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Laboratory Science

Drug Screening to Treat Early-Onset Eye Diseases: Can Zebrafish Expedite the Discovery?

Zhang, Liyun MD, PhD*; Chong, Leelyn MS*; Cho, Jin*; Liao, Pin-Chao*; Shen, Feichen*; Leung, Yuk Fai PhD*†

Author Information
The Asia-Pacific Journal of Ophthalmology: November/December 2012 - Volume 1 - Issue 6 - p 374-383
doi: 10.1097/APO.0b013e31827a9969
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Abstract

Early-onset eye diseases, including photoreceptor dystrophy, retinitis pigmentosa (RP), glaucoma, cataract, and corneal dystrophy, generally lead to visual impairment in children. These young patients will have a lifelong inconvenience and an expensive health care cost ahead of them. For example, it is estimated that patients with RP consumes $7317 more in annual expenditure than people without RP.1 Although surgery can treat some of these diseases such as cataract, there are not many effective treatments available for retinal disorders because the damaged retina has no ability to regenerate itself once injured. Nonetheless, some retinal degenerative diseases are progressive, and the visual loss is a gradual process; thus, this has opened a valuable therapeutic window for intervention. In particular, any drugs or therapies that may slow the progression of disease and preserve the residual vision can potentially improve the quality of life of patients and alleviate the financial burden on the health care system. Unfortunately, the process of drug discovery and development involves a significant amount of time and cost2; therefore, it would be particularly helpful to establish new approaches that can identify novel drug candidates in an economical and convenient manner.

Traditionally, high-throughput drug screening involves assay plate preparation and reaction observation in solution and in cultured cells.3,4 Large libraries of tens to hundreds of thousands of chemicals are applied to the testing system.5 A number of assays can be done to reveal and determine the effects of a drug on protein subcellular localization, protein-protein interaction, and signal transduction.6 Combined with automated microscopy and advanced computational analysis, cell-based screening can rapidly reveal the possible function(s) of novel drug candidates.7,8 Nonetheless, analyzing a drug’s effect in vitro cannot entirely replace the need of in vivo screening. This is because a drug’s action and metabolism are not only far more complicated in a multicellular organism, but the efficacy of a drug also depends on its capacity to restore and/or improve an impaired function. The latter issue is particularly true for screening drugs to improve vision, which is the output of multiple cell types in the eye in response to light stimuli. Therefore, it is essential to use in vivo models to screen drugs that can have a therapeutic value in vision improvement. Research that involves traditional laboratory animal models for drug screening is often costly, but the zebrafish model can potentially bridge this gap and expedite drug discovery and development.9

Zebrafish Model for Rapid Drug Discovery

There are 6 major reasons for supporting zebrafish as a good vertebrate model for drug screening, specifically on visual problems: (1) zebrafish’s eye, particularly the retina, is anatomically similar to many vertebrates, including human.10 In fact, zebrafish is a diurnal animal and thus possesses richer color vision than the other classic models such as mice and rats. This feature facilitates research that is focused on diseases affecting cones; (2) zebrafish vision develops early during embryogenesis,10 and thus, visual problems can be examined rapidly; (3) zebrafish can be easily raised in the laboratory at a large scale in an economical manner11; (4) the fecundity of zebrafish is high compared with other animal models, and each pair of fish can lay up to 100 to 200 embryos at a weekly interval. These 2 reasons allow a large number of embryos to be obtained for drug screening; (5) the embryo size is small, and this enables the analysis of individual drug response in a 96-well plate; and (6) various drugs can be simply added to the water and absorbed by the larvae through their skin.12 These last 2 reasons indicate that a large number of drugs can be rapidly administered to the zebrafish embryos.

The feasibility of high-throughput drug screening with zebrafish was first demonstrated by a screening of 1100 small chemicals that could affect various aspects of embryogenesis,13 including heart-patterning defects.14 There was also a screening of 5000 small molecules that successfully revealed 2 suppressors of congenital aortic coarctation in a zebrafish grl mutant,15 demonstrating the possibility of finding novel drugs to treat disease through the use of zebrafish mutants in high-throughput screening.

Visual Behavior of Zebrafish as a Means to Study Visual Impairment and Find Treatment

The behavior of an animal will change when there is a visual impairment. Thus, the characterization of visual behavior is an effective way to assess the presence of visual problem and visual improvement after drug treatment. To identify drugs that can improve vision in zebrafish mutants, it is necessary to identify a robust visual behavior that can be reliably characterized in a high-throughput manner. As a highly visual animal, zebrafish’s visual behaviors have been studied and applied frequently for genetic analysis of vision.16,17 However, many of these behaviors are not suitable for high-throughput drug screening. For example, optokinetic response (OKR), a stereotypic eye movement in response to movement in the environment,18–20 has been used in 4 genetic studies21–24 that identified many mutants with visual problems. Nonetheless, OKR comes with a drawback because it requires frequent transfers of larvae to viscous methylcellulose for immobilization. Although the contemporary implementation of computer-assisted analysis25 may speed up the analyzing process, it is still not versatile enough for a high-throughput drug screening that requires the examination of different drugs at different concentrations, let alone the analysis of multiple fish lines. Recently, a photomotor response (PMR) was used to screen 14,000 drugs in 30 hours postfertilization (hpf) embryos to identify neuroactive drugs.26 Photomotor response consists of a stereotypic series of motor behaviors elicited by a high-intensity light stimulus. However, the retina of 30 hpf embryos is immature, and the first detectable visual response will not occur until at least 68 hpf.27 Thus, PMR is less likely to be compatible for screening zebrafish mutants that have visual impairments.

A novel visual behavior assay has recently been used in a screen of 4000 drugs for identifying candidates that could alter sleeping behavior of fish larvae.28 In this assay, zebrafish larvae were placed individually in each well of a 96-well plate, treated with different drugs, and monitored concurrently by ZebraBox (ViewPoint Life Sciences, Lyon, France), an automated video-tracking system.29 The embryos treated with different drugs elicited different locomotor behaviors in the light and dark phases. Remarkably, it was observed that a drastic response could be induced during the transition between light on and off. This led to the subsequent development of visual-motor response (VMR),30 a special adaptation of the aforementioned assay, which is particularly suitable for high-throughput screening of drugs to improve vision.

Potential Application of Visual-Motor Response for High-Throughput Screening of Drugs that can Improve Vision in Larval Zebrafish

In the VMR assay, larvae that are at least 5 days postfertilization (dpf) would be first acclimatized to the machine environment for 3 hours, before being subjected to 30 minutes of light on and 30 minutes of light off for a total of 3 trials.31 Different variations of the assay have been reported since the initial development of VMR.32,33 Although they were all based on the same light-on and light-off principle, there were differences in the acclimatization time, duration of the light on and off, intensity of light, length of the whole assay, and definition of activity. The VMR of visual mutants has been shown to be different from wild-type (WT) zebrafish.30 In particular, the VMR of an eyeless chk mutant was completely abolished, suggesting that the response was mediated by vision. The VMR in another nrc mutant, which had defects in the ON ganglion cells, was attenuated. By testing the VMR in enucleated larvae, Fernandes et al32 have recently demonstrated that there were 2 additional photosensitive brain regions that contributed to this response in addition to the eye. These included the pineal gland and a domain in the hypothalamus that is specified by the orthopedic transcription factor. Intriguingly, this study found that the chk mutant elicited a VMR by the light-off stimulus, albeit it was substantially lower than the normal siblings. Although it was proposed that the difference in the VMR parameters, including the activity definition and the length of the assay, might have contributed to the observed difference, few interpretations about VMR can be drawn: (1) VMR is consisted of a vision-mediated component and a non-vision-mediated component of VMR; and (2) the vision-mediated component mediates a distinctive fast response, including a large-angle turn (O-bend) that is the most prominent in the first 2 minutes, whereas the non–vision-mediated components mediate another slower response, including a smaller-angle routine turn (R-turn) that is sustained for at least 10 minutes.

The vision-mediated component of the VMR is also supported by an early enucleation experiment that studied the development of larval startle response, defined as an increase in body twitch in 2 seconds after the interruption of light illumination.27 This is essentially the early light-off phase of the VMR. Specifically, the enucleated embryos in this study did not show any startle response despite having an intact pineal photoreceptor and brain. It should be noted that the light illumination in this study was substantially lower (∼7 μW cm−2) than the ones used in Zebrabox (69–8330 and 9032 μW cm−2), suggesting that the vision-mediated component of the startle response/VMR is more sensitive than the other non–vision-mediated components. Together, these observations indicate that analyzing the immediate part of the VMR and/or using a lower light intensity may maximize the difference in VMR that is mediated by the vision-mediated component.

The usage of this VMR assay for screening drugs to improve vision of visual mutants has several advantages: (1) it measures the improvement in an activity output that is substantially mediated by vision and hence directly shows the potential therapeutic value of a drug candidate; (2) it does not require a prior knowledge of disease mechanism; (3) it alleviates the need to implement and use advanced microscopy for screening histologic improvement; and (4) it is scalable and can be conducted in a high-throughput manner, which can facilitate testing of multiple drug libraries with numerous visual mutants. Our laboratory is currently using the VMR assay to screen drugs that can potentially improve the altered response in a visual mutant (Fig. 1) with photoreceptor degeneration (Fig. 2). This mutant has been specifically chosen because it carries a point mutation that is also found in human patients having early-onset photoreceptor degeneration. Although the photoreceptors first develop normally in this mutant, they degenerate by 5 dpf.

FIGURE 1
FIGURE 1:
Visual-motor response is altered in a zebrafish mutant that has retinal degeneration. The VMR of 24 WT (black trace) and 24 visual mutants (red trace) with photoreceptor degeneration (Fig. 2) were recorded at 5 dpf. The activity is defined as the fraction of a second that a larva moves. Each activity trace is an average of the response of 24 larvae over 3 consecutive trials. In this example, light illumination was turned on (A) or off (B) at 60 seconds (illustrated by a white and a black bar above the graph, respectively). (A) The light-on stimulus elicited an immediate and intense response in WT; then, the larvae promptly adapted and returned to a baseline activity level. The visual mutant, on the other hand, only showed a minuscule immediate response to the light-on stimulus compared with WT. (B) The light-off stimulus again triggered an instantaneous and strong response that sustained for more than 2 minutes in WT. The activity gradually diminished and returned to the baseline level by the end of a 30-minute light-off phase; and the first 60 seconds in A illustrates this baseline activity level immediately before the change of light stimulus at 60 seconds. On the contrary, the visual mutant did not respond to the light-off stimulus.
FIGURE 2
FIGURE 2:
A zebrafish model of retinal degeneration. Immunohistologic analysis of the visual mutant inFigure 1 demonstrated that the photoreceptors were degenerating. Top row, WT retinas stained with anti-zpr1 for red/green double cones (left, yellow) and anti-zpr3 for rods (right, yellow). Bottom row, retinas of the visual mutant stained with anti-zpr1 (left, yellow) and anti-zpr3 (right, yellow). The cell nuclei were stained with 4’,6-diamidino-2-phenylindol (DAPI) (blue). These immunostaining analyses were conducted with 8 dpf embryos with standard procedures.119 The mutant embryos collected at 5 dpf revealed similar photoreceptor degeneration.

Despite its advantages, it should be noted that the VMR assay is not suitable for screening drugs for every type of eye diseases. Because the current format of the VMR assay requires the usage of larvae, it may not be applicable to screening drugs for eye diseases in which the histologic and VMR defects are late onset. Specifically, zebrafish requires feeding to survive after 9 dpf; thus, the high-throughput application of VMR is not easily amenable after 9 dpf and is the most compatible with drugs that may show a fast response. Although it is possible to extend the screen beyond 9 dpf in a smaller-scale study by feeding the larvae, one has to consider the confounding factor caused by the possible variation in nutrients of the feed. A standardized diet may help alleviate this problem34 and may potentially extend the use of VMR to later larval stages. Nonetheless, this does not negate the use of the zebrafish model for studying late-onset eye diseases and determining the long-term protective effects of the identified drugs in a smaller scale or the possible development of comparable high-throughput visual-behavioral assay for adult fish in the future. Because it was also described previously that there are non–vision-mediated components in the VMR, it is essential to further characterize the promising drug candidates by additional methods (see “Conclusions”). Together, these suggest that VMR is very suitable for a large-scale drug screening on visual mutants that manifest a visual defect at an early stage.

Generation of Zebrafish Models of Early-Onset Eye Diseases for Drug Screening

The establishment of the VMR assay allows for the screening of drugs that can improve vision with visual mutants of zebrafish. A number of these mutants have been generated through ethylnitrosourea11 and retroviral35 mutagenesis. As discussed previously, many mutants have been discovered by OKR and other visual-behavioral screens.21,23,24 In addition, the same mutagenesis approaches have generated a great number of mutants that have morphological defects in the visual systems.36–38 Many of these mutants affect early eye development and have detectable defects by 5 dpf; thus, they serve as valuable models for drug screening with the VMR assay and studying the underlying mechanisms for various eye diseases.39,40 There is an ongoing effort by the Zebrafish Mutaion Project (http://www.sanger.ac.uk/Projects/D_rerio/zmp/) to knockout every gene in the zebrafish genome. The newly generated mutants have been deposited in the Zebrafish International Resource Center (http://zebrafish.org), a public repository of reagents. Therefore, it is anticipated that more visual mutants would become publicly available for translational research in the future. It is believed that the first type of mutants that is useful for drug screening would be those that carry mutations in genes that cause human visual impairment and have similar histologic defects and alteration in visual behavior.

In the meantime, there are 2 excellent approaches for generating targeted gene perturbation if a mutant is not available. The first one is transcription activator-like effector nuclease (TALEN),41 which can be used to generate targeted disruption in the zebrafish genome,42,43 and the second one is Tol2 transgenesis, which can be used to drive the expression of an exogenous gene by various promoters.44 The transgenesis is made efficient by the Tol2 transposon element originally isolated from medaka.45 Thus, the former approach allows for a rapid targeted disruption of the disease-causing genes identified from human genetics research, whereas the latter enables the generation of transgenic fish carrying a transgene that contains the same disease-causing mutation as in human and/or the mutated gene isolated from patients. Nonetheless, it should be noticed that the transgenic fish still contain 2 normal zebrafish alleles in the genome, which may complicate the downstream analysis; hence, this type of mutants requires careful histologic confirmation to show clear signs of ocular impairments before their usage in drug screening.

Finding Novel Drugs, Especially From Naturally Derived Chemicals, for Treating Eye Disorders

As described previously, 1 major advantage of VMR-based drug screening is that it is possible to identify new drugs that can improve vision before the dissection of disease mechanism and drug function. Hence, this assay can be applicable to the screening of established Western drug/chemical libraries and a plethora of naturally derived chemicals from Eastern medical literature and nutrition sources. The contemporary high-throughput screening with the large Western drug/chemical libraries has identified a number of drugs that have been approved by the Food and Drug Administration for treating different diseases over the years.5 It is very likely that new drugs for eye disorders will be discovered from these libraries.

A number of failures of Western drugs after years of research46 have prompted the search of additional new drug leads. Another promising group of chemicals that may benefit the eyes is naturally derived compounds that have been consumed by many populations for good vision and/or have proven therapeutic values based on Eastern medical literature and nutritional concepts. This category of compounds includes traditional Chinese medicines (TCMs), herbal medicines, and nutritional therapies/supplements. They present as a novel source of chemicals for drug development.47,48 Although many of these naturally derived chemicals have been characterized because of their ability to act as antioxidants (see Appendix 1), it should be noted that their therapeutic values reported in the Eastern medical literature involved other possible properties or actions exerted in vivo. In particular, many TCMs and nutritional therapies aim at achieving a balance of the living system, so that desired health effects can be reached. Moreover, effective TCMs from the literature are often administered in complex formulations. The characterization and treatment philosophy of this type of chemicals constitute a whole body of knowledge and challenges49 that is out of the scope of this review. Nonetheless, VMR-based screening, which does not require prior knowledge of drug action, can potentially be an excellent tool to expedite the characterization of these compounds.

In the following, we will provide a brief review of several promising candidates, which we plan to screen and determine their capability on vision improvement with zebrafish mutants of retinal degeneration.

Antioxidants Recognized With Protective Effects on Photoreceptors

There are at least 3 dietary antioxidants that have been investigated and established for their ability to protect photoreceptors from damages including oxidative stress in rat cell culture and whole animal. These include docosahexaenoic acid (DHA), lutein (LUT), and zeaxanthin (ZEA). Docosahexaenoic acid is the major polyunsaturated fatty acid in the retina, which makes up 35 to 60 percent of the photoreceptor outer segment.50 It has been demonstrated to protect rat retinal photoreceptors from oxidative stress51 and ceramide-induced apoptosis52 in vitro. This antiapoptotic effect was shown to be mediated by the extracellular-signal-regulated kinases/mitogen-activated protein kinase (ERK/MAPK) pathway.53 Interestingly, DHA not only was found to prevent photoreceptors from undergoing apoptosis but also was shown to enhance their survival during development54 and promotes differentiation.55–57 Notably, DHA has been demonstrated to enhance opsin expression and axonal outgrowth without affecting the expression of Crx, a transcription factor that determines the fate of rods and cones.56 This supports DHA’s role in late-stage differentiation, which was mediated by the ERK/MAPK pathway.53 Thus, this further suggests that photoreceptors use the same signal transduction pathway for controlling differentiation and apoptosis. For clinical applications, DHA supplementation on patients with RP receiving vitamin A treatment was reported to slow the disease progression for 2 years,58 whereas dietary intake of eicosapentaenoic acid (EPA) and DHA was demonstrated to decrease the likelihood of age-related macular degeneration (AMD).59

Lutein and ZEA are 2 major components in the macula that possess antioxidant capability.60,61 Treatment with LUT, ZEA, and DHA has been indicated to protect rat photoreceptors from apoptosis induced by oxidative stress.61 Long-term dietary supplementation with ZEA has also been shown to reduce photoreceptor death in a light-damaged Japanese quail model.62 Furthermore, ZEA and LUT promoted photoreceptor differentiation by increasing opsin expression and promoting development of outer segment.61 There exists epidemiologic evidence of intakes of LUT and ZEA can protect patients from developing various eye disorders such as AMD and cataracts.63,64 It appears that a dietary supplement of LUT/ZEA could decrease the likelihood of AMD.65

Vitamin C and Vitamin E

Vitamin C or ascorbic acid serves as a cofactor in 8 important enzymatic reactions, including collagen synthesis, carnitine synthesis, biosynthesis of norepinephrine from dopamine, addition of amide groups to peptide hormones, and modulation of tyrosine metabolism.66 It also has the antioxidant activities against oxidative stress because it possesses the capability to donate its electrons to prevent other compounds from oxidation. Vitamin E is the term for 8 lipophilic compounds that consist of 4 tocopherols and 4 tocotrienols.67 It is mainly known for its ability to act as a chain-breaking antioxidant that terminates the propagation of lipid peroxidation. Vitamin C and E may directly interact with each other under conditions of oxidative stress as studies have indicated that ascorbic acid can either save α-tocopherol from undergoing oxidation68–70 or allow α-tocopherol to regenerate from its oxidized form of α-tocopheroxyl radical.71 Because of their antioxidant capabilities, vitamin C and E perhaps can be used simultaneously to improve the conditions of photoreceptor death. In fact, in 2 recent in vivo studies, treatment with a mixture of α-tocopherol, ascorbic acid, Mn(III) tetrakis porphyrin (MnTBAP), and α-lipoic acid was found to reduce death of photoreceptors and preserve function of cones in the retinal degeneration 1 (rd1) mice72 and both rods and cones in the retinal degeneration 10 (rd10) mice.73 When a single treatment of α-tocopherol was applied, cone survival was still promoted in the rd1 mice.72 Therefore, combining various antioxidants together as a treatment may provide protective advantages for the photoreceptors. Despite these positive results in the laboratory research, it should be noted that there are clinical trials and epidemiologic studies that reported different outcomes. These include a positive74 and no effect65,75–77 with vitamin A and C supplement on AMD progression and a deleterious effect with vitamin E supplement on RP progression.78 (See Appendix 1 for a thorough discussion). These clinical observations indicate that not all antioxidants will necessarily act on all retinal diseases in the same manner.

Resveratrol

Resveratrol (RSV) is a compound present abundantly in the Japanese medicinal plant Polygonum cuspidatum and grapevines.79 It is also found in peanuts, pines, and red wines. It possesses antioxidant properties that may offer health benefits, including reducing the risk of cardiovascular disease and eye disorders. For example, treatment of retinal pigment epithelium (RPE) cell culture by RSV has been shown to protect these cells from H2O2-induced cell death.80 This protection was mediated through the inhibition of MAPK. In the meantime, the same treatment also reduced RPE cell proliferation. In a study of light-induced retinal degeneration in mice, it has been demonstrated that oral-administration of RSV suppressed the deleterious effects on retinal structure and function by light damage.81 Furthermore, intraocular injection of RSV has been indicated to suppress retinal vascular degeneration caused by ischemia-reperfusion (I/R) injury in mice, whereas orally administered RSV could reduce capillary degeneration induced by endoplasmic reticulum stress.82 Interestingly, 2 recent studies have also shown that RSV could inhibit endothelial cell proliferation in vitro83 and pathologic retinal neovascularization in very low-density-lipoprotein receptor mutant mice.84 These findings suggest that RSV played multiple roles in maintaining the health of retinal vasculature.

Schisandrin A and B

Schisandrin A and B (Sch A and B) are 2 active components of Fructus Schisandrae, a fruit that is commonly consumed by the Chinese for sustaining health and vision. In Chinese medicine, the fruit is believed to provide nourishment and therapeutic actions. For example, Sch B has been demonstrated to possess antioxidant activity and protect heart cells against oxidative damage in hypoxia/reoxygenation-induced apoptosis85 and in rats that had I/R injury.86 This protective effect was mediated by the activation of glutathione antioxidant87 and heat shock88 responses. The activation of the protective genes in the glutathione antioxidant response has also been determined to be regulated by the ERK/Nrf2 pathway.89 Despite these good protective effects against oxidative stress, the extent to which these schisandrins inhibit retinal degeneration is unclear. Our group is currently conducting a preliminary investigation of Sch B on improving the histology and visual behavior of the retinal degeneration mutant as shown in Figures 1 and 2.

Lycium

Lycium barbarum is a plant that produces a fruit that is commonly referred to as goji/gouqi berry or wolfberry. The fruit is harvested for health food and supplement purposes, specifically for good vision. It has been known for its powerful antioxidant properties and potential benefits for cardiovascular system and inflammation. Furthermore, its extract has been extensively characterized to have neuroprotective, neurogenic, and antioxidative effects in both retinal and nonretinal systems.90–95 In the retinal system, it has been tested that oral administration of Lycium promoted the survival of retinal ganglion cells (RGCs), the target cell type that is affected by glaucoma, in an ocular-hypertension rat model.90 Notably, the RGCs were protected, although the elevated intraocular pressure was not significantly altered. This protective effect on RGCs was mediated by the up-regulation of βB2-crystallin.92 Pretreatment with Lycium before I/R injury has also been demonstrated to protect the retinas from oxidative damage and apoptosis.93

Flavonoids

Isoliquiritigenin is a flavonoid existing in licorice. It exhibits many desirable properties such as antioxidant, anti-inflammatory, antibacterial, antiviral, and antitumor activities.96 It has been shown to suppress neovascularization in experimental ocular angiogenesis models97 and further proposed to work as a plausible therapy for the wet form of AMD, which exists as an exuberant growth of blood vessels. Catechins are flavonoids that present richly in green tea leaves. They have been confirmed to possess antioxidative property in eye research.83,98 In particular, epigallocatechin gallate (EGCG) is a commonly used catechin in research, and there are a number of reports demonstrating its protective role on retina. It produced a protective effect on RGCs after optic nerve axotomy,99 optic nerve crush,100 and intraocular pressure increase101 when administered via intraperitoneal, intraperitoneal and oral, and oral route, respectively. Moreover, orally administered EGCG attenuated photoreceptor damage induced by a light insult and preserved the electrophysiologic properties of the retina.102 A similar protective effect against light-induced damage was uncovered in RGCs.101 Intraperitoneally administered EGCG also protected the retina after I/R injury,103,104 which could be mediated through a suppression of nitric oxide synthase expression.104 Furthermore, EGCG also protected retina105 and RGCs103 against oxidative stress induced in vivo and in vitro, respectively. There is also a report on EGCG’s protective effect on Müller cells against catechol-induced toxicity in vitro.106 In humans, a short-term oral administration of EGCG for 3 months improved the pattern-evoked electroretinogram in patients with open glaucoma.107 Thus, these studies have established EGCG as a good candidate for retinal protection. In addition to EGCG, other catechin isoforms have been demonstrated to reach a higher concentration in the eye in vivo.98 These isoforms are potentially useful therapeutic agents for further characterizations.

Bioavailability of Drugs and Therapeutic Window in Zebrafish

Drug absorption and distribution are 2 important issues to consider in drug development and screening. Eye drugs are usually delivered by topical or intravenous administration or intravitreal injection.108 The first 2 routes are more amenable to drug screening and are the likely mechanisms through which the zebrafish larvae absorb the drugs into the eyes. The drugs that are dissolved in water can potentially diffuse through the larval body and get into the blood circulation and/or through the ocular surface as if it is applied topically. In the latter case, they may diffuse into the eye through the permeation of cornea, conjunctiva, and sclera, which are exposed to the water continuously.

Although it is currently not clear about the bioavailability of drugs in larval eye, which has to be determined case by case, a few investigations have laid down important foundations for its characterization. First, there are studies that have attempted to determine the bioavailability of the drugs in the whole embryo.109,110 In particular, these studies showed that the hydrophobicity of the drug, as calculated by the logarithm of the octanol:water partition coefficient (LogP), could be a good indicator of the general bioavailability. Specifically, drugs with LogP value greater than 1 have been proposed to be used in general screen111 because they were readily absorbed by fish embryos and showed specific response in a screen of drugs that cause bradycardia.110 The drugs that had a LogP below 1, although bioactive, required microinjection to elicit the effect. It can be speculated that these hydrophobicity rules can potentially be applicable to the drugs that are diffusing into the eye directly. Second, drugs that diffuse into the body and get into the general circulation have to pass through blood-retinal barrier (BRB) and/or blood-aqueous barrier (BAB) before they can elicit their effect. Zebrafish have functional BRB112,113 and blood-brain barrier112–114 that are analogous to the other mammals and begin to function at around 3 dpf, whereas the BAB has not been characterized in fish yet. It should be noted that a drug that does not show an alteration in VMR can be a false negative caused by a lack of diffusion and/or a problem in getting through the BRB and BAB. If necessary, the specific bioavailability in the eye can be determined by methods such as liquid chromatography–mass spectroscopy (LC-MS).109

With regard to the therapeutic window, it has to be determined case by case as well. For example, the study of retinal degeneration has to take into account of the process of photoreceptor development. Zebrafish photoreceptors begin to differentiate at 48 to 50 hpf,10,115 and the first visual behavior is detected at 68 hpf.27 To avoid unnecessary drug exposure during early embryogenesis, treatment starting at approximately 2 or 3 dpf is a logical choice. Alternatively, treatment can begin at the stage when the first detectable degeneration occurs. For the visual mutant that we are currently characterizing, 5 dpf would be a reasonable choice.

Conclusions

The VMR assay of zebrafish is potentially a powerful approach for screening drugs that can affect visual behavior. It also has a huge potential in characterizing naturally derived chemicals, in particular TCMs, for improving vision for various eye disorders. Because it has become feasible to generate targeted knock-out in zebrafish, mutants of human eye diseases can be created for rapid characterization of potential drug therapies. Although zebrafish is certainly not easily amenable to the ultrahigh throughput screening that involves hundreds of thousands of compounds, it can be envisioned that the zebrafish in vivo drug screening could complement with the in vitro biochemical and/or cellular-based screening by following up leads that are identified from these other faster screens. In the meantime, a screen with a few thousand chemicals with the zebrafish model is definitely feasible.28

In addition, the effect of drugs that show a positive effect in the zebrafish VMR screening can be further characterized at different levels: (1) at the behavioral level, the drug-treated larvae can be tested by OKR; (2) at the physiologic level, the drug-treated retinas can be analyzed by electroretinogram,116,117 a measurement of the electrical activity of various retinal cell type under light stimulus because this can help localize the functional improvement to specific cell type(s); (3) at the cellular level, the drug-treated eyes can be analyzed by various histologic and immunohistochemical methods118–120; (4) at the pharmacologic level, the bioavailability of the drug in vivo can be determined by LC-MS109; and (5) at the molecular level, the components in the disease-causing gene network that are affected by the drug treatment can potentially be identified by expression studies. Our group has not only developed unique microdissection and expression analysis approaches for retinas and RPE121–123 but also successfully used them to analyze a retinal dystrophic mutant.118,119,124 Together, they have created an efficient pipeline to discover and analyze novel drugs for better vision in zebrafish.

ACKNOWLEDGMENTS

The authors thank Robert Ko, Calvin Pang, Kwok-Fai So, Raymond Chang, Amy Lo for sharing their thoughts and experience in studying TCMs for good vision; David Prober and Jason Rihel for sharing their experience on using VMR of zebrafish for high-throughput drug screening; and Viewpoint Life Sciences for their technical support.

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      Appendix 1—Antioxidative Therapies for Retinal Degenerative Diseases

      Because of the constant exposure of photoreceptors to light, which can induce free radical formation and a high–oxygen-tension environment, oxidative stress/damage has been proposed as one of the theories for photoreceptor death in retinal degeneration.72,125–127 In fact, hyperoxia has been shown to cause photoreceptor death in the P23H retinal-degeneration rats128 and normal mice.129 In addition, oxidative damage has been reported to be the underlying cause of photoreceptor death in the Pro347Leu-rhodopsin transgenic pigs.130 This oxidative stress/damage theory has led to the proposal that antioxidative treatment could delay late-stage photoreceptor dystrophy.126 As described in the review, there are promising successes in using antioxidants on improving vision of 2 models of RP mice.72,73 Nonetheless, the clinical trials of different types of antioxidants on treating human retinal degeneration have mixed outcomes. For example, a number of long-term clinical trials have indicated that vitamin E supplement did not decrease the incidence and/or slow the progression of AMD.75–77 One of these studies has also suggested that vitamin C supplement had no effect on AMD incidence.75 The failure of preventing the progression of AMD by vitamins E and C has also been indicated by an epidemiology study on dietary intake.65 However, a combination supplement of vitamin C, E, carotene, and zinc reduced the development of advanced AMD in another trial.74 In addition, DHA supplemented with EPA and dietary LUT/ZEA intake decreased the likelihood of AMD in 2 epidemiologic studies.59,65 This type of mixed outcomes has also been illustrated by a classic clinical trial of RP,78 in which vitamin A supplement slowed the progression of disease, whereas vitamin E had the opposite effect. A subsequent trial conducted by the same group of authors on DHA supplement in conjunction with vitamin A initially showed no effect131; however, a careful analysis of the subgroups indicated that if the patient had not been taking vitamin A beforehand, addition of DHA slowed the course of disease for 2 years.58 All these studies have indicated that not all antioxidants would work for all diseases. The efficiency depends on the types of disease, dosage of the drugs, time of drug administration, and the underlying mechanism of the antioxidative property. The latter also suggests that a combinatorial therapy is necessary to achieve some protective effects, an idea is that supported by the aforementioned animal studies72,73 and clinical trials.58,59,65,74 Therefore, it would be critical to test multiple drugs on various disease models in an efficient manner as offered by zebrafish.

      “Some painters transform the sun into a yellow spot, others transform a yellow spot into the sun.”

      - Pablo Picasso

      Figure
      Figure
      Keywords:

      eye diseases; zebrafish; traditional Chinese medicines; naturally derived chemicals; nutrition; drug screening

      © 2012Asia-Pacific Academy of Ophthalmology