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

Area of Science:

• Ophthalmology
• Biochemistry
• Neuroscience

Background:

• Metabolomics in retina and retinal pigment epithelium (RPE) is crucial for understanding retinal metabolism and disease mechanisms.
• Animal models and postmortem donors are key resources for these studies.

Purpose of the Study:

• To investigate the effects of euthanasia methods, isolation procedures, and postmortem delay on retinal and RPE/choroid metabolites.
• To establish optimized protocols for preparing tissue for metabolomics analysis.

Main Methods:

• Utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).
• Compared metabolites in mouse retina and RPE/choroid following CO2 euthanasia versus cervical dislocation.
• Evaluated metabolite changes after different isolation procedures and varying postmortem delays.
• Assessed the impact of storing eyes in cold nutrient-rich medium.

Main Results:

• CO2 euthanasia significantly altered ATP, GTP, glucose metabolism, and amino acid intermediates compared to cervical dislocation.
• Isolation in cold buffer with glucose minimized metabolite changes.
• Postmortem delay time-dependently increased metabolite alterations in both retina and RPE/choroid.
• Storage in cold nutrient medium effectively preserved metabolite profiles.

Conclusions:

• Euthanasia method, tissue isolation, and postmortem interval critically influence metabolomic data.
• Optimized protocols involving specific euthanasia, cold buffer isolation, and timely processing are recommended.
• Cold nutrient medium storage is a viable strategy to mitigate postmortem metabolite degradation.