Nós graciosamente agradecer membros adicionais do Lab Lever que contribuíram para a coleta de dados (Andries Ferreira, Danarae Aleman, Alexis Mok, Kaitlin Flynn, Elizabeth Bearce e Matan Kadosh) e de revisão de artigos (Andries Ferreira, Rebecca Schneider, e Kate Robbins). Reconhecemos também Roderic Schlotzhauer e Edwin Honse do MU Física Oficina para a sua entrada projeto e fabricação dos tubos de observação de roedores utilizados neste estudo. Estamos especialmente agradecidos por (Departamento de Cirurgia da Universidade de Missouri Radiologia Supervisor na Medicina Veterinária e - Faculdade de Medicina Veterinária) Malea Jan Kunkel e Jan Ivey (Gerente da Cath Lab Pesquisa Animal da Universidade de Missouri - School of Medicine) para demonstrar paciência e motivação constante durante a operação os fluoroscopia de alta energia como foi desenvolvido o protocolo VFSS murino. Fontes de financiamento para este estudo incluiu NIH / NIDCD (TE Lever), NIH / NINDS (GK Pavlath), Otolaryngologia - Cirurgia de Cabeça e Pescoço fundos de arranque (TE Lever), do Fundo de MU PRIME (TE Lever), Mizzou Advantage (TE Lever), e do Centro de MU sobre Envelhecimento (TE Lever).

Open Access para este artigo é patrocinado pela Glenbrook.

Disfagia (distúrbio de deglutição) é um sintoma comum de várias condições médicas que afetam as pessoas de todas as idades. Exemplos incluem acidente vascular cerebral, doença de Parkinson, doença de Alzheimer, paralisia cerebral, distrofia muscular, esclerose lateral amiotrófica (ELA), doença de Batten, câncer de cabeça e pescoço, o nascimento prematuro, e idade avançada. A disfagia é altamente correlacionado com a mortalidade, geralmente como consequência de desnutrição ou pneumonia grave que se desenvolve quando bacteriana-laden comida / líquido / saliva é aspirado para os pulmões 1-4. Esta condição médica debilitante e com risco de vida afeta mais de 15 milhões de pessoas a cada ano nos Estados Unidos sozinho 3. Apesar da alta prevalência e os resultados negativos associados, as opções atuais de tratamento para a disfagia são limitados a paliativos (e não curativa) se aproxima, como a modificação da dieta (por exemplo, evitando as consistências de alimentos / líquidos específicos), alterações posturais (por exemplo, Tucking o queixo quando engolir), abordagens a motor (por exemplo, exerce o direccionamento músculos na cavidade oral, faringe, laringe e), abordagens sensoriais (por exemplo, aroma de execução, a temperatura, e / ou de estimulação mecânica), e tubo de alimentação (por exemplo, a nutrição e hidratação administrados via nasogástrica (NG) tubo de gastrostomia endoscópica percutânea ou (PEG) tubo). Estes tratamentos servem apenas como terapia sintomática em vez de mirar as causas subjacentes do problema. Na verdade, uma grande barreira para a descoberta de novos tratamentos eficazes, para a disfagia é o conhecimento científico limitado de mecanismos patológicos responsáveis, que provavelmente diferente para cada doença. Diagnóstico Disfagia é predominantemente feita através de um procedimento radiográfico chamado de deglutição estudo (VFSS), também conhecido como um estudo de bário modificado. Ao longo dos últimos 30 anos ou mais, este teste de diagnóstico tem sido considerada o padrão-ouro para evaluating função andorinha 5-7. Este teste implica ter o paciente se sentar ou ficar no caminho do feixe de raios-X de uma máquina de fluoroscopia enquanto voluntariamente a ingestão de alimentos e consistências líquida misturada com um agente de contraste oral, tipicamente sulfato de bário 8,9 ou iohexol 10. À medida que o paciente engole, alimentos e líquido contendo agente de contraste pode ser observada em tempo real através de um monitor de computador, enquanto viaja a partir da boca para o estômago. Estruturas de tecido mole também são visíveis e podem ser avaliados em relação à estrutura e função. Os doentes são chamados a realizar vários goles de cada alimento e consistência líquida, todos os quais são vídeos gravados para posterior visualização e análise de frame-by-frame para quantificar a presença eo grau de disfagia. Numerosos componentes fisiológicos de engolir normalmente são analisados, como o ponto de disparo anatômica da andorinha da faringe, o tempo de trânsito bolus através da faringe e esôfago, extensão e duração do laryngeal elevação, localização e quantidade de resíduo pós-andorinha, e ocorrência de e razão fisiológica para aspiração 7,11. Aspectos do protocolo VFSS humano foram recentemente adaptado para o estudo ratos livremente comportando; no entanto, os resultados foram limitados porque os ratos não permanecer no campo videofluoroscópica de vista durante o teste 12. VFSS não tenha sido previamente tentada com ratinhos. A adaptação bem sucedida do protocolo VFSS humano para utilização com ratos e ratazanas iria proporcionar um método novo de pesquisa para investigar as centenas de modelos de doenças que são conhecidos por causar disfagia em humanos actualmente existente murino (ratinho e rato). Este novo método (doravante referida como VFSS murina), portanto, acelerar a identificação e validação de modelos murinos de disfagia que são adequados para investigar os mecanismos neurofisiológicos subjacentes no tecido músculos, nervos e cérebro que são patológico e contribuindo para a disfagia in humanos. Além disso, murino VFSS iria permitir a identificação de medidas objetivas (biomarcadores) de capacidade de deglutição / disfunção que poderia ser comparado diretamente com os seres humanos. Estas espécies cruzadas biomarcadores videofluoroscópicos poderia então servir medidas de resultados, como novos para quantificar a eficácia do tratamento em ensaios pré-clínicos com camundongos e ratos, o que melhor traduzem em ensaios clínicos com pessoas. Para esse efeito, o protocolo VFSS murino foi estabelecida utilizando ~ 100 ratinhos de ambos os sexos. Todos os ratos eram ou C57 ou híbridos estirpes C57 / SJL. Os ratinhos C57 não foram geneticamente alterados, enquanto C57 / SJL foi a estirpe de fundo para uma colónia de murganhos transgénicos G93A-SOD1 (ou SOD1), o modelo animal mais largamente utilizado dos ALS. A colônia SOD1 foi um aproximada de 50-50 mix de transgênicos (ou seja, ALS-afetados) ratos e não transgênicas (ou seja, não afetados) ninhada. O protocolo VFSS murino consiste em três componentes: <ol><li&gt; câmaras de observação a pedido de clientes que permitem a alimentação voluntária e deglutição ao estar desenfreado em um espaço confinado dentro de uma máquina de fluoroscopia, </li><li> Receitas que mascaram o sabor aversivo / odor de agentes de contraste oral e produzem radiodensity suficiente para permitir a visualização adequada da deglutição, </li><li> Um protocolo passo-a-passo de teste que maximiza o cumprimento animal, minimiza o tempo total do teste e a exposição à radiação, e permite a quantificação de vários parâmetros de andorinha para cada fase da deglutição (ou seja, oral, faringe, esófago e). </li></ol> O efeito combinado produz um baixo stress, ambiente confortável, auto-alimentando exame que permite a avaliação da alimentação típica e deglutição comportamentos de ratos.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Material/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >Polycarbonate tubing for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >3161T41</td>
			<td>Body of observation tubes, 2&#34;X2&#34; diameter, 0.080&#34; thick wall</td>
		</tr>
		<tr >
			<td >Polycarbonate sheet&#160; for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >9115K71</td>
			<td>End-caps for observation tubes, 2&#34;x12&#34;x3/4&#34;</td>
		</tr>
		<tr >
			<td >Polycarbonate sheet&#160; for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >8574K281</td>
			<td>Peg-bowls for observation tubes</td>
		</tr>
		<tr >
			<td >Silicone O-rings&#160; for end-caps of observation chambers</td>
			<td >McMaster-Carr</td>
			<td >9396K108</td>
			<td>S1138 AS568-029, pack of 25 
			http://www.mcmaster.com/#o-rings/=t0wt5r&#160;</td>
		</tr>
		<tr >
			<td >Silicone stoppers for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >2903K22</td>
			<td>Package of 10 stoppers to plug the oval opening in the top of the observation chamber when using a peg-bowl 
			http://www.mcmaster.com/#catalog/120/3803/=t0y5at</td>
		</tr>
		<tr >
			<td >Centrifuge tubes for sipper tube bottles</td>
			<td >Evergreen Scientific</td>
			<td >222-3530-G80</td>
			<td>30 ml freestanding centrifuge tubes, with caps, sterile 
			https://www.evergreensci.com/labware-catalog/tubes-and-vials/30-and-50-ml-centrifuge-tubes/&#160;</td>
		</tr>
		<tr >
			<td >Silcone stoppers for sipper tube bottles</td>
			<td >Saint-Gobain Performance Plastics</td>
			<td >DX263031-10&#160;</td>
			<td>Number 31D, size: 26 mm bottom, 32 mm top, 30 mm high; 10 pack;&#160; 
			http://www.labpure.com/en/Products.asp?ID=179&#38;PageBrand=STOPPERS</td>
		</tr>
		<tr >
			<td >Stopper borers for sipper tube bottles</td>
			<td >Thomas Scientific</td>
			<td >3276G40</td>
			<td>Cork Borer Set that ranges from 3/16-15/16 inch&#160; 
			http://www.thomassci.com/Supplies/Corks/_/CORK-BORER-SET-316-1516-IN?q=Humboldt</td>
		</tr>
		<tr >
			<td >Drinking tubes for sipper tube bottles</td>
			<td >Ancare</td>
			<td >TD-100&#160;</td>
			<td>2 1/2&#8221; long drinking tubes with 5/16&#8221; opening, straight ball-spout 
			http://www.ancare.com/products/watering-equipment/open-drinking-tubes/straight-tubes-ball-point&#160;</td>
		</tr>
		<tr >
			<td >Iohexol for making oral contrast agent solution</td>
			<td >GE Healthcare</td>
			<td ></td>
			<td>350 mg iodine per ml 
			http://www3.gehealthcare.com/en/products/categories/contrast_media/omnipaque&#160;</td>
		</tr>
		<tr >
			<td >Chocolate syrup for flavoring oral contrast agent</td>
			<td >Herseys</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >10 ml syringe for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >309604</td>
			<td>Luer lock tip syringe without needle, 100 per box 
			http://www.bd.com/hypodermic/products/syringeswithoutneedles.asp</td>
		</tr>
		<tr >
			<td >Catheter tubing for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >427451</td>
			<td>Polyethylene Tubing (Non-Sterile) (PE 240) 100&#39; 
			http://www.bd.com/ds/productCenter/427451.asp&#160;</td>
		</tr>
		<tr >
			<td >Needle for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >427560</td>
			<td>15-gauge needle, fits into PE 240 catheter tubing 
			http://www.bd.com/ds/productCenter/427560.asp&#160;</td>
		</tr>
		<tr >
			<td >Delrin acetal resin rod for syringe delivery system</td>
			<td >McMaster-Carr</td>
			<td >8576K15</td>
			<td>1/2 inch diameter, black 
			http://www.mcmaster.com/#catalog/120/3609/=t0wvaf&#160;</td>
		</tr>
		<tr >
			<td >Acrylic sheeting for scissor lift</td>
			<td >Ponoko</td>
			<td ></td>
			<td>Laser cut 
			http://www.ponoko.com&#160;</td>
		</tr>
		<tr >
			<td >3D printed ABS frame</td>
			<td >Engineering Rapid Prototyping Facility, University of Missouri</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Brass rods for scissor lift</td>
			<td >Amazon</td>
			<td >TTRB-03-12-03</td>
			<td>made into axles 
			http://www.amazon.com/Brass-Seamless-Round-Tubing-Length/dp/B000FN898M</td>
		</tr>
		<tr >
			<td >Drawer slide for scissor lift</td>
			<td >Richelieu</td>
			<td >10292G116</td>
			<td>Attaches to base of scissor lift 
			http://www.lowes.com/pd_380986-93052-T35072G16_0__?productId=50041754</td>
		</tr>
		<tr >
			<td >28BYJ-48 stepper motor for scissor lift</td>
			<td ></td>
			<td ></td>
			<td>2 each</td>
		</tr>
		<tr >
			<td >ULN2003 Darlington transistor array for scissor lift</td>
			<td >Toshiba</td>
			<td >ULN2003APG</td>
			<td>Used as stepper drivers (2 each)</td>
		</tr>
		<tr >
			<td >ATTINY85 microcontroller for scissor lift</td>
			<td >Atmel</td>
			<td >ATTINY85-20PU</td>
			<td>2 each 
			http://www.taydaelectronics.com/attiny85-attiny85-20pu-8-bit-20mhz-microcontroller-ic.html</td>
		</tr>
		<tr >
			<td >Nylon spur gear</td>
			<td >McMaster-Carr</td>
			<td >57655K34</td>
			<td>2 each 
			http://www.mcmaster.com/#57655k34/=t0yaqz</td>
		</tr>
		<tr >
			<td >Nylon spur gear rack</td>
			<td >McMaster-Carr</td>
			<td >57655K62</td>
			<td>2 each 
			http://www.mcmaster.com/#57655k62/=t0ybh9</td>
		</tr>
		<tr >
			<td >4-40 nylon machine screws</td>
			<td >McMaster-Carr</td>
			<td >95133A315</td>
			<td>Lift assembly 
			http://www.mcmaster.com/#95133a315/=t0yd8q</td>
		</tr>
		<tr >
			<td >4-40 nylon hex nuts</td>
			<td >McMaster-Carr</td>
			<td >94812A200</td>
			<td>Lift assembly 
			http://www.mcmaster.com/#94812a200/=t0ye29</td>
		</tr>
		<tr >
			<td >Buna-N O-Ring AS568A Dash No. 104</td>
			<td >McMaster-Carr</td>
			<td >9452K318</td>
			<td>Lift assembly 
			http://www.mcmaster.com/#9452k318/=t0yem7</td>
		</tr>
		<tr >
			<td ></td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

adapting human videofluoroscopic swallow study methods to detect and characterize dysphagia in murine disease models

Este estudo adaptado videofluoroscópica humano deglutição (VFSS) métodos para uso com modelos de doenças murino com a finalidade de facilitar a investigação translacional disfagia. Resultados bem sucedidos dependem três componentes críticos: câmaras de ensaio que permitam a auto-alimentação ao estar desenfreado em um espaço confinado, receitas que mascaram o sabor aversivo / odor de agentes de contraste orais comercialmente disponíveis, e um protocolo de passo-a-passo de teste que permite a quantificação da fisiologia andorinha. A eliminação de um ou mais desses componentes vai ter um impacto negativo nos resultados do estudo. Além disso, o nível de energia de capacidade de o sistema irá determinar que a fluoroscopia engolir parâmetros podem ser investigados. A maioria dos centros de pesquisa têm fluoroscopia de alta energia projetados para uso com pessoas e animais de maior porte, o que resulta em má qualidade de imagem excepcional ao testar ratos e outros pequenos roedores. Apesar dessa limitação, identificamos sete VFSS parâmetros que são consistentemente quantificáveis ​​em ratinhos quando usando um fluoroscópio alta energia em combinação com o novo protocolo VFSS murino. Nós obtivemos recentemente um sistema de baixa fluoroscopia energia com capacidades excepcionalmente de alta resolução de imagem e ampliação que foi projetado para uso com ratos e outros pequenos roedores. Trabalhos preliminares de usar este novo sistema, em combinação com o novo protocolo VFSS murino, identificou 13 parâmetros andorinha que são consistentemente quantificáveis ​​em camundongos, o que é quase o dobro do número obtido usando convencional (ou seja, de alta energia) fluoroscopia. Identificação de parâmetros adicionais andorinha é esperado como otimizar as capacidades deste novo sistema. Os resultados até agora demonstrar a utilidade do uso de um sistema de fluoroscopia de energia de baixo para detectar e quantificar mudanças sutis na fisiologia andorinha que podem passar despercebidas quando se usa fluoroscopia de alta energia para investigar modelos de doenças murino.

Nós projetamos com sucesso um romance e um protocolo específico VFSS-murino replicável que inclui câmaras de ensaio que permitam a auto-alimentação, receitas para aromatização de agentes de contraste orais, e um protocolo de teste passo-a-passo que permite a quantificação da fisiologia andorinha. A capacidade de o nível de energia do sistema de fluoroscopia determinado que engolir parâmetros poderiam ser investigados em ratinhos. Nós inicialmente utilizado fluoroscopia de alta energia projetados para uso com pessoas e animais maiores (por exemplo, GE Advantx, GE OEC 9600, e Omega Cardiac Cath CS-25, cada um com 30 quadros por segundo). No entanto, estes sistemas tinham capacidades de ampliação suficientes para testes ratinhos, o que resultou no enchimento dos animais apenas uma pequena porção do campo de visão (Figura 9). Como resultado, a qualidade de imagem era excepcionalmente fraca, impossibilitando a visualizar a maioria das estruturas do mecanismo de deglutição. Apesar desta limitação, foram identificados 7 VFS objetivoS engolir parâmetros que eram consistentemente quantificável para ratinhos quando se utiliza uma (ou seja, alta energia) fluoroscopia convencional em combinação com o novo protocolo VFSS murino (Tabela 3). Além disso, identificou-se o espaço vallecular como o ponto de disparo anatómica para engolir em ratinhos adultos saudáveis ​​(3-17 meses de idade), bem como condições de ratos com idade avançada (&gt; 18 meses) e ALS em fase terminal. <img alt="Figura 9" src="/files/ftp_upload/52319/52319fig9highres.jpg" width="650" /> Figura 9:. Energia fluoroscopia Sistemas de Alto Esquerda: Imagem representativa de um rato obtidos utilizando alta energia (ou seja, os sistemas convencionais) fluoroscopia. Note-se que o rato preenche apenas uma pequena porção do campo de vista de fluoroscopia, demonstrando assim a capacidade de ampliação insuficiente de fluoroscopia convencional para roedores de imagem. Direita: A mesma imagem ampliada pós-capture usando um programa de software de edição de vídeo. Seta preta: engolir ponto de disparo (valéculas). Seta branca:. Bolus no esôfago distal, imediatamente antes de passar através da junção GE (asterisco branco) <a href="https://www.jove.com/files/ftp_upload/52319/52319fig9highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> PARÂMETROS ANDORINHA </td><td style="width:140px"> Sistema de Alta Energia </td><td style="width:140px"> Low Energy System </td></tr><tr><td> Inter-Swallow Interval (ISI) </td><td> X </td><td> X </td></tr><tr><td> Jaw Excursion Rate (Lamba taxa equivalente) </td><td> X </td><td> X </td></tr><tr><td> Jaw Excursion Distância </td><td> X </td><td> X </td></tr><tr><td> Rácio de lambe-Swallow </td><td> X </td><td> X </td></tr><tr><td> Swallow Taxa </td><td> X </td><td> X </td></tr><tr><td> Pharyngeal Transit Time (PTT) </td><td></td><td> X </td></tr><tr><td> Bolus velocidade pela faringe </td><td></td><td> X </td></tr><tr><td> Esofágico Transit Time (ETT) </td><td></td><td> X </td></tr><tr><td> Bolus velocidade através de Esôfago </td><td></td><td> X </td></tr><tr><td> Bolus velocidade pela faringe e esôfago </td><td> X </td><td> X </td></tr><tr><td> Bolus Area </td><td></td><td> X </td></tr><tr><td> Pharyngeal Resíduos Área </td><td></td><td> X </td></tr><tr><td> Volume de líquidos consumidos </td><td> X </td><td> X </td></tr></tbody></table> Tabela 3: Andorinha Parâmetros Quantificável Usando alta versus baixa energia fluoroscopia Systems. Recentemente, adquiriu um sistema de fluoroscopia baixa ampliação energia chamada The LabScope (Glenbrook Technologies, Randolph, NJ) que foi projetado especificamente para o nosso laboratório para uso comratos e outros roedores pequenos (Figura 10). No entanto, os acentuadamente maiores níveis de ampliação deste sistema tornaram impossível para visualizar todo o mecanismo de deglutição de um rato em um único campo de visão. Em vez disso, são necessárias duas posições de teste, como mostrado na Figura 11. Posição 1 permite a visualização de toda a cabeça e a região proximal torácica. Esta posição é necessário para avaliar as fases oral e faríngea da deglutição. Posição 2 permite a visualização do ponto gatilho andorinha (ie, valéculas) à gastroesofágico (GE). Esta posição é necessário para avaliar a fase esofágica da deglutição. Trabalhos preliminares usando O LabScope em combinação com o novo protocolo VFSS murino identificou 13 parâmetros andorinha objetivas que são consistentemente quantificáveis ​​em camundongos, o que é quase o dobro do número obtido usando alta energia fluoroscopia (ie, convencionais) (Tabela 3). Este o<html> ESULTADOS é atribuído às capacidades de ampliação avançados da LabScope, que permite a visualização de inúmeras estruturas anatômicas (Figura 12), que eram essencialmente invisível quando usando sistemas convencionais: por exemplo, osso hióide, traquéia e vértebras cervicais. Como resultado, nós também foram capazes de analisar os vídeos para a evidência de penetração laríngea e aspiração. Nem penetração aspiração nem foi observado para qualquer rato neste estudo, independentemente das condições de saúde ou doença. <img alt="Figura 10" src="/files/ftp_upload/52319/52319fig10highres.jpg" width="650" /> Figura 10:. O LabScope Esquerda: O LabScope executa como um fluoroscope área de trabalho para pequenos animais. Direita: Close-up vista do LabScope com componentes marcados. A tabela de elevador de tesoura é posicionar uma câmara de observação dentro do campo fluoroscope de vista.<html> tp_upload / 52319 / &quot;target =&quot; _ 52319fig10highres.jpg blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 11" src="/files/ftp_upload/52319/52319fig11highres.jpg" width="650" /> Figura 11: Low Energy. Fluoroscopy Sistema de Imagens de um rato obtidos utilizando um sistema de baixa energia fluoroscopia. Note-se que a capacidade de ampliação elevada impede a visualização de todo o mecanismo de andorinha no interior do campo de visão da fluoroscopia. Esquerda: Posição 1 - permite a visualização de toda a cabeça e região torácica proximal. O ponto de disparo andorinha (seta preta) está essencialmente centrada dentro do campo de visão. Direita: Posição 2 - permite a visualização do ponto gatilho andorinha (seta preta) para a junção GE (asterisco branco). Nota do bolo que passa através do esófago distai (seta branca).<html> &quot;target =&quot; _ g11highres.jpg blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 12" src="/files/ftp_upload/52319/52319fig12highres.jpg" width="650" /> Figura 12:. Usando estruturas anatômicas Visible um Fluoroscopy Sistema Low Energy Mesmo na configuração mais baixa ampliação (à esquerda), estruturas ósseas da cabeça e do pescoço de um rato são facilmente visíveis através do nosso sistema de baixa fluoroscopia energia (ie, A LabScope). As estruturas anatômicas dentro do quadrado preto são mostrados (e rotulados) em maior ampliação para a direita. Melhor visualização das estruturas ósseas permite a quantificação de vários parâmetros andorinha adicionais que eram impossíveis de analisar usando fluoroscopia de alta energia. <a href="https://www.jove.com/files/ftp_upload/52319/52319fig12highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> t &quot;&gt; Swallow taxa e intervalo inter-andorinha são parâmetros representativos VFSS que podem ser quantificados utilizando ou sistemas de baixas ou altas fluoroscopia energia em combinação com o novo protocolo VFSS murino foram quantificados Esses dois parâmetros engolir para três grupos de ratos:. SOD1-G93A (SOD1) ratinhos transgénicos (isto é, um modelo de esclerose lateral amiotrófica), a doença em fase terminal entre 4-5 meses de idade, os ratinhos C57 envelhecidos (18-24 meses de idade), e um grupo de indivíduos saudáveis ​​jovens (4-8 meses de controle de idade) camundongos C57 e irmãos não transgênicas da colônia SOD1. Todos os dados referem-se a bebedouro só, usando um sistema de baixa ou alta fluoroscopia energia. Não foram encontradas diferenças significativas entre os ratos jovens C57 e jovens não transgênico controle) ratos (do SOD1 colônia em relação a estes dois parâmetros andorinha;., portanto, os dados foram combinados em um grupo geral &quot;controle&quot; de camundongos jovens saudáveis ​​para comparação com os ratos e camundongos com idade entre C57 SOD1 em fase terminal taxa de Andorinha (ie,o número de andorinha durante 2 segundos consecutivos de beber ininterrupto) foi significativamente mais lenta para ratos SOD1 em comparação com os ratos e os controlos com idades entre C57. Intervalo de Inter-andorinha (ou seja, o tempo entre duas andorinhas sucessivas) não foi significativamente diferente entre os grupos. Estes resultados suportam a noção de que os perfis de disfagia provável que seja distintamente diferente para cada condição da doença (Figura 13). <img alt="Figura 13" src="/files/ftp_upload/52319/52319fig13highres.jpg" width="650" /> Figura 13:. Resultados preliminares Esta figura mostra resultados preliminares representativos dos dois parâmetros andorinha VFSS quantificados utilizando o protocolo VFSS murino: taxa (à esquerda) e intervalo inter-andorinha (à direita) engolir. Engula taxa foi significativamente mais lento para ratos SOD1 em comparação com camundongos e controles com idades entre C57. Não foram identificadas diferenças significativas entre os grupos para a inter-andorinha entreval. Linhas no topo das barras indicam diferenças estatisticamente significativas (p &lt;0,05) entre os grupos, identificados utilizando comparações de pares de Bonferroni. As barras de erro representam ± 1 SEM. <a href="https://www.jove.com/files/ftp_upload/52319/52319fig13highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models at JoVE.com

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

Este estudo adaptado com sucesso deglutição estudo (VFSS) métodos humanos para uso com modelos de doenças murino com a finalidade de facilitar a investigação translacional disfagia.

Adaptação Humanos videofluoroscópicos Andorinha Métodos de Estudo de detectar e caracterizar Disfagia em modelos de doenças Murino

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

adapting-human-videofluoroscopic-swallow-study-methods-to-detect

Research

JoVE Journal

Medicine

21.1K visualizações.  University of Missouri.  Este estudo adaptado com sucesso deglutição estudo (VFSS) métodos humanos para uso com modelos de doenças murino com a finalidade de facilitar a investigação translacional disfagia. 

Vídeo: Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, recovery from SCI typically involves long term rehabilitation. While recovery of walking ability is a primary goal in many patients early after injury, those with a motor incomplete SCI, indicating partial preservation of volitional control, may have the sufficient residual descending pathways necessary to attain this goal. However, despite physical interventions, motor impairments including weakness, and the manifestation of abnormal involuntary reflex activity, called spasticity or spasms, are thought to contribute to reduced walking recovery. Doctrinaire thought suggests that remediation of this abnormal motor reflexes associated with SCI will produce functional benefits to the patient. For example, physicians and therapists will provide specific pharmacological or physical interventions directed towards reducing spasticity or spasms, although there continues to be little empirical data suggesting that these strategies improve walking ability.
In the past few decades, accumulating data has suggested that specific neuromodulatory agents, including agents which mimic or facilitate the actions of the monoamines, including serotonin (5HT) and norepinephrine (NE), can initiate or augment walking behaviors in animal models of SCI. Interestingly, many of these agents, particularly 5HTergic agonists, can markedly increase spinal excitability, which in turn also increases reflex activity in these animals. Counterintuitive to traditional theories of recovery following human SCI, the empirical evidence from basic science experiments suggest that this reflex hyper excitability and generation of locomotor behaviors are driven in parallel by neuromodulatory inputs (5HT) and may be necessary for functional recovery following SCI. 
The application of this novel concept derived from basic scientific studies to promote recovery following human SCI would appear to be seamless, although the direct translation of the findings can be extremely challenging. Specifically, in the animal models, an implanted catheter facilitates delivery of very specific 5HT agonist compounds directly onto the spinal circuitry. The translation of this technique to humans is hindered by the lack of specific surgical techniques or available pharmacological agents directed towards 5HT receptor subtypes that are safe and effective for human clinical trials. However, oral administration of commonly available 5HTergic agents, such as selective serotonin reuptake inhibitors (SSRIs), may be a viable option to increase central 5HT concentrations in order to facilitate walking recovery in humans. Systematic quantification of how these SSRIs modulate human motor behaviors following SCI, with a specific focus on strength, reflexes, and the recovery of walking ability, are missing.
This video demonstration is a progressive attempt to systematically and quantitatively assess the modulation of reflex activity, volitional strength and ambulation following the acute oral administration of an SSRI in human SCI. Agents are applied on single days to assess the immediate effects on motor function in this patient population, with long-term studies involving repeated drug administration combined with intensive physical interventions.

This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI).

Os métodos para quantificar alterações farmacologicamente induzida na função motora em humanos SCI incompleta

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, ...

Medicina

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

Isolamento de Oligómeros PrP solúveis e insolúveis no cérebro humano normal

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Cariotipagem espectral para o Estudo de Anomalias cromossômicas em humanos e camundongos com doença renal policística

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Modelos de interação gene-ambiente para desmasca mecanismos de suscetibilidade na doença de Parkinson

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modelo murino de imagem não invasivo para detectar e monitorar o cancro do ovário Recorrência

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Isolamento e excisão de Murino Aorta; A técnica versátil no Estudo de Doenças Cardiovasculares

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Usando vírus adeno-associado como uma ferramenta para o estudo da retina Barreiras na Doença

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex Vivo Intestinal Sacs para Avaliar mucosa Permeabilidade de modelos de doença gastrointestinal

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

Métodos para aguda e subaguda Murino dos membros posteriores Isquemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Férricos induzida pelo cloreto modelos murinos Trombose

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...