بتمويل من NINDS ، جمعية القلب الأميركية ، اتحاد أمريكا لبحوث الشيخوخة ، ومارس من الدايمات

1. إعداد قبل البدء ، تجميع الصكوك اللازمة الجراحية ، والتشريح والإعلام والثقافة ، والفئران من العمر 7 أيام أو الجراء الماوس. البروتوكول هو نفسه لفأر والاستعدادات الفئران. (ط) المواد والمعدات <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مقص التشغيل (طول مستقيم 6 &quot;، القط # RS6818 ، Roboz ، غايثرسبيرغ ، MD) </li><li style=";text-align:right;direction:rtl"> الجزئي تشريح مقص ، (4 طول &quot;، القط # RS5910. Roboz ، غايثرسبيرغ ، MD) </li><li style=";text-align:right;direction:rtl"> الكربون الصلب دومون الملقط (القط # RS - 5045.Roboz ، غايثرسبيرغ ، MD) </li><li style=";text-align:right;direction:rtl"> تعديل الزجاج &quot;نقل&quot; ماصات (أسفل ماصة باستير وتلطيف إزالة الحافة) </li><li style=";text-align:right;direction:rtl"> قطع من البلاستيك لفيلم Aclar 2 &quot;في x 2&quot; في المربعات (هانيويل ، # 812071 ، بوتسفيل ، والسلطة الفلسطينية) </li><li style=";text-align:right;direction:rtl"> الإسفنج 4 في تغطية بنسبة 3 في (كندال ، القط # 3157 ، مانسفيلد ، MA) </li><li style=";text-align:right;direction:rtl"> 30 غشاء Millicell إدراج ملم (0.4 ميكرومتر ؛ ميليبور ، بيدفورد ، ماجستير) </li><li style=";text-align:right;direction:rtl"> Mcllwain المروحية الأنسجة مع شفرة ذو حدين (McILwain المروحية الأنسجة ، Vibratome الشركة ، وسانت لويس ، MO) </li><li style=";text-align:right;direction:rtl"> المجهر المقلوب مع كاميرا CCD وصورة برامج التحليل </li></ul> (ب) متوسطة تشريح (DM ، 1 لتر عند pH 7.3) <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> هانك في سولت المتوازن </td><td> 1 فيال (95.2 g/1L) (سيغما : H2387) </td></tr><tr><td> NHCO 3 (4.2 ملم) </td><td> 350 ملغ </td></tr><tr><td> HEPES (10 ملم) </td><td> 2.83 ز </td></tr><tr><td> الجلوكوز (33.3 ملم) </td><td> 6.0 غرام </td></tr><tr><td> البنسلين / الستربتوميسين (100X) </td><td> 10 مل </td></tr><tr><td> BSA (0.3 ٪) </td><td> 300 ملغ </td></tr><tr><td> MgSO 4 - 7H 2 O </td><td> 1.44 ز </td></tr></tbody></table> (ج) الثقافة المتوسطة (400 مليلتر) <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> MEM (Invitrogen 11575-032) </td><td> 200 مل </td></tr><tr><td> *** الملح في ميزان هانك (Invitrogen 24020-117) </td><td> 100 مل </td></tr><tr><td> حصان المصل (Invitrogen 26050-070) </td><td> 100 مل </td></tr><tr><td> HEPES العازلة (1 M ؛ Invitrogen 15630-080) </td><td> 5 مل </td></tr><tr><td> البنسلين / الستبرتوميسين (100X) </td><td> 4 مل </td></tr><tr><td colspan="2"> *** أضف إلى 12.8 الجلوكوز ز ملح 500 مل ميزان هانك قبل اتخاذ مستنبت </td></tr></tbody></table> (رابعا) الحيوانات العمر الفئران لمدة 7 أيام أو بعد الولادة الجراء الماوس ، وعموما لتر واحد لكل تجربة. 2. يوم الثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يجب أن تكون غارقة جميع الأدوات والأفلام تشريح Aclar في ETOH 70 ٪ لمدة 30 دقيقة قبل بداية. ويجب تطهيرها على سطح القلنسوة تشريح ، شفرة ، والمروحية الأنسجة مع الايثانول. </li><li style=";text-align:right;direction:rtl"> إعداد عدة (3-4) 6 زراعة الأنسجة بشكل جيد لوحات ؛ إضافة 1 مل مستنبت لكل بئر ، وادخال ونفاذية الغشاء ميليبور في كل بئر. مكان جيد 6 لوحات في الحاضنة حتى 37 درجة على استعداد لنقل الشرائح. </li><li style=";text-align:right;direction:rtl"> إعداد عدة (4-6) لوحات 60 ملم مع بلدية دبي ومكان على الجليد. </li><li style=";text-align:right;direction:rtl"> قطع رأس بسرعة مع المقص وتراجع 70 ٪ في ETOH ، وفضح بسرعة الدماغ مع شق saggital من الجمجمة. إزالة بعناية الدماغ والمكان على الفور في بلدية دبي الباردة. فصلها إلى نصفين ووضع كل نصف الكرة الجانب الإنسي لأسفل على الإسفنج تغطية لمدة 2-3 ثواني. </li><li style=";text-align:right;direction:rtl"> رفع بلطف ومكان في نصف الكرة على الساحة السينمائية Aclar ومكان المروحية على الأنسجة ؛ خفض الدماغ coronally في 350 ميكرون المقاطع. عند الانتهاء ، واتخاذ بلطف Alcar مربع مع نصف الكرة مقطوع ويغرق في بلدية دبي الباردة. </li><li style=";text-align:right;direction:rtl"> باستخدام مجهر تشريح ، فصل المقاطع الحصين. عن الفئران ، ينبغي أن يكون هناك 5 أقسام تمتد إلى قرن آمون منقاري الذيلية ، والماوس ينبغي أن يكون هناك أقسام 3-4. </li><li style=";text-align:right;direction:rtl"> تأخذ بها لوحات 6 بشكل جيد مع إدراج غشاء من 37 درجة نسيج الثقافة الحاضنة. نقل شرائح الحصين فردي على نفاذية الأغشية باستخدام ماصة نقل الزجاج. إذا كان الكثير من DM يتم تحريرها على الغشاء ، وإزالة الزائدة DM مع ماصة باستور. مكان five شرائح الحصين على كل الأغشية. تسمح على الاقل 3 آبار في الشرط ، أو شرائح 15. </li><li style=";text-align:right;direction:rtl"> لوحات احتضان الثقافة في 37 درجة في حاضنة ثاني أكسيد الكربون بنسبة 5 ٪ لترطيب 12-14 قبل أيام من بداية التجربة السمية. يمكن تغيير مستنبت مرتين في الأسبوع. </li></ol> 3. حمل وتحديد مقدار الضرر متعلق بالخلايا العصبية في قرن آمون الثقافة شريحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد 12-14 يوما في الثقافة ، وإضافة 5 ميكروغرام / مل يوديد propidium (PI) إلى وسائل الإعلام الثقافة. وسوف تسمح هذه الخطوة التصور النوعي والكمي لموت الخلايا القاعدية. </li><li style=";text-align:right;direction:rtl"> في اليوم التالي ، كميا PIمضان في المنطقة دون الإقليمية CA1 (و / أو CA3 المسنن أو إذا رغبت في ذلك) من كل شريحة الحصين والحصول على قياسات يعني PI مضان يمثل موت الخلايا القاعدية (المشار إليها مع مرور الوقت = 0 ساعة ، أو تي 0). لاقتناء الصور والكمي ، ونحن نستخدم OpenLab البرمجيات (الارتجال ، المملكة المتحدة) ، ولكن من الحصول على الصور ويمكن استخدام برامج التحليل لتحديد كثافة مضان. </li><li style=";text-align:right;direction:rtl"> مباشرة بعد الحصول على الصور من بي تي مضان 0 ، تحفز الخلايا العصبية التي اصابة طريقتك في الاختيار (ونحن قد استخدمت 10 ميكرومتر NMDA ، الأوكسجين الجلوكوز الحرمان ، أو LPS لمدة ساعة واحدة للحث على السمية العصبية 2،5). بعد التحفيز السامة ، مع استبدال المتوسطة المتوسطة الطازجة تحتوي على 5 ميكروغرام / مل PI والحفاظ على شرائح بين عشية وضحاها. </li><li style=";text-align:right;direction:rtl"> في يوم 3 ، كميا يعني PI مضان للحالة تجريبية في 24H ، أو تي 24. </li><li style=";text-align:right;direction:rtl"> بعد ذلك الحصول على الصور ، وتغير الحال والمتوسطة وإضافة 10 ميكرومتر NMDA وميكروغرام / 5 مل بي وشرائح احتضان الموت بين عشية وضحاها من أجل تحقيق أقصى قدر من الخلايا العصبية. </li><li style=";text-align:right;direction:rtl"> في يوم 4 ، كميا يعني PI مضان وفاة الخلايا العصبية القصوى ، كما تم تعريفها ماكس تي. حساب موت الخلايا في المئة على النحو التالي : (T 24 -- T 0) / (T - T ماكس 0). </li></ol> 4. ممثل النتائج <img src="/files/ftp_upload/2106/2106fig1.jpg" alt="الشكل 1" /> الشكل 1 (أ) الإطار الزمني لتوليد الإصابة التجريبية الحصين واللاحقة ، والحصول على الصور. (ب) من شرائح الصور الممثل الحصين basally (T 0) ، 24 ساعة بعد التعرض 1 ساعة إلى 10 ميكرومتر NMDA (T 24) ، وبعد 24H ميكرومتر أو 10 للحصول على مزيد من NMDA الاصابة العصبية القصوى (T أقصى).

<ol>
	<li>Stoppin, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-173 (1991).</li><li>McCullough, L., Wu, L., Haughey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+Function+of+the+PGE2+EP2+Receptor+in+Cerebral+Ischemia.">Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia.</a> J Neurosci. 24 (1), 257-257 (2004).</li><li>Czapiga, M., Colton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+microglia+in+organotypic+slice+cultures.">Function of microglia in organotypic slice cultures.</a> J Neurosci Res. 56, 644-644 (1999).</li><li>Newell, D. W., Barth, A., Papermaster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+non-glutamate+receptor+mediated+toxicity+caused+by+oxygen+and+glucose+deprivation+in+organotypic+hippocampal+cultures.">Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures.</a> J Neurosci. 15, 7702-7702 (1995).</li><li>Wu, L., Wang, Q., Liang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+Effects+of+prostaglandin+recetor+signaling+on+neuronal+survival.">Divergent Effects of prostaglandin recetor signaling on neuronal survival.</a> Neuroscience letter. 421, 253-253 (2007).</li><li>Hailer, N. P., Jarhult, J. D., Nitsch, R., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+in+vitro:+Analysis+of+morphology+and+adhesion+molecule+expression+in+organotypic+hippocampal+slice+cultures.">Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures.</a> Glia. 18, 319-319 (1996).</li></ol>

الإعلان عن أي تضارب في المصالح.

هناك نقطتان هامتان لمتابعة لضمان تحقيق نتائج استنساخه ومتناسقة عند تكرار التجارب. أولا ، من المهم السماح للشرائح العمرية 12-14 الحصين لأيام قبل التجريب. مع شريحة العزلة ، وهناك استجابة كبيرة دبقية صغيرة ، وتبقى تنشيط الخلايا الدبقية الصغيرة لبعض الوقت ، مع العودة إلى دولة متشعبة الراحة التي تحدث من قبل 10 أيام في المختبر 3،6. ثانيا ، يعني كثافة PI الاستشعاع النسبي لعدد من الخلايا جرح 4. يقاس مضان متسلسلة في غضون ثلاث نقاط : (ط) في T 0 ، قبل NMDA أو التحفيز Ogd لقياس المستويات القاعدية للموت الخلايا العصبية عفوية ، (الثاني) في تي 24 أو 24 ساعة بعد التحفيز مع NMDA أو التحفيز OGD أو غيرها ، و (ج) في تي ماكس ، بعد المعالجة النهائية للشرائح مع العلاج 24 ساعة قاتلة بين عشية وضحاها مع 10 ميكرومتر النمداوية التي تنتج كمية قصوى للموت الخلايا العصبية. ويتم احتساب ثم موت الخلايا في المئة باستخدام الصيغة (T 24 -- T 0) / (T - T ماكس 0). فقد أمر بالغ الأهمية لتطبيع كل شريحة لنفسها باستخدام هذه الطريقة لأن حجم الحصين والتعبير مستقبلات الغلوتامات وتغير توزيع كبيرة باعتبارها واحدة من منقاري يذهب إلى الدماغ الذيلية ، والفشل في تطبيع كل شريحة على نفسها في البيانات نتيجة مصطنعة .

the organotypic hippocampal slice culture model for examining neuronal injury

عضوي النمط ثقافة شريحة الحصين هو أسلوب في المختبر لدراسة آليات الإصابة العصبية التي يتم الاحتفاظ نسبيا بنية الأساسية وتكوين الحصين 1. منظومة الثقافة عضوي النمط يسمح لفحص الآثار العصبية ، ونجمي دبقية صغيرة ، ولكن كما استعدادا فيفو السابقين ، لا تعالج آثار تدفق الدم ، أو تجنيد خلايا التهابية الطرفية. تحقيقا لهذه الغاية ، وكثيرا ما يستخدم هذا الأسلوب لدراسة الثقافة وإصابة excitotoxic نقص الأوكسجين إلى الخلايا العصبية في قرن آمون الهرمي ، ولكن قد استخدمت أيضا لدراسة الاستجابة الالتهابية. نحن هنا وصف الطرق لتوليد الثقافات شريحة من قرن آمون في الدماغ القوارض بعد الولادة ، وبالإدارة السامة المحفزات للحث على اصابة الخلايا العصبية ، ومعايرة وقياس موت الخلايا العصبية قرن آمون.

Watch this Scientific Journal Video about The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury at JoVE.com

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

وorganoptypic ثقافة شريحة الحصين هو نموذج<em&gt; في المختبر</emتستخدم&gt; نموذج لدراسة اصابة الخلايا العصبية في مجموعة متنوعة من النماذج. في هذه المقالة ، ونحن تصف الأساليب لتوليد شريحة الثقافات وقياس اصابة الخلايا العصبية.

وعضوي النمط الثقافة شريحة الحصين النموذجي لفحص الإصابات متعلق بالخلايا العصبية

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

the-organotypic-hippocampal-slice-culture-model-for-examining

Nanyang Technological University

Research

JoVE Journal

Neuroscience

19.1K Views.  Stanford University School of Medicine. وorganoptypic ثقافة شريحة الحصين هو نموذج في المختبر نموذج لدراسة اصابة الخلايا العصبية في مجموعة متنوعة من النماذج. في هذه المقالة ، ونحن تصف الأساليب لتوليد شريحة الثقافات وقياس اصابة الخلايا العصبية.

Video: The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

الثقافات شريحة عضوي النمط لدراسات ما بعد الولادة تكوين الخلايا العصبية

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

عضوي النمط الثقافات شريحة الحصين

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

excitotoxic المواد تحفيز الدماغ باعتبارها Microslices<em&gt; في المختبر</em&gt; نموذج من السكتة الدماغية

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Medicine

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

الثقافات شريحة عضوي النمط لدراسة ديناميات دبقية قليلة التغصن وتكون الميالين

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

وتحليل وعائية عصبية إعادة عرض في Entorhino-الحصين الثقافات شريحة عضوي النمط

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

أورجانوتيبيك شريحة هيبوكامبال الثقافات كنموذج للدراسة نيوروبروتيكتيون واختزاع الخلايا السرطانية

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

قصيرة الأجل الحرة العائمة شريحة الثقافات من الدماغ البشري الكبار

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

نموذج لتولد الصرع في رينال القشرة-قرن آمون ثقافات شريحة العضوية

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

نموذج ثقافة الخلية لدراسة دور التفاعلات العصبية-غليا في الإقفاري

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

حاد الدماغ الماوس تشريح للتحقيق عفوية نشاط شبكة فرس النهر

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...