This study is supported by the National Natural Science Foundation of China (82101145) and the Beijing Natural Science Foundation (Z200014).

This study was approved by the Institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. HESCs cell line H9 was from the WiCell Research Institute. Pre-warm the cell culture medium at room temperature (RT) for 30 min before the experiment.
1. Generation of human microglia
<ol>
	<li>Culture the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 106 cells in each well.</li>
	<li>Aspirate the stem cell medium and rinse the cells with 1x DPBS (1 mL/well of 6 well plate). The next step requires 2 x 107-1 x 108 cells. If not enough, combine cells of 2-5 wells to do the following steps.</li>
	<li>Dissociate the stem cell colonies using the 0.5 mM EDTA solution for about 5 min in a 37 &#176;C incubator (1 mL/well of 6 well plate).</li>
	<li>Check colony morphology after 5 min. When the colony edges are slightly curled up with loose gaps, cells are ready to be differentiated and aspirate the EDTA. If not, incubate cells at 37 &#176;C for another 1-2 min. Do not incubate for longer than 8 min.</li>
	<li>Gently rinse the cells in each well using 6 mL of Medium A (Table 1) with 6 &#181;L of 10 mM ROCK inhibitor Y-27632 solution. Gently tap the bottom of the dish to help rinse the cells. Set the day as day 0. 
	NOTE: Do not pipette the cells. Cells will form embryoid bodies (EBs) the next day.</li>
	<li>After 24 h, observe the cells under the microscope to confirm the EB formation. Collect the EBs into a 15 mL centrifugal tube with a 10 mL pipet and wait for the EBs to settle to the bottom in 5 min.</li>
	<li>Carefully aspirate the supernatant and resuspend the EBs with 6 mL of fresh Medium A and transfer them into a new low-adhesion well of 6-well plate. Culture in a 37 &#176;C incubator. 
	NOTE: The purpose of the well transferring on day 1 is to reduce the influence of a large number of dead cells during the process of EB formation, which may affect the state of cell growth.</li>
	<li>Refresh the fresh Medium A daily until day 4 (6 mL/well of 6-well plate).</li>
	<li>On day 4, coat a 10 cm dish with 3 mL of 0.1% fish gelatin solution for at least 1 h in a 5% CO2, 37 &#176;C incubator. 
	NOTE: If the coating time is less than 1 h, it is difficult to carry out the following steps.</li>
	<li>Carefully collect the EBs into a 15 mL centrifugal tube with a 10 mL pipet and wait for the EBs to settle to the bottom in 5 min.</li>
	<li>Remove the 0.1% gelatin solution and rinse the 10 cm dish once with 5-6 mL of 1x DPBS. Remove the DPBS.</li>
	<li>Gently resuspend the EBs with 15 mL of Medium B (Table 1) in the 15 mL centrifugal tube and transfer them to the coated 10 cm dish. Culture in a 37 &#176;C, 5% CO2 incubator for 1 week. 
	NOTE: In the first 7 days, do not move the dish.</li>
	<li>Refresh with fresh Medium B once a week until day 49 (15 mL/10 cm dish), and examine the cells under the microscope once a week. 
	NOTE: After day 49, several secretory cells can be found in the supernatant in the culture dish.</li>
	<li>Centrifuge the supernatant cells at 200 x g for 5 min at RT.</li>
	<li>Carefully aspirate the supernatant and resuspend the cells with 2 mL of fresh Medium B and transfer into a new low-adhesion well of 6-well plate. Incubate in a 5% CO2, 37 &#176;C incubator. 
	NOTE: In the next 7 days, do not move the plate. The microglia could adhere to the bottom of the low-adhesion well.</li>
	<li>On day 56, replace with Medium C (Table 1) (2 mL/well of 6-well plate). The microglia adhere to the bottom of the low-adhesion well and will look branched under the microscope. 
	NOTE: By changing Medium C every 3 days (2 mL/well of 6-well plate), the microglia could be cultivated in Medium C for about 15 days.</li>
</ol>
2. Generation of human ROs and co-culture the ROs with microglia
<ol>
	<li>Cultured the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 106 cells in each well.</li>
	<li>Add Dispase (1 mL/well of 6 well plate) to the culture cell. Incubate at 37 &#176;C for 5 min. Aspirate the Dispase solution from the well.</li>
	<li>Add Medium D (Table 1) (1 mL/well of 6 well plate) to the well. Cut the cell into small pieces with a 10 &#181;L pipette.</li>
	<li>Gently collect all the cell pieces and medium in a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge at 200 x g for 5 min and remove the supernatant.</li>
	<li>Gently resuspend the cells in 200 &#181;L of a cold matrix. Move the 1.5 microcentrifuge tube into an incubator for 20 min. Operate fast because the matrix will solidify at room temperature (RT). After 20 min in the incubator, the matrix will solidify.</li>
	<li>Prepare a 10 cm dish with 15 mL Medium D. Resuspend the Matrix with Medium D and shake the Petri dish gently.</li>
	<li>Then, put the dish in an incubator for 5 days. Do not move the plate. Set the day as day 0 (ROs). The cells will adhere in 3 days.</li>
	<li>On day 5 (ROs) and day 10 (ROs), refresh the medium with Medium D.</li>
	<li>On day 12 (ROs), remove the medium and add 3 mL of Dispase to each dish for 5 min.</li>
	<li>Aspirate the Dispase and add 15 mL of Medium E (Table 1).</li>
	<li>Harvest microglia: On day 56 (MGs), gently aspirate the Medium C, add Accutase (1 mL/well of a 6-well plate), and incubate at 37 &#176;C for 3 min.</li>
	<li>Collect the microglia cells into a 15 mL centrifugal tube with a 5 mL pipet. Centrifuge cells at 200 x g at RT for 5 min.</li>
	<li>Gently aspirate the supernatant and suspend the microglia with 1 mL of Medium E. Add microglia to digested ROs on day 12. 
	NOTE: On day 12 (ROs), the cells are adhesive. On day 13 (ROs), the cells are found suspending in Medium E. If the medium changes yellow from day 12 (ROs) to day 19 (ROs), refresh the medium with Medium E.</li>
	<li>On day 19 (ROs), aggregate the organoids to the center of the dish by gently swirling the plate. Gently aspirate Medium E and refresh the medium with Medium F.</li>
	<li>Transfer the organoids with Medium F to a new suspension dish.</li>
	<li>Change the medium once a week and select organoids for experiments.</li>
</ol>

Microglia-Integrated Retinal Organoids: A Model for Studying Retinal Diseases

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(170), e62298 (2021).</li><li>Eiraku, M., 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472 (7341), 51-56 (2011).</li><li>Ginhoux, F., 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=Fate+mapping+analysis+reveals+that+adult+microglia+derive+from+primitive+macrophages.">Fate mapping analysis reveals that adult microglia derive from primitive macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Kierdorf, K., 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=Microglia+emerge+from+erythromyeloid+precursors+via+pu.1-+and+irf8-dependent+pathways.">Microglia emerge from erythromyeloid precursors via pu.1- and irf8-dependent pathways.</a> Nat Neurosci. 16 (3), 273-280 (2013).</li><li>Schulz, C., 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+lineage+of+myeloid+cells+independent+of+myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>O'koren, E. G., 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=Microglial+function+is+distinct+in+different+anatomical+locations+during+retinal+homeostasis+and+degeneration.">Microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration.</a> Immunity. 50 (3), 723-737.e7 (2019).</li><li>Margeta, M. A., 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=Apolipoprotein+E4+impairs+the+response+of+neurodegenerative+retinal+microglia+and+prevents+neuronal+loss+in+glaucoma.">Apolipoprotein E4 impairs the response of neurodegenerative retinal microglia and prevents neuronal loss in glaucoma.</a> Immunity. 55 (9), 1627-1644.e7 (2022).</li><li>Xu, 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=Enhanced+innate+responses+in+microglia+derived+from+retinoblastoma+patient-specific+IPSCs.">Enhanced innate responses in microglia derived from retinoblastoma patient-specific IPSCs.</a> Glia. 72 (5), 872-884 (2024).</li><li>Gosselin, D., 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=An+environment-dependent+transcriptional+network+specifies+human+microglia+identity.">An environment-dependent transcriptional network specifies human microglia identity.</a> Science. 356 (6344), eaal3222 (2017).</li><li>Galatro, T. F., 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=Transcriptomic+analysis+of+purified+human+cortical+microglia+reveals+age-associated+changes.">Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.</a> Nat Neurosci. 20 (8), 1162-1171 (2017).</li><li>Nakano, 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=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10 (6), 771-785 (2012).</li><li>Kim, S., 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=transcriptome+profiling,+and+functional+validation+of+cone-rich+human+retinal+organoids.">transcriptome profiling, and functional validation of cone-rich human retinal organoids.</a> Proc Natl Acad Sci U S A. 116 (22), 10824-10833 (2019).</li><li>Gao, M. L., 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=Functional+microglia+derived+from+human+pluripotent+stem+cells+empower+retinal+organ.">Functional microglia derived from human pluripotent stem cells empower retinal organ.</a> Sci China Life Sci. 65 (6), 1057-1071 (2022).</li><li>Zhang, X., Jin, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruct+human+retinoblastoma+in+vitro.">Reconstruct human retinoblastoma in vitro.</a> J Vis Exp. (188), e62629 (2022).</li><li>Park, D. S., 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=IPS-cell-derived+microglia+promote+brain+organoid+maturation+via+cholesterol+transfer.">IPS-cell-derived microglia promote brain organoid maturation via cholesterol transfer.</a> Nature. 623 (7986), 397-405 (2023).</li><li>Usui-Ouchi, A., 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=Integrating+human+ipsc-derived+macrophage+progenitors+into+retinal+organoids+to+generate+a+mature+retinal+microglial+niche.">Integrating human ipsc-derived macrophage progenitors into retinal organoids to generate a mature retinal microglial niche.</a> Glia. 71 (10), 2372-2382 (2023).</li><li>Chichagova, V., 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=Incorporating+microglia-like+cells+in+human+induced+pluripotent+stem+cell-derived+retinal+organoids.">Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids.</a> J Cell Mol Med. 27 (3), 435-445 (2023).</li></ol>

The authors are not aware of any affiliations, membership, grants, or financial holding that might affect the objectivity of this study.

Due to the restricted availability of the human retina, our current comprehension of retinal inflammatory responses almost comes from animal models. To overcome this limitation, retinal organoids were differentiated. The development of retinal organoid models has been an active area of research, aiming to recapitulate the complexity of the human retina for disease modeling and therapeutic development. Several studies have reported successfully generating retinal organoids from human pluripotent stem cells<sup class="xref...

As the limited source of the human retina, the differentiation of human stem cells into three-dimensional (3D) retinal organoids represents a promising in vitro model for simulating the retina1. It contains different cell types in the retina, including photoreceptors, retinal ganglion cells, bipolar cells, M&#252;ller cells, horizontal cells, and astrocytes2. This model enables the emulation and study of both retinal development mechanisms and the pathogenesis of retinal diseases. However, due to the directional differentiation method, retinal organoids were derived from the neuroectoderm3, lacking many other cell types originating from different germ layers, such as microglia from the yolk sac and perivascular cells from the mesoderm4,5,6.
At present, many retinal diseases, such as retinitis pigmentosa7, glaucoma8, and retinoblastoma9, have been proven to be closely related to microglia within the retina. However, due to the lack of proper research models, specific mechanisms illustrating the relationship between microglia and these diseases still remain unclear. While mice have served as a favorable model for studying retinal diseases, recent studies have highlighted significant differences between mouse and human microglia in terms of lifespan, proliferation rate, and the absence of human homologous genes10,11. These findings suggested that conclusions drawn from mouse models may not be entirely reliable, emphasizing the importance of constructing human retinal organoids containing microglia.
Over the past few decades, various methods for the 3D differentiation of retinal organoids have been developed12,13. To facilitate the co-culture operation of microglia within retinal organoids, we have selected a differentiation method involving a transition from adherent to suspension culture. This approach successfully enables microglia to be incorporated into the retinal organoids, maintaining them for at least 60 days14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acctuase</td>
			<td>Stemcell Technologies</td>
			<td>07920</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Thermo</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CRX(M02)</td>
			<td>abnova</td>
			<td>H00001406-M02</td>
			<td>Antibody; dilution as per the manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Anti-IBA1</td>
			<td>Abcam</td>
			<td>ab5076</td>
			<td>Antibody; dilution as per the manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Life Technologies</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase (1U/mL)</td>
			<td>Stemcell Technologies</td>
			<td>07923</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic</td>
			<td>Gibco</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>C141905005BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biological Industry</td>
			<td>04-002-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G7041-100G</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>H9 cell line</td>
			<td>WiCell Research Institute</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>RD Systems&#160;</td>
			<td>203-IL-050</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-34</td>
			<td>PeproTech</td>
			<td>200-34-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>RD Systems&#160;</td>
			<td>216-MC-500&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem cell medium&#160;</td>
			<td>Stemcell Technologies</td>
			<td>5990</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T-8691-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ViVO</td>
			<td>LONZA</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
	</tbody>
</table>

assembling retinal organoids with microglia

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal the mechanism of retinal development and the occurrence of retinal disease. Microglia (MG) are unique resident macrophages in the retina and central nervous system (CNS), serving crucial immunity functions. However, retinal organoids lack microglia since their differentiation origin is the yolk sac. The specific pathogenesis of microglia in these retinal diseases remains unclear; therefore, the establishment of a microglia-incorporated retinal organoid model turns out to be necessary. Here, we successfully constructed a co-cultured model of retinal organoids with microglia derived from human stem cells. In this article, we differentiated microglia and then co-cultured to retinal organoids in the early stage. As the incorporation of immune cells, this model provides an optimized platform for retinal disease modeling and drug screening to facilitate in-depth research on the pathogenesis and treatment of retinal and CNS-related diseases.

The procedure for generating retinal organoids is described in our previous study15. Here, we show the representative results of microglia and co-culture microglia and retinal organoids.
Here, we demonstrate each stage of microglia differentiation (Figure 1A). Day 0 represents the stage of stem cell culture. Then, the stem cells were digested and cultured for EB formation. In the initial 4 days of the process, cells will form EBs (<strong c...

Generation of Human Microglia to Combine Them with Retinal Organoids for Improved Disease Modeling

Microglia are unique resident immune cells in the retina, playing crucial roles in various retinal degenerative diseases. Generating a co-culture model of retinal organoids with microglia can facilitate a better understanding of the pathogenesis and development progress of retinal diseases.

Assembling Retinal Organoids with Microglia

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal ...

assembling-retinal-organoids-with-microglia

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989 Views.  Capital Medical University. Microglia are unique resident immune cells in the retina, playing crucial roles in various retinal degenerative diseases. Generating a co-culture model of retinal organoids with microglia can facilitate a better understanding of the pathogenesis and development progress of retinal diseases.

Video: Generation of Human Microglia to Combine Them with Retinal Organoids for Improved Disease Modeling

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

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 ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...