ويدعم عملنا من قبل مؤسسة العلوم الوطنية السويسرية (SNF)، والمبادرة السويسرية في بيولوجيا الأنظمة (SystemsX.ch)، والضمور العضلي جمعية الولايات المتحدة الأمريكية (نجمة داود الحمراء)، والفرنسية جمعية مناهضة ليه الاعتلالات العضلية (فؤاد)، والميتوكوندريا المتحدة مرض مؤسسة (UMDF)، وغيبرت في الجبهة المتحدة الثورية مؤسسة برنامج الأمراض النادرة (GRF)، والجمعية السويسرية للأبحاث في أمراض العضلات (SSEM / FSRMM)، والسويسري ايف &quot;Jubiläumsstiftung FÜR Volksgesundheit اوند medizinische Forschung&quot; للأبحاث روش المؤسسة والجامعة من بازل.

1. إعداد الإنسان الأساسية العضلات خلية ثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء خلية عضلة الإنسان الثقافات وفقا لتقنية explantation إعادة explantation-4. أولا، عدم إزالة الأنسجة العضلية من الخزعات. ثم، تضمين 1 explants مم 3 في عضلة خثرة من البلازما، والذي يسمح الليفية للخروج من explants. </li><li style=";text-align:right;direction:rtl"> يتم نقل explants جزءا لا يتجزأ من على طبق من البلازما الجيلاتين المغلفة وتركها لتنمو في المتوسط ​​النمو الطبيعي (MEM تستكمل مع متوسط ​​25٪ 199، 10٪ الجنين المصل البقري [FBS]، 1٪ البنسلين / الستربتومايسين، 10 ميكروغرام / مل الأنسولين، 10 نانوغرام / مل الإنسان عامل نمو البشرة [EGF]، 12.5 نانوغرام / مل عامل نمو الخلايا الليفية الإنسان [جمعية جيل المستقبل]) في أحادي الطبقة. الخلايا التي تخرج من explants في ظل هذه الظروف هي خلايا العضلات. وهذه الخلايا تستمر في النمو في أحادي الطبقة بعد إزالة explants. </li><li style=";text-align:right;direction:rtl"> ثقافة الخلايا aneurally على 0،1 لوحات الجيلاتين المغلفة٪ في متوسط ​​النمو الطبيعي. يمكن للخلايا أن يكونمثقف aneurally وتوسيعها حسب الحاجة. فمن الأفضل أن تستخدم خلايا العضلات في ممر بين 3 و 8 لأداء المشترك الثقافات. من المهم جدا أن يعصب myotubes الشباب، وبعد هذه المرحلة، شارك في والثقافات وارتفاع مخاطر الفشل. وعلاوة على ذلك، ينبغي أن myotubes لا تكون كبيرة جدا وسميكة جدا. </li><li style=";text-align:right;direction:rtl"> قبل يومين من يخطط للتعصيب، لوحة خلايا العضلات على 35 مم أطباق زراعة الأنسجة (أو 6 لوحات أيضا) في مناطق ذات كثافة من 500000 خلية لكل طبق في متوسط ​​النمو الطبيعي. في ذلك الكثافة، كانت الخلايا في confluency المناسب لبدء الاندماج في اليوم التالي، وتعصيب لاحق من اليوم بعد ذلك. باستخدام هذه الظروف، وmyotubes في مرحلة حق لتعصيب الأمثل واللاحقة وبالتالي تقلص الألياف. </li><li style=";text-align:right;direction:rtl"> في اليوم التالي، بدلا من متوسط ​​النمو الطبيعي مع وسيلة اندماج تتألف من MEM تستكمل مع متوسط ​​25٪ 199، 5٪ FBS، الانسولين 10 ميكروغرام / مل و 1٪ من البنسلين / الستربتومايسين عقار. </li></ol> 2. Isolati<html> على من الفأر الجنينية Explants الحبل الشوكي يتم تنفيذ الإجراء بأكمله لعزل explants الحبل الشوكي تحت المجهر مجهر، في الحل هانك في سولت المتوازن (HBSS، Gibco / إينفيتروجن المرجع 14170) التي تحتوي على 10٪ الجنين مصل بقري (FBS). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فمن الأهمية بمكان أن أجنة الفئران ما بين 13 و 14 ED كمرحلة التنمية المختلفة من شأنه أن يعرض للخطر الإجراء بالكامل. تضحية امرأة حامل مع CO 2 وجمع سلسلة جنين كامل في المتوسط ​​HBSS. عزل كل جنين والقضاء على راس منهم للقتل الرحيم. </li><li style=";text-align:right;direction:rtl"> تشريح الحبل الشوكي من جنين وباقي الجسم في قطعة واحدة وإزالة العضلات المحيطة بها، والنسيج الضام. نكون حذرين للغاية للحفاظ على ganglions الجذر الظهري (DRGs) تعلق على الحبل الشوكي لضمان تعصيب وظيفي. </li><li style=";text-align:right;direction:rtl"> كل شريحة الحبل الشوكي عرضيا بطريقة كل يزدرع يحتوي على الأقل 2 DRG مرفقة به (explants من 1 ملم 3). </li></ol><html> ove_title &quot;&gt; 3. تعصيب من خلايا العضلات الإنسان مع Explants الجرذ الحبل الشوكي الجنينية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة المتوسطة الانصهار في ثقافة الخلية العضلية، وترك طبقة رقيقة من المتوسط ​​على أحادي الطبقة. </li><li style=";text-align:right;direction:rtl"> وضع explants بعناية على طبقة الخلايا العضلية، وخمسة explants متباعدة بالتساوي في صحن 35 ملم. </li><li style=";text-align:right;direction:rtl"> إضافة ببطء شديد، وانخفاض متوسط ​​الحكمة الانصهار، وعلى كل ازدراع. فمن الضروري إضافة المتوسطة لخلايا العضلات على قيد الحياة، ولكن ليس كثيرا للسماح للexplants الالتزام الأولى، واجراء اتصالات مع الخلايا. إذا لا يتم إضافة المتوسطة بشكل صحيح (أيضا بقوة أو المتوسطة أكثر من اللازم)، فإن explants تطفو، وسوف تفشل في عصب خلايا العضلات. </li><li style=";text-align:right;direction:rtl"> وبمجرد أن تبدأ explants إلى الالتزام بها وتتصدر المتوسطة الانصهار إلى 2 مل لكل طبق 35 ملم، ووضع أطباق بعناية جدا العودة الى الحاضنة. </li></ol> 4. صيانة غيروي العصب العضلات الثقافات المشارك <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تغيير وسيلة اندماج مرتين في الأسبوع. </li></ol>_title &quot;&gt; النتائج الممثل 5. في أقرب وقت ممكن بعد يومين من تعصيب، فمن الممكن أن نرى neurites الخارجة من كل ازدراع وإقامة اتصالات مع خلايا العضلات، ممثلا في هياكل تشبه الازرار (الشكل رقم 1). في أقرب وقت 4-6 أيام بعد تعصيب، فإن بعض الألياف الفردية تبدأ في العقد. وانقباضات تستمر لعدة أشهر، واصلت ببساطة عن طريق تغيير ثقافة المتوسط ​​مرتين في الأسبوع. بعد حوالي 2 أسابيع، ويزدرع الحبل الشوكي نفسه ينحط، ولكن يتم الحفاظ على تعصيب. هذا أمر طبيعي. طبقة رقيقة من عنصر العصب يبقى على سطح عضلة طبقة الخلية. هذه الشبكة العصبية كافيا للحفاظ على وظيفة من وحدة السيارات. إذا كان explants تطفو على المدى المتوسط ​​بعد يوم من تعصيب، فإنها لن تلتزم. هذا يحدث عندما طبقة الخلايا العضلية هييضاف ليس متكدسة بما فيه الكفاية، أو عندما يكون متوسط ​​تقريبا أيضا. في بعض الأحيان، وسوف تلتزم يزدرع neurites لكن لا تظهر. وهذا هو الأرجح بسبب عدم وجود DRG المرتبطة به. في هذه الحالة كذلك، لن تعصيب تنشأ. التلاعب في أطباق بعناية شديدة في الأسبوع الأول، كما يمكن فصل explants لا يزال بسهولة. لتبادل وسائل الاعلام الانصهار، وإضافة السائل التسرب من الحكمة مع سرعة الحد الأدنى لتجنب انفصال الخلية. <img alt="الشكل 1" src="/files/ftp_upload/3616/3616fig1.jpg" /> الشكل 1. الحبل الشوكي explants العضلات الخلية شارك في الثقافات من يوم 1 إلى يوم 15.

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ليس لدينا ما يكشف.

وهناك أداة الفسيولوجية في المختبر لدراسة العضلات وظيفة الخلية في السياق الطبيعي والمرضي هو أعلى من الفائدة للmyologists، وذلك لأن مزارع الخلايا العضلية عادة لا تلخص أهمية خلايا متعددة واتصالات من نوع الخلية. إضافة تنقية الخلايا العصبية الحركية إلى خلايا العضلات لي...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> HBSS </td><td> Gibco / إينفيتروجن </td><td> 14170 </td></tr><tr><td> MEM </td><td> Gibco / إينفيتروجن </td><td> 31095 </td></tr><tr><td> متوسط ​​199 </td><td> Gibco / إينفيتروجن </td><td> 31153 </td></tr><tr><td> الأبقار مصل الجنين </td><td> استنساخ الأجنة Perbio </td><td> SH30066.03 </td></tr><tr><td> أنسولين </td><td> سيغما </td><td> I9278 </td></tr><tr><td> الإنسان EGF </td><td> سيغما </td><td> E9644 </td></tr><tr><td> الإنسان جمعية جيل المستقبل </td><td> SIGMA </td><td> F0291 </td></tr><tr><td> البنسلين / الستربتومايسين حل </td><td> Gibco </td><td> 15140 </td></tr></tbody></table>

a functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells

خلايا عضلة الابتدائي تربيتها في aneurally أحادي الطبقة عقد نادرا لأن عفويا، في ظل عدم وجود عنصر العصب، ويقتصر تمايز الخلايا وتنشيط الخلايا العصبية الحركية مفقود 1. هذه القيود تعوق الدراسة في المختبر من العديد من الأمراض العصبية والعضلية في خلايا العضلات مثقف. الأهم من ذلك، يمكن التغلب على المعوقات التجريبية من monolayered، مثقف خلايا العضلات بواسطة الغدد العرقية وظيفية من myofibers مع explants الحبل الشوكي في الثقافات المشتركة. هنا، نعرض مختلف الخطوات اللازمة لتحقيق فعال وتعصيب السليم للخلايا عضلة الأولية، مما أدى إلى إكمال التفاضل والألياف الانكماش وفقا للطريقة التي وضعتها Askanas 2. للقيام بذلك، وخلايا العضلات شارك في تربيتها مع explants الحبل الشوكي لأجنة الفئران في ED 13.5، مع العقد الجذرية الظهرية لا تزال تعلق لشرائح الحبل الشوكي. بعد بضعة أيام، في عضلة فايbers بدء العقد وتصبح في نهاية المطاف عبر مخططة من خلال تعصيب بواسطة neurites الوظيفية تتوقع من explants النخاع الشوكي الذي يربط إلى خلايا العضلات. يمكن الحفاظ على هذا الهيكل لعدة شهور، وذلك ببساطة عن طريق التبادل المنتظم للثقافة المتوسط. تطبيقات هذا أداة لا تقدر بثمن هي عديدة، كما أنه يمثل نموذج وظيفي لتحاليل متعددة التخصصات للتنمية البشرية وتعصيب العضلات. في الواقع، مجموعة كاملة من جديد تركيب الموصل العصبي العضلي يحدث في طبق والثقافة، والسماح لقياس سهولة العديد من المعلمات في كل خطوة، في سياق الأساسية والفسيولوجية. ويمكن لمجرد ذكر بعض الأمثلة، الجينومية و / أو دراسات البروتين أن يؤديها بشكل مباشر على الثقافات المشتركة. وعلاوة على ذلك، يمكن أن الآثار قبل وبعد متشابك، على وجه التحديد وبشكل منفصل المقررة في الوصل العصبي العضلي، لأن كلا من مكونات تأتي من مختلف الأنواع،الفئران والبشر على التوالي،. ويمكن أيضا أن العصب العضلات المشاركة في ثقافة يتعين القيام بها مع الخلايا العضلية البشرية المعزولة من المرضى الذين يعانون من الأمراض العصبية والعضلية في العضلات أو 3، وبالتالي يمكن أن تستخدم كأداة لفحص المخدرات مرشح. أخيرا، ليس هناك حاجة إلى معدات خاصة ولكن منتظمة مرفق BSL2 لإعادة إنتاج وحدة محرك وظيفي في صحن الثقافة. هذا الأسلوب هو وبالتالي قيمة لعضلة على حد سواء، فضلا عن الأوساط البحثية العصبي العضلي للدراسات الفسيولوجية والميكانيكية وظيفة الاعصاب، في سياق طبيعي والمرض.

Watch this Scientific Journal Video about A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells at JoVE.com

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

خلايا العضلات مثقف هي نموذج غير كافية لتلخيص العضلات معصب<em&gt; في الجسم الحي</em&gt;. يمكن استنساخها وحدة محرك وظيفي<em&gt; في المختبر</em&gt; من جانب تعصيب من متباينة الإنسان الخلايا العضلية الأولية باستخدام الفئران explants الجنين الحبل الشوكي. توضح هذه المقالة كيفية إنشاء شارك في ثقافات explants النخاع الشوكي وخلايا العضلات.

وحدة للسيارات وظيفية في صحن الثقافة: شارك في ثقافة Explants النخاع الشوكي وخلايا العضلات

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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Neuroscience

15.2K Views.  University of Basel. خلايا العضلات مثقف هي نموذج غير كافية لتلخيص العضلات معصب في الجسم الحي. يمكن استنساخها وحدة محرك وظيفي في المختبر من جانب تعصيب من متباينة الإنسان الخلايا العضلية الأولية باستخدام الفئران explants الجنين الحبل الشوكي. توضح هذه المقالة كيفية إنشاء ...

Video: A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

تشريح والثقافة من الخلايا العصبية من النخاع الشوكي صواري الجنينية

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

تشريح العقدة والثقافة والفرخ Statoacoustic Explants النخاع الشوكي في الهلام الكولاجين لفحوصات ثمرة Neurite

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

الدجاج الأجنة شريحة الحبل الشوكي الثقافة البروتوكول

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

تحليل وضع الأسنان جرثومة تعصيب استخدام أجهزة ميكروفلويديك المشارك الثقافة

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

الزرد<em&gt; في الموقع</em&gt; الحبل الشوكي التحضير للتسجيلات الكهربية من العمود الفقري الحسية والحركية العصبية

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

استئصال الصفيحة الفقرية وغرس نافذه الحبل الشوكي في الماوس

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

تحديد علامات سطح الخلية من الجذعية العصبية الأولية والخلايا السلف عن طريق وضع العلامات الأيضية من Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

الحقن المباشر لناقل لينتيفيرال يسلط الضوء على عدة مسارات الحركية في الحبل الشوكي الفئران

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

تحويل الخلايا الجذعية مستحث البشرية المستحثة (iPSCs) في العمود الفقري الوظيفي الشوكي والجمجمة الحركية باستخدام متجات البكالوريا

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

التسليم داخل الشرايين للخلايا الجذعية العصبية إلى الدماغ الفئران والفأر: تطبيق على الإقفاري الدماغي

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...