Name: glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina
Uploaded: 2015-03-22T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

This paper is dedicated to my beloved wife Maren and our little Karl.

Sığır Eyes 1. Hazırlık <ol><li> Hayvan kesildiği hemen sonra sığır gözleri edinin. </li><li> &quot;Sickel çözeltisi&quot; korunan gözleri Taşıma 120 mM NaCI, 2 mM KCI, 0.1 mM MgCl2, 0.15 mM CaCl2, 1,5 mM NaH 2 PO 4, 13.5 mM Na 2 HPO 4 ve 5 mM glikoz de ihtiva eden özel bir ortam RT. </li><li> Loş bir kırmızı ışık ile koyu adapte koşullar altında retina hazırlanmasını gerçekleştirin. </li><li> Gözün ön kısmını çıkarın. Bir ekvator kesi ca. gerçekleştirin Limbusa 4 mm arka. Bundan sonra tek parça halinde kornea, iris, siliyer cisim ve merceği çıkarın. Sickel-çözeltide retina tutun. </li><li> Mekanik retina yüzeyine vitreus ekleri gevşetin ve açık göz fincan vitrözü çıkarın. </li><li> Daha sonra dört parçaya bölünür göz bölmek ve yaklaşık alanlarında yuvarlak dışarı yumruk Bir trefin kullanarak 7 mm çaplı. </li><li> Yavaşça retina ayrıpigment epitel ve ışıktan korunan bir kutu içinde bir kayıt cihazına yerleştirin. kayıt cihazı ortada bir ağ ile plastik bir koruyucu ile oluşur; örgü üzerine retina yerleştirin ve elektrotlar üzerinde doğrudan bir plastik halka ile düzeltmek sonra. NOT: Plastik sürdürme ortamının sürekli bir akış sağlamak için iki kanal vardır. </li></ol> 2. Kayıt Elektroetinogram (ERG) <ol><li> , Elektroretinogram kayıt retina her iki tarafında iki gümüş / gümüş klorür elektrodu ve yaklaşık sabit bir perfüzyon hızı ile retina serpmek için 1 ml / dakika ve 37 ° C sabit bir sıcaklıkta. Oksijen ile doymuş &quot;Sickel-çözüm&quot; kullanın. </li><li> Ölçüm başlamadan önce, karanlık bir retina (tüm ölçümler sırasında ışıktan korumak) uyum ve beş dakika uyaran aralıkları kullanın. Retina yüzeyinde 6.3 MLX ayarlanmış bir yoğunluk ile uyarılması için bir 1 Hz tek beyaz Xenon flaşı kullanın. </li&gt; <li> Kullanım nötral yoğunluk filtreleri ve optimal yanıtları sahip olmak için bir zamanlayıcı tarafından kontrol 10 mikro saniye olduğu bir ışık uyaranı kalibre. </li><li> Ölçmek ve verileri işlemek, ERG filtre ve bir Grass RPS312RM Amplifikatör kullanarak (100 Hz yüksek geçiren filtre, 50 Hz çentik filtresi, 100,000 x büyütme) yükseltmek için. Sinyali bozabilir olası rahatsız edici frekansları filtrelemek için deneyin. Verileri işlemek için, bir masaüstü bilgisayar (PC uyumlu) bir analog-dijital veri toplama tahtası kullanın. </li><li> Istikrarlı b-dalgası genlikleri kaydedilir kadar sürekli perfüzyon altında karanlık adaptasyonu döneminden sonra, elektrik sinyali genlikleri ölçün. NOT: Beş tek ölçümler ortalama değerine ulaşmak ve daha az% 10 sapma durumunda Amplitüdleri stabil olarak kabul edilir. Tek ölçümlerindeki iyi bir örnek, Şekil 1 &#39;de verilmiştir. </li><li> Test başlatmak için ya saf azot saf oksijen yerine (tek deneylerin sayısı, n = 5) hipoksi test etmek içinveya 250 um glutamat (n = 5). </li><li> Elektrik yanıtları 45 dakika boyunca her 5 dakika kaydedin. </li><li> Test döneminden sonra, 75 dakika boyunca oksijen ile doymuş standart ortam ile retina serpmek ve b-dalgası genlik değişiklikleri bakmak. Bu arınma aşamadır. B-dalgası zirve bir dalga çukur b-dalga amplitüdü ölçün. </li><li> Scotopic koşullar altında fotoreseptör potansiyeli hipoksi veya glutamat etkisini araştırmak için, besin çözeltisine 1 mM ekleyerek b-dalga bastırmak. </li><li> 30 dakika boyunca sabit bir fotoreseptör potansiyeli kaydedildikten sonra, 1 mM aspartat ile farklı sulama çözümlere retina 45 dakika maruz, daha önce olduğu gibi işlem yapmak. Daha önce belirtildiği gibi, aynı arınma dönemi (adım 2.8) kullanın. </li></ol> 3. Veri Analizi <ol><li> Istatistiksel verileri değerlendirmek amacıyla, Kolmogorov-Smirnov kullanarak, örneğin tüm veriler için normal bir dağılım sağlamakTest 15. </li><li> Fuar öncesi son ölçüm karşılaştırıldığında maruz aşamasından sonra yüzdelerle a- ve b-dalgası genliklerinin azalma hesaplayın. 45 dakika sonra ERG-genliklerinin azalma karşılaştırın - ERG için uygulamadan önce ölçülen - fuar dönemi sonunda. </li><li> Olası bir kurtarma incelemek için fuar öncesinde ilgili genlik arınma fazının sonunda a- b- ve dalga karşılaştırın. </li><li> İstatistiksel analiz için, yazılım JMP istatistik yazılımı veya SPSS yazılımını kullanın. Ortalama ± standart sapma olarak boyunca verileri hesaplayın. Uygun istatistiksel testi ile önemini tahmin. NOT: Bu testler deneysel kapsamına bağlı olarak farklı olabilir. Bu ortamda, eşli t-testi kullanın. </li></ol>

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The authors have nothing to disclose.

Bu çalışmada, hipoksi, 45 dakika sonra, b-dalgası amplitüd üzerinde önemli bir etkisi bulunmuştur. Bu azalma hala arınma aşamasından sonra anlamlı oldu. Fotoreseptör potansiyeli benzer bir etki gözlenmemiştir. sonuçlar diğer yayınlanmış veriler 16 ve bize hipoksi sonra mümkün nöroprotektif etkilerini incelemek için fırsat vermek tarafından desteklenmektedir. 250 uM glutamat 45 dakika fuar sonra, biz sadece arınma döneminin s...

Nöroproteksiyon geçmiş yıllarda göz araştırma soruşturma güçlü bir alan olmuştur. Retina oksijenasyonu önemli ölçüde bağlıdır ve onun çevresindeki hücrelerin metabolizması tarafından şiddetle etkilenir son derece hassas nöronal ağ. Sinir hücre hasarına bağlı Binbaşı oküler patolojiler retinal vasküler tıkanıklıklar, glokom, retina dekolmanı ve vardır. Retina arter oklüzyonu, retinal damar oklüzyonu için bir örnek olarak, bağlı iç retina 1 hipoksi ani görme kaybına neden olur. Genellikle görme keskinliği önemli ölçüde 1 kurtarma hastaların sadece% 8 ile, kalıcı görme kaybı 1 genel damar patolojileri 2 ve yol ile ilişkilidir. Arteriyel fıbrinoliz bir tedavi seçeneği olarak öne sürülmüştür rağmen, fayda randomize klinik çalışmada 3 gösterilen olamazdı. Glokom ve retina dekolmanıHer iki glutamat konsantrasyonu 4-6 artış var. Fizyolojik koşullar altında Glutamat, tüm santral sinir sistemi boyunca bir uyarıcı verici ve iç retina 7,8 olarak karşımıza çıkmaktadır. Artmış glutamat seviyeleri, sadece glokom ve retina dekolmanı 5,6 değil, aynı zamanda, proliferatif diyabetik retinopati 9 tespit edilmiştir. Glutamat bir artış muhtemelen, bu nedenle, sinir hücresi harabiyetini 10 nörotoksitisi yol açmaktadır. Retina dekolmanı ve retina (pars plana vitrektomi) proliferatif diyabetik retinopati ameliyat bazı durumlarda Çoğu durumda gereklidir. Pars plana vitrektomi mekanik manipülasyon sırasında, optik fiber veya uzun işlemler sırasında sulama çözümleri yüksek akış oranları tarafından uygulanan kesme stresi parlak ışık retina 11,12 ek bir stres uygular. Tüm söz konusu hastalıklar patoloji Retin lokalize olduğu ortakBir yalnız ve bir nörosensorial sistemi olarak retina korumak için yollar bulmak için bir meydan okuma ile göz topluluk oluşturmaktadır. elektroretinogram (ERG), in vivo fotoreseptör fonksiyonu (a-dalgası) ve iç retina (b-dalgası) fonksiyonunun değerlendirilmesi için standart bir yöntemdir. ERG kornea içine gümüş elektrotlar ile ölçülür ve gözler çubuk veya koni veya iç retina kusurları tespit etmek ışığın bir çok artan düzeyde tarafından teşvik ediliyor. Retinada farklı kusurlar genlik (tepkisinin gücü) veya ERG gecikme (zaman-tepki aralığı) değişim ile saptanabilir. Farklı ERG protokolü ve ölçüm yöntemleri (desen-ERG, multifokal ERG-veya parlak bir alan ERG) kusurların daha farklılaşmasını sağlar. izole retina tekniği mümkün bir çalışmada hayvansal en örn etkilenmeyen retina üzerine etkilerini değerlendirmek için yapım, son zamanlarda girmiştirGenel reaksiyonlar 13,14. Bu değerlendirme ve superfused izole retina üzerinde hipoksi ve glutamat stres tanımlanmış ve standart stres modeli tanıtmak için bu çalışmanın amacı idi. Böylece, biz bazı ajanlar veya göz içi sulama çözümleri nöroprotektif etkileri gelecekteki çalışmalar için temellerini umuyor.

<table><tbody><tr><td>120 mM NaCl&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,064,041,000</td><td></td></tr><tr><td>2 mM KCl,&amp;nbsp;&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,050,010,250</td><td></td></tr><tr><td>0.1 mM MgCl2,&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>58,330,250</td><td></td></tr><tr><td>0.15 mM CaCl2</td><td>Merck Pharma, Germany</td><td>111 TA106282</td><td></td></tr><tr><td>1.5 mM NaH2PO4/13.5 mM Na2HPO4&amp;nbsp;&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,065,860,500</td><td></td></tr><tr><td>5 mM glucose</td><td>Merck Pharma, Germany</td><td>40,741,000</td><td></td></tr></tbody></table>

glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina

Nöroproteksiyon geçmiş yıllarda göz araştırma soruşturma güçlü bir alan olmuştur ve glokom, retina damar tıkanıklığı, retina dekolmanı ve diyabetik retinopati gibi hastalıklar etkilemektedir. Gelecekteki preklinik tedavi test için bir standart stres modeli tanıtmak için bu çalışmanın amacı idi. Sığır retina hazırlanmış ve bir oksijen doymuş standart solüsyonu ile perfüze ve ERG kaydedildi edildi. Sabit b-dalgalar, hipoksi (saf N 2) ya da glutamat stresi (250 mikron glutamat) kaydettikten sonra 45 dakika boyunca sarf edildi. Yalnız fotoreseptör fonksiyonu üzerindeki etkilerini araştırmak için, 1 mM aspartat bir-dalgaları elde eklendi. ERG kurtarma 75 dakika boyunca izlendi. Hipoksi için, 87.0% bir dalga amplitüdünde bir azalma, 45 dakika (yıkama p = 0.03 sona erdikten sonra 36.5% azalma) bir fuar süre sonra (p &lt;0.01) tespit edilmiştir. Buna ek olarak, bir başlangıç ​​DECRistatistiksel anlamlılık ulaştı, 87,23% kaydedildi b-dalgası genlikleri kolaylık (p &lt;0.01, arınma sonunda, p = 0.03 olarak% 25.5 azalma). 250 mikron glutamat,% 1,9 (p&gt; 0.05) bir azalma ardından dalga genlikleri (p&gt; 0.05) bir başlangıç% 7,8 azalma için. B-dalgası genlikleri (p &lt;0.01) not edildi% 83.7 azalma; 75 dakikalık bir yıkanmasından sonra azalma% 2.3 (p = 0.62) olmuştur. Bu çalışmada, bir standart strestir gelecekte mümkün nöro-koruyucu etkilerini belirlemek için yararlı olabilir sunulmuştur.

Oksijen-doymuş standart solüsyonu (Şekil 1A ve B) ile retina preparatların perfüzyon 1 saat sonra ERG-genlikleri istikrar ve tek ölçümler arasındaki genliklerinin daha az değişim göstermiştir. pH değeri, ozmotik basınç, sıcaklık ve (hipoksi testleri için hariç) pO 2 tüm testler için sabit tutulmuştur. Iç retina sinyalinden fotoreseptör sinyali izole edilmesi için, 1 mM aspartat b-dalgası (Şekil 1A) bastırmak için standart çözelt...

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Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Bu çalışma ile, gelecekteki klinik öncesi tedavi test için izole Süperfüzyonlu sığır retina için bir standart stres modeli tanıtmak. a- ve b-dalgası genlikleri ile temsil retina fonksiyonu ya hipoksi (saf N 2) ya da glutamat stres (250 uM glutamat) etkisi değerlendirildi.

İzole Perfüze Omurgalı Retina için bir Stres Modeli olarak Glutamat ve hipoksi

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

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7.8K Görüntüleme.  University Eye Hospital Tübingen.  Bu çalışma ile, gelecekteki klinik öncesi tedavi test için izole Süperfüzyonlu sığır retina için bir standart stres modeli tanıtmak. a- ve b-dalgası genlikleri ile temsil retina fonksiyonu ya hipoksi (saf N 2) ya da glutamat stres (250 uM glutamat) etkisi değerlendirildi. 

Video: Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Fare Retina kullanma CNS&#39;deki Vasküler Rejenerasyon Değerlendirilmesi

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Nörobilim

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.

Içinde Neurovascular Yaptırma Analizi Entorhino-hipokampal Organotipik Dilim Kültürler

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

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Bir oksijen-Glukoz Yoksunluk ve reoksijenizasyonun<em&gt; In Vitro</em&gt; İskemi-Reperfüzyon Modeli incelenmesi Kan-Beyin Bariyeri Disfonksiyon için

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Tıp

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Biomimetic kimyasal Neuromodulation, sıçan retina nörotransmitter Glutamat Vitro ile için metodoloji

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

Biyomühendislik

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Astrocyte reaktivite ve nükleer silahların yayılmasına karşı Vitro iskemik benzeri koşullar altında izleme

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Göz İçi Basıncı Yükselmesi ile Retina İskemi-Reperfüzyon Hasarı Fare Modeli

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

Tedavi Kan Retina Bariyer ihlali ve Potansiyel İlaçlar değerlendirilmesi için bir Akut Retina Modeli

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

Kemirgenlerde İskemik Retina Hastalıkları için Oksijen Kaynaklı Retinopati Modeli

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Geçici Bilateral Ortak Karotid Arter Tıkanması sonucu İndüklenen İskemik Retina Yaralanmasının Murine Modeli

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Retinal Nörovasküler Hastalıkları İncelemek için Ex Vivo Modeli Olarak Yetişkin Fare Retinasının Retinal Eksplantı

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

İzole Perfüze Omurgalı Retina için bir Stres Modeli olarak Glutamat ve hipoksi

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Fare Retina kullanma CNS'deki Vasküler Rejenerasyon Değerlendirilmesi