Apoiado por subvenções dos Institutos Canadenses de Pesquisa em Saúde, a Rede canadense Stroke, e os Alberta Heritage Foundation for Medical Research.

Rotorod aparelho andando O rotorod era um cilindro rotativo, 4 cm de diâmetro, 35 centímetros fixo acima do solo e cercado por acrílico transparente. O cilindro rotorod estava coberto de revestimento de borracha texturizada, o que facilita a tração. O piso do aparelho foi coberto com uma camada de espuma, para evitar lesões quando os animais caíram. Um pequeno motor elétrico desde o poder para transformar o rotorod através de uma correia de borracha. O rotorod foi criado para rodar uma vez a cada 5 s. Gravações de vídeo e cinemática A alta velocidade da câmera de vídeo digital foi usada para filmar os animais, usando uma velocidade do obturador de um 1000 de um segundo. A frente, lateral, traseira e pontos de vista foram filmados enquanto o animal estava caminhando na rotorod. A dois braços Nikon, MII fonte de luz fria e um conjunto de fibra óptica luzes foram usadas para fornecer iluminação adequada durante a gravação. As fitas foram analisadas quadro a quadro, usando uma Sony DSR 20 DV CAM baralho. Frames de vídeo foram capturados usando uma placa de captura e computador Macintosh. Procedimento de teste e análise , A fim de avaliar as diferenças entre os movimentos a pé da 6-OHDA e ratos de controle, os registros de vídeo de pós-cirúrgicas dia 15-30 (após uma recuperação da lesão pode vir a ser em grande parte concluído) foram examinados por dois independentes observadores cegos para as condições experimentais. A lesão 6-OHDA nigroestriatal foi induzida no hemisfério direito, resultando em prejuízos duradouros na frente e deixou-membros posteriores. A escala de classificação foi desenvolvido para taxa de postura dos ratos e os movimentos aprofundamento dos membros anteriores (Whishaw et al., 2003). Cinco componentes de pisar foram classificados: <ol><li> postura, a classificação da postura foi feita através da análise da posição do rato sobre o tambor; </li><li> elevador e solte, a porção do ciclo em que pisar a pata foi levantada a partir do tambor; </li><li> transportar, a parte do ciclo de pisar no qual o membro foi mantido acima do tambor como os ratos se movimentavam para a frente; </li><li> antecedência, a porção do alcance no qual o membro foi estendido para recuperar o contato com o tambor, e </li><li> colocação, em que a pata foi colocado de volta no tambor. </li></ol> Os seguintes critérios foram utilizados para definir um movimento normal: <ul><li> Postura: O corpo do rato foi orientado sobre o tambor com a cabeça erguida. </li><li> Elevador e release: O ombro, cotovelo e punho são flexionados levantar a pata a partir da superfície do tambor. </li><li> Carry: A pata é mantida acima da superfície do tambor, os dígitos são vagamente flexionados, e as pontas dígitos são centrados na linha média do corpo. Antecedência: O membro é estendido para a frente com os dígitos que se deslocam de uma posição semiflexed para uma posição estendida e aberta. </li><li> Colocação: O membro é estendida no ombro, cotovelo e punho e os dígitos são abertos para colocar a pata para a frente e na superfície do tambor, com 2 dígitos (segundo dígito da linha média), localizado a aproximadamente o centro do de rato corpo. </li></ul> Cada movimento foi avaliada em uma escala de 3 pontos: 1 = / o movimento parecia o de um rato controle, 0,5 = / o movimento apareceu ligeiramente anormal, e 0 = / o movimento foi claramente anormal (Whishaw et al, 2003).. Esta escala de classificação foi então utilizado para avaliar cinco movimentos pisar de cada rato em dias pós-operatório de 1, 3, 5, 7, 9 e 15. As pontuações das cinco medidas foram calculados para cada rato em cada dia, e os resultados submetidos a uma ANOVA com dias como medida repetida. Visão geral do rotorod andar em 6-OHDA e ratos controle A escala de classificação movimento foi aplicado para as sessões de teste pós-operatório para análise do movimento qualitativo. Durante as sessões de teste, o grupo lesão 6-OHDA receberam pontuação significativamente menor do que o grupo controle, F (1,8) = 33,2, p &lt;0,001, mas não houve efeito significativo do dia do teste, nem existia um grupo significativo pela interação dia do teste. As pontuações de pisar foram então analisados ​​separadamente com os membros anteriores como uma variável (para os ratos 6-OHDA no membro anterior contralateral à lesão versus o membro anterior ipsilateral à lesão, para os ratos de controle, a pata esquerda contra a pata direita). Houve um efeito significativo do Membro Anterior, F (1,40) = 15,8, p &lt;0,01, e houve uma interação significativa do Grupo por Membro Anterior, F (1,40) = 15,0, p &lt;0,01. Follow-up t-testes indicaram que o membro anterior contralateral-to-lesão do grupo 6-OHDA receberam pontuação significativamente menor do que o membro anterior ipsilateral-to-lesão ou forelimbs dos ratos de controle, que não diferiram. A fim de determinar quais os componentes do movimento contribuiu para a pontuação pobres obtidos pelo grupo 6-OHDA, as medidas de posturae os componentes individuais do ciclo de pisar do forelimbs ipsilateral e contralateral do grupo 6-OHDA foram comparados aos valores obtidos a partir do controle de ratos. O grupo 6-OHDA diferiam em postura e todos os componentes pisar feita pelo membro contralateral, mas nenhum dos componentes do membro ipsilateral (p &lt;0,05). Diferenças qualitativas e descritivo de postura no controle e 6-OHDA ratos Em um rato controle, a cabeça é realizada em uma posição horizontal, e os membros anteriores de apoio e membros posteriores straddle o tambor formando um arco de apoio. Os ratos 6-OHDA normalmente andava com a cabeça para baixo com suas patas dianteiras prejudicada (de frente para o espectador) posicionados mais próximos e mais acima na superfície do tambor do que ocorreu para os membros ipsilateral ou os membros de ratos controle (veja o vídeo de um rato controle com pontuação &quot;1&quot; para a postura, e um animal de lesão 6-OHDA com uma pontuação de &quot;0&quot;). Tipicamente, a anormalidade de postura pode ser visto de relance a partir da posição relativa de uma cabeça de rato. Forelimb pisar Na seqüência de controle, o membro anterior pisar é levantada com a flexão do ombro, cotovelo e punho. Como o membro é levantada, os dígitos são vagamente flexionados, eo membro é levantado bem acima da superfície do tambor. Quando o membro é avançado, é estendido na altura dos ombros, cotovelo e punho e como ele é colocado os dígitos são estendidas e abertas. Em comparação aos ratos controle, a amplitude de todos os componentes dos ratos DA-esgotados contralateral à lesão do membro é atenuado. O membro libera o tambor mais tarde, é levantada menos, e é avançado uma distância menor do que o membro anterior de controle. Além disso, quando o membro é levantada, os dígitos são flexionados mais, e quando o membro é colocado, os dígitos são estendidos e abriu menos do que os de um rato controle. O membro anterior de controle é levantada quando ela atinge o topo do tambor, o pulso é flexionado para levantar a pata para cima e para frente, eo pulso é prolongado com o avanço do membro. Por outro lado, o rato 6-OHDA é contralateral à lesão é realizado forelimb mais caudalmente pelo movimento do tambor, e, portanto, versões posteriores com o pulso ainda estendida. Flexão do punho parece ser passiva e ocorre como os dígitos são levados para trás, o movimento do tambor, e quando o membro anterior é trazido para a frente, a pata eo antebraço não estão claramente fora levantada a superfície do tambor. O membro anterior de um rato controle estende bem para a frente e os dígitos aberto com o avanço do membro. Como o membro é colocado, ele é colocado com um movimento arpejo, com 5 dígitos contato com a superfície da primeira bateria, seguido sucessivamente por dígitos 4, 3, e 2. Por outro lado, o membro do rato 6-OHDA é levantada e estendida menos, os dígitos são estendidos menos, e entram em contato com a superfície do tambor quase simultaneamente com um &#39;tapa&#39;. O vídeo mostra forelimb movimentos pisar em um animal de controle que foram marcados como &quot;1&quot;, e forelimb movimentos pisar em um animal de lesão 6-OHDA que foram principalmente classificada como &quot;0&quot;. Membro posterior pisar Havia também deficiências nos membros contralateral-to-lesão traseiras dos ratos 6-OHDA. Como o membro de controle traseiras se move para trás, parece empurrar a tambor a rodar e como ele faz isso, os dedos do pé girar para fora. Como o membro se move para frente, o pé é oscilado para fora. Do membro posterior do rato 6-OHDA move-se diretamente para trás com rotação do tambor e se alguma coisa, vira um pouco para dentro. Como o membro se move para frente, o pé é feita diretamente para a frente. Ao pé da balança desratização para fora e como ela é avançada, os dedos são mantidos acima do tornozelo, e os dedos dos pés estender. Durante a colocação dos ratos controle, toe 5 contatos a superfície do primeiro tambor eo peso é distribuído entre os dedos dos pés e palma. Em contraste, os dedos dos ratos 6-OHDA são flexionados durante o transporte e colocação é feita sem um movimento de arpejo. Os dedos dos ratos controle são liberados em um padrão de arpejo invertido como toe 5 é levantado primeiro e um dedo do pé é levantado passado. Esse movimento estava faltando no rato lesão 6-OHDA, enquanto o tornozelo mantido a sua postura de flexão durante o movimento de transportar. Mais uma vez os movimentos antes de o rato 6-OHDA são atenuadas, com o membro menos exorotated, levantou menos, e avançou menos do que o do rato controle. Como ratos controle colocar seus dedos, que são estendidas e abertas, tais toe que 5 pontos lateralmente em relação ao corpo do rato. Toe 5 contatos o primeiro tambor seguido pelo restante dos pés de tal forma que o rato suportado seu peso na ponta dos dedos. Para o rato 6-OHDA, os dedos são um pouco como o pé flexionado é levado adiante, os dedos dos pés são mais baixos em relação a horizontal até o tornozelo, há menos abertura dos dedos do pé quando o pé é colocado no tambor. Comprimento da passada também é um pouco menor do que o dos ratos controle. A fim de quantificar as diferenças entre control e 6-OHDA movimentos dos membros posteriores, os componentes de sustentação e liberação, transporte, promoção e colocação foram classificados em uma escala de 3 pontos, assim como os membros anteriores. As medidas foram feitas a partir de registros vídeo feito no dia cirúrgico pós 20, e três ciclos etapa foram classificados para cada rato. Houve diferenças significativas Grupo, F (1,8) = 47,6, p &lt;0,001. Follow-up t-testes dos componentes indicou que todos os componentes do grupo controle receberam pontuação significativamente maior do que os componentes dos ratos 6-OHDA. O vídeo mostra os movimentos dos membros posteriores pisar em um animal de controle que foram marcados como &quot;1&quot;. Membro posterior pisar movimentos no 6-OHDA mostrado no vídeo eram principalmente avaliado com uma pontuação de &quot;0&quot;.

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O objetivo do presente estudo foi examinar se o teste para a habilidade rotorod locomoção de roedores pode ser usado para a análise qualitativa da postura, pisando forelimb e dos membros posteriores em ratos pisar sistema nervoso lesionado. Para a análise, ratos com depleção DA unilateral e ratos controle foram de vídeo gravado a partir de vistas frontal, lateral e posterior. A escala de classificação da postura e movimentos forelimb indicou que os movimentos estavam pisando cronicamente prejudicada após a cirurgia. Exame dos movi...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

O teste rotorod, em que os animais andar sobre um tambor rotativo, é amplamente utilizado para avaliar o estado do motor em roedores de laboratório. O desempenho é medido pela duração que um animal permanece em cima do tambor, em função da velocidade do tambor. Aqui nós relatamos que a tarefa fornece uma rica fonte de informações sobre aspectos qualitativos dos movimentos a pé. Porque os movimentos são realizados em um local fixo, eles podem ser facilmente analisados ​​usando métodos de alta velocidade de gravação de vídeo. O presente estudo foi realizado para examinar o potencial do rotorod para revelar mudanças qualitativas nos movimentos andando de ratos hemi-Parkinson analógico, produzido por injeção de 6 hidroxidopamina (6-OHDA) no feixe direito nigroestriatal de dopamina nigroestriatal a esgotar (DA ). Início no dia seguinte a cirurgia e depois periodicamente ao longo dos próximos dois meses, os ratos foram filmados a partir de vistas frontal, lateral e posterior, enquanto caminhavam no rotorod. Comportamento foi analisado pelo quadro a quadro-repetição dos registros de vídeo. Escalas de avaliação de pisar comportamento indicou que os ratos hemi-Parkinson foram cronicamente prejudicada em sua postura e no uso dos membros contralateral à depleção de DA. Os membros contralateral não apenas disponibilizados alterações posturais e de movimento, que participaram menos de iniciar e manter a propulsão do que os membros ipsilateral. Estes resultados não só revelam déficits nova unilateral secundária à depleção de DA, mas também mostram que a rotorod pode fornecer uma ferramenta robusta para a análise qualitativa do movimento.

Watch this Scientific Journal Video about Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat at JoVE.com

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

O teste rotorod é usado para avaliar o estado do motor no movimento de caminhar de ratos hemi-Parkinson analógica.

Uso de Rotorod como um método de análise qualitativa de Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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12.9K visualizações.  University of Lethbridge. O teste rotorod é usado para avaliar o estado do motor no movimento de caminhar de ratos hemi-Parkinson analógica.

Vídeo: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Um método simples para genotipagem Accurate

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biologia

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

A Estrutura do Membro Anterior Skilled Chegar no Rato: A Escala de Avaliação Movimento

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

O Ladder Rung Walking Tarefa: um sistema de pontuação e sua aplicação prática.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Análise do desenvolvimento de um fenótipo morfológico como uma função da concentração de proteínas em levedura de brotamento

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Microinjeção de embriões Medaka para uso como um organismo modelo genético

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Um método simples para criação de imagens Arabidopsis Folhas Usando Perfluorodecalin como um meio de imagem infiltrativas

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

O Uso de quimiostatos em Microbial Biologia de Sistemas

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Um método de extração de DNA híbrido para a Avaliação de comunidades bacterianas Qualitativa e Quantitativa de amostras de produção de aves de capoeira

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

A amostragem de sangue a partir da veia lateral da cauda do rato

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...