Effect of l-theanine tablets in reducing stress-related emotional signs in cats: an open-label field study.

BACKGROUND: L-theanine is an aminoacid found in tea leaves which has relaxing effects in humans and animals. It is a structural analogue of glutamate which can bind glutamate receptors. Although the relaxing action of L-theanine has been shown in humans, laboratory animals and dogs, it has never been published in cats. The goal of this open-label, multicentre and prospective trial was to determine whether an L-theanine based oral supplement (Anxitane®, Virbac, France) could attenuate manifestations of stress in cats under field conditions. CASE PRESENTATION: Thirty-three privately owned cats presenting signs associated with stress or fear (inappropriate urination/defecation, fear-induced aggressiveness, hypervigilance/tenseness or physical/functional manifestations of stress) for at least 1 month, were included in the study. They were given L-theanine (Anxitane®, 25 mg twice a day) for 30 days and 20 stress-related parameters were scored at Days 0, 15 and 30. The evolution of some parameters was also rated relative to Day 0. All median scores of the 20 parameters were significantly reduced at D30, and 30/33 cats (91%) had a reduced global score at the end of the study, including 21/33 with ≥50% score reduction. The median (IQR) global scores went from 18 (13-23) at D0 to 11 (8-13) at D15 and 5 (3-12) at D30 (p < 0.0001; Friedman test; significant reduction starting from D15). All the stress-related signs were significantly improved compared to D0, according to the owners, especially inappropriate elimination. Tablet palatability was judged good or very good in 94% of cases with spontaneous intake by cats when given by hand or in food. Tolerance was satisfactory as well, and no side effects were reported, so that most owners (27/33; 82%) were satisfied with the product. CONCLUSIONS: Despite the lack of a placebo group, it can be concluded that L-theanine (Anxitane®) helped to improve the undesirable manifestations of stress in cats in as soon as 15 days, though better results could be seen after 30 days of administration. These encouraging results show that L-theanine can help manage stress-related behaviour, but additional trials with a placebo group should be run to confirm this effect.

Protection of Rpe65-deficient mice identifies rhodopsin as a mediator of light-induced retinal degeneration.

Light-induced apoptosis of photoreceptors represents an animal model for retinal degeneration. Major human diseases that affect vision, such as age-related macular degeneration (AMD) and some forms of retinitis pigmentosa (RP), may be promoted by light. The receptor mediating light damage, however, has not yet been conclusively identified; candidate molecules include prostaglandin synthase, cytochrome oxidase, rhodopsin, and opsins of the cones and the retinal pigment epithelium (PE). We exposed to bright light two groups of genetically altered mice that lack the visual pigment rhodopsin (Rpe65-/- and Rho-/-). The gene Rpe65 is specifically expressed in the PE and essential for the re-isomerization of all-trans retinol in the visual cycle and thus for the regeneration of rhodopsin after bleaching. Rho-/- mice do not express the apoprotein opsin in photoreceptors, which, consequently, do not contain rhodopsin. We show that photoreceptors lacking rhodopsin in these mice are completely protected against light-induced apoptosis. The transcription factor AP-1, a central element in the apoptotic response to light, is not activated in the absence of rhodopsin, indicating that rhodopsin is essential for the generation or transduction of the intracellular death signal induced by light.

New views on RPE65 deficiency: the rod system is the source of vision in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is the most serious form of the autosomal recessive childhood-onset retinal dystrophies. Mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium, account for 10-15% of LCA cases. Whereas previous studies of RPE65 deficiency in both animal models and patients attributed remaining visual function to cones, we show here that light-evoked retinal responses in fact originate from rods. For this purpose, we selectively impaired either rod or cone function in Rpe65-/- mice by generating double- mutant mice with models of pure cone function (rhodopsin-deficient mice; Rho-/-) and pure rod function (cyclic nucleotide-gated channel alpha3-deficient mice; Cnga3-/-). The electroretinograms (ERGs) of Rpe65-/- and Rpe65-/-Cnga3-/- mice were almost identical, whereas there was no assessable response in Rpe65-/-Rho-/- mice. Thus, we conclude that the rod system is the source of vision in RPE65 deficiency. Furthermore, we found that lack of RPE65 enables rods to mimic cone function by responding under normally cone-isolating lighting conditions. We propose as a mechanism decreased rod sensitivity due to a reduction in rhodopsin content to less than 1%. In general, the dissection of pathophysiological processes in animal models through the introduction of additional, selective mutations is a promising concept in functional genetics.

The absence of c-fos prevents light-induced apoptotic cell death of photoreceptors in retinal degeneration in vivo.

Apoptotic cell death in the retina was recently demonstrated in animal models of the hereditary human retinal dystrophy known as retinitis pigmentosa. Although recent evidence indicates that the proto-oncogene c-fos is a mediator of apoptosis, its precise role is unclear. In fact, under some conditions, c-fos may even protect against apoptotic cell death. In the retina, c-fos is physiologically expressed in a diurnal manner and is inducible by light. We previously observed a light-elicited, dose-dependent apoptotic response in rat photoreceptors. To determine whether c-fos is involved in the light-induced apoptotic pathway we have used control mice and mice lacking c-fos. We found that following dark adaptation and two hours of light exposure both groups of animals exhibited only a few apoptotic cells. However, at 12 and 24 additional hours after light exposure, apoptosis increased dramatically in controls but was virtually absent in those mice lacking c-fos. Therefore, c-fos is essential for light-induced apoptosis of photoreceptors. Notably, c-fos is continuously upregulated concomitant with apoptotic photoreceptor death in our system and in animal models of retinitis pigmentosa (Agarwal, N. et al., Invest. Ophthalmol. Vis.Sci. Suppl. 36, S638 and Rich, K.A. et al., Invest. Ophthalmol. Vis. Sci. Suppl. 35, 1833). Inhibition of c-fos expression might therefore represent a novel therapeutic strategy to retard the time course of retinal dystrophies and light-induced retinal degeneration.

Effects of a topically applied preparation of epidermal lipids on the stratum corneum barrier of atopic dogs.

Canine atopic dermatitis (AD) is characterized ultrastructurally by disorganization of the lamellar lipids (LLs) in the stratum corneum (SC), similar to that seen in the human disease. This study, based on the examination of biopsy samples, was designed to investigate the expression of canine epidermal lipids and to evaluate quantitatively, by means of electron microscopy and ruthenium tetroxide post-fixation, the effect of a new topical skin lipid complex (SLC) on the structural deficit in the skin of five dogs with AD. The non-lesional skin of atopic dogs differed from the skin of healthy dogs in that the LLs were reduced in number and highly disorganized. After repeated applications of SLC to the non-lesional skin of dogs with AD, numerous LLs were observed in the deepest part of the SC, occupying 74% of the inter-corneocyte space, while they accounted for only 31.8% of the inter-corneocyte space in comparable biopsy samples from untreated (control) skin of the same dogs. In contrast, the LLs filled 89.5% of the deepest inter-corneocyte spaces in the SC of healthy dogs. Many keratinosomes were observed at the interface between living epidermis and SC after treatment of non-lesional AD skin. Stacks of short LL discs represented 57.6% of the total LLs found in the newly formed SC compactum in the treated atopic dogs. It is suggested that the treatment with SLC stimulated the production and secretion of endogenous SC lipids, contributing to the formation of an improved epidermal barrier.

Effect of S-adenosylmethionine tablets on the reduction of age-related mental decline in dogs: a double-blinded, placebo-controlled trial.

Oral S-adenosylmethionine (SAMe) tosylate supplementation (Novifit tablets, Virbac) was evaluated as a dietary aid for the management of age-related mental impairment in dogs. Thirty-six dogs older than 8 years that had displayed signs of cognitive dysfunction for at least 1 month were selected for the study. The dogs were administered 18 mg/kg SAMe tosylate (n=17) or identical placebo tablets (n=19) for 2 months. Concurrent behavioral treatment was forbidden. A 14-item standardized questionnaire evaluated behavior and locomotion difficulties. Compared with the placebo group, SAMe-treated dogs showed greater improvement in activity (41.7% versus 2.6% after 4 weeks, P<.0003; 57.1% versus 9.0% after 8 weeks, P<.003) and awareness (33.3% versus 17.9% after 4 weeks, P<.05; 59.5% versus 21.4% after 8 weeks, P<.01). The aggregate mental impairment score was reduced by more than 50% in 41.2% and 15.8% of dogs treated with SAMe and placebo, respectively, at week 8. SAMe tosylate tablets proved safe and effective in improving signs of age-related mental decline in dogs.

A field trial evaluation of the prophylactic efficacy of amitraz-impregnated collars against canine babesiosis (Babesia canis rossi) in South Africa.

South African canine babesiosis caused by Babesia canis rossi is a common clinical disease in dogs in South Africa and remains a significant cause of domestic dog mortality. To determine whether tick-repellent, 9% amitraz-impregnated tick collars (Preventic-Virbac) could prevent tick-borne exposure to B. canis rossi, 50 dogs were assigned to two groups. Group 1 (20 dogs), polymerase chain reaction (PCR)--and reverse line blot (RLB)-negative for B. canis rossi, were fitted with amitraz collars and blood samples collected monthly, over a 6-month period, and analysed for B. canis rossi. Group 2 (30 dogs) included 5 dogs selected on a month-by-month basis from a population of dogs from the same geographical area as the group 1 dogs, but with no history of previous tick control, which were blood-sampled together with the treatment group and analysed for B. canis rossi by PCR and RLB, to serve as the control group. Eight of the 30 control dogs (26.6%) were PCR/RLB positive for B. canis rossi, indicating high pathogen exposure during the trial period. All twenty of the treatment group dogs remained negative for B. canis rossi throughout the 6 months of the trial. These results suggest that the use of amitraz-impregnated collars had a significant effect on reducing infection with B. canis rossi.

The efficacy of an antiseptic and microbial anti-adhesive ear cleanser in dogs with otitis externa.

A new antimicrobial ear cleanser was evaluated for the treatment of bacterial and yeast ear infection in dogs. Forty-five dogs with erythemato-ceruminous or purulent otitis externa were randomly allocated to two treatment groups: reference ear cleanser (Epiotic, Virbac) or test ear cleanser (Epiotic Advanced, Virbac). Ear cleansing was performed twice daily for 2 weeks, and no other treatment was allowed. By week 2, clinical (exudate quantity, erythema, stenosis, excoriation, and odor) and discomfort (pain, ear scratching, and head shaking) scores were significantly decreased (P < .0001 for all) and no microbial overgrowth could be detected in 25 (64.1%) and 32 (68.1%) ears treated with Epiotic and Epiotic Advanced, respectively. The new pH-balanced, propylene glycol-free test ear cleanser, which incorporates microbial adhesin-blocking carbohydrates, proved as effective as the reference acidic formula.

The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration.

Excessive light can cause retinal degeneration and may be an environmental cofactor accelerating retinal dystrophies and age-related diseases. In rodent models, the light damage susceptibility (LDS) of the retina is determined genetically. In two mouse strains, with different degrees of LDS, a Leu450Met variation in the pigment epithelial protein RPE65 was shown recently to cosegregate with low LDS. Because light damage is rhodopsin-mediated, and RPE65 is essential for the regeneration of rhodopsin in the visual cycle, we analyzed this variation regarding rhodopsin metabolism and LDS in four mouse strains. We found that, in contrast to previous assertions, LDS does not correlate with the maximal retinal content of rhodopsin present after dark adaptation. Instead, LDS correlated positively with the kinetics of rhodopsin regeneration, which determine rhodopsin availability during light exposure. Light damage occurred after absorption of a threshold dose of photons and thus fast regeneration, as observed in those two strains having Leu at position 450 of RPE65, was correlated with the occurrence of photoreceptor apoptosis after short exposure. In contrast, mice with the Leu450Met variation of Rpe65 regenerated rhodopsin with slow kinetics and showed an increased resistance to light-induced retinal degeneration. In these mice, RPE65 protein levels were reduced by a post-transcriptional mechanism. F(1) hybrid mice, carrying one normal and one variant Rpe65 gene, had intermediate levels of the corresponding protein and showed intermediate rhodopsin regeneration kinetics and an intermediate LDS. Thus, none of the two variants of Rpe65 had a dominant effect.

c-fos controls the "private pathway" of light-induced apoptosis of retinal photoreceptors.

White light (5 klux for 2 hr) induces apoptosis of rod photoreceptors in wild-type mice (c-fos(+/+)) within 24 hr, whereas rods of c-fos knock-out mice (c-fos(-/-)) are protected (). The range of this protection was tested by analyzing retinas of c-fos(+/+) and c-fos(-/-) mice up to 10 d after exposure to threefold increased light intensities (15 klux for 2 hr). In c-fos(-/-) mice, rods were unaffected, whereas they were destroyed in c-fos(+/+) mice. After light exposure, mitochondrial damage in rods was observed exclusively in c-fos(+/+) mice. Electroretinograms recorded 48 hr after exposure revealed a decrease of all components in c-fos(+/+) mice but indicated no light-induced loss of function in c-fos(-/-) mice. Thus, in c-fos(-/-) mice, light-induced apoptosis is blocked or its threshold is elevated more than threefold. Increased activity of the transcription factor activator protein-1 (AP-1) in retinas of light-exposed c-fos(+/+) mice indicated an acute contribution of AP-1 to apoptosis induction. AP-1 activity increased already during exposure and peaked approximately 6 hr thereafter, coinciding with the appearance of major morphological signs of apoptosis. Activated AP-1 mainly consisted of c-Fos/Jun heterodimers. In c-fos(-/-) mice, AP-1 activity remained unchanged, indicating that no other Jun- or Fos-family member could substitute for c-Fos. Like damaging light, N-methyl-N-nitrosourea (MNU) induced AP-1 containing c-Fos in c-fos(+/+) mice and did not induce AP-1 in c-fos(-/-) mice. In contrast to light, however, MNU induced apoptosis in rods of c-fos(-/-) mice. Thus, c-Fos is essential for a specific premitochondrial "private apoptotic pathway" induced by light but not for the execution of apoptosis induced by other stimuli.

AP-1 mediated retinal photoreceptor apoptosis is independent of N-terminal phosphorylation of c-Jun.

Apoptosis is essential for retinal development but it is also a major mode of cell loss in many human retinal dystrophies. High levels of visible light induce retinal apoptosis in mice and rats. This process is dependent on the induction of the transcription factor AP-1, a dimeric complex composed of c-Fos and c-Jun/JunD phosphoproteins. While c-Fos is essential, JunD is dispensable for light-induced photoreceptor apoptosis. Here we show that N-terminal phosphorylation of c-Jun, the other main partner of c-Fos in induced AP-1 complexes is not required for programmed cell death during retinal development in vivo and is also dispensable for photoreceptor apoptosis induced by the exogenous stimuli "excessive light" and N-nitroso-N-methylurea (MNU). Mice expressing a mutant c-Jun protein (JunAA) that cannot be phosphorylated at its N-terminus are apoptosis competent and their retina is not distinguishable from wild-type mice. Accordingly, Jun kinase, responsible for phosphorylation of wild-type c-Jun protein is at best only marginally induced by the apoptotic stimuli "light" and MNU. Complex composition of light-induced AP-1 complexes is similar in wild-type and JunAA mice. This shows that the mutant c-Jun protein can be part of the DNA binding complex AP-1 and demonstrates that induction of the DNA binding activity of AP-1 after light insult does not depend on N-terminal phosphorylation of c-Jun. Our results suggest that transactivation of target genes by phosphorylated c-jun/AP-1 is not required for MNU- or light-induced apoptosis of photoreceptor cells.

Apoptotic cell death in retinal degenerations.

Apoptosis is a regulated mode of single cell death that involves gene expression in many instances and occurs under physiological and pathological conditions in a large variety of systems. We briefly summarize major features of apoptosis in general and describe the occurrence of apoptosis in the retina in different situations that comprise animal models of retinitis pigmentosa, light-induced lesions, histogenesis during development, and others. Apoptosis can be separated into several phases: the induction by a multitude of stimuli, the effector phase in which the apoptotic signal is transmitted to the cellular death machinery, the excecution period when proteolytic cascades are activated, and the phagocytic removal of cellular remnants. Control mechanisms for retinal apoptosis are only beginning to be clarified. Potential apoptotic signal transducers were investigated in our laboratory, including metabolites of arachidonic acid and downstream mediators of signaling molecules such as transcription factors. Work in our laboratory revealed an essential role of the immediate-early gene product c-Fos in light-induced apoptosis. c-Fos is a member of the AP-1 family of transcription factors and, together with other members of this family, it may regulate apoptosis in the central nervous system. Expression of the c-fos gene in the retina can be evoked by light exposure and follows a diurnal rhythm. Future studies will have to clarify how light can control the expression of specific genes, and specifically, the role of c-fos and other genes of retinal apoptosis including potential target genes and signaling pathways.

[Survival factors in the treatment of hereditary retinal degeneration]. / Uberlebensfaktoren in der Therapie erblicher Netzhautdegenerationen.

Hereditary retinal degeneration is characterized by apoptotic photoreceptor loss, a process governed by intricate molecular interplay and initiated when proapoptotic signals predominate in the individual cell. Identification of molecules involved and their actions has paved the way for testing the ones with anti-apoptotic functions in models of inherited retinal degeneration. Many of these factors are able to slow the course of the degeneration. However, to date no such treatment has been able to stop or even prevent the devolution of the disorder. Moreover, preservation of morphology does not necessarily correlate with preservation of ERG function. Deepened understanding of the pro- and anti-apoptotic networks is clearly needed for survival factors to be feasible for therapy in humans. In comparison, in a dog model of Leber's congenital amaurosis gene therapy could establish retinal function, thus supplying proof of efficacy of the method.

The visual input stage of the mammalian circadian pacemaking system: II. The effect of light and drugs on retinal function.

Acute light pulses as well as long-term light exposure may not only modulate photoreceptive properties, but also induce reversible or irreversible damage to the retina, depending on exposure conditions. Illuminance levels in laboratory animal colonies and manipulations of lighting regimens in circadian rhythm research can threaten retinal structure and physiology, and may therefore modify zeitgeber input to the central circadian system. Given the opportunity to escape light at any time, the nocturnal rat self-selects a seasonally varying "naturalistic skeleton photoperiod" that protects the eyes from potential damage by nonphysiological light exposures. Both rod rod-segment disk shedding and behavioral circadian phase shifts are elicited by low levels of twilight stimulation. From this vantage point, we hypothesize that certain basic properties of circadian rhythms (e.g., Aschoff's rule and splitting) may reflect modulation of retinal physiology by light. Pharmacological manipulations with or without the addition of lighting strategies have been used to analyze the neurochemistry of circadian timekeeping. Drug modulation of light input at the level of the retina may add to or interact with direct drug modulation of the central circadian pacemaking system.

The visual input stage of the mammalian circadian pacemaking system: I. Is there a clock in the mammalian eye?

Threads of evidence from recent experimentation in retinal morphology, neurochemistry, electrophysiology, and visual perception point toward rhythmic ocular processes that may be integral components of circadian entrainment in mammals. Components of retinal cell biology (rod outer-segment disk shedding, inner-segment degradation, melatonin and dopamine synthesis, electrophysiological responses) show self-sustaining circadian oscillations whose phase can be controlled by light-dark cycles. A complete phase response curve in visual sensitivity can be generated from light-pulse-induced phase shifting. Following lesions of the suprachiasmatic nuclei, circadian rhythms of visual detectability and rod outer-segment disk shedding persist, even though behavioral activity becomes arrhythmic. We discuss the converging evidence for an ocular circadian timing system in terms of interactions between rhythmic retinal processes and the central suprachiasmatic pacemaker, and propose that retinal phase shifts to light provide a critical input signal.

Autophagy in frog visual cells in vitro.

Isolated frog retinas were incubated in a medium free of serum and amino acids under dim white incandescent light of 20 lux/m2. After 1, 2, 6, and 9 hr of incubation, six retinas at each time point were fixed for electron microscopic investigation. Histochemical staining of acid phosphatase was performed in control and experimental tissues. Autophagic vacuoles in visual cell inner segments were counted and compared with the incidence of vacuoles in control tissues. The ratio of newly formed: old autophagic vacuoles was assessed in incubated retinas, and the number of autophagic vacuoles per rod cell and per cone cell was evaluated. The results indicated that the number of autophagic vacuoles was significantly increased from 1 to 9 hr of incubation, the ratio of newly formed: old autophagic vacuoles was constant over this period, and the amount of autophagy occurring in rods and cones was similar. In a second group of experiments, retinas were incubated under the same conditions but at two different levels of illumination. One series of retinas was incubated in dim red incandescent light of 5 lux/m2, the other series was incubated at bright white fluorescent light of 300 lux/m2. The total numbers of autophagic vacuoles showed a consistent elevation of 20% in bright white light material as compared wot dim red light material. Autophagic vacuoles per cone were significantly higher in retinas incubated in white light than in retinas incubated in red light. Autophagic vacuoles per rod were about equal in both groups. Our observations indicated that visual cells contain an intracellular mechanism of degradation, which is increased under changed metabolic conditions and modified as a function of different levels of illumination.

The effects of hibernation on cone visual cells in the ground squirrel.

The cone visual cells of active, hibernating, and aroused 13-line ground squirrels have been studied by microscopy and autoradiography. Major changes occur throughout the cells during hibernation. The outer segments are shortened, and the diameters of the membranous discs may be reduced. Mitochondria are diminished in size and number, ribosomes are depleted, and the Golgi complex is fragmented into vesicles. Calycal processes are thickened, and synaptic ribbons become aggregated ectopically within the synaptic body. When hibernation is terminated, the cells recover rapidly. First, the basic synthetic machinery (mitochondria, ribosomes, Golgi complex) is regenerated,, and then the outer segments are repaired. This process is completed within 1 week. Many of the structural changes observed during hibernation are interpreted as effects of a temporary metabolic imbalance in which degradative mechanisms, including autophagy, are emphasized. In contrast, recovery is achieved by a comparable imbalance in which there is a transient accentuation of formative mechanisms. The recovered cells thereafter maintain a steady state of continuous self-renewal, in which formation and degradation are in balance.

Dietary deficiency of N-3 fatty acids alters rhodopsin content and function in the rat retina.

PURPOSE: To investigate the possibility that previously demonstrated reductions in photoreceptor sensitivity to light in n-3 fatty-acid-deficient rats can be explained by alterations in rhodopsin content and/or function. METHODS: Sprague-Dawley rats were reared throughout gestation, lactation, and up to 24 weeks of age on a diet containing safflower oil (n-3 fatty-acid-deficient) or soybean oil as the sole source of lipids. Dark-adapted content and in vivo regeneration of rhodopsin after bleaching were measured by detergent extraction. The regeneration rate constants and number of photons absorbed by rhodopsin under steady-state bleach conditions were calculated from these values. The rate of metarhodopsin II (MII) formation in vitro was determined by flash bleaching extracted pigment and native rod outer segment membranes. Rod outer segment length and photoreceptor cell density were determined in histologic sections through the inferior central retina. RESULTS: Dark-adapted rhodopsin content of retinas from rats reared on safflower oil was 12% to 15% higher than that of rats raised on soybean oil at every age measured. The rate of rhodopsin regeneration was significantly slower in rats reared on safflower oil while the level of steady-state bleach was the same. This meant that the rats reared on safflower oil absorbed about one half as many photons during light exposure. The rate of metarhodopsin II formation in vitro was unaffected by n-3 fatty acid deficiency. No difference in either rod outer segment length or cell number was detected. CONCLUSION: A reduced capacity for photon absorption by rhodopsin could play a role in lowering retinal sensitivity to light in n-3 fatty-acid-deficient rats.

Protective effect of halothane anesthesia on retinal light damage: inhibition of metabolic rhodopsin regeneration.

PURPOSE: To determine whether the volatile anesthetic halothane protects against light-induced photoreceptor degeneration in the rodent retina. METHODS: Albino mice and rats were anesthetized with halothane and exposed to high levels of white or blue light. Nonanesthetized animals served as controls. Retinal morphology was assessed by light microscopy, and apoptosis of photoreceptor cells was verified by detection of fragmented genomic DNA and in situ staining of apoptotic nuclei (TUNEL assay). Rhodopsin regeneration after bleaching was determined by measuring rhodopsin levels in retinas of mice or rats at different time points in darkness. RESULTS: Halothane anesthesia reversibly inhibited metabolic rhodopsin regeneration and thus prevented rhodopsin from absorbing high numbers of photons during light exposure. Consequently, photoreceptors of mice and rats anesthetized with halothane were completely protected against degeneration induced by white light. In remarkable contrast, however, halothane anesthesia did not protect against blue-light-induced photoreceptor cell death. CONCLUSIONS: After the initial bleach, halothane impeded photon absorption by rhodopsin by inhibiting metabolic rhodopsin regeneration. Apparently, the rhodopsin-mediated uptake of the critical number of photons to initiate white light-induced retinal degeneration was prevented. In contrast, halothane did not protect the retina against blue light. Blue light can efficiently restore functional rhodopsin from bleaching intermediates through a process termed photoreversal of bleaching. This process does not depend on the visual cycle via the pigment epithelium but nevertheless enables rhodopsin molecules to absorb the critical number of photons required to induce retinal degeneration.

Blue light's effects on rhodopsin: photoreversal of bleaching in living rat eyes.

PURPOSE: To determine whether blue light induces photoreversal of rhodopsin bleaching in vivo. METHODS: Eyes of anesthetized albino rats were exposed to either green (550 nm) or deep blue (403 nm) light, and the time course of rhodopsin bleaching was determined. Rhodopsin was isolated from whole retinas by detergent extraction and measured photometrically. To inhibit photoreversal of bleaching, rats were perfused with 70 mM hydroxylamine (NH(2)OH), a known inhibitor of photoreversal. To determine whether blue-absorbing, photoreversible photoproducts were formed, rhodopsin was bleached to near completion with green light and then exposed to blue light. Finally, experimental results were simulated on a computer by means of a simple, three-component model involving a long-lived photoreversible photoproduct. RESULTS: Photoreversal of bleaching in blue light occurs in vivo as evidenced by the following: In the absence of NH(2)OH, bleaching of rhodopsin by blue light was slow and complex. In the presence of NH(2)OH, however, blue light bleached rhodopsin very fast with a simple, pseudo-first-order kinetic. A long-lived bleaching intermediate produced by green light exposure was photoreversed to rhodopsin by exposure to blue light. The three-component computer model, invoking a blue-absorbing, photoreversible, long-lived intermediate accurately described the data. CONCLUSIONS: Because of the instantaneous, nonmetabolic regeneration of rhodopsin by the process of photoreversal of bleaching, blue light exposure permits the absorption of large numbers of photons by rhodopsin and by a photoreversible intermediate of bleaching in vivo. These data may have an important impact on resolving mechanisms of blue light-mediated damage to the retina.