Near Infrared (NIR) Light Therapy of Eye Diseases: A Review.

Near infrared (NIR) light therapy, or photobiomodulation therapy (PBMT), has gained persistent worldwide attention in recent years as a new novel scientific approach for therapeutic applications in ophthalmology. This ongoing therapeutic adoption of NIR therapy is largely propelled by significant advances in the fields of photobiology and bioenergetics, such as the discovery of photoneuromodulation by cytochrome c oxidase and the elucidation of therapeutic biochemical processes. Upon transcranial delivery, NIR light has been shown to significantly increase cytochrome oxidase and superoxide dismutase activities which suggests its role in inducing metabolic and antioxidant beneficial effects. Furthermore, NIR light may also boost cerebral blood flow and cognitive functions in humans without adverse effects. In this review, we highlight the value of NIR therapy as a novel paradigm for treatment of visual and neurological conditions, and provide scientific evidence to support the use of NIR therapy with emphasis on molecular and cellular mechanisms in eye diseases.

Bioenergetic Crosstalk between Mesenchymal Stem Cells and various Ocular Cells through the intercellular trafficking of Mitochondria.

Rationale: Mitochondrial disorders preferentially affect tissues with high energy requirements, such as the retina and corneal endothelium, in human eyes. Mesenchymal stem cell (MSC)-based treatment has been demonstrated to be beneficial for ocular degeneration. However, aside from neuroprotective paracrine actions, the mechanisms underlying the beneficial effect of MSCs on retinal and corneal tissues are largely unknown. In this study, we investigated the fate and associated characteristics of mitochondria subjected to intercellular transfer from MSCs to ocular cells. Methods: MSCs were cocultured with corneal endothelial cells (CECs), 661W cells (a photoreceptor cell line) and ARPE-19 cells (a retinal pigment epithelium cell line). Immunofluorescence, fluorescence activated cell sorting and confocal microscopy imaging were employed to investigate the traits of intercellular mitochondrial transfer and the fate of transferred mitochondria. The oxygen consumption rate of recipient cells was measured to investigate the effect of intercellular mitochondrial transfer. Transcriptome analysis was performed to investigate the expression of metabolic genes in recipient cells with donated mitochondria. Results: Mitochondrial transport is a ubiquitous intercellular mechanism between MSCs and various ocular cells, including the corneal endothelium, retinal pigmented epithelium, and photoreceptors. Additionally, our results indicate that the donation process depends on F-actin-based tunneling nanotubes. Rotenone-pretreated cells that received mitochondria from MSCs displayed increased aerobic capacity and upregulation of mitochondrial genes. Furthermore, living imaging determined the ultimate fate of transferred mitochondria through either degradation by lysosomes or exocytosis as extracellular vesicles. Conclusions: For the first time, we determined the characteristics and fate of mitochondria undergoing intercellular transfer from MSCs to various ocular cells through F-actin-based tunneling nanotubes, helping to characterize MSC-based treatment for ocular tissue regeneration.

Noninvasive Electrical Stimulation Improves Photoreceptor Survival and Retinal Function in Mice with Inherited Photoreceptor Degeneration.

Purpose: Neurons carry electrical signals and communicate via electrical activities. The therapeutic potential of electrical stimulation (ES) for the nervous system, including the retina, through improvement of cell survival and function has been noted. Here we investigated the neuroprotective and regenerative potential of ES in a mouse model of inherited retinal degeneration. Methods: Rhodopsin-deficient (Rho-/-) mice received one or two sessions of transpalpebral ES or sham treatments for 7 consecutive days. Intraperitoneal injection of 5-ethynyl-2'-deoxyuridine was used to label proliferating cells. Weekly electroretinograms were performed to monitor retinal function. Retinal morphology, photoreceptor survival, and regeneration were evaluated in vivo using immunohistochemistry and genetic fate-mapping techniques. Müller cell (MC) cultures were employed to further define the optimal conditions of ES application. Results: Noninvasive transpalpebral ES in Rho-/- mice improved photoreceptor survival and electroretinography function in vivo. ES also triggered residential retinal progenitor-like cells such as MCs to reenter the cell cycle, possibly producing new photoreceptors, as shown by immunohistochemistry and genetic fate-mapping techniques. ES directly stimulated cell proliferation and the expression of progenitor cell markers in MC cultures, at least partially through bFGF signaling. Conclusions: Our study showed that transpalpebral ES improved photoreceptor survival and retinal function and induced the proliferation, probably photoreceptor regeneration, of MCs; this occurs via stimulation of the bFGF pathways. These results suggest the exciting possibility of applying noninvasive ES as a versatile tool for preventing photoreceptor loss and mobilizing endogenous progenitors for reversing vision loss in patients with photoreceptor degeneration.

Retinal Wnt signaling defect in a zebrafish fetal alcohol spectrum disorder model.

Fetal alcohol spectrum disorder caused by prenatal alcohol exposure includes ocular abnormalities (microphthalmia, photoreceptor dysfunction, cataracts). Zebrafish embryos exposed to ethanol from gastrulation through somitogenesis show severe ocular defects, including microphthalmia and photoreceptor differentiation defects. Ethanol-treated zebrafish had an enlarged ciliary marginal zone (CMZ) relative to the retina size and reduced Müller glial cells (MGCs). Ethanol exposure produced immature photoreceptors with increased proliferation, indicating cell cycle exit failure. Signaling mechanisms in the CMZ were affected by embryonic ethanol exposure, including Wnt signaling in the CMZ, Notch signaling and neurod gene expression. Retinoic acid or folic acid co-supplementation with ethanol rescued Wnt signaling and retinal differentiation. Activating Wnt signaling using GSK3 inhibitor (LSN 2105786; Eli Lilly and Co.) restored retinal cell differentiation pathways. Ethanol exposed embryos were treated with Wnt agonist, which rescued Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. Our results illustrate the critical role of Wnt signaling in ethanol-induced retinal defects.

Analysis and characterization of anthocyanins and carotenoids in Japanese blue tomato.

Tomato (Solanum lycopersicum) is rich in anthocyanins, which are polyphenolic pigments. This study aimed to analyze and characterize the anthocyanin composition in cultivated blue tomato in Japan. The extracts of peel, seed, and pulp of tomatoes were purified following which anthocyanins and lycopene contents were analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry. Eleven types of anthocyanins were identified, including delphinidin, petunidin, and malvidin. Further, the antioxidant activity of anthocyanins was evaluated using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt radical quenching assays and electron spin resonance. "Blue tomato" extracts exert antioxidant activity. Thus, we showed that petunidin was present in the "blue tomato" peel while lycopene was present in the peel and pulp. Additionally, the blue tomato peel extract was found to significantly inhibit H2O2-induced cell death in vitro. This is the first study on cell protective effects of Japanese blue tomato extract and petunidin in murine photoreceptor cells.

Neuroprotective effects of low level electrical stimulation therapy on retinal degeneration.

Low-level electrical stimulation applied to the eye has been shown to have neuroprotective effects on photoreceptors and retinal ganglion cells. In this review, we compare the effects of Subretinal Electrical Stimulation (SES), Transcorneal Electrical Stimulation (TES), and Whole Eye Stimulation (WES) on preserving retinal structure and function, and visual acuity, in retinal degeneration. Similarities and differences in stimulus parameters, targeted cells and growth factor expression will be discussed with emphasis on studies that have translated laboratory findings into clinical trials.

[Effect and mechanisms of wenyang yiqi huoxue recipe on the apoptosis of photoreceptor cells in retinal degeneration slow mice].

OBJECTIVE: To observe the effect of Wenyang Yiqi Huoxue Recipe (WYHR) on the apoptosis of photoreceptor cells in retinal degeneration slow (RDS) mice, and to investigate its molecular mechanisms. METHODS: RDS mice were randomly divided into the model group and the Chinese medicine group,and C57BL/6J mice were selected as the normal control group. Each group consisted of 4 female mice and 2 male mice. Mice in the Chinese medicine group were administered with WYHR (10 mg/g) by gastrogavage since mating. Baby mice drunk WYHR decoction instead of drinking water once they were born. The offspring were administrated with low dose WYHR decoction by gastrogavage from the 7th postnatal day, and the dose was increased to that for adult mice from the 21st postnatal day. Physiological saline was administrated to mice in the model group and the normal control group by gastrogavage. At 18, 28 and 48 postnatal days, electroretinogram (ERG) was used to evaluate the retina functional variation, and the apoptotic rate of photoreceptor cells was determined by TUNEL staining. HE staining was performed. The number of photoreceptor cells of the outer nuclear layer was calculated. Furthermore, effect of WYHR on Rhodopsin and basic fibroblast growth factor (bFGF) expression was examined using immunochemical assay. RESULTS: Compared with the model group, a- and b-wave latency and amplitude, as well as the bFGF expression sharply increased in the Max-ERG of the Chinese medicine group (P < 0.05, P < 0.01) at 18 postnatal days. At 28 and 48 postnatal days, a- and b-wave latency and amplitude sharply increased, photoreceptor cell layer numbers of the outer nuclear layer obviously increased, the apoptosis rate of retinal photoreceptor cells obviously decreased, expressions of Rhodopsin and bFGF in the Chinese medicine group significantly increased (P < 0.05, P < 0.01). CONCLUSION: WYHR could effectively inhibit the apoptosis of photoreceptor cells in RDS mice, which might be attributed to up-regulating bFGF expression.

The protective effects of bilberry and lingonberry extracts against UV light-induced retinal photoreceptor cell damage in vitro.

Bilberry extract (B-ext) and lingonberry extract (L-ext) are currently used as health supplements. We investigated the protective mechanisms of the B-ext and L-ext against ultraviolet A (UVA)-induced retinal photoreceptor cell damage. Cultured murine photoreceptor (661W) cells were exposed to UVA following treatment with B-ext and L-ext and their main constituents (cyanidin, delphinidin, malvidin, trans-resveratrol, and procyanidin). B-ext, L-ext, and constituents improved cell viability and suppressed ROS generation. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and protein kinase B (Akt) were analyzed by Western blotting. B-ext and cyanidin inhibited phosphorylation of p38 MAPK, and B-ext also inhibited phosphorylation of JNK by UVA. L-ext, trans-resveratrol, and procyanidin alleviated the reduction of phosphorylated Akt levels by UVA. Finally, a cotreatment with B-ext and L-ext showed an additive effect on cell viability. Our findings suggest that both B-ext and L-ext endow protective effects against UVA-induced retinal damage.

Arachidonic acid supplementation during gestational, lactational and post-weaning periods prevents retinal degeneration induced in a rodent model.

Fatty acids and their derivatives play a role in the response to retinal injury. The effects of dietary arachidonic acid (AA) supplementation on N-methyl-N-nitrosourea (MNU)-induced retinal degeneration was investigated in young Lewis rats during the gestational, lactational and post-weaning periods. Dams were fed 0·1, 0·5 or 2·0% AA diets or a basal (< 0·01% AA) diet. On postnatal day 21 (at weaning), male pups received a single intraperitoneal injection of 50 mg MNU/kg or vehicle, and were fed the same diet as their mother for 7 d. Retinal apoptosis was analysed by the terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labelling (TUNEL) assay 24 h after the MNU treatment, and retinal morphology was examined 7 d post-MNU. Histologically, all rats that received MNU and were fed the basal and 0·1% AA diets developed retinal degeneration characterised by the loss of photoreceptor cells (disappearance of the outer nuclear layer and the photoreceptor layer) in the central retina. The 0·5 and 2·0% AA diets rescued rats from retinal damage. Morphometrically, in parallel with the AA dose (0·5 and 2·0% AA), the photoreceptor ratio significantly increased and the retinal damage ratio decreased in the central retina, compared with the corresponding ratios in basal diet-fed rats. In parallel with the increase in serum and retinal AA levels and the AA:DHA ratio, the apoptotic index in the central retina was dose-dependently decreased in rats fed the 0·5 and 2·0% AA diets. In conclusion, an AA-rich diet during the gestation, lactation and post-weaning periods rescued young Lewis rats from MNU-induced retinal degeneration via the inhibition of photoreceptor apoptosis. Therefore, an AA-enriched diet in the prenatal and postnatal periods may be an important strategy to suppress the degree of photoreceptor injury in humans.

Caffeic acid phenethyl ester protects 661W cells from H2O2-mediated cell death and enhances electroretinography response in dim-reared albino rats.

PURPOSE: Caffeic acid phenethyl ester (CAPE), an active component of honeybee propolis, has a wide range of beneficial properties. The purpose of this study was to test the protective role of CAPE in 661W cells (in vitro) against H(2)O(2)-mediated cell death and in albino rats (in vivo) against various light conditions. METHODS: The 661W cells were pretreated with CAPE and then stressed with H(2)O(2). Cell death was measured with lactate dehydrogenase (LDH) release assay, and mRNA and proteins were analyzed. Sprague Dawley rats were raised on either a control or CAPE (0.02%) diet and exposed to various light conditions for short or long periods. Retinal histology, mRNA, protein, lipid composition, and retinal function by electroretinography (ERG) were measured at the end of feeding. RESULTS: Pretreatment of 661W cells with CAPE reduced H(2)O(2)-mediated cell death in a dose-dependent manner and induced expression of heme oxygenase-1 (Ho1). Albino rats fed with CAPE had greater expression of Ho1 and intercellular adhesion molecule 1 (Icam1), less expression of FOS-like antigen (Fosl) and lipoxygenase 12 (Lox12) genes in the retina, less translocation of nuclear factor kappaB protein to the nucleus, and a lower molar ratio of n-3 polyunsaturated fatty acids. Further, the ERGs of the retinas of CAPE-fed rats were significantly higher than those of the control-fed rats when raised in dim light. CONCLUSIONS: CAPE can activate the antioxidative gene expression pathway in retinal cells in vitro and in vivo. Feeding CAPE to albino rats can enhance ERG responses and change the lipid profile in the rats' retinas.

Ceramide-1-phosphate, a new mediator of development and survival in retina photoreceptors.

PURPOSE. Simple sphingolipids control crucial cellular processes in several cell types. Previous work demonstrated that sphingolipids, such as ceramide, sphingosine, and sphingosine-1-phosphate, are key mediators in the regulation of survival, differentiation, and proliferation of retina photoreceptors. Ceramide-1-phosphate (C1P) regulates growth and survival in several cell types; however, little is known concerning its functions in the retina. Whether C1P also participates in controlling photoreceptor development was also explored. METHODS. Rat retina neuronal cultures were supplemented with 1 to 10 µM C1P. Proliferation was determined by evaluating 5-bromo-2-deoxyuridine (BrdU) uptake and the number of mitotic figures and differentiation by evaluating opsin and peripherin expression by immunocytochemistry and Western blot. Apoptosis was inhibited with the pan caspase inhibitor ZVADFMK and evaluated by TUNEL assay, propidium iodide/annexin V, and DAPI labeling. Preservation of mitochondrial membrane potential was evaluated. RESULTS. C1P enhanced BrdU uptake and increased mitosis in retinal progenitors. C1P addition advanced photoreceptor differentiation, enhancing opsin and peripherin expression and stimulating development of the apical processes in which these proteins were concentrated. In the absence of these trophic factors, photoreceptors degenerated after 4 days in vitro, and at day 6, almost 50% of photoreceptors were apoptotic. C1P decreased photoreceptor apoptosis, reducing this percentage by half. Inhibiting caspase activity reduced photoreceptor apoptosis in the controls, but did not increase opsin expression, implying that C1P has separate effects on differentiation and survival. CONCLUSIONS. These results suggest for the first time that C1P is a novel mediator that has multiple functions in photoreceptors, initially regulating their proliferation and then promoting their survival and differentiation.

Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo.

The present study describes a robust 50-fold increase in rhodopsin gene transcription by cAMP in cultured retinal precursor cells of chicken embryo. Retinal cells isolated at embryonic day 8 (E8) and cultured for 3 days in serum-supplemented medium differentiated mostly into red-sensitive cones and to a lesser degree into green-sensitive cones, as indicated by real-time RT-PCR quantification of each specific opsin mRNA. In contrast, both rhodopsin mRNA concentration and rhodopsin gene promoter activity required the presence of cAMP-increasing agents [forskolin and 3-isobutyl-1-methylxanthine (IBMX)] to reach significant levels. This response was rod-specific and was sufficient to activate rhodopsin gene transcription in serum-free medium. The increase in rhodopsin mRNA levels evoked by a series of cAMP analogs suggested the response was mediated by protein kinase A, not by EPAC. Membrane depolarization by high KCl concentration also increased rhodopsin mRNA levels and this response was strongly potentiated by IBMX. The rhodopsin gene response to cAMP-increasing agents was developmentally gated between E6 and E7. Rod-specific transducin alpha subunit mRNA levels also increased up to 50-fold in response to forskolin and IBMX, while rod-specific phosphodiesterase-VI and rod arrestin transcripts increased 3- to 10-fold. These results suggest a cAMP-mediated signaling pathway may play a role in rod differentiation.

Sphingosine-1-phosphate is a key regulator of proliferation and differentiation in retina photoreceptors.

PURPOSE: Identifying the cues required for the survival and development of photoreceptors is essential for treating retinal neurodegeneration. The authors previously established that glial-derived neurotrophic factor (GDNF) stimulates proliferation and that docosahexaenoic acid (DHA) promotes photoreceptor survival and differentiation. Later findings that ceramide triggers photoreceptor apoptosis suggested sphingolipids might also control photoreceptor development. The present study investigated whether sphingosine-1-phophate (S1P), which promotes survival and differentiation in several cell types, regulates photoreceptor proliferation and differentiation and whether it is a mediator in GDNF and DHA effects. METHODS: Rat retina neuronal cultures were supplemented at day 0 or 1 with S1P, GDNF, or DHA and were treated with DL-threo-dihydrosphingosine to inhibit S1P synthesis or with brefeldin A (BFA) to block intracellular trafficking. Proliferation was quantified to determine bromodeoxyuridine uptake and number of mitotic figures. Opsin, peripherin, and sphingosine kinase (SphK), the enzyme required for S1P synthesis, were quantified by immunocytochemistry and Western blot analysis. RESULTS: S1P increased the proliferation of photoreceptor progenitors. It also stimulated the formation of apical processes, enhanced opsin and peripherin expression, and promoted their localization in these processes; DHA had similar effects. BFA prevented S1P and DHA enhancement of apical process formation without affecting opsin expression. GDNF and DHA enhanced SphK expression in photoreceptors, while inhibiting S1P synthesis blocked GDNF mitogenic effects and DHA effects on differentiation. CONCLUSIONS: The authors propose S1P as a key regulator in photoreceptor development. GDNF and DHA might upregulate SphK levels to promote S1P synthesis, which would initially promote proliferation and then advance photoreceptor differentiation.

Epidermal growth factor promotes the differentiation of stem cells derived from human umbilical cord blood into neuron-like cells via taurine induction in vitro.

Results of recent investigations have demonstrated the plasticity of mesenchymal stem cells (MSC) can differentiate into neural lineages. In this study, we explored the experimental condition of differentiation into neuron-like cells or rhodopsin (RHOS)-positive cells induced by epidermal growth factor (EGF) and taurine in vitro and to investigate their biological characteristics. MSC were obtained from umbilical cord blood (UCB) of term deliveries. Cultured cells were treated with Dulbecco's modified Eagle's medium/F12 (pH 7.0-7.2) supplemented with 30 ng/ml EGF. After the third cell passage, the cells were trysinized and analyzed with a flow cytometer using the following monocloned antibodies: CD90, CD29, CD34, CD44, and CD45. Taking another MSC of the third passage, its basal medium was replaced with alpha minimum essential medium supplemented with taurine (50 micromol/L). Cells were cultured for an additional 8-10 d, fixed, and then immunocytochemically analyzed. Primary antibodies included the following: neuron-specific enolase (NSE), RHOS, and nestin. In our study, we isolated a cell population derived from UCB, which possesses morphological characteristics similar to those of MSC isolated from bone marrow. In the cytometric analysis, MSC did not present labeling for the hematopoietic line (CD34 and CD45) and were positive for CD29, CD44, and CD90. After induction by taurine, 80.5 +/- 16.2% of the cell population expressed NSE, 36.8 +/- 9.6% expressed RHOS, and 29.6 +/- 9.3% expressed Nestin, while only 7.9 +/- 3.5% expressed NSE in the control group. This study demonstrates that partial MSC induced by taurine and EGF can differentiate into neuron-like cells or RHOS-positive cells in vitro, which may provide a promising therapeutic strategy for the treatment of some forms of retinal degeneration.

Photoreceptor-specific expression, light-dependent localization, and transcriptional targets of the zinc-finger protein Yin Yang 1 in the chicken retina.

The zinc-finger transcription factor Yin Yang 1 (YY1) is a multifunctional protein that plays a critical role in embryonic development. Although it has been shown to play a role in eye development, its expression in the retina was not previously described. Here, we investigated YY1 expression in chicken tissues and we identified the neural retina as one of the tissues with highest YY1 protein levels. Immunohistochemical detection of YY1 in the retina revealed a clear-cut photoreceptor specificity and day/night differences in the cytoplasmic localization of the protein. YY1 was also present at high concentration in the nuclei of some photoreceptors. Gel-shift assays indicated YY1 bound to regulatory regions of several genes specifically expressed in photoreceptors. One of these genes, hydroxyindole-O-methyltransferase (EC 2.1.1.4), encodes the last enzyme of the melatonin synthesis pathway. Although over-expression of chicken YY1 was not sufficient to activate the chicken hydroxyindole-O-methyltransferase promoter in HEK293 cells, the YY1-binding site contained in this promoter was clearly required for full transcriptional activity in chicken embryonic retinal cells. These results suggest a role of YY1 in regulating the melatoninergic function of retinal photoreceptors.

Lutein and zeaxanthin protect photoreceptors from apoptosis induced by oxidative stress: relation with docosahexaenoic acid.

PURPOSE: Oxidative stress has been proposed as a major pathogenic factor in age-related macular degeneration (AMD), the leading cause of vision loss among elderly people of western European ancestry. Lutein (LUT) and zeaxanthin (ZEA), major components in macular pigment, are among the retinal antioxidants. Though xanthophyll intake may reduce the likelihood of having advanced AMD, direct evidence of neuroprotection is lacking. Prior work has shown that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, delays apoptosis and promotes differentiation of photoreceptors. This study was conducted to investigate whether LUT, ZEA, and beta-carotene (BC), major dietary carotenoids protect photoreceptors from oxidative stress and whether this protection is synergistic with that of DHA. METHODS: Pure rat retinal neurons in culture, supplemented with LUT, ZEA, or BC, with or without DHA, were subjected to oxidative stress induced with paraquat and hydrogen peroxide. Apoptosis, preservation of mitochondrial membrane potential, cytochrome c translocation, and opsin expression were evaluated. RESULTS: Pretreatment with DHA, LUT, ZEA, and BC reduced oxidative stress-induced apoptosis in photoreceptors, preserved mitochondrial potential, and prevented cytochrome c release from mitochondria. ZEA and LUT also enhanced photoreceptor differentiation. In control cultures, photoreceptors failed to grow their characteristic outer segments; addition of DHA, ZEA, or LUT increased opsin expression and promoted the development of outer-segment-like processes. CONCLUSIONS: These results show for the first time the direct neuroprotection of photoreceptors by xanthophylls and suggest that ZEA and LUT, along with DHA, are important environmental influences that together promote photoreceptor survival and differentiation.

Localization of ZnT7 and zinc ions in mouse retina--immunohistochemistry and selenium autometallography.

Zinc transporter 7 (ZnT7, Slc30a7), a member of the Slc30 family, is involved in mobilizing zinc ions from the cytoplasm into the Golgi apparatus. In the present study, we examined the distribution and localization of ZnT7 and the labile zinc ions in the mouse retina using immunohistochemistry and in vivo zinc-selenium autometallography (ZnSe(AMG)). Our results showed that ZnT7 is abundantly expressed in the ganglion cells and pigment epithelial cells of the mouse retina. ZnT7 is also expressed in the amacrine cells and the layer of optic fibers of the mouse retina, but to a lesser extent. Weak staining of ZnT7 was detected in the inner plexiform layer, outer plexiform layer, and outer segment of the photoreceptors. However, ZnT7 was not detected in the outer nuclear layer and inner segment of the photoreceptors. A high level of labile zinc pool was detected in the pigment epithelial cells, the inner segment of the photoreceptors, and the marginal region of the inner nuclear layer. Less amount of labile zinc ions were detected in the ganglion cells of the retina. These observations strongly suggest that ZnT7 may play critical roles in retinal zinc homeostasis and that chelatable zinc pools may have multiple functions in the retina.

Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursor cells.

PURPOSE: Photoreceptor differentiation involves the activation of two specific sets of genes; those encoding the proteins of the phototransduction cascade and those encoding the enzymes of the melatonin synthesis pathway, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT). The purpose of the present study was to examine the conditions of AANAT and HIOMT gene activation, relative to that of selected phototransduction markers (alpha-transducin and opsins), in both in vivo and in vitro differentiating photoreceptors of the chicken retina. METHODS: Neural retina RNA was obtained between embryonic day 7 (E7) and posthatch day 8 (P8) and analyzed on northern blots with cDNA probes to AANAT, HIOMT, visinin, alpha-transducin, rhodopsin, and the four cone opsins. Cell cultures were prepared from E7 chicken neural retina and incubated for two to four days in vitro, either in basal medium or in serum-supplemented medium or in medium containing an insulin-based supplement. RNA from the cultured cells was analyzed on northern blots as above. Real time RT-PCR was used to confirm in vitro changes in HIOMT and red opsin mRNA levels. The cultured cells were transfected with promoter-reporter plasmids for direct analysis of HIOMT promoter regulation by the dual luciferase method. RESULTS: The different mRNAs composing the photoreceptor phenotype appeared at E7 (visinin), E10 (alpha-transducin), E14 (HIOMT), E15 (rhodopsin, red opsin, and green opsin), E16 (AANAT), E17 (blue opsin), and E18 (violet opsin). In the early differentiating cones of the central retina, HIOMT mRNA appeared two days earlier than red opsin and green opsin mRNAs (E12 rather than E14). In cultured embryonic neural retina cells, basal medium was sufficient to activate alpha-transducin gene transcription, an insulin-based supplement was sufficient to activate HIOMT gene transcription, whereas serum was required for red opsin gene transcription after two days in vitro. All serum batches were able to activate red opsin gene transcription, whereas some of them failed to activate HIOMT gene transcription. Activation of the HIOMT gene promoter by an insulin-based supplement and by serum was confirmed after transfection of chicken embryonic neural retina cells with promoter-reporter plasmids. CONCLUSIONS: Activation of the melatonin synthesis genes in vivo takes place in a time window very close to that of early opsins. However, a 24-48 h lead of HIOMT gene expression over early opsins was clearly observed. Our in vitro experiments indicate that different exogenous signals are required to activate the different genes encoding photoreceptor specific functions. Significantly, marker genes for light sensitivity (red opsin) and for melatonin synthesis (HIOMT) appear to be activated in response to different signals.

Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid.

PURPOSE: In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. METHODS: Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. RESULTS: Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [(3)H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. CONCLUSIONS: These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.