Gostaríamos de agradecer um contrato (WS1763368) da Pfizer Pharmaceuticals para o apoio neste trabalho. Gostaríamos também de agradecer à Arábia Saudita Ministério do Ensino Superior (Mohe) para seu apoio.

1. O Exercício <ol><li> O músculo de interesse para este experimento foi os flexores do cotovelo (bíceps braquial). </li><li> A força muscular foi medida para cada participante para ser capaz de dar a cada indivíduo uma resistência adequada. Isso foi determinado pelo teste de cada participante para a sua máxima resistência (RM). </li><li> Para testar a RM, foi utilizado um dispositivo medidor de tensão a um computador através de um módulo amplificador Biopac (DA-100C) bioelétrica (Biopac Systems, Goleta, CA) para medir a força muscular. O módulo foi conectado a um MP-100 analógico para amostragem conversor digital em uma freqüência de 1.000 hertz por segundo, e com uma resolução de 24 bits (Fig. 10). </li><li> O dispositivo medidor de tensão foi fixada a um banco em um ângulo de 45 °. Os sujeitos foram orientados a sentar-se atrás do dispositivo e descansar os cotovelos sobre a área acolchoada, de modo que a força é o esforço através de seus pulsos. Esta foi a melhor maneira de garantir que o assunto não serát recrutar qualquer outro músculo do que o bíceps (Fig. 11). </li><li> Força foi determinada em 3 ocasiões, com cada contração sendo 3 segundos de duração, com aproximadamente 45 segundos separando as contrações. A média das três medidas foi a RM. </li><li> Após a determinação da RM para o músculo bíceps de cada participante, a sessão destinada do exercício foi realizado com 35% de sua RM. </li><li> Todos os sujeitos foram submetidos ao mesmo exercício utilizando halteres ponderada adequada para induzir a dor muscular (DOMS). Este foi realizado, fazendo 4 séries de 25 repetições de bíceps curls concentração enquanto estiver sentado em uma cadeira, e com os cotovelos apoiados nas coxas (Fig. 12). </li><li> Cada sujeito foi dado um prazo de 90 segundo de descanso entre cada série. Assuntos tanto fez todo o conjunto de 25 repetições, ou foram instruídos a parar se eles não conseguiram controlar o peso de forma constante durante o exercício. </li></ol> 2. Infra-Red Camera preparação e Setup <ol><li> A sala onde a imagem infra-vermelha tem lugar foi fixado em uma temperatura constante para minimizar qualquer viés externo de diferenças na temperatura ambiente, o que poderia levar a falsas leituras térmicas. Para o propósito desta experiência que tivemos uma sala com temperatura controlada, que foi mantida em aproximadamente 23 ° C (+ / - 0,5 ° C). </li><li> A câmera foi fixada em uma distância de 1 metro de distância, e em um ângulo perpendicular à pele que está sendo medido (Fig. 9a) *. </li><li> Após a distância necessária foi criada, os indivíduos foram aconselhados a ficar parado até que a imagem tenha sido tomada. Isto não deve levar mais de um par de segundos, mas é muito crítico para minimizar o movimento para assegurar a precisão da imagem obtida. </li><li> É preferível que o quarto tem mais escura tinta colorida, ao invés de cores mais claras, para minimizar qualquer interferência infra-vermelho. </li><li> Iluminação também é fundamental quando se lida com infra-vermelho imagens, because fonte de luz que emitem infra-vermelho ondas como iluminação fluorescente ou tungstênio poderia dar falsas leituras de altura. A melhor opção de iluminação seria uma sala equipada com uniforme luzes LED, como luzes LED dificilmente produzirá qualquer interferência infra-vermelho (Fig. 9b) *. </li></ol> * Uma série de testes foram feitos em nossos laboratórios usando o FLIR 660 IR Camera (Fig. 8), onde compararam as imagens da pele em diferentes ângulos (0 (perpendicular), 15, 30, 45 e 60 graus), e a distâncias diferentes (1, 2 e 5 metros) a partir da pele, para detectar com precisão a temperatura da pele. Todas as imagens foram comparados com termopares calibrados, ea melhor correlação entre as imagens e as leituras termopar foi em um ângulo perpendicular ea uma distância de 1 metro de distância da pele (r = 0,93). Os ângulos e distâncias diferentes causou uma perda de pixilation, e diminuiu a correlação geral entre as imagens e as leituras do termopar. 3. Imagem Acquirement <ol><li> Para efeitos do presente experimento, a imagem do músculo exercitado foi tomada antes do exercício, e em 24, e 48 horas pós-exercício. </li><li> O calor do corpo de outras fontes que o alvo poderia perturbar a imagem térmica e dar leituras falsas. Por esta razão, ninguém deve estar de pé ao lado ou atrás do alvo pretendido. </li><li> Nesta investigação, as imagens de ambos os braço do exercido e não exercidas foram tomadas para fins de comparação. Nós exercido um dos braços, como foi mencionado anteriormente, e outro braço foi utilizado como controle (Fig. 4 e 5). </li><li> Números das imagens da câmera de infravermelho foram registrados imediatamente em uma planilha separada, como poderia ser difícil identificar qual a imagem pertence a quem. </li></ol> 4. Processamento de Imagem &amp; Análises <ol><li> As imagens adquiridas IR foram processadas utilizando o &quot;ThermoVision ExaminIR&quot; Versão do software: 1.10.2. </li><li> Após selecionar a imagem necessária parar análise, quatro regiões de interesse foram identificadas na imagem adquirida do braço usando caixas de estatísticas sobre a interface de software (Fig.6). </li><li> Quando as regiões necessárias em todo o braço ter sido localizado, o software mostra as médias e desvios padrão das temperaturas para cada uma das regiões selecionadas. Podemos, então, se cruzam comparar cada região individualmente ou obter uma temperatura média de todo o braço (Fig. 7). </li></ol> 5. Escala Analógica Visual e Análise de sangue <ol><li> A escala visual analógica (EVA) foi utilizada para avaliar a dor subjetiva do braço. A escala teve a 10 cm (100mm) longa linha marcada &quot;nenhuma dor&quot; em uma extremidade, e &quot;extremamente doloridos&quot; na extremidade oposta. Cada participante foi orientado a fazer uma marca ao longo da linha de 10 cm para indicar a sua resposta à dor. </li><li> O VAS foram administrados aos sujeitos antes do exercício, 24 horas após o exercício, e em 48 horas. </li><li> Sangue periférico foi colselecionado dos sujeitos para medir os níveis de concentração de mioglobina no sangue. </li><li> O sangue foi retirado da veia antecubital assuntos antes do exercício, a 30 minutos após o exercício foi superior, e em 48 horas. </li><li> O sangue foi centrifugado a 4000 rpm por 10 min para separar o soro das células. As amostras foram então armazenadas a -80 ° C até as análises de mioglobina foi feito. </li><li> Mioglobina foi medida usando um TOSOH &quot;AIA-360&quot; Analyzer Imunoensaio automatizado enzima (TOSOH Corp, Tokyo, Japão). Os kits de mioglobina Assay (Myo 025297, ST AIA-PACK mioglobina) foram utilizados de acordo com a fabrica instruções. </li></ol> 6. Resultados representante Os resultados do IR imagens térmicas tomadas durante esta investigação estão claramente representados na figura 1. Imagens tiradas nos 3 períodos de tempo (pré-exercício, 24 horas pós-exercício, e 48 horas pós-exercício) para os braços exercida dos 41 indivíduos, mostrouum notável aumento de temperatura no dia 2 (24 horas pós-exercício) quando comparado ao pré-exercício temperaturas, e as temperaturas tomadas em 48 horas. Como mostrado na figura 1, a temperatura média foi de 32,80 pele + / - 1,03 ° C para o dia 1 (pré-exercício), e 33,96 + / - 1,46 ° C para o dia 2 (24 horas pós-exercício), e 32,82 + / - 1,29 para o dia 3 (48 horas pós-exercício). Esta diferença na temperatura da pele do dia 1 ao dia 2 foi significativa (ANOVA p &lt;0,01). No entanto, para o braço não-exercido, as mudanças entre os três períodos de tempo não eram evidentes. A Figura 1 mostra que a temperatura média foi de 33,08 pele + / - 0,83 ° C para o dia 1 (pré-exercício), e 32,79 + / - 1,42 ° C para o dia 2 (24 horas pós-exercício), e 33,17 + / - 0,95 para o dia 3 (48 horas pós-exercício). Esta diferença na temperatura da pele durante os 3 dias não foi significativa (ANOVA p = 0,38). Os resultados das leituras dor da VAS são mostrados na figura 2. Como visto na figA Figura 2, a dor relatada teve um aumento dramático nos dias 2 e 3. Níveis de dor do músculo exercitado aumentou de 3.6 + / - 6,1 no dia 1, para 36,3 + / - 22,8 no dia 2, e 37,5 + / - 25,3 no dia 3. Este aumento desde o dia 1 foi significativa (ANOVA p &lt;0,01). Os resultados dos níveis de concentração de mioglobina são mostrados na figura 3. Como pode ser visto nesta figura, não havia quase nenhuma mudança entre as duas concentrações de mioglobina no dia 1 (pré, pós-exercício e 30 minutos). Mas no dia 3, o aumento da mioglobina foi muito grande. Este aumento no dia 3 foi de cerca de 147 nanogramas por mililitro (ng / mL) de sangue quando comparado com os 2 primeiros concentrações no dia 1. Concentrações de mioglobina foram 30,12 + / - 7,66 ng / mL no início do estudo, 31,66 + / - 11,89 ng / mL 30 minutos pós-exercício, e 178,96 + / - 249,51 ng / mL no dia 3. Este aumento no dia 3 foi altamente significativa (ANOVA p &lt;0,01). A análise de correlação foi feita entre as temperaturas da peleobtidos a partir das imagens IR, e os níveis de VAS dor. Verificou-se que havia uma correlação significativa entre as leituras de VAS no dia 2, ea medição da temperatura da pele no dia 2. Essa correlação foi significativa (r = 0,312, p &lt;0,05). No entanto, não houve correlação evidente entre as leituras VAS e as temperaturas da pele no dia 3. Essa correlação foi insignificante (r = 0,047, p = 0,77). <img src="/files/ftp_upload/3551/3551fig1.jpg" alt="Figura 1"/> Figura 1. Um gráfico representativo das diferenças de temperatura da pele nos braços exercidas (Diamonds), e não-exercido braços (quadrados) dos 41 temas ao longo do período de 3 dias. <img src="/files/ftp_upload/3551/3551fig2.jpg" alt="Figura 2"/> Figura 2. Um gráfico representativo das diferenças de dor muscular percebido medida com o VAS durante o período de tempo três dias para todos os 41 indivíduos. Figura 3. Um gráfico representativo das diferenças nas concentrações de mioglobina para todos os 41 temas ao longo dos três períodos de tempo. <img src="/files/ftp_upload/3551/3551fig4.jpg" alt="Figura 4"/> Figura 4. A) imagem de um típico IR do braço exercido um sujeito antes do exercício. B) uma imagem IR do braço mesmos assuntos 24 horas após o exercício. <img src="/files/ftp_upload/3551/3551fig5.jpg" alt="Figura 5"/> Figura 5. A) imagem de um típico IR no braço de um sujeito não-exercidos antes do exercício. B) uma imagem IR do braço mesmos assuntos 24 horas após o exercício. <img src="/files/ftp_upload/3551/3551fig6.jpg" alt="Figura 6"/> Figura 6. Uma ilustração das 4 regiões de interesse para a análise da thermal imagem do braço. <img src="/files/ftp_upload/3551/3551fig7.jpg" alt="Figura 7"/> Figura 7. Interface Software para o &quot;ThermoVision ExaminIR&quot; mostrando as quatro caixas de juros sobre uma imagem IR de um braço exercido. Também são mostradas as interpretações estatísticas para cada caixa. <img src="/files/ftp_upload/3551/3551fig8.jpg" alt="Figura 8"/> Figura 8. A câmera IR térmico utilizado para esta investigação (FLIR 660). <img src="/files/ftp_upload/3551/3551fig9.jpg" alt="Figura 9"/> Figura 9. A) A instalação da Câmara IR 1 metro de distância do braço assuntos. B) As luzes LED usada no laboratório onde as imagens foram tiradas. <img src="/files/ftp_upload/3551/3551fig10.jpg" alt="Figura 10"/> Figura 10. A) Os Módulos Biopac utilizado para medir a força muscular. B) O dispositivo medidor de tensão fixa para a 45 ° banco inclinado e ligado ao sistema Biopac. <img src="/files/ftp_upload/3551/3551fig11.jpg" alt="Figura 11"/> Figura 11. Um exemplo de tema exercer força no dispositivo medidor de tensão. <img src="/files/ftp_upload/3551/3551fig12.jpg" alt="Figura 12"/> Figura 12. Um assunto submetidos ao protocolo de exercícios para induzir a dor muscular.

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O objetivo principal desta investigação foi avaliar a utilidade da imagem IR térmico em detectar e medir a dor muscular após o exercício extenuante, e nossos resultados sugerem que IR imagem poderia ser uma técnica válida para detectar DOMS, especialmente nas primeiras 24 horas de exercício. Isto não é surpreendente, como Pennes 18 forneceu um modelo muito detalhado do fluxo de calor dos músculos para a pele nos membros. Este modelo prevê que o calor em tecidos mais profundos, como músculos pode ...

<table border="1"><tbody><tr><td> Nome do dispositivo </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Infra-Red Camera Thermal </td><td> FLIR </td><td> FLIR SC660 </td><td></td></tr><tr><td> Software de análise térmica Infra-Vermelho </td><td> ThermoVision ExaminIR </td><td> Software Versão 1.10.2 </td><td></td></tr><tr><td> Módulo Amplificador bioelétrica </td><td> Biopac </td><td> DA100C </td><td> O DA100C fornece ajustes de ganho variável, e referências de tensão ajustável. </td></tr><tr><td> Analógico para o Módulo Conversor Digital </td><td> Biopac </td><td> MP100 </td><td></td></tr><tr><td> Analisador de imunoensaio automatizado enzima </td><td> TOSOH </td><td> AIA -360 </td><td> Este dispositivo foi utilizado para analisar as amostras de sangue, e obter as leituras mioglobina. </td></tr>
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the use of thermal infra-red imaging to detect delayed onset muscle soreness

Dor muscular de início retardado (DOMS), também conhecido como exercício lesão muscular induzida (EIMD), é comumente experimentada em indivíduos que tenham sido fisicamente inativos por períodos prolongados de tempo, e começar com um ataque inesperado de exercício 1-4, mas também pode ocorrer em atletas que se exercitam além dos seus limites normais de treinamento 5. Os sintomas associados a este fenômeno doloroso pode variar de sensibilidade muscular leve, a dor debilitante severa 1,3,5. A intensidade desses sintomas e os aumentos desconforto relacionado nas primeiras 24 horas após o término do exercício, e picos entre 24-72 horas pós-exercício 1,3. Por esta razão, DOMS é uma das formas mais comuns recorrentes de lesões esportivas que podem afetar o desempenho de um indivíduo, e se tornar intimidante para 1,4 muitos. Para as últimas 3 décadas, o fenômeno DOMS ganhou uma quantidade considerávelde interesse entre os pesquisadores e especialistas em fisiologia do exercício, esportes e campos de reabilitação 6. Tem havido uma série de estudos publicados investigando esta ocorrência dolorosa no que diz respeito a seus mecanismos subjacentes, intervenções terapêuticas e estratégias preventivas 1-5,7-12. No entanto, é evidente a partir da literatura que DOMS não é uma patologia fácil de quantificar, como há uma enorme quantidade de variabilidade entre as ferramentas de medição e os métodos utilizados para quantificar esta condição 6. É óbvio que nenhum acordo foi feito em uma melhor medida de avaliação para DOMS, o que torna difícil verificar se uma intervenção específica realmente ajuda a diminuir os sintomas associados a este tipo de dor ou não. Assim, a dor tardia pode ser visto como algo ambíguo, porque muitos estudos dependem de medição dor usando uma escala visual analógica (VAS) 10,13-15, que é uma medida subjetiva, em vez de um objetivo. Mesmo que a agulhabiópsias do músculo, e os níveis sanguíneos de proteínas myofibre pode ser considerado um padrão-ouro para cerca de 6, grandes variações em algumas dessas proteínas do sangue têm sido documentados 6,16, além dos altos riscos, por vezes, associada a técnicas invasivas. Portanto, no presente inquérito, testamos uma térmica infra-vermelha técnica de imagem (IR) da pele acima do músculo exercitado para detectar a dor muscular associada. Infra-vermelho termografia tem sido usada, e encontraram para ser bem sucedido na detecção de diferentes tipos de doenças e infecções desde 1950, 17. Mas, surpreendentemente, quase nada tem sido feito em DOMS e mudanças na temperatura da pele. O principal objectivo desta investigação era examinar mudanças nos DOMS usando esta técnica segura e não invasiva.

Watch this Scientific Journal Video about The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness at JoVE.com

The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

O objetivo desta pesquisa foi avaliar se usando uma câmera infra-vermelha térmica é uma ferramenta válida para detectar e quantificar a dor muscular após o exercício.

O Uso de Imagens Infra-Vermelho térmico para detectar dor muscular de início retardado

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically ...

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Research

JoVE Journal

Medicine

19.1K visualizações.  Loma Linda University. O objetivo desta pesquisa foi avaliar se usando uma câmera infra-vermelha térmica é uma ferramenta válida para detectar e quantificar a dor muscular após o exercício.

Vídeo: The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

Ablação térmica para o tratamento de tumores abdominais

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

Medicina

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

Visualization quantitativos e Detecção de Câncer de Pele Usando imagens térmicas dinâmico

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Uso de Modelo Animal de Sepse para avaliar novas terapias à base de plantas

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

O Uso de desafio-Farmacológicas fMRI em pesquisa pré-clínica: aplicação ao sistema 5-HT

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Imagem Molecular para atingir as células progenitoras transplantadas musculares

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

High-throughput Citometria de Fluxo Ensaio Celular para detectar anticorpos em N-metil-D-aspartato ou dopamina-2 Receptor em Soro Humano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

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 ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitativa Ressonância Magnética da doença do músculo esquelético

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Calcificação das células do músculo vascular liso e de imagem da aorta calcificação e inflamação

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.