Novel diagnosis of fungal endophthalmitis by broad-range real-time PCR detection of fungal 28S ribosomal DNA

Aim

To detect the fungal genome in the ocular fluids of patients with fungal endophthalmitis by using a novel broad-range polymerase chain reaction (PCR) system.

Methods

After informed consent was obtained, ocular fluid samples (aqueous humor or vitreous fluids) were collected from 497 patients (76 patients with infectious endophthalmitis including clinically suspected bacterial and fungal endophthalmitis and 421 patients with infectious or non-infectious uveitis). Forty ocular samples from non-infectious patients without ocular inflammation were collected as controls. Fungal ribosomal DNA (28?S rDNA) was measured by a quantitative real-time PCR assay.

Results

Fungal 28?S rDNA of the major fungal species, such as Candida, Aspergillus, and Cryptococcus, were detected by novel broad-range real-time PCR examination (>10¹ copies/ml). Fungal 28?S rDNA was detected in the ocular fluids of 11 patients with endophthalmitis or uveitis (11/497, 2.2%). All 11 positive samples were detected in the infectious endophthalmitis patients (11/76, 14.5%). These PCR-positive ocular fluids had high copy numbers of fungal 28?S rDNA (range, 1.7 × 10³ to 7.9 × 10⁶ copies/ml), which indicated the presence of fungal infection. Of the 11 patients who were PCR positive, further examinations led to a diagnosis of fungal endophthalmitis in ten patients. The fungal 28?S rDNA was detected in one non-infectious case (a false-positive case). In addition, there were two PCR false-negative cases that were clinically suspected of having fungal endophthalmitis.

Conclusions

This novel quantitative broad-range PCR of fungal 28?S rDNA is a useful tool for diagnosing endophthalmitis related to fungal infections.     

Role of aquaporin-1 in trabecular meshwork cell homeostasis during mechanical strain

Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemm's canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24 h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to β-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8 h (n = 4, p < 0.05) and 24 h (n = 6, p < 0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n = 4, p < 0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r² = 0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.     

Kinetics of polymorphonuclear leukocytes in an experimental hypopyon model

Regarding the process of uveitis development, many past studies have used the experimental autoimmune uveoretinitis (EAU) and other animal models to observe histologically the infiltration of inflammatory cells and the process of lesion progression. However, no detailed study of the process of clearance of infiltrated inflammatory cells from the eye has been reported. The purpose of this study was to investigate the process of clearance of polymorphonuclear leukocytes (PMNs) using an experimental hypopyon model. PMNs obtained from ascites of SD rat were injected into the anterior chamber of SD rats. The process of PMNs clearance was evaluated by serial photography and 3D optical coherence tomography (3D-OCT), and histological changes were observed simultaneously. The hypopyon heights regressed from 1.04 ± 0.06 mm at 1 h (day0) to 0.45 ± 0.07 mm at day1, and 0 mm at day3 after PMNs injection. When the hypopyon heights at the three time points were compared, significant differences were found between groups (P < 0.05). The hypopyon volumes also decreased from 1.46 ± 0.07 mm³ at 1 h to 1.16 ± 0.09 mm³ at 2 h, and 0.83 ± 0.04 mm³ at 3 h after PMN injection. When the hypopyon volumes at the three time points were compared, significant differences were found between groups (P < 0.05). Light micrographs of inferior segment of the eyeball revealed dense PMNs in the chamber angle at 1 h after PMNs injection and many PMNs in the iris stroma and vessels, as well as at the episcleral and subconjunctival tissues around limbus at 3 h and day1 after PMNs injection. Light micrographs of superior segment of the eyeball at 3 h after injection revealed PMNs in the episcleral and subconjunctival vessels. Electron micrographs of inferior segment of the eyeball at 3 h after PMNs injection revealed dense PMNs with slightly condensed nuclei in the anterior chamber, as well as in the iris stroma and vessels. In conclusion, in the experimental hypopyon model, PMNs injected into the anterior chamber were cleared from the eye mainly through the iris stroma and vessels, as well as the episcleral and subconjunctival tissues around limbus.     

Retinal ganglion cell population in adult albino and pigmented mice: A computerized analysis of the entire population and its spatial distribution

In adult Swiss albino and C57 pigmented mice, RGCs were identified with a retrogradely transported neuronal tracer applied to both optic nerves (ON) or superior colliculi (SCi). After histological processing, the retinas were prepared as whole-mounts, examined and photographed under a fluorescence microscope equipped with a motorized stage controlled by a commercial computer image analysis system: Image-Pro Plus^® (IPP). Retinas were imaged as a stack of 24-bit color images (140 frames per retina) using IPP with the Scope-Pro plug-in 5.0 and the images montaged to create a high-resolution composite of the retinal whole-mount when required. Single images were also processed by specific macros written in IPP that apply a sequence of filters and transformations in order to separate individual cells for automatic counting. Cell counts were later transferred to a spreadsheet for statistical analysis and used to generate a RGC density map for each retina. Results: The mean total numbers of RGCs labeled from the ON, in Swiss (49,493 ± 3936; n = 18) or C57 mice (42,658 ± 1540; n = 10) were slightly higher than the mean numbers of RGCs labeled from the SCi, in Swiss (48,733 ± 3954; n = 43) or C57 mice (41,192 ± 2821; n = 42), respectively. RGCs were distributed throughout the retina and density maps revealed a horizontal region in the superior retina near the optic disk with highest RGC densities. In conclusion, the population of mice RGCs may be counted automatically with a level of confidence comparable to manual counts. The distribution of RGCs adopts a form of regional specialization that resembles a horizontal visual streak.     

Diagnostic anterior chamber paracentesis in uveitis: a safe procedure?

BACKGROUND—Differentiation between infectious and non-infectious uveitis is of crucial value for accurate management of patients with uveitis. Tests performed on aqueous humour yield more relevant information than those done in serum. The objective of this study was to evaluate whether the aqueous humour tap for diagnostic purposes is a safe procedure to perform in uveitis patients.
METHODS—In this retrospective study 361 patients with uveitis, who underwent a diagnostic anterior chamber paracentesis in an outpatient clinic, were investigated. 72 of the 361 patients were examined 30 minutes after the puncture. The site of the paracentesis, the depth of the anterior chamber, and cells in the anterior chamber were examined. All 361 patients were evaluated within 2 weeks after the paracentesis was performed. The final follow up period varied from 6 months to more than 3 years. The clinical data were analysed with the emphasis on the occurrence of cataract and a history of corneal infections or endophthalmitis.
RESULTS—In this series no serious side effects such as cataract, keratitis, or endophthalmitis were observed. The depth of the anterior chamber of all evaluated patients was restored after 30 minutes. In five out of 72 cases (three AIDS patients with cytomegalovirus retinitis and two patients with anterior uveitis due to herpes simplex virus) a small hyphaema was observed 30 minutes after the paracentesis took place.
CONCLUSION—Anterior chamber paracentesis appears to be a safe procedure in the hands of an experienced ophthalmologist.

     

Spectral sensitivity of single cones in rainbow trout (Oncorhynchus mykiss): A whole-cell voltage clamp study

The UVS cone mechanism is known to light adapt at low intensities in comparison to other cones. We were interested in whether this property was related to higher sensitivity in UVS cones or to network adjustments in sensitivity. We investigated spectral sensitivity of 107 individual cone photoreceptors in rainbow trout (Oncorhynchus mykiss) using a whole-cell voltage clamp technique. Mean time-to-peak response was 339 ± 90 ms and flash sensitivity for a 100 ms flash was 4.37 × 10⁻³ ± 2.50 × 10⁻³ pA photons⁻¹ μm², with no significant differences between the UVS, SWS, MWS and LWS cone classes. The spectral sensitivity of the UVS, SWS and LWS cones conformed to the expression of SWS1, SWS2 and LWS opsin genes. The spectral sensitivity of MWS cones, however, showed clear evidence of co-expression of RH2a and RH2b opsin pigments. The fish used in this study bridged the ontogenetic stage where the MWS cones shift their expression from RH2b to RH2a.     

Diagnostic Utility of Quantitative Polymerase Chain Reaction versus Culture in Endophthalmitis and Uveitis

Purpose: To compare genetic testing for microbes in infectious endophthalmitis or uveitis to culture.Methods: This was a retrospective, single-center case series that enrolled patients with clinically suspected endophthalmitis or uveitis of unknown etiology. Aqueous humor or vitreous was collected and sent for routine cultures and genetic testing.Results: In total, 46 patients were enrolled. Genetic testing was positive in 32/46 (70%) cases and culture 6/46 cases (13%). Five of 16 uveitis cases had a final clinical diagnosis of infectious uveitis, and polymerase chain reaction (PCR) was positive in 4/5 cases (80%), versus 0% for culture. In uveitis cases, PCR was 80% sensitive and 82% specific, and culture had 0% sensitivity. The overall sensitivity and specificity of PCR for all cases were 85% and 67%, respectively, compared with 17% and 100% for culture.Conclusion: Genetic assays are inexpensive ($25/case) and more sensitive than culture for identifying intraocular pathogens in endophthalmitis and uveitis.     

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.     

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.     

C-X-C Chemokines Influence Intraocular Inflammation During Bacillus Endophthalmitis

PurposeThe purpose of this study was to explore the C-X-C chemokines CXCL2 and CXCL10 as potential anti-inflammatory targets for Bacillus endophthalmitis.Methods Bacillus endophthalmitis was induced in C57BL/6J, CXCL2^−/−, and CXCL10^−/− mice. At specific times postinfection, eyes were analyzed for Bacillus, retinal function, and inflammation. The efficacies of intravitreal anti-CXCL2 and anti-CXCL10 with or without gatifloxacin in B. cereus endophthalmitis were also assessed using the same techniques.ResultsDespite similar Bacillus growth in eyes of C57BL/6J, CXCL2^−/−, and CXCL10^−/− mice, retinal function retention was greater in eyes of CXCL2^−/− and CXCL10^−/− mice compared to that of C57BL/6J mice. Neutrophil migration into eyes of CXCL2^−/− and CXCL10^−/− mice was reduced to a greater degree compared to that of eyes of C57BL/6J mice. Infected CXCL2^−/− and CXCL10^−/− mouse eyes had significantly less inflammation compared to that of C57BL/6J eyes. Retinal structures in infected eyes of CXCL2^−/− mice were preserved for a longer time than in CXCL10^−/− eyes. Compared to untreated eyes, there was less inflammation and significant retention of retinal function in eyes treated with anti-CXCL2 and anti-CXCL10 with or without gatifloxacin.ConclusionsFor Bacillus endophthalmitis, the absence of CXCL2 or CXCL10 in mice resulted in retained retinal function and less inflammation. The absence of CXCL2 led to a better clinical outcome than the absence of CXCL10. The use of anti-CXCL2 and anti-CXCL10 limited inflammation during B. cereus endophthalmitis. These results highlight the utility of CXCL2 and CXCL10 as potential targets for anti-inflammatory therapy that can be tested in conjunction with antibiotics for improving treating Bacillus endophthalmitis.     

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.     

A diagnostic dilemma in a patient with delayed onset endophthalmitis

We report a case of nonpainful uveitis nine months after an uncomplicated phacoemulsification cataract surgery. Chronic postoperative endophthalmitis was suspected. Diagnostic vitrectomy and partial capsular bag removal was performed, but the specimens cultured in microbiology laboratory showed no pathogens. Systemic workup came positive for skin Tuberculosis test, and presumed intraocular tuberculosis treatment was started accordingly. Inflammation persisted, so a repeat vitrectomy was performed with removal of the lens implant with the capsule, and this time bedside culture inoculation was performed in operating room, revealing Pseudomonas infection. Delayed-onset postoperative endophthalmitis typically progresses slowly and therefore can be confused with uveitis and treated with steroid and immunosuppressant treatment regimes. Our case confirms both the value of immediate bacterial inoculation and the necessity of aggressive surgical treatment in chronic postoperative endophthalmitis cases.     

Histological protection by cilnidipine, a dual L/N-type Ca channel blocker, against neurotoxicity induced by ischemia-reperfusion in rat retina

Although a blockade or lack of N-type Ca²⁺ channels has been reported to suppress neuronal injury induced by ischemia-reperfusion in several animal models, information is still limited regarding the neuroprotective effects of a dual L/N-type Ca²⁺ channel blocker, cilnidipine. We histologically examined the effects of cilnidipine on neuronal injury induced by ischemia-reperfusion, intravitreous N-methyl-d-aspartate (NMDA) (200 nmol/eye) and intravitreous NOC12 (400 nmol/eye), an nitric oxide donor, in the rat retina, and compared its effects with those of ω-conotoxin MV IIA, an N-type Ca²⁺ channel blocker and amlodipine, an L-type Ca²⁺ channel blocker. Morphometric evaluation at 7 days after ischemia-reperfusion showed that treatment with cilnidipine (100 μg/kg, i.v. or 0.5 pmol/eye, intravitreous injection) prior to ischemia dramatically reduced the retinal damage. Treatment with ω-conotoxin MV IIA before ischemia (0.1 pmol/eye, intravitreous injection) significantly reduced the retinal damage. However, amlodipine (30-100 μg/kg, i.v. or 0.1-1 pmol/eye, intravitreous injection) did not show any protective effects. Treatment with cilnidipine (100 μg/kg, i.v.) reduced the retinal damage induced by intravitreous NMDA, but not NOC12. These results suggest that cilnidipine reduces Ca²⁺ influx via N-type Ca²⁺ channels after NMDA receptors activation and then protects neurons against ischemia-reperfusion injury in the rat retina in vivo. Cilnidipine may be useful as a therapeutic drug against retinal diseases which cause neuronal cell death, such as glaucoma and central retinal vessel occlusion.     

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.     

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.     

Elevated serum IL-8 levels are associated with disease activity in idiopathic intermediate uveitis

AIM—To find a laboratory indicator for systemic involvement in intermediate uveitis.
METHODS—Interleukin 8 (IL-8) and C reactive protein (CRP) serum levels were measured in patients with idiopathic intermediate uveitis (n=61), uveitis controls (n=143), and normal controls (n=29). The records of those with intermediate uveitis were reviewed with the emphasis on disease activity and severity as characterised by the presence of cystoid macular oedema, vitreous exudates or snowbank formation, papillitis, and periphlebitis.
RESULTS—Increased serum IL-8 (20 pg/ml) was found in 27 out of 61 patients with intermediate uveitis (p< 0.01), 12 of 27 patients with sarcoid uveitis (p<0.05), in 19 of 30 patients with HLA-B27 associated acute anterior uveitis (p<0.05), and in five of 29 healthy controls. Raised IL-8 levels in intermediate uveitis were significantly associated with active disease (p<0.001) and the presence of vitreous exudates (p<0.001), papillitis, and periphlebitis (p<0.01). Elevated CRP levels were found in 12 of the 143 uveitis controls but in none of the intermediate uveitis patients or normal controls. During follow up an associated systemic disease was more frequently noticed in patients with an elevated serum IL-8 at entry into the study.
CONCLUSIONS—Elevated IL-8 serum levels were found in patients with active intermediate uveitis of unknown origin. An elevated IL-8 level seems to predispose the patient to a later development of associated systemic disease.

Keywords: C reactive protein; interleukin 8; uveitis; multiple sclerosis; sarcoidosis     

Improved semiautomatic method for morphometry of angiogenesis and lymphangiogenesis in corneal flatmounts

Purpose of the study was to describe a novel semiautomatic, quantitative image analysis method based on threshold analysis for morphometry of corneal (lymph)angiogenesis and to test its validity, reliability and objectivity. Murine corneas were vascularized by using a suture-induced neovascularization assay. For immunohistochemistry, flatmounts of the vascularized corneas were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and with CD31 as panendothelial marker. Morphometry of corneal hem and lymphangiogenesis was performed semi-automatically on digital images using image analysis software. Data were analyzed by a paired t-test, intraclass-correlation and systemic difference analysis compared to a manual method. The semiautomatic method based on threshold analysis was more valid in measuring the area covered by blood or lymphatic vessels. Both methods had a good reproducibility with respect to both vessel types (blood vessels: manual: 0.969, semiautomatic: 0.982; lymphatic vessels: manual: 0.951, semiautomatic: 0.966), whereas the systemic difference was significant for both groups measuring lymphatic vessels (manual: p < 0.003; semiautomatic: p < 0.035) and for the manual method measuring blood vessels (manual: p < 0.0001; semiautomatic: p < 0.419). The new semiautomatic morphometry method based on threshold analysis provides higher accuracy, is more valid than and at least as reproducible and objective as the manual outlining method. Therefore the semiautomatic method can be used to detect even small effects on hem and lymphangiogenesis in murine corneal flatmounts with greater precision.     

Behavioural assessment of flicker fusion frequency in chicken Gallus gallus domesticus

To interact with its visual environment, an organism needs to perceive objects in both space and time. High temporal resolution is hence important to the fitness of diurnally active animals, not least highly active aerial species such as birds. However, temporal resolution, as assessed by flicker fusion frequency (FFF; the stimulus frequency at which a flickering light stimulus can no longer be resolved and appears continuous) or critical flicker fusion frequency (CFF; the highest flicker fusion frequency at any light intensity) has rarely been assessed in birds. In order to further our understanding of temporal resolution as a function of light intensity in birds we used behavioural experiments with domestic chickens (Gallus gallus domesticus) from an old game breed ‘Gammalsvensk dvärghöna’ (which is morphologically and behaviourally similar to the wildtype ancestor, the red jungle fowl, G. gallus), to generate an ‘Intensity/FFF curve’ (I/FFF curve) across full spectrum light intensities ranging from 0.2 to 2812 cd m⁻². The I/FFF curve is double-branched, resembling that of other chordates with a duplex retina of both rods and cones. Assuming that the branches represent rod and cone mediated responses respectively, the break point between them places the transition between scotopic and photopic vision at between 0.8 and 1.9 cd m⁻². Average FFF ranged from 19.8 Hz at the lowest light intensity to a CFF 87.0 Hz at 1375 cd m⁻². FFF dropped slightly at the highest light intensity. There was some individual variation with certain birds displaying CFFs of 90-100 Hz. The FFF values demonstrated by this non-selected breed appear to be considerably higher than other behaviourally derived FFF values for similar stimuli reported for white and brown commercial laying hens, indicating that the domestication process might have influenced temporal resolution in chicken.     

The effect of modulating ocular depth of focus upon accommodation microfluctuations in myopic and emmetropic subjects

The magnitude of accommodation microfluctuations increases in emmetropic subjects viewing low luminance targets or viewing a target through small artificial pupils. Larger microfluctuations reported in myopia may result from an abnormally large depth of focus (DoF). The effect of modulating the size of the DoF has not been investigated in myopic subjects and may help to explain the cause of the increased DoF. Accommodation microfluctuations were recorded under two experimental conditions. Firstly, 12 emmetropes (EMMs), and 24 myopes (MYOs) viewed a Maltese Cross target with luminance levels of 0.002, 0.2, 6 and 600 cd/m² and in darkness, and second, 14 EMMs and 16 MYOs viewed a Maltese Cross target through pupil diameters of 0.5, 1, 2, 3, 4 and 5 mm presented in Maxwellian view. The magnitude of the accommodation microfluctuations increased significantly with a target luminance of 0.002 cd/m² (p < .03) and pinhole diameters of <2 mm (p < .05). For all other luminance levels and pupil diameters the magnitude was constant. For both conditions, MYOs had significantly larger microfluctuations than EMMs (p < .01). Considerable inter-subject variability was observed in the degree to which the magnitude of the microfluctuations increased, for both the 0.002 cd/m² luminance and 0.5 mm pupils, however, this was not correlated with refractive error. The increase in the magnitude of the microfluctuations while viewing a low luminance target (0.002 cd/m²) may be due to a shallower contrast gradient in the cortical image, with a consequent increase in DoF. The microfluctuations also increase when viewing through small pupils (<2 mm), which increases the DoF without altering the contrast gradient. The larger microfluctuations found in the MYOs consolidates the theory that MYOs have a larger DoF than EMMs and therefore have a higher threshold for retinal image blur.     

Naltrexone and insulin are independently effective but not additive in accelerating corneal epithelial healing in type I diabetic rats

Patients with diabetes are at increased risk for developing corneal disorders, termed diabetic keratopathy. Treatments for diabetic keratopathy are limited. Preclinical studies have demonstrated that topical administration of either naltrexone (NTX) or insulin (INS) accelerates corneal re-epithelialization in type I diabetic rats. This study determined whether the combination of NTX and INS would have additive effect(s) on the re-epithelialization of corneal abrasions in diabetic male Sprague-Dawley rats beyond either agent alone. Type 1 diabetes (DB) (glucose levels > 400 mg/dl) was induced with streptozotocin; glycemic levels were not controlled with INS. Eight weeks after induction of diabetes, a 5 mm diameter circular abrasion was created in the center of the cornea in one eye of each rat. Eye drops (0.05 ml) of INS [1 U (∼6 nM)] and NTX (10⁻⁵ M) in Vigamox were administered separately 4 times daily for 7 days (NTX/INS); DB control rats received drops of sterile vehicle (DB SV) 4 times daily. Two other groups of rats were given only NTX (DB NTX) or only INS (DB INS). Re-epithelialization was monitored by fluorescein staining, and images were recorded with a CCD camera. Areal measurements were made using Optimas software, and the percentage of epithelial defect over a 40 h period was calculated. Twenty-four hour after formation of an abrasion (∼21.7 ± 0.4 mm² area), corneal wounds in DB rats treated with NTX, INS, or NTX/INS were significantly smaller (p < 0.001) than those in DB SV rats, with reductions in the size of the defect ranging from 24 to 84%. DB rats treated with NTX or INS alone also were observed to have reductions in wound size of 22 and 29%, respectively, from subjects in the DB SV group at 16 h. At 16 h both the DB NTX and DB INS groups had defects that were 13 and 27%, respectively, smaller than those for the DB NTX/INS group, and at 40 h the DB INS animals had 78% smaller corneal wounds than in the DB NTX/INS group. Therefore, the DB NTX/INS group exhibited some slight delays in wound repair compared to the DB NTX and DB INS groups. Topical application of NTX and/or INS to the cornea had no effect on non-invasive measures that included ocular morphology, intraocular pressure, or corneal thickness. These data demonstrate that although NTX or INS accelerates wound healing, concomitant application of NTX and INS to corneal abrasions in diabetic animals does not have an additive effect on re-epithelialization.