Cargando…

A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy

PURPOSE: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. METHODS: Murine corneal biopsies were obtained using a biopsy punch immediately after eut...

Descripción completa

Detalles Bibliográficos
Autores principales: Liang, Wentao, Huang, Li, Yuan, Tian, Cheng, Rui, Takahashi, Yusuke, Moiseyev, Gennadiy P., Karamichos, Dimitrios, Ma, Jian-Xing
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Association for Research in Vision and Ophthalmology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10476441/
https://www.ncbi.nlm.nih.gov/pubmed/37642632
http://dx.doi.org/10.1167/iovs.64.11.33
_version_ 1785100933508628480
author Liang, Wentao
Huang, Li
Yuan, Tian
Cheng, Rui
Takahashi, Yusuke
Moiseyev, Gennadiy P.
Karamichos, Dimitrios
Ma, Jian-Xing
author_facet Liang, Wentao
Huang, Li
Yuan, Tian
Cheng, Rui
Takahashi, Yusuke
Moiseyev, Gennadiy P.
Karamichos, Dimitrios
Ma, Jian-Xing
author_sort Liang, Wentao
collection PubMed
description PURPOSE: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. METHODS: Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. RESULTS: Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. CONCLUSIONS: The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models.
format Online
Article
Text
id pubmed-10476441
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher The Association for Research in Vision and Ophthalmology
record_format MEDLINE/PubMed
spelling pubmed-104764412023-09-05 A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy Liang, Wentao Huang, Li Yuan, Tian Cheng, Rui Takahashi, Yusuke Moiseyev, Gennadiy P. Karamichos, Dimitrios Ma, Jian-Xing Invest Ophthalmol Vis Sci Cornea PURPOSE: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. METHODS: Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. RESULTS: Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. CONCLUSIONS: The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models. The Association for Research in Vision and Ophthalmology 2023-08-29 /pmc/articles/PMC10476441/ /pubmed/37642632 http://dx.doi.org/10.1167/iovs.64.11.33 Text en Copyright 2023 The Authors https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License.
spellingShingle Cornea
Liang, Wentao
Huang, Li
Yuan, Tian
Cheng, Rui
Takahashi, Yusuke
Moiseyev, Gennadiy P.
Karamichos, Dimitrios
Ma, Jian-Xing
A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy
title A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy
title_full A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy
title_fullStr A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy
title_full_unstemmed A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy
title_short A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy
title_sort method for real-time assessment of mitochondrial respiration using murine corneal biopsy
topic Cornea
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10476441/
https://www.ncbi.nlm.nih.gov/pubmed/37642632
http://dx.doi.org/10.1167/iovs.64.11.33
work_keys_str_mv AT liangwentao amethodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT huangli amethodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT yuantian amethodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT chengrui amethodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT takahashiyusuke amethodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT moiseyevgennadiyp amethodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT karamichosdimitrios amethodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT majianxing amethodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT liangwentao methodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT huangli methodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT yuantian methodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT chengrui methodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT takahashiyusuke methodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT moiseyevgennadiyp methodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT karamichosdimitrios methodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy
AT majianxing methodforrealtimeassessmentofmitochondrialrespirationusingmurinecornealbiopsy