Retinal metabolic events in preconditioning light stress as revealed by wide-spectrum targeted metabolomics

Introduction Light is the primary stimulus for vision, but may also cause damage to the retina. Pre-exposing the retina to sub-lethal amount of light (or preconditioning) improves chances for retinal cells to survive acute damaging light stress. Objectives This study aims at exploring the changes in retinal metabolome after mild light stress and identifying mechanisms that may be involved in preconditioning. Methods Retinas from 12 rats exposed to mild light stress (1000 lux × for 12 h) and 12 controls were collected one and seven days after light stress (LS). One retina was used for targeted metabolomics analysis using the Biocrates p180 kit while the fellow retina was used for histological and immunohistochemistry analysis. Results Immunohistochemistry confirmed that in this experiment, a mild LS with retinal immune response and minimal photoreceptor loss occurred. Compared to controls, LS induced an increased concentration in phosphatidylcholines. The concentration in some amino acids and biogenic amines, particularly those related to the nitric oxide pathway (like asymmetric dimethylarginine (ADMA), arginine and citrulline) also increased 1 day after LS. 7 days after LS, the concentration in two sphingomyelins and phenylethylamine was found to be higher. We further found that in controls, retina metabolome was different between males and females: male retinas had an increased concentration in tyrosine, acetyl-ornithine, phosphatidylcholines and (acyl)-carnitines. Conclusions Besides retinal sexual metabolic dimorphism, this study shows that preconditioning is mostly associated with re-organisation of lipid metabolism and changes in amino acid composition, likely reflecting the involvement of arginine-dependent NO signalling. Electronic supplementary material The online version of this article (doi:10.1007/s11306-016-1156-9) contains supplementary material, which is available to authorized users.

Evidence has been provided for carnitine uptake and oxidation of FA released from phagocytized photoreceptors by RPE cells (Adijanto et al. 2014;Tini 2002). Our results suggest a relatively decreased fatty acid (FA) oxidation rate in male compared to female retinas. That is, FA released from phagocytized photoreceptors in RPE cells may have outpaced mitochondrial oxidative capacity, eventually leading to an increased acyl carnitine accumulation in male retinas. Nevertheless, the activity of CPT-1, as measured by the (C16+C18)-to-C0 ratio, was not significantly higher in female compared to male retinas. It is possible that other "post-CPT-1 events" such as an increased mitochondria number or overall mitochondrial activity could have led to an increase FAO capacity in females, a pattern similar to that found in human skeletal muscle (Kim et al. 2000).
As a matter of fact, a likely cause of the sexual dimorphism in lipid metabolism is the increased estrogen-mediated mitochondrial biogenesis in female retinas. Estrogens increase mitochondrial biogenesis by upregulating nuclear and mitochondrial transcription factors like the Nuclear Respiratory Factor 1 and 2 (NRF 1 and NRF 2), the mitochondrial transcription factor (Tfam) and the three members of the PPARγ coactivator 1 (PGC-1) family, PGC-1α, PGC-1β and PGC-1 related coactivator (PRC) (Klinge 2009;Mattingly et al. 2008;Tcherepanova et al. 2000). The interaction between PGC-1α and members of the peroxisome proliferator-activated receptor (PPARs) family has been shown to increase not only mitochondria biogenesis but also FAO in muscle and liver (Finck and Kelly 2006). It has also been shown that PPARγ expression in RPE cells is selectively enhanced after photoreceptor phagocytosis (Ershov and Bazan 2000). In addition, the peak in PGC-1α expression in mouse RPE cells has been found to coincide with maximal RPE phagocytosis activity (Stone et al. The expression of both subtypes of estrogen receptors alpha and beta (ER α and ER β) and androgen receptor (AR) in rat and human retinas has been well documented (Kobayashi et al. 1998;Munaut et al. 2001;Ogueta et al. 1999;Rocha 2000;Wickham et al. 2000).
Furthermore, the retina, as well as other structures of the central nervous system (CNS), is capable of steroidogenesis with synthesis of estradiol (E2) from cholesterol and from testosterone aromatization (reviewed in (Cascio et al. 2015)). Taken as a whole, the hypothesis of a stimulation of mitochondria biogenesis and FAO capacity by estrogens in females is likely. We nevertheless recognize that further research has to be conducted to examine the specific involvement of estrogens in facilitating lipid recycling after photoreceptor phagocytosis.
In addition to lipids, some nitrogen-containing compounds have been found to be sexually dimorphic. In fact, male retinas had a relatively increased concentration in acetyl-Supplementary Method S1. Tissue collection, histological and immunohistochemistry (IHC) analysis and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) analysis.

Tissue collection
The superior aspect of each eye was marked for orientation. Each eye was enucleated with curved forceps and fixed in 4% paraformaldehyde. Fixed eyes were washed with 1M PBS and cryoprotected in 15% sucrose overnight at 4 o C. All eyes were embedded in OCT medium (Leica Biosystems, Australia) prior to being frozen in acetone and dry ice. Cryosections were 12µm thick, cut in the parasagittal plane (superior-inferior) (CM1850; Leica); with the same orientation on Superfrost UltraPlus glass slides (ThermoScientific, Australia). Sections were then dried overnight at 37 o C and stored at -20 o C. Only sections containing the optic nerve head were used for analysis.
Retinas were visualized with the A1 Nikon confocal microscope and images were taken using NIS-Elements Advanced Research software (Nikon) at 10x magnification. Areas of interest were marked using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA) and their corrected total cell fluorescence (CTCF) was calculated as follows: CTCF= Integrated density -(Area of interest x Mean fluorescence)

Histology
Cryosections were fixed with 10% neutral buffered formalin (Sigma-Aldrich, Cat# HT501128-4L) then hydrated sequentially through decreasing concentrations of ethanol and Milli-Q water (Millipore, USA). Sections were then stained with Harris' Haematoxylin for 10 minutes then washed with tap water. After a brief rinse with Milli-Q water, the sections were counter-stained with Eosin-Y solution (Sigma-Aldrich, Cat# HT110216-500ML) for 1 minute. The sections were dehydrated through increasing concentrations of ethanol (70%, 90% and 100% ethanol). They were then cleared in xylene for 5 minutes and coverslipped with Micromount (Leica Biosystems, Australia) and clear nail varnish. Staining was visualised using the brightfield function of the A1 Nikon confocal microscope and images were taken using NIS-Elements Advanced Research software (Nikon) at 10x magnification.
Sections were then incubated in primary antibodies overnight at 4 o C. To control for nonspecific binding by secondary antibodies, primary antibodies were omitted in designated negative control slides. All primary antibodies were diluted in 1% NGS. After the sections were washed in 0.1M PBS, there were incubated with secondary antibodies. Sections stained for COX5a were incubated with their secondary antibody, Mouse IgG-AlexFluor 594 conjugate (1:500; Life Technologies, Australia, Cat# A31623) for 2 hours at RT, while sections stained for IBA-1 were incubated with secondary antibody, Rabbit IgG-AlexFluor 488 conjugate (1:500; Life Technologies, Cat# A31627) for 4 hours at RT. All secondary antibodies were diluted with 0.1M PBS and the addition of all antibodies to slides were done in the dark. Sections were washed in 0.1M PBS before they were incubated in 0.05% Sudan Black B for 10 minutes to prevent autofluorescence. After another wash in 0.1M PBS, the blue fluorescent dye, bisbenzimide (BBZ) (1:1000; Calbiochem, La Jolla, CA), was used to visualise cellular layers as it stains A-T rich regions. After a final wash in 0.1M PBS, sections were coverslipped with Aquamount (Polysciences, Warrington, PA, Cat# 18606) and clear nail varnish. Immunofluorescence was visualised with the A1 Nikon confocal microscope and images were taken using NIS-Elements Advanced Research software (Nikon) at 10x magnification.

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)
Cryosections used for the TUNEL assay were permeabilised using Triton (0.1% w/ PBS) for 2 minutes at RT. Following a wash in 0.1M PBS, the sections were placed in 1M TdT buffer for 10 minutes at RT. TUNEL reaction mixture was made up according to the manufacturer's instructions (contained fresh and contained 89.11% Milli-Q water (Millipore), 10.51% 10M TdT buffer, 0.25% biotin-dUTP and 0.13% terminal transferase). Sections were marked with a PAP pen before incubation with TUNEL reaction mixture for 1 hour at 37 o C with humidity. The reaction was stopped using 2M saline sodium citrate buffer (SSC) for 15 minutes before incubation in 10% normal goat serum for 10 minutes at RT. Streptavidin-AlexFluor 594 conjugate (1:500; Life Technologies, Australia, Cat# S32356) was applied to sections under dim conditions and then incubated for 1 hour at 37 o C with humidity. Sections were washed in 0.1M PBS before being stained with BBZ for 2 minutes to visualise cellular layers. After a final wash in 0.1M PBS, sections were coverslipped with Aquamount and clear nail varnish. In TUNEL and ONL thickness analyses, n = 6 animals were used per condition and at least 4 samples taken from each of the superior and inferior retina. Measurements were averaged for each of the superior and inferior retina for all samples and animals in the same experimental group.                       C3= propionyl-L-carnitine; lyso-PC= lysophosphatidylcholine; PC= phosphatidylcholine; SM= sphingomyelin; MUFA= mono-unsaturated fatty acid; PUFA= poly-unsaturated fatty acid; SFA= saturated fatty acid; CPT-1= carnitine palmitoyltransferase I. Asterisks (*) stand for significant ratios using a constant non-adjusted P-value threshold of 0.05. Black circles (•) stand for significant ratios using adjusted P-values to keep a false discovery rate less than 10% upon multiple comparisons. 0.32 PUFA/SFA 5.6*10 -3 5.7*10 -3 4.5*10 -3 0.048