המחברים מודים אנשי המעבדה Lwigale לביקורת של כתב היד. SLG נתמך על ידי ל &#39;רות Kirschstein NRSA אחוות מן הלאומי Eye Institute (F32 EY02167301). PYL נתמך על ידי National Eye Institute (EY018050).

החוקרים אין לי מה לחשוף.

השתלת של הצינור העצבי שליו לעוברי חומוס המארחות המתוארת כאן היא טכניקה פשוטה וזולה לאיתור subpopulations ספציפיות של העברת NCCs שמקורם באזורים שונים לאורך ציר הזנב rostro-21,67-69. שיטה זו מנצלת את הקלות של גישה עוברי העופות (לעומת עוברי יונקים) ו ניתן בשילוב עם טכניקות אחרות, ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> חומוס ביצים </td><td> שונים - אנו משתמשים טקסס A &amp; M University עופות המדע של המחלקה, טקסס. </td><td></td></tr><tr><td> שליו ביצים </td><td> שונים - אנו משתמשים Ozarks החברה ביצה, מיזורי. </td><td></td></tr><tr><td> ביצה חממה (הצג הדיגיטלי 1502 ספורטאי החממה w / לחות 110-120 וולט AC) </td><td> <a href="http://www.poultrysupply.com" target="_blank">www.poultrysupply.com</a> </td><td> 1502 </td></tr><tr><td> דומון AA מלקחיים, Inox אפוקסי מצופה </td><td> מדע כלים עדינים </td><td> 11210-10 </td></tr><tr><td> נייר דבק </td><td> כל חנות לציוד משרדי </td><td></td></tr><tr><td> עקום איריס מלקחיים </td><td> מדע כלים עדינים </td><td> 11065-07 </td></tr><tr><td> אניndia דיו </td><td> כל ההיצע בחנות האמנות </td><td></td></tr><tr><td> פן / דלקת (פניצילין, סטרפטומיצין) פתרון </td><td> VWR הבינלאומי </td><td> 101447-068 </td></tr><tr><td> סרט אריזה ברור </td><td> כל חנות לציוד משרדי </td><td></td></tr><tr><td> מנגנון משיכת המחט </td><td> Narashige, יפן </td><td> PE-21 </td></tr><tr><td> זכוכית מחט הוציא, עשה 1.5-1.8 צינור x 100mm בורוסיליקט זכוכית נימי </td><td> קימבל מרדף </td><td> 34500 99 </td></tr><tr><td> משך זכוכית פיפטה, עשוי 5 ¾ &quot;פיפטה פסטר </td><td> פישר סיינטיפיק </td><td> 13-678-6A </td></tr><tr><td> הפה פיפטה המנגנון (צינור aspirator הרכבה על פיפטה microcapillary מכויל) </td><td> Sigma-Aldrich </td><td> A5177-52 א </td></tr><tr><td> דומון # 5 מלקחיים </td><td> מדע כלים עדינים </td><td> 11251-30 </td></tr><tr><td> חבית גדולהחוט gsten, קוטר 0.1mm </td><td> VWR הבינלאומי </td><td> AA10404-H2 </td></tr><tr><td> מחזיקי מחט (מצופה ניקל בעל קוד אישי) </td><td> מדע כלים עדינים </td><td> 26018-17 </td></tr><tr><td> QCPN antiserum </td><td> התפתחותית מחקרים Hybridoma בנק, אוניברסיטת איווה </td><td> QCPN </td></tr><tr><td> Alexa פלואוריד משני נוגדנים (למשל, Alexa פלואוריד 594 עיזים נגד העכבר IgG1) </td><td> Invitrogen </td><td> A21125 </td></tr><tr><td> הצלצול של פתרון (2L): <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 14.4g NaCl </li><li style=";text-align:right;direction:rtl"> 0.34g CaCl 2 </li><li style=";text-align:right;direction:rtl"> 0.74g KCl </li><li style=";text-align:right;direction:rtl"> 0.230g Na 2 HPO 4 </li><li style=";text-align:right;direction:rtl"> 0.04g KH 2 PO 4 </li><li style=";text-align:right;direction:rtl"> DDH 2 O ל-2L </li><li style=";text-align:right;direction:rtl"> סינון החיטוי </li></ul></td><td> כל ריאגנטים של פישר סיינטיפיק </td><td><ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 7647-14-5 </li><li style=";text-align:right;direction:rtl"> 10043-52-4 </li><li style=";text-align:right;direction:rtl"> 7447-40-7 </li><li style=";text-align:right;direction:rtl"> 7558-79-4 </li><li style=";text-align:right;direction:rtl"> 7778-77-0 </li></ul></td></tr></tbody></table>

analysis of neural crest migration and differentiation by cross-species transplantation

עוברי העופות לספק פלטפורמה ייחודית לחקר תהליכים התפתחותיים רבים החוליות, בשל גישה קלה של עוברים בתוך הביצה. עוברי העופות chimeric, שבו הרקמה שליו התורם הושתלו העובר חומוס ב לג&#39;ימי, לשלב את העוצמה של תיוג גנטי של אוכלוסיות תאים בל יימחה בקלות מניפולציה שהוצגו על ידי העובר העופות. שליו, חומוס מפלצות הם כלי קלאסי עבור מעקב אחר תאים נודדים לפסגה עצביים (NCCs) 1-3. NCCs הם אוכלוסייה נודדות חולפות של התאים בעובר, אשר מקורם באזור הגבי של 4 בפיתוח בצינור העצבי. הם עוברים אפיתל המעבר mesenchymal ובהמשך נודדים לאזורים אחרים של העובר, שם הם להתמיין לסוגי תאים שונים, כולל 5-13 סחוס, מלנוציטים 11,14-20, הנוירונים גליה 21-32. NCCs הם multipotent, ואת גורלם האולטימטיבי שלהם משפיעיםenced לפי אזור 1) של הצינור העצבי שבו הם נובעים לאורך ציר rostro-הזנב של העובר 11,33-37, 2) לאותות ששיגרו התאים הסמוכים כשהם נודדים 38-44, ו - 3) microenvironment של האולטימטיבי שלהם היעד בתוך העובר 45,46. מעקב אחר התאים האלה מנקודת המוצא אל הצינור העצבי, למיקום הסופי וגורלו בתוך העובר, מספק תובנה חשובה לתוך התהליכים ההתפתחותיים המווסתים דפוסים ו organogenesis. השתלת אזורים משלימים של הצינור העצבי התורם (homotopic השתלת) או באזורים שונים של הצינור העצבי התורם (heterotopic השתלת) יכול לגלות הבדלים במפרט מראש של NCCs לאורך ציר rostro-הזנב 2,47. טכניקה זו ניתן להתאים עוד יותר להשתיל תא החד צדדית של הצינור העצבי, כך שצד אחד נגזר רקמות התורם, ואת שרידי בצד הנגדי מוטרד בעובר המארח, ייelding הבקרה הפנימית בתוך מדגם זהה 2,47. זה גם יכול להיות מותאמים עבור השתלת קטעי מוח עוברי מאוחר יותר, לאחר HH10, כאשר בצינור העצבי הקדמי סגר 47. כאן אנו מדווחים על טכניקות להפקת שליו, חומוס מפלצות באמצעות השתלת בצינור העצבי, אשר מאפשרים מעקב אחר NCCs נודדים שמקורם קטע נפרד של הצינור העצבי. מינים ספציפיים תיוג של התורם הנגזרות תאים עם נוגדן ספציפי QCPN שליו 48-56 מאפשרת לחוקר להבחין התורם תאים המארחות בנקודת סיום הניסוי. טכניקה זו היא פשוטה, זולה, ויש לה יישומים רבים, כולל מיפוי, גורל, השושלת תא מעקב, זיהוי דפוסים מראש אירועים לאורך ציר הזנב rostro-45. בגלל הקלות של גישה העובר העופות, הטכניקה שליו, חומוס שתל עשוי להיות משולב עם מניפולציות אחרות, כולל אך לא רק, אבלציה העדשה 40, הזרקה של מולקולות מעכבות 57,58 או מניפולציה גנטית דרך electroporation של פלסמידים הביטוי 59-61, כדי לזהות את התגובה של זרמים נודדות מיוחדות של NCCs על הפרעות בתוכנית ההתפתחות של העובר. יתר על כן, שיטה זו השתלת יכול לשמש גם כדי לייצר אחרים עוברים interspecific chimeric כמו שליו, ברווז מפלצות ללמוד תרומה NCC כדי המורפוגנזה Craniofacial או עכבר חומוס מפלצות לשלב את כוחה של הגנטיקה העכבר בקלות מניפולציה של העובר העופות . 62

Watch this Scientific Journal Video about Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation at JoVE.com

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

הגישה לניתוח הגירה גורלו הסופי של העופות לפסגה תאים עצביים שליו, חומוס עוברים chimeric מתואר. שיטה זו היא טכניקה פשוטה וישירה לאיתור תאים עצביים לפסגה במהלך הגירה והבחנה שקשה אחרת להבחין בתוך העובר גוזל unmanipulated.

ניתוח של הגירה קרסט בידול עצבית ועל ידי חוצה מינים להשתלות

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

analysis-neural-crest-migration-differentiation-cross-species

Research

JoVE Journal

Neuroscience

22.7K Views.  Rice University. הגישה לניתוח הגירה גורלו הסופי של העופות לפסגה תאים עצביים שליו, חומוס עוברים chimeric מתואר. שיטה זו היא טכניקה פשוטה וישירה לאיתור תאים עצביים לפסגה במהלך הגירה והבחנה שקשה אחרת להבחין בתוך העובר גוזל unmanipulated.

Video: Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

הפקה של חילוץ העובר צ&#39;יק לגידול Murine בתאי גזע עצביים קרסט

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

אפיון פיזיולוגיים, מורפולוגי והעצבית של נוירונים מווסתת על ידי תנועת

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

בידוד ותרבות של תאי גזע עצביים קרסט מזקיקי שיער אנושיים

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-מבוך לסירוגין בכפייה של שמאל וימין משימות אפליה להערכת עבודה וזיכרון העזר עכברים

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

ניתוח של נדידת תאים עצביות קרסט טרונק באמצעות Assay השתנה Zigmond הקאמרי

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

זיהוי של היפוקסיה Microregional בקליפת עכבר מוחין על ידי הדמיה של שני הפוטונים אנדוגני NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

עכבר לשחזור Sciatic עצב קראש והערכה לאחר ההתחדשות של ניתוח הר שלם שריר

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

בידוד והתרבות של תאים עצביים לפסגה מהצינור עובריים עצבית Murine

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Assay Galvanotaxis לניתוח של קינטיקס נדידת תאים העצביים המבשר בשדה חשמלי נוכחי לשימוש חיצוני ישיר

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Assay נדידת תאים כמותיות לMurine אבות עצביים enteric

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...