Brain-derived neurotrophic factor gene delivery to muller glia preserves structure and function of light-damaged photoreceptors

PURPOSE: To test the hypothesis that adenovirus (Ad)-mediated gene delivery of brain-derived neurotrophic factor (BDNF) to Müller cells can protect photoreceptors from light-induced retinal degeneration. METHODS: Adult Sprague-Dawley rats received an intraocular injection of Ad.BDNF, control Ad containing the green fluorescent protein (GFP) gene, or BDNF recombinant protein. Animals were then exposed to 5, 10, or 16 days of constant light. The effect of Ad.BDNF on photoreceptor survival was examined histologically, by measuring the outer nuclear layer (ONL) thickness, and functionally, by measuring the electroretinographic (ERG) response. RESULTS: Ad.BDNF mediated sustained expression of bioactive neurotrophin by Müller cells that lasted for at least 30 days after viral vector administration. BDNF gene delivery to Müller glia markedly increased the survival and structural integrity of light-damaged photoreceptors. For example, after 10 days of exposure to light, the average percentage of ONL preservation in the superior central retina of eyes that received Ad.BDNF was 71%, compared with 46% in eyes that received a control Ad.GFP or 15% in contralateral eyes. Of importance, retinas exposed to Ad.BDNF had more photoreceptor nuclei than retinas that received a single intraocular injection of BDNF recombinant protein. The neuroprotective effect of Ad.BDNF was accompanied by preservation of the ERG response of the treated eyes. CONCLUSIONS: These data provide proof of the concept that BDNF gene transfer into Müller cells is an effective strategy for preserving structure and function of photoreceptors in retinal degeneration.     

Retardation of photoreceptor degeneration in the detached retina of rd1 mouse

PURPOSE: To study the neuroprotective effect of experimental retinal detachment (RD) on photoreceptor degeneration in rd1 mice. METHODS: RD was produced in the eyes of rd1 mice at postnatal day (P) 9. These eyes were collected and compared to controls without RD. The effects of RD on retinal degeneration were evaluated by histochemical staining of nuclei in the outer nuclear layer (ONL), rod and cone photoreceptors, and retinal vessels at P30 in retinal sections and flatmounts. Apoptotic photoreceptors were detected by TdT-mediated dUTP nick-end labeling (TUNEL) at P15. Mice with or without RD were also reared in darkness and evaluated immunohistochemically at P30. RESULTS: The numbers of rhodopsin-positive (rod), peanut agglutinin-positive (cone), and diamino-2-phenyl-indol-stained (rod-plus-cone) cells in the ONL were increased by 2.0-fold, 1.3-fold, and 1.2-fold, respectively, in the rd1 eyes with RD compared to those without RD at P30. In the detached retina, the cone photoreceptor inner/outer segment structures and the deep retinal vessels surrounding the inner nuclear layer and the ONL, but not the ganglion cell layer, were preserved. At P15, TUNEL-positive cell numbers in the ONL were significantly reduced in the eyes with RD. Light exposure had no effect on photoreceptor degeneration in the eyes with or without RD. CONCLUSIONS: RD mediates the preservation of cone and rod photoreceptors in the ONL and surrounding vascular structures by reducing the rate of apoptosis of photoreceptors in rd1 mice. Light deprivation does not appear to be one of the mechanisms of photoreceptor protection in the detached retinas in these mice.     

Melatonin increases photoreceptor susceptibility to light-induced damage.

Melatonin is an indolamine hormone synthesized in the retina and pineal gland. It is thought to act as a paracrine neurohormone in the mammalian retina. Pinealectomy has been shown to protect photoreceptors from light-induced damage, and melatonin treatment has been reported to increase the degree of photoreceptor damage in albino rats. To determine how melatonin influences photoreceptor survival, the effect of melatonin administration on light-induced retinal damage was studied. Melatonin was administered to albino rats by intraperitoneal injections at various times before or after light exposure. The rats were exposed to high-intensity illumination (1600 lux) for 24 hr to induce photodamage, then returned to cyclic lighting for 12 days. After this, they were killed, and their eyes were removed and examined histologically. Measurements of the outer nuclear layer (ONL) thickness were taken at 12 different loci around the circumference of the retinal sections. The animals that received daily melatonin injections (100 micrograms) in the late afternoon (3 hr before lights off) for 1-3 days before photodamage showed an approximate 30% greater reduction compared with sham control animals in ONL thickness in the superior quadrant, the area most susceptible to light damage. Melatonin injections given after the photodamage did not affect ONL thickness. Although retinal susceptibility to light damage varied with time of day, the degree to which melatonin increased the degree of damage appeared unaffected by the time of day. These results suggest that melatonin may be involved in some aspects of photoreceptor sensitivity to light damage.     

Protective effect of TEMPOL derivatives against light-induced retinal damage in rats

PURPOSE: OT-551 (1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride), a TEMPOL-H (OT-674) derivative, is a new catalytic antioxidant. In the present study, the efficacy of OT-551 and OT-674 in retinal neuroprotection was tested in a model of light-induced photoreceptor degeneration. METHODS: Albino rats were intraperitoneally injected with OT-551, OT-674, or water, approximately 30 minutes before a 6-hour exposure to 2700-lux white fluorescent light. Retinal protection was evaluated histologically by measuring the thickness of the outer nuclear layer (ONL) and functionally by electroretinogram (ERG) analysis, 5 to 7 days after exposure to light. Levels of protein modification by 4-hydroxynonenal (4-HNE) and 4-hydroxyhexenal (4-HHE), which are end products of the nonenzymatic oxidation of n-6 and n-3 polyunsaturated fatty acids, respectively, were measured by Western dot blot analysis immediately after exposure to light. RESULTS: After exposure to light, water-treated animals had a 77% loss of ERG b-wave amplitudes and a 26% and 56% loss of mean ONL thickness in the inferior and superior hemispheres, respectively. Compared with water-treated rats, ERG b-wave amplitudes in light-exposed eyes were significantly higher in 25 (P < 0.05)-, 50 (P < 0.05)-, and 100 (P < 0.001)-mg/kg OT-551-treated rats. Mean ONL thickness in the superior hemisphere was significantly higher in 25 (P < 0.01)-, 50 (P < 0.01)-, and 100 (P < 0.001)-mg/kg OT-551-treated, light-exposed eyes and in 100 mg/kg (P < 0.05) OT-674-treated eyes. No decrease of ONL thickness was observed in the light-protected covered fellow eyes in any animal. Increased levels of 4-HNE- and 4-HHE-protein modifications after exposure to light in water-treated eyes were completely counteracted by 100 mg/kg OT-551. CONCLUSIONS: Systemic administration of OT-551 and OT-674 provides both functional and morphologic photoreceptor cell protection against acute light-induced damage, most likely by inhibiting lipid peroxidation. The protection by OT-551 was greater than OT-674.     

Gene therapy for detached retina by adeno-associated virus vector expressing glial cell line-derived neurotrophic factor

PURPOSE: To examine the protective effect of glial cell line-derived neurotrophic factor (GDNF) on retinal detachment (RD)-induced photoreceptor damage by using gene delivery. METHODS: Gene delivery to photoreceptors was achieved by subretinal injection of recombinant adeno-associated virus expressing GDNF (rAAV-GDNF) in the right eyes and AAV expressing Escherichia coli LacZ (rAAV-LacZ) in the left eyes of Lewis rats. RD in bilateral eyes was induced with subretinal injection of high-density vitreous substitute in the temporal retina 3 weeks after gene delivery. The synthesis and accumulation of GDNF within the retina was monitored 3 weeks after RD by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. The rescue of photoreceptors was evaluated by monitoring the preservation of the thickness of photoreceptor outer segment (OS) and outer nuclear layer (ONL). Apoptosis in the photoreceptors was studied using the TdT-dUTP terminal nick-end labeling (TUNEL) method 2 days after RD. Müller cell activity was checked using the immunohistochemistry with glial fibrillary acidic protein (GFAP) antibody 28 days after RD. RESULTS: Gene delivery was demonstrated by immunohistochemical study. The results of ELISA confirmed that high levels of neurotrophic factors were produced in retinas. Photoreceptor OS degeneration and the gradual shortening of the ONL were noted after RD in all the eyes. However, rAAV-GDNF-treated eyes retained longer OS than rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD. ONL was also longer in rAAV-GDNF-treated eyes than in rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD. GDNF-treated eyes had statistically less apoptotic cells than control eyes in photoreceptor layer (P = 0.043). Subretinal proliferation of Müller cells was suppressed in the GDNF-treated group, indicating less scar formation. CONCLUSIONS: GDNF is a potential factor that can protect photoreceptors from degeneration. In addition to preserving the OS and ONL structures, GDNF may exert its protective action by preventing the apoptosis of photoreceptors after RD. GDNF gene therapy may be a valuable adjuvant to current treatments in certain complicated forms of RD.     

Loss of BCL-XL in rod photoreceptors: Increased susceptibility to bright light stress

PURPOSE: BCL-X(L), an anti-apoptotic member of the BCL-2 family proteins and a cell death/survival checkpoint regulator, was shown to be upregulated in bright light-stressed mouse photoreceptors during an investigation of bright light-induced protein expression. To investigate the significance of BCL-X(L) upregulation in the bright light damage model, the Bcl-x gene was disrupted specifically in mouse rod photoreceptors, and the effect of Bcl-x disruption was characterized on retinal apoptosis, function, and morphology. METHODS: Rod-specific Bcl-x knockout mice, generated by mating mouse opsin promoter-controlled Cre mice with floxed Bcl-x mice, were subjected to bright light stress. Retinal apoptosis in the bright light-stressed conditional Bcl-x knockout mice was characterized with TUNEL, DNA fragmentation, and nuclear staining assays. Photoreceptor structural and functional integrity in the bright light-stressed conditional Bcl-x knockout mice was determined by measuring photoreceptor outer nuclear layer (ONL) thickness and electroretinography amplitudes. RESULTS: Disruption of Bcl-x in rod photoreceptors caused increased photoreceptor apoptosis, decreased retinal function, and decreased ONL thickness in bright light-stressed mice. CONCLUSIONS: The loss of BCL-X(L) increased rod photoreceptor susceptibility to bright light stress. Although the biochemical mechanism(s) of BCL-X(L) in photoreceptor death or survival has not been investigated extensively, results of the present study suggest that BCL-X(L), a cell survival/death checkpoint regulator, is involved in photoreceptor survival under bright light stress.     

Caspase-dependent apoptosis in light-induced retinal degeneration

PURPOSE: To study the apoptotic mechanism involved in our model of light-induced retinal degeneration. METHODS: Rats were injected intravitreally with PBS, 2% dimethyl sulfoxide (DMSO), caspase inhibitor Z-VAD-FMK (1.06 mM), Z-YVAD-FMK (0.16 mM), or Z-DEVD-FMK (2 mM) before they were placed in constant light (3400 lux) for 24 hours. Additional controls included rats that were uninjected or were punctured with a dry needle. Electroretinograms were recorded before injection and 1 day after the cessation of exposure to constant light. A group of rats was killed for apoptotic cell detection in the outer nuclear layer. Fifteen days later, the remaining rats were killed for histology, and the outer nuclear layer (ONL) thickness was measured. Caspase-1, caspase-3, and calpain activities were measured before and 1 day after exposure to the damaging light. RESULTS: ZVAD, YVAD, and DEVD inhibited caspase-1 and -3 activities, but not calpain activity, from the beginning and up to 1 day after light exposure. In untreated, dry needle-punctured, PBS, DMSO, and YVAD groups, light exposure significantly reduced retinal function and ONL thickness and increased by 51-fold the number of apoptotic cells. ZVAD and DEVD preserved retinal function to 86% and 78%, respectively, and reduced by three times the number of apoptotic photoreceptors. ONL thickness was more preserved in ZVAD (to 72%) than in DEVD (to 56%). CONCLUSIONS: In the authors' model of retinal degeneration, photoreceptor cells die through a caspase-dependent mechanism. However, the molecular events involved during and after light exposure seemed to implicate different proteases.     

Neuroprotective effects of naloxone against light-induced photoreceptor degeneration through inhibiting retinal microglial activation

PURPOSE: To determine the role of microglial activation in light-induced photoreceptor degeneration and the neuroprotective effects of naloxone as a novel microglial inhibitor. METHODS: Sprague-Dawley rats were exposed to intense blue light for 24 hours. Daily intraperitoneal injection of naloxone or PBS as a control was given 2 days before exposure to light and was continued for 2 weeks. Apoptotic cells were detected by the TUNEL assay, and anti-OX42 antibody was used to label retinal microglia. Western blot was applied to evaluate the retinal interleukin (IL)-1beta protein levels. Retinal histologic examination and electroretinography (ERG) were also performed to evaluate the effects of naloxone on light-induced photoreceptor degeneration. RESULTS: TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina as early as 2 hours and peaked at 24 hours after exposure to light. OX42-positive microglia occurred in the ONL and subretinal space at 6 hours, peaked at 3 days, and changed morphologically from the resting ramified to the activated amoeboid. Expression of IL-1beta protein was also significantly increased at 3 days. Compared with the control, the number of microglia in the outer retina was significantly decreased in the naloxone-treated group at 3 days, and the thickness of ONL and the amplitudes of dark-adapted a- and b-waves were also well preserved at 14 days. CONCLUSIONS: The activation and migration of microglia and the expression of neurotoxic factor (IL-1beta) coincide with photoreceptor apoptosis, suggesting that activated microglia play a major role in light-induced photoreceptor degeneration. Inhibiting microglial activation by naloxone significantly reduces this degeneration.     

Neuroprotection of photoreceptors by minocycline in light-induced retinal degeneration

PURPOSE: Microglial cells have been found to play pivotal roles in various neuronal degenerative diseases such as Parkinson's and Alzheimer's diseases. Minocycline, a microglial inhibitor, has recently been shown to be neuroprotective in various models of cerebral ischemia and degenerative diseases of the brain. This study was conducted to evaluate the neuroprotective effect of minocycline and the role of microglia in light-induced retinal degeneration. METHODS: BALB/cJ mice were exposed to intense green light for 3 hours and observed during 1, 3, or 7 days of dark recovery. The animals received intraperitoneal injections of minocycline or vehicle 1 day before exposure to light for 2, 4, or 8 days, depending on the periods of survival. Morphologic, morphometric, immunohistochemical, and electrophysiological studies were performed to evaluate the efficacy of minocycline in the amelioration of light-induced retinal degeneration and the possible involvement of microglial cells. RESULTS: Minocycline treatment provided marked amelioration in the loss of photoreceptors in light-induced retinal degeneration, as evidenced by morphologic, morphometric, and electrophysiologic criteria. Morphologically, the minocycline-treated group showed markedly better preservation of the outer retina after exposure to light. Morphometrically, at 7 days after exposure to light, in the minocycline-treated animals, 89.1% of the normal-appearing photoreceptor nuclei remained, but in the retinas of the vehicle-control group only 38.0% of these nuclei remained. This difference was statistically significant (P < 0.001). At 7 days after exposure to light electroretinography (ERG) showed that minocycline significantly preserved the amplitudes of dark-adapted a- and b-wave and light-adapted b-wave, which were all significantly reduced after exposure to light. Concomitant with this protective effect, at 3 days after exposure to light, the CD11b(+) microglial cells in the outer nuclear layer (ONL) and subretinal space in the minocycline-treated group were significantly decreased (by 63.5%) when compared with those in the light-exposed, vehicle-treated control group (P < 0.01). CONCLUSIONS: Minocycline is neuroprotective against light-induced loss of photoreceptors, possibly through the inhibition of retinal microglial activation.     

Systemic administration of phenyl-N-tert-butylnitrone protects the retina from light damage

PURPOSE. This study was conducted to test the hypothesis that phenyl-N-tert-butylnitrone (PBN), a spin-trapping agent known to cross the blood-brain barrier and protect the brain from ischemia-reperfusion injury, is incorporated into the retina after intraperitoneal injection and protects photoreceptor cells from the damaging effects of constant visible light. METHODS. Albino rats were injected intraperitoneally with PBN (aqueous solution) or water, or were not injected, and then were placed in constant light (2700 lux) for 24 hours. The incorporation of PBN into the retina was determined by high-performance liquid chromatography. Electroretinograms (ERGs) were recorded before light treatment and 1 and 15 days after the cessation of exposure to constant light. Eyes were taken for histology at each time point and outer nuclear layer (ONL) thickness determined. RESULTS. PBN was incorporated into the retina after intraperitoneal injection. Both control (water-injected and uninjected) groups exposed to constant light maintained only 28% of ONL thickness and 20% of retinal function, compared with the unexposed control group. In contrast, the PBN-treated animals maintained 80% of ONL thickness and exhibited 87% of retinal function. CONCLUSIONS. PBN protects the albino rat retina from the damaging effects of constant light stress. That light-induced and hereditary retinal degenerations share certain morphologic hallmarks and follow a similar apoptotic mechanism of degeneration raises the possibility of pharmacologic therapy for hereditary and environmentally induced neurodegenerative disorders.     

频域OCT对正常人和青光眼光感受器细胞层厚度的检测

背景青光眼以视网膜内层的神经节细胞丢失为主要病理特征,但其是否累及视网膜外层尚有争议。部分研究认为青光眼将导致视网膜外层（光感受器）功能的异常,而病理学研究得出了不同的结论。目的用频域OCT测量正常人和青光眼患者光感受器细胞层的厚度,探讨青光眼对光感受器细胞层厚度的影响。方法采用病例对照研究。用频域OCT（SDOCT）对正常人38例38眼和青光眼患者48例48眼的黄斑区进行扫描,由一位检测者采用Sigma图像分析软件盲法测量黄斑中心凹和旁中心凹处（中心凹外1．5mm）视网膜光感受器层的厚度。同时采用时域OCT（Stratus OCT）测量所有检测眼的视盘周围视网膜神经纤维层（RNFL）厚度,比较正常组和青光眼组光感受器细胞核层的平均厚度,分析光感受器细胞层厚度与RNFL厚度的关系。结果正常组和青光眼组在黄斑中心凹光感受器细胞核层厚度分别是（96．7±10．7）μm、（103．7±13．3）μm,差异有统计学意义（P=0．011）;中心凹光感受器内节和外节层厚度分别是（59．3±5．5）μm、（59．5±5．5）μm,差异无统计学意义（P=0．890）。正常组和青光眼组在中心凹外3mm处光感受器细胞核层厚度分别是（70．9±14．0）μm、（68．7±10．7）μm,光感受器内节和外节层厚度分别为（45．2±6．4）μm,（43．6±5．5）μm,差异均无统计学意义（P=0．410,P=0．228）。黄斑中心凹处光感受器细胞核层厚度和RNFL厚度两者有二元线性关系（γ=-0．019X。＋2．73X＋10．34,R^2=0．211,P=0．005）。结论青光眼的黄斑中心凹光感受器细胞核层显著增厚,并随病程的变化而改变。     

Encapsulated cell-based intraocular delivery of ciliary neurotrophic factor in normal rabbit: dose-dependent effects on ERG and retinal histology

PURPOSE: ERG and histologic changes were investigated in normal rabbits after intravitreal implantation of encapsulated cell technology (ECT) devices releasing ciliary neurotrophic factor (CNTF). METHODS: Fifteen adult New Zealand White albino rabbits had ECT devices secreting CNTF at 22, 5, or 0 ng/d implanted in the superior temporal quadrant of the left eye. The low dose has been shown to produce substantial rescue of photoreceptors in the rcd1 canine model of retinal degeneration. Right eyes were untreated. Ganzfeld dark- and light-adapted ERGs and clinical observations were performed at 5, 15, and 25 days after implantation. Rod a-waves and rod and cone b-waves and outer nuclear layer (ONL) morphology were evaluated at 25 days. RESULTS: Clinical examination showed minimal changes in a few CNTF-treated eyes, including vitreous membranes and engorgement of iris vessels at day 25. Retinas appeared normal. CNTF did not significantly affect the rod a- or b-waves, although the b-wave amplitude tended to be larger in CNTF-treated retinas at low flash intensities. The cone b-wave amplitude was significantly reduced in high-dose eyes at some flash intensities. The ONL area in high-dose eyes was significantly greater because of increased thickness than in fellow retinas. ONL cell size was significantly increased, and staining density decreased in CNTF-treated retinas. CONCLUSIONS: CNTF, given by intravitreal ECT device at doses that protect photoreceptors in a canine model of retinal degeneration (5 ng/d), did not adversely affect either rod or cone ERG function of normal rabbit retina. The cone ERG was more sensitive to suppression being reduced, at low flash intensities, by 22 ng/d. Dose-related changes in the ONL and photoreceptor cell nuclei did not represent a toxic effect, because they were not associated with deficits in the rod ERG over a broad range of intensities.     

Optimal conditions for long-term photoreceptor cell rescue in RCS rats: the necessity for healthy RPE transplants.

Retinal pigment epithelial (RPE) cells from normal Long-Evans, healthy Royal College of Surgeons (RCS) pigmented congenic (rdy+p+) and RCS pigmented dystrophic rats were transplanted into retinas of RCS non-pigmented dystrophic rats at post-natal days 17 and 26 (P17 and P26). When examined at P60, rescued photoreceptor cells were found in all transplanted groups. In addition, a small, but significant increase in the outer nuclear layer (ONL) thickness was detected in retinas injected with vehicle (sham control); however, the ONL thickness was reduced to control levels by 3-5 months after injections in both the sham controls and the group grafted with RCS pigmented dystrophic RPE cells. Likewise, large volumes of vehicle were injected into the subretinal space resulting in no long lasting beneficial effects. Also, there was no significant difference between the ONL thickness in retinas grafted with RCS congenic RPE and those grafted with Long-Evans RPE. When donor RPE cells from Long-Evans rats at neonatal and adult stages were transplanted into P26 RCS hosts, photoreceptor cell rescue could be found; however, the younger RPE cells affected a better rescue than those derived from adult eyes. In addition, when RPE cells from 6- to 9-day-old Long-Evans rats were transplanted into retinas of RCS dystrophic rats at 10, 17 and 26 days, significant rescue of photoreceptor cells was observed. Of these transplantation times, day 17 appeared to affect the best rescue of photoreceptor cells up to 1 yr. In contrast, little or no rescue was observed in the retinas of those RCS dystrophic rats when RPE cell transplantation was performed at P38, P43 and P48. The photoreceptor cell rescue was also found to be dependent on the concentration of RPE cells injected, which was maximal at 60,000-120,000 cells microliters-1. These findings lead us to conclude that in order to affect long-term, up to 1 yr, rescue of photoreceptor cells in the RCS rat, young, healthy RPE cells are required and transplantation needs to be performed at early stages of the disease process.     

光损伤导致视网膜感光细胞凋亡的实验研究

目的探讨视网膜光损伤后感光细胞病理学改变的特征及其发生机制。方法以白色强光持续照射的方法制成大鼠视网膜光损伤模型并采用常规HE染色与TUNEL技术对光损伤后视网膜感光细胞的病理学改变进行动态观察研究。结果白色强光照射后视网膜感光细胞发生进行性的变性,TUNEL标记结果显示光损伤后视网膜外核层出现大量阳性着色细胞。结论持续高强度白光照射可选择性地导致视网膜感光细胞发生进行性的变性而凋亡是感光细胞退行性变性的重要发生机制。     

Neuroprotection in the juvenile rat model of light-induced retinopathy: evidence suggesting a role for FGF-2 and CNTF

PURPOSE: In a former study, it was demonstrated that the retina of juvenile Sprague-Dawley (SD) rat has a remarkable intrinsic resistance to light-induced retinopathy compared with the adult retina. The purpose of the present study was to investigate the cellular and molecular mechanisms underlying this endogenous resistance to light-induced damage. METHODS: Juvenile SD rats were exposed for 6 (from P14 to P20) or 14 (from P14 to P28) days to a bright, cyclic, luminous environment of 10,000 lux. Retinal histology was examined immediately after exposure to light or at 2 months of age, and photoreceptor cell death was quantified by measuring the thickness of the outer nuclear layer (ONL) and by TUNEL assays. Changes in protein levels and cellular localization of fibroblast growth factor (FGF)-2, ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF) were determined by Western blot analysis and retinal immunohistochemistry, respectively. RESULTS: The data demonstrate that although the rate of photoreceptor loss was different after 6 and 14 days of exposure to light, similar ONL thickness was reached at 2 months of age--that is, 4 to 5 weeks after exposure to light. A large number of TUNEL-positive photoreceptors was visualized immediately after 6 and 14 days of exposure to light, reflecting the intense cell death that was occurring in the ONL. Western blot analysis showed that exposure to light induced a strong upregulation of the neurotrophic factors FGF-2 and CNTF in juvenile retinas, whereas no change in BDNF protein expression was noted. Of interest, after exposure to light, endogenous FGF-2 and CNTF were selectively upregulated in Müller cells. CONCLUSIONS: The results show that endogenous expression of FGF-2 and CNTF by Müller glia may play a role in protecting the juvenile retina from light-induced damage.     

Effects of macrophage and retinal pigment epithelial cell transplants on photoreceptor cell rescue in RCS rats

The effects of macrophage transplants on photoreceptor cell survival in retinas of Royal College of Surgeons (RCS) dystrophic rats were contrasted with RPE-cell transplants, sham-injection and surgical controls. The effects of these different treatments on the thickness and total area of the outer nuclear layer (ONL) were evaluated by light and electron microscopy at 1, 2 and 5 months after transplantation or surgical manipulations. Macrophage transplants into dystrophic retinas, although significantly reducing the debris zone thickness (p less than 0.01), had little effect on photoreceptor cell survival (2-3 cells thick ONL) after two months. In contrast, two months after RPE-cell transplantation, retinas exhibited an 8-10 cell thick ONL. Also, inner and outer segments of rescued photoreceptor cells were present, especially in areas directly beneath RPE-cell transplants. At the same time period, retinas injected with saline had a 2-3 cell thick ONL with no organized inner or outer segments. Furthermore, the affected ONL area in macrophage-transplanted or saline-injected retinas was significantly smaller than that seen in RPE-cell transplanted retinas (p less than 0.0001). Surviving photoreceptor cells were found only in the RPE-cell transplanted retinas five months after treatment. No effect on photoreceptor cell survival was seen in saline-injected, needle-inserted or incision-only retinas. Thus, transplantation of healthy RPE cells is an effective long-term therapeutic approach to correct the genetic defect in retinas of RCS dystrophic rats.     

Protective effects of soft acrylic yellow filter against blue light-induced retinal damage in rats

Recently, a yellow intraocular lens (IOL) was developed for the purpose of reducing potential blue light-induced retinal damage after cataract surgery. However, the effect of yellow filters on retinal protection remains to be clarified. To test the protective effects of yellow filters on blue light-induced retinal damage, a yellow and a clear soft acrylic filter were attached to the right and left eyes, respectively, of albino rats and exposed to 4.5 k lux blue fluorescent lights with peak wavelength at 420 nm (ranging 380-500 nm; short blue) or 446 nm (ranging 400-540 nm; long blue) for 6h. To assess retinal damage, the electroretinogram (ERG) was recorded at 7 days, outer nuclear layer (ONL) thickness and area were measured at 7 days, apoptosis was analyzed by TUNEL staining at 24 h, and the level of lipid peroxidation in retinas was assessed by Western dot blots using specific antibodies against 4-hydroxynonenal (4-HNE)- and carboxyethylpyrrole (CEP)-modified proteins immediately after light exposure. After short blue light exposure, a- and b-wave ERG amplitudes and the ONL thickness at 1-2.5 mm inferior and 0.5-2.5 mm superior to optic nerve head (ONH) were significantly reduced. TUNEL staining in the ONL at 0-2 mm inferior and 1-2 mm superior to the ONH, and retinal levels of 4-HNE- and CEP-modified proteins were significantly increased in the clear filter-covered eyes compared to yellow filter-covered eyes. After long blue light exposure, the only difference seen was a greater ONL thickness at 1.5 mm superior to the ONH in yellow filter-covered eye. Transmission of light through the yellow filter was 58% for short blue and 89% for long blue compared to the clear filter. The ONL area was not different between clear filter-covered and -uncovered eyes after exposure to short or long blue light. Given the results, yellow IOL material protects the retina against acute shorter wavelength blue light exposure more effectively than the clear IOL material.     

Minocycline partially inhibits caspase-3 activation and photoreceptor degeneration after photic injury

PURPOSE: To evaluate the possible role of caspase-3 in retinal photic injury, and to investigate whether minocycline can ameliorate light-induced photoreceptor degeneration. METHODS: Retinal photic injury was induced in rats by exposure to intense light. Expression of caspase-3 was studied using Western blot analysis, immunohistochemical staining and enzyme activity assay. Apoptotic photoreceptor cells were detected by the TdT-dUTP terminal nick-end labeling (TUNEL) method. Minocycline (15, 30 or 45 mg/kg) was administered before or after photic injury in rats randomly assigned to pretreatment and posttreatment groups. Minocycline and vehicle-treated retinas subjected to photic injury were compared with respect to Western blotting, enzyme activity assay, quantitative counts of TUNEL stains, morphometry of the outer nuclear layer (ONL) thickness and histopathological examination. RESULTS: After light exposure, active caspase-3 and poly-adenosine diphosphate-ribose-polymerase were upregulated in the retinas and increased caspase-3 immunoreactivity was observed in the ONL. Caspase-3 enzyme activity increased in the retinas that underwent photic injury, and this increase was significantly reduced in minocycline pretreated (30 and 45 mg/kg) and posttreated (45 mg/kg) groups. Intraperitoneal administration of minocycline before or after photic injury in rats also resulted in less TUNEL-positive photoreceptors, as assessed by the quantitative TUNEL counts. The degree of retinal degeneration, measured by the ONL thickness 14 days after photic injury, was significantly improved in minocycline pretreatment (45 mg/kg) rats. CONCLUSIONS: We demonstrate that increased caspase-3 activities localize specifically within the ONL after photic injury, and that minocycline partially inhibits caspase-3 activation and photoreceptor degeneration in this animal model.     

Photoreceptor rescue in the RCS rat without pigment epithelium transplantation

Transplantation of normal retinal pigment epithelium (RPE) to the subretinal space has been reported to rescue photoreceptors in the RCS rat. Moreover, the rescue effect was surprisingly large considering the relatively small number of RPE cells transplanted. The reason for this widespread rescue of photoreceptors is not known, nor is the mechanism for outer segment phagocytosis in photoreceptors not apposed to the transplanted RPE cells. This suggests that the rescue effect may not be solely mediated by the transplanted cells. We therefore wished to test whether the transplantation surgery itself might contribute to the rescue of RCS photoreceptors. For these control experiments, we performed the surgery on juvenile RCS rats as described by others for the transplantation of RPE but instead of injecting RPE, we injected saline. We sacrificed the RCS control operates two months following surgery. In the area of the surgery (superior retinal quadrant) the outer nuclear layer (ONL) was up to 8-10 photoreceptor cells thick, while at the extreme inferior margin of the retina the ONL was almost eliminated. To investigate the role of temporary retinal detachment in photoreceptor rescue we repeated the above experiment using our trans-corneal approach to the subretinal space. This procedure results in a large temporary retinal detachment and little or no damage to the choroid and sclera.(ABSTRACT TRUNCATED AT 250 WORDS)