Name: fluorescent dye labeling of erythrocytes and leukocytes for studying the flow dynamics in mouse retinal circulation
Uploaded: 2017-07-03T12:30:00
Description: Watch this Scientific Journal Video about Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation at JoVE.com

The research project was funded under New Investigator grant from the National Medical Research Council (NMRC), Singapore. The team will like to acknowledge the research training provided to Dr Agrawal at Institute of Ophthalmology (IoO), University College London (UCL) under National Medical Research Council (NMRC) Overseas Research Training Fellowship from November 2012 till October 2014 under mentorship of Prof. David Shima. Dr. Agrawal acquired the concept and skills for labelling the cells and live imaging in Dr. Shima&#39;s lab. The team will hence like to acknowledge supervision and guidance during the training fellowship from Prof. David Shima, Pro.f Kenith Meissner, Dr. Peter Lundh and Dr. Daiju Iwata.

The animal protocols used in this study were approved by the Institutional Animal Care and Use Committee of SingHealth, Singapore and are in accordance to the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Labeling of Erythrocytes and Leukocytes with Fluorescent Dyes
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	<li>Preparation of reagents
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		<li>Prepare ICG (1.5 mg/mL) by dissolving 3 mg of ICG in 1800 &#181;L of sterilized dist...

Studying the hemodynamics in the retinal and choroidal circulation is vital for understanding the pathophysiology of many ocular diseases. The blood flow dynamics in the retinal circulation can be studied by Fourier-domain optical coherence tomography (FD-OCT), laser speckle flowgraphy (LSFG) and retinal oximetry. Although these methods use different approaches to study the total blood flow in the retinal circulation40,41,42<sup...

Studying the flow dynamics of the blood cells in the retinal and choroidal circulation is imperative to understanding the pathogenesis of potentially vision-threatening ocular diseases and other ocular inflammatory conditions. However, the conventional angiography techniques, which involve the binding of fluorescent dyes to plasma proteins, do not provide any information regarding the dynamics of the erythrocytes or leukocytes1. The erythrocyte retinal flow dynamics are important for studying metabolically efficient circulation in the retina, and the leukocyte flow dynamics, for understanding the cell migration, recognition, adhesion and destruction in various inflammatory conditions2. There are several fluorescent molecules used in the identification and characterization of various cell types3. The hemodynamics of the blood cells can be measured by staining them with the appropriate fluorescent dyes and applying the proper imaging techniques4.
The presence of inflammatory responses in intraocular diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) involve the accumulation of lymphocytes in the diseased area5,6. Tracking the immune cells in the tissues can help understand the complex events involved in the mechanism of disease pathogenesis. Radioactive isotopes like 51Cr and 125I were used as cell tracers in early studies. These dyes are toxic and affect the cell viability. Although the radioactive markers 3H and 14C are less toxic to the cells, due to their lower emission energies, it is difficult to detect their signals in the system7,8. A number of fluorochrome dyes were introduced to overcome the potential problems associated with radioactive markers and track lymphocyte migration in vitro using fluorescent microscopy and flow cytometry9,10. Hoechst 33342 and thiazole orange are DNA binding fluorescent dyes, which are used to track lymphocytes in vivo. Hoechst 33342 binds to AT-rich regions in the DNA, is membrane permeable, retains fluorescent signals for 2 - 4 days and is resistant to quenching9,10. The disadvantages of Hoechst 33342 and thiazole orange are the inhibition of lymphocyte proliferation11 and the short half-life, respectively9.
Calcein-AM, fluorescein diacetate (FDA), 2&#8242;,7&#8242;-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM), 5-(and-6)-carboxyfluorescein diacetate (CFDA), and 5-(and-6)-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) are the cytoplasmic fluorescent dyes used for lymphocyte migration studies. However, FDA, CFDA and CFDA-AM have lower retention in the cells9. BCECF-AM reduces the proliferative response and influences the chemotaxis and superoxide production9,12. Calcein-AM is a fluorescent dye and useful for short-term in vivo lymphocyte migration studies. It emits strong fluorescent signals, does not interfere with most of the cellular functions and retains fluorescent signals for up to 3 days12,13. Fluorescein isothiocyanate (FITC) and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) are covalent coupling fluorescent dyes, which are used for lymphocyte migration studies. FITC exhibits no effect on the cell viability and has a stronger affinity with B lymphocytes than T lymphocytes14,15. The CFDA-SE labeled lymphocytes can be tracked in vivo for more than 8 weeks and up to 8 cell divisions9,16. C18 DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate), DiO (3,3&#39;-dioctadecyloxacarbocyanine perchlorate), Paul Karl Horan (PKH)2, PKH3, and PKH26 are membrane-inserting fluorescent lipophilic carbocyanine dyes used to label leukocytes and erythrocytes. C18 Dil and DiO exhibit higher signals when incorporated into the cell membrane and are relatively non-toxic12,17. PKH2, PKH3 and PKH26 labeled cells exhibit a good retention of the fluorescent signals with less toxicity18,19,20,21,22. However, PKH2 down regulates the CD62L expression and reduces the lymphocyte viability23.
Most of the above-mentioned studies have been performed for tracking the lymphocyte migration and proliferation in the lymphatics and studying the labeled erythrocytes in the non-ocular circulation. There are very few studies applying the labeling techniques to study the blood cells in the ocular circulation. Application of scanning laser ophthalmoscopy (SLO) has a great advantage in studying the labeled cells in the retinal and choroidal circulation in vivo by fundus angiography24. There are several fluorescent dyes, such as ICG, acridine orange, FITC, sodium fluorescein, and CFDA that are used to study the leukocytes in the retinal circulation by SLO25,26,27,28,29,30,31,32,33,34. The phototoxicity and carcinogenicity of acridine orange26,27, the interference of FITC with the cellular activity, and the requirement of an intravascular contrast agent for the resolution of the retinal and choroidal blood vessels limits their application in in vivo animal experiments29. Sodium fluorescein and ICG are non-toxic, approved by the Food and Drug Administration, and safe for testing on humans32,35. Most of the flow dynamic studies are related to the labeling of the leukocytes or erythrocytes and its visualization in the retinal and choroidal blood vessels36,37,38,39. Here, we describe a standardized protocol of ICG labeling of the erythrocytes, sodium fluorescein labeling of the leukocytes, and tracking the visualized labeled cells in the mouse retinal circulation using SLO.

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			<td height="42" style="height:42px;width:287px;">10X Phosphate-buffered saline (PBS) Ultra Pure Grade</td>
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			<td height="21" style="height:21px;width:287px;">Bovine serum albumin</td>
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			<td height="42" style="height:42px;width:287px;">Microtainer tubes with K2E (K2EDTA) - EDTA concentration - 1.8 mg/mL of blood</td>
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			<td style="width:179px;">Eppendorf</td>
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			<td style="width:179px;">Insta BioAnalytik pte. ltd</td>
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			<td height="42" style="height:42px;width:287px;">ILIUM XYLAZIL-20 (Xylazine hydrochloride 20mg/Ml)</td>
			<td style="width:179px;">Troy Laboratories PTY. Limited</td>
			<td style="width:119px;">LI0605</td>
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			<td height="42" style="height:42px;width:287px;">1% Mydriacyl 15 mL (Tropicamide 1%)</td>
			<td style="width:179px;">Alcon&#160;Laboratories, Inc. USA</td>
			<td style="width:119px;">NDC&#160;0998-0355-15</td>
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			<td height="42" style="height:42px;width:287px;">2.5% Mydfrin 5 mL (Phenylephrine hydrochloride 2.5%)</td>
			<td style="width:179px;">Alcon&#160;Laboratories, Inc. USA</td>
			<td style="width:119px;">NDC&#160;0998-0342-05</td>
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			<td height="42" style="height:42px;">Terumo syringe with needle 1cc/mL Tuberculin</td>
			<td style="width:179px;">Terumo (Philippenes) Corporation, Philippines</td>
			<td style="width:119px;">SS-01T2613</td>
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			<td height="63" style="height:63px;width:287px;">Vidisic Gel 10G</td>
			<td style="width:179px;">Dr. Gerhard Mann, Chem.-Pharm, Fabrik Gmbh, Berlin, Germany</td>
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			<td height="42" style="height:42px;width:287px;">Alcohol swabs</td>
			<td style="width:179px;">Assure medical disposables</td>
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			<td height="63" style="height:63px;width:287px;">Confocal laser scanning angiography system (Heidelberg Retina Angiograph 2)</td>
			<td style="width:179px;">Heidelberg Engineering, GmbH, Heidelberg, Germany</td>
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			<td height="63" style="height:63px;width:287px;">Hiedelberg Spectralis Viewing Module software, v4.0</td>
			<td style="width:179px;">Heidelberg Engineering, GmbH, Heidelberg, Germany</td>
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			<td height="42" style="height:42px;">Fluorescent microscope</td>
			<td style="width:179px;">ZEISS</td>
			<td style="width:119px;">Model: axio imager z1</td>
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fluorescent dye labeling of erythrocytes and leukocytes for studying the flow dynamics in mouse retinal circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Erythrocytes labeled with 1.5% ICG were visualized in the retinal circulation of C57BL/6J mice (wild type). Both 1% and 5% hematocrit of 1.5% ICG labeled erythrocytes were distinguishable in the retinal circulation. However, the individual labeled cells were more clearly visualized with 1% hematocrit of 1.5% ICG labeled erythrocytes (Figure 1). In 5% hematocrit, due to the large number of labeled cells in the retinal vessels, it was not possible to mark the i...

Watch this Scientific Journal Video about Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation at JoVE.com

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

The overall goal of this procedure is to visualize retinal circulation of blood cells in the mouse using a scanning laser ophthalmoscope. This method can help answer key questions about retinal diseases such as what is the retinal floor dynamics of WBCs and RBCs and how can their flow dynamics contribute to disease pathogenesis. The main advantages of this technique is that it can refelct on different disease phenotypes and it is also very easy to perform.This procedure will be demonstrated by our team members, Praveen, Bo Bo, and Neha. To isolate the mouse blood cells layer freshly collected whole blood on top of a 1:1 polysucrose and sodium diatrizoatem solution in a two milliliter tube. Then centrifuge the tube for 30 minutes.Next aspirate and discard the plasma supernatant. Then carefully collect the buffy coat layer composed of leukocytes and the underlying layer of erythrocytes in separate tubes. Next wash the erythrocytes with 1XPBS to remove any contaminants and resuspend them in one volume of PBS.Then transfer half milliliter aliquots of the suspension into microcentrifuge tubes. Spin down the aliquots, discard the supernatant and add about 200 microliters of 40%1XPBS to the pelleted erythrocytes. Mix them well.And incubate them at room temperature for five minutes. After five minutes, add 100 microliters of ICG to each suspension. Mix the tubes using inversions and then incubate them at room temperature for five minutes.Next add 300 microliters of 1XPBS and incubate for an hour with gentle agitation at 37 degree Celsius. Then centrifuge the suspensions, keep the pelleted cells and resuspend them in one milliliter of 1XPBS. Keep washing the cells with 1XPBS until the supernatant is clear.After decanting the supernatant add back one volume of 1%BSA and PBS to achieve 50%hematocrit of ICG labeled erythrocytes. Next process the buffy coat collection. First wash the cells once with 10 milliliters of 1XPBS to remove any contaminants.Centrifuge the samples again and then resuspend the pellet in 900 microliters of 1XPBS. Next add 100 microliters of 10%sodium fluorescein and incubate the cells at room temperature for two minutes. Finally wash the labeled lymphocytes three times with 10 milliliters of 1XPBS.Centrifuge and resuspend them in 100 microliters of 1XPBS. To prepare the ICG labeled erythrocytes for injection first dilute an aliquot for injection down to a 5%or 1%hematocrit. Anesthetize the mouse and dilate its pupils.Then apply ophthalmic ointment to both eyes. And place a contact lens over one of the eyes. Now image the mouse using a confocal SLO camera with a positive 25 diopter lens to compensate for the eye's refraction.Fresh perfectly direct the cornea to the optical head of the SLO machine. Next switch on the imaging module and to maneuver it until the optic nerve is at the center of the imaging screen. Then at a 30 degree angle take the infrared fundus images.Next take an infrared fundus video. Turn on the 790 nanometer ICG filter set the camera mode to high speed addition keep the view angle at 15 to 30 degrees. Then set the ICG intensity to 85 for all readings.On the control panel, click on the acquire button to take a baseline video at 8.8 to 15 frames per second for one minute. Next load 100 microliters of ICG labeled erythrocytes into an insulin syringe with a 30 gauge needle and inject the cells into the tail vein or inject them retroorbitally. Immediately after the injection, take a new infrared fundus image and a new ICG filter video.After acquiring the videos export them in TIFF format for image analysis. Process the images using the freely available imaging software package, MHJ. After opening the file, set the frame interval of the image video for velocity measurements.Then open the MtrackJ plugin. From the menu, select tracking and toggle the option for moving to next timeframe index after adding a point. Now locate a cell of interest and track through each frame.From the menu, click add to add a new track and then click on the chosen cell. Then continue clicking on the current position of the cell as images advance from frame to frame. Once tracked change the display option for the trajectory from the displaying choices and choose to display only the track points at the current time.Continue clicking until the trajectory of the cell is displayed and then save the track. Now proceed with tracking the next cell starting with the add command and proceeding as with the first cell. After tracking all the cells of interest open the display of all the track measurements using the measure option.Then save the output for further analysis. Erythrocytes labeled with 1.5%ICG were visualized in the retinal circulation of C57 black 6J mice using 1%or 5%hematocrit, cells were distinguishable but in the 1%hematocrit the labeled cells were more easily visualized. With either injected concentration some erythrostasis was observable in the parapapillary region and it was more visible with the 5%hematocrit.Leukocytes were labeled with 1%sodium fluorescein and visualized as green labeled cells using fluorescence microscopy. Some clumping was observed. Once mastered, this technique can be done in six hours if performed properly.While attempting this procedure, it is important to dilate the eyes to acquire quality retinal images and videos.

fluorescent-dye-labeling-erythrocytes-leukocytes-for-studying-flow

Research

JoVE Journal

Medicine

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such as meningitis. A method is presented here using intrathecal administered fluorescein in a clinical case of bilateral congenital ear malformation. A fluorescent dye is administered intrathecally to achieve intraoperative visualization of CSF leaks. The dye is applied 20 min before surgery, and concentration of 5% is used. Per every 10 kg of body weight, 0.1 mL of the fluid is applied intrathecally. The fluorescein is visualized using a fully digital microscope. The origin of the fluid leak is identified in the stapes footplate. During primary surgery, it is sealed, and cochlea implantation is performed for hearing restoration. In this specific case, 6 weeks later, the implant was explanted due to acute meningitis, and the electrode array was left as a spacer. Postoperatively, in the aural smear, &#946;-transferrin was detected. During a revision mastoidectomy, dislocated coverage of the leak was found. The stapes was removed and oval window sealed. Five days after revision surgery, no &#946;-transferrin was detected in the aural smear. During the revision of cochlea implantation 6 months later, intact coverage of the oval niche was observed. Thus, intrathecal fluorescein application proves to be a reliable tool for the detection of CSF leaks. It facilitates the orientation in malformations and complicated or unknown surgical situs. In the literature, its use is described for CSF fistulas in endonasal surgery but is rarely described in skull base and mastoid surgeries. The method has been used successfully in several cases with CSF leaks, and the results confirm the feasibility of safely accessing the origin of the leak.

Intrathecally applied fluorescein is used to achieve intraoperative visualization of CSF leaks. This protocol describes a lumbar puncture, the application of 5% fluorescein, and intraoperative visualization using a fully digital microscope.

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such ...

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.

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

Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies

Self-assembly is a simple yet reliable method for constructing nanoscale drug delivery systems. Photoactivatable prodrugs enable controllable drug release from nanocarriers at target sites modulated by light irradiation. In this protocol, a facile method for fabricating photoactivatable prodrug-dye nanoparticles via molecular self-assembly is presented. The procedures for prodrug synthesis, nanoparticle fabrication, physical characterization of the nanoassembly, photocleavage demonstration, and in vitro cytotoxicity verification are described in detail. A photocleavable boron-dipyrromethene-chlorambucil (BC) prodrug was first synthesized. BC and a near-infrared dye, IR-783, at an optimized ratio, could self-assemble into nanoparticles (IR783/BC NPs). The synthesized nanoparticles had an average size of 87.22 nm and a surface charge of -29.8 mV. The nanoparticles disassembled upon light irradiation, which could be observed by transmission electronic microscopy. The photocleavage of BC was completed within 10 min, with a 22% recovery efficiency for chlorambucil. The nanoparticles displayed enhanced cytotoxicity under light irradiation at 530 nm compared with the non-irradiated nanoparticles and irradiated free BC prodrug. This protocol provides a reference&#160;for the construction and evaluation of photoresponsive drug delivery systems.

This protocol describes the fabrication and characterization of a photoresponsive prodrug-dye nanoassembly. The methodology for drug release from the nanoparticles by light-triggered disassembly, including the light irradiation setup, is explicitly described. The drugs released from the nanoparticles following light irradiation exhibited excellent anti-proliferation effects on human colorectal tumor cells.

Self-assembly is a simple yet reliable method for constructing nanoscale drug delivery systems. Photoactivatable prodrugs enable controllable drug release ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...