Comparative evaluation of methods for estimating retinal ganglion cell loss in retinal sections and wholemounts
To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of...
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| creator | Mead, Ben Thompson, Adam Scheven, Ben A Logan, Ann Berry, Martin Leadbeater, Wendy |
| description | To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a(+), βIII-tubulin(+) and Islet-1(+) RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG(+) and Brn3a(+) RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin(+) RGCs was greater than Brn3a+ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage. |
| doi_str_mv | 10.1371/journal.pone.0110612 |
| format | Article |
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Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a(+), βIII-tubulin(+) and Islet-1(+) RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG(+) and Brn3a(+) RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin(+) RGCs was greater than Brn3a+ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0110612</identifier><identifier>PMID: 25343338</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Albinism ; Amacrine cells ; Animals ; Astrocytes ; Biology and Life Sciences ; Brn-3 protein ; Cell Count ; Cell Culture Techniques - methods ; Cell Death ; Comparative analysis ; Dentistry ; Diagnosis ; Eye ; Eye (anatomy) ; Female ; Ganglion cysts ; Gene expression ; Glaucoma ; Harvesting ; Hypertension ; Image detection ; Islet-1 protein ; LIM-Homeodomain Proteins - metabolism ; Macrophages ; Medicine ; Medicine and Health Sciences ; Methods ; Microglia ; Microscopy - methods ; Nerves ; Neurobiology ; Neurosciences ; Optic nerve ; Rats ; Rats, Sprague-Dawley ; Research and Analysis Methods ; Retina ; Retinal ganglion cells ; Retinal Ganglion Cells - cytology ; Rodents ; Staining and Labeling ; Transcription Factor Brn-3A - metabolism ; Transcription Factors - metabolism ; Tubulin ; Tubulin - metabolism</subject><ispartof>PloS one, 2014-10, Vol.9 (10), p.e110612-e110612</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Mead et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2014 Mead et al 2014 Mead et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c758t-9f126199fc1a3090d32f6e6f989af4a638b6fc9e702f24fd50724f7c62ed4c523</citedby><cites>FETCH-LOGICAL-c758t-9f126199fc1a3090d32f6e6f989af4a638b6fc9e702f24fd50724f7c62ed4c523</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208790/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208790/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23847,27903,27904,53769,53771,79346,79347</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25343338$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Badea, Tudor C.</contributor><creatorcontrib>Mead, Ben</creatorcontrib><creatorcontrib>Thompson, Adam</creatorcontrib><creatorcontrib>Scheven, Ben A</creatorcontrib><creatorcontrib>Logan, Ann</creatorcontrib><creatorcontrib>Berry, Martin</creatorcontrib><creatorcontrib>Leadbeater, Wendy</creatorcontrib><title>Comparative evaluation of methods for estimating retinal ganglion cell loss in retinal sections and wholemounts</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a(+), βIII-tubulin(+) and Islet-1(+) RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG(+) and Brn3a(+) RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin(+) RGCs was greater than Brn3a+ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mead, Ben</au><au>Thompson, Adam</au><au>Scheven, Ben A</au><au>Logan, Ann</au><au>Berry, Martin</au><au>Leadbeater, Wendy</au><au>Badea, Tudor C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparative evaluation of methods for estimating retinal ganglion cell loss in retinal sections and wholemounts</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2014-10-24</date><risdate>2014</risdate><volume>9</volume><issue>10</issue><spage>e110612</spage><epage>e110612</epage><pages>e110612-e110612</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a(+), βIII-tubulin(+) and Islet-1(+) RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG(+) and Brn3a(+) RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin(+) RGCs was greater than Brn3a+ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25343338</pmid><doi>10.1371/journal.pone.0110612</doi><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
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| issn | 1932-6203 1932-6203 |
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| subjects | Albinism Amacrine cells Animals Astrocytes Biology and Life Sciences Brn-3 protein Cell Count Cell Culture Techniques - methods Cell Death Comparative analysis Dentistry Diagnosis Eye Eye (anatomy) Female Ganglion cysts Gene expression Glaucoma Harvesting Hypertension Image detection Islet-1 protein LIM-Homeodomain Proteins - metabolism Macrophages Medicine Medicine and Health Sciences Methods Microglia Microscopy - methods Nerves Neurobiology Neurosciences Optic nerve Rats Rats, Sprague-Dawley Research and Analysis Methods Retina Retinal ganglion cells Retinal Ganglion Cells - cytology Rodents Staining and Labeling Transcription Factor Brn-3A - metabolism Transcription Factors - metabolism Tubulin Tubulin - metabolism |
| title | Comparative evaluation of methods for estimating retinal ganglion cell loss in retinal sections and wholemounts |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-28T05%3A24%3A30IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Comparative%20evaluation%20of%20methods%20for%20estimating%20retinal%20ganglion%20cell%20loss%20in%20retinal%20sections%20and%20wholemounts&rft.jtitle=PloS%20one&rft.au=Mead,%20Ben&rft.date=2014-10-24&rft.volume=9&rft.issue=10&rft.spage=e110612&rft.epage=e110612&rft.pages=e110612-e110612&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0110612&rft_dat=%3Cgale_plos_%3EA417844007%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1616172193&rft_id=info:pmid/25343338&rft_galeid=A417844007&rft_doaj_id=oai_doaj_org_article_fae10a0428964b848ef6c681622cb203&rfr_iscdi=true |