Este trabalho foi suportado por concessões do Governo federal belga (IUAP P6/28), a Fundação de Pesquisa de-Flandres (FWO) (G.0565.07 e G.0686.09), o Prêmio Fundação Europeia Astellas 2009 e do Conselho de Pesquisa da KU Leuven (GOA 2009/07, e EF/95/010 PFV/10/006). PU e WE são bolsistas de doutorado da Fundação de Pesquisa de-Flandres (FWO). MB é um companheiro Marie Curie. DDR companheiro fundamental clínica do FWO.

1. Animais de Laboratório <ol><li> Os animais (ratos, ratazanas) estão alojados numa instalação para animais com um especializado 12 horas de ciclo claro-escuro e acesso ad libitum a água e alimentos granulados convencionais. Tanto a idade e sexo do animal, são parâmetros importantes, que devem ser normalizadas, de acordo com as necessidades. Nós normalmente executam cistometria em 10 - 12 semanas de idade fêmeas 5,6. </li><li> Todos os experimentos com animais são realizadas de acordo com as orientações comunitárias da União Europeia do Conselho e aprovado pelo comitê de ética local. </li></ol> 2. Anestesia <ol><li> Anestesia com isoflurano é utilizado para a realização de pequenas intervenções cirúrgicas, uma vez que é fácil de dose devido ao seu curto tempo de meia-vida. Gás adequado de lixo de limpeza é necessário quando se utiliza o isoflurano. <ol class="lalpha"><li> Os animais são colocados dentro de uma caixa fechada, gaseificada com 5% de isoflurano em oxigénio puro (1 l / min). </li><li> Após indução da anestesia, a l apropriadaevel é mantido por uma pequena máscara (um corte seringa de 10 ml) por 0,8-1 l / min de oxigénio com 1 - 1,5% de isoflurano. </li></ol></li><li> Para ratos, anestesia com uretana é usado durante as gravações funcionais (cfr. Infra). Anestesia com uretana é utilizado durante a gravação funcionais, porque, em contraste com a maioria dos outros tipos de anestésicos, o reflexo de micção não é suprimida por este agente anestésico 4. Uretano 1,2 g de peso corporal / kg é administrada por via subcutânea (sc) 60 min antes do início da gravação. Se necessário, a dose de uretano necessária para a obtenção de anestesia adequada pode ser ajustada através da administração de doses de reforço (0,1 g / kg), por via subcutânea. Deve-se notar, no entanto, que a anestesia adicional pode alterar a eficiência de esvaziamento e, consequentemente, a frequência de micção 7,8. Assim, as gravações cistométrica devem ser cuidadosamente monitorizados e descartados sempre que desvios significativos do comportamento de controle são observados. Anestésicos não deve ser administrado durante o testeOs compostos são aplicados para evitar uma má interpretação dos dados. </li></ol> 3. Procedimento cirúrgico - Implantação do Cateter Bexiga <ol><li> Os instrumentos cirúrgicos são esterilizados em autoclave antes da sua utilização. Raspar o abdômen do animal, desinfectar com álcool 70% e executar uma laparotomia mediana baixo para expor a bexiga. </li><li> Sob um microscópio cirúrgico, colocar uma sutura em bolsa na cúpula da bexiga com uma sutura não absorvível, monofilamentos (tamanho 6-0). </li><li> Realizar uma cistostomia pequenos (preferencialmente utilizar um agulha 18 G) no interior do fio de bolsa e inserir um cateter de polietileno PE 50, com uma pequena braçadeira no final (obtido aquecendo com cuidado a tubagem) através deste orifício. Nas nossas mãos, os tubos mais finos (PE 10) ter uma resistência mais elevada e mais variável. A tubagem pode ser desinfectado, colocando-o em uma solução de etanol a 70% ou esterilizados em autoclave. Antes da implantação, a tubagem deve ser lavada com solução salina. </li><li> Fixe o str bolsação em torno do tubo, com um nó cirurgião. Puxar o cateter delicadamente para fora, até que a ponta em forma de sino é posicionado logo abaixo da sutura. Empurrar um pedaço de 18 G para o cateter de sutura para evitar fugas. </li><li> Infundir um pequeno volume de solução salina (100 - 200 ul) com cuidado para verificar a existência de fugas. Se os vazamentos estão presentes, uma sutura adicional deve ser colocado. </li><li> Feche os músculos abdominais, utilizando suturas de monofilamento, deixando uma passagem para o cateter na parte superior da incisão. </li><li> Túnel do tubo na região interescapular usando uma haste oca de metal, evitando assim os animais para morder o tubo durante a experiência. </li><li> A pele é fechada com fios não absorvíveis, monofilamentos. O cateter de implante é lavado com solução salina para verificar a sua permeabilidade. </li><li> Antes de os animais acordar, analgésicos são administradas por via subcutânea (buprenorfina, 0,05 mg / kg). </li></ol> 4. Configuração e cistometria <ol><li> Dependendo do questi experimentalem (e das espécies utilizadas) é realizada de cistometria acordada (retenção ou incontinência) ou de uretano-anestesiadas animais. Considerando cistometria é perfeitamente viável em ratos conscientes, que tendem a manter a calma em um ambiente contido, que normalmente executam cistometria sob anestesia com uretana (1,2 mg / kg, por via subcutânea) quando utilizando camundongos 3,5,6,9. </li><li> Em ratos anestesiados, a temperatura do corpo é constantemente monitorizado e mantido a 36,5 ° C ± 0,2 ° C por meio de uma sonda de temperatura e uma lâmpada de aquecimento. </li><li> A configuração é calibrado antes de cada experiência de acordo com as instruções dos fabricantes. </li><li> O cateter de implante é ligado por um topo luer a uma torneira de 3 vias, que está ligado ao transdutor de pressão (Edwards Lifesciences) de um lado e uma bomba de perfusão, por outro (Harvard Apparatus). O transdutor de pressão é ligado através de um amplificador (78534c monitor, Hewlett Packard) com o sistema de aquisição de dados (DI-730-USB, Dataq instrumentos) e um co-mputer. Windaq / Lite software pode ser utilizado para gravação. </li><li> A bomba de infusão é iniciado, permitindo a infusão de solução salina ou PBS a, por exemplo, 100 ul / min (em ratos) ou 20 ^ L / min (em ratos). Como tal a bexiga será preenchida a uma velocidade constante, enquanto a pressão intravesical é gravado. </li><li> A Figura 5 representa um traço típico de pressão: durante a infusão de fluidos, existe uma acumulação de pressão lento na bexiga, até um certo volume / pressão é alcançada, o limiar de micturição. Uma vez que esse limite for atingido, a bexiga e, posteriormente, irão contrair o esfíncter urinário se abre, permitindo a passagem da urina através da uretra. Como tal, a bexiga é esvaziada, a contracção irá parar e a pressão irá cair de novo para o nível &quot;basal&quot;. </li><li> À medida que o rato é colocado numa gaiola metabólica e, acima de uma balança digital, do volume vazio pode ser medido ao mesmo tempo, dando informações sobre o volume vazio. Devido às pequenas quantidades anuladas em milhasce, pode ser muito difícil usar este sistema. Portanto, o volume anulada é determinada utilizando pequenos filtros de papel pesado antes e após a micção. </li><li> Tipicamente, é permitir que o sistema equilibrar durante 30 min, seguido de um período de registo de 30 minutos. Em seguida, a droga pode ser administrada sistemicamente ou intravesical e mais 30 min são gravadas. </li><li> Os dados gravados podem ser exportados como arquivos. CSV que podem ser importados para um software especializado para análise quantitativa. Normalmente usamos Origem (Origin Corporation, MA, EUA). </li><li> Depois de experiências, os animais são sacrificados por deslocamento cervical (ratos) ou intoxicação CO 2 (ratos). </li></ol> 5. Resultados representativos <img alt="Figura 1" src="/files/ftp_upload/3869/3869fig1.jpg" /> Figura 1. Visão geral laparotomia. A) Coloque o rato na posição supina. B) Shave e antiseptically preparar o local da cirurgia. Incisão C) da pele. D) Incisão of os músculos abdominais, e exposição da bexiga. <img alt="Figura 2" src="/files/ftp_upload/3869/3869fig2.jpg" /> Figura 2. Uma sutura em bolsa. <img alt="Figura 3" src="/files/ftp_upload/3869/3869fig3.jpg" /> Figura 3. Implantação do cateter. <img alt="Figura 4" src="/files/ftp_upload/3869/3869fig4.jpg" /> Figura 4. Escavação de túneis do cateter. Exemplos de medidas da pressão intravesical obtidos durante a perfusão de uma solução salina em ratos conscientes e um rato anestesiado são mostrados na Figura 5. Vários parâmetros podem ser extraídos a partir do sinal de pressão (por exemplo, o intervalo de intercontractile, a pressão de linha de base e a pressão de limiar). Para descrições completas desses parâmetros, consulte Andersson et al. (Ref 4) e Yoshiyama et al. (<html> ref 10). Nós temos utilizado recentemente cistometria para identificar os alvos moleculares de óleo de mostarda (MO), um composto muito reactivo que tem sido muito utilizada em modelos experimentais de inflamação e a hiperalgesia de órgãos viscerais, como a bexiga urinária 11,12. Infusão intravesical de 10 mM PB induziu um forte aumento da frequência de micção (diminuição do intervalo intercontractile) em ratinhos do tipo selvagem (Figura 6A, B) e uma diminuição do volume vazio 6. Curiosamente, MO induziu alterações semelhantes em ratos deficientes do receptor de MO TRPA1. Em contraste, MO induziu alterações muito mais fracos em parâmetros cistométricos em ratinhos KO TRPV1 que em ratinhos WT e não tinha qualquer efeito nos ratinhos KO Trpa1/Trpv1. Juntamente com as medições de libertação do gene da calcitonina Related Peptide (CGRP), 6, estes dados indicam que demonstram TRPV1 pode desempenhar um papel-chave na irritação visceral induzida por MO. ve_content &quot;&gt; <img alt="Figura 5" src="/files/ftp_upload/3869/3869fig5.jpg" /> Figura 5. Traços representativos de pressão intravesical registados num rato consciente fêmea (A) e num rato anestesiado fêmea (B). A menor pressão é definida como a &quot;pressão de linha de base&quot; (setas vermelhas). A pressão no final da fase de enchimento é marcada com setas azuis. O volume de fluido administrado entre estes pontos, dividida pela diferença de pressão, permite o cálculo da complacência da parede da bexiga (compliance = infusão volume / (pressão limite -. Pressão de linha de base) O &quot;intervalo intercontractile&quot; (ICI) é o tempo entre duas contrações micção. <img alt="Figura 6" src="/files/ftp_upload/3869/3869fig6.jpg" /> Figura 6. Efeitos da aplicação intravesical de óleo de mostarda sobre o padrão de cistometria no tipo selvagem e camundongos knockout TRPA1, TRPV1 e Trpa1/Trpv1.(A) Os exemplos representativos de mudanças na pressão intravesical gravados no WT, KO TRPA1, KO TRPV1 e ratinhos KO Trpa1/Trpv1 em resposta à infusão de solução salina e 10 mM MO. (B) Tempo de decurso da frequência de micção instantâneo médio antes e durante a infusão intravesical de MO. Para todos os ratinhos, os dados foram normalizados para a frequência média obtida durante a infusão de solução salina. Estes dados são adaptados a partir de Everaerts et al. (Ref 6), com a permissão da Elsevier.

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A técnica aqui apresentada permite cistometria realizar as medições in vivo da função da bexiga em modelos animais. Os ratos são, provavelmente, o modelo animal mais utilizado. Os ratinhos são mais difíceis de manusear, mas oferece a vantagem de utilizar animais geneticamente manipulados. Por causa da dificuldade técnica de usar ratos conscientes, que tendem a ser muito ativos, resultando em afrouxamento do cateter implantado e alterações nas pressões intra-abdominais que podem influenciar ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Nome comercial, a empresa, </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> uretano </td><td> Uretano, Sigma-Aldrich </td><td> 315419 </td><td> grupo 2B cancerígena </td></tr><tr><td> isoflurano </td><td> Isoba, Saúde Animal Schering-Plough </td><td></td><td></td></tr><tr><td> cateter de polietileno </td><td> Intramedic tubagem de polietileno PE50, Becton Dickinson </td><td> 427411 </td><td></td></tr><tr><td> microscópio cirúrgico </td><td> Op-Mi 6, Carl Zeiss </td><td> Op-Mi 6 </td><td></td></tr><tr><td> bolsa-corda sutura </td><td> Prolene 6/0, Ethicon </td><td> 8610H </td><td></td></tr><tr><td> fáscia e sutura da pele </td><td> Ethilon 4/0 e 5/0, Ethicon </td><td> 662G ou 661G </td><td></td></tr><tr><td> analgésicos no pós-operatório </td><td> Temgesic, Saúde Animal Schering-Plough </td><td></td><td> dosagem para ratos: 0,05 mg / kg </td></tr><tr><td> amplificador </td><td> 78534c monitor, Hewlett Packard </td><td></td><td></td></tr><tr><td> balanças analíticas e equilíbrio de software de aquisição de dados </td><td> FZ 300i, A &amp; D </td><td> FZ-300i </td><td></td></tr><tr><td> bombas de infusão </td><td> bomba 33, Harvard Apparatus </td><td> HA33 </td><td></td></tr><tr><td> sistema de gravação cistometria </td><td> Instrumentos Dataq, DI-730 e série Windaq / Lite </td><td> DI-730-USB Windaq / Lite </td><td></td></tr><tr><td> Registro de temperatura </td><td> Fluke 52 KJ termômetro </td><td> 52 KJ </td><td></td></tr><tr><td> transdutores de pressão </td><td> Edwards Lifesciences, conjunto de monitoramento de pressão </td><td> T322247A </td><td></td></tr></tbody></table>

the use of cystometry in small rodents: a study of bladder chemosensation

<p class="jove_content"&gt; As trato urinário inferior (LUT) funciona como um reservatório dinâmico que é capaz de armazenar e urina de forma eficiente expeli-lo em um momento conveniente. Embora o armazenamento da urina, no entanto, a bexiga é exposta por períodos prolongados em produtos residuais. Ao agir como uma barreira forte, o revestimento epitelial da LUT, o urotélio, evita re-absorção de substâncias nocivas. Além disso, os produtos químicos nocivos estimular inervação nociceptiva da bexiga e iniciar a micção contrações que expelem conteúdo da bexiga. É interessante notar que a sensibilidade da bexiga para produtos químicos nocivos tem sido utilizado com sucesso na prática clínica, por infusão intravesical a capsaicina agonista TRPV1 para tratar hiperactividade da bexiga neurogénica<sup&gt; 1</sup&gt;. Isso ressalta a vantagem de ver a bexiga como um órgão chemosensory e pede para a investigação clínica adicional. No entanto, questões éticas limitam seriamente as possibilidades de realizar, em seres humanos, as medidas invasivas que são necessários para desvendar as bases moleculares da farmacologia clínica LUT. Uma forma de ultrapassar esta limitação é a utilização de vários modelos animais<sup&gt; 2</sup&gt;. Aqui descreve-se a aplicação de cistometria em ratinhos e ratos, uma técnica que permite a medição da pressão intravesical, em condições de perfusão controlada bexiga.</p&gt;<p class="jove_content"&gt; Após laparotomia, um cateter é implantado na cúpula da bexiga e encapsulado por via subcutânea na região interescapular. Em seguida, a bexiga pode ser enchido a uma velocidade controlada, ao passo que a uretra é deixado livre para a micção. Durante os ciclos repetitivos de enchimento e esvaziamento, a pressão intravesical pode ser medida através do cateter implantado. Como tal, as variações de pressão podem ser quantificados e analisados. Além disso, a medição simultânea do volume anulado permite distinguir contrações esvaziamento de não esvaziamento contrações<sup&gt; 3</sup&gt;.</p&gt;<p class="jove_content"&gt; Importante, devido às diferenças no controlo da micção entre roedores e humanos, as medições cistométrica nestes animais tem apenas um valor limitado de translação<sup&gt; 4</sup&gt;. No entanto, eles são muito instrumental no estudo da fisiopatologia da bexiga e em farmacologia pré-clínicos experimentais configurações. Uma pesquisa recente usando esta técnica revelou o papel chave das novas jogadores molecular nas propriedades mecânico-químico-sensoriais e da bexiga.</p

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The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Cistometria é uma técnica eficiente para medir a função da bexiga em animais de pequeno porte<em&gt; In vivo</em&gt;. A bexiga é infundida continuamente a taxas controladas por meio de um cateter intravesical, ao passo que a uretra é deixado livre para a micção. Isto permite a repetição de enchimento e esvaziamento da bexiga, enquanto a pressão intravesical e volume vazio são gravadas.

O Uso de cistometria em pequenos roedores: Um Estudo de Chemosensation Bexiga

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

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Research

JoVE Journal

Medicine

22.3K visualizações.  KU Leuven, Belgium. Cistometria é uma técnica eficiente para medir a função da bexiga em animais de pequeno porte In vivo. A bexiga é infundida continuamente a taxas controladas por meio de um cateter intravesical, ao passo que a uretra é deixado livre para a micção. Isto permite a repetição de enchimento e esvaziamento da bexiga, enquanto a pressão intravesical e volume vazio são gravadas.

Vídeo: The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Injeção do mouse parede da bexiga

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos Linhas tumor neuroendócrino de células como um modelo tridimensional para o estudo da terapia de tumores humanos Neuroendócrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Respostas Bexiga Distensão Evocado Visceromotor como um modelo para Bexiga dor em camundongos

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Membro Remoto Pré-Condicionamento Isquêmico: A Técnica neuroprotetor em roedores

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

Realizando Subretinal Injeções em roedores para entregar epitélio pigmentar da retina células em suspensão

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Avaliação da Eficácia da<em&gt; H. pylori</em&gt; Protein HP-NAP como uma ferramenta terapêutica para o tratamento de cancro da bexiga em um Modelo murino Orthotopic

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Uso de ultra-alta MRI campo em pequeno roedor modelos de doença renal policística para<em&gt; In Vivo</em&gt; Fenotipagem and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

Uso de um Modelo de Leitão para o Estudo da Neurotoxicidade de Desenvolvimento induzida por Anestesismo (AIDN): Uma Abordagem de Neurociências Translacionais

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

-Nervo poupadores Mid-obstrução uretral (Nemo) em fêmeas pequenos roedores

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...