Chapter 17

The Role of Oxidative Stress

in the Retinal Lesion of Ccl2/Cx3cr1 Defic iency Mouse on rd8Background

Jingsheng Tuo

Abstract The Ccl2−/−/Cx3cr1−/− deficiency mouse on rd8 background spontaneously develops a broad spectrum of age-related macular degeneration (AMD)-like lesions including abnormal retinal pigment epithelium, focal photoreceptor degeneration, and lipofuscin accumulation. Studies suggest that disturbance of multiple factors/ pathways is involved in the pathogenesis of the retinal lesion of Ccl2−/−/Cx3cr1−/−/rd8, which include endoplasmic reticulum stress, inflammatory response, malfunction of microglia, and oxidative stress. This chapter summarizes the retinal pathological/ molecular features of Ccl2−/−/Cx3cr1−/−/rd8; presents the evidences of enhanced oxidative stress relevant molecules in the retina of Ccl2−/−/Cx3cr1−/−/rd8; reports two studies that directly or indirectly manipulated the status of oxidative stress to intervene the development of the retinal lesion of Ccl2−/−/Cx3cr1−/−/rd8; and suggests that the oxidative stress plays an important role in causing the mouse retinal lesions that mimic human AMD lesions.

17.1Introduction

Age-related macular degeneration (AMD) is the leading cause of blindness in Western countries among the elderly [1]. Strong evidence indicates the involvement of oxidative stress in AMD [2, 3]. Oxidative stress, which refers to cellular damage caused by reactive oxygen/nitrogen species (ROS/RNS), has been implicated in many disease processes, especially age-related disorders. The retina is particularly susceptible to oxidative stress because of its high consumption of oxygen, its high proportion of polyunsaturated fatty acids, and its exposure to visible light. In vitro

J. Tuo (*)

Laboratory of Immunology, National Eye Institute, 10/10N103, NIH/NEI, 10 Center Dr, Bethesda, MD 20892-1857, USA

e-mail: tuoj@nei.nih.gov

in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-61779-606-7_17, © Springer Science+Business Media, LLC 2012

studies have consistently shown that photochemical retinal injury is attributable to oxidative stress and that the antioxidant vitamins A, C, and E protect against this type of injury. In vivo, oxidative stress is significant in AMD pathogenesis by interfering with retinal pigment epithelium (RPE) function, decreasing RPE junction integrity, enhancing RPE expression of proinflammatory and proangiogenic cytokines, and/or triggering RPE apoptosis. ROS could activate the metabolism of arachidonic acid thus alter cellular redox balance, which is implicated in the pathogenesis of AMD. Additionally, several studies have successfully demonstrated associations between AMD and various single nucleotide polymorphisms (SNPs). Some of these SNPs are within genes encoding molecules for oxidative stress and oxidative damage repair molecules [4–8].

Considerable efforts have been made to establish AMD animal models [9–14]. Even though mice have no macula, existing mouse AMD models have been shown to develop cardinal pathological features of the disease [10, 15, 16]. CX3CR1 is the receptor for CX3CL1/fractalkine chemokine and is expressed in the brain microglia and the eye [17–19]. In a functional study, two CX3CR1 SNPs resulted in a decreased number of CX3CL1 binding sites and reduced ligand binding affinity on peripheral blood mononuclear cells [20]. It was reported that these SNPs are associated with AMD [21–23]. Furthermore, we have demonstrated a decreased number of CX3CR1 transcripts and protein in AMD maculae compared to the maculae of normal eyes [21, 24]. AMD-like ocular abnormalities are reported in Cx3cr1 deficiency senescent mice [22]. In addition to CX3CR1, CCL2 (MCP-1, a CC chemokine) has also been suggested to play a homeostatic and immunoregulatory role in AMD pathogenesis [25]. Aged mice with deficient Ccl2 or Ccr2, the corresponding receptor, develop features of AMD [15]. We hypothesized that deficiencies in both CX3CR1 and CCL2 may induce typical AMD pathological features in mice more consistently and at an earlier age of onset than existing animal models. We cross-bred Ccl2 and Cx3cr1 single knockout mice [26, 27] and established an inbred strain of Ccl2/Cx3cr1 deficiency mouse on rd8 background (Ccl2−/−/Cx3cr1 −/−/rd8) [28].

17.2Retinal Pathological/Molecular Features of Ccl2−/−/Cx3cr1−/−/rd8

17.2.1Fundoscopy

Sequential fundoscopy examinations found all 6–9-week-old Ccl2−/−/Cx3cr1−/− mice spontaneously developed deep retinal lesions characterized by heterogeneous, round or domed-shaped, soft-bordered, yellowish deposits within the subretinal space. With aging, these lesions enlarged or flattened and became confluent (Fig. 17.1). Some of the lesions progressed to form chorioretinal scars and depigmented atrophic areas. In comparison, none of the single knockout Ccl2−/− or Cx3cr1−/− mice developed retinal lesions at such young ages [15, 22].

within the RPE (hypopigmentation), a thickened Bruch’s membrane with both basal linear and laminar deposits, and disorganization or atrophy of the photoreceptors. In addition, loss of tight junctions and cellular membrane folding were noted in some RPE cells. The ultrastructural findings indicated drusen formation and degeneration of the RPE and photoreceptors. These findings are reminiscent of the ultrastructural changes observed in human AMD cases [29]. Moreover, these abnormalities were more severe in the older mice, indicating a progressive degenerative process during aging. In contrast, none of the abovementioned abnormalities were found in the eyes of the age-matched wild type mice.

17.3 The Detection of Molecules Relevant to Oxidative Stress in the Retina of Ccl2−/−/Cx3cr1−/−/rd8 Mice

17.3.1Lipofuscin (A2E)

Accumulation of lipofuscin is an early pathological feature observed in human AMD [30]. Studies have shown that short-wavelength visible light induces retinal injury and may be a risk factor for AMD. A2E is a blue light absorbing retinal chromophore that accumulates with age, is one of the major fluorophores of lipofuscin, and is derived from retinoid metabolism in the retina and RPE. The generation of A2E is mediated by a series of steps of photoreaction. The oxidation of the intermediates of the rhodopsin regeneration is required for the formation of A2E [31]. Although A2E itself has a low phototoxic efficiency, the further oxidation of A2E can form highly toxic derivatives in the presence of visible light, which contributes to observed RPE photodamage [32].

The retinal accumulation of A2E in Ccl2−/−/Cx3cr1−/−/rd8 can be detected by HPLC. We measured A2E levels within the retina and found a significant increase of over threefold in 15 week-old and older Ccl2−/−/Cx3cr1−/−/rd8 mice (3.4 pmol of A2E per eye) as compared to the age-matched WT (~1 pmol). The quantification of ocular A2E has become our standard endpoint for assessing the effect of intervention to the retina lesion of Ccl2−/−/Cx3cr1−/−/rd8 [33].

17.3.2The Peroxisome Proliferator-Activated Receptors

The peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that function in the regulation of in lipid degeneration, oxidative stress, and vascular endothelial growth factor (VEGF). PPARs are implicated in the pathophysiology AMD. There are three different subtypes of PPARs: a, b, and g.