Cargando…
An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes
PURPOSE: The molecular mechanisms controlling aqueous humor (AQH) outflow and IOP need much further definition. The mouse is a powerful system for characterizing the mechanistic basis of AQH outflow. To enhance outflow studies in mice, we developed a perfusion system that is based on human anterior...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Association for Research in Vision and Ophthalmology
2016
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054733/ https://www.ncbi.nlm.nih.gov/pubmed/27701632 http://dx.doi.org/10.1167/iovs.16-19481 |
_version_ | 1782458657261223936 |
---|---|
author | Kizhatil, Krishnakumar Chlebowski, Arthur Tolman, Nicholas G. Freeburg, Nelson F. Ryan, Margaret M. Shaw, Nicholas N. Kokini, Alexander D. M. Marchant, Jeffrey K. John, Simon W. M. |
author_facet | Kizhatil, Krishnakumar Chlebowski, Arthur Tolman, Nicholas G. Freeburg, Nelson F. Ryan, Margaret M. Shaw, Nicholas N. Kokini, Alexander D. M. Marchant, Jeffrey K. John, Simon W. M. |
author_sort | Kizhatil, Krishnakumar |
collection | PubMed |
description | PURPOSE: The molecular mechanisms controlling aqueous humor (AQH) outflow and IOP need much further definition. The mouse is a powerful system for characterizing the mechanistic basis of AQH outflow. To enhance outflow studies in mice, we developed a perfusion system that is based on human anterior chamber perfusion culture systems. Our mouse system permits previously impractical experiments. METHODS: We engineered a computer-controlled, pump-based perfusion system with a platform for mounting whole dissected mouse eyes (minus lens and iris, ∼45% of drainage tissue is perfused). We tested the system's ability to monitor outflow and tested the effects of the outflow-elevating drug, Y27632, a rho-associated protein kinase (ROCK) inhibitor. Finally, we tested the system's ability to detect genetically determined decreases in outflow by determining if deficiency of the candidate genes Nos3 and Cav1 alter outflow. RESULTS: Using our system, the outflow facility (C) of C57BL/6J mouse eyes was found to range between 7.7 and 10.4 nl/minutes/mm Hg (corrected for whole eye). Our system readily detected a 74.4% Y27632-induced increase in C. The NOS3 inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) and a Nos3 null mutation reduced C by 28.3% and 35.8%, respectively. Similarly, in Cav1 null eyes C was reduced by 47.8%. CONCLUSIONS: We engineered a unique perfusion system that can accurately measure changes in C. We then used the system to show that NOS3 and CAV1 are key components of mechanism(s) controlling outflow. |
format | Online Article Text |
id | pubmed-5054733 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | The Association for Research in Vision and Ophthalmology |
record_format | MEDLINE/PubMed |
spelling | pubmed-50547332016-10-11 An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes Kizhatil, Krishnakumar Chlebowski, Arthur Tolman, Nicholas G. Freeburg, Nelson F. Ryan, Margaret M. Shaw, Nicholas N. Kokini, Alexander D. M. Marchant, Jeffrey K. John, Simon W. M. Invest Ophthalmol Vis Sci Glaucoma PURPOSE: The molecular mechanisms controlling aqueous humor (AQH) outflow and IOP need much further definition. The mouse is a powerful system for characterizing the mechanistic basis of AQH outflow. To enhance outflow studies in mice, we developed a perfusion system that is based on human anterior chamber perfusion culture systems. Our mouse system permits previously impractical experiments. METHODS: We engineered a computer-controlled, pump-based perfusion system with a platform for mounting whole dissected mouse eyes (minus lens and iris, ∼45% of drainage tissue is perfused). We tested the system's ability to monitor outflow and tested the effects of the outflow-elevating drug, Y27632, a rho-associated protein kinase (ROCK) inhibitor. Finally, we tested the system's ability to detect genetically determined decreases in outflow by determining if deficiency of the candidate genes Nos3 and Cav1 alter outflow. RESULTS: Using our system, the outflow facility (C) of C57BL/6J mouse eyes was found to range between 7.7 and 10.4 nl/minutes/mm Hg (corrected for whole eye). Our system readily detected a 74.4% Y27632-induced increase in C. The NOS3 inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) and a Nos3 null mutation reduced C by 28.3% and 35.8%, respectively. Similarly, in Cav1 null eyes C was reduced by 47.8%. CONCLUSIONS: We engineered a unique perfusion system that can accurately measure changes in C. We then used the system to show that NOS3 and CAV1 are key components of mechanism(s) controlling outflow. The Association for Research in Vision and Ophthalmology 2016-10 /pmc/articles/PMC5054733/ /pubmed/27701632 http://dx.doi.org/10.1167/iovs.16-19481 Text en http://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. |
spellingShingle | Glaucoma Kizhatil, Krishnakumar Chlebowski, Arthur Tolman, Nicholas G. Freeburg, Nelson F. Ryan, Margaret M. Shaw, Nicholas N. Kokini, Alexander D. M. Marchant, Jeffrey K. John, Simon W. M. An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes |
title | An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes |
title_full | An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes |
title_fullStr | An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes |
title_full_unstemmed | An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes |
title_short | An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes |
title_sort | in vitro perfusion system to enhance outflow studies in mouse eyes |
topic | Glaucoma |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054733/ https://www.ncbi.nlm.nih.gov/pubmed/27701632 http://dx.doi.org/10.1167/iovs.16-19481 |
work_keys_str_mv | AT kizhatilkrishnakumar aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT chlebowskiarthur aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT tolmannicholasg aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT freeburgnelsonf aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT ryanmargaretm aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT shawnicholasn aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT kokinialexanderdm aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT marchantjeffreyk aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT johnsimonwm aninvitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT kizhatilkrishnakumar invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT chlebowskiarthur invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT tolmannicholasg invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT freeburgnelsonf invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT ryanmargaretm invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT shawnicholasn invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT kokinialexanderdm invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT marchantjeffreyk invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes AT johnsimonwm invitroperfusionsystemtoenhanceoutflowstudiesinmouseeyes |