Epithelial dysplasia in pterygium postoperative granuloma.

Pterygium postoperative granuloma (PPG) is one of the common complications of pterygium surgery. In order to provide the structural features of PPG, and to further explore its pathogenetic mechanism, we analyzed clinical and pathological characteristics of 12 PPG cases. New blood vessels were observed under a slit lamp in PPG and peripheral conjunctival tissues. In vivo confocal imaging showed that there was extensive neovascularization in the stroma, accompanied by infiltration of dendritic cells and inflammatory cells. Dense fibrous structures were observed in some PPG tissues. H&E staining results confirmed neovascularization and inflammatory cells in PPG tissues. In addition, H&E staining exhibited epithelioid tissue covering some PPG tissues. The immunofluorescence results demonstrated that the PPG epithelium was negative for K19, K10 and Muc5AC. Compared with the normal conjunctiva and pterygium, the expression of collagen IV in PPG basement membrane decreased, the expression of pan-cytokeratin (PCK), claudin 4 and E-cadherin in PPG epithelium was significantly lower, while the expression of vimentin, α-SMA and Snail was significantly increased. Therefore, our results suggest that the expression of epithelial keratin markers and goblet cell specific mucin marker is downregulated in the PPG tissues, and it likely is associated with the occurrence of EMT in granulomatous tissues.

Loss of tenascin X gene function impairs injury-induced stromal angiogenesis in mouse corneas.

To determine the contribution by tenascin X (Tnx) gene expression to corneal stromal angiogenesis, the effects were determined of its loss on this response in TNX knockout (KO) mice. In parallel, the effects of such a loss were evaluated on vascular endothelial growth factor (VEGF) and transforming growth factor ß1 (TGFß1) gene and protein expression in fibroblasts and macrophages in cell culture. Histological, immunohistochemical and quantitative RT-PCR changes determined if Tnx gene ablation on angiogenic gene expression, inflammatory cell infiltration and neovascularization induced by central corneal stromal cauterization. The role was determined of Tnx function in controlling VEGF-A or TGFß1 gene expression by comparing their expression levels in ocular fibroblasts and macrophages obtained from wild-type (WT) and body-wide Tnx KO mice. Tnx was up-regulated in cauterized cornea. In Tnx KO, macrophage invasion was attenuated, VEGF-A and its cognate receptor mRNA expression along with neovascularization were lessened in Tnx KOs relative to the changes occurring in their WT counterpart. Loss of Tnx instead up-regulated in vivo mRNA expression of anti-angiogenic VEGF-B but not VEGF-A. On the other hand, TGFß1 mRNA expression declined in Tnx KO cultured ocular fibroblasts. Loss of Tnx gene expression caused VEGF-A expression to decline in macrophages. Tnx gene expression contributes to promoting TGFß1 mRNA expression in ocular fibroblasts and VEGF-A in macrophages, macrophage invasion, up-regulation of VEGF-A expression and neovascularization in an injured corneal stroma. On the other hand, it suppresses anti-angiogenic VEGF-B mRNA expression in vivo.

Frontline Science: Aspirin-triggered resolvin D1 controls herpes simplex virus-induced corneal immunopathology.

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye, caused by HSV-1 infection, and a common cause of vision impairment in humans. The inflammatory lesions in the cornea are primarily caused by neutrophils with the active participation of CD4+ T cells. Therefore, the targeting of these immune cell types and their products represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin D1 (RvD1) and its epimer aspirin-triggered RvD1 (AT-RvD1) are lipid mediators derived from docosahexaenoic acid (DHA) and were shown to promote resolution in several inflammatory disease models. In this report, we examined whether AT-RvD1 administration, begun before infection or at a later stage after ocular infection of mice with HSV-1, could control the severity of SK lesions. Treatment with AT-RvD1 significantly diminished the extent of corneal neovascularization and the severity of SK lesions. AT-RvD1-treated mice had fewer numbers of inflammatory cells that included neutrophils as well as Th1 and Th17 cells in the infected cornea. The mechanisms by which AT-RvD1 acts appear to be multiple. These include inhibitory effects on proinflammatory mediators, such as IL-1ß, IL-6, IL-12, CXCL1, MCP-1, MIP-2, vascular endothelial growth factor (VEGF)-A, matrix metalloproteinase 9 (MMP-9), and proinflammatory miRNA, such as miR-155, miR-132, and miR-223, which are involved in SK pathogenesis and corneal neovascularization. In addition, AT-RvD1 attenuated STAT1, which plays an important role in Th1 cell differentiation and IFN-γ expression. These findings demonstrate that AT-RvD1 treatment could represent a useful strategy for the management of virus-induced immunopathological lesions.

Factors Influencing Intraocular Pressure Changes after Laser In Situ Keratomileusis with Flaps Created by Femtosecond Laser or Mechanical Microkeratome.

The aim of this study is to describe factors that influence the measured intraocular pressure (IOP) change and to develop a predictive model after myopic laser in situ keratomileusis (LASIK) with a femtosecond (FS) laser or a microkeratome (MK). We retrospectively reviewed preoperative, intraoperative, and 12-month postoperative medical records in 2485 eyes of 1309 patients who underwent LASIK with an FS laser or an MK for myopia and myopic astigmatism. Data were extracted, such as preoperative age, sex, IOP, manifest spherical equivalent (MSE), central corneal keratometry (CCK), central corneal thickness (CCT), and intended flap thickness and postoperative IOP (postIOP) at 1, 6 and 12 months. Linear mixed model (LMM) and multivariate linear regression (MLR) method were used for data analysis. In both models, the preoperative CCT and ablation depth had significant effects on predicting IOP changes in the FS and MK groups. The intended flap thickness was a significant predictor only in the FS laser group (P < .0001 in both models). In the FS group, LMM and MLR could respectively explain 47.00% and 18.91% of the variation of postoperative IOP underestimation (R2 = 0.47 and R(2) = 0.1891). In the MK group, LMM and MLR could explain 37.79% and 19.13% of the variation of IOP underestimation (R(2) = 0.3779 and 0.1913 respectively). The best-fit model for prediction of IOP changes was the LMM in LASIK with an FS laser.

Mosaic chromosome 18q partial deletion syndrome with bilateral full-thickness corneal disease: surgical intervention and histopathology.

A 2-month-old boy diagnosed with a mosaic chromosome 18q partial deletion syndrome was referred for bilateral cloudy corneas. The abnormal metaphases had a terminal deletion of the long arm of chromosome 18 as clonal abnormality. The cytogenetics findings were 46,XY, del (18)(q21.2)[12]/46,XY[20]. Ocular findings included bilateral microcornea with dense opacification and unilateral iris and chorioretinal coloboma. Penetrating keratoplasty (PK) was performed on both eyes. Histopathology of the host corneal button showed complete loss of Bowman's layer, hyperkeratosis of the epithelium, stromal neovascularization, and leukocyte infiltration. Descemet's membrane and endothelium were irregular in both specimens. CD45 stain for leukocytes confirmed perivascular and epithelial leukocytes infiltration. Mosaic chromosome 18q deletion syndrome is a rare genetic abnormality with a variable phenotype - including ocular findings - and hence, warrants an ophthalmic evaluation and genetic counseling.

Ranibizumab injection for corneal neovascularization refractory to bevacizumab treatment.

Vascular endothelial growth factor inhibitor is an emerging therapeutic modality for various ocular diseases with neovascularization (NV). However, for corneal NV, controversy remains regarding whether bevacizumab or ranibizumab is superior. A 32-year-old female diagnosed with herpetic keratoconjunctivitis with refractory corneal NV despite two previous subconjunctival and intrastromal bevacizumab injections, received two subconjunctival and intrastromal ranibizumab injections. Six months postoperatively, there was significant regression of the neovascular area and vessel caliber. Here, the authors report a case of improvement in corneal NV with subconjunctival and intrastromal ranibizumab injections, which was previously refractory to bevacizumab injection. The findings may suggest a new prospect in treating corneal NV.

Alkali burn versus suture-induced corneal neovascularization in C57BL/6 mice: an overview of two common animal models of corneal neovascularization.

The purpose of the present study was to quantify and compare corneal hem- and lymphangiogenesis between alkali burn and suture-induced corneal neovascularization (CNV) in two commonly used mouse strains. A retrospective analysis was performed on C57BL/6 and FVB neovascularized corneas. CNV was induced by surface caustication with NaOH or intrastromal placement of three 10.0 nylon sutures. Hemangiogenesis and lymphangiogenesis extent was calculated on whole mounted corneas by CD31 and LYVE1 immunofluorescence analysis. Blood vessel growth was similar between alkali burn and suture-induced CNV in C57BL/6 mice, and between C57BL/6 and FVB sutured strains. On the contrary, corneal lymphangiogenesis was more pronounced in the C57BL/6 sutured mice versus the alkali burn group, and in the FVB strain versus both C57BL/6 models. These results indicate that significant differences occur in lymphangiogenesis, but not hemangiogenesis, in the alkali burn and suture-induced models in C57BL/6 mice. Furthermore, lymphangiogenesis is more pronounced in the albino (FVB) strain after suture placement. We suggest that the suture model has a number of advantages and may be preferentially used to study corneal lymphangiogenesis.

Equine deep stromal abscesses (51 cases - 2004-2009)--Part 2: the histopathology and immunohistochemical aspect with attention to the histopathologic diagnosis, vascular response, and infectious agents.

PURPOSE: To investigate histopathologic and immunohistochemical aspects of equine deep stromal abscesses (DSA) with a focus on the histopathologic diagnosis, presumptive etiology, and the immunohistochemical expression of three angiogenesis-related factors: vascular endothelial growth factor-A (VEGF-A), pigment epithelium-derived factor (PEDF), and interleukin-1 receptor antagonist (IL-1ra). SAMPLE POPULATION: Paraffin-embedded biopsy samples from 51 DSA. The biopsies were collected from full-thickness penetrating keratoplasty or split-thickness lamellar keratoplasty surgeries at the University of Florida Veterinary Medical Center in the period from 2004 to 2009. PROCEDURE: The histopathologic and immunohistochemical findings were tested for association between each other. Prevalence calculation and test for association with qualitative data analysis was used for data evaluation. RESULTS: Fungal hyphae were found histologically in 47.1% (n = 24) of the DSA cases. Histopathologically, most fungal DSA showed suppurative keratitis (n = 34; 66.7%) and little to no stromal vascularization infiltrating the abscess (negative association, P = 0.005). All three angiogenesis-related factors were expressed to some degree in DSA tissue. A negative association between VEGF-A and PEDF when compared to the presence of fungal hyphae (P < 0.001, P = 0.023) indicated that cases positive for these two factors will most probably not have fungal hyphae present. CONCLUSION: Abnormally decreased VEGF-A expression is suggested as the reason for the slow vascularization and delayed resolution of fungal DSA, whereas PEDF and IL-ra did not seem to have any influence on the vascularization process. Clinical and histopathologic characteristics of DSA make it possible to suggest an etiology for an equine DSA with an unknown etiology.

Ranibizumab Injection for Corneal Neovascularization Refractory to Bevacizumab Treatment

Vascular endothelial growth factor inhibitor is an emerging therapeutic modality for various ocular diseases with neovascularization (NV). However, for corneal NV, controversy remains regarding whether bevacizumab or ranibizumab is superior. A 32-year-old female diagnosed with herpetic keratoconjunctivitis with refractory corneal NV despite two previous subconjunctival and intrastromal bevacizumab injections, received two subconjunctival and intrastromal ranibizumab injections. Six months postoperatively, there was significant regression of the neovascular area and vessel caliber. Here, the authors report a case of improvement in corneal NV with subconjunctival and intrastromal ranibizumab injections, which was previously refractory to bevacizumab injection. The findings may suggest a new prospect in treating corneal NV.

Effect of erythropoietin on healing of corneal epithelial defects in rabbits.

BACKGROUND/AIM: Corneal epithelial defects may heal slowly in patients with diabetes, limbal stem cell deficiency, extensive chemical burns or anesthetized corneas. Studies have shown that erythropoietin, a glycoprotein hormone that promotes red blood cell proliferation and inhibits apoptosis of erythroid progenitors, may also exert a cytoprotective, antiapoptotic effect on nonhematopoietic cells. The aim of the study was to examine the effect of erythropoietin on the healing process of corneal epithelial erosions in rabbit eyes. METHODS: Fifteen New Zealand albino rabbits were divided into 3 groups following induction of unilateral uniform corneal epithelial erosions. The first group received local treatment with erythropoietin-containing cellulose-based gel 4 times daily; the second group received treatment with cellulose-based gel without erythropoietin 4 times daily, and the third group received no treatment. The healing process was monitored twice daily using cobalt-blue-filtered slit lamp photography and digital images of fluorescein-stained corneas until complete re-epithelization was achieved. Following re-epithelization, corneas were removed for histologic processing. One-way analysis of variance and Mann-Whitney tests were used for statistical analysis. RESULTS: Mean ± SD time to complete re-epithelization was 55 ± 2.19 h in the group treated with erythropoietin-containing cellulose-based gel, 66.5 ± 14.25 h in the group treated with gel only and 62.2 ± 9.09 h in the untreated group (p = 0.16, not significant). There was no significant difference among the groups in the time to complete re-epithelization or the rate of epithelial healing. Histologic corneal evaluation revealed stromal vascularization in 2 of the 6 erythropoietin-treated rabbits and in neither of the control groups. CONCLUSION: Erythropoietin has no beneficial effect on the rate of healing of corneal epithelial erosions in rabbit eyes, and corneal stroma neovascularization seems to be a significant adverse effect.

Combined scraping, coagulation, and subconjunctival bevacizumab in Descemet stripping automated endothelial keratoplasty for bullous keratopathy.

PURPOSE: This study evaluated the clinical outcomes of a combined method of scraping corneal epithelium, coagulating vessels, and subconjunctival bevacizumab in Descemet stripping automated endothelial keratoplasty (DSAEK) for bullous keratopathy with corneal neovascularization (NV).
 METHODS: The study included patients with bullous keratopathy undergoing DSAEK. Indications for DSAEK were advanced pseudophakic bullous keratopathy with superficial and deep corneal vascularization and failed corneal grafts. Patients were treated by scraping the corneal epithelium and lightly coagulating the corneal superficial stromal NV and the feeding vessels in the sclera, with a subconjunctival bevacizumab injection at the end of surgery. Subconjunctival and perilimbal bevacizumab dose of 2.5 mg/0.1 mL/affected quadrant was injected at the site of NV in each patient at the end of surgery. One or 2 injections were applied. At each visit, a full eye examination with photographic documentation was performed. Mean follow-up period was 32 (24-36) months.
 RESULTS: Eight eyes of 8 patients with high-risk corneal transplantation and corneal NV were included in this noncomparative interventional case series. The original corneal NV disappeared in all patients immediately after surgery. No patient in the series had recurrent corneal NV or rejection during at least 24 months of follow-up. 
 CONCLUSIONS: . The combination of scraping, coagulating, and bevacizumab injection in DSAEK is an effective method to treat corneal NV in corneal transplantation for bullous keratopathy.

Stromal rejection following deep anterior lamellar keratoplasty: implications for postoperative care.

PURPOSE: To better characterize stromal rejection in the context of deep anterior lamellar keratoplasty (DALK) to improve early diagnosis and proper management. METHODS: The clinical records of 22 patients undergoing DALK by 2 surgeons between October 2006 and January 2008 were reviewed to identify patients who experienced stromal rejection. The diagnosis was made after the demonstration of acute stromal edema and/or stromal neovascularization in the absence of confounding preoperative conditions, such as herpetic keratitis. The incidence and clinical features of stromal rejection were compared with other descriptions found in the literature. RESULTS: Five of 20 eligible patients experienced stromal rejection within 12 months. Two patients were on low-dose corticosteroids when diagnosed. Four of the 5 patients were treated aggressively with q1-3 hourly prednisolone acetate 1% eye drops. The fifth was treated less aggressively with a maximum dose of only q6 hourly prednisolone acetate 1% and subsequently experienced a second rejection episode less than 5 months later. All episodes resolved completely with treatment. CONCLUSIONS: The incidence of stromal rejection in DALK is clinically significant, suggesting that these patients may benefit from corticosteroid regimens similar to those used in penetrating keratoplasty and implies that stromal rejection may be more common in penetrating keratoplasty than previously reported. If misdiagnosed or left untreated, stromal rejection can compromise graft clarity but prompt recognition and aggressive treatment can result in good anatomic and visual outcomes.

Spontaneous hemorrhagic Descemet membrane detachment causing pupillary block.

PURPOSE: To present a unique case of a 65-year-old man using warfarin who presented with acute unilateral loss of vision due to hemorrhagic Descemet membrane detachment (DMD) with pupillary block and elevated intraocular pressure and its subsequent treatment and challenges. METHOD: Case report. RESULTS: Clinical examination showed a visual acuity of finger counting, central DMD with near contact to the iris and premembrane hemorrhage, an intraocular pressure (IOP) of 19 mmHg, and normal pupillary reaction. An International Normalized Ratio (INR) of 4.9 was treated with dose reduction and vitamin K. Twelve hours later the patient re-presented with an acute increase in pain and an IOP of 78 mmHg with pupillary block and iris bombé. YAG-laser membranotomy, anterior chamber paracentesis, and maximal topical and systemic therapy were unsuccessful in reducing the IOP. Surgical management, including irrigation and aspiration of blood, led to a normalization of the IOP. Descemet stripping automated endothelial keratoplasty (DSAEK) resulted in a visual acuity of 0.3. Deep stromal/pre-Descemet membrane neovascularization was found bilaterally, suspicious for a previous interstitial keratitis. CONCLUSIONS: The previously unreported complication of pupillary block following a pre-Descemet membrane hemorrhage was treated successfully for the first reported time, in a 2-step DSAEK. This indicates that DSAEK could be considered as a treatment option for DMD, especially in traumatic circumstances.

Histopathological study of delayed regraft after corneal graft failure.

PURPOSE: To evaluate the histopathological features of corneal graft failures over time. METHODS: A single-center retrospective analysis was performed on corneal specimens diagnosed as corneal graft failure retrieved from The Henry C. Witelson Ophthalmic Pathology Laboratory and Registry (Montreal, Canada) over a 9-year period. The corneal buttons were divided into 3 different groups according to the time between the diagnosis of corneal graft failure and regraft. Corneal specimens obtained during keratoplasty were subjected to hematoxylin and eosin and periodic acid-Schiff. Five different histopathological findings were evaluated in each specimen. RESULTS: Overall, the most common histopathological finding was endothelial decompensation (97.2%). Subepithelial pannus (38.9%), vessels in the corneal stroma (11.1%), and anterior synechiae (2.8%) were the other present findings. The inflammatory reaction was considered discrete in 83.3% of the cases. The only significant histopathological finding correlated with time was the presence of vessels in the corneal stroma (P = 0.0092). CONCLUSIONS: Corneal neovascularization, represented by the presence of vessels in the corneal stroma, was the only histopathological finding correlated with time. Because it is a known factor of poor prognosis, our findings strongly support that early regraft has higher chances of success.

Deep intrastromal bevacizumab injection for management of corneal stromal vascularization after deep anterior lamellar keratoplasty, a novel technique.

PURPOSE: To report the effect of intrastromal injection of bevacizumab in an eye with extensive corneal stromal vascularization after deep anterior lamellar keratoplasty. METHODS: Bevacizumab (2.5 mg/1 mL) was injected into the deep corneal stroma of an eye with severe and extensive stromal vascularization. RESULTS: Corneal vascularization regressed dramatically after deep stromal injection of bevacizumab with no recurrence after 6 months. Visual acuity was improved, and the patient's complaints subsided. CONCLUSIONS: Corneal intrastromal injection of bevacizumab can be considered for management of intrastromal vascularization after deep anterior lamellar keratoplasty.

Impaired angiogenic response in the cornea of mice lacking tenascin C.

PURPOSE: This study investigated the effects of loss of tenascin C (TNC) in the development of neovascularization in a corneal stroma in mice. Cell culture study was also conducted to clarify the roles of TNC in the expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)ß1 in fibroblasts and macrophages. METHODS: Ocular fibroblasts and macrophages from wild-type (WT) and TNC-null (KO) mice were used to study the role of TNC in the expression of VEGF and TGFß1. The effects of the absence of TNC on angiogenic gene expression, inflammatory cell invasion, and cornea neovascularization in the corneal stroma were then evaluated after cauterization of the center of the cornea in mice. Histologic, immunohistochemical, and mRNA expression analyses were performed. RESULTS: Absence of TNC suppressed expression of VEGF and counteracted upregulation of TGFß1 by exogenous TGFß1 in ocular fibroblast culture. Such effects of the absence of TNC were not observed in cultured macrophages. Absence of TNC attenuated expression of both VEGF and TGFß1 mRNA as well as neovascularization into the stroma after cauterization at the center of the cornea in mice. Absence of TNC suppressed macrophages, but not neutrophils, invading the cauterized cornea. CONCLUSIONS: TNC is involved in angiogenic gene expression in ocular fibroblasts in vitro and in vivo and is required for macrophage invasion and neovascularization of injured corneal stroma.

Impaired angiogenic response in the corneas of mice lacking osteopontin.

PURPOSE: To investigate the effects of loss of osteopontin (OPN) in the development of neovascularization in corneal stroma in mice. Cell culture study was also conducted to clarify the effects of OPN in transforming growth factor (TGF) beta1-driven cell signaling and expression of vascular endothelial growth factor (VEGF). METHODS: Ocular fibroblasts from wild-type and OPN-null mice were used to study the role of OPN in TGFbeta1 signal and VEGF expression. The effect of the absence of OPN on corneal neovascularization was evaluated in mice. RESULTS: In ocular fibroblast culture, loss of OPN attenuated TGFbeta1 signals (Smad3 and p38) and reduced expression of VEGF. Loss of OPN attenuated neovascularization in corneal stroma in mice. CONCLUSIONS: OPN is involved in VEGF expression in cultured fibroblasts and is required for neovascularization in corneal stroma in vivo.

Expression of fibroblast activation proteins in corneal stromal neovascularization.

PURPOSE: To observe changes of the factors in corneal stroma during corneal neovascularization and to investigate the mechanism of corneal neovascularization. METHODS: Forty-eight Wistar rats were used, including eight in the control group. Corneal neovascularization was induced by alkali burns in 40 rats. Frozen sections, which were cut across the corneal center, were prepared on days 1, 3, and 7 post-burn, respectively. Transforming growth factor-beta 1 (TGF-beta 1) was examined by immunohistochemistry, and fibroblast activation protein (FAP) and a-smooth muscle actin (a-SMA) were detected by double-labeling fluorescent immunohistochemistry. Blood vessel endothelium was identified for PECAM-1 (CD31). Expressions of FAP in the cornea with or without neovascularization were monitored with reverse transcription-polymerase chain reaction on days 3 and 7. RESULTS: In the alkali-burned eyes, TGF-ss1 first expressed in the corneal stroma, and some stromal cells expressed a-SMA and FAP. The FAP(+) keratocytes were found around the CD31(+) endothelium of angiogenesis. FAP was expressed in the corneas with neovascularization, but not in those without neovascularization. CONCLUSION: Factors in corneal stroma may change when corneal neovascularization occurs. The stromal keratocytes can express FAP(+) cells surrounding the endothelium of angiogenesis.

Regression of severe corneal stromal neovascularization with topical cyclosporine 0.05% after penetrating keratoplasty for fungal corneal ulcer.

PURPOSE: To report regression of corneal stromal neovascularizations with the use of topical cyclosporine 0.05% in a corneal transplant patient performed for fungal corneal ulcer. DESIGN: Case report. METHODS: A 14-year-old boy treated for fungal corneal ulceration developed 360 degrees corneal stromal neovascularization peroperatively. Topical cyclosporine 0.05% was used to decrease the risk of rejection. RESULTS: The neovascularizations regressed totally within 2 months and no signs of graft rejection were present at 6 months follow up. CONCLUSION: Topical cyclosporine 0.05% may result in regression of stromal corneal neovascularizations and help to reduce the risk of graft rejection in selected cases.