Abstract
Purpose :
Mitochondrial abnormalities are reported in retinal pigment epithelial (RPE) cells of patients with age-related macular degeneration (AMD). These include macular mitochondrial DNA mutations, respiratory chain protein dysfunction and gross structural changes in mitochondria size and cristae. Mitochondria in mouse models with features of macular degeneration may share similar pathology. We present a novel method analyzing mitochondrial respiratory parameters of relevant mouse eye tissues.
Methods :
C57BL/6 male mouse eyes were enucleated and maintained on ice in basic cell culture media. Retina was dissected and separated from the posterior eye cup (PEC) which consists of the RPE, Bruch’s membrane and choroid/sclera. PEC tissues were dissected into different sizes (whole, 1/2, 1/4, 1/8, 1/16) and oxygen consumption rates (OCR) in pmols/min were assessed in standard Seahorse XF96 Cell Culture Microplates. Glucose, pyruvate and glutamine were supplied as metabolic substrates. PEC were sequentially treated with: 1) 10 µM of oligomycin (ATP synthase inhibitor) 2) 1 µM carbonilcyanide p-triflouromethoxyphenylhydrazone FCCP (mitochondrial uncoupler) and 3) 5 uM each of rotenone and antimycin A (complex I and III inhibitors, respectively). Analysis was performed with different amounts of HEPES buffer. Mice were dosed with systemic sodium iodate (7.5 – 75 mg/kg) or vehicle and eyes were collected 24 hrs post-dose for analysis. The two-tailed t-test was used for statistical analyses.
Results :
Large PEC (≥ 1/4 size) became hypoxic in the microchambers. Tissue size 1/16 were selected for further study as OCR readings were within the range to respond dynamically to treatment. Oligomycin treatment decreased OCR level; uncoupling with FCCP elicited OCR readings greater than basal OCR, and rotenone/antimycin A depressed all mitochondrial OCR. ATP synthase inhibition prior to uncoupling did not impede maximal respiration as has been reported in some cell cultures. Buffering with 10 mM HEPES improved the metabolic profile. PECs isolated from mice treated with sodium iodate at 75mg/kg inhibited PEC basal OCR (↓59%, p < 0.001) as compared to vehicle.
Conclusions :
The Seahorse XF analyzer can be used to assess mitochondrial function of ocular tissues and investigate mitochondrial dysfunction in mouse AMD models.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.