Impact of euthanasia, dissection and postmortem delay on metabolic profile in mouse retina and RPE/choroid

Abstract

Metabolomics studies in the retina and retinal pigment epithelium (RPE) in animal models or postmortem donors are essential to understanding the retinal metabolism and to revealing the underlying mechanisms of retinal degenerative diseases. We have studied how different methods of euthanasia (CO or cervical dislocation) different isolation procedures and postmortem delay affect metabolites in mouse retina and RPE/choroid using LC MS/MS and GC MS. Compared with cervical dislocation, CO exposure for 5 min dramatically degrades ATP and GTP into purine metabolites in the retina while raising intermediates in glucose metabolism and amino acids in the RPE/choroid. Isolation in cold buffer containing glucose has the least change in metabolites. Postmortem delay time-dependently and differentially impacts metabolites in the retina and RPE/choroid. In the postmortem retina, 18% of metabolites were changed at 0.5 h (h), 41% at 4 h and 51% at 8 h. However, only 6% of metabolites were changed in the postmortem RPE/choroid and it steadily increased to 20% at 8 h. Notably, both postmortem retina and RPE/choroid tissue showed increased purine metabolites. Storage of eyes in cold nutrient-rich medium substantially blocked the postmortem change in the retina and RPE/choroid. In conclusion, our study provides optimized methods to prepare fresh or postmortem retina and RPE/choroid tissue for metabolomics studies.

Proline mediates metabolic communication between retinal pigment epithelial cells and the retina

“…4, D and E). Purine metabolites are sensitive to the availability of glucose in RPE (31). As expected, xanthine, guanosine, and inosine dramatically increased in RPE cells (Fig.…”

“…The metabolites harvested from retinal explants and human RPE were analyzed with GC MS or LC MS as we described in detail before (31,70). For medium metabolites, 10 l of medium or plasma was mixed with 40 l of cold methanol to extract metabolites for analysis with GC MS or LC MS.…”

“…At the end of infusion, the mouse was euthanized by cervical dislocation. Retinas and RPE/choroid were quickly dissected with a cut and pick method (31) and snap-frozen in liquid nitrogen for metabolite analysis.…”

Suppressing endoplasmic reticulum stress-related autophagy attenuates retinal light injury

“…The RPE/choroid mixture was separated according to a previous protocol [ 54 ]. Briefly, the cornea and lens from enucleated eyeballs were carefully removed under a surgery microscope, and then the retina and the soft tissue surrounding the sclera were removed.…”

Negative chronotropic and inotropic effects of lubiprostone on iPS cell-derived cardiomyocytes via activation of CFTR

“…Briefly, 17-day pregnant BALB/cAJc1 mice (CLEA Japan) were euthanized by cervical dislocation, and the uterus was isolated. Euthanasia by cervical dislocation has been widely used in animal experiment, since it can cause a fast and painless death and avoid damage to the fetus [27][28][29][30][31][32][33][34]. Furthermore, it does not chemically contaminate tissues.…”