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

1. Vorbereitung der Rinderaugen <ol><li> Erhalten Rinderaugen direkt nach der Schlachtung des Tieres. </li><li> Transportieren die geschützten Augen &quot;sichel Lösung&quot; ein spezielles Medium, enthaltend 120 mM NaCl, 2 mM KCl, 0,1 mM MgCl 2, 0,15 mM CaCl 2, 1,5 mM NaH 2 PO 4, 13,5 mM Na 2 HPO 4 und 5 mM Glucose bei RT. </li><li> Führen Sie die Vorbereitung der Netzhaut unter dunklen Bedingungen angepasst mit einem dim rotes Licht. </li><li> Entfernen Sie den vorderen Teil des Auges. Führen Sie einen äquatorialen Schnitt ca. 4 mm hinter dem Limbus. Hornhaut, Iris, Ziliarkörper und Linse Danach entfernen Sie in einem Stück. Halten Sie die Netzhaut in Sickel-Lösung. </li><li> Mechanisch lösen Sie die Glaskörper-Anhänge an den Netzhautoberfläche und entfernen Sie die Glaskörper aus der offenen Augenmuschel. </li><li> Danach teilen das Auge in vier Quadranten und Punch-Out runden Flächen von ca. 7 mm Durchmesser mit einem Hohlbohrer. </li><li> Trennen die Netzhaut vorsichtigaus dem Pigmentepithel und auf ein Aufnahmegerät in einem Karton vor Licht geschützt. Die Aufzeichnungsvorrichtung besteht aus einem Kunststoff mit einer Maschen Betreuers in der Mitte; legen Sie die Netzhaut auf dem Netz und dann fix mit einem Kunststoffring direkt auf den Elektroden. HINWEIS: Die Kunststoff Maintainer hat zwei Kanäle, um eine konstante Strömung des Mediums zu ermöglichen. </li></ol> 2. Aufnahme der Elektroretinogramm (ERG) <ol><li> Um das Elektroretinogramm aufzunehmen, verwenden zwei Silber / Silberchlorid-Elektroden auf jeder Seite der Netzhaut und versorgen die Retina bei einer konstanten Geschwindigkeit von ca. Perfusion 1 ml / min, und konstanter Temperatur von 37 ° C. Verwenden Sie die &quot;Sickel-Lösung&quot; mit Sauerstoff gesättigt. </li><li> Vor Beginn der Messung, dunkel Anpassung der Netzhaut (Schutz vor Licht bei allen Messungen) und verwenden Sie Stimulus Abständen von fünf Minuten. Verwenden Sie eine 1 Hz einzelne weiße Xenon-Blitz für die Stimulation mit einer Intensität von 6,3 mlx an der Netzhautoberfläche gesetzt. </li&gt; <li> Verwenden kalibrierten Neutralfilter und einen Lichtreiz von 10 us durch einen Timer, um optimale Antworten haben gesteuert. </li><li> Zur Messung und Verarbeitung der Daten, filtern die ERG und verstärken sie (100 Hz Hochpassfilter, 50 Hz Notch-Filter, 100.000 x Verstärkung) mit einem Gras RPS312RM Verstärker. Versuchen herausfiltern möglich Störfrequenzen, die das Signal stören können. Um die Daten zu verarbeiten, verwenden Sie einen Analog-zu-Digital-Datenerfassungskarte auf einem Desktop-Computer (PC-kompatibel). </li><li> Nach der Dunkeladaptation Zeitraum konstant Perfusion messen die Amplituden des elektrischen Signals, bis stabile b-Wellenamplituden erfasst werden. HINWEIS: Die Amplituden gilt als stabil, wenn fünf Einzelmessungen erreicht einen Mittelwert abweichen und weniger als 10%. Ein gutes Beispiel für Einzelmessungen wird in Abbildung 1 wiedergegeben. </li><li> Um den Test zu starten, wechseln reinem Sauerstoff entweder durch reinen Stickstoff (Anzahl der Einzelversuche, n = 5), um eine Hypoxie testenoder 250 &amp; mgr; m Glutamat (n = 5). </li><li> Notieren Sie die elektrischen Antworten alle 5 min für 45 min. </li><li> Nach der Testphase durchströmen die Netzhaut mit Standardmedium mit Sauerstoff gesättigt 75 Minuten und Blick auf die Veränderungen der b-Wellenamplitude. Dies ist der Auswaschphase. Messen des b-Wellenamplitude von der Wanne der a-Welle auf die Spitze der b-Welle. </li><li> Um die Wirkung von Hypoxie oder Glutamat an der Photorezeptor-Potentials unter skotopischen Bedingungen zu untersuchen, unterdrücken die b-Welle, die durch Zugabe von 1 mM in die Nährlösung. </li><li> Nach der Aufnahme einer stabilen Photorezeptor-Potentials für 30 Minuten, führt das Verfahren nach wie vor Freilegung der Netzhaut 45 Minuten zu den verschiedenen Spüllösungen mit 1 mM Aspartat. Verwenden Sie den gleichen Zeitraum Auswaschen (Schritt 2.8), wie oben erwähnt. </li></ol> 3. Datenanalyse <ol><li> Um die Daten statistisch auszuwerten, sorgen für eine Normalverteilung für alle Daten, beispielsweise unter Verwendung des Kolmogorov-SmirnovTest 15. </li><li> Berechnen Sie die Reduzierung der a- und b-Wellenamplituden in Prozent nach der Belichtungsphase im Vergleich zur letzten Messung vor der Ausstellung. Vergleichen Sie die Reduzierung der ERG-Amplituden nach 45 Minuten - am Ende der Ausstellung Zeitraum - zum ERG gemessen vor der Anwendung. </li><li> Vergleichen der A- und B-Welle am Ende der Auswaschphase zur entsprechenden Amplitude vor Exposition, um eine mögliche Wiederherstellung zu prüfen. </li><li> Für die statistische Analyse, die Software JMP Statistik-Software SPSS oder Software. Berechnen von Daten im gesamten als Mittelwert ± Standardabweichung. Schätzwert von der entsprechenden statistischen Grundlage. HINWEIS: Diese Tests können unterschiedlich sein, je nach Versuchsumfang. Bei dieser Einstellung verwendet der Student-gepaarten t-Test. </li></ol>

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The authors have nothing to disclose.

In dieser Studie wurde eine signifikante Auswirkung auf die b-Wellenamplitude nach 45 min von Hypoxie gefunden. Diese Reduktion war nach der Auswaschphase signifikant. Eine ähnliche Wirkung auf die Photorezeptor-Potentials beobachtet werden. Die Ergebnisse werden von anderen veröffentlichten Daten 16 und geben Sie uns die Gelegenheit, mögliche neuroprotektive Effekte nach Hypoxie Studium unterstützt. Nach 45 min Exposition von 250 &amp; mgr; M Glut...

Neuroprotektion ist ein starker Forschungsgebiet in der ophthalmologischen Forschung in den letzten Jahrzehnten. Die Retina ist ein hochempfindliches neuronalen Netzwerk, hängt signifikant von Sauerstoffversorgung und wird stark durch den Stoffwechsel des ihn umgebenden Zellen beeinflusst. Die wichtigsten Augenkrankheiten zu Schädigungen von Nervenzellen im Zusammenhang stehen retinalen Gefäßverschlüssen, Glaukom und Netzhautablösung. Arterienverschluss, als Beispiel für die Netzhautgefäßverschluss führt zu einem plötzlichen Verlust des Sehvermögens durch Hypoxie des inneren Netzhaut ein. Es wird häufig mit allgemein vaskulären Pathologien 2 und führt zu einer anhaltenden Sehverlust 1 zugeordnet sind, wobei nur 8% der Patienten nach der Sehschärfe signifikant 1. Obwohl arteriellen Fibrinolyse wurde als Behandlungsoption vorgeschlagen worden, könnte der Nutzen nicht in einer randomisierten klinischen Studie 3 angezeigt. Glaukom und Netzhautablösungbeide haben eine Erhöhung der Glutamatkonzentration 4-6. Glutamat unter physiologischen Bedingungen als exzitatorische Transmitter im gesamten Zentralnervensystem und der inneren Retina 7,8 angetroffen. Erhöhte Glutamatspiegel nicht nur bei Glaukom und Netzhautablösung 5,6, sondern auch in proliferative diabetische Retinopathie 9 gefunden. Eine Erhöhung Glutamat führt möglicherweise zu Exzitotoxizität und daher Nervenzellschädigung 10. In den meisten Fällen von Netzhautablösung und in einigen Fällen der proliferativen diabetischen Retinopathie Operation auf der Netzhaut (Vitrektomie) notwendig. Während der Vitrektomie mechanische Manipulation, helles Licht von der optischen Faser oder durch hohe Durchflussraten von Spüllösungen bei langen Operationen ausgeübten Scherspannung üben eine zusätzliche Belastung auf der Netzhaut 11,12. Alle genannten Erkrankungen ist gemeinsam, dass die Pathologie zur retin lokalisiertenein allein und stellen die ophthalmologische Gemeinschaft vor der Herausforderung, Wege, um die Netzhaut als neurosensorische System zu schützen. Das Elektroretinogramm (ERG) ist das Standardverfahren für die Bewertung der in vivo-Photorezeptor-Funktion (a-Welle), und die Funktion der inneren Retina (b-Welle). Die ERG wird durch Silber-Elektroden in die Hornhaut eingeführt, gemessen und die Augen sind durch eine zunehmende Licht stimuliert, um Defekte in Stäbchen oder Zapfen oder in der inneren Netzhaut erkennen. Unterschiedliche Fehler in der Netzhaut kann durch Veränderungen in der Amplitude (der Stärke der Antwort) oder die Latenz (die Zeit bis zur Antwort-Intervall) des ERG festgestellt werden. Verschiedene ERG-Protokoll und Messverfahren (Muster-ERG, multifokalen ERG-oder Hellfeld-ERG) ermöglichen eine weitere Differenzierung von Mängeln. Die Technik des isolierten Netzhaut wurde vor kurzem eingeführt wurde, wodurch es möglich wird, um Effekte auf die Netzhaut ohne Interferenzen von zB eine Studie Tieres zu evaluierenAllgemeinreaktionen 13,14. Es war das Ziel dieser Studie zu bewerten, und die Einführung einer definierten und standardisierten Spannungsmodell für Hypoxie und Glutamat Belastung des Superfusion isoliert Retina. So hoffen wir, die Grundlagen für künftige Studien über neuroprotektive Effekte von bestimmten Agenten oder intraokularen Spüllösungen legen.

<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

Neuroprotektion ist in der ophthalmologischen Forschung in den letzten Jahrzehnten eine starke Untersuchungsfeld und wirkt sich Krankheiten wie Glaukom, Netzhautgefäßverschluss, Netzhautablösung, und diabetische Retinopathie. Es war das Ziel dieser Studie, einen standardisierten Stress-Modell für zukünftige therapeutische präklinischen Tests einzuführen. Rindernetzhäute wurden präpariert und mit einem Sauerstoff gesättigt Standardlösung durchströmt, und der ERG wurde aufgezeichnet. Nach dem Aufzeichnen stabil b-Wellen, Hypoxie (reines N 2) oder Glutamat Belastung (250 &amp; mgr; m glutamat) wurde für 45 min ausgeübt wird. Um die Auswirkungen auf allein Photorezeptorfunktion zu untersuchen, wurde 1 mM zugegeben, um eine Aspartat-Wellen erhalten. ERG-Erholung wurde für 75 min überwacht. Hypoxie wurde eine Abnahme in einer Wellenamplitude von 87,0% nach einer Expositionszeit von 45 min (Abnahme von 36,5% nach dem Ende der Auswaschphase p = 0,03) angemerkt (p &lt;0,01). Zusätzlich wird ein Anfangs decrLeichtigkeit der b-Wellenamplituden von 87,23% erfasst wurde, dass die statistische Signifikanz erreichte (p &lt;0,01, Abnahme von 25,5% am Ende der Auswaschphase, p = 0,03). Für 250 &amp; mgr; m Glutamat, einem anfänglichen 7,8% Reduktion der a-Wellen-Amplituden (p&gt; 0,05), gefolgt von einer Reduktion von 1,9% (p&gt; 0,05). Eine Verringerung von 83,7% der b-Wellenamplituden (p &lt;0,01) wurde festgestellt; nach einer Auswaschung von 75 min war die Reduktion von 2,3% (p = 0,62). In dieser Studie wird eine standardisierte Spannungsmodell dargestellt, die nützlich für mögliche neuroprotektive Wirkung in der Zukunft identifiziert werden kann.

Nach 1 Stunde der Durchblutung der Netzhautpräparate mit sauerstoffgesättigten Standardlösung (1A und B) ERG-Amplituden zeigten Stabilisierung und weniger Variation der Amplituden zwischen Einzelmessungen. pH-Wert, osmotischer Druck, Temperatur und pO 2 (außer für Hypoxie testing) wurden für alle Versuche konstant gehalten. Um die Photorezeptorsignal aus dem Signal der inneren Retina zu isolieren, wurde 1 mM Aspartat zu der Standardlösung zugesetzt, um die b-W...

Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

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

Mit dieser Studie stellen wir einen standardisierten Stress-Modell für die isolierte superfundiert Rinderretina für zukünftige therapeutische präklinischen Tests. Die Wirkung von entweder Hypoxie (reines N 2) oder Glutamat Stress (250 uM Glutamat) auf Netzhautfunktion von a- und b-Wellenamplituden dargestellt wurde ausgewertet.

Glutamat und Hypoxie als Stress-Modell für die isolierten perfundierten Wirbel Retina

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

glutamate-hypoxia-as-stress-model-for-isolated-perfused-vertebrate

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.9K Aufrufe.  University Eye Hospital Tübingen.  Mit dieser Studie stellen wir einen standardisierten Stress-Modell für die isolierte superfundiert Rinderretina für zukünftige therapeutische präklinischen Tests. Die Wirkung von entweder Hypoxie (reines N 2) oder Glutamat Stress (250 uM Glutamat) auf Netzhautfunktion von a- und b-Wellenamplituden dargestellt wurde ausgewertet. 

Serie: 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.

Beurteilung der Gefäß Regeneration im ZNS Verwenden der Maus Retina

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

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Neurowissenschaften

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.

Die Analyse der neurovaskuläre Remodeling in Entorhino-Hippocampus Organotypischen Kulturen

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.

Sauerstoff-Glukose Deprivation und Reoxygenierung als<em&gt; In-vitro-</em&gt; Ischämie-Reperfusion Injury Modell zum Studium der Blut-Hirnschranke Dysfunction

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

Medizin

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.

Methodik für biomimetische Chemie Neuromodulation der Ratte Netzhaut mit dem Neurotransmitter Glutamat In-vitro-

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

Biotechnik

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma auslösende Verfahren in ein<em&gt; In Vivo</em&gt; Rat Modell und Whole-mount Retina Vorbereitung

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

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.

Überwachung der Astrozyten Reaktivität und Proliferation in Vitro unter ischämischen-ähnlichen Bedingungen

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.

Ein Maus-Modell der Netzhautischämie-Reperfusionsschaden Durch Erhöhung des intraokularen Druck

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.

Eine akute Retinal Modell zur Bewertung von Blut Retinal Barrier Breach und potenzielle Medikamente für die Behandlung

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

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.

Ein murines Modell der ischämischen Netzhautverletzung induziert durch transiente bilaterale gemeinsame Carotis ArterieNverschluss

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.

Netzhautexplantat der adulten Mausnetzhaut als Ex-vivo-Modell zur Untersuchung neurovaskulärer Netzhauterkrankungen

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

Glutamat und Hypoxie als Stress-Modell für die isolierten perfundierten Wirbel Retina

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