<meta charset="utf8"></head><body>Immunocolorazione e conteggio basato sull'intelligenza artificiale delle RGC nella retina a montaggio intero di topo

<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>

<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 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.

<meta charset="utf8"></head><body>Autore in primo piano: Conteggio efficiente delle cellule gangliari retiniche in modelli murini di glaucoma per la valutazione del trattamento

Immunocolorazione a montaggio intero e conteggio automatico delle cellule gangliari retiniche di topo

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

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

577 Views.  University of Electronic Science and Technology of China. Il nostro scopo di ricerca è quello di utilizzare il topo come modello animale per gli studi sul glaucoma. Per valutare con precisione la condizione patologica del glaucoma nella retina del topo, dobbiamo determinare quante cellule gangliari retiniche ci sono nella retina. Ci sono circa 50.000 cellule gangliari retiniche in un'intera retina di topo.Se li contiamo manualmente, potrebbero essere necessarie dalle otto alle 10 ore. I metodi di conteggio a...

Video: Autore in primo piano: Conteggio efficiente delle cellule gangliari retiniche in modelli murini di glaucoma per la valutazione del trattamento

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 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.