Postnatal visual deprivation in rats regulates several retinal genes and proteins, including differentiation-associated fibroblast growth factor-2.

Little is known about the retinal cellular basis of amblyopia, which is a developmental disease characterized by impaired visual acuity. This study examined the retinal transcripts associated with experimentally induced unilateral amblyopia in rats. Surgical tarsorrhaphy of the eyelids on one side was performed in pups prior to eye opening at postnatal day 14, thereby preventing any visual experience. This condition was maintained for over 2 months, after which electroretinograms (ERGs) were recorded, the retinal ganglion cell (RGC) arrangement and number were determined using neuroanatomical tracing, the retinal transcripts were studied using microarray analysis, regulated mRNAs were confirmed with quantitative reverse-transcriptase PCR, and proteins were stained using Western blotting and immunohistochemistry. An attenuated ERG was found in eyes that were deprived of visual experience. Retrograde neuroanatomical staining disclosed a larger number of RGCs within the retina on the visually deprived side compared to the non-deprived, control side, and a multilayered distribution of RGCs. At the retinomic level, several transcripts associated with retinal differentiation, such as fibroblast growth factor 2 (FGF-2), were either up- or downregulated. Most of the transcripts could be verified at the mRNA level. To unravel the role of a differentiation-associated protein, we tested FGF-2 in dissociated postnatal retinal cell cultures and found that FGF-2 is a potent factor triggering ganglion cell differentiation. The data suggest that visual experience shapes the postnatal retinal differentiation, whereas visual deprivation induces changes at the functional, cellular and molecular levels within the retina.

Association between congenital nasolacrimal duct obstruction and delivery by cesarean section.

OBJECTIVE: Congenital nasolacrimal duct obstruction (CNDO) is the most common cause of neonatal epiphora. Persistence can lead to chronic dacryocystitis and amblyopia. This study analyzed the association between the incidence of CNDO and delivery by cesarean section. STUDY DESIGN: This was a retrospective cohort study of 386 children with CNDO (born between 2000 and 2008). The incidence of the delivery mode in patients with CNDO was compared with data from a corresponding population derived from annual birth statistics. RESULTS: There was no statistically significant association between the overall cesarean section rate and the incidence of CNDO, but primary cesarean section was significantly more frequent among patients with CNDO (73.15%, p < 0.05). The difference was significant for both genders for the period from 2000 to 2008 (p < 0.05%). The relative risk for CNDO was 1.7-fold increased in children delivered by primary cesarean section. CONCLUSION: Primary cesarean section may be a risk factor for CNDO.

Angiogenic gene therapy does not cause retinal pathology.

BACKGROUND: The potential negative influence of angiogenic gene therapy on the development or progression of retinal pathologies such as diabetic retinopathy (DR) or age-related macular degeneration (AMD) has led to the systematic exclusion of affected patients from trials. We investigated the role of nonviral fibroblast factor 1 (NV1FGF) in two phase II, multinational, double-blind, randomized, placebo-controlled, gene therapy trials (TALISMAN 201 and 211). METHODS: One hundred and fifty-two subjects with critical limb ischemia or claudication were randomized to receive eight intramuscular injections of 2.5 ml of NV1FGF at 0.2 mg/ml or 0.4 mg/dl or placebo. One hundred and fifty-two patients received a plasmid dose of NV1FGF of up to 32 mg or placebo. All patients underwent a systematic ophthalmologic examination at baseline and at 3, 6 or 12 months following gene therapy. Twenty-six of these patients (Münster subgroup) received a retinal fluorescence angiography at baseline and at final examination. RESULTS: Among those 26 patients, four of nine patients with diabetes suffered from nonproliferative DR. Three patients showed non-exsudative AMD. No change of retinal morphology or function was observed in Münster subgroup of both TALISMAN trials independent of the intramuscular NV1FGF dosage applied. CONCLUSIONS: Angiogenic gene therapy using NV1FGF is safe even in diabetics.

Bacterial spectrum and antimicrobial susceptibility patterns in acquired and connatal lacrimal duct stenosis.

PURPOSE: (1) To determine the current bacteriological spectrum in connatal and acquired lacrimal duct obstruction (cLDO and aLDO, respectively) and (2) to analyze the antimicrobial susceptibility patterns of the recovered isolates. MATERIALS AND METHODS: In a prospective study, 463 samples (30% bilateral LDO) were obtained from the lacrimal ducts of 132 infants and 192 adult patients with symptomatic LDO between 2007 and 2012 at a tertiary eye-care center. The samples were cultured for aerobic and anaerobic bacteria, which were subsequently identified using standard microbiological techniques. Antimicrobial susceptibility testing was performed for each isolate using the disk diffusion method. Data were analyzed using SPSS and chi-square test for significance testing. RESULTS: (1) Among 463 samples investigated, 333 samples were positive, i.e. at least one bacterial isolate was recovered. A total of 72% were recovered (97% of samples from children and 56% of samples from adults), yielding a total of 654 bacterial isolates. Co-colonization with up to five different bacterial species was observed in a large proportion of the samples from children (87%), but in only 20% of those from adults and with a maximum of three different bacteria. Gram-positive bacteria were identified in 72% of the positive samples in both aLDO and cLDO. The most common Gram-positive species in cLDO was Streptococcus pneumoniae (29%), while that in cLDO was Staphylococcus aureus (60%). The most prevalent Gram-negative species were Moraxella catarrhalis (8%) and Haemophilus influenzae (9%) in cLDO and Pseudomonas aeruginosa in aLDO (12%). (2) Susceptibility testing revealed chloramphenicol to be the most active antibiotic with resistance rates of 3% in cLDO and 6% in aLDO, followed by ciprofloxacin (1% and 6%). Erythromycin and gentamicin were the least active of all, with resistances of 41% and 22%, respectively, in cLDO, and 23% and 11% in aLDO. CONCLUSIONS: Bacterial colonization occurs regularly in LDO, with Gram-positive bacteria being found in 97% of cLDO samples and 56% of aLDO samples. A remarkable number of different species were found to co-colonize in cLDO. The most common bacteria in LDO are highly susceptible in vitro to chloramphenicol and ciprofloxacin.

Effect of botulinum toxin A on the intraocular pressure and the retina in an animal model.

OBJECTIVE: The purpose of our study was to investigate the effect of an inadvertent intravitreal injection of botulinum toxin A (BTA) on the intraocular pressure (IOP) and the retina in an animal model. METHODS: BTA was injected intravitreally in normotensive rats. IOP was measured preoperatively as well as 1, 2, and 4 weeks postoperatively. Retinas were stained in vivo using a retrograde labelling technique and the density of retinal ganglion cells (RGCs) was determined. Immunohistochemistry was performed for rhodopsin and retinal glial fibrillary acidic protein (GFAP). RESULTS: Significant temporary IOP elevation occurred in all groups in the immediate postoperative period (ANOVA, p < 0.05). IOP changes in the intermediate period were not statistically significant (ANOVA, p > 0.05). The differences in the density of RGCs after BTA injection were not statistically significant (ANOVA, p > 0.05). All retinas displayed the same immunostaining pattern for rhodopsin and GFAP. CONCLUSION: Our findings indicate that BTA has probably no severe impact on IOP and the retina after an inadvertent intravitreal injection. However, temporary rise of IOP may possibly occur in the immediate postoperative period due to a volume-effect.

Microbiological findings and predisposing risk factors in corneal ulcers.

BACKGROUND: To identify the current bacterial spectrum, the specific resistances to commonly used antibiotics, and the predisposing risk factors causing bacterial keratitis. METHODS: We reviewed the microbiological results of corneal scrapes and predisposing risk factors from 346 patients with bacterial keratitis. Infectious bacteria was isolated from corneal samples and sensitivity testing was subsequently performed. RESULTS: In total, 346 samples were obtained from 174 female and 172 male patients (median age: 64 years; range 5-105 years). A positive culture was obtained in 43% (148/346), recovering 199 isolates. Staphylococcus aureus was the most frequently occurring Gram-positive strain (32%) and Pseudomonas aeruginosa was the predominant Gram-negative strain (10%). Patients with specific local and systemic predisposing factors had an elevated risk for bacterial keratitis with a specific risk for certain strains. The sensitivity testing revealed that chloramphenicol and fusidic acid were the most effective monotherapy drugs, with overall resistances of 0 and 12%, respectively, followed by ciprofloxacin (22%), tobramycin (23%), and levofloxacin (24%). CONCLUSIONS: The bacterial spectrum is changing. The most effective drugs are chloramphenicol and fusidic acid, followed by ciprofloxacin. Specific systemic and local predisposing factors promote the risk of bacterial keratitis.

Long-term outcome after treatment with 5% topical imiquimod cream in patients with basal cell carcinoma of the eyelids.

BACKGROUND: Basal cell carcinoma (BCC) is the most common malignancy of the skin and of the eyelid in the Caucasian population. Our study evaluated the long-term results of 5% imiquimod cream therapy for nodular BCC of the eyelid as an alternative to surgical approaches. METHODS: Five patients suffering from histologically proven nodular BCC of the eyelid who had refused surgical treatment were included in this interventional off-label use study. The patients applied 5% imiquimod cream topically five times a week for 6 weeks at the site of the tumorous lesion. Patients were followed up regularly for up to 7 years to check for tumor disappearance or recurrence, and for local and systemic side-effects. RESULTS: Complete long-term clinical clearance was obtained in four of the five patients, with no tumor recurrence after 7 years of follow-up. Cosmetic results were excellent. One patient refused to continue the treatment 2 weeks after therapy onset, due to significant subjective discomfort. No serious local side-effects, and no systemic side-effects at all were observed following treatment. CONCLUSIONS: Imiquimod cream (5%) provides an effective alternative therapy for the treatment of nodular BCC of the eyelid, although surgical treatment remains the gold standard at the present time. This study is the first to assess the long-term efficacy and safety of this approach. Trial studies are necessary.

A coculture assay to visualize and monitor interactions between migrating glioma cells and nerve fibers.

Glioma-cell migration is usually assessed in dissociated cell cultures, spheroid cultures, acute brain slices and intracranial implantation models. However, the interactions between migrating glioma cells and neuronal tracts remain poorly understood. We describe here a protocol for the coculture of glioma cells with myelinated axons in vitro. Unlike other methods, this protocol allows the creation of in vitro conditions that largely mimic the complex in vivo environment. First, long retinal axons from embryonic chicken are formed in an organotypic culture. Glioma cells are then positioned in the vicinity of the explants to allow them to contact the axons, interact with them and eventually migrate along them. High-resolution video microscopy and confocal microscopy can be used to monitor the migratory behavior. This protocol, which takes about 5 days to complete, could be applied to different types of tumor cells that interact with neurites, and is suitable for pharmacological and genetic approaches aimed at elucidating mechanisms underlying tumor migration.

Systemic adverse events: a comparison between topical and peribulbar anaesthesia in cataract surgery.

AIMS: To evaluate the safety of topical anaesthesia (TA) versus peribulbar anaesthesia (PBA) in patients undergoing routine cataract surgery on the basis of systemic adverse events. METHODS: In this retrospective study, a total of 2,020 consecutive cases of cataract surgery performed by one surgeon on 1,621 patients with PBA (n = 1,010; between 1998-1999) or TA (n = 1,010; between 1999-2001) were evaluated on the basis of intra-operative and early postoperative adverse events requiring medical intervention. RESULTS: The rate of pre-existing risk factors in patients undergoing cataract surgery is high (97%). Complications are significantly less frequent in TA than in PBA in the intra-operative (p < 0.001) and postoperative (p = 0.022) courses. The incidence of intra-operative complications is higher in elderly patients (>or=65 years of age) than in younger patients (p < 0.001). CONCLUSION: The results from the present study indicate that intra-operative complications are less likely in patients that receive TA, suggesting the use of TA for routine cataract surgery both in young patients and particularly in elderly patients when there are no contraindications in the individual case.

Treatment of basal cell carcinoma of the eyelids with 5% topical imiquimod: a 3-year follow-up study.

BACKGROUND: The aim of this study was to determine the efficacy and safety of 5% topical imiquimod, and the long-term results following its use, in the treatment of nodular basal cell carcinoma (BCC) of the eyelid. METHODS: Imiquimod cream (5%) was applied topically to five individuals affected by nodular BCC of the eyelid. The patients were followed up during the 6 weeks of treatment and for another 3 years after treatment. Local side effects and evidence of tumour regression or recurrence were noted. RESULTS: Complete clinical clearance of the tumour was obtained in four patients, with no response in the fifth patient. Therapy was typically accompanied by significant discomfort due to local side effects, which disappeared following completion of the treatment. None of these patients showed any local recurrence after 3 years. CONCLUSIONS: Topical imiquimod applied in the form of a 5% cream proved to be a safe, efficacious and sustainable treatment option for nodular BCC of the eyelid in our selected cases.

Growth-associated protein-43 expression in the lens of rats and primates.

Growth-associated protein-43 is a specific neuronal protein that regulates differentiation, growth and plasticity. In the present study, growth-associated protein-43 expression was studied in the lens of rats and primates (including man) at different postnatal ages by immunoblotting, immunohistochemistry and quantitative real-time polymerase chain reaction. Growth-associated protein-43 was expressed at all ages in primates and in developing, but not in adult rats. We demonstrate that the lens - a tissue that is devoid of nerves - expresses growth-associated protein-43 throughout life in primates, and in rats during development but not in adulthood. These results suggest that growth-associated protein-43 is involved in differentiation processes also outside the nervous system.

Pathological changes in human retinal ganglion cells associated with diabetic and hypertensive retinopathy.

BACKGROUND: To examine whether systemic diseases like diabetes and arterial hypertension, which frequently cause retinopathies leading to blindness effect the morphology of retinal ganglion cells (RGC). METHODS: Histological retina material with a history of being untreated, or laser-coagulated (LC) diabetic retinopathy (DR), or arterial hypertensive retinopathy (AHR) was used. The RGC were labeled by introducing crystals of the fluorescent carbocyanine dye DiI into the nerve fiber layer, which contains ganglion cell axons. RESULTS: The typical silhouettes of both major types of RGC, parasol and midget cells, were identified. The axons in DR and AHR retinas showed morphology changes such as irregular swelling and beading. Dendritic field sizes were significantly reduced in RGC of both the hypertonic and diabetic retinas. A significant reduction in branching frequency was evident in both the diabetic and hypertonic retinas, in both the midget and the parasol cells. In LC retinas, both parasol and midget RGC were observed within the LC spots, although their numbers were dramatically decreased compared with normal retinas. CONCLUSIONS: The data suggest that diabetes and arterial hypertonia have similar effects on the morphology of RGC, in addition to causing microvascular alterations and bleeding. Therefore, therapeutic measures and prognostic outcomes in diabetic and hypertensive retinopathy should also consider regressive changes in retinal neurons.

Morphometric examination of human and monkey retinal ganglion cells within the papillomacular area.

PURPOSE: To examine the morphology of the retinal ganglion cells (RGCs) in the lesser characterized area lying between the optic disk and the macula that consists of the central papillomacular area (PMA) and the arcuate papillomacular bundle (PMB). METHODS: Nineteen human and 10 monkey (Macaca fascicularis) retinas obtained after death were used in the study. Perikaryal, axonal, and dendritic silhouettes were examined by postvital application of the fluorescent dye DiI, which specifically labeled RGCs when placed onto the optic fiber layer. The retinas were freed from surrounding tissue, prepared as flat mounts on a nitrocellulose filter, and fixed overnight in 4% paraformaldehyde. DiI diffuses along the membranes of ganglion cell axons, thereby completely labeling them, their cell bodies, and dendrites, which enables the RGCs to be examined with fluorescence microscopy. RESULTS: In both species, midget cells represented most of the RGCs within the PMA (96.15%) and possessed small, umbrella-like dendrites oriented toward the deeper retinal layers. Parasol cells were less abundant in both species and had small, typical symmetric dendrites. Also along the PMB, midget cells represented most cells (91.52%), whereas only 8.47% could be categorized as parasol cells. In both species, parasol cells of the PMB extended dendrites, which were oriented perpendicular to the axons. CONCLUSIONS: The data show that the PMA and PMB mainly contain small midget cells of typical morphology and size but with atypically oriented dendrites, which are only characteristic for this retinal area.

Intraocular pressure changes during high-altitude acclimatization.

BACKGROUND: To evaluate the relationship between hypobaric hypoxia acclimatization and intraocular pressure (IOP) during ascent, acclimatization, and descent between 2286 m and 5050 m. METHODS: The following acclimatization-indicative physiological parameters were compared daily with IOP changes in eight healthy climbers of the 2003 Greek Karakorum expedition in altitude stages between 500 m and 5050 m: hemoglobin oxygen saturation (PO2), resting heart rate, blood pressure, retinal findings, and the Lake Louise score for acclimatization grading. RESULTS: IOP decreased significantly in the ascent phase (0.58 mmHg/100 m) and recovered (0.71 mmHg/100 m) during acclimatization and descent. A direct proportional correlation between decreases in PO2 and IOP was evaluated. Arterial blood pulse and pressure increased during acclimatization, while IOP decreased. No retinal hemorrhages were observed in well-acclimatized and incompletely acclimatized climbers. CONCLUSIONS: Every new active exposure to hypobaric hypoxia in the ascent phase induced a decrease in the IOP parallel to the PO2 decrease and to the level of acclimatization. The results from our study suggest that IOP changes are related to hypoxia-induced respiratory alkalosis and acclimatization stage, which could be used as a simple mobile screening test for acclimatization level to reveal acute mountain sickness and its severe consequences.

Multifocal electroretinography changes in the macula at high altitude: a report of three cases.

BACKGROUND: To evaluate the short- and long-term effects of high-altitude hypobaric hypoxia on macula morphology and function during ascents, acclimatizations, and descents between 500 m and 5,650 m, macula function was evaluated in three healthy climbers of a trekking expedition. METHODS: Macula physiology was tested with multifocal electroretinography (MF ERG), near and farvisual acuity, and Amsler grid tests. Macula morphology was tested with optical coherence tomography (OCT) and with stereoscopic fundoscopy obtained 1 week before ascent, as well as 1 week and 2 weeks after high-altitude exposure. The following physiological parameters indicative of acclimatization were compared daily during the expedition at altitudes between 500 m and 5,050 m: hemoglobin, oxygen saturation, resting heart rate, retinal findings, and the Lake Louise score of acclimatization. RESULTS: The central macula MF ERG responses were significantly reduced 1 week after high-altitude exposure, and had recovered by the follow-up examination performed during the following week. Near visual acuity and Amsler grid tests remained unaffected at both follow-up examinations. No significant changes were found in the follow-up OCT and daily fundoscopy examinations in all three well-acclimatized climbers. CONCLUSIONS: High-altitude hypobaric hypoxia affects the function of the highly sensitive macula region. This suggests that the exposure of persons with macula diseases such as age-related macula degeneration, tapetoretinal degeneration, or diabetic retinopathy to high altitudes may influence the disease progression. For this reason, this population should avoid prolonged and unnecessary high-altitude exposure without proper acclimatization.

Lens epithelium supports axonal regeneration of retinal ganglion cells in a coculture model in vitro.

The purpose of this study was to determine whether the lens epithelium influences the survival or axonal growth of regenerating retinal ganglion cells. The optic nerves of adult albino rats were injured in order to induce axonal regeneration, and axon growth was then studied in retinal explants in the presence of cocultivated lens capsules carrying living epithelial cells. In the first series of experiments, cocultivation of retinal explants with lens epithelium in immediate proximity resulted in penetration of fibers into the lens epithelium, indicating that it supported axonal growth. In the second series of experiments, co-explants were placed 0.5-1.0mm from each other. The numbers of outgrowing retinal axons were determined both with respect to the retinal eccentricity and the topological relationship with the lenticular co-explant. The Wilcoxon matched-pairs signed-rank test was used to determine if the numbers of axons differed significantly between four regions of the explants. Significantly more axons grew out from the retinal edge facing the lenticular explant than from its opposite side, indicating that the lens epithelium supports axon growth. The numbers of surviving retinal ganglion cells in culture were determined after retrograde prelabelling with a neuroanatomical tracer. The number of fluorescent ganglion cells within the retinal explants did not significantly differ between the groups (Mann-Whitney test). These findings indicate that the lens epithelium influences both the amount of axonal regeneration and the direction of growth without affecting the survival rate of retinal ganglion cells in vitro.

Ultrasonic visualization of the effect of blinking on the lacrimal pump mechanism.

BACKGROUND: The role of the lacrimal sac (LS) and the medial canthal tendon in the lacrimal pump mechanism is controversial. This study used ultrasonic visualization to analyze this phenomenon. METHODS: Movements of the LS and the medial canthal tendon during blinking were visualized with sonography. In addition, the maximal profile area of the LS was measured before and after blinking using 15-MHz sonography in 14 individuals with a normal lacrimal drainage system and in six patients with lacrimal duct obstruction. RESULTS: The upper part of the LS could be located as an echolucent structure between the lacrimal bone and the medial canthal tendon. The medial canthal tendon appeared to compress the LS during lid closure and release the LS during lid opening. The measured profile area of the visible normal LS at the compression time decreased by 50%. The dilated LS of patients with obstruction could also be compressed by the orbital muscle on blinking, but the maximum area decrease was only 15.5%. CONCLUSION: The findings imply that the lacrimal part of the orbicularis muscle contracts during blinking, with the medial canthal tendon compressing the LS in a cranial direction. Completion of lid closure then compresses both canaliculi and LS, forcing the intrasacral fluid through the drainage system. The expansion of the LS during the opening phase of the blink causes suction, and after opening of the punctal areas the canaliculi and LS vacuum breaks to reload with tear fluid. These findings demonstrate the importance of the orbicularis muscle and the medial canthal tendon for the lacrimal pump mechanism during blinking.

Presurgical and postsurgical ultrasound assessment of lacrimal drainage dysfunction.

PURPOSE: In addition to the obligatory clinical tests, imaging of the lacrimal drainage system (LDS) is useful in its clinical evaluation. The purpose of this study was to examine the usability and reliability of ultrasonography in the evaluation of the lacrimal drainage system. DESIGN: Observational cohort study. METHODS: A prospective study was conducted at a single institution. We performed ultrasound examinations on 17 patients with epiphora before and after surgery, and on 17 asymptomatic volunteers, to visualize and evaluate the anatomic and functional condition or pathologic abnormalities of the LDS. RESULTS: Echographic evaluation of the LDS was possible in all individuals. Pathologic abnormalities (canaliculitis, diverticulitis, concretion, or dilation of the lacrimal sac, and reduced functionality of the orbicular muscle and/or lacrimal sac pump) could be well demonstrated. In the postsurgical course, functional patency of the dacryocystorhinostomy opening could be verified in all cases. CONCLUSIONS: Sonography of the LDS appears to represent a reliable diagnostic technique supplementary to clinical tests in the presurgical and postsurgical examination of patients with epiphora. Pathologic abnormalities that may not be apparent in routine x-ray dacryocystography can be demonstrated with ultrasound techniques. Patients also benefit from the avoidance of exposure to ionizing radiation. However, ultrasound is not suitable for imaging the lower part of the lacrimal sac and the lacrimal duct because of the presence of overlying bony structures.