Marco Centofanti

Experimental and clinical evidence of neuroprotection by nerve growth factor eye drops: Implications for glaucoma

Elevated intraocular pressure (IOP) in glaucoma causes loss of retinal ganglion cells (RGCs) and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. We evaluated the effects of NGF eye drops in a rat model of glaucoma. We also treated 3 patients with progressive visual field defects despite IOP control. Glaucoma was induced in rats through injection of hypertonic saline into the episcleral vein. Initially, 2 doses of NGF (100 and 200 μg/mL) were tested on 24 rats, and the higher dose was found to be more effective. Glaucoma was then induced in an additional 36 rats: half untreated and half treated with 200 μg/mL NGF QID for 7 weeks. Apoptosis/survival of RGCs was evaluated by histological, biochemical, and molecular analysis. Three patients with advanced glaucoma underwent psychofunctional and electrofunctional tests at baseline, after 3 months of NGF eye drops, and after 3 months of follow-up. Seven weeks of elevated IOP caused RGC degeneration resulting in 40% cell death. Significantly less RGC loss was observed with NGF treatment (2,530 ± 121 vs. 1,850 ± 156 RGCs/mm2) associated with inhibition of cell death by apoptosis. Patients treated with NGF demonstrated long lasting improvements in visual field, optic nerve function, contrast sensitivity, and visual acuity. NGF exerted neuroprotective effects, inhibiting apoptosis of RGCs in animals with glaucoma. In 3 patients with advanced glaucoma, treatment with topical NGF improved all parameters of visual function. These results may open therapeutic perspectives for glaucoma and other neurodegenerative diseases.

Correlation between optical coherence tomography, pattern electroretinogram, and visual evoked potentials in open-angle glaucoma patients

OBJECTIVE [corrected] To correlate the nerve fiber layer (NFL) thickness and the visual function evaluated by electrophysiologic retinal and cortical responses assessed in open-angle glaucoma (OAG) eyes. DESIGN Prospective case-control study. PARTICIPANTS Thirty glaucoma patients (mean age, 47.1 +/- 7.15 years; refractive error range, +/- 2 spherical equivalent) with a mean deviation of computerized static perimetry (24/2 Humphrey, Dublin, CA) from -5 to -28 dB and intraocular pressure less than 21 mmHg on pharmacologic treatment and 14 age-matched control participants. METHODS Nerve fiber layer thickness was measured by optical coherence tomography. Retinal and visual pathway function was assessed by simultaneously recording pattern electroretinograms (PERGs) and visual evoked potentials (VEPs) using high-contrast (80%) checkerboard stimuli (the single check edges subtend 15 minutes of the visual arc) reversed at the rate of two reversals per second. Linear regression analyses were adopted to establish the correlation between NFL thickness and PERG and VEP parameters. MAIN OUTCOME MEASURES Nerve fiber layer thickness measurements in each quadrant (superior, inferior, nasal, and temporal) were taken and then averaged (12 values averaged) and identified as NFL overall, whereas the data obtained in the temporal quadrant only (three values averaged) were identified as NFL temporal. PERG P50 implicit time and P50-N95 amplitude and VEP P100 implicit time and N75-P100 amplitude were also measured. RESULTS In OAG eyes, we found a significant (P < 0.01) reduction in NFL thickness in both NFL overall and NFL temporal evaluations with respect to the values observed in control eyes. PERG and VEP parameters showed a significant (P < 0.01) delay in implicit time and a reduction in peak-to-peak amplitude. In OAG eyes, the NFL overall and NFL temporal values were significantly correlated (P < 0.01) with the PERG P50 implicit time and P50-95 peak-to-peak amplitude. No correlations (P > 0.01) between NFL values and VEP parameters were found. CONCLUSIONS There is a correlation between PERG changes and NFL thickness, but there is no correlation between VEP changes and NFL thickness in patients affected by OAG.

Nerve growth factor (NGF) reduces and NGF antibody exacerbates retinal damage induced in rabbit by experimental ocular hypertension

Abstract• Background: It has been shown that intravitreal injection of NGF inhibits ganglion cell degeneration after optic nerve transection and ischemic injury. The aim of our study was to investigate the presence of NGF in aqueous humor and its involvement in retinal damage during ocular hypertension. • Methods: We used an experimental model of ocular hypertension in rabbit. Before treatment and 4, 10 and 15 days after induction of hypertension, we evaluated histological retinal damage and NGF levels in aqueous humor using an immunoenzymatic assay. Polyclonal anti-NGF antibodies were injected intravitreally into one eye of each rabbit (n = 6), and the animals were killed after 4 days of hypertension. Another group of rabbits (n =12) was injected retro-ocularly with NGF and killed after 10 or 15 days of treatment for histologic evaluation of the retina. • Results: Our data show that experimental ocular hypertension in adult rabbits induces retinal damage and enhances local NGF levels. The highest NGF value was found after 4 days of intraocular hypertension; high levels persisted, though to a lesser extent, for up to 15 days. Histological examination revealed that the number of retinal ganglion cells (RGC) remained unchanged during the first 4 days but decreased at 10 days. These studies also showed that retro-ocular administration of NGF reduced RGC loss, whereas intraocular injection of NGF antibodies, which inhibited the endogenous NGF, exacerbated the retinal insult. • Conclusion: These findings demonstrate a protective effect of NGF on RGC damaged by ocular hypertension and prompt further investigations to evaluate a possible therapeutic use of NGF to retard RGC death in humans.

New proteins as vascular biomarkers in primary open angle glaucomatous aqueous humor.

PURPOSE The aim of this study was to investigate the expression level of several biomarkers in the in the aqueous humor of 14 patients with primary open angle glaucoma who underwent glaucoma surgery, and 11 nonglaucomatous normals who underwent cataract extraction surgery. METHODS The aqueous humor proteome of 25 patients was analyzed using an antibody microarray. Fourteen patients with uncontrolled intraocular pressure-despite profound therapeutic interventions-who underwent filtering procedures and 11 control subjects who underwent surgery for senile cataracts were included in the present study. Protein expression was evaluated using Cy3/Cy5 labeling, column purification, and hybridization on antibody-spotted glass microarrays. Fluorescent signals were detected by fluorescence laser scanning. RESULTS The levels of 13 proteins were significantly increased in the aqueous humor of glaucomatous patients compared with expression levels in healthy controls. One of the 13 proteins (ELAM 1) was involved in inflammation. Two of these proteins (apolipoprotein B and E) were involved in the delivery of cholesterol to cells. Five of the 13 proteins (myotrophin, myoblast determination protein 1, myogenin, vasodilator-stimulated phosphoprotein, and ankyrin-2) were involved in muscle cell differentiation and function. Three proteins (heat shock 60 kilodaltons (kDa) and 90 kDa proteins, and ubiquitin fusion degradation 1-like) were involved in stress response and the removal of damaged proteins; and two proteins (phospholipase C β and γ) were involved in signal transduction and neural development. CONCLUSIONS The expressions of these proteins in the aqueous humor of glaucomatous patients reflect the damage occurring in anterior chamber endothelia, mainly including the trabecular meshwork, which is the main structure of this ocular segment injured by glaucoma.

Estrogen and Progesterone Receptors in Vernal Keratoconjunctivitis

PURPOSE Sex-related influences have been implicated in the pathogenesis of vernal keratoconjunctivitis (VKC), an allergic eosinophilic disease. METHODS The authors evaluated tarsal and bulbar conjunctival biopsies from seven patients with severe and symptomatic VKC for the presence of estrogen and progesterone receptors by using monoclonal antibodies with a peroxidase-antiperoxidase technique. RESULTS Both the epithelium and subepithelium of the tarsal and bulbar conjunctiva of patients with VKC, but not those of four nonatopic control subjects, showed intense positive staining for estrogen and progesterone receptors. Immunofluorescence colocalization of both estrogen and progesterone receptors with eosinophil cationic protein showed that approximately 70% of positive cells were eosinophils. CONCLUSIONS Sexual hormones, through their receptors, may influence the activity of eosinophils in patients with VKC.

Do sex and hormonal status influence choroidal circulation

AIMS To investigate the relation between pulse amplitude (PA), pulsatile ocular blood flow (POBF), and sex and hormonal status. METHODS Measurements of POBF and PA were obtained by ocular blood flow tonography in 76 healthy subjects: 32 males and 44 females (age range 17–77 years). Females were divided into two age groups: group 1 (premenopausal) 17–42 years, and group 2 (post-menopausal) 55 years old and over. Two groups of age matched males served as controls. RESULTS Premenopausal females demonstrated a significantly higher rate of POBF and PA than age matched males and post-menopausal females. CONCLUSION Sex and hormonal status were shown to influence choroidal circulation.

Influence of Disc Size on Optic Nerve Head versus Retinal Nerve Fiber Layer Assessment for Diagnosing Glaucoma

PURPOSE To explore and compare the influence of optic disc size on the diagnostic accuracy of retinal nerve fiber layer (RNFL) thickness and optic nerve head (ONH) quantitative assessment. DESIGN Observational, cross-sectional evaluation of diagnostic tests. PARTICIPANTS We included 120 eyes from 50 normal subjects and 70 glaucomatous patients classified by the presence of a repeatable visual field defect for the analysis. TESTING The RNFL thickness was measured by scanning laser polarimetry with variable corneal compensator (GDx-VCC, Carl-Zeiss Meditec, Dublin, CA) and spectral-domain optical coherence tomography (Cirrus HD-OCT, Carl Zeiss Meditec, Inc). We obtained ONH imaging by means of confocal scanning laser ophthalmoscopy (HRT3; Heidelberg Engineering, GmbH, Dossenheim, Germany). MAIN OUTCOME MEASURES Sensitivity and specificity for normative classifications, sensitivity at fixed specificity and area under the receiver operating characteristics curve (AUC) for continuous parameters. A logistic marginal regression model and coefficients of variation (CoV) have been used to test and quantify the influence of optic disc size on the diagnostic accuracy of the 3 technologies under investigation. RESULTS Among continuous parameters average RNFL thickness for Cirrus HD-OCT, nerve fiber indicator for GDx-VCC and cup shape measure for the HRT3 showed the best diagnostic accuracy with an AUC of 0.97, 0.94, and 0.94, respectively. Among normative classifications, the highest sensitivity and specificity were found for OCT average RNFL thickness (75.8% and 94.7%), for GDx superior thickness (77.1% and 97.5%), for HRT3 Moorfields regression analysis result (89.4% and 73.7%) and for HRT3 GPS global (92.3% and 76.5%). The diagnostic performance of HRT3 parameters seemed to be significantly influenced by optic disc size, although the same was not true for Cirrus HD-OCT and GDx VCC. The most steady performers for each imaging device across disc size groups were Cirrus HD-OCT average thickness (CoV, 1.6%), GDx-VCC inferior thickness (CoV, 2.5%), and HRT3 GPS temporal and nasal (CoV, 21.4%). CONCLUSIONS The diagnostic accuracy of quantitative RNFL assessment as performed by Cirrus HD-OCT and GDx-VCC is high and virtually unaffected or only minimally affected by the size of the optic disc and may provide more consistent diagnostic outcomes across small and large discs than ONH assessment as performed by HRT3.

The role of Humphrey Matrix testing in the early diagnosis of retinopathy in type 1 diabetes

Aim: The aim of the study was to investigate the role of Humphrey Matrix threshold testing in the detection of early functional retinal impairment in subjects with type 1 diabetes mellitus (DM1) without any signs of retinal vasculopathy, and to investigate the relationship between both functional and structural retinal parameters and metabolic control. Methods: Thirty eyes of 30 subjects with DM1, with no sign of retinal vasculopathy, and 30 eyes of 30 age- and sex-matched healthy subjects were enrolled in this cross-sectional clinical study. Functional testing included Humphrey Matrix perimetry and white-on-white Humphrey visual field perimetry (HFA), while retinal nerve fibre layer (RNFL) thickness was measured by scanning laser polarimetry with variable corneal birefringence compensator (GDx VCC). Results: Matrix mean deviation (MD) was found to be significantly reduced in subjects with DM1 compared with controls (−1.10 (SD 2.98; 95% CI −2.21 to 0.01) vs 1.37 (SD 2.11; 95% CI 0.58 to 2.16), p = 0.0005). HFA MD and pattern standard deviation (PSD) were significantly worse in subjects with DM1 compared with controls (p = 0.010 and p = 0.013 respectively). Among structural parameters, average peripapillary RNFL thickness was reduced in DM1 subjects (p = 0.006). Matrix MD and HFA MD and PSD, and average peripapillary and superior RNFL, were significantly reduced in subjects with DM1 with HbA1c ⩾7% compared with controls. Conclusions: Functional and structural retinal testing by Humphrey Matrix and GDx VCC could be useful for the identification of early retinal impairment in people with DM1 with no sign of retinal vasculopathy.

Tear tryptase levels and allergic conjunctivitis

We measured tryptase, a neutral prolease stored in the secretory granules of mast cells, by solid‐phase radioimmunoassay in tears of 12 subjects with vernal keratoconjunctivitis (VKC) during remission phases, nine subjects with seasonal or perennial allergic conjunctivitis, and eight healthy controls. Mean values of tear tryptase levels were significantly (P<0.02) increased in VKC patients (14.5 ± 13 pg/l) when compared to those measured in patients with seasonal or perennial allergic con, junctivitis (0.6 ± 0.1 pgA) and in controls (3.3 ± 3.2 μg/l). In subjects with allergic conjunctivitis, the levels of tryptase, almost undetectable before allergen conjunctival challenge, showed a significant increase in the challenged eye 20 min ‐ but not 6 h ‐ after provocation in 5/9 cases. Our results indicate that VKC, a severe ocular disease characterized by an increased number and abnormal distribution of mast cells in the conjunctiva, also shows elevated levels of tryptase in tears even during remission phases. Evidence of mast‐cell activation, as revealed by a significant increase of tryptase levels in tears, is documented during the early‐phase reaction, but not during the late‐phase reaction, of allergic conjunctivitis patients challenged topically by specific allergen.

Sector-Based Analysis with the Heidelberg Retinal Tomograph 3 Across Disc Sizes and Glaucoma Stages: A Multicenter Study

PURPOSE To investigate the ability of sectorial analysis using the Heidelberg Retinal Tomograph 3 (HRT3) to discriminate between healthy and glaucomatous eyes and to determine whether this is affected by disc size and glaucoma severity. DESIGN Multicenter, cross-sectional evaluation of diagnostic tests. PARTICIPANTS Two hundred thirty-three eyes from 137 normal subjects and 96 glaucoma patients classified by the presence of a repeatable visual field defect. TESTING Participants underwent imaging with the HRT3, and the diagnostic accuracy of stereometric parameters, Moorfields regression analysis (MRA), and glaucoma probability score (GPS) were analyzed sectorially by glaucoma stage and optic disc size. MAIN OUTCOME MEASURES Sensitivity, specificity, positive and negative predictive values, and the area under the receiver operating characteristics curve (AUC). RESULTS Of stereometric parameters, the cup-to-disc area ratio of the inferotemporal sector had the largest AUC (AUC, 0.74). Both MRA and GPS algorithms showed the best diagnostic accuracy in the inferotemporal sector, with a sensitivity and specificity of 63% and 88% for MRA and 80% and 62% for GPS, respectively. In small discs, sectorial MRA analysis had higher diagnostic accuracy than the global optic nerve head (ONH) analysis (sensitivity of 70% and specificity of 82% in the inferonasal sector), and these findings were confirmed in very large discs (sensitivity of 74% and specificity of 85% in the inferotemporal sector). Similarly, stereometric parameters discriminated better sectorially rather than globally, with different parameters giving the best results in different optic disc size subgroups. MRA sensitivity was weak in the early-glaucoma stage, with slightly higher figures if considered sectorially rather than globally. GPS diagnostic accuracy was very consistent across ONH sectors in each disc size and glaucoma stage subgroup, with no single sector demonstrating better diagnostic accuracy than the global analysis. CONCLUSIONS Heidelberg Retinal Tomograph 3 sectorial analysis showed moderate diagnostic performance and may offer potential advantages over global analysis in the clinical diagnostic process. Small discs are classified more accurately by examining the inferonasal sector, whereas larger discs are classified more accurately by examining the inferotemporal sector. Neither HRT parameters nor classification algorithms seem to be good at the earlier stage of the disease. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.