Bizim araştırma NIH, Ulusal Göz Enstitüsü İntramural Araştırma Programı tarafından desteklenmektedir.

Kullanılan tüm ekipmanların ve reaktifler sterildir. Hayvan Bakım tarafından onaylanmış ve tüm hayvan deneyleri (hayvan çalışması protokol NEI-570) NEI / NIH Komitesi (ACUC) kullanın ve NIH kurallar ve düzenlemelere göre yapılmıştır. 1. Gün 1 Retina Ganglion Hücreleri At Temel Retrograd Etiketleme (RGCs) Bu prosedürün amacı, optik sinir ezilme yaralanması üç gün önce farelerin üstün colliculus bir sinir tracer boya enjekte edilerek retrograd retina ganglion hücrelerinin etiket. Boya retrograd retina ganglion hücrelerinin tarafından ele alınan ve yaşam RGCs (Şekil 1) için bir belirteç sağlar olacak. Bu yaklaşım, az bir değişiklik 1-5 ile yaşayabilir RGCs tekrarlanabilir etiketleme verir . <ol><li> Fareyi derinden uyutmak. Başın tepesinde saç temizleyin. Fare, küçük bir stereotaksik aparat yerleştirin. Koruyucu oinment her iki göze uygulanır. Kafa derisi Dezenfekte iyodofor ve% 70 alkol fırçalayın kullanarak her üç kez. </li><li> Kafatası açığa çıkarmak için deride bir kesi yapın ve% 3 hidrojen peroksit kullanılarak kuru ve temiz tutmak. </li><li> Belirlenmesi ve bregma işareti. Bregma arkasında 2.9 mm ve 0.5 mm, her yarımkürenin 7 orta hatta yan üstün colliculus üzerinde bir delik delin . Sondaj sırasında, ikincil ısı hasarı önlemek için delik delinmiş site için tuzlu su uygulamak. </li><li> Stereotaksik bir ölçüm cihazı ve Hamilton şırınga kullanarak, beynin kemik yüzeyinden 1.6 mm derinlikte, her yarımkürenin üstün colliculus içine çok yavaş FluoroGold nöral tracer boya enjekte (% 3 1 ul salin içinde Florokrom). </li><li> Kesi yeri dikin. Analjezi için buprenorfin subkutan enjeksiyon verin. </li><li> Sıcak ve kuru bir alana taşıyın ve dik bir duruş, bakım ve evde kafes dönmek için mümkün olduğu kadar izlemek. Sistemik analjeziklerin (örneğin, buprenorfin) ve topikal antibiyotikli merhem, etiketleme prosedürü sonra ilk üç gün boyunca, günde iki defa verilir ve fare yakından izlenmelidir. </li></ol> 2. 4. Gün, Optik Sinir Ezilme Yaralanması Bu yöntemde, optik sinir, retina ganglion hücrelerinin yavaş yavaş bir ölüme neden olacaktır aksonları birincil hasar (Şekil 2), neden bir ezilme yaralanması geçerli olacaktır. <ol><li> Tracer boya uygulaması üç gün sonra, fare derin anestezi. Diseksiyon mikroskop kullanarak, bir gözün konjonktiva bir çift yaylı makas kullanılarak 04:00 konumu hakkında çizi. </li><li> Yörünge kasları hafifçe saptırmak ve onları bir kenara koymak. Göz küresi çıkışında beyaz optik sinir Açığa. Herhangi bir kan damarlarının zarar vermemek için çok dikkatli olun. </li><li> Bir çift çapraz eylem forseps yardımı ile yaklaşık 3 saniye boyunca yaklaşık 2 mm göz küresi, optik sinir ezilme yaralanması geçerlidir. Kanamaya neden oftalmik arter zarar vermemek için çok dikkatli olun. </li><li> Ezilme yaralanması tamamlanmasından sonra, kesi sütüre edilir. </li><li> Fare anestezi kurtarır önce analjezi için buprenorfin bir subkütan enjeksiyon vermek. </li><li> Dik bir duruş korumak ve kendi ev kafesine geri gelene kadar, sıcak ve kuru bir alana taşıyın ve izlemek. Sistemik analjeziklerin (örneğin, buprenorfin) ve topikal antibiyotikli merhem, ezilme yaralanması prosedürü sonrası ilk üç gün boyunca, günde iki defa verilir ve fare yakından izlenmelidir. </li></ol> 3. Retinae Hasat ve Gün 11 yaşında RGC Survival Analiz Bu prosedürün amacı, retinaeto retina ganglion hücre sağkalım analiz hasat için. <ol><li> 11 (günde 7 ONC sonra) gün, fare, CO 2 infüzyonu ve servikal dislokasyon tarafından ötenazi . </li><li> Gözler yörüngesine basınç uygulayarak bir çift forseps yardımı ile enükleasyon. </li><li> Gözler, iki saat boyunca% 4 paraformaldehid çözüm sabit ve PBS içinde üç kere yıkanır. Retina parçalara ayır. Retina diseksiyon prosedür zaten 6 Gustmann, S. ve ark tarafından 2010 yılında vallahi daha önce yayınlanmış olan ve bu nedenle burada açıklanan olmayacaktır . </li><li> Hayatta retina ganglion hücrelerinin Görüntüler retinanın tanımlanan bölgelerde floresan mikroskop kullanılarak alınır, ve retina ganglion hücre yoğunluğu sayılabilir. Optik sinir ezilme yaralanması ya da floresan etiketli retina ganglion hücrelerinin bir temsilcisi sonucu Şekil 3&#39;te gösterilmiştir. </li></ol> 4. Temsilcisi Sonuçlar: Retina ganglion hücre sağkalım, yedi gün sonra optik sinir ezilme yaralanması analiz etmek retinae, sabit, hasat basık ve monte edildi. Retina görüntüler floresan mikroskop kullanılarak alınmıştır. Yaralanmamış bir şekilde normal r ile karşılaştırıldığındaetina (Normal, sol), yaşayabilir RGCs sayısı (yeşil, retrograd üstün colliculus enjekte FluoroGold nöral tracer boya etiketli) retinae bir optik sinir ezilme yaralanması (sağ, ONC) (Şekil 3) ile anlamlı derecede düşük . <img src="/files/ftp_upload/2685/2685fig1.jpg" alt="Şekil 1" /> Şekil 1. Retina ganglion hücrelerinin bazal retrograd etiketleme Optik sinir ezilme yaralanması sonrası farklı zaman noktalarında uygulanabilir retina ganglion hücreleri saymak için, retina ganglion hücrelerinin (optik sinirin beyin üç gün içinde üstün colliculus önce bir sinir tracer boya (yeşil renk) enjekte retrograd etiketli kırmızı) ezilme yaralanması. Retina ganglion hücrelerinin aksonları üstün colliculus ikamet yılından bu yana, izleme boya retrograd RGCs tarafından ele alınması ve yaşam RGCs için bir belirteç olacaktır. <img src="/files/ftp_upload/2685/2685fig2.jpg" alt="Şekil 2" /> Şekil 2. Optik sinir ezilme yaralanması Retina ganglion hücrelerinin hayatta kalma durumunu analiz etmek için, bir ezilme yaralanması aksonlar için birincil hasara neden optik sinir (kırmızı) uygulanır. Bu kademeli bir retina ganglion hücrelerinin ölümüne yol açacaktır. Üç gün sonra izleme boya (yeşil) enjeksiyonu, fare anestezi. Bir gözün konjonktiva çizi. Yörünge kasları karsilayin ve onları bir kenara koymak. Göz küresi çıkışında optik sinir Açığa. Ezilme yaralanması göz küresi yaklaşık 2 mm optik sinir bir çift çapraz eylem forseps kullanarak yaklaşık 3 saniye boyunca uygulayın. <img src="/files/ftp_upload/2685/2685fig3.jpg" alt="Şekil 3" /> Şekil 3. Floresan-etiketli optik sinir ezilme yaralanması ya da retina ganglion hücrelerinin Retina ganglion hücre sağkalım, yedi gün sonra optik sinir ezilme yaralanması analiz etmek retinae, sabit, hasat basık ve monte edildi. Retina görüntüler floresan mikroskop kullanılarak alınmıştır. Yaralanmamış bir şekilde normal retina (Normal, sol) ile karşılaştırıldığında, canlı RGCs sayısı (yeşil, retrograd üstün colliculus enjekte FluoroGold nöral tracer boya etiketli) bir optik sinir crush yaralanma (sağ, ONC retinae anlamlı olarak daha düşüktü ) (Şekil 3). Ölçek çubuğu: 50 mikron

<ol>
	<li>Yoles, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1+reduces+injury-induced+metabolic+deficits+and+degeneration+in+the+rat+optic+nerve.">GM1 reduces injury-induced metabolic deficits and degeneration in the rat optic nerve.</a> Investigative ophthalmology &#38; visual science. 33, 3586-3591 (1992).</li><li>Fisher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+for+neuroprotection+in+the+mouse+optic+nerve:+implications+for+optic+neuropathies.">Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies.</a> J Neurosci. 21, 136-142 (2001).</li><li>Levkovitch-Verbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGC+death+in+mice+after+optic+nerve+crush+injury:+oxidative+stress+and+neuroprotection.">RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection.</a> Investigative ophthalmology &#38; visual science. 41, 4169-4174 (2000).</li><li>Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+inhibits+apoptosis+via+VEGFR-1-mediated+suppression+of+the+expression+of+BH3-only+protein+genes+in+mice+and+rats.">VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.</a> The Journal of clinical investigation. 118, 913-923 (2008).</li><li>Tang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+effect+of+PDGF-CC+rescues+neurons+from+apoptosis+in+both+brain+and+retina+by+regulating+GSK3beta+phosphorylation.">Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3beta phosphorylation.</a> The Journal of experimental medicine. 207, 867-880 (2010).</li><li>Gustmann, S., Dunker, N. <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-like+organotypic+murine+retinal+wholemount+culture.">In vivo-like organotypic murine retinal wholemount culture.</a> J Vis Exp. , (2010).</li><li>Franklin, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

Optik sinir ezilme yaralanması fare modeli RGC ölüm ve hayatta kalma sürecini incelemek için yararlıdır. Bu model aynı zamanda sık sık farklı reaktifler ve genlerin RGC apoptoz ve sağkalım üzerine etkilerini araştırmak amacıyla kullanılır. Bu modelin bir diğer avantajı da minimal varyasyonları ile yüksek derecede tekrarlanabilirlik olmasıdır. Ancak bu model birkaç adım, özel bakım gereklidir. İlk olarak, optik sinir ezilme yaralanması yaparken, oftalmik arter v...

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an optic nerve crush injury murine model to study retinal ganglion cell survival

Optik sinir yaralanması ardından, geri dönüşü olmayan görme kaybı sonuçları retina ganglion hücrelerinin (RGCs), bir yavaş yavaş ölüm, aksonal dejenerasyona yol açabilir. Insan, bu tür hastalıkları örnekler travmatik optik nöropati ve glokom, optik sinir dejenerasyonu içerir. Bu, görme kaybı ve körlük önde gelen optik sinir başı tipik değişimler, progresif optik sinir dejenerasyonu, retina ganglion hücrelerinin kaybı, kontrolsüz, ile karakterizedir. Optik sinir ezmek (ONC) yaralanma fare modeli Travmatik optik nöropati için önemli bir deneysel hastalık modeli, bu model glokom, vb optik sinir kademeli retina ganglion hücrelerinin apoptozise neden ezilme yaralanması. Bu hastalık modeli nöronal ölüm ve hayatta kalma, terapötik önlemler geliştirilmesi için gerekli olan genel süreçler ve mekanizmalar çalışma kullanılabilir. Buna ek olarak, farmakolojik ve moleküler yaklaşımlar bu modelde farklı tip optik nöropati tedavisinde potansiyel terapötik reaktifler belirlemek ve test etmek için kullanılır. Burada, 1 gün retina ganglion hücrelerinin Temel retrograd etiketleme (RGCs), (II) 4 gün Optik sinir ezilme yaralanması, (III) retinae Hasat ve gün hayatta kalma RGC analiz (I) tarafından adım adım gösteri 11 ve (IV) Temsilcisi sonucu.

Watch this Scientific Journal Video about An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival at JoVE.com

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Bu protokol, retrograd retina ganglion hücreleri ve sonradan retina ganglion hücre hayatta kalma ve apoptosis analiz etmek için bir optik sinir ezilme yaralanması yapmak nasıl etiket gösterir. Bu glokom da dahil olmak üzere optik nöropati farklı türleri için deneysel bir hastalık modeli.

Retina Ganglion Hücre Survival Eğitim Bir Optik Sinir Crush Yaralanma murin Modeli

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

an-optic-nerve-crush-injury-murine-model-to-study-retinal-ganglion

Research

JoVE Journal

Neuroscience

30.2K Görüntüleme.  NIH. Bu protokol, retrograd retina ganglion hücreleri ve sonradan retina ganglion hücre hayatta kalma ve apoptosis analiz etmek için bir optik sinir ezilme yaralanması yapmak nasıl etiket gösterir. Bu glokom da dahil olmak üzere optik nöropati farklı türleri için deneysel bir hastalık modeli.

Video: An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Optik Sinir transeksiyonu: Santral Sinir Sistemi Yetişkin Nöron Apoptoz Bir Model

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Nörobilim

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Genetik Etiketli Retina Ganglion Hücreleri Işık Tepkisi Record İzole Retina Hazırlık

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Deneysel Optik Sinir demyelinizasyon bir Rat Modelinde Görsel Uyarılmış Potansiyel Kayıt

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

Retinal Ganglion Hücrelerinin Tanımlanmış Alt Kümelerinin Tek Hücreli RNA-Seqi

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Sırt kök ganglion Enjeksiyon ve Dorsal Kök Duyusal Axon Rejenerasyon Modeli Olarak Ezilme Yaralanması

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Kısmi optik sinir transeksiyon Sıçanlarda: ikincil dejenerasyonu retina Ganglion hücrelerinin değerlendirmek için yeni bir operatif yaklaşım ile kurulan bir Model

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

Bir yapay sinir kullanarak köpek Inferior Alveolar sinir rejenerasyon teşvik için etanol kaynaklı servikal sempatik Ganglion bloğu uygulamaları

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Siyatik Sinir Ezilme Hasarının Sıçan Modelinde Fonksiyonel Değerlendirme için 3D Kinematik Analiz

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

Tek Taraflı Siyatik Sinir Ezilme Yaralanması Sonrası Sıçanlarda Motor Davranışın Multifaktöriyel Değerlendirilmesi

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

In Vivo Büyük Hayvanlarda Retina Ganglion Hücre ve Optik Sinir Fonksiyonu ve Yapısını Değerlendirme Yöntemleri

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...