Este estudo foi financiado pela Fundação Mcihael J. Fox para Pesquisa 2011 Innovations Resposta Rápida de Parkinson Awards Program. Os financiadores não tiveram nenhum papel no desenho do estudo, coleta de dados e análise, decisão de publicar ou preparação do manuscrito.

Antes da cirurgia certificar-se de todos os procedimentos a serem feitas são aprovados pelo IACUC e quaisquer autoridades éticas ou legais adicionais e utilizar práticas de tratamento de animais sem crueldade. Isso inclui o uso de cirurgia condições estéreis, anestesiar o mouse usando o método IACUC aprovado, lubrificando ratos olhos com pomada veterinário durante a cirurgia, e prestação de cuidados pós-cirúrgico. Não prossiga com a cirurgia se houver qualquer questão de saber se os aspectos do processo são aprovados. Todos os procedimentos aqui realizados foram aprovados pelo Comitê Institucional Universidade Animal Care e Use o Boston. 1. Preparação de Animais e suprimentos cirúrgicos <ol><li> Autoclave todo instrumento cirúrgico que será usado durante a cirurgia. </li><li> Certifique-se de todas as técnicas que estão a ser realizadas são aprovados pelas agências reguladoras de animais. </li></ol> 2. Colheita da mucosa Graft <ol><li> Escolheu um rato geneticamente idênticos da mesma idade comoo mouse experimental e sacrificá-lo em um método IACUC aprovado (aqui: asfixia isoflurano seguido por deslocamento cervical). </li><li> Usando uma tesoura cirúrgica, retire a pele ao redor da região nasal de cabeça de rato expondo o crânio. </li><li> Com um martelo pneumático, marca com três linhas de dois dos quais lateralmente ladeiam a região nasal e um terceiro em linha com os olhos que conecta as duas linhas perpendiculares. </li><li> Broca para baixo ventralmente, a fim de separar o septo nasal a partir do tecido circundante. Um caminho mais largo, evitará danos à membrana mucosa no entanto, também vai tornar mais difícil para isolar a membrana. Um corte estreito mais perto da linha média é recomendado. </li><li> Use a tesoura para cortar o septo livre de qualquer tecido aderido a ele e armazená-lo em uma solução salina estéril. Neste momento, o enxerto pode ser limpa para remover qualquer tecido ligado. O ideal é ter membranas mucosas intactas exposta em ambos os lados da cartilagem do septo. Um grré pode fornecer membrana para dois ratinhos desde que a área da superfície da membrana é suficiente para cobrir os locais craniotomia. Recomenda-se que o enxerto é utilizada tão depressa quanto possível e o investigador prossegue para o passo 3, logo que o enxerto é isolado. </li></ol> 3. Implantação cirúrgica de mucosa Graft <ol><li> Usando procedimentos padrão, assépticas murino cirúrgicos, anestesiar e montar um mouse no quadro estereológica. Use cerca de 2% de isoflurano em oxigênio puro através de um aparelho de anestesia de roedores. </li><li> Imobilizar o mouse em um aparelho estereotáxico com barras de ouvido e um suporte de nariz. Aplicar pomada oftálmica para os olhos e esfregue o couro cabeludo com betadine e álcool 75% por três rodadas. Usando qualquer uma tesoura ou um aparador de cabelo, retire a pele na cabeça. Exponha o crânio com uma lâmina de barbear e nível da cabeça. Realizar uma craniotomia sobre o local do cérebro que vai ser administrado. Por exemplo, quando visando o striatum cortar 1,25 milímetrosdiâmetro do furo circular no crânio (centrado no AP: 1.00 mm; ML: 0,88 mm) usando um martelo pneumático. Molhar a área perfurada com solução salina estéril e utilizar uma lâmina de barbear para remover o crânio. </li><li> Remova cuidadosamente a dura usando a ponta de uma agulha. Além disso, isto pode ser conseguido pela aplicação de uma quantidade mínima de adesivo para o tecido da superfície da dura-máter húmido. Uma vez que esta camada tenha endurecido, com um movimento lateral da ponta da lâmina de barbear pode ser usado para remover a membrana. </li><li> Colocar a membrana mucosa acima da superfície do cérebro, tendo cuidado para manter o lado epitelial virada para fora da ferida. Isto é melhor realizado por transferência de todo o septo sobre a superfície do crânio adjacente ao local da craniotomia com pinças. Com a ponta de um par de tesouras cirúrgicas, puxar a membrana fora da cartilagem e para a superfície do crânio e no cérebro. Não deixe que a membrana secar ou tocá-lo com qualquer material absorvente. O enxerto deve sobrepor-se generosamente todas as extremidades ósseas da craniotomia site. </li><li> Cubra o enxerto com um pedaço estéril de borracha nitrílica. Isto actua de modo a evitar a aderência da pele no enxerto durante a cicatrização. O nitrilo precisa de ser grande o suficiente para cobrir toda a membrana mucosa. Apare membrana excessiva, se necessário. Evite qualquer movimento do nitrilo, uma vez que tenha entrado em contato com o enxerto. </li><li> Feche a pele com um 5-0 sutura estéril correndo e deixar o mouse recuperar para 3-7 dias antes de prosseguir para a próxima etapa. Tome cuidado para não perturbar a barreira nitrílica ou o enxerto gengival durante o fechamento da pele. </li></ol> 4. Administração da Solução de Dosagem <ol><li> Depois de garantir o rato anestesiado no quadro estereotáxico, corte a sutura com uma tesoura e retire o excesso de pele ao redor do crânio. </li><li> Retirar a barreira nitrilo e limpar a superfície do crânio. Use soro fisiológico e cotonetes estéreis para limpar a área até que o enxerto é visível. Pode ser necessário cortar o enxerto com uma lâmina de barbear se tem crescido muito mais do que a desired área de superfície. </li><li> Se a experiência será mais longo do que alguns dias, é sábio para implantar pelo menos dois parafusos crânio para reforçar o implante cabeça. </li><li> Colocar o bem acima do enxerto, de modo que as extremidades estão em contacto com o crânio. Aplique cola de cianoacrilato na junção entre o bem eo crânio. Encha o bem com solução salina estéril e verifique se não há vazamentos. Wells são feitos de agulhas de corte de seringa. </li><li> Aplicar cimento ósseo no crânio para garantir o bem no lugar. </li><li> Remover a solução salina a partir do poço com uma pipeta. Lavar as bem várias vezes para verificar que o adesivo não vazou dentro Adicionar a solução desejada; neste caso, 50 ul de dextrano fluorescente é usado. Espera-se que os compostos solúveis em água de uma polaridade semelhante que se comportam da mesma como dextrano. Entrega de compostos hidrofóbicos ou suspensões não foi explorada com este método. </li><li> Tapar o topo do poço, usando um pedaço circular de nitrilo fixada aotopo usando adesivo de cianoacrilato. Certifique-se de que o adesivo não entra em contacto com o conteúdo também. </li></ol> 5. Análise de entrega transmucosa <ol><li> Depois da quantidade desejada de tempo tiver passado, anestesiar o rato e trocar o conteúdo do poço com uma solução de corante azul de Evan. Este corante é usado para verificar que o enxerto estava intacta. </li><li> Após 30 min, anestesiar o rato fortemente, remover a solução de corante, e a eutanásia através de decapitação. </li><li> Manualmente remover o implante e retire o cérebro usando uma tesoura cirúrgica. Tenha cuidado para manter o enxerto no lugar. </li><li> Uma vez removido, flash congelar o cérebro em solução de isobutano arrefecido em um banho de gelo seco. </li><li> Incorporar o cérebro em solução (OCT) e fatia de 50 mm Temperatura ideal de corte. </li><li> Coloque as fatias desejadas diretamente em uma lâmina de microscópio. </li><li> Imagem da fatia utilizando um microscópio de fluorescência, logo que a solução tenha secado outubro. </li></ol>

<ol>
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Benjamin S. Bleier MD é inventor liderança de métodos provisórias de administração de medicamentos para o sistema nervoso central de cobertura de patentes.

A etapa mais difícil do processo aqui descrito é o sucesso da transferência de uma membrana mucosa de tamanho adequado para a superfície do cérebro. Este passo é feita significativamente mais fácil se o septo nasal colhido é suficientemente grande e bem limpos. Se a porção ventral do septo é truncado, um novo enxerto deve ser obtida. O ângulo de perfuração deve ser perpendicular à cabeça de rato para assegurar que a membrana da mucosa não é danificado pela broca. Se um caminho mais largo do que a perfu...

O tratamento da doença neurológica e psiquiátrica é severamente impedida pela presença da barreira sangue-cérebro (BBB), que impede que mais de 95% de todos os potenciais agentes farmacêuticos de atingir o sistema nervoso central 1-3. Por exemplo, factor neurotrófico derivado da glia (GDNF) foi demonstrado ser eficaz no tratamento da doença de Parkinson, quando injectado directamente no cérebro, no entanto, não é eficaz quando entregues sistemicamente porque não pode penetrar a BBB 4-6. Numerosos abordado foram desenvolvidas para tentar ultrapassar este problema. Melhoria na administração sistémica de neurotheraputics foi demonstrada usando conjugados de medicamentos contendo anticorpos selectivos para as proteínas de transporte localizados no endotélio capilar cerebral; no entanto, este método não tem sido mostrado para ser aplicável a uma ampla gama de produtos farmacêuticos 7,8. Além disso, a abertura osmótica da BHE foi usado clínicaaliado, no entanto, este método sofre de dosagem sistémica de fármacos, em oposição a uma distribuição mais directa para a região do cérebro de interesse 9. Esforço considerável tem sido posta em otimização de entrega transnasal na esperança de atacar directamente o cérebro 10-12. Embora tenha sido alcançado algum sucesso, resultados conclusivos só foram obtidos para as drogas que possuem receptores endógenos, como a insulina 13,14. Além disso, o mecanismo de entrega transnasal tem sido controversa com indícios de entrada indireta para o cérebro por meio de captação de neurônio olfativo ou através da corrente sanguínea 11. A entrega directa, transcraniana utilizando cateteres implantados tem sido conseguida, no entanto este processo é altamente invasivo e associada a numerosas complicações 15,16. Até à data, não existe um método geral, minimamente invasiva para entregar os compostos de elevado peso molecular para o cérebro. Aqui apresentada é um procedimento cirúrgico murinoque cria uma interface semipermeável com o cérebro. Isto é conseguido por um enxertando explante membrana mucosa 17 ao longo de um defeito craniotomia cirúrgica em um rato. Usando este procedimento, tem sido demonstrado que os compostos solúveis de até 500 kDa pode ser entregue no sistema nervoso central (directamente no parênquima cerebral, assim como em fluido cerebrospinal), em ambos tempo e um peso molecular dependente da forma 18. Este método de contornar o BBB é um modelo para reparos de defeitos da base do crânio em seres humanos, que utiliza enxertos vascularizados da mucosa para reparar buracos no crânio após a cirurgia endoscópica transnasal 19,20.

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="849">
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	<tbody>
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			<td height="21" style="height:21px;width:247px;">Name of Material/ Equipment</td>
			<td style="width:220px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:263px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">mice</td>
			<td style="width:220px;">Taconic</td>
			<td>C57BL/6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isoflurane</td>
			<td style="width:220px;">Piramal Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Student Fine Scissors</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91461-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">pneumatic drill</td>
			<td style="width:220px;">MTI Dental</td>
			<td style="width:119px;">333-CB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">drill bit</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">forcepts</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">0.9% Sodium Chloride Injection USP</td>
			<td style="width:220px;">Abbott Laboratories</td>
			<td>4925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Polystyrene Detri Dish</td>
			<td style="width:220px;">Fisher</td>
			<td>08-757-12</td>
			<td>for temporarily storing graft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead Sterilizer</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen/Isoflurane System</td>
			<td style="width:220px;">SurgiVet</td>
			<td>V720100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature Control System</td>
			<td style="width:220px;">Physitemp</td>
			<td style="width:119px;">TCAT-2LV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Stereotaxic Instrument</td>
			<td style="width:220px;">KOPF</td>
			<td style="width:119px;">Model 940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye Ointment</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Electric shaver</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cotton-Tipped Applicators</td>
			<td style="width:220px;">Fisher</td>
			<td>23-400-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5% Provadone Iodine</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>Betadine surgical scrub</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical Blade Stainless</td>
			<td style="width:220px;">Feather</td>
			<td style="width:119px;">2976#10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Scalpel Handle - #3</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3% Hydogen Peroxide</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>for cleaning the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetbond Tissue Adhesive</td>
			<td style="width:220px;">3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">needles</td>
			<td>Becton, Dickinson and Company</td>
			<td>305176</td>
			<td>needle tip cut off and used as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">nitrile gloves</td>
			<td style="width:220px;">Denville Scientific Inc</td>
			<td>G4162</td>
			<td>for well closure and protection of graft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Nylon Suture Thread</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Student Halsey Needle Holder</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cyanoacrylate adhesive</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>commecially available super glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental Cement Kit, 1 lb, Pink Opaque</td>
			<td style="width:220px;">Stoelting</td>
			<td>51458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isobutane (2-methylbutane)</td>
			<td style="width:220px;">Aldrich</td>
			<td>M32631</td>
			<td>for dry ice bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">dry ice</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

heterotopic mucosal engrafting procedure for direct drug delivery to the brain in mice

A entrega de agentes terapêuticos para o cérebro é impedida pela presença da barreira sangue-cérebro (BBB), o que restringe a passagem de compostos polares e de elevado peso molecular a partir da corrente sanguínea e no tecido cerebral. Algum sucesso entrega direta em seres humanos tem sido alcançado através de implantação de cateteres transcraniana; no entanto, este método é altamente invasivo e associado a várias complicações. Uma alternativa menos invasiva seria a dose do cérebro através de um implantado cirurgicamente, membrana semi-permeável, tal como a mucosa nasal, que é usado para reparar defeitos da base do crânio seguinte cirurgia endoscópica tumor remoção em humanos. Transferência da droga que esta membrana seria efectivamente desviar o BBB e difundem directamente no cérebro e no fluido cerebrospinal. Inspirado por essa abordagem, uma abordagem cirúrgica em ratos foi desenvolvido que utiliza uma membrana mucosa septal doador enxertada sobre um defeito extracraniana BBB cirúrgico. Este modelo tem sido demonstrado eficazpermitir a passagem de compostos de alto peso molecular para o cérebro. Desde inúmeros candidatos a fármacos são incapazes de atravessar a certificação, este modelo é valiosa para a realização de testes pré-clínicos de novas terapias para doenças neurológicas e psiquiátricas.

A obtenção de um grande o suficiente explante septo nasal é fundamental para as etapas subseqüentes. Isto pode ser conseguido através da perfuração no local no crânio do rato doador mostrada na Figura 1a. Corte ao longo deste caminho irá produzir um explante de tamanho suficiente, como mostrado na Figura 1b. Se a profundidade da perfuração não é profundo o suficiente, o enxerto será truncado e será difícil obter uma grande membrana suficiente para cobrir a superfície do...

Watch this Scientific Journal Video about Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice at JoVE.com

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

Um modelo do rato de reconstrução endoscópica da base do crânio humano foi desenvolvido que cria uma interface semipermeável entre o cérebro eo nariz com enxertos mucosas nasais. Este método permite que os investigadores a estudar a libertação no sistema nervoso central de terapêutica de elevado peso molecular, que são de outro modo excluídos pela barreira sangue-cérebro, quando administrado sistemicamente.

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 ...

heterotopic-mucosal-engrafting-procedure-for-direct-drug-delivery-to

Research

JoVE Journal

Medicine

10.7K visualizações.  Boston University.  Um modelo do rato de reconstrução endoscópica da base do crânio humano foi desenvolvido que cria uma interface semipermeável entre o cérebro eo nariz com enxertos mucosas nasais. Este método permite que os investigadores a estudar a libertação no sistema nervoso central de terapêutica de elevado peso molecular, que são de outro modo excluídos pela barreira sangue-cérebro, quando administrado sistemicamente. 

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

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 ...

Medicina

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

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 diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

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 ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Direto Rato Trauma / Burn Modelo de ossificação heterotópica

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Heterotopic Cervical Transplante Cardíaco em Ratos

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

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 ...

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 ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Na Vivo Photolabeling de células no cólon para avaliar o potencial migratória das células hematopoiéticas em camundongos Neonatais

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Novos métodos para a Administração Intranasal sob anestesia por inalação para avaliar a entrega da droga de nariz-de-Brain

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...