Name: purkinje cell survival in organotypic cerebellar slice cultures
Uploaded: 2019-12-18T12:00:00
Description: Watch this Scientific Journal Video about Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures at JoVE.com

وأجريت اعمال التصوير في مركز جامعه نورث وسترن لمجهر المتقدمة بسخاء بدعم من الكلية CCSG P30 CA060553 منحت لمركز روبرت H لوري الشامل للسرطان. نشكر شون ماكدرمت لمساعدته الفنية ودعمه ، ومايا ابستاين للرسوم التوضيحية المرسومة باليد المبينة في الشكل 1.

وأجريت جميع التجارب المتعلقة بالماشية وفقا للجنة الدراسات الحيوانية بجامعه نورث وسترن.
1. الاعداد قبل الثقافات شريحة المخيخ الشكل العضوي
<ol><li>في خليه الثقافة هود رش مع 70 ٪ الايثانول مسبقا ، واعداد 200 mL من الوسط الثقافي في فلتر فراغ زجاجه 250 mL تعلق علي جهاز استقبال زجاجه معقم?...

<ol>
	<li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Gahwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+monolayer+cultures+of+nervous+tissue.">Organotypic monolayer cultures of nervous tissue.</a> Journal of Neuroscience Methods. 4 (4), 329-342 (1981).</li><li>Yamamoto, N., Kurotani, T., Toyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+connections+between+the+lateral+geniculate+nucleus+and+visual+cortex+in+vitro.">Neural connections between the lateral geniculate nucleus and visual cortex in vitro.</a> Science. 245 (4914), 192-194 (1989).</li><li>Dusart, I., Airaksinen, M. S., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purkinje+cell+survival+and+axonal+regeneration+are+age+dependent:+an+in+vitro+study.">Purkinje cell survival and axonal regeneration are age dependent: an in vitro study.</a> Journal of Neuroscience. 17 (10), 3710-3726 (1997).</li><li>Wiegreffe, C., Feldmann, S., Gaessler, S., Britsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+Confocal+Imaging+of+Migrating+Neurons+in+Organotypic+Slice+Culture+of+Embryonic+Mouse+Brain+Using+In+Utero+Electroporation.">Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation.</a> Journal of Visualized Experiments. (125), e55886 (2017).</li><li>Daviaud, N., 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=Modeling+nigrostriatal+degeneration+in+organotypic+cultures,+a+new+ex+vivo+model+of+Parkinson's+disease.">Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease.</a> Neuroscience. 256, 10-22 (2014).</li><li>Ghoumari, A. M., 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=Mifepristone+(RU486)+protects+Purkinje+cells+from+cell+death+in+organotypic+slice+cultures+of+postnatal+rat+and+mouse+cerebellum.">Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (13), 7953-7958 (2003).</li><li>Labombarda, F., 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=Neuroprotection+by+steroids+after+neurotrauma+in+organotypic+spinal+cord+cultures:+a+key+role+for+progesterone+receptors+and+steroidal+modulators+of+GABA(A)+receptors.">Neuroprotection by steroids after neurotrauma in organotypic spinal cord cultures: a key role for progesterone receptors and steroidal modulators of GABA(A) receptors.</a> Neuropharmacology. 71, 46-55 (2013).</li><li>Gao, P., 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=P2X7+receptor-sensitivity+of+astrocytes+and+neurons+in+the+substantia+gelatinosa+of+organotypic+spinal+cord+slices+of+the+mouse+depends+on+the+length+of+the+culture+period.">P2X7 receptor-sensitivity of astrocytes and neurons in the substantia gelatinosa of organotypic spinal cord slices of the mouse depends on the length of the culture period.</a> Neuroscience. , 195-207 (2017).</li><li>Sundstrom, L., Morrison, B., Bradley, M., Pringle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cultures+as+tools+for+functional+screening+in+the+CNS.">Organotypic cultures as tools for functional screening in the CNS.</a> Drug Discovery Today. 10 (14), 993-1000 (2005).</li><li>Jankowski, J., Miething, A., Schilling, K., Baader, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+purkinje+cell+death+is+spatiotemporally+organized+in+the+developing+mouse+cerebellum.">Physiological purkinje cell death is spatiotemporally organized in the developing mouse cerebellum.</a> Cerebellum. 8 (3), 277-290 (2009).</li><li>Ghoumari, A. M., Wehrle, R., Bernard, O., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+Bcl-2+and+Caspase-3+in+age-related+Purkinje+cell+death+in+murine+organotypic+culture:+an+in+vitro+model+to+study+apoptosis.">Implication of Bcl-2 and Caspase-3 in age-related Purkinje cell death in murine organotypic culture: an in vitro model to study apoptosis.</a> European Journal of Neuroscience. 12 (8), 2935-2949 (2000).</li><li>Ghoumari, A. M., Wehrle, R., De Zeeuw, C. I., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protein+kinase+C+prevents+Purkinje+cell+death+but+does+not+affect+axonal+regeneration.">Inhibition of protein kinase C prevents Purkinje cell death but does not affect axonal regeneration.</a> Journal of Neuroscience. 22 (9), 3531-3542 (2002).</li><li>Ghoumari, A. M., 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=Neuroprotective+effect+of+mifepristone+involves+neuron+depolarization.">Neuroprotective effect of mifepristone involves neuron depolarization.</a> FASEB Journal. 20 (9), 1377-1386 (2006).</li><li>Repici, M., Wehrle, R., Antoniou, X., Borsello, T., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Jun+N-terminal+kinase+(JNK)+and+p38+play+different+roles+in+age-related+Purkinje+cell+death+in+murine+organotypic+culture.">c-Jun N-terminal kinase (JNK) and p38 play different roles in age-related Purkinje cell death in murine organotypic culture.</a> Cerebellum. 10 (2), 281-290 (2011).</li><li>Rakotomamonjy, J., Ghoumari, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-Derived+Neurotrophic+Factor+Is+Required+for+the+Neuroprotective+Effect+of+Mifepristone+on+Immature+Purkinje+Cells+in+Cerebellar+Slice+Culture.">Brain-Derived Neurotrophic Factor Is Required for the Neuroprotective Effect of Mifepristone on Immature Purkinje Cells in Cerebellar Slice Culture.</a> International Journal of Molecular Sciences. 20 (2), (2019).</li><li>Stoppini, 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> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Linkert, M., 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Uesaka, N., 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=Organotypic+coculture+preparation+for+the+study+of+developmental+synapse+elimination+in+mammalian+brain.">Organotypic coculture preparation for the study of developmental synapse elimination in mammalian brain.</a> Journal of Neuroscience. 32 (34), 11657-11670 (2012).</li><li>Falsig, J., 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=Prion+pathogenesis+is+faithfully+reproduced+in+cerebellar+organotypic+slice+cultures.">Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.</a> PLoS Pathogens. 8 (11), 1002985 (2012).</li><li>Bouslama-Oueghlani, L., Wehrle, R., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+loss+of+the+ability+of+Purkinje+cells+to+regenerate+their+axons+occurs+in+the+absence+of+myelin:+an+in+vitro+model+to+prevent+myelination.">The developmental loss of the ability of Purkinje cells to regenerate their axons occurs in the absence of myelin: an in vitro model to prevent myelination.</a> Journal of Neuroscience. 23 (23), 8318-8329 (2003).</li><li>Apuschkin, M., Ougaard, M., Rekling, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+calcium+waves+in+granule+cells+in+cerebellar+slice+cultures.">Spontaneous calcium waves in granule cells in cerebellar slice cultures.</a> Neuroscience Letters. 553, 78-83 (2013).</li><li>Audinat, E., Knopfel, T., Gahwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+to+excitatory+amino+acids+of+Purkinje+cells'+and+neurones+of+the+deep+nuclei+in+cerebellar+slice+cultures.">Responses to excitatory amino acids of Purkinje cells' and neurones of the deep nuclei in cerebellar slice cultures.</a> Journal of Physiology. 430, 297-313 (1990).</li></ol>

الثقافة شريحة المخيخ هو أداه قويه لدراسة الموت المبرمجة الخلية Purkinje اثناء التنمية بعد الولادة. يمكن استخدام هذه التقنية لفحص الجزيئات المرشحة بسرعة لإمكاناتها المحصنة. الميزة الرئيسية هي ان الاعداد بسيط وفعال من حيث التكلفة جدا ، ويتطلب فقط استثمار متواضع في المعدات (يمكن ان تكلف الذبذب?...

النمذجة في المختبر هو أداه أساسيه في البحوث الطبية الحيوية. وهو يسمح للمحققين بدراسة ومراقبه أليات محدده في أنواع الخلايا المقيدة ، أو في النظم/الاجهزه المعزولة. ثقافة شريحة التنميط العضوي هي تقنيه تستخدم علي نطاق واسع في المختبر ، وخاصه في مجال علوم الأعصاب1. تاسست هذه الطريقة لأول مره من قبل Gähwiler ، الذي مثقف شرائح الدماغ باستخدام تقنيه أنبوب الاسطوانه2، وتعديلها في وقت لاحق من قبل ياماموتو وآخرون ، الذي ادخل استخدام غشاء ميكرومساميه لأداء الثقافات شريحة القشرية3. بالمقارنة مع ثقافات الخلايا الاوليه ، تقدم ثقافات الشريحة العضوية ميزه الحفاظ علي البنية الخلوية للانسجه ، فضلا عن اتصالات خلايا الخلايا الاصليه في الجزء الخاص بالانسجه.
وقد تم استزراع شرائح التنميط العضوي من أجزاء كثيره من الجهاز العصبي المركزي ، مثل الحصين4، اللحاء5، سترياتوم6، المخيخ4،7، والحبل الشوكي8،9، من بين أمور أخرى. وقد ثبت انها أداه قويه في دراسات اكتشاف المخدرات10. يمكن تقييم اثار الجزيئات العصبية بطرق عديده: البقاء علي قيد الحياة والضمور العصبي باستخدام اختبارات المناعة والكيمياء الحيوية ، وتشكيل دوائر الخلايا العصبية ، أو التعطيل باستخدام الفيزيولوجيا الكهربية والتصوير الحي.
والهدف من هذا العمل هو وصف طريقه بسيطه لأداء ثقافة شريحة المخيخ المجسمة ، والتي من المعروف انها نموذج مناسب لتقليد التطور المخيخ في المختبر. وعلي وجه الخصوص ، ركزنا علي دراسة وفاه الخلية Purkinje النمو. في الجسم المجري, خلايا Purkinje الخضوع للخلايا المبرمج خلال الأسبوع الأول بعد الولادة, ذروتها في اليوم بعد الولادة 3 (P3)11. لوحظ نفس النمط في الثقافة شريحة المخيخ ، مع الخلايا العصبية purkinje الموت من قبل المبرمج عندما تؤخذ المخيخ من الحيوانية بين P1 و P8 ، مع ذروه في P34،12. وقد سمح استخدام الثقافات شريحة المخيخ الشكل العضوي لتحديد عده جزيئات نيوروبروتكتيف7,13, فضلا عن فهم جزء من أليات المعنية في هذه الخلية المبرمجة الموت14,15,16. هنا ، ونحن وصف بروتوكول يستند إلى دراسة ستوبيني وآخرون17 في الحصين ، وتكييفها مع المخيخ من قبل dusart وآخرون4 ويشمل تشريح السريع وتقطيع المخيخ بعد الولادة. تشريح الثقافة علي الخلية الثقافة ادراج تحتوي علي غشاء ميكرومساميه ، مع أو بدون علاج نيوروبروتكتيف ؛ وتلطيخ المناعي لتقييم بقاء الخلايا العصبية.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody</td>
			<td>ThermoFisher scientific</td>
			<td>A21203</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal Medium Eagle (BME)</td>
			<td>ThermoFisher scientific</td>
			<td>21010046</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet Class II, Type A2</td>
			<td>NuAire</td>
			<td>NU-540-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Calbindin D-28K antibody (CB-955)</td>
			<td>Abcam</td>
			<td>ab82812</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NuAire</td>
			<td>NU-5700</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom 6-well Cell Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge blades</td>
			<td>Fisher Scientific</td>
			<td>50949411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-127</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100X</td>
			<td>ThermoFisher scientific</td>
			<td>25030149</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose solution</td>
			<td>ThermoFisher scientific</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks' Balanced Salt Solution</td>
			<td>ThermoFisher scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum, heat inactivated, New Zealand origin</td>
			<td>ThermoFisher scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>Fisher Scientific</td>
			<td>NC9914528</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprobes</td>
			<td>Fisher Scientific</td>
			<td>08-850</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Inserts</td>
			<td>Millipore Sigma</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL</td>
			<td>ThermoFisher scientific</td>
			<td>168-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal laser microscope system</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Imaging Software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>41678-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8403</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker Belly Dancer</td>
			<td>IBI Scientific</td>
			<td>BDRLS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish, 60 mm w/ grip ring</td>
			<td>Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 well</td>
			<td>Falcon/Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes, sterile</td>
			<td>ThermoFisher scientific</td>
			<td>13-711-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

purkinje cell survival in organotypic cerebellar slice cultures

ثقافة شريحة التنميط العضوي هو نموذج قوي في المختبر الذي يقلد في ظروف الجسم الآخر عن كثب من الثقافات الخلية الاوليه المنفصلة. في مرحله ما بعد الولادة المبكرة ، من المعروف ان خلايا المخيخ Purkinje تمر خلال فتره الضعف ، والتي تمر خلالها بموت الخلايا المبرمج. هنا ، ونحن نقدم بروتوكولا مفصلا لأداء الماوس الظاهرية شريحة المخيخ الثقافة خلال هذا الوقت الحرج. وتصنف الشرائح كذلك لتقييم بقاء الخلية Purkinje وفعالية العلاجات نيوروبروتكتيف. هذه الطريقة يمكن ان تكون قيمه للغاية للشاشة لجزيئات نيواكتيف جديده.

وكما هو مبين في الشكل 4، ينتج هذا البروتوكول الثقافات المجسمة لشريحة المخيخ التي يمكن تقييم بقاء خليه Purkinje فيها بعد الخطوات المناعية والحصول علي الصورة. تم تسميه الخلايا purkinje مع مزيج من المضادة-Calbindin د-28K (تخفيف 1/200) و Alexa594 مكافحه الماوس (تخفيف 1/300) الأجسام المضادة. وقد تم خياط...

Watch this Scientific Journal Video about Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures at JoVE.com

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

الثقافات شريحة التنميط العضوي هي أداه قويه لدراسة العمليات العصبية أو التنكسية/التجدد. هنا ، ونحن وصف البروتوكول الذي نماذج وفاه النمو العصبي للخلايا Purkinje في الثقافات شريحة الفئران المخيخ. هذا الأسلوب قد تستفيد البحوث في اكتشاف المخدرات نيوروبروتكتيف.

البقاء علي قيد الحياة خليه purkinje في الثقافات شريحة المخيخ الوراثية

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

تعتبر ثقافة شريحة Organotypic أداة قوية لدراسة العمليات العصبية النمائية والتنكسية أو التجديدية. ويمكن استخدام هذه التقنية لفرز بسرعة الجزيئات المرشحة لإمكاناتها العصبية. هذه الطريقة تحاكي عن كثب في ظروف الجسم الحي ، مقارنة بثقافات الخلايا الأولية المنفصلة ، حيث يتم الحفاظ على بنية مجموعة الأنسجة واتصالات الخلايا الخلوية الأصلية داخل مستويات الأقسام.هنا نبرهن على دراسة موت خلايا بوركينجي في المخيخ النامي. ولكن ثقافة شريحة الأعضاء هي مناسبة على قدم المساواة لنموذج الأمراض العصبية في كل منطقة الجهاز العصبي المركزي تقريبا. إثبات الإجراء مع جنيفر Rakotomamonjy سيكون شون ماكديرموت، وهو فني من مختبري.قبل حصاد شرائح cerebellar، في مجلس الوزراء السلامة البيولوجية معقمة، ملء كل بئر من لوحة ثقافة ستة بئر مع ملليلتر واحد من فراغ تصفية الثقافة المتوسطة، وإضافة عامل الدوائية ذات الفائدة لآبار العلاج، وحجم متساو من السيارة إلى آبار التحكم. باستخدام ملقط معقمة، وضع واحد 0.4 ميكرومتر المسام حجم PTFE غشاء مرشح إدراج في كل بئر، مع الحرص على تجنب فقاعات في واجهة كل غشاء والمتوسطة، وتكليل المتوسطة في 37 درجة مئوية وحاضنة ثاني أكسيد الكربون 5٪ لمدة ساعتين. لحصاد المخيخ، استخدم ملقط خلع الملابس مباشرة لفهم رأس الجرو من الأنف، واستخدام مقص العين مباشرة لقطع فتح فروة الرأس من النهاية الخلفية في الجانب الآخر إلى خط الوسط.قطع فتح الجمجمة بنفس الطريقة، مشيرا إلى نصائح مقص إلى الخارج لتجنب إتلاف المخيخ، واستخدام ملعقة لنقل الدماغ إلى طبق 60 ملليمتر تحتوي على HBSS الباردة وخمسة ملليغرام لكل ملليلتر من الجلوكوز. استخدام ملقط خلع الملابس على التوالي لتشريح بعناية خارج المخيخ، واستخدام ملقط ناعم منحني معقمة لوضع الأنسجة على قرص بلاستيكي على طاولة القطع من المروحية الأنسجة. تدوير الجدول القطع لتوجيه الأنسجة للسماح لاقتناء المقاطع parasagittal، وسحب مقبض الطاولة الإفراج إلى اليمين لنقل الجدول القطع حتى يتم وضع شفرة على حافة الأنسجة.ثم ضبط سمك شريحة إلى 350 ميكرومتر، وسرعة شفرة إلى متوسطة، وبدء المروحية. عندما يتم تقطيع المخيخ بأكمله، قم بإيقاف تشغيل المروحية. باستخدام ملقط معقمة، عقد القرص على طبق جديد 60 ملليمتر.استخدام ماصة نقل لطرد HBSS بالإضافة إلى الجلوكوز على القرص بحيث تقع شرائح في الطبق. ثم، لمس العينات بأقل قدر ممكن، استخدم microprobe لفصل الشرائح. استخدام ماصة نقل لتحديد أجزاء من المخيخ على مقربة من vermis على إدراج ثقافة الخلية الفردية في لوحة ستة جيدا، واستخدام microprobe لوضع شرائح في مركز كل إدراج.عندما تم وضع جميع الشرائح، وpireate بعناية أي HBSS الزائدة بالإضافة إلى الجلوكوز، والعودة لوحة إلى الحاضنة ثقافة الخلية. لطخة الفلوروس المناعية، وإزالة افرنح من كل بئر، وغسل إدراج مع برنامج تلفزيوني. إصلاح شرائح مع ملليلتر واحد من 4٪ paraformaldehyde الباردة في بئر كل إدراج، و 500 ميكرولترات على رأس كل إدراج لمدة ساعة واحدة.في نهاية التثبيت، وغسل إدراج أربع مرات لمدة 10 دقائق مع ملليلتر واحد من برنامج تلفزيوني جديد تحت كل إدراج و 500 ميكرولترات من برنامج تلفزيوني جديد على رأس كل إدراج لكل غسل على شاكر المدارية. قبل ملء كل بئر من لوحة 24-well مع 500 ميكرولترات من برنامج تلفزيوني TB، واستخدام فرشاة الطلاء لنقل شرائح cerebellar من كل ثقافة الخلية إدراج في الآبار الفردية من لوحة 24 جيدا. Permeabilize وحجب شرائح في درجة حرارة الغرفة لمدة ساعة واحدة.في نهاية الحضانة، وتسمية الخلايا مع 200 ميكرولترات من الأجسام المضادة الأولية من الفائدة المخففة في برنامج تلفزيوني - السل في البئر بين عشية وضحاها في أربع درجات مئوية على شاكر المدارية. في صباح اليوم التالي، وغسل شرائح أربع مرات لمدة 10 دقائق و 500 ميكرولترات من برنامج تلفزيوني الطازجة لكل غسل على شاكر، قبل وضع علامة على العينات مع الأجسام المضادة الثانوية الفلوروومي المناسبة مترافق لمدة ساعتين في درجة حرارة الغرفة على شاكر، محمية من الضوء. في نهاية الحضانة، و counterstain أقسام مع 500 microliters من وصمة عار النووية المناسبة في بئر لمدة 10 دقيقة في درجة حرارة الغرفة، محمية من الضوء.واستخدم ماصة نقل لتركيب الشرائح على الشرائح الزجاجية. ثم اسمحوا الهواء أقسام جافة تماما قبل إعادة الجفاف مع برنامج تلفزيوني وتركيب الأنسجة مع أغطية مغلفة مع ما يقرب من 80 ميكرولترات من المتوسط المتصاعدة. بمجرد أن يتم شفاء المتوسطة المتصاعدة ، تصبح أقسام cerebellar جاهزة للصور.في اليوم التالي للولادة ستة، بقاء خلية بوركيني منخفض في عينات التحكم، بما يتفق مع نافذة الضعف المعروفة. يزداد البقاء على قيد الحياة مع تقدم الحيوان المانح في السن وخروجه من هذه الفترة الحرجة. علاج شرائح cerebellar مع تركيز عال من كلوريد البوتاسيوم النتائج في الحث الناجح من إزالة القطب والبقاء على قيد الحياة.للحصول على نتائج متناسقة وقابلة لإعادة إنتاجها، من الضروري تحديد أقسام سيريبيلار صحية، وإجراء إعداد الثقافة بأكبر قدر ممكن من الكفاءة. تتجاوز تطبيقات ما بعد الاستزراع الفلورات المناعية. كما يمكن استخدام شرائح organotypic لدراسات التعبير عن بروتين الجينوم، ورصد نشاط الدوائر العصبية باستخدام الفيزيولوجيا الكهربائية والكالسيوم التصوير الحي.

purkinje-cell-survival-in-organotypic-cerebellar-slice-cultures

Research

JoVE Journal

Neuroscience

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

عضوي النمط من الثقافة شريحة GFP - معربا عن أجنة الفئران لريال مدريد في الوقت ثمرة تصوير الأعصاب الطرفية

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

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 ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

مخيخي الثقافات عضوي النمط : التحديات والكشف عن أفكارك

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

تسجيل خلية كاملة من إعداد عضوي النمط شريحة من القشرة المخية الحديثة

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

الثقافات شريحة عضوي النمط من المخ الأوسط بطني الجنينية : نظام لدراسة التنمية متعلق بالخلايا العصبية الدوبامينية في المختبر</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

على تحليل من الخليوي بركينيي علم الصرف شجيرى في الثقافات شريحة عضوي النمط

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

بعد تضمين التعريف Immunogold من البروتينات متشابك في الثقافات شريحة الحصين

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

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 ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

استنفاد الانتقائي للالدبقية الصغيرة من مخيخي الحبيبة الثقافات الخلية باستخدام L-ليسين استر الميثيل

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...