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.
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#215; PBS</td>
			<td>Servicebio</td>
			<td>G4202</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa594-conjugated Goat Anti-Rabbit IgG&#160; (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&#160;</td>
		</tr>
		<tr>
			<td>Cloud disk</td>
			<td>Baidu link</td>
			<td>Extraction code: g44k</td>
			<td>https://pan.baidu.com/s/1lccg1OVbeudsp2VtnqxWZg&#160;</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.

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

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

Our research scope is to use the mouse as an animal model for studies of glaucoma. To precise assess the disease condition of glaucoma in the mouse retina, we need to determine how many retinal ganglion cells are in the retina. There around the 50, 000 retinal ganglion cells in an entire mouse retina.If we count them manually, it may take up to eight to 10 hours. Automatic counting methods can get it done in minutes and save people lots of time and effort. In the future, our lab will focus on glaucoma treatment study.We are going to use different mouse models for glaucoma to test a variety of treatment methods. The method described in this protocol is particularly useful for treatment evaluation.

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...

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.

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

384 Görüntüleme.  University of Electronic Science and Technology of China. Araştırma kapsamımız, fareyi glokom çalışmaları için bir hayvan modeli olarak kullanmaktır. Fare retinasındaki glokomun hastalık durumunu kesin olarak değerlendirmek için, retinada kaç tane retinal ganglion hücresi olduğunu belirlememiz gerekir. Bütün bir fare retinasında yaklaşık 50.000 retinal ganglion hücresi var.Bunları manuel olarak sayarsak, sekiz ila 10 saat kadar sürebilir. Otomatik sayma yöntemleri bunu dakikalar içi...