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 (CO₂ 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, CO₂ 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.

Keywords: Euthanasia; Metabolite; Postmortem; RPE; Retina.

Fig. 1.. The impact of euthanasia with CO₂ on metabolites in retina and RPE/choroid.

(A) Schematic for experimental procedures. The mice were sacrificed by either cervical dislocation (Dislocation) or CO₂ for 5 min followed by cervical dislocation (CO₂ 5 min Dislocation). The retina and RPE/choroid were isolated in cold HBSS and extracted metabolites were analyzed by mass spectrometry. (B–C) CO₂ decreased mitochondrial metabolites and promoted purine degradation in retina. Data were ion intensity normalized by those in Dislocation. *P < 0.05 with t-test, N = 3. High Energy, high energy phosphates. (C) A schematic for purine degradation pathway. Green represents decrease and red represents increase. (D) CO₂ impacts multiple metabolic pathways in RPE/choroid. Data were ion intensity normalized by those in Dislocation. 3 PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate, 2-HG, 2-hydroxyglutarate; SSA, Succinic semialdehyde; N-ASP, N-acetylaspartate; GSH, glutathione. *P < 0.05 with t-Test, N = 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2.. The impact of isolation on metabolites in retina and RPE/choroid.

(A) Schematic for different isolation procedures (See details in the methods). (B) The impact of isolation procedures on metabolites in retina. Data were normalized by the HBSS group with Enucleate & Dissect. *P < 0.05, N = 3. (C–D) The impact of short delay in cold HBSS after enucleation on metabolites in retina. The enucleated eye balls were left in cold HBSS for 0, 2.5 or 5 min before isolation in cold HBSS. *P < 0.05 vs 0 min group and ^#P < 0.05 vs 2.5 min group. (D) The impact of isolation procedures on metabolites in retina. Data were normalized by the HBSS group with Enucleate & Dissect. *P < 0.05, N = 3.

Fig. 3.. The impact of postmortem delay on metabolites in retina.

(A) Schematic on postmortem delay. After enucleation, the eyeballs were kept in cold moisture for different time intervals before isolation. (B) Heat map of significantly changed metabolites vs. 0 h group by One-Way ANOVA test (P < 0.05, n = 3). The data was normalized by the average from each row and clustered for similar patterns using Metaboanalyst 3.0 software. (C) The number of metabolites that were significantly changed vs. 0 h group. (D) The top changed metabolites with fold change > 10 over 0 h group. N = 3.

Fig. 4.. The impact of postmortem delay on metabolites in RPE/choroid.

(A) The number of metabolites that were significantly changed vs. 0 h group in the RPE/choroid. (B) Heat map of significantly changed metabolites vs. 0 h group by One-Way ANOVA test (P < 0.05, n = 3). The data was scaled by the average from each row and clustered for similar patterns using Metaboanalyst 3.0 software. (C) The top changed metabolites with fold change > 3 in the 8 h over 0 h group. N = 3.

Fig. 5.. D/F12 medium partially rescued the change in metabolite by postmortem delay.

(A) Schematic on experiment procedures. The enucleated eyeballs were placed in either a cold and moist 96-well plate or cold D/F12 medium for 8 h before isolation. (B) D/F12 medium rescued changes of some metabolites in the retinas from storage for 8 h *P < 0.05 vs the retinas dissected at 0 h and ^#P < 0.05 vs the retinas storage in the moisture for 8 h by One-Way ANOVA test. N = 3. (C) D/F12 medium rescued changes of some metabolites in the RPE/choroid from stored for 8 h *P < 0.05 vs the RPE/choroid dissected at 0 h and #P < 0.05 vs the RPE/choroid stored in the moisture for 8 h by One-Way ANOVA test N = 3.