A high-resolution RNA expression atlas of Retinitis Pigmentosa genes in the human and mouse retinas

PURPOSE. Retinitis pigmentosa (RP) is one of the leading causes of visual handicap in the world population and is characterized by high genetic heterogeneity. The study of the disease mechanisms and the development of efficient therapeutic approaches have mostly relied on the availability of animal models for this condition, so far. Nevertheless, little information is available about the RNA expression profiles of RP genes in the human retina. An expression atlas of 34 known RP genes in human and murine retinas was generated to overcome this lack of information. METHODS. Appropriate templates were retrieved for 34 RP genes that were used to perform RNA in situ hybridization studies on human and murine adult eyes. RESULTS. Most of the genes displayed similar patterns between human and mouse retina. Different expression patterns were observed for the CNGB1, USH2A, and FSCN2 genes, compared with those in previously reported profiles. In addition, different expression profiles were detected for the RPGR, CA4, PAP1, RGR, and RLBP1 genes in human and mouse retinas. CONCLUSIONS. The first gene expression atlas has been generated of RP genes in human and murine retinas. Differences observed in the expression patterns of some genes in humans and mice, will open new perspectives on the function of these genes and their putative roles in disease pathogenesis

Eye bank issues: II. Preservation techniques: warm versus cold storage

Most of the tissue used for penetrating keratoplasty is issued through eye banks that store the corneoscleral button either in hypothermic storage at 2–6°C or in organ culture at 31–37°C

Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium

Objective To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. Methods Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16 S and 18 S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. Results In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. Conclusion Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turn-around time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice

Bacterial contamination of human organ-cultured corneas

Purpose: This study was designed to define the risk of contamination of human corneas preserved by the organ-culture method. Methods: We examined the microbial contaminations in 3,100 corneoscleral rims cultivated in our eye bank. Microbiologic tests were performed in the preservation medium 5 days after the beginning of cornea cultures and in the last day of culture (21.5 \ub1 8.1 days), when the corneas were transferred to the deswelling medium. In 1,029 corneas a microbiologic test also was performed 1 day after the beginning of deswelling procedure. Results: We found 206 microbial contaminations (6.65% of total) after 5 days and 17 (0.55%) at the end of the preservation period. The total number of contaminated samples during the cornea culture was 223 corresponding to 7.2% of the samples (95% confidence interval, 6.3-8.1). The 1,029 tests performed during the deswelling step disclosed 26 contaminated cornea cultures despite apparent sterility of the medium (2.5%; 95% confidence interval, 1.5-3.5). Conclusions: The observation of microbial contaminations in a time close to the transplant (i.e., at the end of the preservation period and in the deswelling step) showed that a fast microbial tests during the deswelling procedure may prevent the grafting of a contaminated cornea. The appearance of bacteria in the deswelling medium despite a negative culture medium suggests that bacteria penetrate the corneal tissues during the culture to be subsequently extruded when the internal fluids move outward

Morphological Aspect of LASEK. Lasek & Asa History Technique Long-term Results.

The quality of the results of a photoablative refractive surgery procedure is determined by a number of factors. From amongst these, two anatomo-physiological factors play a critical role in influencing firstly the action of the laser beam and secondly, the repair reaction started up by the stromal- epithelial complex, as a response to the wound. These factors reside in the degree of smoothness of the surface on which the laser is applied and in the protective system of the post-photoablation residual stroma. The connecting link between these two aspects is the method used for preparing the stromal bed to be treated, which represents the first stage of the keratorefractive operation with the laser. There are two basic procedures for preparing the stromal bed: the creation of an epithelialstromal flap (LASIK technique) and the corneal disepithelialization (PRK technique). The LASEK technique was proposed with the goal of combining the advantages of both methods and of eliminating the possible risks associated with them at the same time. It provides for the removal of the epithelium, as an integral epithelial flap, to be repositioned on the treated stroma at the end of the laser treatment. With the aim of ascertaining the efficacy of the LASEK method, a large number of pre-clinical and clinical studies have been conducted to evaluate the two critical factors cited at the beginning, i.e. the surface on which the laser is applied and the consequent repair reaction of the stromal-epithelial complex