Die Finanzierung wurde von Fight for Sight und Forschung zur Erblindung zu verhindern.

1. Anterior Segment Dissektion. <ol><li> Skleralgewebe hinter dem Limbus ist mit einem 0,22 ForceP und der ganzen Welt (Augapfel) stabilisiert wird erfasst. </li><li> Eine mikrochirurgische Klinge wird verwendet, um eine lineare Schnitt in der Hornhaut von Limbus zu Limbus zu machen. </li><li> Einer 0,12 Colibri ForceP wird dann verwendet, um die Hornhaut an den Einschnitt zu begreifen. </li><li> Die Vorderkammer Flüssigkeit wird dann mit einer Weck-Cel chirurgischen Speer absorbiert. </li></ol> 2. Objektiv Ausnehmen. <ol><li> Einer 0,12 Colibri ForceP Greifen der Hornhaut wird verwendet, um den Globus zu stabilisieren. </li><li> Eine feine gebogene Nadel-Halter (oder gebogenen Kornzange) ist hinter der Linse in Richtung der hinteren Seite des Globus eingesetzt. Der Nadelhalter ist teilweise geschlossen, und nach vorne gezogen. Druck ausgeübt wird, mit der Pinzette auf der äußeren Oberfläche der Sklera, der die Linse schiebt nach vorne durch die Hornhaut-Schnitt, während die Augen Wand intakt. Der Glaskörper wird als durchscheinende Gel und ist teilweise Anhänger der Linse. </li><li> Das Objektiv-Glaskörpergewebe wird dann in einer gefilterten Zentrifugenröhrchen mit 20 Mikroliter Protease Inhibitor Cocktail (Roche) in PBS gelöst platziert. </li></ol> 3. Retina Ausnehmen. <ol><li> Weiter um den Globus mit 0,12 Colibri Pinzette greifen die Hornhautschnitt stabilisieren. </li><li> Eine feine gebogene Nadel-Halter (oder gebogenen Kornzange) ist so weit hinter den Globus wie möglich platziert, in der Nähe des Sehnervs. Der Nadelhalter ist teilweise geschlossen, und nach vorne gezogen. Druck ausgeübt wird, mit der Pinzette auf der äußeren Oberfläche der Lederhaut, die die Netzhaut drängt nach vorne durch die Hornhaut-Schnitt. Der Glaskörper wird als durchscheinende Gel Anhänger der Netzhaut. Die Netzhaut wird visualisiert in Form eines gelben, vaskularisierten Gewebe. Jeder Streifen von pigmentierten Gewebe entfernt mit einer Pinzette abgezogen werden. </li><li> Die Netzhaut-Glaskörper Gewebe wird dann in den gefilterten Zentrifugenröhrchen mit dem Objektiv-Glaskörpergewebe platziert. </li></ol> 4. Gefilterte Zentrifugation. <ol><li> Die gefilterte Zentrifugenglas wird in einer Tischzentrifuge gestellt. Spin bei 14.000 x G für 12 Minuten. </li><li> Saugen Sie das Eluens aus der unteren Kammer, die den Glaskörper. Die Netzhaut bleibt in der oberen Kammer. Diese Proben können für Protein-Untersuchungen genutzt werden. </li></ol> 5. Repräsentative Ergebnisse Vitreous Proben wurden durch SDS-PAGE (Abbildung 2) analysiert. Die Proben wurden auch für eine enzymatische Aktivität Assay (Abbildung 3). <img src="/files/ftp_upload/2795/2795fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Maus Augendiagramm. Querschnitts-Schema des humanen und Maus-Auge zeigt die relative kleinere Glaskörper Volumen in der Maus Auge aufgrund seiner größeren Objektiv. <img src="/files/ftp_upload/2795/2795fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Eindimensionale SDS-PAGE von Maus Glaskörper und Netzhaut. Protein-Profile von Maus Glaskörper, 13,6 mg / mL (Bahn 1), und der Netzhaut, 11,35 mg / mL (Spur 2). Gel-Elektrophorese wurde bei 150 kV für 45 Minuten durchgeführt, gefärbt mit Flamingo fluoreszierende Gel färben und visualisiert mit Hilfe eines VersaDoc Imaging Systems (BioRad, Hercules, CA). <img src="/files/ftp_upload/2795/2795fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Superoxiddismutase (SOD) Enzymaktivität in der Maus und Mensch Glaskörper. Messung von insgesamt SOD-Aktivität (SOD Aktivität kolorimetrischen aasay Kit, # AB65354, Abcam, Inc, Cambridge, MA) wurde am Glaskörper und Netzhaut durch das Ausnehmen von DBA und Albino-Mäusen gesammelt wurden. Dies wurde SOD-Aktivität in menschlichen Glaskörper Kern und menschliche Netzhaut aus post-mortem menschlichen Spender Augen gesammelt, um die relative Aktivität zu bestimmen. Das unverwässerte Glaskörper Kernproben wurden manuell abgesaugt mit einer 23-Gauge-Nadel, 11 und Netzhaut war von blühenden das Auge und Schneiden des Gewebes entfernt von der RPE / Aderhaut-Komplex gesammelt. Obwohl der Test nicht zwischen SOD-Isoformen unterscheiden, wird der Glaskörper SOD-Aktivität wahrscheinlich SOD3 reflektieren, da sie die einzige extrazelluläre Isoform ist 12 Die Netzhaut SOD-Aktivität wird wahrscheinlich alle drei SOD-Isoformen spiegeln:. Intrazellulären SOD1, mitochondriale SOD2 und extrazellulären SOD3. 12 Für hochsensible enzymatischen Assays, einschließlich dieser SOD-Assay wird die Möglichkeit einer Kreuzkontamination von Proteinen möglich und bedarf weiterer Untersuchung. Fehlerbalken stellen SEM.

<ol>
	<li>Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+macromolecules+and+supramolecular+organisation+of+the+vitreous+gel.">Structural macromolecules and supramolecular organisation of the vitreous gel.</a> Prog. Retin. Eye Res. 19, 323-344 (2000).</li><li>Gao, B. B., Chen, X., Timothy, N., Aiello, L. P. &. a. m. p. ;. a. m. p., Feener, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+vitreous+proteome+in+diabetes+without+diabetic+retinopathy+and+diabetes+with+proliferative+diabetic+retinopathy.">Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy.</a> J. Proteome Res. 7, 2516-2525 (2008).</li><li>Izuta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+SOD+and+VEGF+are+increased+in+vitreous+bodies+from+proliferative+diabetic+retinopathy+patients.">Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients.</a> Mol. Vis. 15, 2663-2672 (2009).</li><li>Sebag, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pharmacologic+vitreolysis.">Molecular biology of pharmacologic vitreolysis.</a> Trans. Am. Ophthalmol. Soc. 103, 473-494 (2005).</li><li>Smith, R. S., Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Systematic evaluation of the mouse eye: Anatomy, pathology, and biomethods. , 161-195 (2002).</li><li>Ihanamaki, T., Metsaranta, M., Rintala, M., Vuorio, E., Sandberg-Lall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+abnormalities+in+transgenic+mice+harboring+mutations+in+the+type+II+collagen+gene.">Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene.</a> Eur. J. Ophthalmol. 6, 427-435 (1996).</li><li>Kaarniranta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+Stickler's+syndrome:+ocular+phenotype+of+mice+carrying+a+targeted+heterozygous+inactivation+of+type+II+(pro)collagen+gene+(Col2a1.">A mouse model for Stickler's syndrome: ocular phenotype of mice carrying a targeted heterozygous inactivation of type II (pro)collagen gene (Col2a1.</a> Exp. Eye Res. 83, 297-303 (2006).</li><li>Marneros, A. G. &. a. m. p. ;. a. m. p., Olsen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+iris+abnormalities+in+collagen+XVIII/endostatin+deficient+mice+with+similarities+to+human+pigment+dispersion+syndrome.">Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome.</a> Invest. Ophthalmol. Vis. Sci. 44, 2367-2372 (2003).</li><li>Martin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+persistent+hyperplastic+primary+vitreous+in+mice+lacking+the+arf+tumor+suppressor+gene.">Pathogenesis of persistent hyperplastic primary vitreous in mice lacking the arf tumor suppressor gene.</a> Invest. Ophthalmol. Vis. Sci. 45, 3387-3396 (2004).</li><li>Song, B. J., Tsang, S. H., Lin, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene. Ther. Mol. Biol. 11B, 229-262 (2007).</li><li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Human+Vitreous+Body+Elements+for+Proteomic+Analysis.">Dissection of Human Vitreous Body Elements for Proteomic Analysis.</a> J Vis Exp. , (2011).</li><li>Ciechanowski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+synthesis+is+not+the+reason+for+decreased+activity+of+extracellular+superoxide+dismutase+in+patients+with+diabetes.">Impaired synthesis is not the reason for decreased activity of extracellular superoxide dismutase in patients with diabetes.</a> Arch Med Res. 36, 148-153 (2005).</li></ol>

Tierversuche wurden in Übereinstimmung mit der Association for Research and Vision in Ophthalmology Anweisung für den Einsatz von Tieren in Ophthalmic und Vision Research durchgeführt.

Transgene Mäuse sind ein wichtiges Modell für die Untersuchung der Netzhaut und Glaskörper Krankheit. 6-10 Die Maus Glaskörper, umfasst jedoch einen deutlich kleineren Anteil des Auges im Vergleich zu den menschlichen Glaskörper aufgrund der großen Linse in der Maus Auge. 5 Diese macht es schwierig zu isolieren und zu reinigen der Maus Glaskörper. Das Verständnis der Protein-Veränderungen mit den Glaskörper bei Krankheiten wie proliferative diabetische Retinopathie, altersbedingte Makula-Degeneration, Uveitis und Netzhautablösung assoziiert wird Einblick in die Mechanismen geben, durch die sie Fortschritte. Diese visualisiert experimentelle Protokoll bietet die Möglichkeit zu erhalten und zu reinigen der Maus Glaskörper, bei gleichzeitiger Maximierung der Ausbeute an Protein für die enzymatische und Proteom-Analysen.

<table border="1"><tbody><tr><th> Name </th><th> Firma </th><th> Catalog Number </th><th> Kommentare </th></tr><tr><td> 15 °, BD Beaver Mikrochirurgische Klinge </td><td> Becton-Dickinson </td><td> 374881 </td><td></td></tr><tr><td> PBS, pH 7,4 </td><td> Invitrogen </td><td> 70011-044 </td><td></td></tr><tr><td> 0,22 Fine-Castroviejo Naht Zange </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0,12 Colibri Zange </td><td> Storz Ophthalmics </td><td> 2 / 132 </td><td></td></tr><tr><td> Microcon Zentrifugalfilter Ultracel YM-100 oder YM-50 </td><td> Millipore </td><td> 42412, 42415 </td><td></td></tr><tr><td> SOD Aktivität kolorimetrischen Assay Kit </td><td> Abcam </td><td> Ab65354 </td><td></td></tr><tr><td> Weck-Cel chirurgischen Speere </td><td> Medtronic </td><td> 0008680 </td><td></td></tr><tr><td> Protease-Inhibitor-Cocktail </td><td> Roche </td><td> 11 836 170 001 </td><td></td></tr><tr><td> Flamingo fluoreszierende Gel Fleck </td><td> Bio-Rad </td><td> 161-04910 </td><td></td></tr></tbody></table>

evisceration of mouse vitreous and retina for proteomic analyses

Während sich die Maus Netzhaut als ein wichtiges genetisches Modell für erbliche Netzhauterkrankung entstanden ist, bleibt die Maus Glaskörper, erkundet zu werden. Der Glaskörper ist eine sehr wässrige extrazellulären Matrix, die über der Netzhaut, wo intraokularen sowie extraokulären Proteine ​​akkumulieren während der Erkrankung. 1-3 Abnormal Interaktionen zwischen Glaskörper und Netzhaut zu Grunde liegen mehrere Krankheiten wie Netzhautablösung, proliferative diabetische Retinopathie, Uveitis und proliferative Vitreoretinopathie. 1 , 4 Die relative Maus Glaskörper Volumen ist deutlich kleiner als die menschliche Glaskörper (Abbildung 1), da die Maus Linse nimmt fast 75% seines Auges. 5 Diese hat biochemische Untersuchungen der Maus Glaskörper gemacht herausfordernd. In diesem Video-Artikel präsentieren wir eine Technik zu sezieren und zu isolieren, die Maus Glaskörpers von der Netzhaut, die ermöglichen die Verwendung von transgenen Mausmodellen, klarer zu definieren, welche Rolle dieser extrazellulären Matrix bei der Entwicklung von vitreoretinale Krankheiten werden.

Watch this Scientific Journal Video about Evisceration of Mouse Vitreous and Retina for Proteomic Analyses at JoVE.com

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

Die Dissektionstechnik zeigt Ausnehmen des Glaskörpers, der Netzhaut und Linse aus dem Auge der Maus, die Trennung durch Zentrifugation und Charakterisierung mit Protein-Assays.

Ausweiden der Maus Glaskörper und Netzhaut für Proteomik-Analysen

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

evisceration-of-mouse-vitreous-and-retina-for-proteomic-analyses

Research

JoVE Journal

Biology

28.9K Aufrufe.  University of Iowa. Die Dissektionstechnik zeigt Ausnehmen des Glaskörpers, der Netzhaut und Linse aus dem Auge der Maus, die Trennung durch Zentrifugation und Charakterisierung mit Protein-Assays.

Serie: Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

Dissection of Human Glaskörper Elemente für Proteomanalyse

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Forschung

Medizin

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

Maus Eye Enukleation für Remote High-throughput Phänotypisierung

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin Digest Protokoll zum Analysieren des retinalen Gefäßsystem eines Maus-Modell

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Neurowissenschaften

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Isolierung von retinen retinalen Endothelzellen für die Sequenzierung der nächsten Generation

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Entwicklungsbiologie

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Vorbereitung von Horizontal Scheiben der erwachsenen Maus Retina für elektrophysiologische Untersuchungen

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemie

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Zwei Peeling-Methoden zur Isolierung von Photorezeptor-Zellkompartimenten in der Netzhaut der Maus zur Proteinanalyse

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

Retinale pathophysiologische Bewertung in einem Rattenmodell

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

<meta charset="utf8"></head><body>Visualisierung von synaptischen Strukturen der Netzhaut in 3D mit Volumenelektronenmikroskopie

Preparation of Retinal Samples for Volume Electron Microscopy

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Retinale neurowissenschaftliche Studien mit Volumenelektronenmikroskopie

This protocol describes the detailed steps for preparing retinal samples for volume electron microscopy, focusing on the structural features of retinal photoreceptor terminals.