Edith Suss-Toby is thanked for her assistance in the time-lapse microscopy and image analysis. Nofar Rada is thanked for the artistic work.

ארבע עד C57BL נקבה שישה בן-שבוע / CJ עכברים (הרלן, ירושלים, ישראל) שימשו בניסוי פי האגודה הערכה וההסמכה של מפרטים טיפול בבעלי חיים מעבדה. כל הפרוצדורות בוצעו בהתאם טיפול בבעלי חיים מוסדיים ועדה השתמש ומח&#39; תקנות חקלאות. 1. קציר חוט השדרה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את העכבר באמצעות תא 2 CO. הימנע נקע בצוואר הרחם כיוון שהוא עלול לגרום נזק שורשי גנגליון בשל כוחות גזירה. מכאן ואילך, לבצע את כל השלבים בתנאים סטריליים. </li><li style=";text-align:right;direction:rtl"> משרים את חיה עם 70% אתנול. שלב זה חשוב עבור עיקור למניעת השיער מגרירת דרך האיברים הפנימיים. </li><li style=";text-align:right;direction:rtl"> השאירו את העכבר להתייבש מן אתנול. מנע כל מגע של אתנול עם האיברים הפנימיים בשל רעילות עצבית. </li><li style=";text-align:right;direction:rtl"> לאחר ייבוש העכבר, מקם אותו באמצעות סיכות אפרקדן(פנים למטה). מניחים את הפינים שעל כל hindlimb ו forelimb. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים, לעשות חתך קו אמצע הארכת craniocaudally מהצוואר האחורי של הגב התחתון. לאחר מכן, להעלות דשי עורי באמצעות מלקחיים בילטרלי ולחשוף הרקמה התת עורית. </li><li style=";text-align:right;direction:rtl"> למשש גולגולת עכבר עד שמגיע לצומת craniocervical. בעזרת מספריים בניצב החיה, בקעה את שרירי צוואר רחם עמוד שדרה בצומת craniocervical (חוליות צוואר הרחם -7), באמצעות מלקחיים כבדים. לנתח את עמוד שדרת caudally, ולהסיר את עמוד השדרה עם מלקחיים כבדים עד שהגיע לרמת המותני (רמת גפיים אחורית). </li><li style=";text-align:right;direction:rtl"> בצע חיתוך רוחב זנב מלא ברמת עמוד השדרה המותני (חוליות 5 th) באמצעות מלקחיים כבדים. </li><li style=";text-align:right;direction:rtl"> מגלגלים את העכבר מעל (עם הפנים כלפי מעלה). אין צורך לכסות את החתך מאז DRG מופק רמות שדרה תחתונות ולא צוואר הרחם. </li><li style=";text-align:right;direction:rtl"> צמצמו על קו האמצע מהצוואר אל הבטן, לחזור בועור רוחבי ואז לחתוך כדי לפתוח את הצפק. </li><li style=";text-align:right;direction:rtl"> הסר את האיברים הפנימיים בתוך הצפק (כבד, טחול, לבלב, קיבה ומעי) כמקשה אחת. לאחר מכן, להסיר את איברי retroperitoneal (כלומר, כליות ולבלב). Cranially, לפתוח את בית החזה. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים להב כירורגי, לחתוך את הצלעות, משאיר 5 מ&quot;מ של צלעות בילטרלי משם מעמוד השדרה. בשלב זה, עמוד השדרה מופרד משאר הגוף. </li><li style=";text-align:right;direction:rtl"> שטפו את עמוד השדרה מדם עם בופר פוספט טרי קר (4 ° C) (PBS) פעמיים. </li></ol> 2. בידוד השורש הגבה גרעינים (DRG) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש stereomicroscope עם הגדלה 4X. </li><li style=";text-align:right;direction:rtl"> מקם את עמוד השדרה על פלטפורמה קולטת הלא לא חסידה (Telfa / ניילון). מקם את הפנים עמוד השדרה למעלה על האוריינטציה סקראל הזנב אותו. </li><li style=";text-align:right;direction:rtl"> הסר את כל שרירי עמוד שדרה ורקמות חיבור. השתמש צלעות כציון דרך לעצבים כפי שהםלעזוב את עמוד השדרה. צלעות גם להגן על העצבים מן הכלים כירורגיים. DRG ממוקמים בחלק הצווארי, החזי, ואתרי המותני של החוליות. </li><li style=";text-align:right;direction:rtl"> לאחר מכן, מספריים באמצעות לחתוך את הגוף בחוליות קו האמצע ולחשוף את חוט השדרה ושורשי DRG ידי הכחשה לרוחב עדינה של הגוף בחוליות. </li><li style=";text-align:right;direction:rtl"> במרחב צלע, בצע את העצבים ההיקפיים מדיאלית לאורך לרוחב צלע אל DRG. </li><li style=";text-align:right;direction:rtl"> זהה את DRG. זה נראה כמו חלמון של &#39;ביצת עין&#39; שוכב על העצב. </li><li style=";text-align:right;direction:rtl"> חותכים את דיסטלי עצב צלעי אל DRG עוזב 2-3 מ&quot;מ של דיסטלי החוצפה הגרעינים, לשמש הכחשה. זה &quot;זנב&quot; יוסר לאחר הקטיף. </li><li style=";text-align:right;direction:rtl"> אחוז העצב באמצעות מלקחיים. צביטת הגוף DRG עצמו יגרום נזק לתאים עצביים יש להימנע. </li><li style=";text-align:right;direction:rtl"> מתקרב DRG proximally יחד התקשרותו אל חוט השדרה, באמצעות הקדמי שלה ושורשים אחוריים. </li><li style=";text-align:right;direction:rtl"> החל הכחשה עדינה על DRG ידי משייכת EFעצב ferent (גדם עצב צלעי) רוחבי, וחתך את הקדמי ואת האחורי שורשים הקרוב DRG. </li><li style=";text-align:right;direction:rtl"> שמור את DRG בצלחת פטרי 35 מ&quot;מ מלא DMEM טרי קר, קרח בתוספת 10% נסיוב עגל עוברית (FCS), 1%, סטרפטומיצין פניצילין, 1% חומצות אמינו לא חיוניות, ו פירובט סודיום 1%. </li></ol> 3. השרשה בצלחת פטרי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בצע את הצעדים הבאים על הקרח באמצעות פיפטה טיפים מראש מקורר כדי למנוע התמצקות ECM. </li><li style=";text-align:right;direction:rtl"> תחת סטראו בהגדלה 2-4X, מניחים צלחת פטרי תחתית זכוכית 35 מ&quot;מ על רשת נייר. חשוב: עבודה על קרח כדי לשמור על ECM במצב נוזלי. צור נקודה בקוטר 1 מ&quot;מ (כ 20 μl) של ECM מדולדל גורם הגדילה במרכז הרשת. </li><li style=";text-align:right;direction:rtl"> תחת להדמיה ישירה, למקם את DRG במרכז המקום ECM קרוב לתחתית של המנה. הערה: התאים הסרטניים בשימוש בפרוטוקול זה הם תאים סרטניים בעכברי לבלב (KPC) establisהד מ דגימות הגידול מבודדים טרי מעכברים KPC כמתואר במקום אחר 19. </li><li style=";text-align:right;direction:rtl"> קציר תאים 40,000 סרטן מתרבויות ומחוברות, לשטוף אותם פעם אחת עם PBS, resuspend אותם 40 ECM μl על הקרח. </li><li style=";text-align:right;direction:rtl"> תחת ראיה ישירה של הרשת באמצעות מיקרומטר סטראו מידה 500 בכל כיוון מן DRG. בשלב זה לאט זרע 10,000 תאים / 10 μl ECM. השתמש טיפ 2-10 μl precooled להזריק את התאים קרובים לתחתית הצלחת, הימנעות ההתפשטות שלהם במטריצה ​​או הניתוק של מטריקס. (איור 1). </li><li style=";text-align:right;direction:rtl"> משאיר את הכלי בתוך מכסה מנוע זרימה למינרית במשך 10-15 דקות כדי לחזק. הימנע ייבוש ECM. </li><li style=";text-align:right;direction:rtl"> לאט לאט להוסיף DMEM, שהוכן כאמור, על הקיר הצדדי של הצלחת. הוסף מספיק בינוני כדי לכסות את ECM (כ 2 מ&quot;ל). </li><li style=";text-align:right;direction:rtl"> שים את הצלחת בחזרה אל האינקובטור ב 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> חלף בינוני עם בינוני טרי ביום הבא. </li><li style=";text-align:right;direction:rtl"> החלף את המדיום כל 2 ימים. &lt;/ Li&gt; </ol> 4. רכישת נתונים, זמן לשגות Videomicroscopy <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ביום 7 לאחר ההשתלה, לקחת את הצלחת עבור מיקרוסקופיה זמן לשגות. שים לב תאים מסוימים עשויים לחייב רכישה מוקדם יותר בגלל הגירה מהר. </li><li style=";text-align:right;direction:rtl"> במהלך ההדמיה לחיות, במקום צלחת בסביבה סגורה בטמפרטורה ממוצעת של 37 ± 0.1 מעלות צלזיוס, 5% CO 2. </li><li style=";text-align:right;direction:rtl"> רשום את התאים באמצעות מצלמת המכשיר מצמידים תשלום מושם על המיקרוסקופ. </li><li style=";text-align:right;direction:rtl"> קח תמונות דיגיטציה כל 10 דקות עד 72 שעות כדי לעקוב אחרי תנועת תא בין שלושה לשישה תאים שונים. </li><li style=";text-align:right;direction:rtl"> חלף בינוני לאחר 24 שעות. </li><li style=";text-align:right;direction:rtl"> בצע רכישת נתונים וניתוח פשוט באמצעות תוכנת מיקרוסקופ. </li></ol> ניתוח 5. נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עבור יישומים מתקדמים יותר (כלומר, מעקב תא 2D או 3D) להשתמש בתוכנה מיוחדת (כגון Imaris 4D). הערה: תוכנה זו קובעת את קואורדינטות XY של תאבכל רגע נתון, יחד עם המרחק ממקור, ומהירות תנועה. </li><li style=";text-align:right;direction:rtl"> לחשב את המהירות הממוצעת על ידי מדידת מרחק הנסיעה בין עמדות שלאחר מכן ב 10 מרווחי דקות. חישוב מדד ההגירה הקדמי (FMI) המהווה את המרחק של חזית התא מן neurite ומתאר את התנועה חד כיווני של התאים כלפי DRG. </li></ol>

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This research was supported by the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a grant from the US-Israel Binational Science Foundation. No other financial disclosures from the authors are to be made.

מאמר זה מציג מודל במבחנה כי שחזר במייקרו-הסביבה של הסרטני בגומחה העצבית, מודל DRG. הסרטון מדגים את כל השלבים החל מהכרה ציוני דרך אנטומיים כגון DRG בתוך העכבר, החילוץ שלה, ולבסוף, culturing שלה ECM. שיתוף culturing DRG לצד עם תאים סרטניים מוצג גם. אין מודלים אחרים עבור במבחנת ?...

גידולים מוצקים להפיץ בשלוש דרכים עיקריות: פלישה ישירה, ממרח הלימפה, והתפשטות hematogenic. עם זאת, יש אמצעי הרביעי של התפשטות סרטן זה התעלם לעתים קרובות, הפצה לאורך עצבים. פלישה עצבית סרטנית (CNI) היא מסלול ידוע של התפשטות הסרטן, במיוחד סרטן של הראש והצוואר, 1 הערמונית 2, 3 ולבלב. CNI 4-8 מתרחש בלמעלה מ 80% של אנשים עם אדנוקרצינומה של לבלב, מוביל כדי גידול retroperitoneal התפשט עצבי גנגליון צליאק. יש תאים סרטניים neurotropic אלה יכולת ייחודית להעביר בכיוון אחד לאורך עצבים כלפי מערכת העצבים המרכזית (CNS). 9 ממצא זה מצביע על כך microenvironment perineural יכול להיות מנוצלת על ידי תאי סרטן, מתן הגורמים התומכים גידול ממאיר. אחד הדגמים כמה במבחנה למחקר CNI הוא הגרעינים השורש הגבי (DRG) / דגם תאים סרטניים. mod זהאל משמש לעתים קרובות כדי לחקור את האינטראקציה בין תאי paracrine stroma והסרטן עצביים. 10-18 במודל זה, עכבר או שורות תאים סרטניים אנושיות גדלות תאי מטריקס (ECM) סמכו הכנות של DRG המתורבת טרי ניתק. מאמר וידאו זה מציג את היישום של במבחנה CNI ב אדנוקרצינומה ductal הלבלב.

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in vitro modeling of cancerous neural invasion: the dorsal root ganglion model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

<html> לדימות באמצעות מיקרוסקופיה וידאו, DRG ניתן לראות הנבטה neurites 5-7 ימים לאחר ההשתלה בעוד תאים סרטניים נודדות הרחק ממושבות שלהם כלפי DRG. עד היום ה -7 לאחר ההשתלה, התאים הסרטניים לבוא במגע עם neurites (איור 2). <p class="jove_content" fo:keep-together.within-page="1" style...

Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

במבחנת דוגמנות של פלישה העצבית סרטנית: מודל גנגליון השורש הגבה

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

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Neuroscience

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

9.0K Views.  Rambam Medical Healthcare Campus. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

Video: In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

דוגמנות איתות החיסון עצבית של מיגרנה אפיזודי כרוני באמצעות הפצת דיכאון במבחנה</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

הדמיה חיה של אקסונים לאחר רוט הגבי Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

המחקר הגנטי של הדור הבא אקסון עם נוירונים בתרבית למבוגרים הגנגליון הגב שורש

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

מדידת השדרה Presynaptic עיכוב בעכברים על ידי הקלטה פוטנציאלית הגבי רוט<em&gt; בVivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

גב שורש הגרעינים עצב ותאי גזע שמקורם בשומן Differentiated:<em&gt; במבחנה</em&gt; משותף תרבות דגם לחקר התחדשות של עצבים היקפיים

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

גישת שפר מערך myelination של נוירונים שורש הגבה גנגליון

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

שחול הידראולי של קורד ובידוד השדרה של גרעיני שורש הגבה במכרסמים

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

הזרקת גנגליון שורש הגבה ו שורש הגבה Crush פגיעה כמודל להתחדשות החושית אקסון

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomy גישה גנגליון המותני שורש העזוב, הזרקה של החזירים

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

ניתוח של תאים חיסוניים העצבים ממוגלה יחיד, גנגליון העזוב השורש של עכבר אחת באמצעות Cytometry זרימה

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

במבחנת דוגמנות של פלישה העצבית סרטנית: מודל גנגליון השורש הגבה

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דוגמנות איתות החיסון עצבית של מיגרנה אפיזודי כרוני באמצעות הפצת דיכאון במבחנה