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

Abstract

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.

Figure 1.

Overall workflow of the research procedure. Corneal biopsies were obtained using disposable biopsy punches after euthanizing the mice. The biopsies were loaded into a Seahorse XFe96 spheroid microplate, one per well. The metabolic profile and mitochondrial respiration rates of the biopsies were assessed using a Seahorse XFe96 pro analyzer.

Figure 2.

Metabolic profile of murine corneal biopsy. (A) ATP production from glycolysis (Glyco) and mitochondria (Mito) were measured in 1.5-mm diameter corneal punches with the epithelium side up from 8-week-old male C57BL/6J mice (n = 10 corneal biopsies from 5 mice) using real-time ATP rate assay. Values are expressed as mean ± standard deviation. (B) The average percentages of ATP production from glycolysis and oxidative phosphorylation in murine corneal biopsy (n = 10 corneal biopsies from 5 mice).

Figure 3.

The impact of different euthanasia methods on mouse corneal oxygen consumption. (A) Mitochondrial stress test in the corneal biopsies from male C57BL/6J mice euthanized by carbon dioxide asphyxiation (CO₂) and an overdose of the anesthetic drug (overdose). (B–D) Statistic analysis graphs of basal, maximal, and spare mitochondrial respiration capacities. Values are expressed as mean ± standard deviation, n = 8 corneal biopsies from 4 mice. FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ns, nonsignificant.

Figure 4.

The impact of punch sizes on mouse corneal oxygen consumption. (A) Plot showing OCR curves of individual corneal punches in 1.0-mm (red) and 1.5-mm (blue) diameter. (B, C) Mitochondrial stress test using corneal punches of 1.0-mm diameter (n = 10 corneal biopsies from 5 mice) and 1.5-mm diameter (n = 8 corneal biopsies from 4 mice) before normalization (B) and after normalization by the area of punches (C). (D–F) OCR values in the corneal punches of 1.0-mm diameter (n = 10 corneal biopsies from 5 mice) and 1.5-mm diameter (n = 8 corneal biopsies from 4 mice) after normalization by the area of punches. Values are expressed as mean ± standard deviation. ns, nonsignificant. **P < 0.01; ***P < 0.001. FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone.

Figure 5.

The impact of biopsy orientation and biopsy locations on mouse corneal oxygen consumption. (A) A representation figure of the orientation of corneal biopsies with the epithelium (epi) and endothelium (endo) facing the cartridge sensor. (B–E) The OCR values in the murine corneal biopsy with the epithelial layer (epi) and endothelium (endo) facing upward (n = 8 corneal biopsies from 4 mice). (F) A diagram showing the location of biopsies in the central or off-central regions of the cornea. A star symbol (★) represents the center of the cornea. (G–J) The mitochondrial OCR in the punches from the central or off-central areas of the corneas. Values are expressed as mean ± standard deviation, n = 8 corneal biopsies from 4 mice. FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ns, nonsignificant.

Figure 6.

The impacts of sex, strain, and age on murine corneal OCR values. (A–D) Mitochondrial stress test in the corneal punches from male and female C57BL/6J mice (n = 8 corneal biopsies from 4 mice). (E–H) OCR levels in the cornea punches from C57BL/6J and BALB/cJ mice (n = 8 corneal biopsies from 4 mice). (I–L) Mitochondrial stress test in the corneas from 4-, 8- and 32-week-old male C57BL/6J mice (n = 8 corneal biopsies from 4 mice). Values are expressed as mean ± standard deviation. FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ns, nonsignificant.

Figure 7.

Mitochondrial respiration measurement in wounded corneas. (A–D) Mitochondrial stress test in the unwounded cornea and corneas at 0 d and 3 d after wound (n = 8 corneal biopsies from 4 mice). Values are expressed as mean ± standard deviation. FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ns, nonsignificant; **P < 0.01; ***P < 0.001; ****P < 0.0001.