Forschung von der Natural Sciences and Engineering Research Council of Canada unterstützt

Das Experiment beinhaltet Ausbildung Ratten, um nach Nahrung zu erreichen und dann die Dreharbeiten für den Bewegungen für das Erreichen verwendet. <ol><li> Long Evans Laborratten sind leicht von Lebensmitteln für 10 Tage entzogen, dann auf eine Box mit einem Schlitz, durch den sie für Futterpellets erreichen kann angepasst. </li><li> Nach etwa 10 Tagen der Ausbildung die Ratten konsequent zu erreichen und zeigt eine Bevorzugung einer Pfote. </li><li> Das Erreichen Akt gefilmt mit einer kurzen Verschlusszeit (1.000 m / sec) und fiming ist aus einer Reihe von Perspektiven gemacht. </li><li> Das Erreichen handeln wird anschließend wiederholt mit Frame für Frame wiedergeben. </li><li> Die Bewegungen sind in den beigefügten Video beschrieben. </li></ol>

<ol>
	<li>Whishaw, I. Q., Pellis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+skilled+forelimb+reaching+in+the+rat.">The structure of skilled forelimb reaching in the rat.</a> Bev. Br. Res. 41, 49-59 (1990).</li><li>Whishaw, I. Q., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The behavior of the laboratory rat. , 162-170 (2005).</li></ol>

Frühere Studien des Körpers verwenden bei der Ratte haben die Bewegung in Bezug auf die Endpunkte, Erfolg oder Misserfolg, oder in Form eines kartesischen Flugbahn der Pfote auf das Ziel aus. Die vorliegende Studie beschreibt die Bewegung in Bezug auf die Beziehungen zwischen Gliedmaßensegmente, andere Körperteile und das Ziel.

the structure of skilled forelimb reaching in the rat: a movement rating scale

Skilled griff nach Lebensmitteln ist ein entwicklungsgeschichtlich sehr alt zu handeln und wird von vielen Tierarten, einschließlich derjenigen, in der Schwester Subtypen von Nagern und Primaten angezeigt. Das Video beschreibt eine Testsituation, die Verfilmung von wiederholten Handlungen zu erreichen für Lebensmittel, die von der Ratte, die milde Nahrung beraubt werden können. Eine Ratte ist ausgebildet, um durch einen Schlitz in eine Holding-Box für Lebensmittel Pellet, dass es begreift und legt dann in den Mund zum Essen zu erreichen. Erreichen wird in der Hauptsache von proximal angetrieben Bewegungen des Körpers, sondern distalen Bewegungen der Gliedmaßen sind für Pronation der Pfote, Greifen der Nahrung und die Freigabe der Nahrung in den Mund eingesetzt durchgeführt. Jeder zu erreichen, ist in mindestens 10 Bewegungen der vorderen Gliedmaßen und dem Erreichen Akt wird durch Haltungs-Anpassungen erleichtert unterteilt. Jede der Bewegungen beschrieben und Beispiele für die Bewegungen werden aus einer Reihe von Anzeigen Perspektiven gegeben. Durch die Bewertung jeder Bewegung Element auf einer 3-Punkte-Skala, kann die Reichweite quantifiziert werden. Eine Reihe von Studien haben gezeigt, dass die Bewegung Elemente durch motorische System Schäden, einschließlich Schäden an den motorischen Cortex, Basalganglien, Hirnstamm und Rückenmark sind verändert. Die Bewegungen sind auch in neurologischen Erkrankungen, die in der Ratte, einschließlich Morbus Parkinson und Chorea Huntington modelliert werden kann verändert werden. Somit ist das Rating-Skala sinnvoll für die Quantifizierung motorischen Beeinträchtigungen und die Effektivität der neuronalen Restaurierung und Sanierung. Da das Erreichen Akt für die Ratte ist sehr ähnlich wie die von Menschen und nichtmenschlichen Primaten angezeigt wird, kann der Maßstab für vergleichende Zwecke verwendet werden. aus einer Reihe von Anzeigen Perspektiven. Durch die Bewertung jeder Bewegung Element auf einer 3-Punkte-Skala, kann die Reichweite quantifiziert werden. Eine Reihe von Studien haben gezeigt, dass die Bewegung Elemente durch motorische System Schäden, einschließlich Schäden an den motorischen Cortex, Basalganglien, Hirnstamm und Rückenmark sind verändert. Die Bewegungen sind auch in neurologischen Erkrankungen, die in der Ratte, einschließlich Morbus Parkinson und Chorea Huntington modelliert werden kann verändert werden. Somit ist das Rating-Skala sinnvoll für die Quantifizierung motorischen Beeinträchtigungen und die Effektivität der neuronalen Restaurierung und Sanierung. Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von der University of Lethbridge Animal Care Committee in Übereinstimmung mit den Bestimmungen des Canadian Council on Animal Care Set durchgeführt.

Watch this Scientific Journal Video about The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale at JoVE.com

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

Der gelernte Erreichen Skala teilt die Bewegung durch ein Vorderbein in einer Reichweite für Lebensmittel handeln zu Verbundelementen von denen jede mit einem Drei-Punkte-Skala bewertet werden. Die Rating-Skala ist für eine normale Ratte beschrieben und können nach Auswertung neurologischen Bewegungsstörungen angewandt werden.

The Structure of Skilled Vorderbein Reaching in den 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 ...

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Research

JoVE Journal

Biology

12.8K Aufrufe.  University of Lethbridge. Der gelernte Erreichen Skala teilt die Bewegung durch ein Vorderbein in einer Reichweite für Lebensmittel handeln zu Verbundelementen von denen jede mit einem Drei-Punkte-Skala bewertet werden. Die Rating-Skala ist für eine normale Ratte beschrieben und können nach Auswertung neurologischen Bewegungsstörungen angewandt werden.

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

Use of Rotorod as a Method for the Qualitative Analysis of 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 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.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

Verwendung von Drehstabtest als Methode für die qualitative Analyse von 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 ...

Forschung

Biologie

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Die Implantation von Engineered Tissue in der Ratte Herz

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&amp;#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Imaging von Estrogen Receptor-α in Rat Pial Arteriolen mit einem Digital-Immunfluoreszenz-Mikroskop

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolierung von Ratten-Portal Fibroblasten durch<em&gt; In-situ</em&gt; Leberperfusion

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Die Kultivierung der menschlichen Nasen Epithelzellen an der Air flüssig-Grenzfläche

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

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 ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

Quantifizierung der Mikrogefäßdurchlässigkeit im Einzel isolierten Blutdurchbluteten Lungen Rat Vorbereitung

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Ein Verfahren zur Auswahl Struktur-Schalt Aptamere zu einem Farbmetrik Gold-Nanopartikeln Assay angewandt

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

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.

Blutprobenentnahme aus der laterale Schwanzvene der Ratte

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

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Milchsammlung in der Ratte Mit Kapillarröhrchen und Schätzung der Milchfettgehalt von Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...