Clinical characteristics and prognostic factors of open globe injuries in a North Spain population: a 10-year review.

OBJECTIVES: To describe the epidemiologic and clinical characteristics of open globe injuries (OGIs) treated in a tertiary hospital and analyse predictors of visual outcome. METHODS: This retrospective observational study included all patients with OGIs admitted to Cruces University Hospital between 2010 and 2020. The descriptive analysis included demographic data, type of injury classified as "rupture", "penetration", "perforation", or "intraocular foreign body", trauma mechanism and setting, injury zone, Ocular Trauma Score, delay to surgery, length of hospital stay, antibiotic prophylaxis, initial and final best corrected visual acuity (BCVA), complications and further surgery. Univariate analysis and logistic regression were performed to identify prognostic factors, based on final BCVA. RESULTS: Overall, 207 OGI cases were reported. The most common type of injuries were ruptures caused by domestic falls. Notably, 44.4% of eyes developed phthisis bulbi. In the univariate analysis, the following variables were significantly linked to visual outcome: age > 60 years, "rupture", "fall", posterior and/or combined zones of injury, lens damage, retinal/choroidal detachment, initial BCVA of no light perception, and Ocular Trauma Score ≤ 2 (p < 0.001). Delay to surgery, length of stay and further surgery did not have prognostic value. In the logistic regression, initial BCVA of no light perception (p < 0.001) and injury zone III (p = 0.005) remained significant predictors of poor outcome. CONCLUSIONS: In the population studied, most OGIs were caused by domestic falls usually affecting elderly patients with comorbidities. Visual outcome depended on patients´ specific characteristics and the nature of the trauma itself, whereas environmental factors failed to show any prognostic value.

Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin ß4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

Cytocompatibility and Suitability of Protein-Based Biomaterials as Potential Candidates for Corneal Tissue Engineering.

The vision impairments suffered by millions of people worldwide and the shortage of corneal donors show the need of substitutes that mimic native tissue to promote cell growth and subsequent tissue regeneration. The current study focused on the in vitro assessment of protein-based biomaterials that could be a potential source for corneal scaffolds. Collagen, soy protein isolate (SPI), and gelatin films cross-linked with lactose or citric acid were prepared and physicochemical, transmittance, and degradation measurements were carried out. In vitro cytotoxicity, cell adhesion, and migration studies were performed with human corneal epithelial (HCE) cells and 3T3 fibroblasts for the films' cytocompatibility assessment. Transmittance values met the cornea's needs, and the degradation profile revealed a progressive biomaterials' decomposition in enzymatic and hydrolytic assays. Cell viability at 72 h was above 70% when exposed to SPI and gelatin films. Live/dead assays and scanning electron microscopy (SEM) analysis demonstrated the adhesion of both cell types to the films, with a similar arrangement to that observed in controls. Besides, both cell lines were able to proliferate and migrate over the films. Without ruling out any material, the appropriate optical and biological properties shown by lactose-crosslinked gelatin film highlight its potential for corneal bioengineering.

Multicentre study: reliability and repeatability of Scheimpflug system measurement in keratoconus.

PURPOSE: To assess the repeatability and reliability of the most important tomographic parameters for characterising keratoconus measured with a Pentacam HR (high resolution). METHODS: Overall, 230 eyes in 158 patients with keratoconus were analysed. We performed five consecutive corneal tomography examinations for each eye with a Pentacam HR in patients with keratoconus. Study eyes were classified into three groups depending on the maximum posterior elevation (max_BFS_post): grade 1 for cases of keratoconus with a max_BFS_post of 40 µm; grade 2 for those with a max_BFS_post of between 41 and 75 µm and grade 3 for those with a max_BFS_post of over 75 µm. We calculated the intraclass correlation coefficients (ICCs) and repeatability limits of parameters from tomography and aberrometry. RESULTS: All the parameters were found to have excellent ICCs (0.9). The repeatability limits for the key parameters were higher than 0.5D for the power parameters, 20° for the axis of corneal astigmatism and 10 µm for the thinnest corneal thickness. Further, we obtained repeatability limits of above 0.1 µm for the aberrometry values and overall greater than 15° for the coma axis. All the values increase with the severity of keratoconus, except for that of the coma axis which falls with keratoconus grade. CONCLUSIONS: The reliability indicated by ICCs supports the view that the Pentacam HR is useful for the diagnosis of keratoconus. The repeatability limits suggest that new criteria should be established for monitoring progression taking into account the real measurements that can be made using this system.

Hyaluronic Acid Combined with Serum Rich in Growth Factors in Corneal Epithelial Defects.

The aim of this study is to assess if an adhesive biopolymer, sodium hyaluronate (NaHA), has synergistic effects with s-PRGF (a serum derived from plasma rich in growth factors and a blood derivative that has already shown efficacy in corneal epithelial wound healing), to reduce time of healing or posology. In vitro proliferation and migration studies, both in human corneal epithelial (HCE) cells and in rabbit primary corneal epithelial (RPCE) cultures, were carried out. In addition, we performed studies of corneal wound healing in vivo in rabbits treated with s-PRGF, NaHA, or the combination of both. We performed immunohistochemistry techniques (CK3, CK15, Ki67, ß4 integrin, ZO-1, α-SMA) in rabbit corneas 7 and 30 days after a surgically induced epithelial defect. In vitro results show that the combination of NaHA and s-PRGF offers the worst proliferation rates in both HCE and RPCE cells. Addition of NaHA to s-PRGF diminishes the re-epithelializing capability of s-PRGF. In vivo, all treatments, given twice a day, showed equivalent efficacy in corneal epithelial healing. We conclude that the combined use of s-PRGF and HaNA as an adhesive biopolymer does not improve the efficacy of s-PRGF alone in the wound healing of corneal epithelial defects.

Serum from plasma rich in growth factors regenerates rabbit corneas by promoting cell proliferation, migration, differentiation, adhesion and limbal stemness.

PURPOSE: To evaluate the regenerating potential and the mechanisms through which the autologous serum derived from plasma rich in growth factors (s-PRGF) favours corneal wound healing in vitro and in vivo. METHODS: We compared the effect of various concentrations of s-PRGF versus fetal bovine serum (FBS) and control treatment in rabbit primary corneal epithelial and stromal cells and wounded rabbit corneas. Cell proliferation was measured using an enzymatic colorimetric assay. In vitro and in vivo wound-healing progression was assessed by image-analysis software. Migration and invasion were evaluated using transfilter assays. Histological structure was analysed in stained sections. Protein expression was evaluated by immunohistochemistry. RESULTS: s-PRGF promoted the robust proliferation of epithelial cultures at any concentration, similar to FBS. Likewise, s-PRGF and FBS produced similar re-epithelialization rates in in vitro wound-healing assays. In vivo, s-PRGF treatment accelerated corneal wound healing in comparison with control treatment. This difference was significant only for 100% s-PRGF treatment in our healthy rabbit model. Histological analysis confirmed normal epithelialization in all cases. Immunohistochemistry showed a higher expression of cytokeratins 3/76 and 15, zonula occludens-1 and alpha-smooth muscle actin proteins as a function of s-PRGF concentration. Notably, keratocyte density in the anterior third of the stroma increased with increase in s-PRGF concentration, suggesting an in vivo chemotactic effect of s-PRGF on keratocytes that was further confirmed in vitro. CONCLUSION: s-PRGF promotes proliferation and migration and influences limbal stemness, adhesion and fibrosis during corneal healing.

Corneal wound healing promoted by 3 blood derivatives: an in vitro and in vivo comparative study.

PURPOSE: The aim of this study was to compare the effect on corneal wound healing of 3 differently manufactured blood derivatives [autologous serum (AS), platelet-rich plasma, and serum derived from plasma rich in growth factors (s-PRGF)]. METHODS: Scratch wound-healing assays were performed on rabbit primary corneal epithelial cultures and human corneal epithelial cells. Additionally, mechanical debridement of rabbit corneal epithelium was performed. Wound-healing progression was assessed by measuring the denuded areas remaining over time after treatment with each of the 3 blood derivatives or a control treatment. RESULTS: In vitro data show statistically significant differences in the healing process with all the derivatives compared with the control, but 2 of them (AS and s-PRGF) induced markedly faster wound healing. In contrast, although the mean time required to complete in vivo reepithelization was similar to that of AS and s-PRGF treatment, only wounds treated with s-PRGF were significantly smaller in size from 2.5 days onward with respect to the control treatment. CONCLUSIONS: All 3 blood derivatives studied are promoters of corneal reepithelization. However, the corneal wound-healing progresses differently with each derivative, being faster in vitro under AS and s-PRGF treatment and producing in vivo the greatest decrease in wound size under s-PRGF treatment. These findings highlight that the manufacturing process of the blood derivatives may modulate the efficacy of the final product.

Novel molecular diagnostic system of limbal stem cell deficiency based on MUC5AC transcript detection in corneal epithelium by PCR-reverse dot blot.

PURPOSE: To evaluate the sensitivity and specificity of a PCR-strip system based on reverse dot blot for detection of MUC5AC mRNA in corneal epithelium samples from patients with limbal stem cell deficiency (LSCD), and to determine the correlation with clinical diagnosis. METHODS: We obtained 87 corneal impression cytology (IC) samples from 55 subjects (37 patients clinically diagnosed with LSCD and 18 control subjects). Total RNA was extracted from each IC sample and retrotranscribed to cDNA. MUC5AC mRNA transcript was amplified by a customized RT-PCR assay and detected in PCR strips based on reverse dot blot hybridization and in agarose gels. Conjunctival IC samples were used as positive controls. RESULTS: Forty-four of 45 corneal IC samples obtained from patients clinically diagnosed with LSCD were positive for MUC5AC, whereas 34 of 42 corneal ICs from healthy subjects were negative for MUC5AC. Four healthy corneas were found MUC5AC positive, and four rendered inconclusive results. A correlation of 91.4% (P < 0.001) between molecular testing and clinical diagnosis was found. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR-strip system were 98%, 89%, 92%, and 97%, respectively. CONCLUSIONS: Corneal epithelium MUC5AC transcript detection by reverse dot blot PCR-strip is a highly sensitive technique for LSCD diagnosis. The test results strongly correlate with clinical diagnosis of characterized LSCD cases. The PCR-strip system may be used for early detection, and for the detection of mild cases of LSCD, and constitutes an objective clinical tool for the monitoring of treatments and surgical decisions.

In vitro effects of three blood derivatives on human corneal epithelial cells.

PURPOSE: We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. METHODS: The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. RESULTS: We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. CONCLUSIONS: PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

Comparative study of limbal stem cell deficiency diagnosis methods: detection of MUC5AC mRNA and goblet cells in corneal epithelium.

PURPOSE: To evaluate a limbal stem cell deficiency (LSCD) diagnosis method based on the detection of the MUC5AC transcript by reverse transcription-polymerase chain reaction (RT-PCR) in comparison with the standard diagnostic method based on goblet cell detection by periodic acid-Schiff (PAS)-hematoxylin staining, using samples obtained from corneal epithelium impression cytology (IC). DESIGN: Transversal, comparative case series. PARTICIPANTS: We studied 59 eyes from 43 patients clinically diagnosed with LSCD. METHODS: Impression cytology was used to gather cells from corneal and conjunctival epithelium from the same eye. The presence of goblet cells in the cornea was determined by PAS-hematoxylin staining, whereas the presence of the MUC5AC transcript was detected by RT-PCR using a custom-designed primer pair. MAIN OUTCOME MEASURES: Goblet cells in the corneal epithelium were detected by light microscopy, and the MUC5AC transcript was detected as the corresponding PCR amplicon in agarose gels. RESULTS: Our study included 59 corneal samples, together with their respective conjunctival samples for RT-PCR assays. Of these, 47 samples were also available for comparative PAS-hematoxylin staining. The MUC5AC amplicon was detected in 56 of 59 (94.9%) corneal epithelium samples. In contrast, conventional IC staining detected goblet cells in only 17 of 47 (36.2%) samples; these were not found in 27 of 47 (57.4%) samples (negative results), and 3 of 47 (6.4%) showed inconclusive results. CONCLUSIONS: The detection of the MUC5AC transcript in corneal epithelium is a more sensitive method to diagnose LSCD than the conventional PAS-hematoxylin method, although a minimum RNA concentration of 1.2 ng/µl is required for negative results to be reliable. Moreover, RT-PCR is a highly specific and more objective technique. Overall, these findings indicate that molecular analysis facilitates a more precise clinical diagnosis of LSCD, thereby reducing the risk of surgical failure.

Plasma rich in growth factors as a therapeutic agent for persistent corneal epithelial defects.

OBJECTIVE: To evaluate the efficacy of topically applied autologous plasma rich in growth factors (PRGF) as a treatment for persistent epithelial defects (PEDs) of the cornea. METHODS: A series of prospective noncomparative cases. PARTICIPANTS: Twenty eyes from 18 patients with PED with various underlying etiopathologies: neurogenic, iatrogenic, associated with burning or secondary to severe dry eye. Patients were treated with a PRGF eyedrop solution. Serial photographs of the cornea were taken until epithelialization was complete. We had previously characterized the levels of a panel of growth factors (platelet-derived growth factor, epithelial growth factor, vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor, and nerve growth factor) in the PRGF of 11 of these patients. The following variables were additionally recorded: (1) duration of PED before treatment, (2) previous treatments, (3) time for complete epithelialization, and (4) treatments required concomitantly with PRGF. RESULTS: Epithelial defects healed in 17 of 20 cases (85%), with a mean therapeutic time of 10.9 weeks (range 2-39 weeks). Mean progression time before treatment was 26.7 weeks (range 2-104 weeks). Growth factor concentrations were platelet-derived growth factor 12645.9 +/- 1690.0 pg/mL, epithelial growth factor 468.9 +/- 97.6 pg/mL, vascular endothelial growth factor 204.5 +/- 119.4 pg/mL, hepatocyte growth factor 149.5 +/- 173.5 pg/mL, fibroblast growth factor 82.6 +/- 95.9 pg/mL, and nerve growth factor 37.7 +/- 18.6 pg/mL. CONCLUSION: PRGF, when applied as eyedrops, is a highly effective therapeutic agent for the treatment of a broad etiopathological spectrum of corneal PEDs.

Diagnóstico microbiológico de las infecciones oculares / Microbiological diagnosis of ocular infections

El reciente aumento del intervencionismo quirúrgico, junto con un incremento de usuarios de lentes de contacto, han producido un mayor número de infecciones oculares graves. El tratamiento inadecuado de la infección ocular puede llevar a la pérdida irreversible de la visión. El diagnóstico microbiológico es fundamental cuando las lesiones son inespecíficas, recurrentes o falla el tratamiento empírico, pero presenta la dificultad de que analiza muestras de pequeño volumen que contienen un bajo inóculo.En este documento se revisan las infecciones más importantes según su localización ocular, se describen las etiologías más frecuentes y las situaciones en las que se recomienda un diagnóstico microbiológico. Se incluye información sobre los tipos de muestras con la forma de obtención, transporte, tratamiento y procesamiento en el laboratorio, con especial atención a los medios de cultivo líquidos. Se detalla el rendimiento que se puede esperar del examen microscópico y del cultivo de cada tipo de muestra. Finalmente, se describen los métodos moleculares que recientemente se han introducido para el diagnóstico de infección fúngica ocular y por Acanthamoeba spp (AU)

The recent increase in the practice of ocular surgery and widespread use of contact lenses have led to an increase in the incidence of severe ocular infections. Inadequate management of these infections can result in irreversible loss of vision. Microbiological diagnosis is essential when the lesions are non-specific, recurrent, or unresponsive to antibiotic therapy, but is hampered by the difficulty of analyzing limited sample volumes containing small inocula. This document presents a review of the most important ocular infections according to the structure affected, and describes the most common causes of these infections and the situations in which a microbiological diagnosis is recommended. Information is included on the sample type and sampling methods, sample transport to the laboratory, and laboratory management and processing techniques, with special attention to liquid culture media. The yield of smear examination and culture of each type of ocular specimen is specified. Lastly, the molecular methods recently developed for the diagnosis of ocular fungal infections and Acanthamoeba infections are described (AU)

[Microbiological diagnosis of ocular infections]. / Diagnóstico microbiológico de las infecciones oculares.

The recent increase in the practice of ocular surgery and widespread use of contact lenses have led to an increase in the incidence of severe ocular infections. Inadequate management of these infections can result in irreversible loss of vision. Microbiological diagnosis is essential when the lesions are non-specific, recurrent, or unresponsive to antibiotic therapy, but is hampered by the difficulty of analyzing limited sample volumes containing small inocula. This document presents a review of the most important ocular infections according to the structure affected, and describes the most common causes of these infections and the situations in which a microbiological diagnosis is recommended. Information is included on the sample type and sampling methods, sample transport to the laboratory, and laboratory management and processing techniques, with special attention to liquid culture media. The yield of smear examination and culture of each type of ocular specimen is specified. Lastly, the molecular methods recently developed for the diagnosis of ocular fungal infections and Acanthamoeba infections are described.

Intravenous immunoglobulin therapy for refractory ocular cicatricial pemphigoid: case report.

PURPOSE: To report the favorable outcome of a patient with severe ocular cicatricial pemphigoid, which was refractory to conventional treatment but recovered after intravenous immunoglobulin therapy. METHODS: A case report. RESULTS: Conventional therapy failed to control the ocular symptoms of this patient, which are typical of the disease. However, the patient improved remarkably in response to pulsed intravenous human immunoglobulin therapy. CONCLUSIONS: We achieved stabilization of the disease around the seventh intravenous immunoglobulin cycle, thus contributing to satisfactory postoperative evolution after limbal allograft transplantation and facilitating the performance of intraocular surgical procedures, such as phacoemulsification, with improvement in best-corrected visual acuity.

Use of bortezomib in the management of chronic graft-versus-host disease among multiple myeloma patients relapsing after allogeneic transplantation.

We report on the use of bortezomib for the management of chronic graft versus host disease (cGVHD) among 8 multiple myeloma (MM) patients who relapsed after reduced-intensity conditioning (RIC) allogeneic transplantation. Five patients (62%) responded to bortezomib demonstrating anti-myeloma effect. Four patients had active cGVHD, including 3 patients with severe punctate keratopathy, at the time of bortezomib administration. All showed an improvement in their condition. This is the first report showing that bortezomib may be useful in the management of cGVHD and related ocular involvement.