Individual set-point and gain of emmetropization in chickens

During the developmental process of emmetropization evidence shows that visual feedback guides the eye as it approaches a refractive state close to zero, or slightly hyperopic. How this “set-point” is internally defined, in the presence of continuous shifts of the focal plane with different viewing distances and accommodation, remains unclear. Minimizing defocus blur over time should produce similar end-point refractions in different individuals. However, we found that individual chickens display considerable variability in their set-point refractive states, despite that they all had the same visual experience. This variability is not random since the refractions in both eyes were highly correlated - even though it is known that they can emmetropize independently. Furthermore, if chicks underwent a period of experimentally induced ametropia, they returned to their individual set-point refractions during recovery (correlation of the refractions before treatment versus after recovery: n = 19 chicks, 38 eyes, left eyes: slope 1.01, R = 0.860; right eyes: slope 0.85, R = 0.610, p < 0.001, linear regression). Also, the induced deprivation myopia was correlated in both eyes (n = 18 chicks, 36 eyes, p < 0.01, orthogonal regression). If chicks were treated with spectacle lenses, the compensatory changes in refraction were, on average, appropriate but individual chicks displayed variable responses. Again, the refractions of both eyes remained correlated (negative lenses, n = 18 chicks, 36 eyes, slope 0.89, R = 0.504, p < 0.01, positive lenses: n = 21 chicks, 42 eyes, slope 1.14, R = 0.791, p < 0.001). The amount of deprivation myopia that developed in two successive treatment cycles, with an intermittent period of recovery, was not correlated; only vitreous chamber growth was almost significantly correlated in both cycles (n = 7 chicks, 14 eyes; p < 0.05). The amounts of ametropia and vitreous chamber changes induced in two successive cycles of treatment, first with lenses and then with diffusers, were also not correlated, suggesting that the “gains of lens compensation” are different from those in deprivation myopia. In summary, (1) there appears to be an endogenous, possibly genetic, definition of the set-point of emmetropization in each individual, which is similar in both eyes, (2) visual conditions that induce ametropia produce variable changes in refractions, with high correlations between both eyes, (3) overall, the “gain of emmetropization” appears only weakly controlled by endogenous factors.     

Genomic response of hypoxic Müller cells involves the very low density lipoprotein receptor as part of an angiogenic network

Müller cells have recently been found to produce select angiogenic substances. In choosing a more comprehensive approach, we wanted to study the genomic response of Müller cells to hypoxia to identify novel angiogenic genes. An established Müller cell line (rMC-1) was exposed to standard or hypoxic conditions. We analyzed gene expression with three independent microarrays and determined differential expression levels compared to normoxia. Selected genes were confirmed by real-time PCR (RTPCR). Subcellular localization of proteins was examined by immunocytochemistry. A network-based pathway analysis was performed to investigate how those genes may contribute to angiogenesis. We found 19?004 of 28?000 known rat genes expressed in Müller cells. 211 genes were upregulated by hypoxia 1.5 to 14.9-fold (p < 0.001, FDR ≤ 5%) and 220 genes were downregulated 1.5-4.6-fold (p < 0.001, FDR ≤ 5%). Unexpectedly, expression patterns of cell proliferation, differentiation and organogenesis were increased besides predictable declines in cell function. Very low density lipoprotein receptor (VLDLR) and tribbles 3 (TRIB3) were further analyzed because of recent implication in retinal neovascularization and macular degeneration (VLDLR) and in ocular mesodermal development and differentiation (TRIB3), respectively. VLDLR was upregulated 3.1-fold (p = 0.001, RTPCR 3.0-fold) and TRIB3 2.8-fold (p = 0.025, RTPCR 5.1-fold). VEGF was increased 3.1-fold (p = 0.003, RTPCR 8.3-fold) and apelin, a novel factor of retinal angiogenesis, 5.6-fold (p = 0.006, RTPCR 8.7-fold). A network of interacting angiogenic genes was identified in silico that included VLDLR as a surface receptor. VLDLR protein localized to the perinucleus, cytoplasm and cell membrane, while TRIB3 was found in nucleoli, the nucleus and cytoplasm. We conclude that hypoxia triggers an angiogenic network response in Müller cells with VLDLR as a novel node and gene expression patterns of proliferation, differentiation and organogenesis.     

Viral delivery of heme oxygenase-1 attenuates photoreceptor apoptosis in an experimental model of retinal detachment

This study was designed to evaluate the efficacy of subretinal injection of recombinant adeno-associated virus vector expressing heme oxygenase-1 (rAAV-HO-1) in attenuating photoreceptor apoptosis induced by experimental retinal detachment (RD) in Sprague-Dawley rats. Our results disclosed that subretinal rAAV-HO-1 delivery achieved localized high HO-1 gene expression in retinal outer nuclear layer (ONL) compared with rAAV-lacZ-injected eyes and eyes with RD left untreated both at 2 (p = 0.003) and 28 (p = 0.007) days of RD. The ONL thickness (p = 0.018) and mean photoreceptor nuclei count (p = 0.009) in eyes receiving rAAV-HO-1 injection was significantly higher than in rAAV-lacZ-injected or eyes with RD left untreated at 28 days of RD. There were fewer apoptotic photoreceptor nuclei at 2 (p = 0.008) and 5 (p = 0.018) days of RD and less activated caspase-3 expression (p = 0.008) at 2 days of RD in rAAV-HO-1 treated eyes than in control eyes. These data supported that gene transfer approach might attenuate photoreceptor apoptosis caused by RD with a resultant better ONL preservation.     

Investigating structural and biochemical correlates of ganglion cell dysfunction in streptozotocin-induced diabetic rats

The aim of this study was to determine whether inner retinal dysfunction in diabetic rats is correlated with structural and/or biochemical changes in the retina and optic nerve. Using the electroretinogram (ERG; −5.83 to 1.28 log cd.s.m⁻²) retinal function (photoreceptor, bipolar, amacrine and ganglion cell components) was measured in control (n = 13; citrate buffer) and diabetic (n = 13; streptozotocin, STZ, 50 mg kg⁻¹) rats, 12 weeks following treatment. Retinae and optic nerves were analyzed for structural changes and retinae were assessed for alterations in growth factor/cytokine expression using quantitative real-time PCR. We found that phototransduction efficiency was reduced 12 weeks after STZ-induced diabetes (−30%), leading to reduced amplitude of ON-bipolar (−18%) and amacrine cell (−29%) dominated responses; ganglion cell dysfunction (−84%) was more profound. In the optic nerve, nerve fascicle area and myelin sheath thickness were reduced (p < 0.05), whereas the ratio of blood vessels and connective tissue to total nerve cross-sectional area was increased (p < 0.05) in diabetic compared to control rats. In the retina, connective tissue growth factor (CTGF), transforming growth factor beta, type 2 receptor (TGFβ-r2) mRNA and platelet-derived growth factor B (PDGF-B) mRNA were increased (p < 0.035). Reduced ganglion cell function was correlated with increased CTGF and TGFβ-r2, but not PDGF-B mRNA. In summary, the ganglion cell component exhibited the greatest level of dysfunction within the ERG components examined after 12 weeks of STZ-induced diabetes; the level correlated with increased CTGF and TGFβ-r2 mRNA, but not with gross morphological changes in the retina or optic nerve.     

Cabergoline: Pharmacology, ocular hypotensive studies in multiple species, and aqueous humor dynamic modulation in the Cynomolgus monkey eyes

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT_2A/B/C), 5HT_1A, 5HT₇, α_2B, and dopamine-2/3 (D_2/3) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT₂, 5HT_1A and D_2/3 receptors but an antagonist at 5HT₇ and α₂ receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC₅₀ = 19 ± 7 nM in TM; 76 nM in h-CM) and intracellular Ca²⁺ ([Ca²⁺]_i) mobilization (EC₅₀ = 570 ± 83 nM in h-TM; EC₅₀ = 900 ± 320 nM in h-CM). Cabergoline-induced [Ca²⁺]_i mobilization in h-TM and h-CM cells was potently antagonized by a 5HT_2A-selective antagonist (M-100907, K_i = 0.29-0.53 nM). Cabergoline also stimulated [Ca²⁺]_i mobilization more potently via human cloned 5HT_2A (EC₅₀ = 63.4 ± 10.3 nM) than via 5HT_2B and 5HT_2C receptors. In h-CM cells, cabergoline (1 μM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1 μM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300-500 μg) in Dutch-belted rabbit eyes yielded 4.5 μM and 1.97 μM levels in the aqueous humor 30 min and 90 min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6 μg at 4 h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6 ± 3.6% with 50 μg at 3 h post-dose; 30.4 ± 4.5% with 500 μg at 7 h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5 h after the second topical ocular dose (300 μg) of cabergoline by 23% (p < 0.02) and 35% (p < 0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69 ± 0.7 μL/min-1.61 ± 0.97 μL/min, n = 13; p < 0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT₂, 5HT_1A, and D_2/3 receptors. Since 5HT_1A agonists, 5HT₇ antagonists, and α₂ antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT₂ and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.     

Corneal epithelial proliferation and thickness in a mouse model of dry eye

Although several studies have previously focused on the conjunctival epithelial response to surface dryness, little is known about the effect of a dry environment on corneal epithelium, which is the most clinically significant tissue affected in dry eye. The aim of this study was to quantitatively evaluate the effect of desiccating stress on the number of proliferating corneal epithelial cells and corneal epithelial thickness in mice placed in a controlled-environment chamber (CEC) that induces dry eye. Corneal epithelial cell proliferation and thickness were studied in 8- to 12-week-old female BALB/c mice placed in the CEC (temperature: 22.3 ± 0.7 °C; relative humidity: 22.5 ± 4.5%; airflow: 15 L/min) for 7 days and compared to a control group of mice with no dry eye. Actively proliferating cells were identified by immunofluorescence using a FITC-conjugated antibody against the Ki-67 protein, a cell proliferation marker expressed during active phases of the cell cycle. To detect the spatial distribution of proliferative cells, Ki-67⁺ cells were counted in three areas of the epithelium: center, periphery, and limbus. Corneal epithelial thickness was evaluated in the central cornea after staining with hematoxylin-eosin. Results from each experimental group were compared using the Mann-Whitney test. The number of Ki-67⁺ cells observed in the corneal epithelium of mice exposed to the CEC was significantly higher in each area (center: 32.1 ± 1.1; periphery: 94.2 ± 5.3; limbus: 4.0 ± 1.5) than in the control group (center: 13.2 ± 1.0, p = 0.02; periphery: 42.9 ± 2.3, p = 0.02; limbus: 0.0, p = 0.01). In mice subjected to desiccating stress, a significant number of Ki-67⁺ positive cells were detected in the basal and suprabasal cell layers (central area 46%; periphery 30.8%: limbus 0%), whereas in the control group the cells were exclusively distributed through the basal cell layer. Ki-67⁺ cells were not found in the corneal stroma or endothelium in any group. The corneal epithelium was found to be significantly thicker in dry eye mice (54.94 ± 6.09 μm) as compared to the controls (43.9 ± 6.23 μm, p < 0.0001) by a mean of 25%. These results demonstrate that desiccating stress increases corneal epithelial turnover and thickness, similar to what is observed in other chronic inflammatory states of other epithelialized surfaces. The CEC can facilitate the study of the regulation of epithelial cell function and turnover at the molecular and cellular levels under desiccating stress conditions.     

Axial myopia induced by a monocularly-deprived facemask in guinea pigs: A non-invasive and effective model

This study evaluated the efficacy of a facemask, a non-invasive and potentially more reliable method, in inducing axial myopia in guinea pigs. Thirty-six animals were randomly assigned to 3 groups: MDF (monocularly-deprived facemask, n=6), lid-suture (eyelids sutured monocularly, n=24) and normal control (free of form deprivation, n=6). All the groups underwent biometric measurement (refraction, corneal curvature and axial length) prior to the experiment. All animals in the MDF group underwent biometric measurement at each of the 4 timepoints (2, 4, 6 and 8 weeks of form deprivation). In the lid-sutured group, the animals were randomly assigned to 4 subgroups (n=6 each) and each subgroup underwent biometric measurement at one of the timepoints matching those of the MDF group. In the normal control group, all animals underwent biometric measurement at each of the timepoints matching those of the 2 experimental groups. Placement of a facemask on an animal took approximately 10 sec and all the facemasks remained in place at all timepoints. The procedure of lid-suture took at least 20 min for an animal and rupture of the sutures occurred in 50% of the animals after 4 weeks. The MDF eyes developed myopia from −2.21±2.11D (Mean±s.d.) at 2 weeks to −4.38±2.14 at 8 weeks (p<0.05 at all timepoints, compared to the contralateral eyes) with a lengthening of the vitreous chamber from 0.17±0.05 mm at 2 weeks to 0.29±0.12 mm at 8 weeks (p<0.01 at all timepoints, compared to the contralateral eyes). The lid-sutured eyes developed myopia from −2.38±1.21D at 2 weeks to −4.75±1.39D at 8 weeks (p<0.05 at all timepoints, compared to the contralateral eyes) with a lengthening of the vitreous chamber from 0.13±0.02 mm at 2 weeks to 0.30±0.10 mm at 8 weeks (p<0.05 at 2, 4, 8 weeks, but >0.05 at 6 weeks, compared to the contralateral eyes) and an increase in the radius of the corneal curvature (0.20±0.07 mm at 4 weeks, p<0.01; 0.17±0.05 mm at 8 weeks, p<0.05; compared to the contralateral eyes). Both the MDF and lid-sutured groups had a similar development in myopia and vitreous length (MDF vs lid-suturing: p>0.05 at all timepoints, one-way ANOVA with Bonferroni correction). This development was significantly faster than in the normal control group (MDF or lid-suture vs normal control: p<0.05 to <0.01 from 2 to 8 weeks, one-way ANOVA with Bonferroni correction). The radius of corneal curvature in the lid-sutured group was significantly greater than in either the MDF group or the normal control group since 4 weeks of form deprivation (p<0.05, one-way ANOVA with Bonferroni correction). Treatment with MDFs is as effective as the lid-suture in inducing axial myopia in guinea pigs. This method is non-invasive and allows evaluation of the same group of animals at different timepoints so that the number of animals required could be minimized without affecting the accuracy of the results.     

Comparing mfERGs with estimates of cone density from in vivo imaging of the photoreceptor mosaic using a modified Heidelberg retina tomograph

The spatial variation in central retinal function determined from mfERG was compared to co-localised measurements of cone density in two normal subjects. Individual cone cells in the parafoveal region of the retina were identified from 1° × 1° images of the photoreceptor mosaic using a modified Heidelberg retina tomograph (HRT). The variation in cone density compared well with previous histology and retinal imaging studies and was strongly linearly correlated (r = 0.98, p < 0.001) with mfERG amplitude within the central retina. Retinal function determined from mfERG amplitude appears to directly reflect the density of the cone cells in this region.     

Age-dependence of the optomechanical responses of ex vivo human lenses from India and the USA, and the force required to produce these in a lens stretcher: the similarity to in vivo disaccommodation

The purpose of this study was to study the age-dependence of the optomechanical properties of human lenses during simulated disaccommodation in a mechanical lens stretcher, designed to determine accommodative forces as a function of stretch distance, to compare the results with in vivo disaccommodation and to examine whether differences exist between eyes harvested in the USA and India.Postmortem human eyes obtained in the USA (n = 46, age = 6-83 years) and India (n = 91, age = 1 day-85 years) were mounted in an optomechanical lens stretching system and dissected to expose the lens complete with its accommodating framework, including zonules, ciliary body, anterior vitreous and a segmented rim of sclera. Disaccommodation was simulated through radial stretching of the sectioned globe by 2 mm in increments of 0.25 mm. The load, inner ciliary ring diameter, lens equatorial diameter, central thickness and power were measured at each step. Changes in these parameters were examined as a function of age, as were the dimension/load and power/load responses.Unstretched lens diameter and thickness increased over the whole age range examined and were indistinguishable from those of in vivo lenses as well as those of in vitro lenses freed from zonular attachments. Stretching increased the diameter and decreased the thickness in all lenses examined but the amount of change decreased with age. Unstretched lens power decreased with age and the accommodative amplitude decreased to zero by age 45-50. The load required to produce maximum stretch was independent of age (median 80 mN) whereas the change in lens diameter and power per unit load decreased significantly with age.The age related changes in the properties of human lenses, as observed in the lens stretching device, are similar to those observed in vivo and are consistent with the classical Helmholtz theory of accommodation. The response of lens diameter and power to disaccommodative (stretching) forces decreases with age, consistent with lens nuclear stiffening.     

Axial myopia induced by hyperopic defocus in guinea pigs: A detailed assessment on susceptibility and recovery

This study investigated whether adolescent guinea pigs can develop myopia induced by negative lenses, and whether they can recover from the induced myopia. Forty-nine pigmented guinea pigs (age of 3 weeks) were randomly assigned to 4 groups: 2-week defocus (n = 16), 4-week defocus (n = 9), 2-week control (n = 15) and 4-week control (n = 9). A −4.00 D lens was worn in the defocus groups and a plano lens worn in the control groups monocularly. The lenses were worn from 3 weeks to 5 weeks of age in the 2-week treatment groups with the biometry measured at 2, 4, 6, 10 and 14 days of lens wear. The lenses were worn from 3 weeks to 7 weeks of age in the 4-week treatment groups with the biometry measured immediately and at 2, 4, 6, 10 and 14 days after lens removal. Refractions in the defocused eyes developed towards myopia rapidly within 2 days of lens wear, followed by a slower development. The defocused eyes were at least 3.00 D more myopic with a greater increase in vitreous length by 0.08 mm compared to the fellow eyes at 14 days (p < 0.05). The estimated choroidal thickness of the defocused eyes decreased rapidly within 2 days of lens wear, followed by a slower decrease over the next 4 days. Relative myopia induced by 4 weeks of negative-lens treatment declined rapidly following lens removal. A complete recovery occurred 14 days after lens removal when compared to the fellow controls. The refractive changes during the recovery corresponded to a slower vitreous lengthening and a rapid thickening of the choroid. The plano-lens wearing eyes showed a slight but significant myopic shift (<−0.80 D) with no associated biometrical changes. Guinea pigs aged 3 weeks can still develop negative lens induced myopia and this myopia is reversible after removal of the lens. The myopia and recovery are mainly due to changes in vitreous length and choroidal thickness.     

Retinal thinning in tree shrews with induced high myopia: optical coherence tomography and histological assessment

This study determined retinal thinning in a mammalian model of high myopia using optical coherence tomography (OCT) and histological sections from the same retinal tissue. High myopia was induced in three tree shrews (Tupaia belangeri) by deprivation of form vision via lid suture of one eye, with the other eye a control. Ocular biometry data was obtained by Ascan ultrasonography, keratometry and retinoscopy. The Zeiss StratusOCT was used to obtain Bscans in vivo across the retina. Subsequently, eyes were enucleated and retinas fixed, dehydrated, embedded and sectioned. Treated eyes developed a high degree of axial myopia (−15.9 ± 2.3 D; n = 3). The OCT analysis showed that in myopic eyes the nasal retina thinned more than the temporal retina relative to the disc (p = 0.005). Histology showed that the retinas in the myopic eyes comprise all layers but were thinner than the retinas in normal and control eyes. Detailed thickness measurements in corresponding locations of myopic and control eyes in superior nasal retina using longitudinal reflectivity profiles from OCT and semithin vertical histological sections showed the percentage of retinal thinning in the myopic eyes was similar between methods (OCT 15.34 ± 5.69%; histology 17.61 ± 3.02%; p = 0.10). Analysis of retinal layers revealed that the inner plexiform, inner nuclear and outer plexiform layers thin the most. Cell density measurements showed all neuronal cell types are involved in retinal thinning. The results indicate that in vivo OCT measurements can accurately detect retinal thinning in high myopia.     

A computerized analysis of the entire retinal ganglion cell population and its spatial distribution in adult rats

In adult albino (SD) and pigmented (PVG) rats the entire population of retinal ganglion cells (RGCs) was quantified and their spatial distribution analyzed using a computerized technique. RGCs were back-labelled from the optic nerves (ON) or the superior colliculi (SCi) with Fluorogold (FG). Numbers of RGCs labelled from the ON [SD: 82,818 ± 3,949, n = 27; PVG: 89,241 ± 3,576, n = 6) were comparable to those labelled from the SCi [SD: 81,486 ± 4,340, n = 37; PVG: 87,229 ± 3,199; n = 59]. Detailed methodology to provide cell density information at small scales demonstrated the presence of a horizontal region in the dorsal retina with highest densities, resembling a visual streak.     

Latanoprost protects rat retinal ganglion cells from apoptosis in vitro and in vivo

We investigated whether latanoprost has a direct anti-apoptotic effect in retinal ganglion cell (RGC) line and RGCs in the rat. RGC-5 cells were induced to undergo apoptosis by serum deprivation and exogenous glutamate. The level of cell death with or without latanoprost acid was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in the level of intracellular calcium ([Ca²⁺]i) were measured with fluo-4 fluorescence. The XTT assay revealed that latanoprost acid increased RGC-5 cell viability. Latanoprost acid significantly reduced caspase-3 positive cells and suppressed [Ca²⁺]i evoked by glutamate. U0126, a mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 inhibitor, partially blocked the rescue effect of latnanoprost acid (p = 0.013). In vivo, rat RGCs were degenerated by optic nerve crush. After topical instillation of latanoprost for 7 days, RGCs labeled with fluorogold were significantly. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells. TUNEL-positive cells were significantly decreased in eyes with topically instilled latanoprost (p = 0.015). These data suggest that latanoprost has an neuroprotective ability in RGCs.     

The impact of macular pigment augmentation on visual performance in normal subjects: COMPASS

This study was conducted to investigate whether augmentation of macular pigment (MP) enhances visual performance (VP). 121 normal subjects were recruited. The active (A) group consumed 12 mg of lutein (L) and 1 mg of zeaxanthin (Z) daily. MP optical density (MPOD) was assessed by customized heterochromatic flicker photometry. VP was assessed as best corrected visual acuity (BCVA), mesopic and photopic contrast sensitivity (CS), glare disability, photostress, and subjective visual function. Subjects were assessed at baseline; 3; 6; 12 months (V1, V2, V3 and V4, respectively). Central MPOD increased significantly in the A group (p < 0.05) but not in the placebo group (p > 0.05). This statistically significant increase in MPOD in the A group was not, in general, associated with a corresponding improvement in VP (p > 0.05, for all variables), with the exception of a statistically significant time/treatment effect in “daily tasks comparative analysis” (p = 0.03). At V4, we report statistically significant differences in mesopic CS at 20.7 cpd, mesopic CS at 1.5 cpd under high glare conditions, and light/dark adaptation comparative analysis between the lower and the upper MP tertile groups (p < 0.05) Further study into the relationship between MP and VP is warranted, with particular attention directed towards individuals with low MP and suboptimal VP.     

Transgenic expression of AQP1 in the fiber cells of AQP0 knockout mouse: Effects on lens transparency

Mutations and knockout of aquaporin 0 (AQP0) result in dominant lens cataract. To date, several functions have been proposed for AQP0; however, two functions, water permeability and cell-to-cell adhesion have been supported by several investigators and only water channel function has been readily authenticated by in vitro and ex vivo studies. Lens shifts protein expression from the more efficient AQP1 in the equatorial epithelial cells to the less efficient water channel, AQP0, in the differentiating secondary fiber cells; perhaps, AQP0 performs a distinctive function. If AQP0 has only water permeability function, can the more efficient water channel AQP1 transgenically expressed in the fiber cells compensate and restore lens transparency in the AQP0 knockout (AQP0^−/−) mouse? To investigate, we generated a transgenic wild-type mouse line expressing AQP1 in the fiber cells using αA-crystallin promoter. These transgenic mice (TgAQP1^+/+) showed increase in fiber cell membrane water permeability without any morphological, anatomical or physiological defects compared to the wild type indicating that the main purpose of the shift in expression from AQP1 to AQP0 may not be to lessen the membrane water permeability. Further, we transgenically expressed AQP1 in the lens fiber cells of AQP0 knockout mouse (TgAQP1^+/+/AQP0^−/−) to determine whether AQP1 could restore AQP0 water channel function and regain lens transparency. Fiber cells of these mice showed 2.6 times more water permeability than the wild type. Transgene AQP1 reduced the severity of lens cataract and prevented dramatic acceleration of cataractogenesis. However, lens fiber cells showed deformities and lack of compact cellular architecture. Loss of lens transparency due to the absence of AQP0 was not completely restored indicating an additional function for AQP0. In vitro studies showed that AQP0 is capable of cell-to-cell adhesion while AQP1 is not. To our knowledge, this is the first report which uses an animal model to demonstrate that AQP0 may have an additional function, possibly cell-to-cell adhesion.     

Parvalbumin-immunoreactive neurons in the inner nuclear layer of zebrafish retina

The purpose of this investigation is to characterize parvalbumin-immunoreactive (IR) neurons in the inner nuclear layer (INL) of zebrafish retina through immunocytochemistry, quantitative analysis, and confocal microscopy. In the INL, parvalbumin-IR neurons were located in the inner marginal portion of the INL. On the basis of dendritic stratification in the inner plexiform layer (IPL), at least two types of amacrine cells were IR for parvalbumin. The first one formed distinctive laminar tiers within s4 (PVs4) of the IPL, and the second within s5 (PVs5). The average number of PVs4 cells was 8263 cells per retina (n = 3), and the mean density was 1671 cells/mm². The average number of PVs5 cells was 1037 cells per retina (n = 3), and the mean density was 210 cells/mm². Quantitatively, 88.9% of anti-parvalbumin labeled neurons were PVs4 cells and 11.1% were PVs5 cells. Their density was highest in the midcentral region of the ventrotemporal retina and lowest in the periphery of the dorsonasal retina. The average regularity index of the PVs4 cell mosaic was 4.09, while the average regularity index of the PVs5 cell mosaic was 3.46. No parvalbumin-IR cells expressed calretinin or disabled-1, markers for AII amacrine cells, in several animals. These results indicate that parvalbumin-IR neurons in zebrafish are limited to specific subpopulations of amacrine cells and the expressional pattern of parvalbumin may not correspond to AII amacrine cells in several other animals. Their distribution suggests that parvalbumin-IR neurons are mainly involved in ON pathway information flow.     

Oxidative injury to blood vessels and glia of the pre-laminar optic nerve head in human glaucoma

Glaucoma is a leading cause of irreversible world blindness. Oxidative damage and vascular injury have been implicated in the pathogenesis of this disease. The purpose of this study was to determine in human primary open angle glaucoma whether oxidative injury occurs in pre-laminar optic nerve blood vessels and glial cells. Following IRB approval, sections from post-mortem primary open angle glaucoma eyes (n = 5) with mean age of 77 ± 9 yrs (±SD) were compared to normal control eyes (n = 4) with mean age 70 ± 9 yrs (Eye Bank of Canada). Immunostaining with nitrotyrosine, a footprint for peroxynitrite-mediated injury, was performed and sections were double-labeled with markers for vascular endothelial cells, perivascular smooth muscle cells, and astrocytes with CD34, smooth muscle actin (SMA), and glial fibrillary acidic protein (GFAP), respectively. Immunostaining was captured in a masked fashion using confocal microscopy, and defined regions of interest for blood vessels and glial tissue. Intensity measurements of supra-threshold area in pixels as percent of the total number of pixels were calculated using ImageJ (NIH) and compared using two-tailed Mann-Whitney nonparametric tests between glaucoma and control groups. Colocalization coefficients with cell-specific markers were determined and compared with random coefficients of correlation. Increased nitrotyrosine immunoreactivity was observed in pre-laminar optic nerve head blood vessels of primary open angle glaucoma eyes compared to controls and this difference was statistically significant (1.35 ± 1.11% [±SD] vs. 0.01 ± 0.01%, P = 0.016). NT-immunoreactivity was also increased in the glial tissue surrounding the pre-laminar optic nerve head in the glaucoma group and compared to controls, and this difference was statistically significant (18.37 ± 12.80% vs. 0.08 ± 0.04%, P = 0.016). Colocalization studies demonstrated nitrotyrosine staining in vascular endothelial and smooth muscle cells, in addition to astrocytes. Correlation coefficients for CD34, SMA, and GFAP were 0.37, 0.52, and 0.64, respectively. Oxidative injury is present in blood vessels and astrocytes in the pre-laminar optic nerve head in human primary open angle glaucoma. Peroxynitrite-mediated oxidative injury, whether primary or secondary, may contribute to the pathobiology of glaucoma disease.     

Effects of unilateral topical atropine on binocular pupil responses and eye growth in mice

Purpose

Studies on drugs selected to target myopia development often use the vehicle-treated fellow eye as a control. However, it is not clear how much of the drug reaches the fellow eye, rendering it a potentially invalid control. Therefore, in this study, pupil responses were used to probe the effects of atropine in both eyes in mice, after unilateral topical application. In a second experiment, interocular differences in refractive development and axial eye growth were studied while atropine was applied daily to one eye.

Methods

In 20 C57BL/6 (B6) wildtype mice, a single drop of 1% atropine solution was instilled into one eye. Mice were gently restrained by holding their necks while video image processing software detected the pupil and measured its diameter at a sampling rate of 30 Hz. A bright green LED, attached to the photoretinoscope of the video camera, was flashed. Pupil responses were quantified daily over a period of 2 weeks. In another group of 24 mice, one drop of 1% atropine was applied daily for 28 days. Axial length was measured pre- and post-treatment, using low coherence interferometry (the Zeiss AC-Master). Refractive development was measured by infrared photorefraction.

Results

Similar to previous findings with the same device, untreated eyes displayed a pupil constriction of 24.84 ± 1.73% upon stimulation with the green LED. A single drop of 1% atropine caused complete suppression with no significant recovery over the whole observation period of two weeks. The responses in the fellow eye were temporarily reduced to about 75% and then recovered towards baseline. After daily atropine application, there was significant reduction in axial length of the eyes, relative to the saline-treated fellow eyes (3.234 ± 0.186 versus 3.378 ± 0.176 mm, n = 24, p < 0.01, paired t-test) and the refractions became more hyperopic/less myopic (+13.46 ± 2.15 D versus +10.06 ± 2.02 D, n = 24, p < 0.01).

Conclusions

In line with previous findings, one drop of atropine solution caused a long lasting suppression of pupil responses in the mouse eye. New data show that the transfer to the fellow eye was limited, making interocular comparisons feasible. It is also new that topical atropine reduced axial eye growth even when mice had largely normal vision.     

Prognosis of eyelid sebaceous gland carcinoma based on the tumor (T) category of the American Joint Committee on Cancer (AJCC) classification

The purpose of this study was to evaluate the clinical features and prognosis of eyelid sebaceous gland carcinoma (SGC) based on the T category of the American Joint Committee on Cancer (AJCC) classification (7th edition). This is a retrospective interventional case series study. Based on the T category of the AJCC classification, 191 patients with eyelid sebaceous gland carcinoma were classified as T1 (n = 1, 1 %), T2 (n = 111, 58 %), T3 (n = 76, 40 %), and T4 (n = 3, 2 %). Based on multivariate analysis, the factors predictive of regional lymph node metastasis included duration of symptoms >6 months (p = 0.04) and orbital tumor extension (p < 0.001). The factors predictive of systemic metastasis included orbital tumor extension (p < 0.001) and perivascular invasion (p = 0.007). The factor predictive of death due to systemic metastasis included orbital tumor extension (p < 0.001). Kaplan–Meier estimates of regional lymph node metastasis at 5 and 10 years, respectively, were 0 and 0 % for T1, 11 and 11 % for T2, 44 and 59 % for T3, and 100 and 100 % for T4 (p < 0.001). Kaplan–Meier estimates of systemic metastasis at 5 and 10 years, respectively, were 0 and 0 % for T1, 6 and 6 % for T2, 35 and 35 % for T3, and 100 and 100 % for T4 (p < 0.001). Kaplan–Meier estimates of death due to metastasis at 5 and 10 years, respectively, were 0 and 0 % for T1, 3 and 3 % for T2, 30 and 50 % for T3, and 100 and 100 % for T4 (p < 0.001). Primary tumor (T) category of the AJCC classification predicts the prognosis of patients with eyelid SGC. The risk of systemic metastasis and death increases with increasing tumor category.