Delaware Valley Lions Göz Bankası desteğiyle bu iş için çok değerli idi. ICP8 karşı Birincil fare monoklonal antikoru Dr David Knipe (Harvard Tıp Okulu) bir tür hediye oldu. Biz de yararlı tartışmalar ve uzmanlık için Dr Stephen Jennings (Tıp Drexel Üniversitesi) ve Dr Peter Laibson (Wills Göz Enstitüsü) teşekkür ederim.

Tüm reaktifler ve ekipman steril satın alındığı veya prosedür öncesi sterilize edildi. Bu makalede, bir ön-enüklee gözü ile başlayan ex vivo model kurma adımları açıklanmaktadır. Deneylerde, biz genç (8-12 hft) albino tavşanlar (Pel-Freez Biyolojik, Rogers, AR) taze bütün gözbebekleri kullanın. Ayrıca, yerel göz bankasından elde insan korneası kullanın. Enükleasyon kendiniz yapıyorsanız, işlem sırasında kornea veya limbus zarar vermemek için dikkat edin. Tavşan enükleasyon protokolleri başka 2 bulunabilir. Bölüm 1: korneoskleral Düğme eksizyonu <ol><li> PBS ile gazlı bez, dar bir şerit ıslatın ve işlem sırasında tutarak kolaylaştırmak için göz küresi etrafına sarın. </li><li> Yaklaşık 0.5 cm uzaklıkta limbustan, sklera bir teğetsel kesi yapmak için keskin bir neşter kullanın. </li><li> Kesi uzatmak için keskin bir makas kullanın ve c tamamen tüketimorneoscleral düğmesi. </li><li> Korneoskleral düğmesi izole edildikten sonra forseps ile skleral jant tutarken, iris uzak tease. Endotele zarar veya kornea üzerinde aşırı stres uygulamakla kaçının. </li></ol> Bölüm 2: Organotipik Kültür içine Kornea Tanıtımı <ol><li> Eksplante kornealar Temel Olmayan Amino Asitler (1X), L-Glutamin (2 mM), Penisilin (200 U / ml) ve streptomisin (200 mg / ml) ile desteklenmiş Minimum Essential Medium (MEM) kültüre edilmiştir. Gerekirse örneğin amfoterisin gibi bir antifungal ajan, ayrıca ilave edilebilir. </li><li> 55 ° C&#39;de besiyerine% 1 agaroz çözeltisi hazırlayın ve hazır tutmak </li><li> İyice steril PBS içeren penisilin (200 U / ml) ve streptomisin (200 ug / ml) olarak kornea durulama. </li><li> Steril bir nokta plaka bir kuyuya kornea epitel kısmı aşağı gelecek şekilde yerleştirin. </li><li> Için yeterli miktarda, korneanın endotel çukurluk agaroz içeren ortam eklemetamamen doldurunuz. Agaroz için ~ 1 dk katılaşmasını bekleyin. </li><li> Epitel yüzeyini kapsayacak şekilde 35 mm doku kültürü çanak içine jel iskele epitel tarafı destekleyen ve eklemek besiyeri ile kornea yerleştirin. </li><li> Kornea orta değişiklikleri her 48 saat ile bir hafta boyunca bu şekilde kültüre edilebilir. </li></ol> HSV-1 enfeksiyonu ve Deneysel Uyuşturucu Tedavi: Bölüm 3 <ol><li> Bir 35 mm doku kültürü çanağı içine virüs istenen miktarda ihtiva eden orta 1 ml ilave edilir. Biz genellikle zorlanma KOS korneanın başına HSV-1 1x10 4 PFU ile enfekte. Bizim stok titreleri CV-1 mono tabakaları üzerindeki plak deneyleri yapılarak tespit edilir. </li><li> Virüs içeren ortam içine aşağı agaroz iskele epitel kısmı ile birlikte kornea yerleştirin. </li><li> Aralıklı sallanan her 10-15 dakika ile 1 saat enfekte etmek için bir doku kültürü kuluçka makinesi içine kornea yerleştirin. </li><li> 1 saat sonra, virüs-contai aspireorta ning ve herhangi bir kalıntı virüs parçacıkları çıkarmak için PBS ile 2-3 kez yıkayın. </li><li> Geri önceki konumuna kornea yerleştirin ve epitel yüzeyini kapsayacak şekilde taze kültür ortamı ekleyin. </li><li> Deneysel tedavi deney doğasına bağlı olarak, enfeksiyondan önce veya sonra başlatılabilir. </li><li> Kültür ortamı, her 48 saat değiştirilir. </li></ol> Bölüm 4: Örnek Toplama <ol class="lalpha"><li> Plak tahlil analizleri için orta. Sıra orta karıştırın ve -80 plaket tahlil veya mağaza için hemen kullanmaya ° C farklı zaman noktalarında örneklerin toplanması halinde daha sonra kullanmak için. </li><li> DNA, RNA ve protein ya da kornea epitel hücreleri <ol><li> Agaroz iskele çıkarın ve PBS ile de kornea durulayın. </li><li> Yalnız kornea epitel malzeme sağlamak için kornea dan skleral doku Tüketim halka toplanır. </li><li> Ubir neşter şarkı, izole edilmesi için malzemenin türüne bağlı olarak, uygun bir tampon içinde, kornea epitel hücreleri kazıma. DNA ve RNA izolasyonu için, DNeasy Kan ve Doku Kiti ve RNeasy Mini Kit, sırasıyla (QIAGEN, Hilden, Almanya) kullanın. Protein lizatları toplanması için, Laemmli tampon kullanın. Kornea epitel hücrelerinin akış sitometri analizi için, hücreler tripsin gevşetmek için küçük bir miktar içinde korneaya ön-inkübe edilir ve daha sonra bir bisturi ile tripsin içine bunları çıkarmak. Doku eşit miktarda verim değil kornea epitel kazınması yana, örnekler analiz edilirken uygun kontroller dahil etmek önemlidir. Biz Western blot için bir yükleme kontrol olarak bir referans qPCRs gen ve nucleolin gibi qRT-PCR, GAPDH bir referans transkript olarak 18S rRNA kullanın. </li></ol></li><li> Immünohistokimya veya immünofloresan için doku örnekleri <ol><li> Agaroz iskele çıkarın ve mısır durulamaPBS ile de ea. </li><li> Videoda görüldüğü gibi deneysel hedefe bağlı olarak, parafin doku blok veya dondurulmuş doku blok yapmak için kornea kullanın. Kornea doku bölümleri hazırlanırken Biz standart prosedürleri izleyin. </li></ol></li></ol> Temsilcisi Sonuçlar Doku kültür içinde hücreleri incelemek için mevcut yöntemlerin çoğu kolayca HSV-1 ile enfekte olan ex vivo olarak kornea kullanım için uyarlanabilir. Burada gösterilen HSV-1 enfeksiyonu okuyan uygulanan en yaygın teknikler birkaç temsilcisi sonuçları - qPCR (Şekil 1D), qRT-PCR (Şekil 1B), Western Blot (Şekil 1F), plak testi (Şekil 1C) ve doku immünofloresan (Şekil 1E). <img src="/files/ftp_upload/3631/3631fig1.jpg" alt="Şekil 1" /> Şekil 1. HSV-1 ex v ile enfekte kornea Analiziivo. Akut kornea epitel HSV-1 enfeksiyonu ex vivo modelde (A) şematik gösterimi. (BF) Bu video makalede anlatılan yöntemi kullanarak enfekte kornealar analizi. Çıkarılmış tavşan korneası suşu KOS HSV-1 1x10 4 PFU / kornea ile enfekte edilmiş ve varlığında (PAA) ya da phosphonoacetic asit (400 ug / ml), HSV replikasyon bilinen bir inhibitör yokluğunda (Mock) içinde kültüre edildi. (B) Toplam RNA, belirtilen zaman noktalarında toplanmıştır ve viral transkripsiyon HSV-1 polimeraz için primerler ile qRT-PCR ile yapılmıştır. 18S karşı Astarlar rRNA bir referans olarak kullanılmıştır. n = 3. Hata çubukları, ortalama ± SEM göstermektedir. (C) Kültür ortamı 48 hpi toplanmıştır ve bulaşıcı parçacık üretim CV-1 mono tabakaları üzerindeki plak deneyi ile değerlendirilmiştir. (D) Toplam DNA 48 hpi toplanmıştır ve viral genom kopyelenmesi değerlendirilmiştir HSV-1 genomu için primerler ile bir qPCR tahlil. PrGAPDH karşı imers referans olarak kullanılmıştır. n = 3. Hata çubukları ± SEM göstermektedir. (E) Mock-işlenmiş kornealar kesitli 48 hpi, at-flaş donmuş ve enfekte epitel içinde bir viral erken protein (ICP8) varlığı için indirekt immunfloresan tarafından analiz edildi. Çekirdekler Höchst 33258 ile zıt edilir. Proteininin (F) lizatları 48 hpi toplandı ve bir viral protein (ICP0) Western blot ifadesi ile değerlendirildi. BCA tahlil örnekleri arasında eşit yükleme sağlamak için kullanılmıştır. hpi = saat sonrası enfeksiyon.

<ol>
	<li>Kaye, S., Choudhary, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+keratitis.">Herpes simplex keratitis.</a> Prog. Retin. Eye Res. 25, 355-380 (2006).</li><li>Holmberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+exotic+pets.">Enucleation of exotic pets.</a> Journal of Exotic Pet Medicine. 16, 88-94 (2007).</li><li>Remeijer, L., Osterhaus, A., Verjans, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+herpes+simplex+virus+keratitis:+the+pathogenesis+revisited.">Human herpes simplex virus keratitis: the pathogenesis revisited.</a> Ocul. Immunol. Inflamm. 12, 255-285 (2004).</li><li>Foreman, D. M., Pancholi, S., Jarvis-Evans, J., McLeod, D., Boulton, M. E. <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+organ+culture+model+for+assessing+the+effects+of+growth+factors+on+corneal+re-epithelialization.">A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization.</a> Exp. Eye Res. 62, 555-564 (1996).</li><li>Xu, K. P., Li, X. F., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+organ+culture+model+for+assessing+epithelial+responses+to+surfactants.">Corneal organ culture model for assessing epithelial responses to surfactants.</a> Toxicol. Sci. 58, 306-314 (2000).</li><li>Klausner, E. A., Peer, D., Chapman, R. L., Multack, R. F., Andurkar, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+gene+therapy.">Corneal gene therapy.</a> J. Control Release. 124, 107-133 (2007).</li><li>Halford, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICP0+antagonizes+Stat+1-dependent+repression+of+herpes+simplex+virus:+implications+for+the+regulation+of+viral+latency.">ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency.</a> Virol. J. 3, 44 (2006).</li><li>Bhattacharjee, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+treatment+of+ocular+HSK+with+a+human+apolipoprotein+E+mimetic+peptide+in+a+mouse+eye+model.">Effective treatment of ocular HSK with a human apolipoprotein E mimetic peptide in a mouse eye model.</a> Invest Ophthalmol Vis. Sci. 49, 4263-4268 (2008).</li><li>Novitskaya, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difficulties+imaging+herpes+simplex+keratitis+with+fluorescein+isothiocynate-labeled+anti-HSV-1+antibodies+in+an+ex+vivo+model.">Difficulties imaging herpes simplex keratitis with fluorescein isothiocynate-labeled anti-HSV-1 antibodies in an ex vivo model.</a> Cornea. 28, 421-425 (2009).</li><li>Sharma, A., Shimeld, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+immunofluorescence+to+diagnose+herpes+simplex+virus+keratitis+in+mice.">In vivo immunofluorescence to diagnose herpes simplex virus keratitis in mice.</a> Br. J. Ophthalmol. 81, 785-788 (1997).</li></ol>

Çıkar çatışması ilan etti.

Bu video yazıda bir ex vivo modelde akut kornea epitel HSV-1 enfeksiyonu çalışmak için bir yöntem açıklanmaktadır. Bu hücre kültürü sonuçlarının fizyolojik-doğru doğrulama için izin verir ve böylece, yapılmalıdır hayvan deneyleri miktarını sınırlar, çünkü bu model, in vivo modeller in vitro ve arasında yararlı bir adım. Buna ek olarak, ex vivo modelde bozulmamış insan kornea epitel herpes enfeksiyonu incelemek için eşsiz ve paha biçilmez ...

<table border="1"><tbody><tr><td> Reaktifler / Malzemeleri </td><td> Şirket </td><td> Katalog # </td></tr><tr><td> Minimum Essential Medium (MEM) </td><td> Mediatech </td><td> 10-010-CV </td></tr><tr><td> Temel Olmayan Amino Asitler (100x çözüm) </td><td> Mediatech </td><td> 25-025-CI </td></tr><tr><td> L-Glutamine </td><td> Mediatech </td><td> 25-005-CI </td></tr><tr><td> Penisilin G Sodyum Tuzu </td><td> Sigma </td><td> P3032 </td></tr><tr><td> Streptomisin Sülfat Tuz </td><td> Sigma </td><td> S9137 </td></tr><tr><td> Agaroz </td><td> Invitrogen </td><td> 15510-027 </td></tr><tr><td> Fosfat Salin (PBS) Tamponlanmış </td><td> Laboratuarda hazırlanan </td><td> N / A </td></tr><tr><td> Tripsin EDTA (1x) &lt;/ Td&gt; <td> Mediatech </td><td> 25-053-CI </td></tr><tr><td> DNeasy Kan ve Doku Seti </td><td> QIAGEN </td><td> 69504 </td></tr><tr><td> RNeasy Mini Kit </td><td> QIAGEN </td><td> 74104 </td></tr><tr><td> Laemmli Tampon </td><td> Laboratuarda hazırlanan </td><td> N / A </td></tr><tr><td> Optimal Kesme Sıcaklık (OCT) Bileşik </td><td> Tissue-Tek </td><td> 4583 </td></tr><tr><td> Tüm Tavşan Eyes (Genç) </td><td> Pel-Freez </td><td> 41211-2 </td></tr><tr><td> İnsan kornealar ve Tüm Gözler </td><td> Yerel Göz Bankası </td><td> N / A </td></tr><tr><td> 12 Peki Porselen Noktası Tabak </td><td> Avogadro Lab Kaynağı </td><td> 1877 </td></tr><tr><td> 35 mm Doku Kültürü Bulaşık </td><td> Şahin </td><td> 353001 </td></tr><tr><td> Peki 6Hücre Kültürü Tabak </td><td> Greiner Bio-One </td><td> 657160 </td></tr><tr><td> Cryomold Orta </td><td> Tissue-Tek </td><td> 4566 </td></tr></tbody></table>

ex vivo organotypic corneal model of acute epithelial herpes simplex virus type i infection

Herpes keratit, herpes simpleks virüsü tip-1 (HSV-1) ile ilişkili en ciddi patolojilerde biridir. Herpes keratit anda gelişmiş ülkelerde hem kornea kaynaklı ve infeksiyona bağlı körlüğün en önde gelen nedenidir. Herpes keratit Tipik tanıtımı, yara izi gibi kalıcı kornea patolojileri yol açan incelme ve opaklık 1, kornea epitel enfeksiyon ve bazen derin korneal stroma ve endotel içerir. Kornea HSV-1 enfeksiyonu geleneksel deneysel modellerin iki tipte incelenir. Kornea epitel hücrelerinin kültürlü monolayers bir Petri kabındaki enfekte olduğu in vitro modelin de basitlik, tekrarlanabilirliği yüksek düzeyde, hızlı deneyler ve göreceli olarak düşük maliyeti sunmaktadır. Diğer taraftan, böyle bir tavşan veya fare gibi hayvanlarda kornea direkt olarak inoküle edildiği bir in vivo modeli, en son derece gelişmiş bir fizyolojik sunmaktadırsistem, ancak daha yüksek maliyetler, uzun deneyler, gerekli hayvan bakımı ve değişkenliği daha büyük bir derecesi vardır. Bu video yazıda, in vivo modeller in vitro ve hem gücünü birleştiriyor kornea epitel HSV-1 enfeksiyonu yeni bir ex vivo modelde, ayrıntılı bir gösteri sunmak. Ex vivo modelde organotypically kültür içinde muhafaza edilmekte ve HSV-1 ile enfekte olan bozulmamış korneası kullanmaktadır. Ex vivo modelinin kullanımı çok fizyolojik açıdan dayalı sonuçlar sağlar, yine de oldukça pahalı değildir ve in vitro model ile karşılaştırılabilir zaman kararlılık gerektirir.

Watch this Scientific Journal Video about Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection at JoVE.com

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Bu video yazıda yeni bir kullanımı tarif<em&gt; Ex vivo</emAkut herpes&gt; modeli virüsü tip I kornea epitel enfeksiyon simpleks.

Akut Epitel Herpes Ex Vivo Organotipik Kornea Model Virüs Tip I Enfeksiyon Simplex

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

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Neuroscience

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

12.6K Görüntüleme.  Drexel University College of Medicine. Bu video yazıda yeni bir kullanımı tarif Ex vivo modeli virüsü tip I kornea epitel enfeksiyon simpleks.

Video: Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

Tüm ex vivo ekimi ise, Gelişmekte Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Nörobilim

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Bozulmamış Vomeronasal Organ ve Aksesuar Koku Ampul Ex Vivo Hazırlıklar

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Bir<em&gt; Ex vivo</em&gt; Lazer kaynaklı Omurilik Felçlileri Modeli Gerçek zamanlı aksonal Dejenerasyon mekanizmaları değerlendirmek için

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Koroid Pleksus Epitel Hücreleri Silier Hareketi Gözlem<em&gt; Ex Vivo</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Beyin Dilimleri Korku Devre Ex Vivo optogenetic Diseksiyon

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

Solvent-temizlenmiş beyin dokusu kuduz virüs enfeksiyonu yüksek çözünürlüklü 3D görüntüleme

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

Yerinde Akut Ex Vivo Embriyonik Beyin Dilimi Kültürlerinde Akson Büyümesi ve Büyüme Koni Dinamiğinin Görselleştirilmesi

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

İnsan Kök Hücre Nakli ve Validasyonu için Ex vivo Model Olarak Yetişkin İnsan Korteksinin Organotipik Kültürleri

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

AMD Araştırması için İnsan Donör Gözlerinin OCT Tabanlı Multimodal Görüntülemesi

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Yazar Spotlight: Yaşa Bağlı Makula Dejenerasyonu Araştırmaları için İnsan Donör Gözlerinin Ex Vivo OCT Tabanlı Multimodal Görüntülemesi  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

Ex Vivo Yaşa Bağlı Makula Dejenerasyonu Araştırmaları için İnsan Donör Gözlerinin OCT Tabanlı Multimodal Görüntülemesi

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...