The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues
Purpose The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium–Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK). Methods Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endoth...
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| creator | Gisoldi, Rossella Colabelli Lodato, Gemma Balzamino, Bijorn Omar Esposito, Graziana Micera, Alessandra Pocobelli, Augusto |
| description | Purpose
The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium–Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK).
Methods
Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endothelium was tested at 25 s (T0), 1 min (T1), 2 min (T2), and 4 min (T4) from dye addition. Staining intensity and cell counting were compared. Stripped EDM was analyzed for selected apoptotic (AP, caspases, BCL2, BAX) and differentiation (VEGF-A, TGF-β1RI, SMAD3/7, SMA) targets and changes in target expression. Protein extracts were analyzed through SDS-PAGE/IB.
Results
Although trypan blue staining produced the same color intensity of lutein/trypan blue dye in half the time, lutein/trypan blue reached a good and adequate color intensity at T4, which persisted even on excised and washed EDM grafts. Lutein/trypan blue-stained EDM showed a reduced number of blue-stained cells and AP immunoreactivity was significantly reduced in the same samples. An increased BCL2 transcript and a reduced BAX transcript were detected in lutein/trypan blue-stained EDM. No significant changes were observed for the main effector caspases (3/9) upon both treatments and the target genes representative of endothelial cell trans-differentiation (TGF-β1RI, SMAD3/7, SMA). A trend in vascular endothelial growth factor (VEGF-A) regulation was observed in lutein/trypan blue-treated EDM grafts.
Conclusion
Obtained results suggest that lutein/trypan blue dye deserves attention in the DMEK field and support the potential routine use of this dye as a valid alternative to trypan blue for all procedures devoted to the assessment of endothelial cell viability and visualization of EDM graft before DMEK grafting. |
| doi_str_mv | 10.1007/s00417-022-05909-x |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_9707145</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>2807560539</sourcerecordid><originalsourceid>FETCH-LOGICAL-c474t-31d2b982a3e01c3fd3ae463dbd2d9319d9bfb6a1045b67dff3003c7172196fb83</originalsourceid><addsrcrecordid>eNp9kU9v1DAUxC0EokvhC3BAljiHPv9JvOGAhEopiCIuRerNcuLnrausHWyn6t746LjdUuDCyYf5zbyRh5CXDN4wAHWUASRTDXDeQNtD39w8IismRdso4BePyQoUZ81a8IsD8iznK6i8aNlTciA6KVspuxX5eX6JdMnolilgzjQ6Oi0FfTgqaTebQIdpQXrti5mo3SF1MdFSLbkYH3zY3BrGmAJWHYONVZv8sn1LDZ39FEsFF7ujMdAPX0--0DlhSWgKWlp8zgvm5-SJM1PGF_fvIfn-8eT8-FNz9u308_H7s2aUSpZGMMuHfs2NQGCjcFYYlJ2wg-W2F6y3_eCGzjCQ7dAp65wAEKNi9Qf6zg1rcUje7XPnZdiiHTGUZCY9J781aaej8fpfJfhLvYnXulegmGxrwOv7gBR_1OJFX8UlhdpZ8zWotoNW9JXie2pMMeeE7uECA327mt6vputq-m41fVNNr_7u9mD5PVMFxB7IVQobTH9u_yf2F9wvpp8</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2807560539</pqid></control><display><type>article</type><title>The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues</title><source>MEDLINE</source><source>SpringerLink Journals - AutoHoldings</source><creator>Gisoldi, Rossella Colabelli ; Lodato, Gemma ; Balzamino, Bijorn Omar ; Esposito, Graziana ; Micera, Alessandra ; Pocobelli, Augusto</creator><creatorcontrib>Gisoldi, Rossella Colabelli ; Lodato, Gemma ; Balzamino, Bijorn Omar ; Esposito, Graziana ; Micera, Alessandra ; Pocobelli, Augusto</creatorcontrib><description>Purpose
The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium–Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK).
Methods
Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endothelium was tested at 25 s (T0), 1 min (T1), 2 min (T2), and 4 min (T4) from dye addition. Staining intensity and cell counting were compared. Stripped EDM was analyzed for selected apoptotic (AP, caspases, BCL2, BAX) and differentiation (VEGF-A, TGF-β1RI, SMAD3/7, SMA) targets and changes in target expression. Protein extracts were analyzed through SDS-PAGE/IB.
Results
Although trypan blue staining produced the same color intensity of lutein/trypan blue dye in half the time, lutein/trypan blue reached a good and adequate color intensity at T4, which persisted even on excised and washed EDM grafts. Lutein/trypan blue-stained EDM showed a reduced number of blue-stained cells and AP immunoreactivity was significantly reduced in the same samples. An increased BCL2 transcript and a reduced BAX transcript were detected in lutein/trypan blue-stained EDM. No significant changes were observed for the main effector caspases (3/9) upon both treatments and the target genes representative of endothelial cell trans-differentiation (TGF-β1RI, SMAD3/7, SMA). A trend in vascular endothelial growth factor (VEGF-A) regulation was observed in lutein/trypan blue-treated EDM grafts.
Conclusion
Obtained results suggest that lutein/trypan blue dye deserves attention in the DMEK field and support the potential routine use of this dye as a valid alternative to trypan blue for all procedures devoted to the assessment of endothelial cell viability and visualization of EDM graft before DMEK grafting.</description><identifier>ISSN: 0721-832X</identifier><identifier>EISSN: 1435-702X</identifier><identifier>DOI: 10.1007/s00417-022-05909-x</identifier><identifier>PMID: 36445446</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Apoptosis ; BAX protein ; bcl-2-Associated X Protein ; Cell Count ; Cell differentiation ; Cell viability ; Coloring Agents - pharmacology ; Cornea ; Corneal transplantation ; Descemet Membrane - surgery ; Descemet Stripping Endothelial Keratoplasty - methods ; Dyes ; Endothelial cells ; Endothelium ; Endothelium, Corneal - transplantation ; Goats ; Humans ; Immunoreactivity ; Lutein - pharmacology ; Medicine ; Medicine & Public Health ; Ophthalmology ; Pilot Projects ; Smad3 protein ; Staining and Labeling ; Tissue and Organ Harvesting ; Tissue Donors ; Trypan Blue - pharmacology ; Vascular endothelial growth factor ; Vascular Endothelial Growth Factor A - pharmacology</subject><ispartof>Graefe's archive for clinical and experimental ophthalmology, 2023-05, Vol.261 (5), p.1321-1329</ispartof><rights>The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.</rights><rights>2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.</rights><rights>The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c474t-31d2b982a3e01c3fd3ae463dbd2d9319d9bfb6a1045b67dff3003c7172196fb83</citedby><cites>FETCH-LOGICAL-c474t-31d2b982a3e01c3fd3ae463dbd2d9319d9bfb6a1045b67dff3003c7172196fb83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00417-022-05909-x$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00417-022-05909-x$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,776,780,881,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36445446$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gisoldi, Rossella Colabelli</creatorcontrib><creatorcontrib>Lodato, Gemma</creatorcontrib><creatorcontrib>Balzamino, Bijorn Omar</creatorcontrib><creatorcontrib>Esposito, Graziana</creatorcontrib><creatorcontrib>Micera, Alessandra</creatorcontrib><creatorcontrib>Pocobelli, Augusto</creatorcontrib><title>The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues</title><title>Graefe's archive for clinical and experimental ophthalmology</title><addtitle>Graefes Arch Clin Exp Ophthalmol</addtitle><addtitle>Graefes Arch Clin Exp Ophthalmol</addtitle><description>Purpose
The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium–Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK).
Methods
Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endothelium was tested at 25 s (T0), 1 min (T1), 2 min (T2), and 4 min (T4) from dye addition. Staining intensity and cell counting were compared. Stripped EDM was analyzed for selected apoptotic (AP, caspases, BCL2, BAX) and differentiation (VEGF-A, TGF-β1RI, SMAD3/7, SMA) targets and changes in target expression. Protein extracts were analyzed through SDS-PAGE/IB.
Results
Although trypan blue staining produced the same color intensity of lutein/trypan blue dye in half the time, lutein/trypan blue reached a good and adequate color intensity at T4, which persisted even on excised and washed EDM grafts. Lutein/trypan blue-stained EDM showed a reduced number of blue-stained cells and AP immunoreactivity was significantly reduced in the same samples. An increased BCL2 transcript and a reduced BAX transcript were detected in lutein/trypan blue-stained EDM. No significant changes were observed for the main effector caspases (3/9) upon both treatments and the target genes representative of endothelial cell trans-differentiation (TGF-β1RI, SMAD3/7, SMA). A trend in vascular endothelial growth factor (VEGF-A) regulation was observed in lutein/trypan blue-treated EDM grafts.
Conclusion
Obtained results suggest that lutein/trypan blue dye deserves attention in the DMEK field and support the potential routine use of this dye as a valid alternative to trypan blue for all procedures devoted to the assessment of endothelial cell viability and visualization of EDM graft before DMEK grafting.</description><subject>Apoptosis</subject><subject>BAX protein</subject><subject>bcl-2-Associated X Protein</subject><subject>Cell Count</subject><subject>Cell differentiation</subject><subject>Cell viability</subject><subject>Coloring Agents - pharmacology</subject><subject>Cornea</subject><subject>Corneal transplantation</subject><subject>Descemet Membrane - surgery</subject><subject>Descemet Stripping Endothelial Keratoplasty - methods</subject><subject>Dyes</subject><subject>Endothelial cells</subject><subject>Endothelium</subject><subject>Endothelium, Corneal - transplantation</subject><subject>Goats</subject><subject>Humans</subject><subject>Immunoreactivity</subject><subject>Lutein - pharmacology</subject><subject>Medicine</subject><subject>Medicine & Public Health</subject><subject>Ophthalmology</subject><subject>Pilot Projects</subject><subject>Smad3 protein</subject><subject>Staining and Labeling</subject><subject>Tissue and Organ Harvesting</subject><subject>Tissue Donors</subject><subject>Trypan Blue - pharmacology</subject><subject>Vascular endothelial growth factor</subject><subject>Vascular Endothelial Growth Factor A - pharmacology</subject><issn>0721-832X</issn><issn>1435-702X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kU9v1DAUxC0EokvhC3BAljiHPv9JvOGAhEopiCIuRerNcuLnrausHWyn6t746LjdUuDCyYf5zbyRh5CXDN4wAHWUASRTDXDeQNtD39w8IismRdso4BePyQoUZ81a8IsD8iznK6i8aNlTciA6KVspuxX5eX6JdMnolilgzjQ6Oi0FfTgqaTebQIdpQXrti5mo3SF1MdFSLbkYH3zY3BrGmAJWHYONVZv8sn1LDZ39FEsFF7ujMdAPX0--0DlhSWgKWlp8zgvm5-SJM1PGF_fvIfn-8eT8-FNz9u308_H7s2aUSpZGMMuHfs2NQGCjcFYYlJ2wg-W2F6y3_eCGzjCQ7dAp65wAEKNi9Qf6zg1rcUje7XPnZdiiHTGUZCY9J781aaej8fpfJfhLvYnXulegmGxrwOv7gBR_1OJFX8UlhdpZ8zWotoNW9JXie2pMMeeE7uECA327mt6vputq-m41fVNNr_7u9mD5PVMFxB7IVQobTH9u_yf2F9wvpp8</recordid><startdate>20230501</startdate><enddate>20230501</enddate><creator>Gisoldi, Rossella Colabelli</creator><creator>Lodato, Gemma</creator><creator>Balzamino, Bijorn Omar</creator><creator>Esposito, Graziana</creator><creator>Micera, Alessandra</creator><creator>Pocobelli, Augusto</creator><general>Springer Berlin Heidelberg</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7TK</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>5PM</scope></search><sort><creationdate>20230501</creationdate><title>The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues</title><author>Gisoldi, Rossella Colabelli ; Lodato, Gemma ; Balzamino, Bijorn Omar ; Esposito, Graziana ; Micera, Alessandra ; Pocobelli, Augusto</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c474t-31d2b982a3e01c3fd3ae463dbd2d9319d9bfb6a1045b67dff3003c7172196fb83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Apoptosis</topic><topic>BAX protein</topic><topic>bcl-2-Associated X Protein</topic><topic>Cell Count</topic><topic>Cell differentiation</topic><topic>Cell viability</topic><topic>Coloring Agents - pharmacology</topic><topic>Cornea</topic><topic>Corneal transplantation</topic><topic>Descemet Membrane - surgery</topic><topic>Descemet Stripping Endothelial Keratoplasty - methods</topic><topic>Dyes</topic><topic>Endothelial cells</topic><topic>Endothelium</topic><topic>Endothelium, Corneal - transplantation</topic><topic>Goats</topic><topic>Humans</topic><topic>Immunoreactivity</topic><topic>Lutein - pharmacology</topic><topic>Medicine</topic><topic>Medicine & Public Health</topic><topic>Ophthalmology</topic><topic>Pilot Projects</topic><topic>Smad3 protein</topic><topic>Staining and Labeling</topic><topic>Tissue and Organ Harvesting</topic><topic>Tissue Donors</topic><topic>Trypan Blue - pharmacology</topic><topic>Vascular endothelial growth factor</topic><topic>Vascular Endothelial Growth Factor A - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gisoldi, Rossella Colabelli</creatorcontrib><creatorcontrib>Lodato, Gemma</creatorcontrib><creatorcontrib>Balzamino, Bijorn Omar</creatorcontrib><creatorcontrib>Esposito, Graziana</creatorcontrib><creatorcontrib>Micera, Alessandra</creatorcontrib><creatorcontrib>Pocobelli, Augusto</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Neurosciences Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Graefe's archive for clinical and experimental ophthalmology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gisoldi, Rossella Colabelli</au><au>Lodato, Gemma</au><au>Balzamino, Bijorn Omar</au><au>Esposito, Graziana</au><au>Micera, Alessandra</au><au>Pocobelli, Augusto</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues</atitle><jtitle>Graefe's archive for clinical and experimental ophthalmology</jtitle><stitle>Graefes Arch Clin Exp Ophthalmol</stitle><addtitle>Graefes Arch Clin Exp Ophthalmol</addtitle><date>2023-05-01</date><risdate>2023</risdate><volume>261</volume><issue>5</issue><spage>1321</spage><epage>1329</epage><pages>1321-1329</pages><issn>0721-832X</issn><eissn>1435-702X</eissn><abstract>Purpose
The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium–Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK).
Methods
Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endothelium was tested at 25 s (T0), 1 min (T1), 2 min (T2), and 4 min (T4) from dye addition. Staining intensity and cell counting were compared. Stripped EDM was analyzed for selected apoptotic (AP, caspases, BCL2, BAX) and differentiation (VEGF-A, TGF-β1RI, SMAD3/7, SMA) targets and changes in target expression. Protein extracts were analyzed through SDS-PAGE/IB.
Results
Although trypan blue staining produced the same color intensity of lutein/trypan blue dye in half the time, lutein/trypan blue reached a good and adequate color intensity at T4, which persisted even on excised and washed EDM grafts. Lutein/trypan blue-stained EDM showed a reduced number of blue-stained cells and AP immunoreactivity was significantly reduced in the same samples. An increased BCL2 transcript and a reduced BAX transcript were detected in lutein/trypan blue-stained EDM. No significant changes were observed for the main effector caspases (3/9) upon both treatments and the target genes representative of endothelial cell trans-differentiation (TGF-β1RI, SMAD3/7, SMA). A trend in vascular endothelial growth factor (VEGF-A) regulation was observed in lutein/trypan blue-treated EDM grafts.
Conclusion
Obtained results suggest that lutein/trypan blue dye deserves attention in the DMEK field and support the potential routine use of this dye as a valid alternative to trypan blue for all procedures devoted to the assessment of endothelial cell viability and visualization of EDM graft before DMEK grafting.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>36445446</pmid><doi>10.1007/s00417-022-05909-x</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| issn | 0721-832X 1435-702X |
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| source | MEDLINE; SpringerLink Journals - AutoHoldings |
| subjects | Apoptosis BAX protein bcl-2-Associated X Protein Cell Count Cell differentiation Cell viability Coloring Agents - pharmacology Cornea Corneal transplantation Descemet Membrane - surgery Descemet Stripping Endothelial Keratoplasty - methods Dyes Endothelial cells Endothelium Endothelium, Corneal - transplantation Goats Humans Immunoreactivity Lutein - pharmacology Medicine Medicine & Public Health Ophthalmology Pilot Projects Smad3 protein Staining and Labeling Tissue and Organ Harvesting Tissue Donors Trypan Blue - pharmacology Vascular endothelial growth factor Vascular Endothelial Growth Factor A - pharmacology |
| title | The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues |
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