Suprachoroidal delivery of bevacizumab in rabbit in vivo eyes: Rapid distribution throughout the posterior segment.

PURPOSE: To test the in-vivo bio-distribution and safety of bevacizumab delivery into the suprachoroidal space (SCS) using a novel injection system in a large eye model. METHODS: Bevacizumab (1.25 mg) was injected into the vitreous (IVT, 50 µL, n = 12) or the SCS, (150 µL, n = 37) of live rabbits. Immunofluorescence and ELISA were used to assess bevacizumab distribution. Intraocular pressure (IOP) measurements, SD-OCT and fundus imaging, electroretinogram, and histology analysis were performed for safety assessment. RESULTS: Bevacizumab was observed throughout the choroid layers up to the retinal pigment epithelium (RPE), within 1 h following SCS injection. The Cmax of bevacizumab in the retina/choroid was 1043 ± 597 µg/gr tissue (mean ± standard error), 40-fold higher than in IVT injected eyes (p = 0.0339). One day following SCS injection, bevacizumab was detected throughout the posterior pole with a two-fold lower concentration. One week post-SCS injection, bevacizumab concentration in the retina/choroid dropped to 2.36 ± 1.32 µg/gr tissue (p = 0.034 vs. 1 h), with a half-life of 20 h. No suprachoroidal blebs, retinal detachment, hemorrhages, inflammation or changes in retinal function were observed up to 2 months following SCS injection. Elevated IOP (+16 mmHg) was observed two minutes post-SCS injection and spontaneously returned to baseline levels within 10 minutes. CONCLUSIONS: The novel injection system enabled a minimally invasive, safe, and consistent delivery of bevacizumab with rapid distribution throughout the choroid layers up to the RPE in large eyes. Large volumes of anti-angiogenic are delivered in close proximity to the retina due to the high volume distribution.

Chromatic Pupilloperimetry Measures Correlate With Visual Acuity and Visual Field Defects in Retinitis Pigmentosa Patients.

Purpose: To evaluate the ability of chromatic pupilloperimetry to identify visual field (VF) defects in patients with retinitis pigmentosa (RP) and to test the correlation between pupilloperimetry impairment and retinal structural and functional measures. Methods: The pupil responses of 10 patients with RP (mean age, 41.3 ± 16.2 years) and 32 healthy age-similar controls (mean age, 50.7 ± 15.5 years) for 54 focal blue and red stimuli presented in a 24-2 VF were recorded. The pupilloperimetry measures were correlated with Humphrey VF mean deviation, best-corrected visual acuity, and ellipsoid zone area. Results: Substantially lower percentage of pupil contraction and maximal pupil contraction velocity (MCV) were recorded in patients with RP throughout the VF in response to blue and red stimuli. The mean absolute deviation (MADEV) in the latency of MCV (LMCV) was significantly larger in patients compared with controls for blue and red stimuli (P = 1.0 × 10-7 and P = 1.0 × 10-6, respectively). The LMCV MADEV differentiated between patients and controls with high specificity and sensitivity (area under the receiver operating characteristic curve, 0.987 and 0.973 for blue and red, respectively). The MADEV of LMCV for blue stimuli correlated with best-corrected visual acuity (ρ = 0.938, P = 5.9 × 10-5) and ellipsoid zone area (ρ = -0.857; P = 0.002). The MADEV of LMCV for red stimuli correlated with Humphrey VF mean deviation (ρ = -0.709; P = 0.022). Minimizing the test to 15 targets maintained a diagnosis of retinal damage in patients with RP with high sensitivity and specificity (area under the receiver operating characteristic curve, 0.927). Conclusions: The chromatic pupilloperimetry measures significantly correlated with retinal function and structure in patients with RP at various disease stages. Translational Relevance: Chromatic pupilloperimetry may enable objective assessment of visual field defects and visual acuity in RP.

Efficacy and safety of injecting increasing volumes into the extravascular spaces of the choroid using a blunt adjustable depth injector.

PURPOSE: To evaluate the efficacy and safety of injecting increasing volumes into the extravascular spaces of the choroid (EVSC) in rabbit eyes in vivo using a blunt adjustable depth injector. METHODS: Indocyanine green (ICG) was injected in the superior-temporal quadrant, 2 mm posterior to the limbus at increasing volumes (0.1-0.3 ml) into the EVSC of New Zealand rabbit eyes in vivo. Intraocular pressure (IOP) measurements, spectral domain optical coherence tomography (SD-OCT), fundus imaging and histology analysis were performed to assess the safety and efficacy of the injection. RESULTS: Volumes up to 0.3 ml were administered consistently. ICG injection was successfully monitored in vivo using infrared fundus imaging and SD-OCT. ICG was detected across the EVSC compartment, reaching the retinal pigment epithelium, optic nerve head and visual streak. Injection of 0.3 ml yielded maximal dye distribution with a coverage area of 61.8% ± 6.7% (mean ± standard error, SE) of the posterior segment. Maximal IOP elevation was recorded 5 min following injection of 0.2 and 0.3 ml ICG (+ 20.0 mmHg, + 19.4 mmHg, respectively). Twenty minutes post-injection, the IOP was < 15 mmHg in all injection volumes. No retinal detachment or hemorrhages were detected in any of the injected eyes. CONCLUSIONS: This study demonstrates consistent and safe delivery of large volumes within the EVSC using a blunt adjustable depth injector that distributes the dye over 60% of the retinal surface. This injection system may offer a minimally invasive and easy way to deliver large volumes of pharmaceuticals into the posterior segment.

Topical Estrogen Therapy for Hyperopia Correction in Vivo.

Purpose: In vitro studies found that 17ß-estradiol (estrogen) modulates corneal biomechanical properties and reduces tissue stiffness. Therefore we hypothesized that topical estrogen might affect the refractive properties of the cornea, inducing a myopic shift. Methods: Twelve female New Zealand white rabbits 16 weeks old were used. The rabbits were randomly divided to either the treatment group receiving 1.5% (w/v) estrogen eye drops or a control group receiving vehicle only (n = 6 each group). Both groups were given drops (50 µL) to the right eye every 12 hours for 35 days. Ocular examination, pachymetry, intraocular pressure (IOP), keratometry ,and refraction were evaluated at baseline and on a weekly basis. Results: No significant differences were observed between the two groups at baseline in all outcome measures. Both groups displayed corneal flattening and a hyperopic shift. However, the change rate was slower in the treatment group. Repeated measurements analysis revealed a statistically significant difference in keratometry readings between groups (P = 0.034) with steeper keratometry by up to 0.6 diopters in the treatment group. The difference between the two groups diminished and became statistically insignificant after treatment cessation. No significant changes were observed in IOP and pachymetry throughout the study period. No side effects were observed in either group. Conclusions: Estrogen eye drops induced a myopic shift in keratometry readings. These results suggest that corneal refractive power might be manipulated pharmacologically. Further studies on the physiology behind this change are warranted to facilitate a pathway for development of novel pharmacologic treatments to correct refractive errors.

Repetitive magnetic stimulation protects corneal epithelium in a rabbit model of short-term exposure keratopathy.

PURPOSE: To investigate the effect of repetitive magnetic stimulation (RMS) on corneal epithelial permeability in a rabbit model of exposure keratopathy. METHODS: 61 female New Zealand White (NZW) rabbits were treated on one eye with repetitive magnetic stimulation (RMS) at a frequency of 20 Hz for 15 min. The other eye was untreated. Rabbit eyes were kept open for 2 h to induce acute corneal desiccation. The extent of fluorescein corneal staining was evaluated using EpiView software and the concentration of fluorescein in the anterior chamber was determined by a fluorometer. Safety was evaluated by electroretinogram, spectral domain optical coherence tomography (SD-OCT) and histopathology. Expression pattern of corneal cell markers was determined by immunofluorescence. RESULTS: A significant decrease in fluorescein concentration in the anterior chamber (54 ±â€¯8.4 ng/ml vs. 146.5 ±â€¯18.6 ng/ml, p = 0.000001) and in corneal surface fluorescein staining score (1.7 ±â€¯0.2 vs. 4.6 ±â€¯0.6, p = 0.00001) was obtained in RMS-treated eyes compared with control eyes, respectively. RMS treatment reduced by nearly 4 fold the percentage of corneal area with epithelial erosions by anterior segment SD-OCT. The therapeutic effect was maintained for at least 3 months. Increased expression of epithelial tight junction protein Zo-1 was observed in treated eyes. SD-OCT and histopathology analysis revealed no pathological changes in the treated or non-treated eyes. CONCLUSIONS: RMS treatment decreases epithelial corneal erosions in a rabbit model of exposure keratopathy, with no indication of pathological changes. RMS may present a novel treatment for protection of corneal epithelium from desiccation.

Anterior-Segment Optical Coherence Tomography-Guided Measurement Of A Melting Ulcer For Follow-Up Of Corneoscleral Thinning Progression.

PURPOSE: To evaluate the feasibility of using anterior-segment optical coherence tomography (AS-OCT) for three-dimensional assessment of corneoscleral thinning progression in ulcers after pterygium removal. METHODS: A patient with corneoscleral melting after pterygium removal surgery and mitomycin C treatment was evaluated using AS-OCT imaging of the corneoscleral ulcer at five consecutive time points, up to 2 years. AS-OCT scans of 8.3×5.6 mm (15°×10°) containing 41 B-scans spaced 139 µm apart were performed monthly for 4 months and then at 2 years following pterygium removal. A single B-scan was comprised of 768 A-scans. Ten B-scans of the same position were averaged in a single AS-OCT image. The area of ulcer's section (AUS) was measured in seven fixed landmarks through a horizontally aligned plane in order to provide an estimation of the three-dimensional size of the lesion. RESULTS: The AUS in the two superior locations increased during the follow-up period to an average of 114% at 2 years compared to the initial visit. In the other five locations (three midline and two inferior), the AUS decreased and was on average 64% in the midline and 29% in the inferior locations at 24 months. CONCLUSION: AS-OCT provided a readily available assessment of the lesion's three-dimensional size during repeated follow-ups and identification of localized areas at higher risk for perforation. This method may potentially be useful for corneal surface pathologies requiring repeated follow-ups and may aid in decision-making regarding corneal thickness based on an accurate measurement.

Blue Autofluorescence Fundus Imaging for Monitoring Retinal Degeneration in Royal College of Surgeons Rats.

PURPOSE: Development of a method for noninvasive longitudinal follow-up of retinal degeneration in the whole retina for Royal College of Surgeons (RCS) rats, a commonly used model of retinitis pigmentosa associated with mutations in the MER-proto-oncogene tyrosine kinase (MERTK) gene. METHODS: Pigmented RCS rats at postnatal (p) days p28 to p84 were subjected to a biweekly spectral-domain optical coherence tomography (SD-OCT), blue laser fundus autofluorescence (BL-FAF) imaging, and multicolor fundus imaging. Wild-type (WT; Long Evans) rats were tested as control. RESULTS: Hyperautofluorescence developed throughout the fundus at p42, concomitant with a significant increase in SD-OCT thickness and reflectivity of the debris zone (DZ) layer as well as thinning of the photoreceptor outer nuclear layer (ONL). From p56 to p84, discrete hypofluorescent lesions surrounded by hyperfluorescent flecks were demonstrated around the optic disc that gradually spread throughout the retina. The hypofluorescent lesions were associated with loss of ONL and gradual thinning of the DZ layer. No hypofluorescent BL-FAF lesions were observed in WT rats. CONCLUSIONS: This study suggests that BL-FAF imaging may present a new method for noninvasive longitudinal follow-up of retinal degeneration in nearly the whole retina in RCS rats. TRANSLATIONAL RELEVANCE: A clinical test was developed that may be implemented in translational studies in the RCS rat model of MERTK-associated retinitis pigmentosa.

A minimally invasive adjustable-depth blunt injector for delivery of pharmaceuticals into the posterior pole.

PURPOSE: To investigate the feasibility and safety of a novel minimally invasive adjustable-depth blunt injector for pharmaceuticals delivery into the posterior segment. METHODS: Indocyanine green (ICG), sodium fluorescein and iron oxide nanoparticles (IONPs) were injected using the new injector into the extravascular spaces of the choroid (EVSC) compartment of rabbits and cadaver pig eyes. Spectral domain optical coherence tomography (SD-OCT), fundus imaging and histology analysis were performed for assessment of injection safety and efficacy. RESULTS: Indocyanine green, fluorescein and IONPs were detected across the EVSC in rabbit eyes, covering over 80 per cent of the posterior eye surface. Injected IONPs were retained in the EVSC for at least 2 weeks following injection. No retinal detachment, choroidal haemorrhage or inflammation was detected in any of the injected eyes. In cadaver pig eyes, ICG was detected across the EVSC. CONCLUSIONS: This novel minimally invasive delivery system may be used to safely deliver large volumes of pharmaceuticals into a new treatment reservoir compartment - the EVSC which can serve as a depot, in close proximity to the retina, covering most of the surface of the back of the eye without insertion of surgical instruments under the central retina. This system is predicted to enhance the therapeutic effect of treatments for posterior eye disorders.

Cytosolic Hsp60 can modulate proteasome activity in yeast.

Hsp60, an essential oligomeric molecular mitochondrial chaperone, has been subject to rigorous basic and clinical research. With yeast as a model system, we provide evidence for the ability of cytosolic yHsp60 to inhibit the yeast proteasome. (i) Following biological turnover of murine Bax (a proteasome substrate), we show that co-expression of cytosolic yHsp60 stabilizes Bax, enhances its association with mitochondria, and enhances its killing capacity. (ii) Expression of yHsp60 in the yeast cytosol (yHsp60c) inhibits degradation of a cytosolic protein ΔMTS-Aco1 tagged with the degron SL17 (a ubiquitin-proteasome substrate). (iii) Conditions under which Hsp60 accumulates in the cytosol (elevated Hsp60c or growth at 37 °C) correlate with reduced 20 S peptidase activity in proteasomes purified from cell extracts. (iv) Elevated yHsp60 in the cytosol correlate with accumulation of polyubiquitinated proteins. (v) According to 20 S proteasome pulldown experiments, Hsp60 is physically associated with proteasomes in extracts of cells expressing Hsp60c or grown at 37 °C. Even mutant Hsp60 proteins, lacking chaperone activity, were still capable of proteasome inhibition. The results support the hypothesis that localization of Hsp60 to the cytosol may modulate proteasome activity according to cell need.