1: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

The B subunit of Escherichia coli heat-labile enterotoxin inhibits Th1 but not
Th17 cell responses in established autoimmune uveoretinitis.

Raveney BJ, Richards CM, Aknin ML, Copland DA, Burton BR, Kerr EC, Nicholson LB,
Williams NA, Dick AD.

Department of Cellular and Molecular Medicine, University of Bristol, Bristol,
United Kingdom.

Purpose: To investigate the efficacy of B subunit of Escherichia coli
heat-labile enterotoxin (EtxB) in the treatment of ocular autoimmune disease.
Background: Murine experimental autoimmune uveoretinitis (EAU) is an animal
model of autoimmune posterior uveitis initiated by retinal-antigen-specific Th1
and Th17 CD4(+) T cells, which activate myeloid cells, inducing retinal damage.
EtxB is a potent immune modulator that ameliorates other Th1-mediated autoimmune
diseases, enhancing regulatory T cell activity. Methods: EAU was induced in
B10.RIII mice by immunisation with peptide of hIRBP161-180. Disease severity was
measured by clinical and histological assessment and functional responses of
macrophages (Mphi) and T cells were assessed both in vivo and in in vitro
co-cultures. EtxB was administered intranasally daily for 4 days, starting
either 3 days before or 3 days after EAU induction. Results: Pre-immunisation
treatment with EtxB protected mice from EAU, limiting both the number and the
activation status of retinal infiltrating immune cells. Treatment following EAU
induction did not alter disease course, despite suppression of IFN-gamma.
Although EtxB treatment of in vitro co-cultures of T cells and Mphi increased
IL-10 production, EtxB treatment in vivo increased the proportion and numbers of
IL-17-producing CD4(+) cells infiltrating the eye. Conclusion: EtxB
pre-immunisation protects mice from EAU induction by inhibiting Th1 responses,
but the resulting reduction in IFN-gamma responses by EtxB does not effect
infiltration or structural damage in established EAU, where Th17 responses
predominate. These data highlight the critical importance of the dynamics of T
cell phenotype and infiltration during EAU when considering immunomodulatory
therapy.

PMID: 18469197 [PubMed - as supplied by publisher]

2: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Reprogramming progeny cells of embryonic RPE to produce photoreceptors:
development of advanced photoreceptor traits under the induction of neuroD.

Liang L, Yan RT, Li X, Chimento M, Wang SZ.

Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, United
States.

Purpose. To address issues critical to the prospect of producing functional
photoreceptors by reprogramming the differentiation of RPE progeny cells, this
study examines whether reprogrammed cells can develop highly specialized
ultrastructural and physiological traits that characterize retinal
photoreceptors. Methods. Cultured chick RPE cells were reprogrammed to
differentiate along the photoreceptor pathway by ectopic expression of neuroD.
Cellular ultrastructure was examined with electron microscopy. Cellular
physiology was studied by monitoring cellular, free calcium (Ca++) levels in
dark-adapted cells in response to light and in light-bleached cells in response
to 9-cis-retinal. Results. Reprogrammed cells were found to localize red opsin
protein appropriately to the apex. These cells developed inner segments rich in
mitochondria, and while in culture some formed rudimentary outer segments,
analogous to those of developing photoreceptors in the retina. In response to
light, reprogrammed cells reduced their Ca++ levels, as observed with developing
retinal photoreceptors in culture. Further, upon exposure to 9-cis -retinal,
light-bleached, reprogrammed cells increased their Ca++ levels, reminiscent of
the visual cycle recovery. Conclusions. These results indicate the potential of
reprogrammed cells to develop advanced ultrastructural and physiological traits
of photoreceptors and point to reprogramming progeny cells of embryonic RPE as a
possible alternative in producing developing photoreceptors.

PMID: 18469196 [PubMed - as supplied by publisher]

3: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Cultured Porcine Trabecular Meshwork Cells Display Altered Lysosomal Function
when Subjected to Chronic Oxidative Stress.

Liton PB, Lin Y, Luna C, Li G, Gonzalez P, Epstein DL.

Ophthalmology, Duke University, AERI 4004, Durham, North Carolina, 27710, United
States.

Purpose: To investigate the effects of chronic oxidative stress on lysosomal
function in trabecular meshwork (TM) cells. Methods: Confluent cultures of
porcine TM cells were grown for two weeks at physiological (5% O2) or hyperoxic
conditions (40% O2) in the presence or absence of the protease inhibitor
leupeptin (10 microM). The following parameters were quantified using the
fluorogenic probes indicated within parentheses: autofluorescence, intracellular
ROS (H2DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content
(mitotracker red), lysosomal content (acridine orange and lysotracker red),
autophagic vacuoles content (MDC), SA-beta-galactosidase (FDG), and cathepsin
activities (z-FR-AMC). Cathepsin levels were quantified by qPCR and Western-blot
analysis. Ultrastructural analysis was performed by transmission electron
microscopy. Results: Chronic exposure of porcine TM cells to a hyperoxic
environment led to an increase in ROS production and oxidized material. Electron
micrographs revealed the cytoplasmic accumulation of lipofuscin-loaded
lysosomes. Augmented lysosomal and autophagic vacuoles content was confirmed
using specific fluorophores. The mRNA and protein levels of several cathepsins
were upregulated with oxidative stress. This upregulated expression did not
correlate with increased lysosomal activity. Conclusions: Our results indicate
that chronic exposure of TM cells to oxidative stress causes the accumulation of
nondegradable material within the lysosomal compartment leading to diminished
lysosomal activity. Since the lysosomal system is responsible for the continuous
turnover of cellular organelles, impaired lysosomal activity may lead to
progressive failure of cellular TM function with age.

PMID: 18469195 [PubMed - as supplied by publisher]

4: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Low spatial frequency channels and the spatial frequency doubling illusion.

Rosli Y, Petratchkov SM, Maddess T.

DEPT. OF BIOMEDICAL SCIENCE, FACULTY OF ALLIED HEALTH SCIENCES, UNIVERSITI
KEBANGSAAN MALAYSIA, KUALA LUMPUR, Wilayah Persekutuan, Malaysia; ARC CoE in
Visual Sciences, RSBS, CVS, Australian National University, Canberra, Australia.

Purpose: This study examined the number and nature of spatio-temporal channels
in the region where the frequency-doubling (FD) illusion would be expected to
occur at 8 locations spanning the central 30 degrees of the visual field.
Methods: The probability of seeing the FD illusion was examined in 17 subjects.
Stimuli were presented at 5 octaves of low spatial frequencies, at each of 7
flicker frequencies in the range 5.65 to 27.95 Hz. In a single trial subjects
matched the apparent spatial frequency of the flickering test pattern using a 2
Alternative Forced Choice METHOD: Thirteen subjects were examined for stimuli
presented at contrast 0.95. Three or 4 subjects were examined at each of the
contrasts 0.2, 0.4 and 0.8. A factor analysis was conducted on the psychometric
functions, quantifying the number and possible spatio-temporal tuning of neural
channels present. Results: At contrast 0.95 three factors were able to explain
79.3% of the total variance in the psychometric responses to the 35 test
conditions. This simple form of three broad spatiotemporal channels was also
found at the other contrasts, and for different subjects. The Factor Scores
showed differential distribution of the Factors onto the 8 different visual
field locations. Thus the expression of the 3 channels differed somewhat across
the visual field. Conclusions: The results support earlier reports, that several
low spatial frequency channels exist below 1 cpd in the periphery. The results
may have implications for the FDT and Matrix perimeters.

PMID: 18469194 [PubMed - as supplied by publisher]

5: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Interactions Between Trabecular Meshwork Cells and Lens Epithelial Cells ; A
Possible Mechanism in Infantile Aphakic Glaucoma.

Michael I, Shmoish M, Walton DS, Levenberg S.

Biomedical Engineering Faculty, Technion-Israel institute of technology, Haifa,
Israel.

PURPOSE: Infantile aphakic glaucoma may develop as a postoperative complication
of early childhood cataract surgery. It has been associated with risk factors
including surgery in early life and retained lens material; however, its cause
and mechanism to date are poorly understood. This study focused on the potential
role of retained lens material (specifically, exposed lens epithelial cells
(LECs)) to be responsible for undesired changes of the trabecular meshwork (TM)
structure and function. METHODS: Interactions between LECs and TM cells were
studied by analyzing structural changes and differential gene and protein
expression in TM cells co-cultured with LECs. RESULTS: Subjecting normal TM
cells to the presence of LECs resulted in changes in their structural features
(such as increase in volume and size, and decrease in cell-cell interactions),
as well as in their protein expression (mainly cytoskeletal) and gene expression
(such as genes related to organ and cell morphogenesis, inflammatory response,
response to stimulus, ion homeostasis, and several signaling pathways).
CONCLUSIONS: Many of the changes observed in TM cells following exposure to LECs
resemble alternations seen in primary open-angle glaucoma. This strengthens the
suspected role of LECs in the development of aphakic glaucoma.

PMID: 18469193 [PubMed - as supplied by publisher]

6: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Epithelial Membrane Protein 2 (EMP2) Controls Collagen Gel Contraction in
ARPE-19 Cells by Modulating FAK Activation.

Morales SA, Mareninov S, Wadehra M, Zhang L, Goodglick L, Braun J, Gordon LK.

Department of Pathology and Laboratory Medicine, University of California, Los
Angeles , 650 Charles E. Young Dr. South, Los Angeles, California, 90095, United
States; Department of Ophthalmology, University of California, Los Angeles , Los
Angeles, California, United States.

Purpose: Proliferative vitreoretinopathy (PVR) occurs in approximately 10% of
patients following retinal detachment. PVR results from a multi-phase process
that leads to an aberrant wound-healing strategy with contractile cellular
forces and retinal detachment (TRD). Epithelial membrane protein 2 (EMP2)
controls cell surface expression and function of integrin isoforms associated
with cellular contraction in many cell types. Since EMP2 is highly expressed in
retinal pigment epithelium, this study investigates the role of EMP2 in collagen
gel contraction. Methods: EMP2 expression was recombinantly modified in the
ARPE-19 cell line. Cell surface integrin expression was assessed by flow
cytometry. Collagen gel contraction was assessed using an in vitro assay and
percent contraction quantified using NIH Image software. Proliferation and
migration was measured by BrdU incorporation and a wound healing assay
respectively. Cellular invasion was investigated using polycarbonate membranes
coated with collagen. Results: EMP2 expression levels correlate positively with
the ability to contract collagen gels. As compared to wildtype ARPE-19, cells
with increased EMP2 expression exhibited enhanced contraction (p=0.02) and
decreased EMP2 expression concomitantly resulted in decreased contraction
(p=0.002). EMP2 over expression resulted in reduced proliferation, migration,
and integrin alpha1 and alpha2 integrin expression. EMP2 overexpression was
associated with a 70% increase in FAK activation (p=0.0003) and relative
resistance of gel contraction to inhibitors of FAK/Src activation. Conclusions:
ARPE-19-mediated collagen gel contraction is a multi-step process which requires
integrin ligation and activation of the FAK/Src complex. EMP2 positively
modulates collagen gel contraction by ARPE-19 cells through increased FAK
activation.

PMID: 18469192 [PubMed - as supplied by publisher]

7: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Use of visual search in the assessment of pattern-related visual stress (PRVS)
and its alleviation by coloured filters.

Allen PM, Gilchrist JM, Hollis J.

Department of Optometry and Ophthalmic Dispensing, Anglia Ruskin University,
Cambridge, United Kingdom.

Purpose: Visual search measures have been used to evaluate the effects of
pattern-related visual stress (PRVS), and its alleviation by coloured filters,
but tasks and results have varied between studies. We measure performance with a
high-difficulty search task in individuals having low- and high-PRVS
susceptibility. Methods: Two PRVS groups (low & high) were formed on the basis
of participants responses to a visual symptoms questionnaire and perceptions of
a high-contrast grating pattern. Each participant searched for multiple
instances of a single-digit number (target) within an array of similar numbers
(distractors). Performance was measured by Response Time and Error Count. A
3-factor mixed factorial ANOVA design was employed to investigate the effects
of: i) PRVS GROUP, ii) a high-contrast background PATTERN, iii) an overlay of an
individually-selected COLOUR. Results: Individuals classified with high visual
stress were found to experience significantly greater improvement in reading
performance (F(1,26) = 24.579, p 0.001) and reduction in the number of errors
(F(1,26) = 9.502, p = 0.005) when performing the Wilkins Rate of Reading Test
using a coloured overlay than those with low visual stress. When performing a
visual search task Error Count was significantly higher in the high-PRVS group
in the absence of either PATTERN or COLOUR, but Response Time was not
significantly different. Neither Response Time nor Error Count was significantly
affected by background PATTERN and/or COLOURed overlay. Conclusions: Results of
this and previous studies confirm that visual search measures may be helpful in
the assessment of PRVS, but a number of important methodological issues may
limit their application in this context.

PMID: 18469191 [PubMed - as supplied by publisher]

8: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Abnormal Reactivity of Mueller Cells After Retinal Detachment in Mice Deficient
in GFAP and Vimentin.

Verardo M, Lewis GP, Takeda M, Linberg KA, Byun J, Luna G, Wilhelmsson U, Pekny
M, Chen DF, Fisher SK.

Neuroscience Research Institute, University of California Santa Barbara, Santa
Barbara, California, United States; Center for Bio-Image Informatics, University
of California Santa Barbara, Santa Barbara, California, United States.

Purpose. To determine the roles of glial fibrillary acidic protein (GFAP) and
vimentin in Mueller cell reactivity. Methods. Retinal detachments were created
in mice deficient for GFAP and vimentin (GFAP-/-vim-/-) and age-matched
wild-type (wt) mice. The reactivity of the retina was studied by
immunofluorescence and electron microscopy. Results. Mueller cell morphology was
different and glutamine synthetase immunoreactivity was reduced in the
undisturbed GFAP-/-vim-/- retinas. Following retinal detachment, Mueller cells
formed subretinal glial scars in the wt mice. In contrast, such scars were not
observed in GFAP-/-vim-/- mice. Mueller cells which normally elongate and
thicken in response to detachment appeared compressed, thin and "spikey" in the
GFAP-/-vim-/- mice. The endfoot region of Mueller cells in the GFAP-/-vim-/-
mice often sheared away from the rest of the retina during detachment,
corroborating earlier results showing decreased resistance of this region in
GFAP-/-vim-/- retinas to mechanical stress. In regions with end-foot shearing,
ganglion cells showed intense neurite sprouting, as revealed by
anti-neurofilament labeling, a response rarely observed in wt mice. Conclusions.
Mueller cells are subtly different in the GFAP-/-vim-/- mouse retina before
detachment. The endfoot region of these cells may be structurally reinforced by
the presence of the intermediate filament cytoskeleton, and our data suggest a
critical role for these proteins in the Mueller cell\'s reaction to retinal
detachment and participation in subretinal gliosis.

PMID: 18469190 [PubMed - as supplied by publisher]

9: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

The effect of acute superior oblique palsy on vertical pursuit in monkeys.

Tian J, Shan X, Ying H, Walker MF, Tamargo RJ, Zee DS.

Neurology, The Johns Hopkins University, School of Medicine, Baltimore,
Maryland, United States.

Purpose: To investigate vertical smooth pursuit eye movements in monkeys with
acute acquired superior oblique palsy (SOP). Methods: The trochlear nerve was
severed intracranially in two rhesus monkeys. After surgery, the paretic eye was
patched for 6 or 9 days, and then binocular viewing was allowed. Eye movements
were measured with binocular, dual search coils, before and after surgery, under
monocular viewing conditions. Vertical pursuit movements along the midline were
elicited using triangular-wave (20 degrees /s, +/-20 degrees ) or step-ramp (20
degrees /s) stimuli at a distance of 66 cm. Results: During the early
post-lesion period, before binocular viewing was allowed, pursuit velocity of
the paretic eye during triangular-wave tracking was lower than that of the
normal eye. When the viewing eye crossed straight ahead, the changes in pursuit
velocity conjugacy were similar for upward and downward tracking. After habitual
binocular viewing was allowed differences between upward and downward pursuit
emerged. When measured ~30 days post-lesion this directional asymmetry was less
during the open-loop period of step-ramp tracking than during triangular-wave
tracking. Conclusions: Rhesus monkeys with acute acquired SOP show
characteristic changes in vertical pursuit, with deficits for both upward and
downward tracking, and differences between the initiation of step-ramp pursuit
and the sustained response during triangular-wave tracking. The habitual viewing
condition (monocular vs. binocular) also affected the pattern of deficit.

PMID: 18469189 [PubMed - as supplied by publisher]

10: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Dietary omega-3 fatty acids modify ganglion cell function.

Nguyen CT, Vingrys AJ, Bui BV.

Optometry and Vision Sciences, The University of Melbourne, Cnr Keppel &
Cardigan St, Carlton, Victoria, 3053, Australia.

AIM: Dietary induced deficiencies in Omega-3 (omega-3) fatty acids are well
known to alter photoreceptor function. In this study we consider the broader
functional changes in a diversity of retinal neurons. METHODS: Sprague-Dawley
dams were fed either omega-3 sufficient (omega-3(+), n = 21) or deficient
(omega-3(-), n = 19) diets 5 weeks prior to conception with pups continued on
the mothers diet. After 20 weeks of age, electroretinograms (ERGs) were recorded
using protocols that isolate separate cellular generators, including;
photoreceptors (PIII), ON-bipolar cells (PII), and ganglion/amacrine cells
(STR). At brightest energies rod and cone responses were isolated with a paired
flash paradigm. Retinal tissue (omega-3(+), n = 5; omega-3(-), n = 5) was
harvested at 23 weeks of age for fatty acid assays with thin layer and gas
liquid chromatography. RESULTS: Omega-3 deficiency caused 48.6% decrease in
total retinal docosahexaenoic acid (DHA). This change induced a significant
amplitude decreases only in the rod PII (-8.2%) and pSTR components (-27.4%)
with widespread delays in all signals (PIII 5.7%, PII 13.6%, pSTR 7.6%, nSTR
8.3%). Omega-3 deficiency yielded its greatest effects on signals originating in
the inner retina (pSTR). CONCLUSIONS: Increasing dietary omega-3 has beneficial
effects across the retina, with the greatest improvement occurring in ganglion
cell function.

PMID: 18469188 [PubMed - as supplied by publisher]

11: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Identification of Candidate KLF4 Target Genes Reveals the Molecular Basis of the
Diverse Regulatory Roles of KLF4 in the Mouse Cornea.

Swamynathan S, Davis J, Piatigorsky J.

Ophthalmology, Univ. Pittsburgh School of Medicine , 203 Lothrop Street Room
1025, Pittsburgh, Pennsylvania, 15213, United States.

Purpose: Kruppel-like factor KLF4 plays a crucial role in the development and
maintenance of the mouse cornea. Here, we have compared the wild type (WT) and
Klf4-conditional null (Klf4CN) corneal gene expression patterns to understand
the molecular basis of the Klf4CN corneal phenotype. Methods: Expression of more
than 22,000 genes in 10 WT and Klf4CN corneas was compared by microarrays,
analyzed using BRB ArrayTools and validated by Q-RT-PCR. Transient
cotransfections were employed to test if KLF4 activates the aquaporin-3, Aldh3a1
and TKT promoters. Results: Scatter plot analysis identified 740 and 529 genes
up- and down-regulated by more than 2-fold, respectively, in the Klf4CN corneas.
Cell cycle activators were upregulated while the inhibitors were downregulated,
consistent with the increased Klf4CN corneal epithelial cell proliferation.
Desmosomal components were downregulated, consistent with the Klf4CN corneal
epithelial fragility. Downregulation of aquaporin-3, detected by microarray, was
confirmed by immunoblot and immunohistochemistry. Aquaporin-3 promoter activity
was stimulated 7-10 fold by cotransfection with pCI-KLF4. Corneal crystallins
Aldh3A1 and TKT were downregulated in the Klf4CN cornea and their respective
promoter activities were upregulated 16- and 9-fold by pCI-KLF4 in
co-transfections. Expression of epidermal keratinocyte differentiation markers
was affected in the Klf4CN cornea. While the cornea specific keratin-12 was
downregulated, most other keratins were upregulated, suggesting hyperkeratosis.
Conclusions: We have identified functionally diverse candidate KLF4 target
genes, revealing the molecular basis of the diverse aspects of the Klf4CN
corneal phenotype. These results establish KLF4 as an important node in the
genetic network of transcription factors regulating the corneal homeostasis.

PMID: 18469187 [PubMed - as supplied by publisher]

12: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Myopia and the urban environment: Findings for a sample of 12-year old
Australian school children.

Ip JM, Rose K, Morgan I, Burlutsky G, Mitchell P.

Department of Ophthalmology and the Westmead Millennium Institute, Centre for
Vision Research, University of Sydney, Sydney, New South Wales, Australia.

Purpose: To examine associations between myopia and measures of urbanization in
a population-based sample of 12-year-old Australian children Methods:
Questionnaire data on socio-demographic and environmental factors including
ethnicity, parental education, and time spent in near-work and outdoor activity
were collected from 2367 children (75.0% response) and their parents. Population
density data for the Sydney area were used to construct 5 urban regions. Myopia
was defined as spherical equivalent refraction 20 mmHg
IOP elevation. EM analysis showed mitochondrial fission, matrix swelling,
substantially reduced cristae volume, and abnormal cristae depletion in 10
month-old glaucomatous ONH axons. The mean length of mitochondrial cross section
in these axons decreased from 916.6 +/- 768.4 nm in 3 month-old mice to 582.87
+/- 303.3 nm in 10 month-old glaucomatous mice (P<0.001). Moderate reductions of
COX mRNA were observed in the 10 month-old DBA/2J mice optic nerve heads. Larger
reductions of OPA1 immunoreactivity and gene expression were coupled with larger
increases of Dnm1 gene expression in 10 month-old glaucomatous ONH. Subcellular
fractionation analysis indicates increased release of both OPA1 and cytochrome c
from mitochondria in 10 month-old glaucomatous ONs. CONCLUSIONS. IOP elevation
may directly damage mitochondria in the ONH axons by promoting reduction of COX,
mitochondrial fission and cristae depletion, alterations of OPA1 and Dnm1
expression, and induction of OPA1 release. Thus, interventions to preserve
mitochondria may be useful for protecting ON degeneration in glaucoma.

PMID: 18469184 [PubMed - as supplied by publisher]

15: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

A Rapid Separation of Two Distinct Populations of Mouse Corneal Epithelial Cells
with Limbal Stem Cell Characteristics by Centrifugation on Percoll Gradient.

Krulova M, Pokorna K, Lencova A, Fric J, Zajicova A, Filipec M, Forrester JV,
Holan V.

Department of Transplantation Immunology, Institute of Molecular Genetics,
Prague, Czech Republic.

Purpose. To detect and isolate cells with stem cell (SC) characteristics in the
limbus of the mouse. Methods. Limbal tissues from BALB/c mice were
trypsin-dissociated and separated on a Percoll gradient. Several fractions were
isolated and characterized by Real-time PCR for the presence of limbal SC
markers and differentiation markers of corneal epithelial cells, by flow
cytometry for the determination of the side-population (SP) phenotype and for
growth properties in vitro. Results. Cells retained in the lightest fraction
(40% Percoll) and in the densest fraction (80% Percoll) of the gradient were
both enriched for populations with a high expression of SC markers ABCG2 and
Lgr5 and also expressing the SP phenotype. However, the lightest fraction
(representing approximately 12% of total limbal cells) contained cells with the
strongest spontaneous proliferative capacity and expressed the corneal
epithelial differentiation marker K12. In contrast the densest fraction (less
than 7% of original cells) was K12 negative and contained small
non-spontaneously proliferating cells, which instead were positive for p63.
Unexpectedly, cells from this fraction had the highest proliferative activity
when cultured on a 3T3 feeder cell monolayer. Conclusion. These findings
demonstrate the presence of two distinct populations of corneal epithelial cells
with limbal SC characteristics, based on differential expression of the keratin
specific marker K12 and transcription factor p63, and suggest a difference in
developmental stage of the two populations, with the K12- p63+ population being
closer to the primitive limbal SC.

PMID: 18469183 [PubMed - as supplied by publisher]

16: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Soluble gp130, an antagonist of IL-6 trans-signaling, is elevated in uveitis
aqueous humor.

Simon D, Denniston AK, Tomlins PJ, Wallace GR, Rauz S, Salmon M, Murray PI,
Curnow SJ.

Medical School, The University of Birmingham, Institute of Biomedical Research,
Division of Immunity and Infection, Birmingham, United Kingdom; University of
Birmingham, Birmingham and Midland Eye Centre, Academic Unit of Ophthalmology,
Division of Immunity and Infection, Birmingham, United Kingdom.

PURPOSE. To determine the concentrations of soluble gp130, a natural antagonist
of IL-6 trans-signaling, in the serum and aqueous humor (AqH) of patients with
uveitis. METHODS. Serum was obtained from the peripheral blood of patients with
active uveitis and healthy controls. AqH samples were collected from patients
with active uveitis and patients without uveitis undergoing routine cataract
surgery. Samples were centrifuged and the cell-free supernatant frozen at -80
degrees C. Concentrations of sgp130, sIL-6R and IL-6 were determined by a
sandwich ELISA or multiplex bead immunoassay, using standard curves of known
concentrations of recombinant cytokines. RESULTS. Serum concentrations of sgp130
were not significantly different between control individuals and patients with
active anterior uveitis, regardless of the degree of intraocular inflammation
cells. By contrast, the concentration of sgp130 in AqH was very low in patients
with no or little inflammation, but increased significantly with disease
severity. The greatest elevations of AqH sgp130 were found in patients with the
highest cellular activity. Simultaneous measurement of IL-6, sIL-6R and sgp130
revealed a high degree of correlation between the levels of these molecules,
especially for sIL-6R and sgp130. CONCLUSIONS. Soluble gp130 is increased in the
AqH of patients with active uveitis. It is likely that sgp130 partially inhibits
the process of IL-6 trans-signalling during inflammation. However, the
concentration found is still far below that in serum, suggesting that increasing
the level of sgp130 further may assist in reducing the inflammatory changes
induced by IL-6 trans-signalling.

PMID: 18469182 [PubMed - as supplied by publisher]

17: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

In vivo protection against retinal neurodegeneration by the {sigma}R1 ligand
(+)-pentazocine.

Smith SB, Duplantier JN, Dun Y, Mysona B, Roon P, Martin PM, Ganapathy V.

Cellular Biology & Anatomy, Medical College of Georgia, CB 2820, Augusta,
Georgia, 30912-2000, United States.

Purpose: To evaluate the neuroprotective properties of the sigma receptor 1
(sigmaR1) ligand, (+)- pentazocine in an in vivo model of retinal
neurodegeneration. Methods: Spontaneously diabetic Ins2(Akita/+) and wildtype
mice received intraperitoneal injections of (+)-pentazocine for 22 weeks
beginning at diabetes onset. Retinal mRNA and protein were analyzed by RT-PCR
and western blotting. Retinal histological sections were measured to determine
total retinal thickness, thicknesses of inner/outer nuclear and plexiform layers
(INL, ONL, IPL, INL) and the number of cell bodies in the ganglion cell layer
(GCL). Immunolabeling experiments were performed using antibodies specific for
4-hydroxynonenal and nitrotyrosine, markers of lipid peroxidation and reactive
nitrogen species, respectively, and an antibody specific for vimentin to view
radial Muller fibers. Results: sigmaR1 mRNA and protein levels in the
Ins2(Akita/+) retina were comparable to wildtype indicating that sigmaR1 is an
available target during the disease process. Histological evaluation of eyes of
Ins2(Akita/+) mice showed disruption of retinal architecture. By 17-25 weeks
postnatally, Ins2(Akita/+) mice demonstrated ~30% and 25% decreases in IPL and
INL thicknesses, respectively, and 30% reduction in ganglion cells. In the
(+)-pentazocine-treated group, retinas of Ins2(Akita/+) mice showed remarkable
preservation of retinal architecture; IPL and INL thicknesses of
(+)-pentazocine-treated Ins2(Akita/+) mouse retinas were within normal limits.
The number of ganglion cells was 15.6 +/- 1.5 vs. 10.4 +/- 1.2 cells/100 microm
retinal length in (+)- pentazocine-treated vs. non-treated mutant mice. Levels
of nitrotyrosine and 4 hydroxynonenal increased in Ins2(Akita/+) retinas, but
were reduced in (+)-pentazocine-treated mice. Retinas of Ins2(Akita/+) mice
showed loss of the uniform organization of radial Muller fibers; retinas of (+)-
pentazocine-treated mice maintained the radial organization of glial processes.
Conclusion: Sustained (+)-pentazocine treatment in an in vivo model of retinal
degeneration conferred significant neuroprotection, reduced evidence of
oxidative stress and preserved retinal architecture suggesting that sigmaR1
ligands are promising therapeutic agents for intervention in neurodegenerative
diseases of retina.

PMID: 18469181 [PubMed - as supplied by publisher]

18: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Analysis of HRT images: comparison of reference planes.

Poli A, Strouthidis NG, Ho T, Garway-Heath DF.

Eye Clinic, University of Verona, Piazzale Stefani 1, Verona, 37124, Italy;
Glaucoma Research Unit, Moorfields Eye Hospital, London, United Kingdom.

Background: HRT stereometric parameter values depend on the position of a
reference plane (RP), the instability of which results in parameter variability.
The identification of change depends on the stability of the RP position. The
purpose of the study was to evaluate the influence of various RPs on rim area
(RA) values in longitudinal image series. Methods: Longitudinal HRT image series
of 31 ocular hypertensive subjects who developed reproducible visual field loss
and 19 normal subjects were analyzed using 5 different RPs; the Standard RP
(Software Version 3.1.2.0), two 320 microm RPs (Software Versions 3.1.2.0 and
1.7.0), a previously-described Experimental RP and a new \'Moorfields\' RP. The
Moorfields RP is set at the level of the Standard RP at baseline, and is then
fixed relative to the reference ring for subsequent images. Linear regression of
RA over time was performed and the standard error (residual standard deviation;
RSD) of the rim change slope was calculated for each RP. Comparisons between RPs
were made by paired T- test analyses. Results: In progressing eyes, application
of the Standard RP resulted in significantly greater variability (as measured by
the RSD) compared to other RPs (mean 0.057 mm(2) versus 0.035 to 0.038 mm(2)).
There was a trend to faster RA change / time ( mean -0.0123 mm(2) / year) for
the Standard RP and slower (-0.0095 mm(2) / year) for the Experimental RP. There
was a trend for the Moorfields RP to result in the best signal-to-noise ratio
(speed of RA change/variability) - mean RA slope -0.0118 mm(2) / year; mean
global RSD 0.037 mm(2). Conclusions: Compared to the Standard RP, the Moorfields
RP has significantly lower variability and probably provides a greater facility
to discriminate RA change from measurement variability in the analysis of
longitudinal HRT image series.

PMID: 18469180 [PubMed - as supplied by publisher]

19: Invest Ophthalmol Vis Sci. 2008 May 9; [Epub ahead of print] 

Rheology of Tear Film Lipid Layer Spread in Normal and Aqueous Tear Deficient
Dry Eyes.

Yokoi N, Yamada H, Mizukusa Y, Bron AJ, Tiffany JM, Kato T, Kinoshita S.

Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465
Kajiicho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan.

Purpose: To analyze the relationship between tear volume and tear film lipid
layer (TFLL) spread. Subjects and Methods: Twenty-nine eyes from 22 subjects,
including normal eyes and eyes with aqueous tear-deficient dry eye, were
enrolled in this study. In all eyes, the radius of curvature (R: mm) of the
central lower tear meniscus was measured with a video-meniscometer and
interference images from the TFLL were recorded with a video-interferometer. The
interference images were captured as still images every 0.05 seconds, and the
relationship between the acquisition time for each image after a blink and the
averaged heights of the spreading TFLL in the upstroke of the blink, were
calculated. Results: In all cases, the time-dependent changes in TFLL spread
could be described by the expression H(t) - H(0) = rho[1-exp(-t/lambda)] [where
H(t) = averaged height in mm at time = t; H(0) = averaged height at t = 0; rho =
a constant; t = time in seconds; lambda: characteristic time in seconds], and a
statistically significant correlation was found between those changes and the
initial upward velocity of the spreading TFLL [H\'(0) = dH(0)/dt] and R (r =
0.573, p = 0.003). Conclusions: This study demonstrated that the time-dependent
changes of TFLL spread are compatible with the Voigt model of viscoelasticity,
and that the initial velocity of TFLL spread after a blink decreased in
proportion to the decrease of tear volume. There is potential interest in using
this parameter to diagnose and evaluate the severity of aqueous tear deficiency.

PMID: 18469179 [PubMed - as supplied by publisher]

20: Invest Ophthalmol Vis Sci. 2008 Apr 30; [Epub ahead of print] 

Association of LOXL1 Gene Polymorphisms with Pseudoexfoliation in the Japanese.

Ozaki M, Lee KY, Vithana EN, Yong VH, Thalamuthu A, Mizoguchi T, Venkatraman A,
Aung T.

University of Miyazaki, Miyazaki, Japan.

Purpose: Single nucleotide polymorphisms (SNPs) rs1048661, rs3825942 and
rs2165241 within the LOXL1 gene was recently found to confer risk to
pseudoexfoliation glaucoma (XFG) through pseudoexfoliation syndrome (XFS) in
Caucasians. The aim of this study was to test this association in Japanese
subjects with XFS/XFG. Methods: Japanese subjects with clinically diagnosed
XFS/XFG and normal controls were recruited. Genomic DNA was extracted and the 3
SNPs of LOXL1 gene were genotyped by bi-directional sequencing. The association
of individual SNPs with XFG/XFS was evaluated using chi-square and Fishers exact
test. Results: 209 Japanese patients (106XFG and 103XFS) and 172 controls were
studied. Strong associations were observed for all 3 SNPs of LOXL1 for XFS
(OR=13.56, P=3.39x10(-28) for allele T of rs1048661; OR=10.71, P=1.49x10(-7) for
allele G of rs3825942 and OR=4.55, P=5.33x10(-4) for allele C of rs2165241) and
XFG (OR=25.21, P=1.44x10(-34) for allele T of rs1048661; OR=11.02, P=1.40x10(-7)
for allele G of rs3825942 and OR=11.89, P= 4.76x10(-6) for allele C of
rs2165241). The risk-associated alleles of rs1048661 and rs2165241 differed
between the Japanese and Caucasians, whilst allele G of rs3825942 was associated
with disease in both populations. Conditional analysis indicated that rs3825942
was not independent but was highly correlated to rs1048661. The at-risk
haplotype T-G-C was present approximately two times higher (94.7% vs 50.6%,
P=4.22x10(-43)) in cases than controls and conferred a 2.9 (95%CI: 2.357-3.464)
fold increased likelihood of XFS. Conclusions: Polymorphisms in the LOXL1 gene
confer risk to XFS/XFG in the Japanese, but there are different risk-associated
alleles and haplotypes in the Japanese.

PMID: 18450598 [PubMed - as supplied by publisher]

21: Invest Ophthalmol Vis Sci. 2008 Apr 30; [Epub ahead of print] 

Control of Chemokine Gradients by the Retinal Pigment Epithelium.

Shi G, Maminishkis A, Banzon T, Jalickee S, Li R, Hammer J, Miller SS.

NEI, NIH, Bethesda, Maryland, United States.

PURPOSE Pro-inflammatory cytokines in degenerative diseases can lead to the loss
of normal physiology and destruction of surrounding tissues. In the present
study, we sought to determine the physiological responses of human fetal retinal
pigment epithelia (hfRPE) in vitro following polarized activation of
pro-inflammatory cytokine receptors. METHODS Primary cultures of hfRPE were
stimulated with an inflammatory cytokine mixture (ICM): interleukin-1beta
(IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma
(IFN-gamma). Western blots and immunofluorescence were used to determine the
expression/localization of the cytokine receptors on hfRPE. Polarized secretion
of cytokines was measured using Searchlight Technology. A capacitance probe
technique was used to measure transepithelial fluid flow (JV) and resistance
(RT). RESULTS IL-1R1 is mainly localized to the apical membrane and TNFR1 to the
basal membrane, while IFN-gammaR1 is detected at both membranes. Activation by
apical ICM induced a significant secretion of angiogenic and angiostatic
chemokines mainly across the hfRPE apical membrane. Addition of the ICM to the
basal, but not the apical bath significantly increased net fluid absorption (JV)
across the hfRPE within 20 min. Similar increases in JV were produced by 24 hr
exposure to ICM, which significantly decreased total tissue resistance (RT).
CONCLUSIONS Chemokine gradients across the RPE can be altered: (1) through an
ICM-induced change in polarized chemokine secretion; (2) through an increase in
ICM-induced net fluid absorption. In vivo, both of these factors could
contribute to the development of chemokine gradients that help mediate the
progression of inflammation/angiogenesis at the retina/RPE/choroid complex.

PMID: 18450597 [PubMed - as supplied by publisher]

22: Invest Ophthalmol Vis Sci. 2008 Apr 30; [Epub ahead of print] 

Oral supplementation of lutein/zeaxanthin and omega-3 long chain polyunsaturated
fatty acids in persons aged 60 years and older, with and without age-related
macular degeneration.

Huang LL, Coleman HR, Kim J, de Monasterio F, Wong WT, Schleicher RL, Ferris FL,
Chew EY.

Division of Epidemiology and Clinical Research, National Eye Institute/National
Institutes of Health, Bethesda, Maryland, United States.

Background: Increased dietary intake of lutein/zeaxanthin; and omega long-chain
polyunsaturated fatty acids (omega-3 LCPUFA) was found to be associated with
reduced risk of advanced age-related macular degeneration (AMD). Objectives: To
examine the effect of oral supplementation of omega-3 LCPUFA on changes in serum
levels of lutein/zeaxanthin when supplementing with lutein/zeaxanthin in persons
aged 60 years and older, with or without AMD. Design: Forty participants with
varying severity of AMD received lutein (10 mg) and zeaxanthin (2 mg) daily and
were equally randomized to receive omega-3 LCPUFA (350 mg docosahexaenoic acid
(DHA) and 650 mg eicosapentaenoic acid (EPA)) or placebo for 6 months. Serum
levels of lutein, zeaxanthin, and omega-3 LCPUFAs, and macular pigment optical
densities were measured at baseline, 1 week and months 1, 3, 6 and 9. Results:
By month 6, the median serum levels of lutein/zeaxanthin increased by 2 to 3
fold compared with baseline. Increases in serum levels of lutein/zeaxanthin did
not differ by omega-3 LCPUFA treatment (Ps > 0.5). After 1 month, in the omega-3
LCPUFA treated group, the median levels of DHA and EPA increased and the placebo
group had no changes. At month 6, participants with AMD had serum lutein
concentration lower than those without AMD (P < 0.05). Conclusions: The addition
of omega-3 LCPUFA to oral supplementation of lutein/zeaxanthin did not change
the serum levels of lutein and zeaxanthin. A long-term large clinical trial is
necessary to investigate the benefits and adverse effects of these factors for
the treatment of AMD.

PMID: 18450596 [PubMed - as supplied by publisher]

23: Invest Ophthalmol Vis Sci. 2008 Apr 30; [Epub ahead of print] 

Mitochondrial Oxidative DNA Damage In Experimental Autoimmune Uveitis.

Khurana RN, Parikh JG, Saraswathy S, Wu GS, Rao NA.

Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States.

Abstract Purpose: In experimental autoimmune uveitis (EAU), recent work has
demonstrated that retinal damage involves oxidative stress early in the uveitis
before macrophage cellular infiltration. The purpose of this study is to
determine whether oxidative mitochondrial DNA damage occurs early in EAU before
the leukocyte infiltration. Methods: Lewis rats were immunized with S-antigen
mixed with complete Freund\'s adjuvant (CFA) to induce EAU. Nonimmunized animals
and animals injected with CFA served as controls. Animals were killed on days 3,
4, 7, and 12 after immunization. Damage to mitochondrial DNA and nuclear DNA was
assessed using a novel long quantitative polymerase chain reaction (PCR)
technique. TUNEL staining to detect apoptosis and immunohistochemical detection
of leukocyte infiltration in EAU retinas were also performed at the above time
points. Results: Mitochondrial DNA damage occurred early in EAU beginning at day
4 and continued throughout until Day 12. At early phase of EAU, (day 4 through
Day 7), there was no inflammatory cell infiltration. On day 12 there was
infiltration of inflammatory cells in the retina and uvea. Nuclear DNA damage
occurred later in EAU at day 12. There was neither mitochondrial nor nuclear DNA
damage in the controls. TUNEL positive staining for apoptosis was only detected
at day 12 in EAU retina. Conclusions: Oxidative mitochondrial DNA damage begins
at day 4 in EAU. This further supports that oxidative stress selectively occurs
in the mitochondria in the early phase of EAU before leukocyte infiltration.
Such oxidative damage in the mitochondria may be the initial event leading to
retinal degeneration in EAU.

PMID: 18450595 [PubMed - as supplied by publisher]

24: Invest Ophthalmol Vis Sci. 2008 Apr 30; [Epub ahead of print] 

Quantification of Metamorphopsia in Patients with Macular Hole.

Kroyer K, Christensen U, Larsen M, la Cour M.

Department of Ophthalmology, Glostrup Hospital, Glostrup, Denmark.

Purpose. To describe a novel method for the evaluation of metamorphopsia within
the central 10 degrees of visual field in 55 patients with idiopathic macular
hole. Our test evaluates metamorphopsia in terms of interocular disparity
between the two eyes. Methods. We applied semicircular test- and
reference-stimuli of variable diameters in a binocular test that measured
interocular size disparity between perceptually iseikonic stimuli in subjects
with a unilateral macular hole. A group of 11 healthy subjects was used as
reference. Results. In 55 patients with a macular hole, interocular disparity
demonstrated a mean value of 0.71 degrees for stimuli in the range 1.0 - 2.5
degrees in diameter. This number declined to 0.41 degrees for stimuli in the
range of 9.0 - 10.0 degrees in diameter. Both hole diameter and eccentricity had
a significant effect on mean disparity (P < 0.001). Conclusions. Metamorphopsia
level declined as a function of eccentricity and affected the central 10 degrees
of visual field. Macular hole size had an independent effect on interocular
disparity. These results confirm reports that visuospatial distortion in macular
hole, is primarily the result of radial displacement of photoreceptors.

PMID: 18450594 [PubMed - as supplied by publisher]

25: Invest Ophthalmol Vis Sci. 2008 Apr 30; [Epub ahead of print] 

Human Transscleral Albumin Permeability and the Effect of Topographical Location
and Donor Age.

Anderson OA, Jackson TL, Singh J, Hussain AA, Marshall J.

Kings College London Department of Ophthalmology, The Rayne Institute, London,
United Kingdom.

PURPOSE. To quantify the permeability coefficient of albumin across human sclera
and to assess topographical and age-related variation. METHODS. Equatorial
superotemporal scleral tissue from 15 donor eyes (mean age 60 years, range 39-84
years) was mounted in a modified Ussing chamber. Additional tissue was taken
from the anterior and posterior superotemporal regions of 6 eyes, and equatorial
superonasal, and inferotemporal regions of a further 6 eyes. Fluorescein
isothiocyanate (FITC) labeled, 0.412 mM, bovine albumin was placed in one
hemi-chamber facing the internal scleral surface, and the rate of trans-scleral
flux was determined over 24 hours, at 25 degrees C, with a spectrophotometer.
RESULTS. Permeability coefficient for equatorial superotemporal scleral tissue
at 25 degrees C (+/- 1 SD) was 0.83 +/- 0.50 x10(-6) cm.s(-1). The permeability
coefficient adjusted for 37 degrees C (+/- 1 SD) was 1.43 +/- 0.86 x10(-6)
cm.s(-1). The effect of donor age was assessed for the fifteen equatorial
superotemporal samples. Regression analysis showed a significant decline in
scleral diffusion of albumin with increasing donor age (p = 0.0166). There was
no significant difference in diffusion over the different topographical regions
tested. Partition coefficient to albumin also showed a decline with increasing
donor age (p = 0.001). CONCLUSIONS: The permeability and partition coefficients
of human sclera both significantly decline with increasing donor age.
Permeability coefficient shows no significant variation over the different
topographical regions tested. The decrease in albumin permeability with
increasing donor age may have pharmacokinetic implications when considering
transscleral diffuson of high molecular weight compounds.

PMID: 18450593 [PubMed - as supplied by publisher]

26: Invest Ophthalmol Vis Sci. 2008 Apr 30; [Epub ahead of print] 

Comparison of Macular Thickness Measurements between Time Domain and Spectral
Domain Optical Coherence Tomography.

Leung CK, Cheung CY, Weinreb RN, Lee G, Lin DS, Pang CP, Lam DS.

Department of Ophthalmology and Visual Sciences, The Chinese University of Hong
Kong, 3/F, Chinese University of Hong Kong Eye Center, Hong Kong Eye Hospital,
147K Argyle Street, Kowloon City, nil, Hong Kong; Ophthalmology, Hamilton
Glaucoma Center, 9500 Gilman Drive, La Jolla, California, 92093, United States.

PURPOSE: To compare macular thickness measurements obtained from time domain
optical coherence tomography (OCT) and spectral domain OCT and to evaluate their
repeatability and agreement. METHODS: Thirty-five healthy normal subjects were
included. In one randomly selected eye in each subject, three serial macular
measurements were obtained from time domain OCT (Stratus OCT, Carl Zeiss
Meditec, Dublin, CA, USA) and spectral domain OCT (3D OCT, Topcon, Tokyo, Japan)
by an experienced technician in a random order. Total and regional macular
thicknesses obtained by the 2 OCTs were compared. Their agreement was examined
with Bland Altman plots. Repeatability [2.77 x Within subject standard deviation
(Sw)], coefficient of variation (CVw) (Sw / overall mean), and intraclass
correlation coefficient (ICC) were calculated to evaluate repeatability.
RESULTS: Low variability for macular thickness measurements were found in both
time domain and spectral domain OCTs. The range of the respective CVw and ICC
values were 1.6%-3.2% and 0.85-0.91 for Stratus OCT, and 0.6%-2.4% and 0.92-0.99
for 3D OCT. 3D OCT demonstrated better repeatability for total and regional
macular thicknesses (all with p