Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer's disease-linked presenilin 1 A246E mutation can be reversed with cAMP.

Mutation in presenilin 1 (PS1) is one of the leading causes of familial Alzheimer's disease (fAD). PS1 mutation exacerbates the autophagic and lysosomal pathology in AD patients, leading to accumulation of partially degraded material in bloated lysosomes and autophagosomes - a pathology that bears some resemblance to other diseases characterized by elevated lysosomal pH, like age-related macular degeneration. In this study, we examined the effect of the PS1-fAD mutation A246E on lysosomal pH and lysosomal function, and asked whether restoration of lysosomal pH could reverse some of these changes. Lysosomal pH was elevated by 0.2-0.3 pH units in human fibroblasts with the PS1-fAD mutation. The lysosomal alkalization in PS1-fAD fibroblasts was supported by a reduction in the pH-dependent cleavage of cathepsin D and by a reduction in binding of boron-dipyrromethene (BODIPY) FL-pepstatin A to the cathepsin D active site. PS1-fAD cells had increased LC3B-II/-I ratios and p62 levels, consistent with impaired lysosomal degradation and analogous to changes induced by lysosomal alkalinization with chloroquine. PS1-fAD fibroblasts had increased expression of ATP6V1B2, ATG5, BECN1 TFEB mRNA, and of ATP6V1B2, ATG5 and beclin at the protein level, consistent with chronic impairment of autophagic and lysosomal functions in the mutant cells. Critically, cyclic adenosine monophosphate (cAMP) treatment reacidified lysosomal pH in mutant PS1-fAD; cAMP also increased the availability of active cathepsin D and lowered the LC3B-II/-I ratio. These results confirm a small elevation in the lysosomal pH of human PS1-fAD fibroblasts, demonstrate that this lysosomal alkalization is associated with chronic changes in autophagy and degradation, and suggest that treatment to reacidify the lysosomes with cAMP can reverse these changes.

Ultrasound biomicroscopy of Chinese eyes with iridocorneal endothelial syndrome.

AIM: To document the ultrasound biomicroscopic (UBM) findings in Chinese patients with iridocorneal endothelial (ICE) syndrome. METHODS: 21 patients with ICE syndrome and 15 normal subjects underwent UBM. UBM findings of anterior segment were compared between normal subjects and three clinical types of ICE syndrome: progressive iris atrophy (PIA), Chandler's syndrome (CS), and Cogan-Reese syndrome (CRS). RESULTS: Central anterior chamber depth was significantly less in patients with ICE syndrome (2.25 (SD 0.32) mm) than in normal subjects (2.76 (0.32) mm). Peripheral anterior synechiae were observed in all the ICE patients by UBM. Three out of four CRS subjects showed an "arborised" shape of iridocorneal angle. Two eyes out of 10 with CS presented bridge-shaped synechiae. A membrane-like mound was observed in iridocorneal angle in two patients: one with CRS and one with CS. UBM was found to be more effective in detecting peripheral anterior synechiae (PAS) and iris atrophy than slit lamp microscopy and gonioscopy, mainly because of corneal oedema in patients with CS. Four out of 11 patients with unilateral ICE syndrome had shallow or closed anterior chamber angles in their fellow eyes. Two of them successfully responded to laser peripheral iridotomy. CONCLUSIONS: UBM is an effective method to reveal the anterior segment features and provides a useful tool in the diagnosis of ICE syndrome. Different subtypes of ICE syndrome may have different UBM manifestations. UBM can help to identify angle closure in the fellow eye of unilateral ICE syndromes.

Augmented rod bipolar cell function in partial receptor loss: an ERG study in P23H rhodopsin transgenic and aging normal rats.

Physiological consequences of early stages of photoreceptor degeneration were examined in heterozygous P23H rhodopsin transgenic (Tg) and in aging normal Sprague-Dawley rats. Rod photoreceptor and rod bipolar (RB) cell function were estimated with maximum value and sensitivity parameters of P3 and P2 components of the electroretinogram. In both Tg and aging normal rats, the age-related rate of decline of P3 amplitude was steeper than that of the P2 amplitude. Tg rats showed greater than normal sensitivity of the rods. A new model of distal RB pathway connectivity suggested photoreceptor loss could not be the sole cause of physiological abnormalities; there was an additional increase of post-receptoral sensitivity. We propose that changes at rod-RB synapses compensate for the partial loss of rod photoreceptors in senescence and in early stages of retinal degeneration.

Calcium channel blocker D-cis-diltiazem does not slow retinal degeneration in the PDE6B mutant rcd1 canine model of retinitis pigmentosa.

PURPOSE: D-cis-diltiazem, a calcium channel blocker, has been reported to enhance photoreceptor survival in the rd mouse, a model of retinitis pigmentosa (RP) resulting from mutation of the PDE6B gene. We tested the hypothesis that diltiazem treatment would similarly rescue the canine rcd1 model of RP, which is also caused by a null mutation in the PDE6B gene. METHODS: D-cis-diltiazem was delivered orally twice daily to rcd1 affected dogs beginning at 4 weeks of age; untreated age-matched rcd1 dogs served as controls. At 14 weeks, electroretinograms (ERG) were performed on all animals; 14 dogs were euthanized at this age, and 2 dogs at 25 weeks of age. Eyes were enucleated, fixed, and processed for routine histological examination. RESULTS: No significant differences were found in ERG or histopathologic parameters between diltiazem-treated and untreated rcd1 dogs. Neither rcd1 group showed a rod b-wave; ERGs evoked by single white flashes (dark- or light-adapted) and flicker were also identical between groups. Similarly, treated and untreated animals did not differ in the degree of preservation of the photoreceptor layer, confirmed in cell counts within the outer nuclear layer. CONCLUSIONS: Treatment of rcd1 affected dogs with D-cis-diltiazem did not modify the photoreceptor disease when results were assessed using either ERG or histopathologic criteria. The positive photoreceptor-rescue effect of calcium channel blockers reported in the rd mouse was thus not generalizable to another species with retinal degeneration due to mutation in the PDE6B gene. Caution needs to be exerted in extrapolation to the comparable human forms of RP.

Association between intraocular pressure and budesonide inhalation therapy in asthmatic patients.

BACKGROUND: The extent to which inhaled glucocorticoids increase the risk of intraocular pressure elevation has been controversial. OBJECTIVE: The authors attempt to assess such risk attributable to budesonide, an inhaled glucocorticoid for asthma therapy. METHODS: Data were pooled from four prospective, randomized, double-blind, parallel-group, placebo-controlled, multicenter clinical trials of 12 to 20 weeks in duration. One thousand two hundred and fifty-five patients, 6 to 70 years of age whose intraocular pressures (IOPs) were less than 23 mmHg at screening were randomized to receive placebo or inhaled budesonide at doses ranging from 100 to 800 microg, administered twice daily. Intraocular pressure was measured at screening and at the end of double-blind treatment. Intraocular change was compared between budesonide and placebo, accounting for the confounding effects of gender, race, age, history of diabetes, history of hypertension, clinical trial, systemic glucocorticoid use during the trials, ophthalmic glucocorticoid use during the trials, and prior oral glucocorticoid use. RESULTS: No budesonide treatment effect on the IOP was evident either in the crude analysis or after adjustment for possible confounding factors. For patients exposed to budesonide at a total daily dose of 1600 microg for 20 weeks, there was no difference in IOP change compared with the placebo controls. CONCLUSIONS: No association with an increased IOP was observed in asthmatic patients treated with budesonide at daily doses ranging from 200 to 1600 microg for durations of 12 to 20 weeks. The subgroup analysis, which focused on the highest dose and longer term therapy was reassuring, as was the overall result.

No clinically important effects on intraocular pressure after short-term administration of sildenafil citrate (Viagra).

PURPOSE: To assess the short-term effects of sildenafil citrate on intraocular pressure in healthy male volunteers and participants in clinical trials. METHODS: Intraocular pressure and pupil diameter were measured in two placebo-controlled studies. Oral doses of sildenafil citrate (VIAGRA; Pfizer Inc, New York, New York) ranged from 10 mg to 150 mg. RESULTS: No major changes in intraocular pressure or pupillometry were detected at any time (1.0-24 hours) after administration of sildenafil. Additionally, of 36 subjects with a medical history of increased intraocular pressure in the sildenafil safety database, none were reported to have a clinically significant increase of their intraocular pressure. During clinical trials, two glaucoma cases were listed as serious adverse events, but were not considered treatment related. CONCLUSION: No clinical abnormalities were observed in intraocular pressure or pupil diameter in subjects receiving sildenafil. Currently, no evidence suggests that long-term treatment with sildenafil has an effect on intraocular pressure or is associated with the development or worsening of glaucoma.

Choroidal vascular permeability in visually regulated eye growth.

The choroidal thickness fluctuates both diurnally and in response to changes in visual input. The fluctuations may represent a physiologic means of aligning the retinal photoreceptors with the focal position of distant images during the emmetropization process. To evaluate the basis for choroidal thickness changes, we studied the sources of the extravascular fluid in the chick choroid in two visually-regulated ocular growth conditions: accelerated ocular growth in goggle-induced form-deprivation myopia and ocular growth retardation in the recovery from myopia after goggle removal. Two week old chicks, controls, myopic and those recovering from myopia, received fluorescein dextran (MW = 140,000) as a tracer. It was given by intravenous injection to identify a potential vascular pathway and by intracameral injection to identify a potential pathway from the anterior chamber to the suprachoroidal space. Using a microscopically positioned needle, clear fluid was aspirated from the suprachoroidal space of the enucleated chick eye; this fluid presumably corresponds to the contents of the lacunae, prominent lymphatic-like structures of the chick choroid. Plasma, aqueous humor and suprachoroidal fluid were sampled 1 hr after injection and assayed for both protein content and the tracer dye. Sulfated glycosaminoglycans were assayed in the suprachoroidal fluid, choroid and sclera under each experimental condition. In control chicks, aqueous humor and suprachoroidal fluid protein concentrations were about 0.8 and 9% of plasma levels respectively. Aqueous humor protein concentration was unaltered in myopic or recovering eyes. Suprachoroidal fluid protein concentration in myopic eyes fell dramatically to 1.5% of plasma levels (P < 0.001). In contrast, recovery from myopia led to a marked increase in suprachoroidal fluid protein level to 30% of that in plasma (P < 0.001). None of the procedures affected suprachoroidal fluid protein in the contralateral control eyes. In all three groups of chicks, fluorescein dextran distribution in the suprachoroidal fluid at 1 hr after intravenous injection tracked protein levels, with reduced levels in myopic eyes and elevated levels in recovering eyes. After intracameral injection, suprachoroidal fluid dextran levels were higher in injected eyes of control chicks (P < 0.01) and in recovering eyes (P < 0.001) but lower in myopic eyes (P < 0.01), compared to the levels in the respective contralateral non-injected eyes in each group. Sulfated glycosaminoglycan levels were at the limits of detection in the suprachoroidal fluid under all conditions and, on a whole choroid basis, were unaltered in the choroid in either myopia or recovery. Suprachoroidal fluid is lymph-like in nature and largely derives from plasma. Sulfated glycosaminoglycan levels do not seem to regulate the fluid content of the choroid in either myopia or recovery. Instead, the changes in protein and marker dye levels in myopic and recovering eyes suggest markedly altered choroidal circulatory dynamics and capillary permeability in both conditions.

Effects of muscarinic cholinergic receptor antagonists on postnatal eye growth of rhesus monkeys.

PURPOSE: To study a potential role for muscarinic receptors in the inhibition of deprivation-induced excessive axial elongation and myopia in a monkey model. METHODS: The right eyes of 20 newborn rhesus monkeys were occluded with a black contact lens. In seven monkeys each, either atropine or pirenzepine was topically applied daily to the occluded eyes. The nonoccluded fellow eyes and both the occluded and nonoccluded fellow eyes of another six monkeys were treated with vehicle solution. RESULTS: After 33 to 39 weeks, in 5 monkeys of the vehicle group, occluded eyes were longer and the myopic shift significantly greater than in the nonoccluded fellow eyes. In six atropine-treated monkeys, axial length and reduction of the initial hyperopia of occluded and nonoccluded fellow eyes were not different statistically. The myopic shift of the occluded eyes was significantly smaller than in the vehicle-treated occluded eyes. In the pirenzepine-treated group, axial length of the occluded eyes was similar to the nonoccluded eyes of controls and the occluded eyes of atropine-treated monkeys. There was a trend of pirenzepine to reduce the myopic shift of the occluded eye. No effect of atropine or pirenzepine was noted on muscarinic receptor density in retina, brain, or heart, but a small increase was observed in iris + ciliary body. CONCLUSIONS: The drug treatment results implicate muscarinic receptors in postnatal eye growth regulation. Because of interanimal differences our data do not indicate whether nonselective or selective muscarinic blockade is more effective in reducing deprivation-induced myopia.

Activation of caspase-3 in the retina of transgenic rats with the rhodopsin mutation s334ter during photoreceptor degeneration.

The role of caspase-3 in photoreceptor degeneration was examined in a line of transgenic rats that carry a rhodopsin mutation S334ter. Photoreceptor degeneration in these animals is rapid. It is detected as early as postnatal day (PD) 8, and by PD 20, only one of the original 12 rows of nuclei remain in the outer nuclear layer. At PD 11 and 12, the number of photoreceptors dying per day reaches a peak of approximately 30% of the total photoreceptors in the retina. Coincident with this rapid degeneration is an increase in caspase-3-like activity as assessed by the cleavage of a fluorescent substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin and an increase in activated caspase-3 as determined by Western blot analysis for its 12 kDa subunit. Intraocular injection of an irreversible caspase-3 inhibitor N-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyk etone partially protected photoreceptors from degeneration. These findings indicate that a caspase-3-dependent mechanism is operative in photoreceptor death in the transgenic rats under investigation.

Autonomic denervations influence ocular dimensions and intraocular pressure in chicks.

Choroidal thickness and axial eye length in the chick undergo day/night fluctuations that can also be modulated by visual experience. In the present study, we tested the effect of parasympathetic and sympathetic denervations on both day/night and image dependent changes in ocular dimensions. We also correlated such changes with fluctuations in intraocular pressure. Parasympathectomy influenced choroidal thickness and its day/night fluctuation, but had no effect on vision dependent choroidal thickness modulation. Parasympathectomy also influenced-to a lesser extent-axial length and reduced the axial growth response to form vision deprivation. Sympathectomy had little effect on ocular dimensions, but reduced the day/night differences in intraocular pressure. We conclude that (a) the parasympathetic nervous system influences both choroidal thickness and axial length and participates in the neural control mechanism leading to form deprivation myopia and, (b) the day/night fluctuations of choroidal thickness and axial length are unlikely to be explained by fluctuations in intraocular pressure. For the regulation of choroidal thickness, we hypothesize the existence of two independent mechanisms. One involves the parasympathetic nervous system; it influences the day/night choroidal thickness fluctuation. The other uses a separate pathway and is driven by visual input.

Ocular safety of Viagra, (sildenafil citrate).

To date, sildenafil citrate (Viagra) gives every evidence of being a safe drug for the eye despite a series of expressed concerns. A review of how its ocular safety profile has been identified offers insights into the strengths and weaknesses of present systems and resources for judging the ocular safety of Viagra or, for that matter, of any new drug. Such insights include: The great value of careful, informed assessment of preclinical information gleaned from laboratory experiments. By and large, such assessments point the way toward appropriate clinical evaluation. For Viagra, early in its development it was noted that besides exerting a major inhibitory effect on the intended target, the vascular-associated enzyme phosphodiesterase 5 (PDE5), the drug also exerts a lesser but definite inhibitory effect on the closely related PDE6, located in the retina. For this reason, preclinical evaluation of the drug included electroretinography plus postmortem histology. In addition, an extended eye examination was incorporated into clinical protocols. The often chaotic but invaluable information stream that becomes available once marketing approval has been gained and large populations begin to use a drug. False alarms, misattribution, and erroneous information are the order of the day. Nevertheless, as information accumulates, patterns of response clarify and the true nature of special susceptibility for subpopulations, if any, becomes apparent. A role for the astute clinician remains: Subtle changes or unusual risks for subpopulations can be missed entirely for long periods of time. A manifest need for improvement in evaluation of postmarketing side-effects. This need has led to the establishment of a new discipline: pharmacoepidemiology. In ophthalmology, the National Registry of Drug Induced Ocular Side-Effects maintains a constant and invaluable surveillance. Examples are supplied to illustrate each of these major points: Our presentation will include data gleaned from clinical trials plus postmarketing information on the incidence, duration, and type of color vision defects observed at different doses of Viagra.

Induction of axial eye elongation and myopic refractive shift in one-year-old chickens.

Depriving the eyes of neonatal animals of form vision induces axial eye elongation and ipsilateral myopia. We studied one-year-old chickens, an age at which full body growth has been attained, to learn if form deprivation myopia can develop at a later stage. We found that ocular reactivity to visual form deprivation continues in one-year-old chickens; but both the growth stimulation and the myopic shift in refraction are attenuated compared with newly hatched birds. Neurochemical changes in visually deprived eyes of one-year-old chickens parallel those in newly hatched chicks: ipsilateral decreases in retinal dopamine and in the activity of ciliary ganglion and uveal choline acetyltransferase. These findings strengthen the relevance of the form deprivation model to more common human myopia and suggest a common eye growth control mechanism at both ages.

Alpha2-adrenergic agonists selectively activate extracellular signal-regulated kinases in Müller cells in vivo.

PURPOSE: Alpha2-adrenergic agonists have specific and selective effects on the retina to induce expression of basic fibroblast growth factor and to protect photoreceptors. This work explores the signaling pathway that mediates these effects. METHODS: Alpha2-adrenergic agonists xylazine and clonidine were administered systemically to male adult Sprague-Dawley rats. The activation state of extracellular signal-regulated kinases (ERKs) in the retina was assessed by immunoblot analysis, using antibodies that specifically recognize the dually phosphorylated forms of p44/p42 ERKs. Localization of phosphorylated ERKs was determined by immunocytochemistry. RESULTS: Intramuscular injection of 6 mg/kg xylazine induced an increase in ERK phosphorylation in the retina within 30 minutes that lasted 3 hours. Xylazine induced ERK phosphorylation at 1 mg/kg and reached a maximum at 10 mg/kg. Injection of clonidine also induced ERK phosphorylation in the retina. Yohimbine, a specific alpha2-adrenergic antagonist, completely prevented the induction of ERK phosphorylation. Immunocytochemical studies showed that the increase in ERK phosphorylation occurred mainly in Müller cells. In the brain, xylazine injection resulted in a decrease in ERK phosphorylation. CONCLUSIONS: Our results indicate that systemically administered alpha2-adrenergic agonists selectively activate ERKs in retinal Müller cells. The induced activation of ERKs in Müller cells is probably one of the early events that result in photoreceptor protection. These results also indicate that Müller cells are unique in response to alpha2-adrenergic agonists and imply a role for Müller cells in alpha2-adrenergic agonist-induced photoreceptor protection.

Ocular axial length and choroidal thickness in newly hatched chicks and one-year-old chickens fluctuate in a diurnal pattern that is influenced by visual experience and intraocular pressure changes.

Low coherence laser Doppler interferometry (LDI) allows high precision measurements of the axial length of the eye and of the thickness of the individual layers of the ocular fundus. Here, we used LDI to monitor diurnal changes in these dimensions in eyes of newly hatched chicks and one-year-old chickens with normal or altered visual input. In chicks and chickens with normal visual experience, axial eye length displays diurnal fluctuations increasing during the light phase. Choroidal thickness also exhibits a diurnal pattern, shrinking during the day and expanding during the night. Retinal thickness does not vary. Based on the pressure compliance of the enucleated chick eye, the diurnal intraocular pressure (IOP) fluctuation could contribute both to the increase in axial length and to daytime choroidal shrinkage. Following deprivation of form vision by unilateral goggle wear, occluded chick eyes demonstrate enhanced axial elongation. Diurnal fluctuations in axial length but not in choroidal thickness are temporarily disrupted. The retina of form deprived eyes thins approximately 10% in five days. In contralateral eyes, the diurnal patterns of both axial length and choroidal thickness fluctuations are also disrupted. Following occluder removal in chicks, choroidal thickness increases for several days during both the light and dark phase, leading to its overall expansion. Retinal thickness returns to baseline. When deprived of form vision for five days, the eyes of year-old chickens do not exhibit measurable axial elongation. Diurnal patterns of fluctuation in axial length and choroidal thickness are however disrupted. After goggle removal, axial length fluctuation is restored to normal, but the diurnal choroidal thickness pattern is inverted. In contralateral eyes, choroidal thickness exhibits normal diurnal fluctuations both during and after form vision deprivation. In conclusion, axial length and choroidal thickness fluctuations are influenced by visual experience in both newborn chicks and one-year-old chickens. In selected instances a binocular interaction regarding axial length and choroidal thickness changes is suggested, the effect weakening with age.

Preconditioning with bright light evokes a protective response against light damage in the rat retina.

Constant exposure to bright light induces photoreceptor degeneration and at the same time upregulates the expression of several neurotrophic factors in the retina. At issue is whether the induced neurotrophic factors protect photoreceptors. We used a preconditioning paradigm to show that animals preconditioned with bright light became resistant to subsequent light damage. The preconditioning consisted of a 12-48 hr preexposure, followed by a 48 hr "rest phase" of normal cyclic lighting. The greatest protection was achieved by a 12 hr preexposure. Preconditioning induces a prolonged increase in two endogenous neurotrophic factors: basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). It also stimulates the phosphorylation of extracellular signal-regulated protein kinases (Erks) in both photoreceptors and Müller cells. These findings indicate that exposure to bright light initiates two opposing processes: a fast degenerative process that kills photoreceptors and a relatively slower process that leads to the protection of photoreceptors. The extent of light damage, therefore, depends on the interaction of the two processes. These results also suggest a role of endogenous bFGF and CNTF in photoreceptor protection and the importance of Erk activation in photoreceptor survival.

Validation of laser Doppler interferometric measurements in vivo of axial eye length and thickness of fundus layers in chicks.

Purpose. Laser Doppler interferometry (LDI) permits the measurement of intraocular distances to a precision of better than 20 microm. The signal complex from the posterior segment of the eye consists of four peaks in the chick, an animal frequently used in ocular development studies. The present study sought to identify anatomical landmarks corresponding to these LDI peaks. Methods. Distances obtained with LDI at the posterior pole were compared to axial length components measured with three independent methods: vernier calipers, tissue sections and high frequency A-scan ultrasound. Results. LDI reflections appear to originate from the retinal inner limiting membrane, Bruch's membrane and the inner and outer scleral surfaces. Conclusions. The non-invasive and highly precise nature of LDI measurements enables repetitive and accurate assessment of intraocular distances. Such measurements should prove particularly useful for the assessment of short-term cyclic variations in intraocular distances as well as post-natal eye growth.

Spectroscopic characterization of bendazac and benzydamine: possible photochemical modes of action.

The involvement of near-UV light in cataract development suggests that potential anti-cataract drugs may display unusual spectroscopic properties. As bendazac impedes certain effects associated with lens opacification, we have characterized the singlet and triplet states of bendazac and its analog, benzydamine, by fluorescence and phosphorescence methods. These compounds have much shorter triplet state lifetimes compared to the triplet state lifetimes observed in proteins. Our results raise the possibility that the photoprotective action of these compounds may result from their ability to dissipate energy through the triplet state. We propose alternative modes for the photoprotective actions of these compounds.

Measurement by laser Doppler interferometry of intraocular distances in humans and chicks with a precision of better than ±20 µm.

A laser Doppler interferometer was built for the precise measurement of intraocular optical distances in humans and chicks. A technique using Purkinje images was developed to position the chick's eye reproducibly. A computer algorithm for the objective analysis of the interference signal and determination of the optical distances is presented. The precision of this noncontact interferometric method for measuring the cornea-retina distance is better than ±20 µm.