Rapid isolation of intact retinal astrocytes: a novel approach
Abstract Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-09-01
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| Series: | Acta Neuropathologica Communications |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s40478-023-01641-7 |
| _version_ | 1797451125002076160 |
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| author | Paul F. Cullen Arpan G. Mazumder Daniel Sun John G. Flanagan |
| author_facet | Paul F. Cullen Arpan G. Mazumder Daniel Sun John G. Flanagan |
| author_sort | Paul F. Cullen |
| collection | DOAJ |
| description | Abstract Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA. |
| first_indexed | 2024-03-09T14:50:24Z |
| format | Article |
| id | doaj.art-704abfc556014771b40c731d4b7ce760 |
| institution | Directory Open Access Journal |
| issn | 2051-5960 |
| language | English |
| last_indexed | 2024-03-09T14:50:24Z |
| publishDate | 2023-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Acta Neuropathologica Communications |
| spelling | doaj.art-704abfc556014771b40c731d4b7ce7602023-11-26T14:31:48ZengBMCActa Neuropathologica Communications2051-59602023-09-0111111710.1186/s40478-023-01641-7Rapid isolation of intact retinal astrocytes: a novel approachPaul F. Cullen0Arpan G. Mazumder1Daniel Sun2John G. Flanagan3Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical SchoolDepartment of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical SchoolDepartment of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical SchoolHerbert Wertheim School of Optometry and Vision Science, University of California at BerkeleyAbstract Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA.https://doi.org/10.1186/s40478-023-01641-7RetinaRetinal astrocytesGlaucomaReactive astrocytesRibotagIsolating astrocytes |
| spellingShingle | Paul F. Cullen Arpan G. Mazumder Daniel Sun John G. Flanagan Rapid isolation of intact retinal astrocytes: a novel approach Acta Neuropathologica Communications Retina Retinal astrocytes Glaucoma Reactive astrocytes Ribotag Isolating astrocytes |
| title | Rapid isolation of intact retinal astrocytes: a novel approach |
| title_full | Rapid isolation of intact retinal astrocytes: a novel approach |
| title_fullStr | Rapid isolation of intact retinal astrocytes: a novel approach |
| title_full_unstemmed | Rapid isolation of intact retinal astrocytes: a novel approach |
| title_short | Rapid isolation of intact retinal astrocytes: a novel approach |
| title_sort | rapid isolation of intact retinal astrocytes a novel approach |
| topic | Retina Retinal astrocytes Glaucoma Reactive astrocytes Ribotag Isolating astrocytes |
| url | https://doi.org/10.1186/s40478-023-01641-7 |
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