RESUMO
Limbal stem cell deficiency (LSCD) is a clinically challenging eye disease caused by damage to limbal stem cells (LSCs). Currently, the international consensus classifies LSCD into three clinical stages based on the disease severity. However, no existing animal models attempt to replicate the varying degrees of LSCD observed in clinical cases. The present study demonstrates an easy-to-create, reproducible, and reliable mouse model of graded LSCD. To achieve mild, moderate, or severe LSCD, filter paper rings with a variety of central angles (90°, 180°, or 270°) are utilized to deliver alkali burns to different sizes of the limbal area (1, 2, or 3 quarters). The animal model has successfully resulted in the development of clinical signs and pathological manifestations in escalating severity that are similarly observed in the three clinical stages of LSCD. Our study thus provides new insights into distinct pathological features underlying different grades of LSCD and serves as a new tool for further exploring the disease mechanisms and developing new effective therapeutics for repairing damaged LSCs.
Assuntos
Queimaduras Químicas , Doenças da Córnea , Modelos Animais de Doenças , Queimaduras Oculares , Limbo da Córnea , Células-Tronco , Animais , Limbo da Córnea/patologia , Camundongos , Células-Tronco/patologia , Doenças da Córnea/patologia , Queimaduras Químicas/patologia , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/patologia , Camundongos Endogâmicos C57BL , Feminino , Deficiência Límbica de Células-TroncoRESUMO
Our objective was to determine the effect of a semi-synthetic sodium alginate hydrogel and its combination with platelet-rich plasma (PRP) on histopathological, biochemical, clinical, and anterior segment optical coherence tomography (AS-OCT) data. Alkali chemical burn of the cornea was induced. Injured rats were randomly divided into five equal groups and topically treated with phosphate-buffered saline (sham), platelet-rich plasma (PRP), 0.5% sodium citrate, a semi-synthetic sodium alginate hydrogel, or a combination of PRP and hydrogel (combined group) three times daily. The degree of corneal opacity (CO), corneal epithelial staining (CES), percentage of corneal epithelial defects (CEDP), degree of ciliary hyperemia (CH), neovascularization size (NVS), and extent of neovascularization (NVE) were evaluated. AS-OCT was performed at nine days, and then rats were sacrificed. Histological examination and enzyme-linked immunosorbent assays were performed to detect the concentrations of IL-1ß and MMP-9 in the cornea. There were no significant differences between the groups regarding CEDP, CO, CES, CH, NVS, or NVE on the first day after corneal alkali burn injury (p > 0,05). At the last examination, CO was significantly lower in the PRP group than in the sham group (p = 0,044), while the CO concentrations were similar in terms of NVS (p > 0,05). Similarly, in terms of tissue MMP-9 levels, there were no significant differences between groups (p > 0,05). However, there was a significant difference in tissue IL-1ß levels between the groups (p < 0,001). In the PRP and combined groups, the level of IL-1ß was significantly lower than that in the sham group (p = 0,043 and p = 0,036, respectively). There was a significant difference in epithelial necrosis between the PRP, and it was the lowest in the combined group (p = 0,003). Epithelial thickness was highest in the combined group (p = 0,002). CEDP was significantly different at the last visit between the groups (p = 0.042). The fastest epithelial closing rate was observed for the combined group (p = 0,026). There was a significant negative correlation between tissue MMP-9 levels and corneal solidity and between tissue MMP-9 levels and the corneal area according to the AS-OCT measurements (p = 0,012 and p = 0,027, respectively). When used alone, topical hydrogel application did not significantly enhance the healing of corneal wounds. However, when combined with PRP, it leads to an increased rate of epithelial closure and neovascularization. This combination did not exacerbate inflammation or corneal opacity compared to PRP alone. The anticoagulant citrate solution in the PRP tube did not prove effective. The synergistic use of PRP and hydrogel could enhance epithelial thickness and reduce epithelial necrosis. The use of new parameters for corneal wound healing assessment was facilitated through AS-OCT image processing.
Assuntos
Alginatos , Queimaduras Químicas , Modelos Animais de Doenças , Queimaduras Oculares , Hidrogéis , Plasma Rico em Plaquetas , Tomografia de Coerência Óptica , Cicatrização , Animais , Queimaduras Químicas/tratamento farmacológico , Queimaduras Oculares/induzido quimicamente , Ratos , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Ratos Sprague-Dawley , Ensaio de Imunoadsorção Enzimática , Interleucina-1beta/metabolismo , Lesões da Córnea/induzido quimicamente , Segmento Anterior do Olho/diagnóstico por imagemRESUMO
Corneal alkali burns often occur in industrial production and daily life, combined with infection, and may cause severe eye disease. Oxidative stress and neovascularization (NV) are important factors leading to a poor prognosis. URP20 is an antimicrobial peptide that has been proven to treat bacterial keratitis in rats through antibacterial and anti-NV effects. Therefore, in this study, the protective effect and influence mechanism of URP20 were explored in a rat model of alkali burn together with pathogenic bacteria (Staphylococcus aureus and Escherichia coli) infection. In addition, human umbilical vein endothelial cells (HUVECs) and human corneal epithelial cells (HCECs) were selected to verify the effects of URP20 on vascularization and oxidative stress. The results showed that URP20 treatment could protect corneal tissue, reduce corneal turbidity, and reduce the NV pathological score. Furthermore, URP20 significantly inhibited the expression of the vascularization marker proteins VEGFR2 and CD31. URP20 also reduced the migration ability of HUVECs. In terms of oxidative stress, URP20 significantly upregulated SOD and GSH contents in corneal tissue and HCECs (treated with 200 µM H2O2) and promoted the expression of the antioxidant protein Nrf2/HO-1. At the same time, MDA and ROS levels were also inhibited. In conclusion, URP20 could improve corneal injury combined with bacterial infection in rats caused by alkali burns through antibacterial, anti-NV, and antioxidant activities.
Assuntos
Infecções Bacterianas , Queimaduras Químicas , Lesões da Córnea , Neovascularização da Córnea , Queimaduras Oculares , Ratos , Humanos , Animais , Queimaduras Químicas/complicações , Queimaduras Químicas/tratamento farmacológico , Queimaduras Químicas/metabolismo , Neovascularização da Córnea/metabolismo , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Peróxido de Hidrogênio/farmacologia , Neovascularização Patológica/metabolismo , Lesões da Córnea/tratamento farmacológico , Células Endoteliais da Veia Umbilical Humana , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/tratamento farmacológico , Queimaduras Oculares/patologia , Modelos Animais de Doenças , Álcalis/toxicidadeRESUMO
Nitrogen mustard (NM) is a potent vesicating chemical warfare agent that is primarily absorbed through skin, inhalation, or ocular surface. Ocular exposure of NM can cause acute to chronic keratopathy which can eventually lead to blindness. There is a current lack of effective countermeasures against ocular exposure of NM despite their imperative need. Herein, we aim to explore the sustained effect of Dexamethasone sodium phosphate (DSP)-loaded polymeric nanoparticles (PLGA-DSP-NP) following a single subconjunctival injection in the management and prevention of corneal injury progression upon exposure to NM. DSP is an FDA approved corticosteroid with proven anti-inflammatory properties. We formulated PLGA-DSP-NP with zinc chelation ion bridging method using PLGA polymer, with particles of approximately 250 nm and a drug loading of 6.5 wt%. Under in vitro sink conditions, PLGA-DSP-NP exhibited a sustained drug release for two weeks. Notably, in NM injured cornea, a single subconjunctival (SCT) injection of PLGA-DSP-NP outperformed DSP eyedrops (0.1%), DSP solution, placebo NP, and saline, significantly mitigating corneal neovascularization, ulceration, and opacity for the two weeks study period. Through PLGA-DSP-NP injection, sustained DSP release hindered inflammatory cytokine recruitment, angiogenic factors, and endothelial cell proliferation in the cornea. This strategy presents a promising localized corticosteroid delivery system to effectively combat NM-induced corneal injury, offering insights into managing vesicant exposure.
Assuntos
Dexametasona , Mecloretamina , Nanopartículas , Dexametasona/análogos & derivados , Animais , Mecloretamina/toxicidade , Modelos Animais de Doenças , Lesões da Córnea/prevenção & controle , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/patologia , Lesões da Córnea/tratamento farmacológico , Glucocorticoides , Substâncias para a Guerra Química/toxicidade , Camundongos , Queimaduras Químicas/prevenção & controle , Queimaduras Químicas/tratamento farmacológico , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/prevenção & controle , Coelhos , Córnea/efeitos dos fármacos , Córnea/patologia , Córnea/metabolismoRESUMO
PURPOSE: Benzalkonium chloride (BAC) is commonly used as a preservative in ophthalmic medications, despite its potential to induce chemical injury. Extensive research has demonstrated that BAC can lead to adverse effects, including injuries to the ocular surface. Our study aimed to elucidate the underlying mechanism of necroptosis induced by BAC. METHODS: Human corneal epithelial (HCE) cells and mouse corneas were subjected to chemical injury, and the necrostatin-1 (Nec1) group was compared to the dimethylsulfoxide (DMSO) group. The extent of damage to HCE cells was assessed using CCK-8 and flow cytometry. Hematoxylin and eosin staining, as well as fluorescein sodium staining, were used to detect and characterize corneal injury. The activation of inflammatory cytokines and necroptosis-related proteins and genes was evaluated using Western blotting, immunofluorescence staining, and quantitative RTâPCR. RESULTS: In our study, the induction of necroptosis by a hypertonic solution was not observed. However, necroptosis was observed in HCE cells exposed to NaOH and BAC, which activated the receptor-interacting protein kinase 1 (RIPK1) - receptor-interacting protein kinase 3 (RIPK3) - mixed lineage kinase domain-like protein (MLKL) signaling pathway. In mouse corneal tissues, BAC could induce necroptosis and inflammation. The administration of Nec1 mitigated the inflammatory response and ocular surface damage caused by BAC-induced necroptosis in our experimental models. Furthermore, our in vivo experiments revealed that the severity of necroptosis was greater in the 3-day group than in the 7-day group. CONCLUSIONS: Necroptosis plays a role in the pathological development of ocular surface injury caused by exposure to BAC. Furthermore, our study demonstrated that the administration of Nec1 could mitigate the pathological effects of necroptosis induced by BAC in clinical settings.
Assuntos
Compostos de Benzalcônio , Epitélio Corneano , Imidazóis , Indóis , Necroptose , Proteínas Quinases , Proteína Serina-Treonina Quinases de Interação com Receptores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Necroptose/efeitos dos fármacos , Animais , Camundongos , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Epitélio Corneano/metabolismo , Indóis/farmacologia , Compostos de Benzalcônio/toxicidade , Compostos de Benzalcônio/farmacologia , Imidazóis/farmacologia , Proteínas Quinases/metabolismo , Humanos , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Western Blotting , Células Cultivadas , Citometria de Fluxo , Transdução de Sinais/efeitos dos fármacos , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/patologia , Masculino , Queimaduras Químicas/patologia , Queimaduras Químicas/metabolismo , Queimaduras Químicas/tratamento farmacológico , Conservantes Farmacêuticos/toxicidadeRESUMO
Severe corneal injury can lead to blindness even after prompt treatment. 14-3-3zeta, a member of an adaptor protein family, contributes to tissue repair by enhancing cellular viability and inhibiting fibrosis and inflammation in renal disease or arthritis. However, its role in corneal regeneration is less studied. In this study, filter disc of 2-mm diameter soaked in sodium hydroxide with a concentration of 0.5 N was placed at the center of the cornea for 30 s to establish a mouse model of corneal alkali injury. We found that 14-3-3zeta, which is mainly expressed in the epithelial layer, was upregulated following injury. Overexpression of 14-3-3zeta in ocular tissues via adeno-associated virus-mediated subconjunctival delivery promoted corneal wound healing, showing improved corneal structure and transparency. In vitro studies on human corneal epithelial cells showed that 14-3-3zeta was critical for cell proliferation and migration. mRNA-sequencing in conjunction with KEGG analysis and validation experiments revealed that 14-3-3zeta regulated the mRNA levels of ITGB1, PIK3R1, FGF5, PRKAA1 and the phosphorylation level of Akt, suggesting the involvement of the PI3K-Akt pathway in 14-3-3zeta-mediated tissue repair. 14-3-3zeta is a potential novel therapeutic candidate for treating severe corneal injury.
Assuntos
Proteínas 14-3-3 , Queimaduras Químicas , Lesões da Córnea , Cicatrização , Animais , Humanos , Masculino , Camundongos , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/biossíntese , Western Blotting , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Queimaduras Químicas/tratamento farmacológico , Movimento Celular , Proliferação de Células , Células Cultivadas , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Lesões da Córnea/genética , Modelos Animais de Doenças , Epitélio Corneano/metabolismo , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/lesões , Queimaduras Oculares/induzido quimicamente , Regulação da Expressão Gênica , Homeostase , Camundongos Endogâmicos C57BL , Hidróxido de Sódio , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
A 40-year-old woman underwent periocular plasma skin regeneration, a cosmetic treatment for periorbital rejuvenation. She subsequently developed bilateral thermal keratitis, manifesting as blurred vision, irritation, and redness, with a vision decrease to 20/60 and 20/50 in her OD and OS, respectively. Examination demonstrated bilateral large, irregular corneal epithelial defects and edema, necessitating treatment with amniotic membrane grafts, bandage contact lenses, and hypertonic saline. One year posttreatment, her visual acuity improved to 20/20 and 20/25, albeit with ongoing symptomatic dryness and bilateral anterior stromal haze. This case, as only the second reported instance of ocular damage from periocular plasma skin regeneration, underscores the need for heightened awareness of potential ocular complications following plasma skin regeneration and reinforces the importance of protective measures during periocular procedures.
Assuntos
Queimaduras Oculares , Humanos , Feminino , Adulto , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/diagnóstico , Ceratite/diagnóstico , Ceratite/etiologia , Ceratite/fisiopatologia , Gases em Plasma/uso terapêutico , Regeneração/fisiologia , Técnicas Cosméticas/efeitos adversos , Acuidade VisualRESUMO
PURPOSE: To evaluate limbal graft transplantation success in pediatric patients with chemical injury-induced limbal stem cell deficiency (LSCD) using the 'LSCD Working Group' staging system. METHODS: Medical records of 11 eyes of 11 children who underwent limbal graft transplantation (limbal autograft/limbal allograft) were included. Surgical success was defined as improvement in the post-operative 1st year LSCD stage. RESULTS: The mean age was 12 ± 5 (4-17) years. Causative agent was alkaline in 4(36.4%) and acid in 3(27.2%) patients. Limbal autograft was performed in 9 (81.8%) eyes with unilateral LSCD, and allograft transplantation was performed in 2 (18.2%) eyes with bilateral LSCD. The mean follow-up time was 33.89 ± 30.73 (12-102.33) months. The overall limbal graft transplantation success rate was 72.7%. Among 9 patients who receive limbal autograft, 8 had improvement in post-operative LSCD stage, 1 had stable LSCD stage. Of the 2 patients who receive limbal allograft, post-operative LSCD stage remained the same in 1 and worsened in 1 patient. The mean time between injury and the surgery was 30.47 ± 30.08 (7-108.47) months. Penetrating keratoplasty was performed in 3 (27.2%) of 11 patients following limbal graft transplantation. CONCLUSION: Management of LSCD in children is challenging and appears to be somewhat different from that of adults. Limited data in the literature indicate that cultivated or simple limbal epithelial transplantations (CLET/SLET) are primarily preferred in children. Although the tendency to take small tissue from the healthy eye is noteworthy, conventional limbal allograft and autograft transplantations also show promising results without any further complications in at least 1 year follow-up period.
Assuntos
Deficiência Límbica de Células-Tronco , Limbo da Córnea , Acuidade Visual , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Queimaduras Químicas/cirurgia , Transplante de Córnea/métodos , Queimaduras Oculares/cirurgia , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/diagnóstico , Seguimentos , Deficiência Límbica de Células-Tronco/induzido quimicamente , Deficiência Límbica de Células-Tronco/diagnóstico , Deficiência Límbica de Células-Tronco/cirurgia , Limbo da Córnea/citologia , Estudos Retrospectivos , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Transplante Autólogo , Resultado do TratamentoRESUMO
Alkali burns are one of the most common injuries used in corneal wound healing studies. Investigators have used different conditions to produce corneal alkali injuries that have varied in sodium hydroxide concentration, application methods, and duration of exposure. A critical factor in the subsequent corneal healing responses, including myofibroblast generation and fibrosis localization, is whether, or not, Descemet's membrane and the endothelium are injured during the initial exposure. After exposures that produce injuries confined to the epithelium and stroma, anterior stromal myofibroblasts and fibrosis are typical, with sparing of the posterior stroma. However, if there is also injury to Descemet's membrane and the endothelium, then myofibroblast generation and fibrosis is noted full corneal thickness, with predilection to the most anterior and most posterior stroma and a tendency for relative sparring of the central stroma that is likely related to the availability of TGF beta from the tears, epithelium, and the aqueous humor. A method is described where a 5 mm diameter circle of Whatman #1 filter paper wetted with only 30 µL of alkali solution is applied for 15 s prior to profuse irrigation in rabbit corneas. When 0.6N, or lower, NaOH is used, then the injury, myofibroblasts, and fibrosis generation are limited to the epithelium and stroma. Use of 0.75N NaOH triggers injury to Descemet's membrane and the corneal endothelium with fibrosis throughout the stroma, but rare corneal neovascularization (CNV) and persistent epithelial defects (PED). Use of 1N NaOH with this method produces greater stromal fibrosis and increased likelihood that CNV and PED will occur in individual corneas.
Assuntos
Queimaduras Químicas , Lesões da Córnea , Queimaduras Oculares , Animais , Coelhos , Substância Própria/patologia , Álcalis/toxicidade , Queimaduras Químicas/patologia , Hidróxido de Sódio/toxicidade , Córnea/patologia , Lesões da Córnea/patologia , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/patologia , Fibrose , Padrões de ReferênciaRESUMO
Alkali burn-induced corneal injury often causes inflammation and neovascularization and leads to compromised vision. We previously reported that rapamycin ameliorated corneal injury after alkali burns by methylation modification. In this study, we aimed to investigate the rapamycin-medicated mechanism against corneal inflammation and neovascularization. Our data showed that alkali burn could induce a range of different inflammatory response, including a stark upregulation of pro-inflammatory factor expression and an increase in the infiltration of myeloperoxidase- and F4/80-positive cells from the corneal limbus to the central stroma. Rapamycin effectively downregulated the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), toll-like receptor 4 (TLR4), nucleotide binding oligomerization domain-like receptors (NLR) family pyrin domain-containing 3 (NLRP3), and Caspase-1, and suppressed the infiltration of neutrophils and macrophages. Inflammation-related angiogenesis mediated by matrix metalloproteinase-2 (MMP-2) and rapamycin restrained this process by inhibiting the TNF-α upregulation in burned corneas of mice. Rapamycin also restrained corneal alkali burn-induced inflammation by regulating HIF-1α/VEGF-mediated angiogenesis and the serum cytokines TNF-α, IL-6, Interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The findings of this study indicated rapamycin may reduce inflammation-associated infiltration of inflammatory cells, shape the expression of cytokines, and balance the regulation of MMP-2 and HIF-1α-mediated inflammation and angiogenesis by suppressing mTOR activation in corneal wound healing induced by an alkali injury. It offered novel insights relevant for a potent drug for treating corneal alkali burn.
Assuntos
Queimaduras Químicas , Lesões da Córnea , Neovascularização da Córnea , Queimaduras Oculares , Camundongos , Animais , Metaloproteinase 2 da Matriz/metabolismo , Queimaduras Químicas/metabolismo , Neovascularização da Córnea/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Álcalis/toxicidade , Córnea/metabolismo , Neovascularização Patológica/metabolismo , Lesões da Córnea/metabolismo , Inflamação/metabolismo , Citocinas/metabolismo , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/tratamento farmacológico , Queimaduras Oculares/patologia , Modelos Animais de DoençasRESUMO
FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.
Assuntos
Queimaduras Químicas , Lesões da Córnea , Neovascularização da Córnea , Queimaduras Oculares , Ratos , Animais , Dissulfiram/farmacologia , Dissulfiram/uso terapêutico , Queimaduras Químicas/tratamento farmacológico , Queimaduras Químicas/metabolismo , Soluções Oftálmicas/farmacologia , Álcalis/farmacologia , Pseudópodes/metabolismo , Córnea/metabolismo , Macrófagos/metabolismo , Lesões da Córnea/tratamento farmacológico , Lesões da Córnea/metabolismo , Neovascularização da Córnea/tratamento farmacológico , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/tratamento farmacológico , Queimaduras Oculares/metabolismo , Modelos Animais de DoençasRESUMO
A characteristic rigid spatial arrangement of collagen fibrils in the stroma is critical for corneal transparency. This unique organization of collagen fibrils in corneal stroma can be impacted by the presence and interactions of proteoglycans and extracellular matrix (ECM) proteins in a corneal microenvironment. Earlier studies revealed that decorin, a leucine-rich proteoglycan in stroma, regulates keratocyte-collagen matrix assembly and wound healing in the cornea. This study investigated the role of decorin in the regulation of stromal fibrillogenesis and corneal transparency in vivo employing a loss-of-function genetic approach using decorin null (dcn-/-) and wild type (dcn+/+) mice and a standard alkali-injury model. A time-dependent ocular examinations with Slit lamp microscope in live animals assessed corneal clarity, haze, and neovascularization levels in normal and injured eyes. Morphometric changes in normal and injured dcn+/+ and dcn-/- corneas, post-euthanasia, were analyzed with Masson's Trichrome and Periodic Acid-Schiff (PAS) histology evaluations. The ultrastructure changes in all corneas were investigated with transmission electron microscopy (TEM). Injury to eye produced clinically relevant corneal haze and neovascularization in dcn-/- and dcn+/+ mice while corneas of uninjured eyes remained clear and avascular. A clinically significant haze and neovascularization appeared in injured dcn-/- corneas compared to the dcn+/+ corneas at day 21 post-injury and not at early tested times. Histological examinations revealed noticeably abnormal morphology and compromised collagen levels in injured dcn-/- corneas compared to the injured/normal dcn+/+ and uninjured dcn-/- corneas. TEM analysis exhibited remarkably uneven collagen fibrils size and distribution in the stroma with asymmetrical organization and loose packing in injured dcn-/- corneas than injured/normal dcn+/+ and uninjured dcn-/- corneas. The minimum and maximum inter-fibril distances were markedly irregular in injured dcn-/- corneas compared to all other corneas. Together, results of clinical, histological, and ultrastructural investigations in a genetic knockout model suggested that decorin influenced stromal fibrillogenesis and transparency in healing cornea.
Assuntos
Lesões da Córnea/metabolismo , Decorina/fisiologia , Colágenos Fibrilares/metabolismo , Organogênese/fisiologia , Cicatrização/fisiologia , Animais , Queimaduras Químicas/metabolismo , Lesões da Córnea/patologia , Proteínas da Matriz Extracelular/metabolismo , Queimaduras Oculares/induzido quimicamente , Colágenos Fibrilares/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Microscopia com Lâmpada de Fenda , Hidróxido de SódioRESUMO
Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-ß (TGF-ß) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-ß was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.
Assuntos
Queimaduras Químicas , Lesões da Córnea , Queimaduras Oculares , Álcalis/toxicidade , Animais , Queimaduras Químicas/metabolismo , Lesões da Córnea/patologia , Modelos Animais de Doenças , Amarelo de Eosina-(YS)/efeitos adversos , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/tratamento farmacológico , Queimaduras Oculares/patologia , Fator 10 de Crescimento de Fibroblastos/farmacologia , Hematoxilina/farmacologia , Hematoxilina/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/uso terapêutico , Coelhos , Fator de Crescimento Transformador beta/metabolismo , Fatores de Crescimento Transformadores/efeitos adversos , CicatrizaçãoRESUMO
The pathological mechanism of corneal injuries mediated by alkali burns are associated with Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 protein (NLRP3)-related corneal sterile inflammation. Whether the executive protein gasdermin D (GSDMD) of pyroptosis mediated by the NLRP3 inflammasome is present in alkali-induced corneal lesions remains unclear. Dexamethasone (Dex) is a commonly used drug for ocular surface diseases that can maintain corneal transparency and anti-inflammatory effects by topical administration. Here, we presented evidence that the effect of Dex on the pyroptosis-related caspase-1/GSDMD pathway in corneal alkali burns (CABs). We assessed the clinical manifestations and histological characteristics of the placebo group, 0.05% Dex group, 0.1% Dex group on day 3 or day 7 postburn and the control group (healthy corneas). The expression of factors (including NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N termini, pro-interleukin(IL)-1ß, IL-1ß, pro-IL-18 and IL-18) involved in the pyroptosis related caspase-1/GSDMD signaling pathway was demonstrated by molecular experiments in CAB. Alkali burns can upregulate the originally relatively dim expression of NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, pro-IL-1ß, pro-IL-18, IL-1ß and IL-18 in the healthy corneal epithelium and stroma. However, Dex can reverse the enhanced expression at the two timepoints. Corneal sterile inflammation can activate the NLRP3 inflammasome through the innate immune response mechanism and then activate the pyroptosis-related caspase-1/GSDMD signaling pathway. In addition, Dex can inhibit pyroptosis through this pathway.
Assuntos
Queimaduras Químicas/prevenção & controle , Caspase 1/metabolismo , Lesões da Córnea/prevenção & controle , Dexametasona/uso terapêutico , Queimaduras Oculares/induzido quimicamente , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose/efeitos dos fármacos , Administração Oftálmica , Animais , Western Blotting , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Dexametasona/administração & dosagem , Modelos Animais de Doenças , Queimaduras Oculares/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Glucocorticoides/administração & dosagem , Glucocorticoides/uso terapêutico , Imuno-Histoquímica , Interleucina-18/genética , Interleucina-1beta/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Soluções Oftálmicas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Hidróxido de SódioRESUMO
BACKGROUND: Apoptosis signal-regulating kinase 1-interacting protein 1 (AIP1) participates in inflammatory neovascularization induction. NADPH oxidase 4 (NOX4) produces reactive oxygen species (ROS), leading to an imbalance in nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) and NLR family pyrin domain containing 6 (NLRP6) expression. The mechanisms of AIP1, NOX4, ROS and inflammasomes in corneal neovascularization were studied herein. METHODS: C57BL/6 and AIP1-knockout mice were used in this study. The alkali burn procedure was performed on the right eye. Adenovirus encoding AIP1 plus green fluorescence protein (GFP) (Ad-AIP1-GFP) or GFP alone was injected into the right anterior chamber, GLX351322 was applied as a NOX4 inhibitor, and then corneal neovascularization was scored. The expression of related genes was measured by quantitative real-time polymerase chain reaction, western blotting and immunofluorescence staining. 2',7'-Dichlorofluorescin diacetate staining was used to determine the ROS levels. RESULTS: The expression of AIP1 was decreased, while that of cleaved interleukin-1ß (clv-IL-1ß) and vascular endothelial growth factor A (VEGFa) was increased after alkali burn injury. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. NLRP3/NLRP6 expression was imbalanced after alkali burns. GLX351322 reversed the imbalance in NLRP3/NLRP6 by reducing the ROS levels. This treatment also reduced the expression of clv-IL-1ß and VEGFa, suppressing neovascularization. CONCLUSIONS: AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burn injury. Based on the pathogenesis of corneal neovascularization, these findings are expected to provide new therapeutic strategies for patients. Corneal alkali burn injury is a common type of ocular injury that is difficult to treat in the clinic. The cornea is a clear and avascular tissue. Corneal neovascularization after alkali burn injury is a serious complication; it not only seriously affects the patient's vision but also is the main reason for failed corneal transplantation. Corneal neovascularization affects approximately 1.4 million patients a year. We show for the first time that AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burns. The expression of AIP1 was decreased, while that of clv-IL-1ß and VEGFa was increased after alkali burns. We tried to elucidate the specific molecular mechanisms by which AIP1 regulates corneal neovascularization. NOX4 activation was due to decreased AIP1 expression in murine corneas with alkali burns. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. Additionally, NLRP3/NLRP6 expression was unbalanced, with NLRP3 activation and NLRP6 suppression in the corneal alkali burn murine model. Eye drops containing GLX351322, a NOX4 inhibitor, reversed the imbalance in NLRP3/NLRP6 by reducing ROS expression. This treatment also reduced the expression of clv-IL-1ß and VEGFa, reducing neovascularization. Therefore, we provide new gene therapeutic strategies for patients. With the development of neovascularization therapy, we believe that in addition to corneal transplantation, new drug or gene therapies can achieve better results. Video Abstract.
Assuntos
Queimaduras Químicas , Lesões da Córnea , Neovascularização da Córnea , Queimaduras Oculares , Proteínas Ativadoras de ras GTPase , Álcalis/efeitos adversos , Animais , Queimaduras Químicas/complicações , Queimaduras Químicas/tratamento farmacológico , Queimaduras Químicas/patologia , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/tratamento farmacológico , Lesões da Córnea/metabolismo , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/complicações , Neovascularização da Córnea/tratamento farmacológico , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/complicações , Queimaduras Oculares/tratamento farmacológico , Humanos , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 4 , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neovascularização Patológica , Espécies Reativas de Oxigênio , Receptores de Superfície Celular , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
This study was to explore the inhibitory effect of bromfenac sodium (BF) / chitosan (CS) nanoparticles (NPs) on corneal neovascularization (CNV). 45 New Zealand white rabbits provided by The First Affiliated Hospital of Jinan University were randomly divided into a control group (group A, n = 15), 0.1% BF aqueous solution treatment group (group B, n = 15), and 0.1% BF/CS-NPs suspension treatment group (group C, n = 15). A rabbit corneal alkali burn model was established. The average particle size of BF/CS-NPs with different BF concentrations was mainly 341.6 ± 12.9 nm - 548.7 ± 15.4 nm; and the Zeta potential distribution was 24.3 ± 2.5 mV - 35.7 ± 4.3 mV. When the initial concentration of BF was 1.5 mg/mL, the maximum drug loading was 57.35 ± 5.26%. The area of CNV in group C was significantly lower than that in groups B and A, and the differences were statistically significant (P < 0.05). At 6, 12, 18, and 24 days after surgery, the mRNA expression levels in cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) gene were compared after standardized by ß-actin; group A had the highest expression level, followed by group B, and group C had the lowest expression level, showing statistically significant differences (P < 0.05). The BF/CS-NPs granules prepared in this study had stable physical and chemical properties and had a good sustained-release effect, and the release duration can be as long as 48 hours. BF/CS-NPs can inhibit the formation of CNV at different time points after alkali burn, and reduce the expression of VEGF and COX-2 in corneal tissue after alkali burn.
Assuntos
Queimaduras Químicas , Neovascularização da Córnea , Queimaduras Oculares , Animais , Benzofenonas , Bromobenzenos , Queimaduras Químicas/tratamento farmacológico , Queimaduras Químicas/metabolismo , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/genética , Neovascularização da Córnea/metabolismo , Ciclo-Oxigenase 2/genética , Modelos Animais de Doenças , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/tratamento farmacológico , Queimaduras Oculares/metabolismo , Coelhos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Ocular surface burns can be caused by chemicals (alkalis and acids) or direct heat. One effect of the burn is damage to the limbal epithelial stem cells of the ocular surface with delayed re-epithelialisation, stem cell failure, and conjunctivalisation of the cornea. Amniotic membrane transplantation (AMT) performed in the acute phase (day 0 to day 7) following an ocular surface burn is claimed to reduce pain and accelerate healing. The surgery involves securing a layer of amniotic membrane (AM) to the eyelid margins as a patch to cover the entire ocular surface. However, there is debate about the severity of an ocular burn that may benefit from AMT and uncertainty of whether AMT improves outcomes. OBJECTIVES: To compare the effect of AMT with medical therapy in the first seven days after an ocular surface burn, compared to medical therapy alone. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL; which contains the Cochrane Eyes and Vision Trials Register; 2021, Issue 9); Ovid MEDLINE; Ovid Embase; LILACS; the ISRCTN registry; ClinicalTrials.gov and the WHO ICTRP. We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 29 September 2021. SELECTION CRITERIA: We included randomised trials that compared an AMT applied in the first seven days following an ocular surface burn in addition to medical therapy with medical therapy alone. The outcome measures were failure of re-epithelialisation by day 21 post injury, visual acuity at final follow-up, corneal neovascularisation, symblepharon, time to re-epithelialisation and adverse effects. DATA COLLECTION AND ANALYSIS: Two review authors independently screened search results, assessed the included studies for risk of bias and extracted relevant data. We contacted trial investigators for missing information. We summarised data using risk ratios (RRs) and mean differences (MDs) as appropriate. MAIN RESULTS: We analysed two RCTs, but excluded individual patients who had been treated outside the acute phase in one of the studies (data provided by study authors). In total, 36 moderate burns from one RCT and 92 severe burns from two RCTs were evaluated separately. For both categories, the certainty of the evidence was downgraded principally as a result of high risks of performance and detection biases, and because of imprecision indicated by very wide confidence intervals. In addition, follow-up was insufficiently frequent to calculate time-to-epithelialisation precisely. Moderate severity ocular burns (Roper-Hall classification II-III) The relative risk of AMT on failure of epithelialisation by day 21 was 0.18 (0.02 to 1.31), and LogMAR visual acuity was 0.32 lower (0.55 to 0.09 lower) in the treatment group (i.e. better), suggesting a possible benefit of AMT. The GRADE assessment for failure of epithelialisation by day 21 was downgraded to very low due to the risk of bias and imprecision (very wide confidence intervals including no effect). The GRADE assessment for visual acuity at final follow-up was downgraded to low due to the risk of bias and imprecision (optimal information size not met). The relative effects of AMT on corneal neovascularisation (RR 0.56; 0.21 to 1.48), symblepharon (RR 0.41; 0.02 to 9.48) and time-to-epithelialisation (13 days lower; 26.30 lower to 0.30 higher) suggest possible benefit of AMT, but the wide confidence intervals indicate that both harm and benefit are possible. GRADE assessments for these outcomes were once again downgraded to very low due to the risk of bias and imprecision. Since adverse effects are rare, the small sample would have fewer occurrences of rare but potentially important adverse effects. The GRADE assessment for adverse effects was therefore considered to be low. Severe ocular burns (Roper-Hall classification IV) The relative risk of AMT on failure of epithelialisation by day 21 was 1.03 (0.94 to 1.12), and LogMAR visual acuity was 0.01 higher (0.29 lower to 0.31 higher) in the treatment group (i.e, worse), indicating no benefit of AMT. GRADE assessments for failure of epithelialisation by day 21 and final outcomes were downgraded to low. The relative effects of AMT on corneal neovascularisation (RR 0.84; 0.66 to 1.06), symblepharon (RR 0.89; 0.56 to 1.42) and time-to-epithelialisation (1.66 days lower; 11.09 lower to 7.77 higher) may include both benefit and harm. GRADE assessments for corneal neovascularisation, symblepharon and time-to-epithelialisation were downgraded to low due to risk of bias and imprecision. For adverse effects, the GRADE assessment was downgraded to low, reflecting the small sample sizes in the RCTs. AUTHORS' CONCLUSIONS: There is uncertain evidence to support the treatment of moderate acute ocular surface burns with AMT in addition to standard medical therapy as a means of preventing failure of epithelialisation by day 21, improving visual outcome and reducing corneal neovascularisation, symblepharon formation and time-to-epithelialisation. For severe burns, the available evidence does not indicate any significant benefit of treatment with AMT.
Assuntos
Neovascularização da Córnea , Queimaduras Oculares , Âmnio , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/cirurgia , Humanos , Acuidade Visual , CicatrizaçãoRESUMO
BACKGROUND: Our previous study revealed that mesenchymal stem cells (MSCs) inhibited angiogenesis via miRNA-mediated repression of prospero homeobox 1 (PROX1). This study aimed to verify whether miR-340-5p participates in the therapeutic effect of MSCs on corneal neovascularization (CNV) via repressing PROX1 and epithelial membrane protein 2 (EMP2). MATERIALS AND METHODS: The rat CNV model was established by corneal alkali burn. The binding relationship between miR-340-5p and 3'-untranslational regions (3'UTRs) of EMP2 and PROX1 was confirmed using dual-luciferase reporter assay. After culturing corneal epithelial cells (CECs) using MSC supernatants, the vascular endothelial growth factor (VEGF) level in CEC supernatants and the CEC viability were detected. The role of miR-340-5p in the therapeutic effect of MSC on CNV was determined via lentivirus-mediated miR-340-5p intervention in vivo. RESULTS: The expression of miR-340-5p was reduced and EMP2 and PROX1 were increased in CNV corneal tissues. The lentivirus-mediated overexpression of miR-340-5p inhibited the expressions of EMP2 and PROX1. The dual-luciferase reporter assay confirmed that miR-340-5p could bind with the 3'UTRs of EMP2 and PROX1. miR-340-5p was enriched in MSC supernatants and the culture of CECs using MSC supernatants increased the miR-340-5p expression in CECs. After being cultured in miR-340-5p-knocking down MSC supernatants, the expressions of EMP2 and PROX1 were increased, and the VEGF level and CEC viability were restored. The in vivo experiments also indicated that the therapeutic effect of MSCs was mediated by miR-340-5p. CONCLUSIONS: miR-340-5p mediates the therapeutic effect of MSCs on CNV via binding and repressing the expressions of EMP2 and PROX1.
Assuntos
Neovascularização da Córnea , Queimaduras Oculares , Células-Tronco Mesenquimais , MicroRNAs , Animais , Neovascularização da Córnea/genética , Neovascularização da Córnea/terapia , Queimaduras Oculares/induzido quimicamente , Proteínas de Homeodomínio/genética , Glicoproteínas de Membrana/genética , MicroRNAs/genética , Ratos , Proteínas Supressoras de Tumor/genética , Fator A de Crescimento do Endotélio VascularRESUMO
BACKGROUND: Corneal neovascularization (CNV) is an important disease that causes blindness. Secretogranin III (Scg3) has emerged as a new influencing factor of neovascularization. This study analyzed the Scg3 antibody's inhibitory effect on CNV and and explored its preliminary mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with Scg3 and anti-Scg3. Cell proliferation, wound healing migration and tube formation assays were performed. Healthy adult New Zealand rabbits were randomly selected to be alkali burned and establish the corneal neovascularization (CNV) model. The rabbits were randomly divided into 3 groups (the high concentration group, low concentration group and control group). Different doses of anti-Scg3 and PBS were administered to the rabbits. Clinical examinations, immunostaining, quantitative real-time polymerase chain reaction (qPCR) and western blotting analyses were performed postoperatively. RESULTS: In the in vitro study, the Scg3 antibody mixture inhibited Scg3-induced endothelial cell proliferation and angiogenesis. In the in vivo study, significant CNV was observed in the control group. Confocal microscopy also revealed considerable active neovascularization in the control group. There was no obvious CNV growth in the high concentration group. Additionally, CD31, LYVE1 and CD45 expression was significantly inhibited after treatment with a high concentration of Scg3 antibody. The qPCR and western blotting analyses revealed that the levels of ERK in the low concentration group and high concentration group were higher than those in the control group at 7 days and 14 days. The levels of VEGF in the control group were significantly increased compared with those in the high concentration group. In all three groups, the levels of Akt were not significantly different at any time point. CONCLUSION: The expression of Scg3 could affect the growth of HUVECs in vitro. Treatment with a high concentration (0.5 µg/mL) of Scg3 antibody reduced the inflammatory response and inhibited the growth of corneal neovascularization after corneal alkali burn injury in rabbits. The MEK/ERK pathway might play an important role in the inhibitory effect of anti-Scg3.
Assuntos
Lesões da Córnea , Neovascularização da Córnea , Queimaduras Oculares , Adulto , Coelhos , Humanos , Animais , Neovascularização da Córnea/tratamento farmacológico , Células Endoteliais , Neovascularização Patológica , Queimaduras Oculares/induzido quimicamente , ÁlcalisRESUMO
OBJECTIVES: To report demographic characteristics, types and grades of injury, regional distribution of injury severity, clinical findings, and long-term results of acute ocular chemical burns. METHODS: Medical records of patients with chemical burns between 2010 and 2020 who were admitted to Ege University less than 72 hr after the injury were reviewed. Age, gender, cause of the burn, injury severity, initial and final best-corrected visual acuity, surgical intervention, and complications were recorded. The injury severity was graded according to Dua classification. RESULTS: A total of 104 patients (137 eyes) were included. The mean age was 42.69±17.39 (7-90) years with a male-to-female ratio of 86:18. The most common causes were home (32.6%) and industrial accidents (45.1%). The causative agent percentages for alkaline, acid, and neutral were 49.0%, 35.5%, and 5.7%, respectively. The percentages of eyes in each grade (1-6) were 16.0%, 16.0%, 15.3%, 16.7%, 17.5%, and 18.2%, respectively. Complications mostly occurred in eyes with grade 2 or higher injuries (83.7%). The relationship between injury grade and limbal stem-cell deficiency was statistically significant (P<0.001). Surgery was mostly needed in grade 4 or higher injuries (44 eyes). CONCLUSION: The severity of the burn is one of the most important prognostic factors in chemical burns. It is important to determine the spreading of the regional injury severity beside the global one to predict complication risk of the injuries. In the present study, the injury distribution was homogeneous in all grades. Limbal stem-cell deficiency development took place mostly in grade 5 and 6 burns as expected.