Name: whole-mount immunostaining and automatic counting of mouse retinal ganglion cells
Uploaded: 2024-10-04T14:00:00.000+00:00
Duration: 5 min 52 s
Description: Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

This research project was supported by the National Natural Science Foundation of China (82371059 (H.Z.)), the Department of Science and Technology of Sichuan Province, China (2023JDZH0002 (H.Z.)), the Chengdu Science and Technology Bureau (2022-YF05-01984-SN (H.Z.)), and Sichuan Provincial People&#39;s Hospital (30320230095 (J.Y.), 30420220062 (J.Y.)).&#160;

The procedure complied with the guidelines of the Association for Research in Vision and Ophthalmology for using animals in research and was approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan Provincial People&#39;s Hospital. C57Bl/6J male mice (2 months old) were used in this study. Figure 1 illustrates the overall procedure described here. The details of the reagents and equipment used are listed in the Table of Materials.
1. Isolation of whole-mount retinas
<ol>
	<li>Animal sacrifice
	<ol>
		<li>Sacrifice the mice using cervical dislocation (following institutionally approved protocols) (Figure 2A). Prior to cervical dislocation, 
		anesthetize the mice by inhaling isoflurane (3%-5% in oxygen) or by intraperitoneal injection of&#160;the ketamine/xylazine mixture.</li>
	</ol>
	</li>
	<li>Enucleation and eyeball processing
	<ol>
		<li>Mark the edge of the cornea on the dorsal side of the eyeball with a blue marker to remember the orientation during subsequent dissection.</li>
		<li>Remove residual muscle tissues attached to the eyeball with scissors. Transfer the enucleated eyeball to a 2 mL round-bottom microcentrifuge tube filled with 1x PBS for a brief rinse (Figure 2C).</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Transfer the eyeball to another 2 mL round-bottom microcentrifuge tube containing freshly made 4% paraformaldehyde (PFA) for fixation at 4 &#176;C for 10 min (Figure 2D).</li>
		<li>Make a small incision (approximately 1-2 mm) on the cornea using fine scissors under a dissecting microscope.</li>
		<li>Place the eyeball back into the fixative for further fixation at 4 &#176;C for 3 h to preserve tissue morphology (Figure 2D).</li>
	</ol>
	</li>
	<li>Removal of cornea and lens1,9
	<ol>
		<li>Make a small incision of approximately 1 mm around the corneal limbus, the border between the cornea and the sclera, using fine surgical scissors.</li>
		<li>Carefully cut around the circumference of the cornea, excising it entirely to expose the underlying structures.</li>
		<li>Use fine forceps to gently grasp and carefully lift the lens from the surrounding tissues to prevent damage to the retina. Maintain the structural integrity of the retina with delicate operation.</li>
		<li>Transfer the eyecup to a microcentrifuge tube for further processing. 
		NOTE: Excise the cornea and extract the lens more efficiently using the &#34;cross-slit&#34; technique10. Make two perpendicular slits across the cornea and use forceps to extract the lens.</li>
	</ol>
	</li>
	<li>Scleral incision
	<ol>
		<li>Cut a precise slit in the sclera with fine ophthalmic scissors to facilitate subsequent dissection steps (Figure 2J).</li>
	</ol>
	</li>
	<li>Retina separation
	<ol>
		<li>Insert forceps with teeth into the incision between the sclera and the retina. Gently grasp the edge of the sclera, avoiding excessive force to prevent tearing the tissue.</li>
		<li>Gradually enlarge the incision by moving the forceps along the boundary between the sclera and the retina with slow, steady hand movements. Use the toothed part of the forceps to carefully separate the sclera from the retina, ensuring the retina is not damaged (Figure 2J).</li>
	</ol>
	</li>
	<li>Retina cutting
	<ol>
		<li>Divide the retina into four roughly equal-sized flaps using sharp scissors. Ensure cuts are approximately halfway towards the optic nerve and oriented at 90&#176; from each other.</li>
	</ol>
	</li>
	<li>Retina cleaning
	<ol>
		<li>Gently unfold the dissected retina using a soft-bristled brush, and meticulously remove debris from its surface (Figure 2K).</li>
	</ol>
	</li>
	<li>Final fixation
	<ol>
		<li>Transfer the isolated retina to a microcentrifuge tube containing fresh 4% PFA using a disposable Pasteur pipette. Allow fixation on ice for an additional 5 h to overnight for tissue preservation.</li>
	</ol>
	</li>
</ol>
2. Immunostaining
<ol>
	<li>Washing
	<ol>
		<li>Wash the isolated whole-mount retina in 1x PBS with gentle agitation for 5 min. Discard the PBS solution. Repeat this process twice to remove residual fixative and debris.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Immerse the washed retina in a blocking buffer solution (1x PBS with 4% normal donkey serum (NDS) and 0.2% Triton X-100) for 30 min to prevent nonspecific antibody binding and enhance permeabilization (Figure 3A).</li>
	</ol>
	</li>
	<li>Primary antibody incubation
	<ol>
		<li>Replace the blocking buffer with primary antibody BRN3A (diluted 1:200 in the blocking buffer). Incubate the retina overnight at 4 &#176;C to allow specific antigen-antibody binding (Figure 3B).</li>
	</ol>
	</li>
	<li>Washing
	<ol>
		<li>Wash the retina three times with 1x PBS for 5 min each to remove unbound primary antibodies and reduce background staining (Figure 3C).</li>
	</ol>
	</li>
	<li>Secondary antibody incubation
	<ol>
		<li>Incubate the retina with Alexa594- or Alexa488-conjugated anti-rabbit secondary antibody (diluted 1:300) for 2 h at room temperature to visualize target antigens (Figure 3D).</li>
	</ol>
	</li>
	<li>Washing
	<ol>
		<li>Perform three consecutive washes with 1x PBS for 5 min each to remove excess secondary antibodies and minimize nonspecific binding.</li>
	</ol>
	</li>
	<li>Mounting
	<ol>
		<li>Gently transfer the immunostained retina onto a glass microscope slide, carefully flattening it to minimize folding or distortion.</li>
		<li>Apply a small drop of anti-fade mounting medium to the center of the retina, ensuring even distribution across the tissue.</li>
		<li>Carefully position a coverslip over the mounted retina, avoiding air bubbles (Figure 3E,F).</li>
	</ol>
	</li>
</ol>
3. Image processing
NOTE: Import the image into the RGC automatic counting software and start counting. The number of BRN3A-positive cells for the entire retina can be obtained in minutes. Download the AutoCount software from GitHub (https://github.com/MOEMIL/Intelligent-quantifying-RGCs). Follow the detailed installation steps for the software below:
<ol>
	<li>Download the code from the GitHub link. One will receive a file named &#34;Intelligent-quantifying-RGCs.zip&#34;. Unzip this file.</li>
	<li>Download the necessary files from the cloud disk (see Table of Materials) and unzip them. The downloaded file is named &#34;yolov5_cpu&#34;. Unzip this package to the current folder to avoid unexpected issues.</li>
	<li>Locate the Config&#160;file from step 1 and modify its content to point to the &#34;yolov5_cpu&#34; path. 
	NOTE: For example, if the downloaded &#34;yolov5_cpu&#34; is placed in &#34;D:\1me\yolov5_cpu_&#34;, change the &#34;config.ini&#34; file in step 1 to &#34;D:/1me/yolov5_cpu&#34; (use / instead of \). (Figure 4A).</li>
	<li>Open the graphical interface by clicking on userinterface.exe. The initial loading may be slow (run as administrator) (Figure 4B).</li>
	<li>After opening the software, verify whether the &#34;pmse_plus.pt&#34; model file is present in the root directory of drive C. If it is not present, copy it to this directory. 
	NOTE: The &#34;pmse_plus.pt&#34; model file is located in the weights folder of the &#34;yolov5&#34; folder, found in the extracted Intelligent-quantifying RGCs folder (Figure 4C).</li>
	<li>Prepare the images to be detected. 
	NOTE: The software allows only one or five pictures to be read at a time. Reading more will result in an error.</li>
</ol>
4. Automated cell counting
<ol>
	<li>Open the counting software (Figure 5A).</li>
	<li>Click on OPEN IMAGE to import the image (Figure 5B).</li>
	<li>Click on RUN to allow the software to automatically count the cells (Figure 5C). 
	NOTE: The software will mark the counted cells with a red square box and display the cell count in the top right corner (Figure 5D).</li>
</ol>

Immunostaining and AI-Based Counting of RGCs in Mouse Whole-Mount Retina

<ol>
	<li>Quigley, H. A., Broman, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+people+with+glaucoma+worldwide+in+2010+and+2020.">The number of people with glaucoma worldwide in 2010 and 2020.</a> Br J Ophthalmol. 90 (3), 262-267 (2006).</li><li>Gallo Afflitto, G., Swaminathan, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial-ethnic+disparities+in+concurrent+rates+of+peripapillary+&amp;+macular+OCT+parameters+among+a+large+glaucomatous+clinical+population.">Racial-ethnic disparities in concurrent rates of peripapillary &amp; macular OCT parameters among a large glaucomatous clinical population.</a> Eye. , (2024).</li><li>Atkinson, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+measure+of+patient+satisfaction+with+ocular+hypotensive+medications:+the+Treatment+Satisfaction+Survey+for+Intraocular+Pressure+(TSS-IOP).">A new measure of patient satisfaction with ocular hypotensive medications: the Treatment Satisfaction Survey for Intraocular Pressure (TSS-IOP).</a> Health Qual Life Outcomes. 1, 67 (2003).</li><li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mouse-fixation+device+for+IOP+measurement+in+awake+mice.">A new mouse-fixation device for IOP measurement in awake mice.</a> Vision Res. 219, 108397 (2024).</li><li>Almasieh, M., Wilson, A. M., Morquette, B., Cueva Vargas, J. L., Di Polo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+retinal+ganglion+cell+death+in+glaucoma.">The molecular basis of retinal ganglion cell death in glaucoma.</a> Prog Retin Eye Res. 31 (2), 152-181 (2012).</li><li>Jeon, C. J., Strettoi, E., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+cell+populations+of+the+mouse+retina.">The major cell populations of the mouse retina.</a> J Neurosci. 18 (21), 8936-8946 (1998).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+counting+of+retinal+ganglion+cells+in+the+entire+mouse+retina+based+on+improved+YOLOv5.">Automatic counting of retinal ganglion cells in the entire mouse retina based on improved YOLOv5.</a> Zool Res. 43 (5), 738-749 (2022).</li><li>Al-Khindi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+Tbx5+regulates+direction-selective+retinal+ganglion+cell+development+and+image+stabilization.">The transcription factor Tbx5 regulates direction-selective retinal ganglion cell development and image stabilization.</a> Curr Biol. 32 (19), 4286-4298 (2022).</li><li>Claes, M., Moons, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cells:+Global+number,+density+and+vulnerability+to+glaucomatous+injury+in+common+laboratory+mice.">Retinal ganglion cells: Global number, density and vulnerability to glaucomatous injury in common laboratory mice.</a> Cells. 11 (17), 2689 (2022).</li><li>Zhang, N., Wang, Z., Lin, P., Xing, Y., Yang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methanol-based+whole-mount+preparation+for+the+investigation+of+retinal+ganglion+cells.">Methanol-based whole-mount preparation for the investigation of retinal ganglion cells.</a> J Vis Exp. (194), e65222 (2023).</li></ol>

The authors have no conflicts to disclose.

This protocol provides a method for determining all retinal ganglion cells (RGCs) in a mouse retina, which can be used to monitor the progression of RGC degeneration in mouse models for glaucoma studies. The mouse retina is a delicate nerve tissue5, and isolating whole-mount retinas from mouse eyes requires repeated practice. During experimentation, it was found that the fixation time significantly affects retina morphology. For mice around 2 weeks of age, a fixation time of 20 min is sufficient t...

Glaucoma is characterized by&#160;the progressive death of ganglion cells, which&#160;poses a significant challenge for sight&#160;restoration1,2. This disease is a major focus of ophthalmic research due to its prevalence and impact on vision3. Mouse models are indispensable in glaucoma 
research due to&#160;their homogeneous genetic background, high&#160;reproductive capacity, and the similarity of&#160;their ganglion cell properties to humans4. The primary goal of this method is to accurately quantify retinal ganglion cells (RGCs) in mouse models, which is essential for understanding the pathogenesis of glaucoma and developing relevant treatments.
The rationale behind developing this technique stems from the need for a reliable and efficient method to assess RGC degeneration in mouse models. Traditional methods, such as labeling RGCs in retinal sections, often provide unreliable results due to the non-uniform distribution of RGCs in the retina5. Quantifying RGCs across the entire retina better reflects changes in their numbers and is crucial for evaluating disease progression and therapeutic interventions.
This method offers several advantages over alternative techniques. For instance, manual counting of retinal ganglion cells (RGCs) in a normal adult mouse retina, which contains 40,000 to 60,000 RGCs, can be time-consuming and error-prone6,7,8. The software for automatic RGC counting that we developed allows for accurate counting in less than 3 min, potentially saving researchers a significant amount of time. Additionally, the AI-based software used&#160;for automatic counting minimizes bias and&#160;enhances reproducibility.
Furthermore, this technique provides a standardized approach to assess RGC degeneration across different mouse models and experimental conditions, contributing valuable data to the field of glaucoma research. The method aligns with other studies that emphasize the importance of whole-mount retina analysis for understanding retinal changes in diseases9.
To help readers determine whether this method is suitable for their application, it is important to note that this technique is particularly advantageous for researchers studying retinal ganglion cell (RGC) degeneration in mouse models of glaucoma or other retinal diseases. The method is adaptable to various experimental setups and offers a high degree of accuracy and efficiency in RGC counting, making it ideal for both small-scale and large-scale studies. Additionally, the protocol&#39;s straightforward design and the availability of user-friendly software make it accessible to researchers with varying levels of expertise in retinal analysis.

<table><tbody><tr><td>1&amp;times; PBS</td><td>Servicebio</td><td>G4202</td><td></td></tr><tr><td>Alexa594-conjugated Goat Anti-Rabbit IgG&amp;nbsp; (H+L)</td><td>ThermoFisher</td><td>A-11012</td><td></td></tr><tr><td>Anti-BRN3A antibody [EPR23257-285]</td><td>Abcam</td><td>ab245230</td><td></td></tr><tr><td>AutoCount software</td><td></td><td></td><td>https://github.com/MOEMIL/Intelligent-quantifying-RGCs</td></tr><tr><td>Cloud disk</td><td>Google drive link</td><td></td><td>https://drive.google.com/file/d/1yOEsBvil6KEdZFa5ENQxB6&amp;nbsp;</td></tr><tr><td>Cloud disk</td><td>Baidu link</td><td>Extraction code: g44k</td><td>https://pan.baidu.com/s/1lccg1OVbeudsp2VtnqxWZg&amp;nbsp;</td></tr><tr><td>Marker</td><td>Sharpie</td><td></td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Biosharp, Labgic</td><td>25030081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Macklin</td><td>P804536</td><td></td></tr><tr><td>ProClean 300</td><td>Beyotime</td><td>ST853</td><td></td></tr><tr><td>Sucrose</td><td>BBI, Sangon</td><td>A610498</td><td></td></tr><tr><td>Triton X-100</td><td>BioFroxx, neoFroxx</td><td>1139ML100</td><td></td></tr></tbody></table>

whole-mount immunostaining and automatic counting of mouse retinal ganglion cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

This protocol details the methodology for whole-mount immunostaining of mouse retinas, ensuring meticulous tissue preparation, precise antibody incubation, and reliable automated cell counting. The procedure facilitates robust labeling and quantification of retinal ganglion cells (RGCs), enabling accurate assessment of cellular populations in various experimental contexts. The method was used to count RGCs in wild-type and glaucoma-modeled mice, yielding consistent results. A representative result shows that the number o...

Author Spotlight: Efficient Retinal Ganglion Cell Counting in Mouse Models of Glaucoma for Treatment Evaluation

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

whole-mount-immunostaining-automatic-counting-mouse-retinal-ganglion

Research

JoVE Journal

Neuroscience

811 Views.  University of Electronic Science and Technology of China. Ons onderzoek is om de muis te gebruiken als diermodel voor studies van glaucoom. Om de ziektetoestand van glaucoom in het netvlies van muizen nauwkeurig te beoordelen, moeten we bepalen hoeveel retinale ganglioncellen zich in het netvlies bevinden. Er zijn ongeveer 50.000 retinale ganglioncellen in een heel netvlies van muizen.Als we ze handmatig tellen, kan het wel acht tot tien uur duren. Automatische telmethoden kunnen het in enkele minuten voor el...

Video: Auteur in de schijnwerpers: efficiënte telling van retinale ganglioncellen in muismodellen van glaucoom voor evaluatie van de behandeling

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Biology

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Mouse Retina Isolation and Methanol Treatment for Studying Retinal Ganglion Cells

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (&#8722;20 &#176;C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (&#8722;20 &#176;C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

Author Spotlight: Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells  

Methanol can be used as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage, which is useful for the investigation of retinal ganglion cells.

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

Assessing Retinal Thickness and Ganglion Cell Layer Counts in Rodent Eyeballs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...

Medicine

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://app.jove.com/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

JoVE Encyclopedia of Experiments

Neuroimaging

Live Imaging & In Vivo Tracking

Quantification of Retinal Ganglion Cell Somas Following Cauterization-Induced Ocular Hypertension 

<a href='https://app.jove.com/v/63442/full-circle-cauterization-limbal-vascular-plexus-for-surgically'>Source: Lani-Louzada, R., et al.&#160; Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022).</a>This video demonstrates the quantification of retinal ganglion cells from chemically fixed rat retinas subjected to full-circle cauterization of the limbal vascular plexus. The process includes permeabilization, antibody staining, and nuclear counterstaining, followed by visualization under an epifluorescence microscope to assess the impact of elevated intraocular pressure on retinal ganglion cell viability.

Source: Lani-Louzada, R., et al.&#160; Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. ...

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

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