Juan E Gallo

Preparation of platelet-rich plasma as a tissue adhesive for experimental transplantation in rabbits

PurposePlatelet-rich plasma (PRP) is an autologous substance with adhesive properties. We aimed at developing and testing the efficacy of a method for PRP preparation in rabbits.Materials and methodsAn in vitro study was carried out to obtain PRP from forty rabbits and to analyze the number of platelets and type of substance needed to trigger platelet activation. To induce platelet activation, 5%, 10%, 25% and 50% CaCl solutions were used. Then, an in vivo study was performed in twelve rabbits to test PRP adhesiveness in lamellar corneal graft. A control group made up of six rabbits underwent corneal transplantation without using PRP.Results5% CaCl was the most effective concentration in activating PRP, with a mean time of 19 minutes. An attached corneal flap was seen 3 months after surgery. A detached corneal button was seen in all controls.ConclusionOur method was able to produce rabbit-derived PRP with suitable properties for soft tissue adhesion. These results could be useful for researchers of the growing fields of tissue repair and experimental transplantation.

Y1 receptor of neuropeptide Y as a glial marker in proliferative vitreoretinopathy and diseased human retina

The Y1 receptor of neuropeptide Y (NPY) has been demonstrated in glial cells of astrocytic lineage in vitro. We have studied the immunohistochemical expression of Y1 receptors in the glia of the diseased human retina, in tissue samples obtained after surgery for proliferative vitreoretinopathy. In this condition, glia and other cell types migrate and form epi‐ or subretinal membranes. Both diseased retinas (n = 8) and PVR membranes (n = 43) contained numerous Y1‐immunoreactive cells. In the diseased retina, the Y1 antiserum labeled cells with the morphological radial pattern characteristic of Müller cells, whereas in the membranes, label appeared in a large population of elongate cells, measuring up to 250 μm. In both retina and membranes, double labeling demonstrated that the vast majority of Y1‐immunoreactive cells were also labeled by a glial fibrillary acidic protein (GFAP) antibody, indicating their glial origin. Retinal regions devoid of GFAP immunoreactivity also lacked the Y1 label. None of these markers was detected in Müller cells of normal retina. Y1 immunoreactivity did not co‐localize with smooth muscle actin immunoreactivity, a marker of myofibroblasts. Expression of Y1 receptors would characterize reactive and proliferating glial cells of the diseased retina and could perhaps be involved in the proliferation of injured glial cells causing regrowth of PVR membranes and the consequent secondary retinal detachments. GLIA 39:320–324, 2002.

Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns.

PURPOSE: To evaluate the efficacy of autologous corneal epithelial sheet implantation in restoring transparency of rabbit corneas severely injured by alkaline and the effect of photocoagulation in arresting corneal neovessel ingrowth. SETTING: Ophthalmology Department, School of Biomedical Sciences, Universidad Austral, Buenos Aires, Argentina. METHODS: Limbal stem‐cell deficiency (LSCD) was induced in 14 rabbits by alkali burns. A limbal cell biopsy was done in the contralateral eye, and the cells were cultured on a fibroblast feeder layer grown on autologous clotted platelet‐poor plasma or commercial fibrin for 21 days. Anterior keratectomy was followed by suturing corneal cell sheets over the stroma. If regrowth of vessels occurred, argon laser photocoagulation was applied to them. Rabbits were killed at 30, 60, 90, 180, and 360 days and the corneas processed for histopathology and inmunohistochemistry. RESULTS: A small (2.5 mm2) limbal biopsy achieved stem‐cell replication in vitro. Corneal clarity and epithelial defects evolved with a trend toward improvement. There was a significant reduction in corneal neovascularization. Histology showed a multilayered stratified epithelium including several epithelial‐like cells with clear cytoplasm in the deepest part. There were no signs of intraepithelial mucin cells on the implanted corneas. Immunohistochemical results showed expression of cytokeratins 3 and 12 in the central corneal epithelium and an absence of cytokeratin 19. CONCLUSIONS: Autologous limbal epithelial cell transplantation improved the corneal surface in eyes with LSCD. Photocoagulation of neovessel ingrowth was effective over the 1‐year follow‐up. Results may facilitate the application of this technique in patients.

Comparative study of scientific publications in Ophthalmology and Visual Sciences in Argentina, Brazil, Chile, Paraguay and Uruguay (1995-2004)

PURPOSE The study aimed to measure the scientific production in Ophthalmology and Vision (O&V) in Argentina, Brazil, Chile, Paraguay and Uruguay over a period of 10 years (1995-2004), in order to find out temporal evolution and variations in this field of research. METHODS PubMed / Medline was used to retrieve records on O&V research literature. The search strategy included keywords, country in the affiliation field and publication date. Data were extracted from each citation and recorded in a spreadsheet. Subsequent analysis focused on type and main topic of publication, journals where articles had been published, and evolution of research done on animals and humans. RESULTS A total of 1,216 citations were retrieved. Brazil had the largest number of authored publications with an average annual production of 82.4, followed by Argentina with 31.0, Chile 6.4, Uruguay 1.6, and Paraguay 0.2. The ratio of articles on O&V relative to publications involving Health Science ranged from 1.0 to 2.3. The frequency of publications almost tripled from 1995 to 2004. Research on humans showed a significant increase in Argentina and Brazil. CONCLUSIONS Results provide initial benchmarks on O&V publication rates in countries in South America that may be useful to follow research trends.

Retinal upregulation of inflammatory and proangiogenic markers in a model of neonatal diabetic rats fed on a high-fat-diet

BackgroundThe contemporary peak of diabetes seems to be related to obesity, sedentary lifestyle and diet. Diabetic retinopathy is the most leading cause of blindness in adulthood in industrialized countries. Our purpose was to evaluate the effect of a high-fat-diet (HFD) on the retina of diabetic rats.MethodsTwo groups of Wistar rats were injected with streptozotocin (STZ) two days after birth using 45 and 90 mg/kg, respectively. At 8 weeks the group on lower doses started to be fed on a HFD. Animals were sacrificed at 37 weeks of diabetes. A control group was made up of non-diabetic rats. Retinal flat mounts were examined using the trypsin digestion technique. Pericytes counts were compared between diabetic and control rats. Cross retinal sections were analyzed by histological techniques and immunohistochemistry and immunofluorescent technique. Primary antibodies against inflammatory and proangiogenic mediators such as RAGE, GFAP, 5-LO, VEGF and TNF-α were used for immunohistochemistry and Western Blot (WB) analyses.ResultsIn the two diabetic groups we observed GFAP-positive cells with a morphology and spatial organization similar to those seen in Müller cells. Both diabetic groups had a significantly lower number of pericytes than non-diabetic animals.Increased retinal immunoreactivity of GFAP, RAGE, TNF-α, VEGF and 5-LO was seen in diabetic animals fed on HFD compared to the other groups of animals. WB analysis revealed a higher expression of 5-LO, VEGF, TNF-α and RAGE in the retina of diabetic rats on HFD than in controls and diabetics fed on a normal diet. The percentage of RAGE-stained ganglion cells and ganglion cells was found to be significantly lower in animals on a HFD than in the other animals.ConclusionsDiabetic animals fed on a HFD showed an increased upregulation of inflammatory and proangiogenic markers. This animal model may be useful to study mechanisms of diabetic retinopathy and therapeutic targets.

Involvement of purinergic P2 receptors in experimental retinal neovascularization.

Purpose: The present study was aimed to investigate the expression of purinergic P2 receptors in oxygen-induced retinal neovascularization. Methods: Immunohistochemistry was used to study the expression of purinergic P2Y2 and P2X2 receptors in the neonatal mouse retina during normal vascular development and after oxygen-induced retinopathy (OIR). The effect of the P2 antagonists, suramin and PPADS, on the extent of oxygen-induced retinal neovascularization was analyzed. Results: In normal mice, the expression of P2Y2 receptors was weak throughout the retina, whereas P2X2 receptor expression was detected in the outer plexiform layer. In mice treated with oxygen, P2Y2 expression was detected in the ganglion and in the nerve fiber layers, whereas P2X2 expression was found in the inner and outer plexiform layers. Oxygen-induced preretinal neovascularization was strongly inhibited by the P2 antagonists, suramin (p < 0.05) and PPADS (p < 0.05), and this was accompanied by a down-regulation of P2X2 receptor expression in the inner plexiform layer in suramin-treated mice. Conclusions: The data suggest that purinergic P2 receptors are involved in neovascularization associated with OIR.

In vivo lamellar keratoplasty using platelet-rich plasma as a bioadhesive

PurposeThe use of standard sutures has been replaced by platelet-rich plasma (PRP), a bioadhesive agent, in several surgical procedures. This prompted us to test PRP efficacy in experimental lamellar keratoplasty.MethodsAfter lamellar anterior keratoplasty, PRP with a mean concentration of 807 564 platelets/mm3 was used to attach the corneal flap to the stromal surface in 12 New Zealand white rabbits. 10-0 nylon sutures were used in one control group of 12 animals and no suture was used in a second control group of six rabbits. Animals were killed at days 2, 7, 30, and 90 for histological and smooth muscle actin (SMA) immunohistochemical analysis.ResultsThe PRP group showed a tight corneal graft from the first postoperative hours until they were killed. A transparent cornea was seen at 30 days and remained clear until the end of the protocol. Histological specimens showed no signs of ocular inflammation in any animal within the PRP group. Electron microscopy showed normal morphological features on the flap and stromal bed, and a clear interface zone without cells or debris. The number of stromal myofibroblasts was lower than that seen in the suture group at 3 months postsurgery. The sutured group showed an attached cornea with signs of inflammation around the knots. All flaps without PRP or sutures were completely detached in the immediate postoperative period.ConclusionsPRP was useful for attaching the corneal flap and it was well tolerated by the rabbit corneal tissue. Corneal healing was satisfactory. Further studies on PRP adhesiveness in grafts with donor corneas should be performed before considering its use in patients.

Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option?

Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.

Corneal Neovascularization: A Combined Approach of Bevacizumab and Suramin Showed Increased Antiangiogenic Effect Through Downregulation of BFGF and P2X2

ABSTRACT Purpose: The objective is to analyze the antiangiogenic mechanism of suramab, a pharmaceutical compound of bevacizumab and suramin, in a rabbit model of corneal angiogenesis. Material and Methods: Corneal neovascularization was induced in four groups of six New Zealand White rabbits by applying a filter paper disk soaked in 1 M Na (OH) on the central cornea. Group one was treated after injury with intravenous suramab at a dose equivalent to 3 mg/kg of bevacizumab and 10 mg/kg of suramin. Group two was treated with intravenous bevacizumab (5 mg/kg). Group three was treated with 10 mg/kg of suramin while the control group received no treatment. Digital photographs were taken at days 9, 15, 21, and 35. Neovessel formation was quantified giving a 0–4 score to each quadrant according to the centripetal growth of the longest vessel (neovessel index, NVI). Animals were sacrificed at day 35. Corneas were processed for histology, immunohistochemistry, and Western-blot using primary antibodies against P2X2, basic fibroblast growth factor (bFGF), LYVE-1, PECAM-1, and vascular endothelial growth factor-A (VEGF-A). Results: Suramab significantly reduced neovessel growth (mean NVI: 4.2) compared to bevacizumab (8.4), suramin (7.22), and control animals (12.2) at 35 days post-injury (p < 0.01). A lower protein expression of P2X2, bFGF, LYVE-1, PECAM-1, and VEGF-A was found in the cornea of suramab animals than in the other groups of animals. Conclusions: Joint downregulation of bFGF, P2X2, bFGF, and LYVE-1 constitutes a mechanism that induces greater and longer inhibition of corneal angiogenesis. Results might be relevant to ophthalmic care. Ocular administration of suramab is currently being investigated.

Transglutaminase binding fusion protein linked to SLPI reduced corneal inflammation and neovascularization.

BackgroundTo study the effect of topical administration of a fusion protein (PF-MC) made up of N-terminal portion of the protease inhibitor Trappin-2 (which is a substrate of transglutaminasa-2) and SLPI (protein with anti-inflammatory, anti-bacterial and anti-viral ability), in an animal model of corneal inflammation and angiogenesis.MethodsAn alkali injury was produced with a filter paper of 3 mm with 1 N NaOH during 40 seconds on the right cornea of 36 male Sprague Dawley rats, under general anesthesia. Animals were divided into three groups according to treatment. Group 1 was treated with 10 ul of PF-MC (200 ug/ml; n = 12), Group 2, with 10 ul of SLPI (200 ug/ml; n = 12) and Group 3 was treated with buffer (10 ul; n = 12) topically administered four times a day for up to 7 days. Half of the animals were sacrificed at day 3 before making a re-epithelialization time analysis with fluorescein staining at 18 and 24 hours. In the remaining animals corneal opacity was studied and digital photographs were taken at day 7 before doing euthanasia. Eyes were processed for histology and immunofluorescence.ResultsCorneal ulcerated area was significantly lower in PF-MC treated animals compared to SLPI and buffer-treated animals at 18 hours and 24 hours postinjury. A clear cornea and fundus red reflex was only found among PF-MC treated animals. Histological analysis revealed a stratified corneal epithelium with at least three layers in all PF-MC animals at day 7. In this group there was a reduced number of PMNs in the corneal stroma at 3 and 7 days of follow-up. Besides, corneal neovascularization was much more extended in SLPI and Buffer animals than in animals treated with PF-MC.ConclusionsThe binding of SLPI with Cementoin to transglutaminase seems to be an effective strategy to treat corneal inflammation and angiogenesis.