We would like to acknowledge Beth Levine for critically reviewing the manuscript, Valerie Tassinari for developing the genotyping protocol, and Matt Ferola and Carlos Maya for their exceptional animal care.

Todos os experimentos são conduzidos de acordo com os protocolos descritos pelos Institutos Nacionais de Saúde Guia para o Cuidado e Uso de Animais e foram aprovados pelo cuidado com os animais institucional e uso comitê do ALS TDI (IACUC). 1. Os animais, habitação e desenho do estudo Nota: O rato colônia SOD1-G93A foi derivado do elevado número de cópias B6SJLTgN (SOD1G93A) estirpe 1Gur originalmente produzido por Gurney et al 7.. A colónia está actualmente a ser mantido pelo cruzamento de machos transgénicos C57BL / 6-SJL com o tipo selvagem fêmeas C57BL / 6-F1 SJL. Os animais F1 são gerados pelo cruzamento de SJL machos com fêmeas B6. Os ratinhos são enviados para a ALS TDI semanal em 35-45 dias de idade. <ol><li> Permitir ratinhos pelo menos uma semana de aclimatação à unidade de animais (12-h luz / escuridão a uma temperatura de 18-23 ° C e 40-60% de humidade) antes de atribuir a um estudo. </li><li> Casa masculino SOD1 G93A-mice um rato por gaiola, desde grupo alojados machos tendem a lutar. Casa ratos fêmea SOD1-G93A até dois por gaiola. Proporcionar um enriquecimento ambiental, na forma de cabanas de plástico. Fornecer alimentos e água ad libitum, a menos que os ratos são parte de um estudo que requer a administração do fármaco em água potável / alimento. Nestes casos, ração e comida registro e água. </li><li> Começar um estudo fármaco-eficácia em torno de 50 dias de idade e incluem 64 camundongos com equilíbrio de gênero de correspondência de maca (32 por coorte). Por fim, garantir que os investigadores realizam os estudos estão cegos para as condições experimentais. Nota: É recomendado que um estudo típico droga-eficácia começa no dia de idade 50 Contudo, com base no tipo de intervenção terapêutica pode ser preferível começar o estudo mais cedo ou mais tarde. Nota: Um estudo com 32 ratos por grupo é alimentado para detectar drogas efeitos específicos de cada sexo. Se não forem necessárias comparações específicas de gênero, 24 ratos por grupo deve ser suficiente para detectar drogas efeitos globais.</li></ol> 2. Peso Corporal <ol><li> Os pesos corporais para todos os ratinhos a ficha em estudos diariamente com uma precisão de um décimo de um grama. Nota: O peso corporal é um indicador sensível da progressão da doença e de qualquer mal-estar que pode resultar do tratamento crônico de drogas. </li><li> Corpo pesos mouse variar consideravelmente durante o curso de um dia, sendo maior no período da manhã, assim gravá-los em torno do mesmo tempo todos os dias. </li></ol> 3. Neurological Scoring System (NeuroScore) <ol><li> Avaliar pontuações neurológicas (NS) para cada rato diariamente, observando o mouse sob as seguintes três condições realizadas sequencialmente: a) o mouse é suspenso pela cauda, ​​b) o mouse é permitido andar, e após o início da paralisia c) a rato é colocado no seu lado. <ol><li> Para o teste de suspensão pela cauda, ​​segurar o rato de cerca de 1,5 &quot;a partir da base da cauda sobre a parte superior do fio da sua gaiola durante 1-2 segundos, para longe da bacia de alimentos, enquanto observing membros posteriores. Repita o teste de suspensão 3 vezes e registar o resultado mais consistente. </li><li> Para o teste de caminhada, coloque o mouse sobre uma superfície limpa que fornece alguma tração e uma curta distância de 25 cm (eg., Uma toalha de papel gravada para baixo para evitar o deslize). Permitir que o mouse para andar um total de 75 cm (comprimento 3X da toalha de papel), observando a marcha. </li><li> Para o teste de &quot;reflexo de endireitamento&quot;, coloque o mouse sobre o lado esquerdo ou direito e usando um cronômetro medir o tempo que leva para se endireitar-se sem ajuda de ambos os lados. Nota: Apenas um estudo deste teste é necessário. </li></ol></li><li> Determine o NS de cada membro posterior (esquerda ou direita) de forma independente em uma escala de 0 a 4. Nota: Por exemplo, uma pontuação de 0, 1 indica que o membro esquerdo NS é 0, enquanto o direito dos membros posteriores NS é 1. </li><li> Atribuir pontuações neurológicas (0-4) que correspondem ao seguinte conjunto de observações (resumo no Quadro 1):<ol><li> Atribuir NS 0 (pré-sintomático) se o seguinte é observado: Quando o mouse é suspenso pela cauda, ​​o membro posterior apresenta um splay normal, ou seja, ele está totalmente estendido longe da linha média lateral e permanece nessa posição por 2 segundos ou mais longo. Quando o mouse é permitido andar, marcha normal é observado. </li><li> Atribuir NS 1 (primeiros sintomas) se o seguinte é observado: Quando o mouse é suspenso pela cauda, ​​o membro posterior apresenta um splay anormal, ou seja, é recolhido ou ruiu parcialmente em direção a linha média lateral ou ela treme durante a suspensão da cauda ou é retraída / apertou. Quando o mouse é permitido andar, marcha normal ou um pouco lento é observado. </li><li> Atribuir NS 2 (início da paresia) se o seguinte é observado: Quando o mouse é suspenso pela cauda, ​​o membro posterior é parcial ou completamente destruídas, não se estendendo muito. (Há ainda pode ser o movimento articular). Quando o mouse é permitido andar, o membro posterior é usado para mot frenteion no entanto enrolar os dedos dos pés para baixo, pelo menos duas vezes durante a 90 centímetros caminhada ou qualquer parte do pé está se arrastando ao longo fundo da gaiola / table. Quando o mouse é colocado em seu lado esquerdo e direito, ele é capaz de endireitar-se dentro de 10 segundos de ambos os lados. </li><li> Atribuir NS 3 (Paralisia) se o seguinte é observado: Quando o mouse é suspenso pela cauda, ​​há paralisia rígida na hindlimb OU movimento articular mínima. Quando o mouse é permitido andar há movimento para a frente no entanto, o membro posterior não está sendo usado para o movimento para a frente. Quando o mouse é colocado em seu lado esquerdo e direito, ele é capaz de endireitar-se dentro de 10 segundos de ambos os lados. Nota: Raramente, após o início da paralisia, umidade urina pode aparecer nos membros posteriores. Umidade urina não tratada pode resultar em urina &quot;queimadura&quot; e lesões de pele. Trate umidade urina por corte o cabelo, aplique água morna para remover absorve a urina, e secar delicadamente. Se as lesões cutâneas estão presentes aplicar antibioTic pomada. </li><li> Atribuir NS 4 (Humane ponto final) se o seguinte é observado: Quando o mouse é suspenso pela cauda, ​​há paralisia rígida nos membros posteriores. Quando o rato é permitido a andar, não há movimento para a frente. Quando o mouse é colocado sobre a sua esquerda e direita ele não é capaz de endireitar-se dentro de 10 segundos a partir do lado também. Ou seja, ausência de reflexo de endireitamento. </li></ol></li></ol> 4. Entrada de Dados e Análise <ol><li> Durante o curso de um estudo de eficácia da droga, inserir os dados coletados para cada rato em um Sistema de Gerenciamento de Informações de Laboratório (LIMS) ou em um software de planilha disponível no mercado. Nota: O software LIMS ALS TDI-desenvolvido é um repositório central de todos os dados coletados a partir de estudos internos, tornando estes dados convenientemente acessível ao longo do tempo para os pesquisadores ALS TDI. LIMS rastreia qualquer ratinho individual, com a sua identificação única, de volta à sua linhagem, peso corporal ou pontuação neurológica em todos os dias de estudo,e inclui quaisquer outras observações feitas durante a sua vida. LIMS acaba por gerar uma planilha que é usado para análises posteriores. </li><li> Após a conclusão de um estudo de drogas eficácia, ou seja, quando todos os ratos atingir o ponto final mais humano, software uso de planilha (consulte a Tabela de Materiais) para calcular a NS combinado para cada rato, para cada dia pela média do membro posterior esquerda e direita NS . Em seguida, calcular a idade média a cada NS para cada rato e do número de dias de cada rato gasta em um determinado NS. </li><li> Analisar as mudanças globais em NS ao longo do tempo, importando os dados do software de planilha eletrônica para o software estatístico (ver Tabela de Materiais). Caber estes dados utilizando um modelo de regressão logística ordinal que estima o NS esperado, com idade média no NS e de tratamento da toxicodependência como efeitos na regressão. Usando o teste do qui-quadrado de log-rank estimar a probabilidade efeito probabilidade e determinar se o efeito da droga-tratamento sobre a progressão NS é estatísticaly significativa (ver arquivo de código suplementar para obter instruções passo-a-passo). Nota: Um valor de p &lt;0,05 é considerado estatisticamente significativo. Nota: Este método de análise estatística respeita a natureza ordinal dos dados NS e à natureza variável das trajetórias NS para ratos individuais. É preferível porque as trajetórias individuais NS pode ser marcadamente diferente resultando em NS médias de grupo associado em tempo que não são suficientemente reflexiva de respostas NS. Além disso, em alguns casos NS pode flutuar, particularmente quando na gama de zero a um. </li><li> Finalmente, o uso de software de gráficos (ver Tabela de Materiais) para gerar &quot;ordinal esperado NS vs Idade mediana no NS&quot; gráficos para animais tratados com a droga e controle (Figura 2; consulte Código Suplementar arquivo para obter instruções passo-a-passo). Use estes gráficos para estimar convenientemente deslocamentos entre os grupos tratados com o fármaco e de controlo pela interpolação do eixo-y a idade média de início da doença (i.e., NS 2) no eixo dos x. Nota: A diferença na idade média entre os dois grupos representa uma mudança induzida por drogas e é considerado como uma medida prática da magnitude do efeito. Nota: desvios para a direita na curva de NS vs idade mediana esperada indicam retardou a progressão da doença sintomática. Por outro lado, os desvios para a esquerda na NS vs a curva idade mediana esperada acelerado indicam a progressão da doença sintomática. </li></ol>

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The authors have nothing to disclose.

Neste relatório, nós descrevemos um protocolo de vídeo rápido e simples que, se aplicada corretamente, é capaz de avaliar de forma confiável a progressão da doença em camundongos SOD1-G93A e identificar as alterações induzidas pela droga. Embora diferentes grupos desenvolveram sistemas de pontuação fenotípicas para ratos SOD1 G93A-8-10, que muitas vezes não fornecem detalhes suficientes sobre o procedimento e são insuficientes para replicação. Como resultado, sistemas de pontuação são rara...

Desde o seu desenvolvimento em meados de 1990, o modelo de camundongo transgênico SOD1-G93A tem sido o modelo animal mais utilizado da esclerose lateral amiotrófica (ELA) 1. Este modelo de ratinho transgénico foi geneticamente manipulada para sobre-expressar uma forma mutante do gene de Cu / Zn superóxido dismutase humana 1 (SOD1) portador da ALS-glicina associada a mutação da alanina no aminoácido 93 (G93A). A mutação G93A é uma das muitas mutações no gene SOD1 que colectivamente make-up de cerca de um quinto casos de ALS familiar 2. O modelo SOD1-G93A tornou-se uma força de trabalho de pesquisa de desenvolvimento de drogas ALS porque, além de sua óbvia ligação genética para ALS, ele recapitula muitas das características patológicas do ALS em seres humanos, como a perda do neurônio motor, fraqueza muscular progressiva e atrofia, com eventual paralisia e morte 3. Embora, como é frequentemente o caso com os modelos animais transgénicos, existe uma bi inerenteological variabilidade associada com os ratos SOD1 G93A-. Scott et ai. ter determinado que as coortes de pelo menos 24 ratinhos com equilíbrio de gênero de correspondência de maca são necessários em projetos de estudo fármaco-eficácia, a fim de superar o ruído criado pela variabilidade biológica 4. O grande número de animais necessários nestes estudos, em combinação com a progressão da doença agressiva e a necessidade de acompanhamento diário proíbe a utilização de, técnicas demoradas e potencialmente estressantes elaboradas para medir força e função neuromuscular (por exemplo, eletromiografia, força de preensão, rotorod , etc). Em vez disso, ele destaca a necessidade de uma ferramenta de triagem in vivo que avalia de forma confiável a função neuromuscular de camundongos SOD1-G93A de uma forma rápida. No ALS TDI foi desenvolvido um protocolo que permite a avaliação confiável da função neuromuscular de camundongos SOD1 G93A-em menos de 30 segundos por rato em média. Este protocolo encompasses avaliação diária da função dos membros posteriores utilizando um sistema simples de pontuação neurológica (NeuroScore), que é descrita neste relatório na imprensa e em vídeo. NeuroScore está focada em função dos membros posteriores porque os déficits dos membros posteriores são os primeiros relatados sinal neurológico da doença nos ratos SOD1 G93A-5,6. Além disso, o protocolo desenvolvido pela ALS TDI fornece uma avaliação imparcial do início da paresia (paralisia leve ou parcial), progressão e gravidade da paralisia e é sensível o suficiente para identificar as alterações induzidas pela droga na progressão da doença.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>B6SJLTgN(SOD1G93A)1Gur strain</td>
			<td>The Jackson Laboratory, Bar Harbor, ME</td>
			<td>002726</td>
			<td>Strain of mice used in this study</td>
		</tr>
		<tr>
			<td>Breeders</td>
			<td>Biomedical Research Models, Inc., Worcester, MA</td>
			<td></td>
			<td>Colony maintainance</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Navigator OHAUS, Parsippany, NJ</td>
			<td>Model N14120</td>
			<td>Used to weigh mice</td>
		</tr>
		<tr>
			<td>Nutra-GelH</td>
			<td>Bio-Serv, Flemington, NJ</td>
			<td>#S4798</td>
			<td>Wet food provided to sick mice</td>
		</tr>
		<tr>
			<td>Irradiated Lab Animal Diet</td>
			<td>Harlan, Indianapolis, IN</td>
			<td>2918-111914M</td>
			<td>Chow diet</td>
		</tr>
		<tr>
			<td>Lab-grade Sani-chips</td>
			<td>Harlan Teklad, Indianapolis, IN</td>
			<td>7090</td>
			<td>Bedding</td>
		</tr>
		<tr>
			<td>JMPH&#160;</td>
			<td>SAS Institute, Inc., SAS Campus Drive, Cary, NC</td>
			<td>&#160;v10.0.2</td>
			<td>Statistical software</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft, One Microsoft Way, Redmond, WA</td>
			<td>Excel 2013 (v 15.0)</td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad software Inc., La Jolla, CA</td>
			<td>v5.4</td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>Mouse Huts</td>
			<td>Bio-Serv, Flemington, NJ</td>
			<td>K3272</td>
			<td>For environmental enrichment</td>
		</tr>
	</tbody>
</table>

a quick phenotypic neurological scoring system for evaluating disease progression in the sod1-g93a mouse model of als

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

Os dados da contagem neurológicos para 90 do sexo masculino e 94 do sexo feminino não-tratados ratos SOD1 G93A-estudou em ALS TDI durante 2014 foram avaliados. Os resultados mostram que os ratos macho têm tipicamente uma progressão da doença mais agressivo do que os ratos do sexo feminino, tal como evidenciado por uma proporção maior da sua vida útil no NS 1 em comparação com as fêmeas. NS 2 e 3 NS ocorrem com praticamente a mesma frequência de diferentes sexos. 0 NS foi excluído da análise, porque é cons...

Watch this Scientific Journal Video about A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS at JoVE.com

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

Um sistema de pontuação neurológica rápida fenotípica para a Avaliação de progressão da doença na SOD1 G93A-Rato modelo de ALS

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening ...

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Research

JoVE Journal

Medicine

19.2K visualizações.  ALS Therapy Development Institute. This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

Vídeo: A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

Desenvolvimento de um Unilateralmente-lesionadas modelo de rato 6-OHDA da Doença de Parkinson

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Medicina

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizando Estimulação Magnética Transcraniana para o Estudo do sistema neuromuscular humano

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

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 ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Testes clínicos e Remoção da Medula Espinhal em um modelo do rato para a Esclerose Lateral Amiotrófica (ELA)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Um método simples de intubação Lung Rato

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Modelo ortotópico bioluminescente de progressão do câncer de pâncreas

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

A colonoscopia flexível em camundongos para avaliar a gravidade da colite e colorretal Tumores Usando um sistema validado endoscópica Scoring

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Criação e Caracterização da UTI e CAUTI em um modelo de rato

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Abordagens ecocardiograma e protocolos de Comprehensive Caracterização fenotípica de doença cardíaca valvular em ratos

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Avaliando o procedimento para realizar cistometria acordada em um modelo de mouse

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...