Retina neural

The retina comprises two principal components, the non-neural retinal pigment epithelium and the neural retina. The retinal pigment epithelium is an essential component of the visual system both structurally and functionally. It is important for the turnover and phagocytosis of photoreceptor outer segments, the metabolism of retinoids, the exchange of nutrients between the photoreceptors, and the choroidal blood vessels and the maintenance of an efficient outer blood-retinal barrier. 

Interestingly, carotenoids more abundant in the blood plasma than zeaxanthin, such as lycopene, P-carotene, and P-cryptoxanthin, do not accumulate in the retina. RPE cells express p,p-carotene 15,15 -monooxygenase (BCO), formerly known as P-carotene 15,l5 -dioxygcnase, an enzyme that catalyzes the oxidative cleavage of P-carotene into two molecules of all-trans-retinal (Aleman et al., 2001 Bhatti et al., 2003 Chichili et al., 2005 Leuenberger et al., 2001 Lindqvist and Andersson, 2002). Therefore it may be suggested that p -carotene transported into RPE-cells is efficiently cleaved into retinal molecules. BCO cleaves also P-cryptoxanthin (Lindqvist and Andersson, 2002), and its absence in the retina may also be explained by its efficient cleavage to retinoids. However, lycopene, often the most abundant carotenoid in human plasma, cannot serve as a substrate for BCO, and yet it is not detectable in the neural retina (Khachik et al., 2002). 

These data suggest that one of possible mechanisms of carotenoid delivery to the neural retina may involve lipoprotein uptake from the basal side of the RPE followed by its retro-endocytosis on the apical site (Lorenzi et al., 2008). Alternatively, the endocytosed lipoprotein may be degraded in the RPE followed by secretion of certain lipophilic components from the lipoprotein at the apical site. Due to low solubility of carotenoids in aqueous solutions, it may be suggested that they are secreted already bound to a protein or that an acceptor protein is available in the interphotoreceptor matrix and/or POS. 

ApoC-I is expressed mainly in liver but also in lung, skin, testis, spleen, neural retina, and RPE. Its multiple functions include the activation of lecithin cholesterol acyltransferase (LCAT) and the inhibition, among others, of lipoprotein and hepatic lipases that hydrolyze triglycerides in particle cores. Notably, both LCAT and lipoprotein lipases are expressed in RPE and choroid (Li et al., 2006). Moreover ApoC-I has been shown to displace ApoE on the VLDL and LDL and thus hinder their binding and uptake via their corresponding receptors (Li et al., 2006). 

ApoC-II is expressed in liver and intestine, and both the neural retina and RPE (Li et al., 2006). In contrast to ApoC-I, it can function as an activator of lipoprotein lipase. Similar to ApoA-I, ApoA-II, and ApoE, in the absence of lipid to stabilize its structure, ApoC-II forms amyloid assemblies. 

ApoJ is another protein component of HDL which is highly expressed by the RPE and neural retina, especially under oxidative stress conditions (Wong et al., 2000, 2001). It can act as a complement regulatory protein, which by binding to and inactivating the membrane-attack complex can prevent cytolysis (Bartl et al., 2001). ApoJ accumulation was identified in drusen in AMD patients (Sakaguchi et al., 2002 Wong et al., 2000). 

The expression of all these apo-lipoproteins by the RPE, and its ability to form lipoprotein particles suggest that these newly formed lipoproteins may be involved in the transport of lipophilic molecules, including carotenoids, from the RPE to the neural retina and/or to the choroidal blood supply. Testing the roles of apolipoproteins and lipoprotein particles in carotenoid secretion from the RPE is another subject awaiting experimental investigation. 

While it may be speculated that in the RPE both lipoprotein and/or scavenger receptors are likely to be involved in carotenoid uptake from the blood, it is not clear what mechanism(s) are responsible for carotenoid transport through the RPE into the neural retina. Also, it is not clear what mechanism(s) are responsible for selective accumulation in the retina of only two carotenoids. 

In addition to its presence in the RPE, ABCA1 has been found to be localized in the neural retina, particularly in the ganglion cell layer and rod photoreceptor inner segments (Tserentsoodol et al., 2006a), suggesting it may be involved in carotenoid transport throughout the retina. 

Apart from SR-BI, SR-BII, CD36, and ABCA1, a microarray analysis of gene expression in human RPE reveals some additional lipid transporters that might potentially be involved in intracellular transport of carotenoids and/or their efflux from the RPE cells into the neural retina or out of the retina into the choroidal blood (van Soest et al., 2007). These include other ABC... 

Altogether, the role of transporters of lipophilic molecules regulating the movement of carotenoids through the RPE and into the neural retina is another area awaiting experimental investigation. 

Altogether, there are many unknowns about carotenoid transport in the retina. However, present knowledge on carotenoid uptake in other cell types and the finding of multiple proteins potentially involved in carotenoid transport in the RPE and adjacent neural retina leads to the suggestion that several hypothetical pathways exist (Figure 15.3). Many such pathways can be easily tested in cultured RPE. 

Hemopexin was first identified as a heme binding P-globin in elec-trophoretograms of plasma of patients with hemolysis (17-19). The protein is synthesized and secreted by the liver (20-22), and during secretion the signal peptide is removed and the protein is glycosylated (23). Tissue forms of hemopexin are expected due to the presence of mRNA in brain (24), peripheral neurons (25), and neural retina (26), pointing to a function of hemopexin in barrier tissues. 

Chick embryo neural retina cells have also been grown in culture (Daston et al., 1995), and studies indicate positive responses to chemicals at concentrations similar to those active in in vivo tests. Reinhardt (1993) has reviewed the use of organ slices, aggregate cell cultures and the micromass techniques for the study of neurodev-elopmental toxicity. Two recent reviews of these in vitro systems, especially the micromass and the chick embryo neural retinal cell system, have been published (Daston, 1996a Mirkes, 1996). 

Daston GP, Baines D, Elmore E, Fitzgerald MP, Sharma S (1995) Evaluation of chick embryo neural retina cell culture as a screen for developmental toxicants. Fundam Appl Toxicol, 26 203-210. 

The ability of Ca2+ to crosslink groups and so stabilize proteins against thermal denaturation or hydrolytic attack is well illustrated by thermolysin, which involves four Ca2+ and a catalytic Zn2+. The function of the calcium is to stabilize the protein structure.404 Calcium also protects surface protein of the neural retina from tryptic cleavage.405... 

Figure 5.1 A schematic showing the neural retina lining the back of the eye and the expanded retina on the right with the macular pigments in the inner layers.

Neural retina 11) Diffusion across neural retina 11) In vitro permeabiUty of human and animal retina in vivo permeabiUty of rabbit retina... 

Retinal pigmental epithelia (RPE) are dopaminergic support cells in the neural retina. RPE cells on gelatin beads, also called Spheramine, produce levodopa (Watts et al., 2003), but there are no data yet on the consistency of dopamine production by these cells. An open label clinical trial of transplantation of Spheramine was conducted in six patients with PD (Bakay et al., 2004). Spheramine was transplanted into the striatum and show ed clinical effects over 24 months. Spheramine is now undergoing double bhnd placebo controlled trials in advanced PD. 

Daniotti JL, Martina JA, Zurita AR, Maccioni HJ (1999) Mouse /11,3-galactosyltransferase (GAl/GMl/GDlb synthase) protein characterization, tissue expression, and developmental regulation in neural retina. J Neurosci Res 58 318-327... 

Due to the limited applicability of in silico SAR approaches for developmental toxicity, there is more reliance on in vitro screening. From what has been publicly disclosed, it is evident that the four in vitro tests used for industrial screening are chick embryonic neural retina (CENR) micromass culture, whole embryo culture (WEC, rodent or rabbit), and mouse embryonic stem cells (EST). Recently, there has been significant interest within the pharmaceutical industry in the use of zebrafish for developmental toxicity testing,30 but because this aspect is in its infancy, there is little that has been publicly disclosed except limited abstracts and slide decks at several workshops.31 Although reviewed in considerable detail elsewhere,30-32 36 each test will be briefly compared and contrasted here. 

Figure 9-1 The highlights of the chick embryonic neural retina micromass cultures are depicted. The tissue is harvested and digested to a single-cell suspension. These are placed into culture, and after 24 h three parameters are measured (a) the number of aggregates that are produced, (b) the size of the aggregates, and (c) their protein content. After five days cortisol is added to precociously induce glutamine synthetase activity. This is measured two days later, after a total of seven days of culture. Each parameter is uniquely sensitive to different agents, but a decrease in any one parameter is considered to be toxicologically relevant.

Chick embryo neural retina cell culture model... 

Figure 9-5 The sensitivity of the conceptus to a theoretical teratogen during rat gestation (modified from 161). The most susceptible window is organogenesis with low levels of vulnerability at the time of implantation and the period of functional maturation. Superimposed are the approximations of when the developmental landmarks that are represented in the four in vitro tests occur. The chick embryo neural retina model (CENR) represents events around GD 10-13. The mouse embryonic stem cell test (EST) corresponds roughly to the period of GD 6-10 in the rat, near the peak of sensitivity. Whole embryo culture (WEC) recapitulates the window at the peak of sensitivity, between GD 9-11 or GD 10-12 depending upon the window within which the culture is conducted. Rabbit cultures are also done between GD 10-12. Represented by the single ( ) and double asterisk ( ), respectively, are the initiation and termination of the dosing period in regulatory compliant preclinical embryo/fetal toxicity studies. Thus, the zebrafish is the only model that permits exposure to test article during this important period.

Figure 9-6 A generic strategy integrating the three facets of developmental toxicity risk assessment namely (a) the risk of pharmacologic modulation of the therapeutic target during gestation, (b) in silico, SAR and (c) in vitro screening. Abbreviations The chick embryo neural retina (CENR) embryonic stem cell test (EST), whole embryo culture (WEC), Good Laboratory Practice (GLP), Embryo/Fetal Developmental Toxicity (EFD) study. "Front-loading" is the conduct of the EFD study prior to Phase lib.

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