Limbal Vessels Research Articles

Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting

To elucidate the temporal sequence of events leading to new capillary sprouting in inflammatory corneal angiogenesis. Angiogenesis was induced by corneal suture placement in Wistar rats. The inflamed region was examined by time-lapse in vivo confocal microscopy for up to 7 days. At 6 and 12 hours and 1, 2, 4, and 7 days, corneas were excised for flat mount immunofluorescence with primary antibodies for CD31, CD34, CD45, CD11b, CD11c, Ki-M2R, NG2, and α-SMA. From days 0 to 4, the in vivo extravasation and expansion characteristics of single limbal vessels were quantified. Starting hours after induction and peaking at day 1, CD45(+)CD11b(+) myeloid cells extravasated from limbal vessels and formed endothelium-free tunnels within the stroma en route to the inflammatory stimulus. Limbal vessel diameter tripled on days 2 to 3 as vascular buds emerged and transformed into perfused capillary sprouts less than 1 day later. A subset of spindle-shaped CD11b(+) myeloid-lineage cells, but not dendritic cells or mature macrophages, appeared to directly facilitate further capillary sprout growth. These cells incorporated into vascular endothelium near the sprout tip, co-expressing endothelial marker CD31. Sprouts had perfusion characteristics distinct from feeder vessels and many sprout tips were open-ended. Time-lapse in vivo corneal confocal microscopy can be used to track a temporal sequence of events in corneal angiogenesis. The technique has revealed potential roles for myeloid cells in promoting vessel sprouting in an inflammatory corneal setting.

ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse

Corneal epithelial abrasion elicits an inflammatory response involving neutrophil (PMN) recruitment from the limbal vessels into the corneal stroma. These migrating PMNs make surface contact with collagen and stromal keratocytes. Using mice deficient in PMN integrin CD18, we previously showed that PMN contact with stromal keratocytes is CD18-dependent, while contact with collagen is CD18-independent. In the present study, we wished to extend these observations and determine if ICAM-1, a known ligand for CD18, mediates PMN contact with keratocytes during corneal wound healing. Uninjured and injured right corneas from C57Bl/6 wild type (WT) mice and ICAM-1 −/− mice were processed for transmission electron microscopy and imaged for morphometric analysis. PMN migration, stromal thickness, and ICAM-1 staining were evaluated using light microscopy. Twelve hours after epithelial abrasion, PMN surface contact with paralimbal keratocytes in ICAM-1 −/− corneas was reduced to ˜ 50% of that observed in WT corneas; PMN surface contact with collagen was not affected. Stromal thickness (edema), keratocyte network surface area and keratocyte shape were similar in ICAM-1 −/− and WT corneas. WT keratocyte ICAM-1 expression was detected at baseline and ICAM-1 staining intensity increased following injury. Since ICAM-1 is readily detected on mouse keratocytes and PMN-keratocyte surface contact in ICAM-1 −/− mice is markedly reduced, the data suggest PMN adhesive interactions with keratocyte-stromal networks is in part regulated by keratocyte ICAM-1 expression.

Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains

Genetic background significantly affects angiogenesis in mice. However, lymphangiogenic response to growth factors (GFs) in different strains has not been studied. We report constitutive expression of corneal lymphatics that extends beyond the limits of normal limbal vessels. In untreated corneas, the total number (P=0.006), the number above blood vessels (P=10(-8)), and the area of preexisting lymphatics (P=0.007) were significantly higher in C57BL/6 than in BALB/c mice. Normal corneas of three other strains, the nu/nu, 129E, and Black Swiss mice, showed in most parameters intermediate phenotypes. FGF-2(-/-) mice showed significantly less preexisting lymphatics than control (P=0.009), which suggests a role for this GF in lymphatic development. VEGF-A-induced corneal lymphangiogenic response was significantly higher in BALB/c mice (P=0.03), but it did not differ significantly in C57BL/6 mice, when compared to PBS-implanted control. FGFR-3 expression was higher in C57BL/6 than BALB/c mice, which suggests GF-receptor heterogeneity as a possible explanation for strain-dependent differences. The heterogeneity of preexisting lymphatic vessels in the limbal area significantly correlated with the extent of corneal lymphangiogenesis (VEGF-A: r=0.7, P=0.01; FGF-2: r=0.96, P=10(-5)) in BALB/c but not in C57BL/6 mice. Removal of conjunctival lymphatics did not affect GF-induced lymphangiogenesis. This work introduces physiological expression of lymphatics without blood vessels, which indicates that angiogenesis and lymphangiogenesis, even though intricately related, may occur independently. Furthermore, we show strain-dependence of normal and GF-induced lymphangiogenesis. These differences may affect disease development in various strains.

Transplanted Corneal Graft With Metastatic Cholangiocarcinoma to the Donor Eye

To report a case of corneal transplantation from a donor died of cholangiocarcinoma with metastasis to the eye. A patient with limbal dermoid received corneal transplant from a donor died of cholangiocarcinoma. Pathologic examination of the remaining donor limbal tissue revealed metastasis of tumor to the limbal vessels. Prompt exchange of the graft was performed. Before the second corneal transplantation, the surrounding tissue of the recipient bed was excised and sent for histopathologic examination. No tumor transmission was noted surrounding the recipient bed. The second graft remained clear and the patient remained cancer free after regular examination for over a year. This is the first case to report that cholangiocarcinoma can metastasize to the corneal limbus. To avoid transmission of malignancies from the donor to the recipient, we suggest that donor tissue with history of malignancy should not be used for limbal allografting, and that frozen-section examination of donor limbal tissue is recommended before the transplantation.

Inhibition of Corneal Neovascularization by Blocking the Angiotensin II Type 1 Receptor

To determine the role of angiotensin II type 1 receptor (AT1R) signaling in corneal neovascularization. Corneal neovascularization was induced by suturing 10-0 nylon 1 mm away from limbal vessels in C57 BJ6 mice. Angiotensinogen and its receptor (AT1R) gene expression levels were evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The expression of angiotensin II (Ang2) and AT1R was confirmed by Western blotting and immunohistochemistry. To investigate the function of Ang2 in corneal neovascularization, infiltrating macrophages in vascularized corneas and the neovascularized area were investigated after intraperitoneal injection of an AT1R antagonist (telmisartan, 10 mg/kg). Further, corneal mRNA of VEGF, MCP-1, IL-6, ICAM-1, and TNF-alpha was examined in control and telmisartan-treated mice. Ang2 and AT1R markedly increased in the neovascularized corneas compared with normal corneas. Ang2 and AT1R were expressed in epithelium and stromal cells (vascular endothelium, infiltrating leukocytes, and keratocytes) in neovascularized cornea at protein levels and were weakly detected in normal corneal epithelium. Infiltrating macrophages were reduced in telmisartan-treated mice on day 7 after suturing. Neovascularized area in the cornea of telmisartan-treated mice was 70% smaller than that of control mice on day 7 after suturing. A PPAR-gamma antagonist partially, but significantly, reversed the suppressive effect of telmisartan on induction of corneal neovascularization. The expression of VEGF, MCP-1, IL-6, and ICAM-1 was significantly inhibited in telmisartan-treated mice. These findings indicate that Ang2, abundantly expressed in neovascularized corneas, has a significant role in inflammation-related driven corneal neovascularization. AT1R may be a therapeutic target for the suppression of corneal neovascularization.

Age-related changes in murine limbal lymphatic vessels and corneal lymphangiogenesis

Important risk factors for graft rejection after corneal transplantation are pathologic corneal lymphangiogenesis and young recipient age. Purpose of this study was to investigate whether there are age-related differences in normal murine limbal and pathologic corneal lymphatic vessels, which could partly explain the unequal outcome of corneal transplantation in young versus old recipients. Furthermore, we investigated whether these observed differences correlate with changes in allograft survival in the murine model of corneal transplantation. Corneal whole mounts from untreated young (aged 6–8 weeks), untreated old (aged 9–15 months) and young and old mice after suture-induced, inflammatory corneal neovascularization were prepared and stained with LYVE-1 as a lymphendothelial marker. Angles of corneal parts with and without a main circumferential limbal lymphatic vessel were measured and then related to the total 360° of corneal circumference. Centrally directed vascular extensions from the main limbal lymphatic vessel (“sprouts”) of previously untreated old mice were counted. Concerning the outgrowth of pathologic lymphatic vessels after inflammatory corneal neovascularization, the area covered with pathologic lymphatic vessels was detected by an algorithm on digitized whole mounts using cell^F ® software. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed with one recipient group being young, the other group being old mice. In young, untreated mice, 70.5% of the total corneal circumference was covered by a main circumferential limbal lymphatic vessel versus 60.8% in old, untreated mice. Comparing the number of centripedal vascular extensions from the main limbal lymphatic vessel (“sprouts”), untreated old mice had significantly less extensions than young, untreated mice ( p < 0.001). After an inflammatory stimulus, old mice had significantly less pathologic corneal lymphatic vessels than young mice (42% less, p < 0.001). Comparing the survival proportions after corneal transplantation, old recipient mice showed a significantly better graft survival 6 weeks after transplantation (65% versus 33%, p < 0.05). Thus, limbal lymphatic vascular sprouts and inflammation-induced pathologic corneal lymphangiogenesis decrease with age. The lower lymphangiogenic potency of older mice may explain the better outcome of corneal transplantations in old recipients, supporting the concept that lymphangiogenesis is an important risk-factor for corneal transplant rejection.

γδ T Cells Are Necessary for Platelet and Neutrophil Accumulation in Limbal Vessels and Efficient Epithelial Repair after Corneal Abrasion

Corneal epithelial abrasion in C57BL/6 mice induces an inflammatory response with peak accumulation of neutrophils in the corneal stroma within 12 hours. Platelets localize in the limbal vessels throughout the same time course as neutrophils and contribute to wound healing because antibody-dependent depletion of platelets retards epithelial division and wound closure. In the present study, T cells in the limbal epithelium were found to predominantly express the gammadelta T-cell receptor (TCR). Corneal abrasion in wild-type, CD11a(-/-), and P-sel(-/-) mice increased the numbers of gammadelta T cells in the limbal and peripheral corneal epithelium and in the corneal stroma adjacent to the limbal blood vessels. Intercellular adhesion molecule (ICAM)-1(-/-) mice exhibited a reduction in gammadelta T-cell accumulation. TCRdelta(-/-) mice exhibited reduced inflammation and delayed epithelial wound healing as evidenced by delayed wound closure, reduced epithelial cell division, reduced neutrophil infiltration, and reduced epithelial cell density at 96 hours after wounding. TCRdelta(-/-) mice also exhibited >60% reduction in platelet localization in the limbus despite similar platelet counts and platelet function assessed with an in vivo thrombosis model. These results are consistent with the conclusion that gammadelta T cells are necessary for efficient inflammation, platelet localization in the limbus, and epithelial wound healing after corneal abrasion.

Contact lens-induced changes in the anterior eye as observed in vivo with the confocal microscope

The availability of the confocal microscope over the past decade has allowed clinicians and researchers to refine their understanding of the physiological and pathological basis of the ocular response to contact lens wear, and to discover previously unknown phenomena. Mucin balls, which form in the tear layer in patients wearing silicone hydrogel lenses, can penetrate the full thickness of the epithelium, leading to activation of keratocytes in the underlying anterior stroma. Epithelial cell size increases in response to all forms of lens wear, with lenses of higher oxygen transmissibility (Dk/t) interfering least with the normal process of epithelial desquamation. A higher density of Langerhans' cells is observed in the layer of the sub-basal nerve plexus among contact lens wearers, suggesting that contact lens wear may be altering the immune status of the cornea. Dark lines and folds are observed in the oedematous cornea in response to contact lens wear. Mechanical stimulation of the corneal surface, due to the physical presence of a contact lens, and the consequent release of inflammatory mediators, is the likely cause of reduced keratocyte density associated with lens wear. Highly reflective stromal 'microdot deposits' are observed throughout the entire stroma in higher numbers in lens wearers. 'Blebs' in the endothelium have a bright centre surrounded by a dark annular shadow; this appearance is explained with the aid of an optical model. The confocal microscope has considerable clinical utility in diagnosing Acanthamoeba and fungal keratitis. At the limbus, contact lenses can induce structural changes such as increases in basal epithelial cell size. An increased number of rolling leucocytes is observed in limbal vessels in response to low Dk/t lenses. It is concluded that the confocal microscope has considerable utility in contact lens research and practice.

Anti-Inflammatory Effect of Blocking the Interaction between Platelet Glycoprotein Ibα and the Leukocyte Integrin αMβ2.

The interaction between the leukocyte integrin Mac-1 (α M β 2) and platelet glycoprotein (GP) Ibα allows leukocytes to firmly adhere to platelet thrombi, enabling their subsequent transmigration into the vessel wall. This interaction is blocked by the GP Ibα monoclonal antibody AP1, which we have previously mapped to the GP Ibα sequence Arg218 to Tyr228. In the current investigation we examined the effectiveness of a GP Ibα-based peptide containing the AP1 epitope in blocking platelet-leukocyte interactions under flow and in an inflammation model based on corneal wound healing. The peptide bound the α M I-domain specifically and saturably and was able to inhibit several GP Ibα-dependent functions under flow:the adhesion of GP Ib-IX complex-expressing CHO cells to immobilized α M I-domain,the adhesion of THP-1 cells - a monocytic cell line expressing α Mβ 2 - to immobilized glycocalicin (soluble extracellular region of GP Ibα ),adhesion of activated THP-1 cells to surface-adherent GP Ib-IX complex-expressing CHO cells, andthe binding of neutrophils to immobilized glycocalicin or a platelet monolayer.We therefore tested whether the peptide could inhibit inflammatory processes that may be dependent on the interactions between platelets and neutrophils. Neutrophils play an important role in corneal wound healing, but because the cornea is avascular, they must migrate to the wound from the limbal vessels at the periphery of the cornea. Therefore we chose this model of mouse corneal wounding as we have previously demonstrated that antibody-induced platelet depletion markedly reduces neutrophil localization and emigration from the limbal vessels and therefore postulated that platelets may directly facilitate neutrophil emigration. In our model, a 2 mm wound was made at the center of the cornea by removing the epithelium without injuring the stroma. Neutrophil infiltration across the cornea, from periphery to center, was assessed at 12 and 30 hr after the injury. Mice were injected with either AP1 peptide or control peptide (scrambled AP1 peptide) three times at 4 hour intervals beginning immediately after corneal wounding. Mice treated with AP1 peptide showed markedly fewer neutrophils infiltrating all regions of the cornea. These results indicate that the interaction between GP Ibα and Mac-1 is required for efficient leukocyte extravasation. They also suggest that drugs targeting the GP Ibα-α Mβ 2 axis could have anti-inflammatory properties without compromising hemostasis, as the peptide had minimal effects on the interaction of GP Ibα with von Willebrand factor.

Platelet Response to Corneal Abrasion Is Necessary for Acute Inflammation and Efficient Re-epithelialization

Adhesion molecules play a critical role in leukocyte emigration to wound sites, but differences are evident in different vascular beds. In this study, the contributions of P-selectin to neutrophil emigration into the cornea after central epithelial abrasion were investigated. Re-epithelialization, neutrophil influx, and platelet accumulation were assessed in C57BL/6 mice after removal of a 2-mm diameter area of central corneal epithelium that did not directly injure the limbal vessels or the avascular stroma of the cornea. Comparisons were made between wild-type (WT) mice and mice with targeted deletions of genes for P-selectin, CD18, or CD54, or mice with antibody-induced neutropenia or thrombocytopenia. After central corneal epithelial abrasion, platelets localized in the limbal vessels and neutrophils emigrated from the limbal vessels to the region of the epithelial wound. There was temporal correspondence of platelet and neutrophil localization, peaking within 12 hours of wounding. Platelet accumulation, neutrophil emigration and corneal epithelial healing as measured by wound closure, basal epithelial cell density, and epithelial cell division were significantly reduced in P-selectin-deficient mice (P-sel(-/-)). Anti-GP1balpha antibody-induced thrombocytopenia in WT mice significantly reduced platelet and neutrophil accumulation and wound healing. Passive transfer of wild-type platelets into P-sel(-/-) mice significantly restored platelet localization in limbal vessels, neutrophil emigration, epithelial cell division, and epithelial cell migration into the abraded region of the cornea. Platelet localization in the limbus of abraded corneas contributes to re-epithelialization, and P-selectin provides a necessary step in this process.

Lymphocyte Function-Associated Antigen-1-Dependent Inhibition of Corneal Wound Healing

Abrasion of murine corneal epithelium induces neutrophil emigration through limbal vessels into the avascular corneal stroma, peaking within 12 to 18 hours after wounding. A central corneal wound closes within 24 hours by epithelial cell migration and division, and during wound closure corneal epithelial cells express intercellular adhesion molecule (ICAM)-1 (CD54). We investigated the contributions of lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) by analyzing wound closure in mice with targeted deletions of CD11a (CD11a-/-) or CD11b (CD11b-/-). In contrast to CD11a-/- mice, CD11b deficiency revealed a much greater delay in epithelial wound closure with >90% inhibition of epithelial cell division at a time when neutrophil accumulation in the cornea was approximately threefold higher than normal. Treating CD11b-/- mice with anti-CD11a monoclonal antibody at the time of epithelial abrasion resulted in significant reductions in neutrophils and significant increases in corneal epithelial cell division and migration. Treating CD11b-/- mice with anti-ICAM-1 significantly increased measures of healing but marginally reduced neutrophil influx. In conclusion, wound healing after corneal epithelial abrasion is disrupted by the absence of CD11b. The disruption is apparently linked to excessive neutrophil accumulation at a time when epithelial division is essential to wound repair, and neutrophils appear to be detrimental through processes involving LFA-1 and ICAM-1.

Flap tearing during lift-flap laser in situ keratomileusis retreatment

A flap tear occurred during laser in situ keratomileusis (LASIK) retreatment using a flap-lifting technique in 1 eye of 2 patients 4 to 5 months after the primary procedure. In the first case, the tear occurred in a decentered, standard thickness flap (168 mum) in a location close to the corneal limbus and limbal vessels. In the second case, the tear occurred in a well-centered thin flap (116 mum) that involved a peripheral corneal pannus. The false track was identified early, and central extension of the tear was averted. After the flap was successfully dissected, retreatment was performed without further complications. This report suggests that flaps with margins near the limbus or a corneal pannus may be prone to an earlier and stronger healing process at the edge that may lead to a flap tear during LASIK retreatment. This may be of increasing importance because of the trend toward larger flap diameters.

Transfection of Cytochrome P4504B1 into the Cornea Increases Angiogenic Activity of the Limbal Vessels

Injury to the ocular surface induces the production of the corneal epithelial-derived 12-hydroxyeicosatetrienoic acid (12-HETrE), which exhibits stereospecific potent inflammatory and angiogenic properties and is formed by a cytochrome P450 (P450) enzyme, CYP4B1. We have cloned the rabbit corneal CYP4B1 into the expression plasmid pIRES2-enhanced green fluorescent protein (EGFP) and examined the effect of CYP4B1 overexpression on corneal inflammation in vivo and limbal vessel sprouting ex vivo. Cultured rabbit corneal epithelial cells transfected with pIRES2-EGFP-CYP4B1 metabolized arachidonic acid to 12-HETrE at a rate five times higher than that of pIRES2-EGFP-transfected cells (3.53 +/- 0.08 versus 0.62 +/- 0.10 nmol/h/10(6) cells; mean +/- S.E.M., n = 6, p < 0.05), indicating a functional expression of the CYP4B1. Injection of either plasmid into the rabbit cornea resulted in EGFP fluorescence in the corneal epithelium. However, corneal neovascularization, as measured by the length of penetrating blood vessels, was significantly greater in the corneas of eyes transfected with the pIRES2-CYP4B1 compared with pIRES2-EGFP. Corneal-limbal explants from eyes transfected with pIRES2-CYP4B1 showed a marked angiogenic activity (46 +/- 10 versus 12 +/- 3 mm capillary length, n = 6, p < 0.05), which correlated with increased levels of 12-HETrE, the CYP4B1-derived angiogenic 12-hydroxyeicosanoid (0.93 +/- 0.18 versus 0.15 +/- 0.02 pmol/explant, n = 6, p < 0.05), and was inhibited (76 +/- 5%) by the P450 inhibitor 17-octadecynoic acid. The results further implicate the corneal CYP4B1 as a component of the inflammatory and angiogenic cascade initiated by injury to the ocular surface and raise the possibility of a new therapeutic target for preventing corneal neovascularization, namely, the CYP4B1-12-HETrE system.

Dehydrated Form of Plasmid Expressing Basic Fibroblast Growth Factor–Polyethylenimine Complex Is a Novel and Accurate Method for Gene Transfer to the Cornea

Purpose: We describe a novel vector system of nonviral gene transfer into the cornea using a dehydrated form of a plasmid expressing basic fibroblast growth factor–polyethylenimine (p-bFGF-PEI) complex to induce angiogenesis. Methods: Corneal neovascularization was evaluated in 48 eyes of Sprague-Dawley rats after implantation of a dehydrated form of PEI containing 1 μ g green fluorescent protein (p-GFP-PEI; control group), or 10 μ g, 1 μ g, or 0.1 μ g of p-bFGF-PEI introduced by spin vacuum at ambient temperature. Neovascularization was observed and quantified from day 1 to day 45. Eighteen kDa bFGF protein expression was analyzed by Western blot and immunohistochemistry. Results: Limbal vessels began to sprout on day 3 in the p-bFGF-PEI groups. The dehydrated form of the p-bFGF-PEI complex induced dose-dependent corneal neovascularization, which reached a maximum on days 24–30 in the 10 μ g bFGF group, days 18–24 in the 1 μ g bFGF group, and days 15–21 in 0.1 μ g bFGF group, and then regressed progressively. No neovascularization was observed in the GFP group. Conclusions: The dehydrated form of the p-bFGF-PEI complex is a novel and precise method for controlling the dose, localizing the reagents, and avoiding loss of liquid form during transfection into corneal tissue.

The rabbit corneal pocket assay for the study of angiogenesis.

The corneal pocket assay consists of the placement of a stimulus (purified growth factor, cell suspension, tumor tissue) into a corneal pocket in order to induce a vascular outgrowth from the limbal vessels toward the implant. This assay, with respect to the other in vivo assays, such as the chorio-allantoic membrane, has the advantage of measuring only new blood vessels, since the cornea is normally avascular. By using rabbits, additional advantages are gained like the easy manipulation of the animals and monitoring of the neovascular growth and inflammatory reaction over time. Measurement of corneal angiogenesis is useful to quantify the effects of angiogenic stimuli and to evaluate the efficacy of potential inhibitors of neovascularization following local delivery or systemic treatment.

Corneal lymphangiogenesis: evidence, mechanisms, and implications for corneal transplant immunology.

The normal cornea is devoid of blood and lymphatic vessels but can become vascularized secondary to a variety of corneal diseases and surgical manipulations. Whereas corneal (hem)angiogenesis, i.e., the outgrowth of new blood vessels from preexisting limbal vessels, is obvious both clinically and histologically, proof of associated corneal lymphangiogenesis has long been hampered by invisibility and lack of specific markers. This has changed with the recent discovery of the lymphatic endothelial markers vascular endothelial growth factor receptor 3, LYVE-1 (a lymphatic endothelium-specific hyaluronan receptor), Prox 1, and Podoplanin. We herein summarize the current evidence for lymphangiogenesis in the cornea and describe its molecular markers and mediators. Furthermore, the pathophysiologic implications of corneal lymphangiogenesis for corneal transplant immunology are discussed. Whereas corneal angiogenesis in vascularized high-risk beds provides a route of entry for immune effector cells to the graft, lymphangiogenesis enables the exit of antigen-presenting cells and antigenic material from the graft to regional lymph nodes, thus inducing alloimmunization and subsequent graft rejection. Antilymphangiogenic strategies may improve transplant survival both in the high- and low-risk setting of corneal transplantation.

The limbal vasculature

The cornea is an avascular structure whose closest point of approach to the systemic blood stream is provided by the limbal vessels. Activity within these structures provides the clinician with a sensitive indicator of contact lens performance and associated problems. In this paper, the anatomy and physiology of the limbal vessels is reviewed with particular attention to the control of capillary perfusion. Mechanisms whereby soft contact lenses can interact with this network of vessels are considered.

An Essential Role for Antibody in Neutrophil and Eosinophil Recruitment to the Cornea: B Cell-Deficient (μMT) Mice Fail to Develop Th2-Dependent, Helminth-Mediated Keratitis

Abstract Invasion of the corneal stroma by neutrophils and eosinophils and subsequent degranulation disrupts corneal clarity and can result in permanent loss of vision. In the current study, we used a model of helminth-induced inflammation to demonstrate a novel role for Ab in mediating recruitment of these inflammatory cells to the central cornea. C57BL/6 and B cell-deficient (μMT) mice were immunized s.c. and injected intrastromally with Ags from the parasitic helminth Onchocerca volvulus (which causes river blindness). C57BL/6 mice developed pronounced corneal opacification, which was associated with an Ag-specific IL-5 response and peripheral eosinophilia, temporal recruitment of neutrophils and eosinophils from the limbal vessels to the peripheral cornea and subsequent migration to the central cornea. In contrast, the corneas of μMT mice failed to develop keratitis after intrastromal injection of parasite Ags unless Ags were injected with immune sera. Eosinophils were recruited from the limbal vessels to the peripheral cornea in μMT mice, but failed to migrate to the central cornea, whereas neutrophil recruitment was impaired at both stages. With the exception of IL-5, T cell responses and peripheral eosinophils were not significantly different between C57BL/6 and μMT mice. Taken together, these findings not only demonstrate that Ab is required for the development of keratitis, but also show that recruitment of neutrophils to the cornea is Ab-dependent, whereas eosinophil migration is only partially dependent upon Ab interactions.

Ocular thrombosis and retinal degeneration induced in female F344 rats by 2-butoxyethanol.

2-butoxyethanol, used extensively for domestic and industrial purposes, was tested in our experiments for its potential to cause damage to female rat ocular tissues. Female rats were previously found to be particularly sensitive to 2-butoxyethanol. A group of eight female F344 rats (2 - 3 months old) were exposed by gavage to 250 mg of 2-butoxyethanol/kg b.w. per day for 3 consecutive days and sacrificed 24 h after the last dose. Eight female rats received the dosing vehicle (water) and served as controls. At necropsy, petechial hemorrhages were noted on the sclera. Microscopic examination revealed treatment-related effects in the eyes, in addition to other known effects of BE exposure such as disseminated thrombosis and necrosis and infarction in various organs. The spectrum of histopathological changes noted in the eyes included hemorrhages localized in the posterior layers of the retina, leading to photoreceptor degeneration. Thrombi were identified in ciliary processes and limbal blood vessels. Histological changes suggestive of the retinal ischemic-infarctive process were also noted. Possible pathogenic mechanisms of 2-butoxyethanol-induced retinopathy are discussed.

Vascular endothelial growth factor C induces angiogenesis in vivo.

Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects in vivo. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas induced by VEGF-C is intensive, with a high density of new capillaries. However, the outgrowth of microvessels stimulated by VEGF-C was significantly longer than that induced by VEGF. In the developing embryo, VEGF-C was able to induce branch sprouts from the established blood vessels. VEGF-C also induced an elongated, spindle-like cell shape change and actin reorganization in both VEGF receptor (VEGFR)-2 and VEGFR-3-overexpressing endothelial cells, but not in VEGFR-1-expressing cells. Further, both VEGFR-2 and VEGFR-3 could mediate proliferative and chemotactic responses in endothelial cells on VEGF-C stimulation. Thus, VEGF-C may regulate physiological angiogenesis and participate in the development and progression of angiogenic diseases in addition to lymphangiogenesis.