International Center for Ocular Physiopathology

Venezia, Italy

International Center for Ocular Physiopathology

Venezia, Italy
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PubMed | University of Perugia, International Center for Ocular Physiopathology, Taibah University and Linköping University
Type: Journal Article | Journal: British journal of pharmacology | Year: 2016

The connexin 43 (Cx43) mimetic peptide Gap27 was designed to transiently block the function of this gap junction. This study was undertaken to investigate the effect of Gap27 on corneal healing, inflammation and neovascularization.The effect of Gap27 on wound healing, inflammation and vascularization was assessed in primary human corneal epithelial cells (HCEC) in vitro and whole human corneas ex vivo, and in an in vivo rat wound healing model.Gap27 enhanced the wound closure of HCEC in vitro and accelerated wound closure and stratification of epithelium in human corneas ex vivo, but did not suppress the corneal release of inflammatory mediators IL-6 or TNF- in vivo. In human corneas ex vivo, F4/80 positive macrophages were observed around the wound site. In vivo, topical Gap27 treatment enhanced the speed and density of early granulocyte infiltration into rat corneas. After 7days, the expressions of TNF- and TGF1 were elevated and correlated with inflammatory cell accumulation in the tissue. Additionally, Gap27 did not suppress VEGF release in organotypic culture, nor did it suppress early or late VEGFA expression or neovascularization in vivo.Gap27 can be effective in promoting the healing of superficial epithelial wounds, but in deep stromal wounds it has the potential to promote inflammatory cell migration and accumulation in the tissue and does not suppress the subsequent neovascularization response. These results support the proposal that Gap27 acts as a healing agent in the transient, early stages of corneal epithelial wounding.

Parekh M.,International Center for Ocular Physiopathology | Ruzza A.,International Center for Ocular Physiopathology | Ferrari S.,International Center for Ocular Physiopathology | Busin M.,Villa Igea Hospital | Ponzin D.,International Center for Ocular Physiopathology
American Journal of Ophthalmology | Year: 2016

Introduction To determine the feasibility of preloading endothelial tissues for Descemet membrane endothelial keratoplasty (DMEK). Design Laboratory investigation. Methods setting: Institutional. participants: Twenty human donor corneas unsuitable for transplantation with endothelial cell density in a range of 1600-2700 cells/mm2. Intervention: The endothelium was punched, stripped (8.5 mm diameter) and manually tri-folded with the endothelial side inward. The excised membranes were gently moved in a 2.2 intraocular lens (IOL) cartridge and pulled further in the funnel using 25 G end-grasping forceps. The cartridge was filled with transport media (TM) (sealed at its funnel and back entrance with a stopper) and the tissue was preserved for 4 days at room temperature in the bottles containing TM. main outcome measures: Success rate of preparation, processing time, endothelial cell loss (ECL), and active metabolism. Results The tissues were peeled and loaded successfully in all cases. Average stripping and loading time was 20 and 4.5 minutes, respectively. ECL after preservation was 4.35% with 3.55% (± 5.89%) mortality and 7.80% (± 14.12%) uncovered areas. A total of 0.55 (± 0.26) mg/mL of glucose was consumed by the cells showing active metabolism. Conclusions Tri-folded (endothelium-in) DMEK grafts can be preloaded using TM in an IOL cartridge and stored up to 4 days with limited endothelial damage. Direct injection of TM should be avoided because of the presence of bovine serum, but the tissue can be washed using balanced salt solution and gently injected. Alternatively, the graft can be easily delivered using a bimanual pull-through technique. Preloading DMEK grafts will simplify the surgery with reproducibility, reduced surgical time, and reduced tissue wastage, cost, and logistical requirements. © 2016 Elsevier Inc.

Ruzza A.,International Center for Ocular Physiopathology | Parekh M.,International Center for Ocular Physiopathology | Ferrari S.,International Center for Ocular Physiopathology | Salvalaio G.,International Center for Ocular Physiopathology | And 5 more authors.
British Journal of Ophthalmology | Year: 2015

Purpose: To design and validate the efficacy of threedimensional (3D) printed smart storage glide (SSG) which is capable of preserving and delivering posterior lenticules for Descemet stripping automated endothelial keratoplasty (DSAEK). Methods: Laboratory investigation (A) was followed by clinical validation (B). Unsuitable corneas for transplantation (n=20) were used for study A. These tissues were cut using a standard microkeratome, punched and loaded into the SSG and preserved for 7 days in transport media. Endothelial cell density (ECD), Trypan blue and Alizarin red staining for endothelial morphology, thickness measurements and glucose uptake, cell apoptosis and immunostaining post preservation were analysed. For study B, clinical grade corneas (n=14) were preloaded in SSG and grafted in patients with indications of Fuchs' dystrophy (n=8), pseudophakic bullous keratopathy (n=3), posterior polymorphous dystrophy (n=2), and previous DSAEK failure (n=1). Standard DSAEK included descemetorhexis under air and bimanual delivery of the tissue under infusion of buffered saline solution through an anterior chamber maintainer placed at the 12 o'clock position. Main outcome measures for study B were less surgical time, best spectacle-corrected visual acuity (BSCVA), speed of visual recovery, and ECD. Results: For study A, an average ECD loss was 2.30 ±3.21%, thickness increased by 30.80±20.85% and one-third of glucose was utilised during the preservation phase. Alizarin red showed hexagonality of the cells. Cell apoptosis was not observed and expression of ZO-1 was noted on the preserved tissues. In study B, 25% ECD loss was observed after 6 months. BSCVA improved to 20/25 or better within 3 months after DSAEK. Mean surgical time recorded was 21 min. Conclusions: This paper describes the development, design, laboratory and clinical validation of a 3D printed SSG which helps to store and deliver posterior lenticules, therefore allowing transportation of quality-controlled precut tissues.

Parekh M.,International Center for Ocular Physiopathology | Ruzza A.,International Center for Ocular Physiopathology | Salvalaio G.,International Center for Ocular Physiopathology | Ferrari S.,International Center for Ocular Physiopathology | And 3 more authors.
American Journal of Ophthalmology | Year: 2014

Purpose To standardize a novel submerged hydro-separation technique for Descemet membrane endothelial keratoplasty (DMEK) graft preparation from donor corneal tissues. Design Experimental study, laboratory investigation. Methods setting: The Veneto Eye Bank Foundation, Venice, Italy. study population: Fifty-four random human donor corneal tissues unsuitable for transplantation. intervention: Donor corneas were laid in a sterile basin partially filled with tissue culture medium. A 25 gauge needle with 1 mL mounted syringe was filled with the tissue culture medium. The needle (with bevel up) was bent to 90 degrees and was inserted in the posterior cornea initiating beneath the trabecular meshwork. It was further advanced toward the midperiphery, ensuring that only the bevel was inserted, considering it as a threshold of insertion. The liquid was injected with a medium to high pressure into the posterior stroma or in the Descemet membrane-stroma interface to create the bubble. The tissues were preserved for 7 days in tissue culture medium at 31°C. Parametrical, physiological and histological analyses were carried out. main outcome measures: Larger-diameter tissue, no tissue wastage, reproducibility, and preshipment evaluation. Results Complete detachment was achieved in all the cases without any tissue wastage. Average diameter of the excised graft was 10.80 (±0.28) mm and endothelial cell loss post preservation was 11.48%. Expression of tight junction protein and regular morphology was observed post preservation. No signs of cell apoptosis were seen. Histological analysis showed elimination of residual stroma in most of the cases. Conclusions The submerged hydro-separation method reduces tissue wastage. It allows preshipment evaluation, thus allowing a validated tissue to be transported from the eye banks to the surgeon. Because of the liquid interface, the peeling of the DMEK graft becomes easy for transplantation. © 2014 by Elsevier Inc. All rights reserved.

PubMed | International Center for Ocular Physiopathology and University of Liverpool
Type: Journal Article | Journal: Stem cells translational medicine | Year: 2016

The cornea forms the front window of the eye, enabling the transmission of light to the retina through a crystalline lens. Many disorders of the cornea lead to partial or total blindness, and therefore corneal transplantation becomes mandatory. Recently, selective corneal layer (as opposed to full thickness) transplantation has become popular because this leads to earlier rehabilitation and visual outcomes. Corneal endothelial disorders are a common cause of corneal disease and transplantation. Corneal endothelial transplantation is successful but limited worldwide because of lower donor corneal supply. Alternatives to corneal tissue for endothelial transplantation therefore require immediate attention. The field of human corneal endothelial culture for transplantation is rapidly emerging as a possible viable option. This manuscript provides an update regarding these developments. Significance: The cornea is the front clear window of the eye. It needs to be kept transparent for normal vision. It is formed of various layers of which the posterior layer (the endothelium) is responsible for the transparency of the cornea because it allows the transport of ions and solutes to and from the other layers of the cornea. Corneal blindness that results from the corneal endothelial dysfunction can be treated using healthy donor tissues. There is a huge demand for human donor corneas but limited supply, and therefore there is a need to identify alternatives that would reduce this demand. Research is underway to understand the isolation techniques for corneal endothelial cells, culturing these cells in the laboratory, and finding possible options to transplant these cells in the patients. This review article is an update on the recent developments in this field.

Salvalaio G.,International Center for Ocular Physiopathology | Parekh M.,International Center for Ocular Physiopathology | Ruzza A.,International Center for Ocular Physiopathology | Ferrari S.,International Center for Ocular Physiopathology | And 2 more authors.
British Journal of Ophthalmology | Year: 2014

Purpose: To describe a novel submerged hydro-separation (SubHyS) technique followed by anterior corneal dissection to prepare a Descemet endothelial graft (DEG) for Descemet's membrane endothelial keratoplasty from human donor corneas. Methods: 30 human donor corneas were immersed in liquid (organ culture (OC) storage medium). Using a 25-gauge needle, approximately 0.3 mL of OC was injected (SubHyS) in the posterior stroma to create a liquid bubble. The bubbled cornea was mounted onto a modified artificial chamber with the epithelial side facing the air. The endothelium was protected with a viscoelastic solution. The anterior cornea was excised with a Barron radial vacuum trephine and the residual peripheral stroma was removed manually using micro-scissors. The DEG was dismounted and washed. The endothelial cell density (ECD) and mortality of the prepared DEG was determined. All the DEGs were preserved in deturgescent medium for 7 days using a cornea claw which was fixed to the sclera. ECD and mortality were checked post preservation. Results: Complete detachment of Descemet's membrane and stroma was achieved in all 30 cases. Bubble burst was observed in five cases (excluded from the study) due to overfilling of the liquid. The average diameter of the excised DEG was 10.96 mm. The average endothelial cell loss post preservation was 27.69%. Histological analysis con firmed elimination of the residual stroma (n=13). Conclusions: The DEGs can be preserved in a deturgescent medium for up to 7 days. The procedure provides a standardised, pre-validated (quality assured), pre-separated, no-touch, ready-to-use tissue and also reduces the preparation time. Further, the tissues can be trephined as per the surgeon's convenience and can either be rolled or a contact lens could be used for final delivery of the DEG using a surgical glide.

Parekh M.,International Center for Ocular Physiopathology | Ferrari S.,International Center for Ocular Physiopathology | Salvalaio G.,International Center for Ocular Physiopathology | Ponzin D.,International Center for Ocular Physiopathology
European Journal of Ophthalmology | Year: 2014

Purpose: To study the effect of a synthetic medium and compare it with a serum-based medium for corneal preservation in organ culture using an overall quality assessment system. Methods: A randomized study with blinded observers was performed comparing parameters such as thickness, transparency, viable endothelial cell density (VECD), morphology, and overall quality (OQ) of the corneal tissues preserved in synthetic and a serum-based medium, respectively. Seven human paired corneas were randomly selected and assessed at day 0 (initial), day 2 (before organ culture), day 30 (before deturgescence/ deswelling storage), and 48 hours post deswelling. Thickness was determined with optical coherence tomography and transparency with a validated, custom device. The morphology and VECD were observed after treating the tissues with trypan blue and sucrose. Data were compared using paired t tests with p<0.05 deemed significant. Results: Parameters were similar at the initial stage between the groups with no statistically significant difference. However, after preservation in the deturgescent medium, the corneas stored in a serum-based medium showed a higher and statistically significant OQ value (p = 0.0317). Conclusions: The OQ of a serum-based medium was higher than that of the synthetic medium. A higher rate of transparency and reduction in thickness was observed in the serum-based medium at the end of the storage. Although complete synthetic media may have distinct advantages of being serum/animal-free, the quality of the cornea is of a reasonable concern when it is deemed for transplantation. © 2014 Wichtig Publishing.

PubMed | International Center for Ocular Physiopathology and Polytechnic of Bari
Type: | Journal: European journal of ophthalmology | Year: 2016

To investigate the adhesive and stiffness properties of prestripped Descemet membrane endothelial keratoplasty (DMEK) lenticules in different preservation conditions (with and without dextran).The study included 3 conditions: (C1) tissues collected from tissue culture media (TCM), stripped and preserved in TCM; (C2) tissues collected from transport media (TM) (TCM supplemented with 6% dextran T-500), stripped and preserved in TM; and (C3) tissues collected from TCM, stripped and preserved in TM. Using a hinge, 9.5-mm stripped DMEK lenticules were restored back on the stroma and preserved for 4 days at room temperature (RT) in different conditions as above. Nine tissues, 3 from each condition, were used to check the adhesive (fibronectin, laminin, and vitronectin) and elastic properties (fibrillin, elastin, and collagen VI) using different antibodies. Six tissues, 2 from each condition, were used to check the stiffness properties after preservation using atomic force microscopy (AFM) nanoindentation method.On the Descemet membrane, fibronectin was strongly expressed in C2 and C3, whereas laminin was intense in C2 postpreservation. Vitronectin was expressed in all the conditions. Elastic proteins were not expressed in either of the conditions apart from collagen VI, which was expressed on the posterior stroma. Atomic force microscopy showed higher stiffness in C3 and an insignificant but lower rigidity in C2 as compared to C1.The tissues from C2 showed expression of adherent proteins and lower stiffness. Dextran may be suitable in preservation of DMEK grafts before and after preparation. Less stiff tissues may help reduce manipulations required in the recipient eye during DMEK surgery.

PubMed | University Institute of Health Sciences and International Center for Ocular Physiopathology
Type: | Journal: Acta ophthalmologica | Year: 2016

To evaluate the difference between endothelium-in and endothelium-out for Descemet membrane endothelial keratoplasty (DMEK) preparation and implantation using injection method.The study was a randomized, comparative, institutional, laboratory investigation. Eighteen human donor corneas were included. A total of 9.5mm DMEK grafts were stripped and restored on the corneal base with a hinge and preserved in the deswelling medium. The grafts were detached from the hinge and either manually tri-folded with the endothelium on the inside, or allowed to spontaneously roll in phosphate-buffered saline (PBS) with endothelium outwards. The corneas were mounted on an artificial anterior chamber (AAC) and four incisions made using a 15 eye knife for the side ports and slit knife to create a 3.0mm incision for delivery of the graft into the AAC. The grafts with endothelium-in (endo-in) were pulled into a cartridge, whereas those with endothelium-out (endo-out) were aspirated using a modified Jones tube. Both were delivered using an injection method. Central and peripheral endothelial cell density (pre- and post-delivery and after removal), time of preparation and unfolding were measured.Endothelial cell loss postimplantation was 10.53%(2.82) with endo-in (n=9) compared to 7.56%(14.74) with endo-out (n=9) (p>0.05). Preparation and unfolding time was 4.43min (3.43) and 0.96min (1.10)for endo-in compared to 1.68min (0.57) and 4.92min (4.21)for endo-out. A statistical significance between endo-in and endo-out for loading (p=0.04) and unfolding (p=0.023) time was observed.Descemet membrane endothelial keratoplasty tissues can be tri-folded (endo-in) with no significantly less cell loss as compared to endo-out. Spontaneous unfolding of endo-in may reduce overall time and surgical manipulation.

PubMed | International Center for Ocular Physiopathology
Type: Journal Article | Journal: The British journal of ophthalmology | Year: 2016

Endothelial Keratoplasty (EK) is a corneal surgical procedure that allows a selective transplantation of the posterior layer of the cornea. Descemets Membrane Endothelial Keratoplasty (DMEK) is one of the EK procedures in which the diseased Descemets Membrane and the endothelium are replaced with a healthy donor tissue. To achieve this, the donor cornea is cut superficially from the endothelial side and the tissue can be separated using specific instruments like Pierse Notched, Acute or Fogla forceps. However, the pressure required to punch the superficial layer has always been a challenge and therefore a calibrated device to punch and excise the required superficial layer has been designed. This new model of punch will help to identify the peripheral edge of the DMEK lenticule which in turn helps to excise the tissue exclusively, further reducing the donor tissue wastage, as seen with the current tissue preparation methods.

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