20. No sistema nervoso central de rato, a resina foi usada apenas no cérebro de 21-23, mas o seu uso no olho do rato não foi documentada. As vantagens da utilização desta resina de copolímero para entregar drogas lentamente a retina através de outros métodos, é que é um composto estável que não cause inflamação ou irritação no olho. Ao contrário das partículas transportadoras, o complexo de droga-resina não iria prejudicar a visão, após o implante, uma vez que normalmente permanece no local da entrega, em vez de flutuar no vítreo. Só seria necessário um processo simples implantação na cavidade vítrea próxima ao limbo do olho, e que não requerem a sutura após o implante. Recentemente, tem havido uma emergência em vários novos sistemas de distribuição, tais comotecnologia de célula encapsulada (ECT) 24,25, hidrogéis 26 e 27 de microfilmes. No entanto, o método utilizado no presente estudo para a preparação e fornecimento do complexo droga-resina é fácil de seguir e de baixo custo, sendo assim mais vantajosa para utilização num ambiente de investigação básica. O desafio de utilizar este complexo para entregar drogas tratamentos de longo prazo é determinar a melhor concentração da droga que irá maximizar os benefícios terapêuticos da redução do número de injeções intravítreas. Este artigo tem como objetivo demonstrar o uso do complexo droga-resina para tratamento a longo prazo da retina de ratos adultos. A eficácia deste modo de entrega é testado usando o análogo do glutamato 2-amino-4-fosfonobutirato (APB) como a droga. Blocos APB a resposta à luz das células bipolares ON imitando glutamato, um neurotransmissor endógeno na retina 28. Quando APB concorre com o glutamato para o seu receptor, ele bloqueia o lighresposta t. APB foi usado em estudos fisiológicos para controlar a função da retina e medir o seu efeito através de métodos electrofisiológicos como electroretinografia (ERG). Em estudos anteriores, APB foi usado para tanto a curto prazo 29 e o tratamento de longo prazo da retina em desenvolvimento; este último envolvido que dá uma dose única, por injecção intra-ocular ao dia durante 30 dias 30. A mesma quantidade de APB (0,092 mg em solução salina estéril, a uma concentração de 50 ^ M) foi usada para todas as injecções, tal como sugerido em 28,29,31 trabalho anterior. Escolhemos APB para testar o complexo droga-resina, como um veículo de libertação lenta para entregar drogas no olho. Os métodos descritos neste estudo são similares aos métodos descritos anteriormente, envolvendo a preparação da droga-resina complexo 16,32; no entanto, também detalhar o seu uso principalmente no olho de um rato adulto. Após a implantação cirúrgica da resina APB-carregado para dentro do olho, ERG foi realizado para determinar se APB abole a rresposta à luz etinal e, portanto, se APB foi lançado com sucesso no vítreo e retina.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Elvax 40W Pellets</td>
			<td>Du Pont, DE, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-amino-4-phosphonobutyric acid (APB)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>A1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite, calcium sulfate</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>238910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine, Ilium Ketamil</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine, Ilium Xylazil-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulphate, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracaine Hydrochloride, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol, Chlorsig ointment</td>
			<td>Aspen Pharma Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital, Lethabarb</td>
			<td>Virbac Australia Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lignocaine Hydrochloride, Ilium Lignocaine-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uni-Core Punch Tool</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Microscope, Zeiss OPMI 99</td>
			<td>Zeiss, West Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G Insulin Needle</td>
			<td>Terumo Corp., Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

slow-release drug delivery through elvax 40w to the rat retina: implications for the treatment of chronic conditions

Doenças da retina, são difíceis de tratar como na retina encontra-se profundamente no interior do olho. Métodos invasivos de entrega de drogas são muitas vezes necessários para tratar essas doenças. Doenças da retina crônicas, tais como edema retinal ou neovascularização normalmente requerem múltiplas injecções intra-oculares para tratar eficazmente a doença. No entanto, os riscos associados a estas injecções aumentar com a repetição de uma entrega da droga. Portanto, métodos alternativos de entrega precisam ser estabelecidos, a fim de minimizar os riscos de reinjeção. Várias outras investigações têm desenvolvido métodos para administrar medicamentos ao longo do tempo prolongado, através de materiais capazes de libertar lentamente produtos químicos para dentro do olho. Na presente investigação, que descrevem a utilização de Elvax 40W, uma resina de copolímero, para actuar como um veículo para a entrega da droga à retina de um rato adulto. A resina é feita e carregados com o fármaco. O complexo de droga-resina é então implantado na cavidade vítrea, que irá libertar o fármaco lentamente durante time. Este método foi testada utilizando 2-amino-4-fosfonobutirato (APB), um análogo de glutamato que bloqueia a resposta à luz da retina. Demonstrou-se que o APB foi libertada lentamente a partir da resina, e foi capaz de bloquear a resposta da retina por 7 dias após a implantação. Isto indica que a entrega de droga de libertação lenta utilizando esta resina de copolímero é eficaz para o tratamento da retina, e pode ser utilizada de forma terapêutica com outros ensaios.

Electroretinografia de campo total (ERG) foi utilizado para detectar o efeito de APB na retina. Para detalhes sobre como o ERG foi realizada, consulte os seguintes estudos 33,34. Em resumo, depois de anestesiar o animal, um eléctrodo de ouro foi colocado sobre a córnea do olho, e o eléctrodo de referência foi colocado no couro cabeludo anterior, entre os olhos, para registar a actividade eléctrica da retina. Os estímulos foram geradas usando uma unidade de flash electrónico e a intensidade variou de <s...

Watch this Scientific Journal Video about Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions at JoVE.com

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Este artigo fornece detalhes sobre como Elvax 40W pode ser utilizado como um método de libertação lenta para entrega da droga à retina de um rato adulto. O protocolo para a preparação, o carregamento e entrega o complexo droga-resina, para o olho é descrito.

De liberação lenta Drug Delivery através Elvax 40W para o Rat Retina: implicações para o tratamento de condições crônicas

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

slow-release-drug-delivery-through-elvax-40w-to-rat-retina

Research

JoVE Journal

Medicine

11.4K visualizações.  University of L'Aquila.  Este artigo fornece detalhes sobre como Elvax 40W pode ser utilizado como um método de libertação lenta para entrega da droga à retina de um rato adulto. O protocolo para a preparação, o carregamento e entrega o complexo droga-resina, para o olho é descrito. 

Vídeo: Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

A Medição e Tratamento de Supressão de ambliopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Medicina

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Um Modelo de Infusão Crónica de nutrientes no Rato

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Osmótica Drug Delivery para hindlimbs isquêmicos e perfusão da vasculatura com Microfil para Micro-tomografia computadorizada

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Heterotópico mucosa enxertando Procedimento para entrega da droga direto para o cérebro em ratos

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Drug Delivery intraluminal para o Rato Fístula Arteriovenosa endotélio

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

Entrega intravascular de Biologics para o rim de rato

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Análise de Redes de forame oval Eletrodo Recordings na epilepsia do lobo temporal Os pacientes resistentes a drogas

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Método otimizado de LC-MS/MS para a análise do elevado-throughput de amostras clínicas de Ivacaftor, seus principais metabolitos e Lumacaftor em fluidos biológicos de pacientes de fibrose cística

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

Entrega de trans-timpânicas drogas para o tratamento da ototoxicidade

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...