Chapter 65

Correlation Between Tissue Docosahexaenoic Acid Levels and Susceptibility to Light-Induced Retinal Degeneration

Masaki Tanito, Richard S. Brush, Michael H. Elliott, Lea D. Wicker, Kimberly R. Henry, and Robert E. Anderson

Abstract In a mouse model of acute light-induced retinal degeneration, positive correlations between the levels of DHA, the levels of n3 PUFA lipid peroxidation, and the vulnerability to photooxidative stress were observed. On the other hand, higher sensitivity of the electroretinogram a-wave response, a measure of the amplification of the phototransduction cascade, was correlated with higher retinal DHA levels. These results highlight the dual roles of DHA in cellular physiology and pathology.

65.1 Introduction

Docosahexaenoic acid (DHA; 22:6n3) is more abundant in rod photoreceptor outer segments (ROS) than in any other mammalian membrane (Fliesler and Anderson 1983). Studies in rodents and monkeys have demonstrated that DHA plays an important role in retinal function (Benolken et al. 1973; Wheeler et al. 1975; Birch et al. 1992; Bush et al. 1994; Jeffrey et al. 2002; Mitchell et al. 2003; Anderson and Penn 2004; Niu et al. 2004). Animals cannot synthesize n3 or n6 fatty acids de novo and must rely on a dietary source of these essential fatty acids. The fat-1 gene, cloned from C. elegans (Spychalla et al. 1997), encodes an n6 desaturase that converts n6 to n3 polyunsaturated fatty acids (PUFA). This transgene has been expressed in mice (Kang et al. 2004), which were found to produce n3 PUFA when fed a diet containing only n6 PUFA.

Acute light exposure to rats and mice causes photoreceptor and retinal pigment epithelial (RPE) cell damage (Tytell et al. 1989). Exposure of the retina to intense light causes lipid peroxidation of retinal tissues (Wiegand et al. 1983; Organisciak

M. Tanito (B)

Department of Ophthalmology, Shimane University Faculty of Medicine, Enya 89-1, Izumo, Shimane, 693-8501, Japan

e-mail: tanito-oph@umin.ac.jp

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_65,C Springer Science+Business Media, LLC 2010

et al. 1992; Tanito et al. 2006a) and lipid peroxidation is propagated by free radicals, especially lipid radicals (De La Paz and Anderson 1992; Winkler et al. 1999). Thus, double bonds in PUFA are target substrates to propagate oxidative stress in photoreceptors.

65.2 Methods

All procedures were carried out according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the University of Oklahoma Health Sciences Center (OUHSC) Guidelines for Animals in Research. The breeding pairs of fat-1 transgenic mice carrying a fat-1 gene of Caenorhabditis elegans and wildtype C57BL/6 J were kindly provided from Dr. Jing Kang (Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA) (Kang et al. 2004). Fat-1 C57BL/6 J mice were bred onto a Balb/c background and both C57BL/6 J and Balb/c fat-1 animals were independently utilized alongside their fat-1 negative wildtype siblings (wt animals). Fat-1 and wt males expressing the fat-1 gene were bred to wild type females that, prior to breeding, had been placed on a semi-synthetic, modified AIN-76A diet (#180465; Dyets, Bethlehem, PA) containing 10% (wt/wt) safflower oil (n6/n3 ratio of 274). Mice were born and raised under a cyclic light environment (< 30 lux, 12 h on/off, 7AM-7PM) in the Dean A. McGee Eye Institute vivarium and weaned onto the SFO diet (SFO animals).

Fatty acid profiles were analyzed in ROS, cerebellum, plasma, and liver from fat-1-SFO and wt-SFO of both C57BL/6 J and Balb/c strains. Purified lipid extracts from plasma, liver, and cerebellum were resolved into neutral lipid classes using one-dimensional thin-layer chromatography (TLC). Fatty acids from scraped TLC spots and from purified lipid extracts from ROS were derivatized to form fatty acid methyl esters (FAMES) and analyzed using gas-liquid chromatography (GLC) (Morrison and Smith 1964).

For the damaging light exposure experiments, 6-week-old Balb/c fat-1 and wt mice fed with a safflower oil diet (fat-1-SFO and wt-SFO, respectively) were exposed to 3,000 lux diffuse, cool, white fluorescent light for 24 h as described previously (Tanito and Anderson 2006) with slight modifications. After light exposure [light (+) animals], the mice were kept under the cyclic light environment (< 30 lux, 12 h on/off, 7AM-7PM) for up to 1 wk, after which electroretinograms (ERGs) were recorded and eyes were enucleated for morphometric and biochemical analyses. The outer nuclear layer (ONL) thickness was measured in retinal sections as described previously (Tanito et al. 2007). TUNEL was performed on paraffinembedded sections using an Apoptag Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA) according to the manufacturer’s instructions. Western dot blot analyses for 4-hydroxynonenal (4-HNE)- and 4-hydroxyhexenal (4-HHE)- modified retinal proteins were performed as previously described (Tanito et al., 2005) with slight modification.

Fig. 65.2 Relative mole percentage of n-3 and n-6 PUFA from total lipid extracts of rod outer segments from C57BL/6 J and Balb/c fat-1 transgenic mice and control siblings all maintained on a diet consisting of 10% safflower oil. p<0.001, p<0.01, p<0.05; Multivariant ANOVA with post-hoc Neuman-Keuls test (n=3). This figure is reproduced from Tanito et al., Journal of Lipid Research 2008 Nov 20 with permission from the American Society for Biochemistry and Molecular Biology

group compared to those in the wt-SFO group (Tanito et al. 2009). The ONL thickness was significantly reduced in fat-1-SFO compared to wt-SFO light (+) animals (Tanito et al. 2009). In light (+) animals, larger numbers of TUNEL positive ONL cell nuclei were observed in retinal sections from the fat-1-SFO group compared to those from the wt-SFO (Tanito et al. 2009).

The levels of proteins modified by 4-HNE and 4-HHE, reactive aldehydes derived from non-enzymatic oxidation of n6 and n3 PUFAs, respectively, were tested in Balb/c wt-SFO and fat-1-SFO retinas from light (–) and light (+) animals by Western dot blot. In light (+) animals, the levels of 4-HNE protein modifications increased significantly in both groups compared to the corresponding light (–) animals, but no differences were detected between the 2 light-exposed groups (Tanito et al. 2009). However, in light (+) animals, the levels of 4-HHE protein modification increased significantly in the fat-1-SFO group compared to corresponding light (–)