עבודה זו נתמכה על ידי מענקים NIH K08NS065157 (כדי TAF) בנוסף, מרכז פן על צ&#39;כית, מספר פרס P30AR050950 מן המכון הלאומי של דלקת פרקים, מחלות השלד והשרירים והעור נתמך בעבודה זו (TAF וסטיבן ס &#39;שרר). לבסוף, שריינרים ילדים מרכז המחקר זרע מימון (TAF) נתמך על העבודה הזאת. ברצוננו להודות לד&quot;ר יאנג, ג&#39;ין בן עבור בתחילה הוכחת הר ההליך כולו ואיימי א &#39;קים על עזרתה בהפקת סקיצות של איור 1.

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אין לנו מה למסור.

הצגנו שיטה להשיג דלוקה מלאה ומהימנה גיד הנשה עם סימון מדויק של אתר דלוק. כאמור, למעוך גיד הנשה הוא מודל נפוץ של פגיעה עצבית היקפית בעכברים וחולדות. למרות כל שיטה של ​​התאהבות יש יתרונות וחסרונות, מצאנו שיטה זו הופק דלוקה שלמה סומן בקלות עם מינימום של ציוד מיוחד (מלחצי...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם (תיאור / כמות) </td><td> ספק </td><td> מספר קטלוגי </td></tr><tr><td> קליפר מיני עם מס &#39;0000 Blade </td><td> Roboz </td><td> RC-5903 </td></tr><tr><td> נאיר מסיר שיער (9 גר &#39;.) </td><td> הכנסייה דווייט ושות&#39; בע&quot;מ </td><td> N / A </td></tr><tr><td> בבטאדין ליטוש כירורגי (1 ליטר) </td><td> פישר סיינטיפיק </td><td> 19066452 </td></tr><tr><td> משחת עיניים (1 גר &#39;.) </td><td> פישר סיינטיפיק </td><td> 19082795 </td></tr></tbody></table> כירורגי כלים: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם (תיאור / כמות) </td><td> ספק </td><td> מספר קטלוגי </td></tr><tr><td> FST 250 חם חרוז מעקר </td><td> מדע כלים עדינים </td><td> 18000-45 </td> &lt;/ Tr&gt; <tr><td> איריס מספריים (11 ס&quot;מ) </td><td> Precision של מכשירים בעולם </td><td> 500216 </td></tr><tr><td> פוטס, סמית מלקחיים (סטרייט, 18cm; 1x2 שיניים) </td><td> מדע כלים עדינים </td><td> 11024-18 </td></tr><tr><td> מקפרסון, Vannas מספריים (5 להבים מ&quot;מ) </td><td> Precision של מכשירים בעולם </td><td> 14124-G </td></tr><tr><td> דומונט # 5 מלקחיים - Dumoxel תקן עצה </td><td> מדע כלים עדינים </td><td> 11252-30 </td></tr><tr><td> דומונט # 5/45 מלקחיים - Dumoxel תקן עצה </td><td> מדע כלים עדינים </td><td> 11251-35 </td></tr><tr><td> Hemostats יפות במיוחד (סטרייט, בתוך משטחים חלקים) </td><td> מדע כלים עדינים </td><td> 13020-12 </td></tr><tr><td> אבקת פחם פעיל (500 גרם) </td><td> פישר סיינטיפיק </td><td> C272-500 </td></tr><tr><td> 6-0 גודל התפרים עם מחט C-1 (משי סטרילי, שחור, קלועה,שאינם נספגים, 18 &quot;אורך; Box של 36) </td><td> Roboz </td><td> SUT-1073-1011 </td></tr><tr><td> רפלקס Applier קליפ (במשך 9 קליפים מ&quot;מ) </td><td> Precision של מכשירים בעולם </td><td> 500345 </td></tr><tr><td> 9 מ&quot;מ נירוסטה רפלקס קליפים (100/box) </td><td> Precision של מכשירים בעולם </td><td> 500346 </td></tr></tbody></table> Animal Care: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם (תיאור / כמות) </td><td> ספק </td><td> מספר קטלוגי </td></tr><tr><td> % 0.9 סודיום כלוריד להזרקה (חינם חומר משמר; 20 מ&quot;ל) </td><td> Hospira </td><td> 0409-4888-20 </td></tr><tr><td> מערכת שלמה שמיכה Homeothermic עם בדיקה גמיש (בינוני, 115 VAC, 60 הרץ) </td><td> הרווארד Apparatus </td><td> 507222F </td></tr></tbody></table> כירורגי Platforמ &#39;ו Retractors: (לרכוש בחנות המקומית לחומרי) <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם (תיאור /) </td><td> כמות </td></tr><tr><td> פלטפורמת פלדה אל חלד (מחורץ, ~ 12 &quot;x12&quot; x1 / 8 &quot;) </td><td> 1 </td></tr><tr><td> לחצן מגנטים (לדוגמה: ליקוי חמה E825) </td><td> 3 </td></tr><tr><td> פלדה אל חלד (3 ברגים &quot;ארוך, קוטר נקבע על ידי מגנטים לחצן) </td><td> 3 </td></tr><tr><td> נירוסטה אגוזים (בהתאמת גודל ברגים) </td><td> 9 </td></tr><tr><td> גומיות (המתח אור) </td><td> 3 </td></tr><tr><td> סיכות חרקים (שני הקצוות בנט טופס הוקס) </td><td> 3 </td></tr></tbody></table> חצי מאריה: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם (תיאור / כמות) </td><td> הספק &lt;/ Strong&gt; </td><td> מספר קטלוגי </td></tr><tr><td> Paraformaldehyde (1 ק&quot;ג) </td><td> Sigma-Aldrich </td><td> P6148-1 ק&quot;ג </td></tr><tr><td> פוספט נתרן Dibasic נטול מים (500 גר &#39;, המשמשים להכנת מאגר פוספט) </td><td> פישר סיינטיפיק </td><td> S375-500 </td></tr><tr><td> פוספט נתרן Monobasic נטול מים (1 ק&quot;ג, המשמשים להכנת מאגר פוספט) </td><td> פישר סיינטיפיק </td><td> AC38987-0010 </td></tr><tr><td> Glutaraldehyde (50%, 10 x 10 מ&quot;ל) </td><td> טד פלה, Inc </td><td> 18431 </td></tr><tr><td> אוסמיום Tetroxide (מימית 4%, 10 x 10 מ&quot;ל) </td><td> טד פלה, Inc </td><td> 18465 </td></tr><tr><td> פרופילן אוקסיד (450 מ&quot;ל) </td><td> טד פלה, Inc </td><td> 18601 </td></tr><tr><td> הטמע 812 (קיט, על בלוקים קשיחים / ניגודיות תמונה גבוהה) </td><td> מיקרוסקופית אלקטרונים למדעים </td><td> 14120 </td></tr><tr><td> Toluidiנה כחול (25 גרם) </td><td> טד פלה, Inc </td><td> 19451 </td></tr></tbody></table> הכנת כל הר שריר ו אימונוהיסטוכימיה: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם (תיאור / כמות) </td><td> ספק </td><td> מספר קטלוגי </td></tr><tr><td> Paraformaldehyde (1 ק&quot;ג) </td><td> Sigma-Aldrich </td><td> P6148-1 ק&quot;ג </td></tr><tr><td> פוספט נתרן Dibasic נטול מים (500 גר &#39;, המשמשים להכנת מאגר פוספט) </td><td> פישר סיינטיפיק </td><td> S375-500 </td></tr><tr><td> פוספט נתרן Monobasic נטול מים (1 ק&quot;ג, המשמשים להכנת מאגר פוספט) </td><td> פישר סיינטיפיק </td><td> AC38987-0010 </td></tr><tr><td> 10% diluent BSA / פתרון חסימת (200 מ&quot;ל) </td><td> Kirkegaard &amp; פרי Laboratories, Inc </td><td> 50-61-00 </td></tr><tr><td> טריטון X-100 (100 מ&quot;ל) </td&gt; <td> דוט מדעי Incorporated </td><td> 9002-93-1 </td></tr><tr><td> גליצין, 98% (1 ק&quot;ג) </td><td> פישר סיינטיפיק </td><td> AC12007-0010 </td></tr><tr><td> רקמת-Tek cryo-אוקטובר מתחם (המקרה של 12, 4 בקבוקי עוז) </td><td> פישר סיינטיפיק </td><td> 14-373-65 </td></tr><tr><td> Sylgard דאו 170 (2 ערכת פאונד) </td><td> פישר סיינטיפיק </td><td> NC9492579 </td></tr><tr><td> נירוסטה סיכות חרקים, גודל 1 (100/pkg) </td><td> מדע כלים עדינים </td><td> 26001-40 </td></tr><tr><td> Tetramethylrhodamine-A-Bungarotoxin (0.5 מ&quot;ג) </td><td> Sigma-Aldrich </td><td> T0195-.5MG </td></tr><tr><td> עכבר נוגדן חד שבטי נגד SMI-312 (0.1 מ&quot;ל) </td><td> Covance </td><td> SMI-312R </td></tr><tr><td> עכבר נוגדן חד שבטי נגד SV2 (0.1 מ&quot;ל) </td><td> התפתחותית מחקרים Hybridoma בנק (DSHB) </td><td> SV2 </td></tr><tr><td> הארנב פולינוגדן נגד משובטים GAP-43 </td><td> נובוס ביולוגיות </td><td> NB300-143 </td></tr><tr><td> Flourescein מצומדות נגד עכבר עיזים IgG, Fcγ סדרה 1 ספציפי </td><td> ג&#39;קסון ImmunoResearch </td><td> <a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=115-095-205&amp;Attributes=Yes&amp;Quantity=1">11 5-095-205</a> </td></tr><tr><td> DyLight 649 מצומדות נגד ארנב חמור IgG </td><td> ג&#39;קסון ImmunoResearch </td><td> <a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=711-495-152&amp;Attributes=Yes&amp;Quantity=1">711-495-152</a> </td></tr><tr><td> 4 &#39;,6-diamidino-2-phenylindole (DAPI, dilactate, 10 מ&quot;ג) </td><td> Invitrogen </td><td> D3571 </TD&gt; </tr><tr><td> Vectashield הרכבה בינונית (10 מ&quot;ל) </td><td> וקטור מעבדות </td><td> H-1000 </td></tr><tr><td> Superfrost פלוס שקופיות מיקרוסקופ (לבן, גודל: 75 x 25 מ&quot;מ, חבילה של 144) </td><td> פישר סיינטיפיק </td><td> 12-550-15 </td></tr><tr><td> Fisherfinest כיסוי פרימיום משקפיים (גודל: 40 x 22 מ&quot;מ; חפיסת עוז 1.) </td><td> פישר סיינטיפיק </td><td> 12-548-5C </td></tr></tbody></table>

reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis

הדור הבא של מערכת העצבים ההיקפית (PNS) נלמדת באופן נרחב גם על הרלוונטיות שלה למחלות האדם להבין את התגובה משובי חזקים רכוב על ידי נוירונים PNS ובכך אולי שהאירו את הכישלונות של התחדשות מערכת העצבים המרכזית 1. למעוך גיד הנשה (axonotmesis) הוא אחד המודלים הנפוצים ביותר של פגיעה עצבית היקפית במכרסמים 2. ריסוק קוטע את כל האקסונים אלא רבדים שוואן התאים הבזליים נשמרים כך התחדשות כי הוא אופטימלי 3,4. זו מאפשרת לחוקר לבחון דווקא את היכולת של האקסון הגוברת לקיים אינטראקציה עם התא הן שוואן ואת רבדים הבסיס 4. החולדות היו בדרך כלל את הדגמים המועדפים על בעלי חיים למחוץ העצב ניסיוני. הם זמינים באופן נרחב ועל גיד הנשה lesioned שלהם מספק קירוב סביר של נגעים עצב אנושיים 5,4. אם כי קטנים יותר מאשר עצב חולדה, עצב העכבר יש תכונות דומות רבות. והכי חשוב אם כי, mousמודלים אלקטרוניים הם בעלי ערך יותר ויותר בגלל הזמינות רחב של קווי מהונדס כעת מאפשרת לנתיחה מפורט של מולקולות בודדות קריטיים עבור התחדשות עצבים 6, 7. חוקרים קודמים השתמשו בשיטות רבות כדי לייצר דלוקה עצב או פציעה כולל מלקחיים זווית פשוטים, מלקחיים, מלקחיים מצוננים עוצר דמום, Clamps כלי הדם, וכן החוקר מלחציים, שנועדו 8,9,10,11,12. החוקרים השתמשו גם שיטות שונות של סימון האתר כולל פגיעה תפר, חלקיקי פחמן חרוזי ניאון 13,14,1. אנו מתארים את השיטה שלנו להשיג דלוקה מלאה reproducibly גיד הנשה עם סימון מדויק ועקבי של האתר, למעוך בעזרת מלקחיים עוצר דמום דקים פחמן לאחר המדליקה האתר סימון. במסגרת תיאור שלנו הליך גיד הנשה למחוץ כללנו גם שיטה פשוטה יחסית של כל שריר לעלות לאחר מכן אנו משתמשים כדי לכמת התחדשות.

Watch this Scientific Journal Video about Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis at JoVE.com

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

בדו&quot;ח זה אנו מתארים שיטה למחוץ העצב העכבר הנשה. שיטה זו משתמשת מלקחיים עוצר דמום זמינים ובקלות reproducibly מייצרת העצב המלא למחוץ הנשה. בנוסף, אנו מתארים שיטה להכין mounts שרירים שלמים מתאימים לניתוח של התחדשות עצבים אחרי למחוץ גיד הנשה.

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 ...

reproducible-mouse-sciatic-nerve-crush-subsequent-assessment

Research

JoVE Journal

Neuroscience

33.6K Views.  Temple University. בדו"ח זה אנו מתארים שיטה למחוץ העצב העכבר הנשה. שיטה זו משתמשת מלקחיים עוצר דמום זמינים ובקלות reproducibly מייצרת העצב המלא למחוץ הנשה. בנוסף, אנו מתארים שיטה להכין mounts שרירים שלמים מתאימים לניתוח של התחדשות עצבים אחרי למחוץ גיד הנשה.

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

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

Axoplasm ניתוק מן החולדה עצב השת

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

הערכת התנהגויות אינטראקציה חברתית

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Dissection העין עכבר על הר שלם של האפיתל פיגמנט רשתית

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

הר הכנה שלמים של המבוגר תסיסנית עצבי חוט הגחון עבור הזרקת סיבים ענק דיי

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

ניתוח של השריר Transversus Abdominis לניתוח צומת נוירומוסקולרי הר שלם

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

הדמיה הר-כל תחזיות החושיות אקסון העובר העכבר

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

ההר שלם Dissection וImmunofluorescence של מבוגרי עכבר השבלול

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In vivo ג&#39;ין העברה בתאי שוואן בעצב מכרסמים Sciatic ידי Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

3D ניתוח קימטי עבור הערכה פונקציונלית במודל חולדה של פציעה העצבים הגיד מעיכה

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

כתמים, הדמיה וניתוח של פטריות, בלוטות טעם של הרכבה מלאה, מקיפים וחיך

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...