Name: production and isolation of axons from sensory neurons for biochemical analysis using porous filters
Uploaded: 2014-07-08T23:45:00.000+00:00
Duration: 12 min
Description: Watch this Scientific Journal Video about Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters at JoVE.com

This project was supported by grant MOP62827 from the Canadian Institutes of Health Research.

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+is+Wallerian+degeneration+in+the+CNS+so+slow.">Why is Wallerian degeneration in the CNS so slow.</a> Annu. Rev. Neurosci. 30, 153-179 (2007).</li><li>Waller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+Section+of+the+Glossopharyngeal+and+Hypoglossal+Nerves+of+the+Frog,+and+Observations+of+the+Alterations+Produced+Thereby+in+the+Structure+of+Their+Primitive+Fibres.">Experiments on the Section of the Glossopharyngeal and Hypoglossal Nerves of the Frog, and Observations of the Alterations Produced Thereby in the Structure of Their Primitive Fibres.</a> Philosophical Transactions of the Royal Society of London. 140, 423-429 .</li><li>Albuquerque, C., Joseph, D. J., Choudhury, P., MacDermott, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection,+Plating,+and+Maintenance+of+Dorsal+Root+Ganglion+Neurons+for+Monoculture+and+for+Coculture+with+Dorsal+Horn+Neurons.">Dissection, Plating, and Maintenance of Dorsal Root Ganglion Neurons for Monoculture and for Coculture with Dorsal Horn Neurons.</a> Cold Spring Harbor Protocols. , (2009).</li><li>Leach, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+primary+motor+and+sensory+neurons+in+defined+media+on+electrospun+poly-L-lactide+nanofiber+scaffolds.">The culture of primary motor and sensory neurons in defined media on electrospun poly-L-lactide nanofiber scaffolds.</a> J Vis Exp. 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The authors have nothing to disclose.

פרמטרים חשובים לקחת בחשבון בעת ​​התאמת טכניקה זו כוללים את האוכלוסייה העצבית שתילמד, מצע, גודל נקבובית של המסנן ועל פני שטח. כל הפרמטרים הללו ישפיעו על הספציפיות, איכות וכמות של הכנת neurite שהושגה, ויש לשקול בזהירות על ידי משתמש הקצה. בדוגמאות שהוצגו בפרוטוקול זה, השימ?...

תא אקסון של נוירונים מראה מידה רבה של עצמאות תפקודית מתא somato-הדנדריטים. בנוירונים הקרנה, אקסונים מכילים את רוב 1,2 החלבון העצבי. חקר הפיסיולוגיה והפתופיזיולוגיה של אקסונים צבר תאוצה בקהילת מדעי המוח, כי מחקרים שנעשה לאחרונה הראו כי תפקוד לקוי של אקסון המוקדם נראה כי תכונה משותפת של מחלות ניווניות והפרעות נוירופסיכיאטריות 3. סביר להניח כי הבנה טובה יותר של מנגנוני אקסון בלעדי בתנאים נורמלים ופתולוגית שתשפוך אור על האירועים המוקדמים המניע את חוסר תפקוד. הפרוטוקול הנוכחי מתאר כיצד להשתמש בתרבות מוסיף נושאות קרום נקבובי (מסנן) כדי להשיג כמויות גדולות של חומר אקסון טהור כי הוא מתאים ביוכימי וimmunocytochemical מנתח. השימוש במסנן מוסיף ללמוד הביולוגיה אקסון יושמה לראשונה על ידי דייל ועמיתים 4 ופותחו עוד יותר על ידי Twiss ועמיתים 5 לתצורתו הנוכחית. הטכניקה היא כעת בשימוש שוטף על ידי קבוצות לימוד היבטים שונים של אקסון והביולוגיה דנדריט, עם שינויים קלים בכל מקרה כדי להתאים לאוכלוסייה של נוירונים למדו, הטיפולים שבוצעו ואת הסוג של ניתוחים ביוכימיים המשמשים 6-9. הפרוטוקול הנוכחי אינו מתכוון להכיל את כל החלופות למגוון כה רחב של יישומים, אלא מספק גישה פשוטה, שמתאימה למרבית היישומים. בפרט, הפרוטוקול המתואר כאן משתמש בציפוי משולש למיקסום תשואת אקסון עבור ניתוחים ביוכימיים והוא מתאים ביותר ללמוד אקסון ציפויי ניוון-אחר המתוארים בספרות המיוצר פחות אקסונים שיחזרו ולהידרדר מהר יותר כאשר נשללו מגורמים תזונתיים 9. בהליך זה, גופי תא עצביים נותרו מבודדים על גבי עלי לעשות סינוןאקסונים ile לעבור הדרך הנקבוביות ולגדול לאורך המשטח התחתון שלה. אקסונים טהורים (ללא גליה ומזהמים גוף תא עצביים) הגדלים על המשטח התחתון של המסנן יכולים להיות שנאספו עבור ניתוחים ביוכימיים או יכולים להיות קבוע באתר ונבחנו על ידי טכניקות immunocytochemical 9. ההליך מסתמך על השימוש בתאי עצב תחושתי עובריים מהגרעינים הגבי שורש (DRG). DRGs עוברי נמצא בשימוש נרחב ללמוד ביולוגיה אקסון בגלל neurites מהתאים אלה עוברות צמיחה מהירה וחזקה במבחנה, כאשר נשמר בגורם גדילה עצבי (NGF), וכי הם במהירות לעבור ניוון כאשר נשללו של גורם זה. כמו כן, הנוירונים DRG חסרי דנדריטים, כך שכל neurites שנאסף על ידי בשיטה זו הן אך ורק אקסונים. מוסיף מסנן מייצג יתרון גדול בהשוואה לגישות אחרות המשמשות ללמוד אקסונים. מחקרים להסתמך על השימוש במיקרוסקופ לבדל תהליכים המתרחשים בתא סומה מאלוxons לספק מידע ביוכימי מוגבל. כדוגמא נוספת, מערכות ממודרות תרבות (למשל, תאי Campenot 10 או מכשירי microfluidic 11) שימושיות לגישות הדמיה ולטיפול בהפרש של תאי תאים אלא לספק רק כמויות קטנות של חומר באקסון המונעות את השימוש בטכניקות אלה לניתוחים ביוכימיים אשר דורשים כמויות גדולות היחסי של מדגם. יתר על כן, השימוש בם דורש הכשרה משמעותית, כמו גם התקנים מיוחדים, והם גוזלים זמן. גישה אחרת המשמשת לעתים קרובות לבודד את האקסונים מ explants היא להסיר באופן ידני מרכז explant (המכיל גופי תא עצביים) לפני איסוף דגימת אקסון. למרות שזה יכול לייצר כמויות גדולות של תכשירים מועשר האקסון, ניתן עטפו אקסונים בתכשיר זה בגליה ומהירות הסרת הגופים של 20 explant ברציפות מ -6 בארות (כדוגמא לניסוי מינימאלי) היא זמן רב ועל גבול כדאיות. לעומת זאת, בשיטה המוצגת כאן מייצרת הכנות אקסון שופעים וטהורות, כי אז יכול להיות מנותחת על ידי כמעט כל טכניקה ביוכימיים המשמש בדרך כלל כדי לנתח lysates כל התא, כמו כתם מערבי, immunoprecipitation 6, כתם צפוני 5 ספקטרומטריית מסה 6, טיהור RNA 7,12,13, בין יתר. יתר על כן, הפרוטוקול יכול להיות מיושם על Immunofluorescence טכניקות (IF), כפי שאפשר לתקן אקסונים גדל בצד התחתון של המסנן כדי לבחון אותם עוד יותר על ידי אם. גישה זו מאוד מקלה על הניתוח של תהליכי אקסון ספציפי מכיוון שאין גופי תא בהכנה הסופית. זהו יתרון משמעותי שכן אות גבוהה immunofluorescent מגופי תא לעתים קרובות פוגעת בבחינה וניתוח של אותות חלשים שמקורם במבנים דקים כגון אקסונים. שני יישומים של שיטת התרבות ללמוד ar ניוון אקסוןדואר תאר; מודל של גיזום התפתחותיים ומודל של ניוון הנגרם פגיעה (המכונות ניוון Wallerian כ). המודלים של גיזום התפתחותי מבוסס על העובדה שתאי עצב תחושתי in vivo להתחרות על הגבלת סכומי NGF-derived יעד ואלו שלא הצליחו לקבל מספיק מנוון תמיכת נוירוטרופי 14. תופעה זו יכולה להיות חיקה בתרבויות DRG העובריים על ידי נסיגת NGF מהתקשורת והתרבות, אשר, כפי שקורה בvivo, יוזם ניוון אקסון אחריו הרס גוף תא. למודל ניוון Wallerian, הצד העליון של המסנן עם גופי התא שרוט משם. אקסונים שצמחו על הצד התחתון של המסנן להיות מופרד פיזי מהגופים התא ולאחר מכן לעבור את התהליך המהיר וסטריאוטיפי ניווניות המכונה ניוון Wallerian 15, על שמו של א &#39;וולר שדיווח על התופעה ראשונה בשנת 1850 16.

<table><tbody><tr><td>CD-1 mouse, E13 timed pregnancy</td><td>Charles River</td><td></td><td>In house timed pregnancies can be done by mating mice and checking for vaginal plugs (Day E0)</td></tr><tr><td>Falcon cell culture inserts</td><td>Corning</td><td>353102</td><td>1 &amp;micro;m pore size, fits 6-well receiving plate</td></tr><tr><td>Falcon cell culture companion plates</td><td>Corning</td><td>353502</td><td>Receiving plates for inserts</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>Used at 1 mg/ml in water. Can re-use once.</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>Used at 10 &amp;micro;g/ml in water. Take 100x stock from -80 &amp;deg;C and thaw O/N at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>PureCol bovine collagen solution</td><td>Advanced Biomatrix</td><td>5005-B</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Invitrogen</td><td>11415-064</td><td>Can be replaced by DMEM</td></tr><tr><td>Neurobasal medium</td><td>Invitrogen</td><td>21103-049</td><td></td></tr><tr><td>B-27 serum-free supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine (FDU)</td><td>Sigma-Aldrich</td><td>F0503</td><td></td></tr><tr><td>NGF (2.5S, beta subunit)</td><td>Cedarlane</td><td>CLMCNET-001</td><td></td></tr><tr><td>Anti-NGF antibody</td><td>Prepared in-house</td><td></td><td>As described in: Acheson, A. &lt;em&gt;et al.&lt;/em&gt; Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF. Neuron. &lt;strong&gt;7&lt;/strong&gt;, 265-75, (1991).</td></tr><tr><td>Alomone Labs</td><td>AN-240</td><td>Commercial option</td><td></td></tr><tr><td>Anti-tubulin antibody</td><td>Millipore</td><td>MAB5564</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse antibody</td><td>Invitrogen</td><td>A-11029</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;DRG culture medium&lt;/strong&gt;</td></tr><tr><td colspan="4">Neurobasal Medium&lt;br/&gt; 2% B27 Neurobasal Supplement&lt;br/&gt; 2 mM l-Glutamine&lt;br/&gt; 1% Penicillin-streptomysin&lt;br/&gt; 10 &amp;micro;M FDU&lt;br/&gt; 15 ng/ml NGF (bottom compartment only)</td></tr><tr><td colspan="4">&lt;strong&gt;1x Laemmli sample buffer&lt;/strong&gt;</td></tr><tr><td colspan="4">2% SDS&lt;br/&gt; 50 mM DTT&lt;br/&gt; 60 mM Tris (pH 6.8)&lt;br/&gt; 5% Glycerol&lt;br/&gt; 0.01% (w/v) Bromophenol blue</td></tr></tbody></table>

production and isolation of axons from sensory neurons for biochemical analysis using porous filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

explants גנגליון שורש הגבי מעכברי E13.5 לגדול בהרחבה על מסננים ברצף מצופה פולי-D-ליזין, laminin וקולגן (איור 1 ב, משמאל), והגיע עד 2 מ&quot;מ בתוך 2 ימים בתרבות. שילוב מצע זה מניב צמיחה במיוחד שופעת; שילובים אחרים יניבו אקסונים שהם הרבה יותר קצרים (איור 1 ב, מימין). יתר ...

Watch this Scientific Journal Video about Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters at JoVE.com

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

הפקה ובידוד של אקסונים מסנסורי ביוכימית ניתוח באמצעות מסננים נקבוביים

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

production-isolation-axons-from-sensory-neurons-for-biochemical

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.2K Views.  Montreal Neurological Institute, McGill University. This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration. 

Video: Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

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 ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

הכנת Synaptoneurosomes מ - עכבר Cortex באמצעות צבע רציפה Percoll-סוכרוז צפיפות

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

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 ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

בידוד של נוירונים חושיים של<em&gt; Aplysia californica</em&gt; להקלטות מהדק תיקון של זרמי glutamatergic

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

שיטת שיתוף התרבות כדי ללמוד Neurite Outgrowth בתגובה שיניים זולה הקרנף סימנים

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Developmental Biology

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

עיצוב הסיליקון הנקבובי סרטים כמו נשאים של גורם הגדילה העצבי

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Bioengineering

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

טיהור של פיברותקיעות ותאי Schwann מפני חושים ומוטוריים עצבים בחוץ גופית

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many neurodegenerative diseases. Given some axons' elaborate architecture and extreme length, it is not surprising that mitochondria in axons can experience different environments compared to their cell body counterparts. Interestingly, dysfunction of axonal mitochondria often precedes effects on the cell body. To model axonal mitochondrial dysfunction in vitro, microfluidic devices allow treatment of axonal mitochondria without affecting the somal mitochondria. The fluidic pressure gradient in these chambers prevents diffusion of molecules against the gradient, thus allowing for analysis of mitochondrial properties in response to local pharmacological challenges within axons. The current protocol describes the seeding of dissociated hippocampal neurons in microfluidic devices, staining with a membrane-potential sensitive dye, treatment with a mitochondrial toxin, and the subsequent microscopic analysis. This versatile method to study axonal biology can be applied to many pharmacological perturbations and imaging readouts, and is suitable for several neuronal subtypes.

The present protocol describes the seeding and staining of neuronal mitochondria in microfluidic chambers. The fluidic pressure gradient in these chambers allows for the selective treatment of mitochondria in axons to analyze their properties in response to pharmacological challenges without affecting the cell body compartment.

דה-פולריזציה סלקטיבית בסיוע מיקרופלואידיקה של מיטוכונדריה אקסונאלית

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many ...

Isolation of Cells from Sensory Ganglia

Source: Kaelberer, M. M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53917">A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice.</a> J. Vis. Exp. (2016).
This video demonstrates the isolation of cells from the sensory ganglia of mice. Ganglia are subjected to enzymatic digestion to dissolve the extracellular matrix. Single cells are then collected via filtering through a cell strainer followed by density gradient centrifugation for debris removal. Finally, washing and centrifugation are done to collect the isolated cells for future analysis.

בידוד תאים מגרעינים חושיים

All procedures involving sample collection have been performed in accordance with the institute&#8217;s IRB guidelines.
1. Sensory Ganglia Dissociation

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