Bardet-Biedl syndrome (BBS) is a rare genetic disorder that features retinal degeneration, obesity, polydactyly, learning disabilities and renal abnormalities. The diagnosis is often missed at birth, the median age at diagnosis being 9 years. In the attempt to shed light on BBS and improve its diagnosis and treatment, we evaluated the genotype-phenotype relationship in patients with a molecular diagnosis of BBS. We analyzed three common BBS genes, BBS1, BBS10 and BBS2, in 25 Italian patients fulfilling the clinical criteria of BBS. In 12 patients, we identified gene-specific biallelic variants and thus correlated genotype to the ophthalmic, renal and audio-vestibular phenotypes. At least one sequence variant was found in 60% of patients. The most common mutated gene was BBS1 followed by BBS10. Of the 17 sequence variants we found, 11 have not previously been associated with BBS. In 12 patients, we identified biallelic pathogenic variants; they had retinitis pigmentosa with early onset of visual impairment. However, retinal dystrophy was less severe in patients with BBS1 than in those with BBS10 variants. Overall, we found a high prevalence of renal dysmorphism and dysfunction. Notably, patients with BBS10 variants had the most severe renal impairment, which resulted in a critical decline in renal function. All the patients who underwent audio-vestibular evaluation had dysfunction of the cochlear outer hair cells, thus confirming the presence of hearing defects. BBS1, BBS2 and BBS10 are major causative genes in Italian BBS patients. BBS10 was associated with the worse outcome in terms of the renal, ocular and audiovestibular phenotypes. Cochlear dysfunction should be included among the hallmarks of BBS.

Purpose: Evaluating the clinical results of trans-epithelial collagen cross-linking (CXL) and standard CXL in patients with progressive keratoconus. Methods: This prospective study comprised 20 eyes of 20 patients with progressive keratoconus. Ten eyes were treated by standard CXL and ten by trans-epithelial cross-linking (TE-CXL, epithelium on) with 1 year of follow-up. All patients underwent complete ophthalmologic testing that included pre- and postoperative uncorrected visual acuity, corrected visual acuity, spherical error, spherical equivalent, corneal astigmatism, simulated maximum, minimum, and average keratometry, coma and spherical aberration, optical pachymetry, and endothelial cell density. Intra- and postoperative complications were recorded. The solution used for standard CXL comprised riboflavin 0.1% and dextran 20.0% (Ricrolin), while the solution for TE-CXL (Ricrolin, TE) comprised riboflavin 0.1%, dextran 15.0%, trometamol (Tris), and ethylenediaminetetraacetic acid. Ultraviolet-A treatment was performed with UV-X System at 3 mW/cm². Results: In both the standard CXL group (ten patients, ten eyes; mean age, 30.4±7.3 years) and the TE-CXL group (ten patients, ten eyes; mean age, 28±3.8 years), uncorrected visual acuity and corrected visual acuity improved significantly after treatment. Furthermore, a significant improvement in topographic outcomes, spherical error, and spherical equivalent was observed in both groups at month 12 posttreatment. No significant variations were recorded in other parameters. No complications were noted. Conclusion: A 1-year follow-up showed stability of clinical and refractive outcomes after standard CXL and TE-CXL.

The impairment of the blood retinal barrier (BRB) represents one of the main features of diabetic retinopathy, a secondary microvascular complication of diabetes. Hyperglycemia is a triggering factor of vascular cells damage in diabetic retinopathy. The aim of this study was to assess the effects of vitamin D₃ on BRB protection, and to investigate its regulatory role on inflammatory pathways. We challenged human retinal endothelial cells with high glucose (HG) levels. We found that vitamin D₃ attenuates cell damage elicited by HG, maintaining cell viability and reducing the expression of inflammatory cytokines such as IL-1β and ICAM-1. Furthermore, we showed that vitamin D₃ preserved the BRB integrity as demonstrated by trans-endothelial electrical resistance, permeability assay, and cell junction morphology and quantification (ZO-1 and VE-cadherin). In conclusion this in vitro study provided new insights on the retinal protective role of vitamin D₃, particularly as regard as the early phase of diabetic retinopathy, characterized by BRB breakdown and inflammation.

Background: Diabetic retinopathy (DR) is a neurovascular disease, characterized by a deficiency of brain-derived neurotrophic factor (BDNF), a regulator of autophagy. Beta-hydroxybutyrate (BHB), previously reported as a protective agent in DR, has been associated with BDNF promotion. Here, we investigated whether systemic BHB affects the retinal levels of BDNF and local autophagy in diabetic mice with retinopathy; Methods: C57BL/6J mice were administered with intraperitoneal (i.p.) streptozotocin (STZ) (75 mg/kg) injection to develop diabetes. After 2 weeks, they received i.p. injections of BHB (25–50–100 mg/kg) twice a week for 10 weeks. Retinal samples were collected in order to perform immunofluorescence, Western blotting, and ELISA analysis; Results: BHB 50 mg/kg and 100 mg/kg significantly improved retinal BDNF levels (p < 0.01) in diabetic mice. This improvement was negatively associated with autophagosome–lysosome formations (marked by LC3B and ATG14) and to higher levels of connexin 43 (p < 0.01), a marker of cell integrity. Moreover, BHB administration significantly reduced M1 microglial activation and autophagy (p < 0.01); Conclusions: The systemic administration of BHB in mice with DR improves the retinal levels of BDNF, with the consequent reduction of the abnormal microglial autophagy. This leads to retinal cell safety through connexin 43 restoration.

Objective techniques to assess the amelioration of vision in patients with impaired visual function are needed to standardize efficacy assessment in gene therapy trials for ocular diseases. Pupillometry has been investigated in several diseases in order to provide objective information about the visual reflex pathway and has been adopted to quantify visual impairment in patients with Leber Congenital Amaurosis (LCA). In this paper, we describe detailed methods of pupillometric analysis and a case study on three Italian patients affected by Leber Congenital Amaurosis (LCA) involved in a gene therapy clinical trial at two follow-up time-points: 1 year and 3 years after therapy administration. Pupillary light reflexes (PLR) were measured in patients who had received a unilateral subretinal injection in a clinical gene therapy trial. Pupil images were recorded simultaneously in both eyes with a commercial pupillometer and related software. A program was generated with MATLAB software in order to enable enhanced pupil detection with revision of the acquired images (correcting aberrations due to the inability of these severely visually impaired patients to fixate), and computation of the pupillometric parameters for each stimulus. Pupil detection was performed through Hough Transform and a non-parametric paired statistical test was adopted for comparison. The developed program provided correct pupil detection also for frames in which the pupil is not totally visible. Moreover, it provided an automatic computation of the pupillometric parameters for each stimulus and enabled semi-automatic revision of computerized detection, eliminating the need for the user to manually check frame by frame. With reference to the case study, the amplitude of pupillary constriction and the constriction velocity were increased in the right (treated eye) compared to the left (untreated) eye at both follow-up time-points, showing stability of the improved PLR in the treated eye. Our method streamlined the pupillometric analyses and allowed rapid statistical analysis of a range of parameters associated with PLR. The results confirm that pupillometry is a useful objective measure for the assessment of therapeutic effect of gene therapy in patients with LCA. ClinicalTrials.gov NCT00516477

The most frequent retinal diseases, such as diabetic retinopathy, age-related macular degeneration and posterior uveitis, are underlined by oxidative stress or aging-induced retinal inflammation, which contributes to vision impairing or loss. Resolution of inflammation is emerging as a critical phase able to counteract the inflammatory process leading to the progression of retinal damage. Particularly, pro-resolving mediators (PMs) play a key role in the modulation of inflammatory exudates and could be considered a new target to be investigated in different inflammatory-autoimmune pathologies. Here, we highlight the most recent studies concerning the role of the main PMs (lipoxins, resolvins, prtectins, maresins and annexins) in retinal inflammation, in order to collect the best evidence in the field of inflammatory retinal damage resolution and to propose novel pharmacological approaches in the management of the most common retinal diseases.