Vitreous M2 Macrophage-Derived Microparticles Promote RPE Cell Proliferation and Migration in Traumatic Proliferative Vitreoretinopathy.

Purpose: To characterize vitreous microparticles (MPs) in patients with traumatic proliferative vitreoretinopathy (PVR) and investigate their role in PVR pathogenesis. Methods: Vitreous MPs were characterized in patients with traumatic PVR, patients with rhegmatogenous retinal detachment (RRD) complicated with PVR, and control subjects by flow cytometry. The presence of M2 macrophages in epiretinal membranes was measured by immunostaining. Vitreous cytokines were quantified by ELISA assay. For in vitro studies, MPs isolated from THP-1 cell differentiated M1 and M2 macrophages, termed M1-MPs and M2-MPs, were used. The effects and mechanisms of M1-MPs and M2-MPs on RPE cell proliferation, migration, and epithelial to mesenchymal transition were analyzed. Results: Vitreous MPs derived from photoreceptors, microglia, and macrophages were significantly increased in patients with traumatic PVR in comparison with control and patients with RRD (PVR), whereas no significance was identified between the two control groups. M2 macrophages were present in epiretinal membranes, and their signature cytokines were markedly elevated in the vitreous of patients with traumatic PVR. Moreover, MPs from M2 macrophages were increased in the vitreous of patients with traumatic PVR. In vitro analyses showed that M2-MPs promoted the proliferation and migration of RPE cells via activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. However, M2-MPs did not induce the expression of fibrotic proteins, including fibronectin, α-smooth muscle actin, and N-cadherin in RPE cells. Conclusions: This study demonstrated increased MP shedding in the vitreous of patients with traumatic PVR; specifically, MPs derived from M2 polarized macrophages may contribute to PVR progression by stimulating RPE cell proliferation and migration.

Connexin43 knockdown accelerates wound healing but inhibits mesenchymal transition after corneal endothelial injury in vivo.

PURPOSE: To explore connexin43 (Cx43) knockdown as an efficient treatment for corneal endothelial injury in an in vivo rat corneal scrape injury model. METHODS: Scrape injury was induced in the corneal endothelium, and immunolabeling (ZO-1, alpha-SMA, Cx43) was performed to analyze changes in Cx43 expression during wound healing. Single injection of Cx43 antisense oligodeoxynucleotide (AS-ODN), small interfering RNA (siRNA), or adenovirus (CMV-Cx43-mRFP1) was applied into the anterior chamber simultaneously with the injury, and wound closure was examined by immunolabeling (ZO-1, Cx43) and propidium iodide staining. Corneal endothelium proliferation on day 1 after injury was studied by Ki67-immunolabeling. Cx43-knockdown treatment was performed also without injury, and its effect on Cx43 expression and Ki67 immunolabeling was examined. The postinjury appearance of myofibroblasts in Cx43 AS-ODN- and sense-ODN-treated corneas was compared by alpha-SMA-immunolabeling. RESULTS: Complete wound closures were observed in five of six corneas on day 3 after injury with either Cx43 AS-ODN or siRNA treatment, whereas no complete closure was observed on day 3 in the control corneas (S-ODN, zero of six; or nonsense siRNA, zero of six). Consistently, Cx43 overexpression using adenovirus delayed wound closure. Cx43 knockdown increased the number of Ki67-positive proliferating cells on day 1, whereas it decreased the number of alpha-SMA-positive myofibroblasts on day 5. Cx43 knockdown without injury decreased Cx43 expression and induced endothelial proliferation in vivo. CONCLUSIONS: These results show that Cx43 knockdown induces corneal endothelium proliferation but inhibits endothelial-mesenchymal transition/transformation after injury, suggesting that Cx43 knockdown is a new therapeutic approach for acceleration of wound closure and for prevention of retrocorneal fibrous membrane formation.

Vascular endothelial growth factor in aqueous humor of patients with perforative eye injuries.

Considering that VEGF is the key factor for angiogenesis stimulation, we wanted to establish if VEGF level is increased in aqueous humor of patients with open globe eye injury. The study included 20 patients with open globe injury. During the surgery, aqueous humor samples were taken out and VEGF levels were measured by ELISA. VEGF levels were significantly higher in the aqueous humor of patients with open globe eye injury and uveitis, in patients with wound bigger than 2 mm and in patients where from injury to surgery passed more than 4 hours. VEGF levels were also higher, but not significantly, in patients with intrabulbar foreign body. Considering that VEGF levels were significantly higher in patients with open globe eye injury with uveitis, wound larger than 2 mm and in patients where from injury to surgery passed more than 4 hours, anti VEGF therapy might have application in these conditions.

[Intraocular ferritin production due to an iron-containing intraocular foreign body]. / Synthèse intraoculaire de ferritine suite à un corps étranger intraoculaire ferrique.

INTRODUCTION: Ocular siderosis is a rare but severe complication of open globe trauma with intraocular retention of a metallic foreign body. CASE STUDY: We report a case of recurrent uveitis in a 37-year-old patient. The ophthalmic examination revealed poor vision in the left eye, lid edema, limbal scleromalacia, hyphema and severe ocular hypertension. Orbital CT showed the presence of a radio-opaque IOFB between the crystalline lens and vitreous body. An aqueous humor sample was obtained for iron and ferritin levels. The results came back 100 and 2000 times higher, respectively, than the serum reference values. DISCUSSION: The very high iron content is the result of a sustained release from the metallic INFB and is responsible for ocular siderosis in our patient. The extremely high ferritin level would be the result of in situ synthesis by the various cells of the ocular structures in order to preserve the components of the eye. Measurement of these two levels would improve the diagnosis, prognosis and treatment of metallic IOFBs.

Angiostatic effect of penetrating ocular injury: role of pigment epithelium-derived factor.

PURPOSE: To characterize the angiostatic effect of penetrating ocular injury and to begin to explore its mechanism, with an emphasis on the role of pigment epithelium-derived factor (PEDF). METHODS: Using the rat model of oxygen-induced retinopathy (OIR), single or multiple dry needle injuries were made, penetrating the globe of one eye; the opposite eye served as a control. Eyes were harvested from rats killed 1, 3, and 6 days after injury, and retinas were dissected and processed for assessment of neovascularization and microglial activation or were processed for genetic and proteomic analysis. Temporal and spatial expression patterns of PEDF were analyzed by in situ hybridization. RESULTS: Penetrating ocular injury resulted in a 30% decrease in neovascular area in the retinas of OIR rats. At day 1 after injury, needle insertion caused a 4.1-fold increase in retinal PEDF mRNA and a 1.5-fold increase in retinal PEDF protein. Vitreous PEDF protein increased 3.4-fold in injured eyes compared with noninjured eyes. In situ hybridization showed an increase in PEDF mRNA in areas surrounding the puncture site. Concentrated vitreous protein from injured eyes caused a 60% decrease in retinal neovascularization when injected into the vitreous cavity of OIR rats. Preincubation of vitreous samples with anti-PEDF partially abolished this efficacy. CONCLUSIONS: The pattern of angiostasis resulting from penetrating ocular injury is consistent with the release of an endogenous antiangiogenic factor from the wound site. Preliminary studies show a possible role for PEDF in this effect. Further characterization of this role and the identification of other factors may lead to new therapeutic strategies for angiogenic eye conditions.

Role of angiotensin II receptor subtypes in conjunctival wound healing.

PURPOSE: To investigate the role of angiotensin II (Ang II) receptor subtypes in subconjunctival injury. METHODS: A wound-healing model was developed by subconjunctival blunt dissection in male wild-type, AT1a receptor-deficient (AT1aKO) and AT2 receptor-deficient (AT2KO) mice. Collagen deposition and cell infiltration were evaluated histologically. Expression of collagen, matrix metalloproteinase (MMP), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by real-time PCR. RESULTS: Subconjunctival injury increased the infiltration of inflammatory cells, collagen deposition in the subconjunctival space, and the expression of collagen type I and type III, TIMP-1 and MMP2. In AT1aKO mice, collagen deposition, cell infiltration, and expression of collagen and TIMP-1 were inhibited, but MMP2 expression was enhanced. In contrast, in AT2KO mice, the increase in collagen deposition, cell infiltration, and expression of collagen and TIMP-1 were further enhanced. CONCLUSIONS: These results indicate that AT1a and AT2 receptor stimulation may in addition to other mechanisms be antagonistically involved in the wound-healing process after subconjunctival injury.

Immunohistochemical Characterization of the Ectopic Epithelium Devoid of Goblet Cells From a Posttraumatic Iris Cyst Causing Mucogenic Glaucoma.

PURPOSE: Mucogenic glaucoma is an unusual form of secondary open-angle glaucoma caused by intracameral ectopic mucus-producing epithelium. To date, only 3 cases have been described in detail. Numerous goblet cells in the specimens indicated a possible conjunctival origin. We immunohistochemically characterized the implanted epithelium from an iris cyst responsible for mucogenic glaucoma. METHODS: A series of immunostaining analyses were performed on a sector-iridectomy specimen derived from an eye with mucogenic glaucoma and a history of limbal penetrating injury. An iris cyst was present in the inferonasal quadrant of the right eye of a 58-year-old man. The anterior chamber was filled with hazy, translucent material, and the chamber angle was gonioscopically open. The cyst was resected due to medically uncontrollable high intraocular pressure. RESULTS: The ectopic epithelium was mostly positive for CK19, a corneal and conjunctival epithelial marker. Negative staining for MUC5AC, a secretory mucin, and positive staining for MUC1, a membrane-bound mucin, corroborated the absence of goblet cells. Ectopic epithelial cells were abundantly positive for CK15, a limbal basal cell marker, but there was patchy immunostaining of CK13, a conjunctival epithelial marker, and sparse labeling with CK12, a corneal epithelial marker. Immunostaining patterns of CK15, CK13, and CK12 were nearly mutually exclusive. CONCLUSIONS: The ectopic epithelium of an iris cyst causing mucogenic glaucoma was most likely to originate from limbal basal cells, which showed dual direction of differentiation toward both the conjunctival and corneal epithelia. The membrane-bound mucin may have caused mucogenic glaucoma in the absence of goblet cells.

Absence of scar formation in human donor cornea with prior laser in situ keratomileusis.

PURPOSE: To investigate transglutaminases (enzymes capable of cross-linking extracellular matrix proteins to proteolysis-resistant complexes during scar tissue formation) in a human donor cornea after successful laser in situ keratomileusis (LASIK) without clinical complications and to compare with the results in a human donor cornea with corneal scarring after corneal injury. SETTING: Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany. METHODS: A donor cornea with prior uneventful LASIK treatment and 1 with corneal scarring after penetrating injury were investigated. Cryostat sections were stained immunohistochemically for tissue transglutaminase (tTG), keratocyte transglutaminase (kTG), and their reaction product epsilon-(gamma-glutamyl)-lysine. RESULTS: With light microscopy, the flap interface of the LASIK-treated eye could hardly be detected, while in the injured eye, infiltration of cells and a clear margin next to the scar formation were present. Immunohistochemistry demonstrated a distinct staining for tTG, kTG, and epsilon-(gamma-glutamyl)-lysine in the corneal scar. In contrast, neither transglutaminase nor epsilon-(gamma-glutamyl)-lysine staining could be observed at the flap margin or in the interface of the LASIK-treated donor eye. CONCLUSIONS: Irreversible protein cross-linking of transglutaminases via epsilon-(gamma-glutamyl)-lysine connections seem to be indicators for scarring in corneal wound healing. The absence of transglutaminases and their reaction product epsilon-(gamma-glutamyl)-lysine in a LASIK-treated cornea supports the idea of missing scar tissue formation after LASIK surgery.

Elevated mRNA expression of brain-derived neurotrophic factor in retinal ganglion cell layer after optic nerve injury.

PURPOSE: Recent studies show that exogenous brain-derived neurotrophic factor (BDNF) can promote retinal ganglion cell survival in vivo and in vitro. BDNF is expressed by a subpopulation of cells in the ganglion cell layer (GCL). To investigate whether endogenous BDNF may play a role in neuronal protection after ganglion cell trauma, BDNF expression in the retina was examined after optic nerve (ON) injury. METHODS: The optic nerve in Sprague-Dawley rats was crushed intraorbitally posterior to the optic disc. For controls, the optic nerve on the opposite side in each animal was similarly exposed but was not crushed. After intervals of 6 hours to 6 weeks, eye tissues were processed for in situ hybridization, Northern blot, and RNase protection assay using radiolabeled rat riboprobes. RESULTS: After ON injury, BDNF expression was significantly elevated in cells restricted to the GCL, and more cells demonstrated expression of BDNF than were observed in the controls. Elevated BDNF expression was first observed at 24 hours, peaked at 48 hours, and declined to the basal level 2 weeks after ON injury. Quantitative analysis showed a fivefold to sixfold increase in the number of BDNF-positive cells and a 54% increase in BDNF signal intensity in individual cells in the GCL 48 hours after ON injury. In control retinas without ON injury, BDNF expression was localized to some cells in the GCL, as was observed in normal eyes without surgery. Northern blot and RNase protection assay demonstrated a 38% elevation in BDNF expression above control levels 48 hours after ON injury. CONCLUSIONS: These results indicate that cells in the GCL can upregulate gene expression of BDNF in response to ganglion cell axonal injury and suggest that endogenous BDNF may contribute to a natural neuroprotective process after ON injury.

Elevation of transforming growth factor alpha in cat aqueous humor after corneal endothelial injury.

PURPOSE: To determine if a scrape injury to cat corneal endothelial cells increases the level of mitogenic proteins such as transforming growth factor alpha (TGF alpha) in aqueous humor. METHODS: Aqueous humor of cats was collected at 0, 2, 6, and 24 hours after wounding the endothelium by contact with a cannula tip. Aqueous humor samples collected from sham-wounded cats served as controls. Aqueous humor samples were analyzed for levels of protein, for mitogenic activity using incorporation of tritiated thymidine by cultures of bovine corneal endothelial cells, and for immunoreactive TGF alpha protein using a specific radioimmunoassay. RESULTS: The average protein level in aqueous humor obtained before wounding was low (0.5 mg/ml), increased 26-fold at 2 hours after injury (13 mg/ml), then progressively decreased at 6 hours (8 mg/ml) and 24 hours (2 mg/ml). Levels of mitogenic activity of aqueous humor samples collected 2, 6, and 24 hours after wounding were 2-fold, 2.5-fold, and 0.6-fold higher, respectively, compared to the level of mitogenic activity measured in aqueous humor collected before wounding (0 hours) or in aqueous humor collected from sham-wounded eyes. TGF alpha concentration in aqueous humor collected before endothelial wounding was low (6.8 ng/ml), increased 14-fold 2 hours after wounding (97.4 ng/ml), then progressively decreased at 6 hours (63.3 ng/ml) and 24 hours (35.5 ng/ml) after wounding. TGF alpha concentrations in aqueous humor collected from sham-wounded eyes at 2 hours (9.5 ng/ml) and 6 hours (5.3 ng/ml) were not significantly different from prewound levels. Detergent extracts of bovine corneal endothelial cells contained substantial levels of TGF alpha immunoreactive protein (20 ng/mg protein). CONCLUSIONS: Wounding of cat endothelium causes a rapid increase in mitogenic proteins in aqueous humor including TGF alpha, which may act by an autocrine mechanism to stimulate endothelial wound healing.

C-fos gene expression in rat retinal cells after focal retinal injury.

PURPOSE: To examine the expression of c-fos proto-oncogene in the rat retina after focal retinal injury. METHODS: A penetrating wound was made by pushing a 22-gauge hypodermic needle through the retina into the vitreous. The retinas were analyzed by in situ hybridization using single-stranded RNA probes for c-fos transcripts and by immunocytochemistry using anti-S100 protein antibody. RESULTS: Thirty minutes after the penetrating wound, c-fos mRNA was expressed in the inner nuclear layer (INL) and the ganglion cell layer (GCL) of the retina. Immunocytochemistry before in situ hybridization demonstrated c-fos mRNA surrounding the S-100 protein immunoreactive cytoplasm in the middle layer of the INL. CONCLUSIONS: Focal retinal injury of the rat retina induced the expression of c-fos mRNA in retinal cells. In the INL, it is suggested that the major cells that express c-fos mRNA after focal retinal injury were the Müller cells. These results suggest that c-fos may be involved in the transcription in the Müller cells after injury to the retina.

Expression of collagen I, smooth muscle alpha-actin, and vimentin during the healing of alkali-burned and lacerated corneas.

PURPOSES: Alkali-burned corneas can seldom heal properly to restore corneal transparency. To provide a better understanding of this devastating corneal injury, we compared the expression of collagen I, smooth muscle alpha-actin (alpha-SMA), and vimentin in lacerated and alkali-burned rabbit corneas. METHODS: A radiolabeled cDNA probe of alpha 1(I) chain was used in slot-blot hybridization to determine the levels of alpha 1(I) mRNA in alkali-burned corneas. In situ hybridization was used to identify the cell types that express the alpha 1(I) chain. Antibodies against collagen I, alpha-SMA, and vimentin were used in immunohistochemical studies to determine the tissue distribution of collagen I and to identify cells expressing alpha-SMA and vimentin. RESULTS: The levels of alpha 1(I) mRNA in alkali-burned corneas increased steadily after the alkali burn and reached a plateau within 2 weeks. One day after alkali burn, specific in situ hybridization signals were detected in stromal cells immediately surrounding the edge of the corneal injury. As the healing proceeded, the fibroblastic cells migrated into the injured stroma, and they showed positive reactions by in situ hybridization and by immunostaining with anti-collagen I probes. In alkali-burned corneas, retrocorneal membranes were formed 1 week after injury. This fibrillar membrane was stained by anti-collagen I antibody, and the fibroblastic cells in the membrane were hybridized by the 3H-labeled alpha 1(I) cDNA probe. No retrocorneal membrane was formed in the lacerated corneas, even after the injured corneas were allowed to heal for 3 weeks. The epithelial cells in the epithelial plug of lacerated corneas were positive by in situ hybridization, whereas the epithelial cells in the regenerated epithelium of alkali-burned cornea was not. Antibodies against alpha-SMA reacted with the migrating fibroblastic cells but did not react with epithelial cells or endothelial cells in the injured corneas. Anti-vimentin antibody reacted with fibroblastic cells, endothelial cells, and keratocytes in normal and injured corneas, and with the basal epithelial cells of injured corneas. CONCLUSIONS: During wound healing, the keratocytes that migrate to injured stroma transform into myofibroblasts. These myofibroblasts express high levels of alpha 1(I) mRNA, alpha-SMA, and vimentin. The healing of alkali-burned corneas differ from that of lacerated corneas in that the retrocorneal membranes are formed in the former but not in the latter. In addition, the epithelial cells of alkali-burned corneas lack alpha 1(I) mRNA, whereas it is found in the epithelium of lacerated corneas. These differences may result from the persistence of inflammatory cells in the alkali-burned corneas.

Rapid upregulation of fibroblast growth factor receptor 1 (flg) by rat photoreceptor cells after injury.

PURPOSE: To determine the mechanism by which basic fibroblast growth factor (bFGF) exerts its neuroprotective effects on degenerating or injured photoreceptors. METHODS: Confocal immunofluorescence microscopy was used to identify sites of bFGF and FGF receptor 1 (FGFR1) expression after focal injury or experimental retinal detachment in adult rats. FGFR1 expression was analyzed immunohistochemically and at the transcription level in single photoreceptor cells, after reverse transcription (RT), using the polymerase chain reaction (PCR). Real time quantitative RT-PCR was used to measure changes in FGFR1 mRNA levels in the retina in response to injury or detachment. RESULTS: Confocal immunofluorescence observations showed that FGFR1 immunoreactivity in the rat retina is concentrated primarily in the perinuclear cytoplasm of photoreceptor cell bodies. Reverse transcription of total RNA derived from dissociated rat photoreceptor cells, followed by amplification of FGFR1 cDNA using the PCR, verified the presence of FGFR1 transcripts in normal rat photoreceptor cells; in contrast, no evidence of bFGF transcription was detected. Collectively, these results provide compelling evidence for FGFR1 gene expression by rat photoreceptors in situ. Within hours after experimental retinal detachment or focal injury, there is a twofold increase in FGFR1 immunoreactivity in the outer nuclear layer that persists for at least 7 days; a similar increase in bFGF immunoreactivity in the interphotoreceptor matrix is also apparent. This increase in FGFR1 protein levels after detachment and injury also was confirmed by western blot analysis. Real time quantitative RT-PCR analyses revealed that a rapid upregulation of FGFR1 mRNA occurred within 12 hours after retinal injury/detachment, but then declined to near baseline levels by 24 hours. CONCLUSIONS: This body of evidence strongly suggests that the photoreceptor rescue effect elicited by retinal injury as well as by injection of exogenous bFGF is mediated, at least in part, by upregulation of the FGFR1 by the photoreceptor cells.

Response of lens epithelial cells to injury: role of lumican in epithelial-mesenchymal transition.

PURPOSE: Lens epithelial cells (LECs) undergo epithelial-mesenchcymal transition (EMT) after injury and transform into myofibroblasts positive for alpha-smooth muscle actin (alphaSMA), an established marker of this process. Lumican is a keratan sulfate proteoglycan core protein. This study was conducted to examine whether human and mouse LECs express lumican after injury. To determine whether lumican may modulate EMT of LECs in response to injury or to exposure to transforming growth factor-beta2 (TGFbeta2), alphaSMA expression by the LECs was examined in lumican (Lum)-knockout mice in vivo and in organ culture. METHODS: Human postoperative capsular specimens and healing, injured mouse lenses at various intervals were immunostained for lumican or alphaSMA. alphaSMA was also immunolocalized in healing, injured lenses of Lum-knockout mice. Finally, expression of lumican and alphaSMA was examined in lenses of Lum-knockout mice incubated with TGFbeta2. RESULTS: Lumican was immunolocalized in matrix in human postoperative capsular opacification. Lumican and alphaSMA were upregulated in mouse LECs from 8 hours and day 5 after an injury, respectively. LECs accumulated adjacent to the capsular break were of epithelial shape in Lum(-/-) mice and fibroblast-like in Lum(+/-) mice during healing. alphaSMA expression by LECs was significantly delayed in Lum(-/-) mice, indicating that lumican may modulate injury-induced EMT in LECs. TGFbeta2-induced EMT appeared to be suppressed in organ-cultured lenses of Lum(-/-) mice compared with those of Lum(+/+) mice. CONCLUSIONS: Human capsular opacification contains lumican, and mouse LECs upregulate lumican and alphaSMA in response to injury. Loss of lumican perturbs EMT of mouse LECs.

Ocular wounding prevents pre-retinal neovascularization and upregulates PEDF expression in the inner retina.

PURPOSE: Perforation injury to the eye can protect against retinal degeneration and pigment epithelial derived factor (PEDF) may play a role in this neuro-protective effect. PEDF has also been shown to possess potent anti-angiogenic properties. The current study has investigated a possible anti-angiogenic effect of penetrating ocular injury in a murine model of oxygen induced proliferative retinopathy (OIR) and determined if such a procedure alters PEDF expression in the retina. METHODS: OIR was produced by exposure of neonatal mice to 75% oxygen between postnatal days 7 and 12 (P7-P12). Mice were separated into various groups, with one group receiving a penetrating injury in a single eye. Pre-retinal neovascularization and intra-retinal ischaemia was quantified at P17 and PEDF protein expression was determined using immunofluorescence in retinal flatmounts and sections. PEDF mRNA was quantified using real-time RT-PCR. RESULTS: Punctured eyes showed less pre-retinal neovascularization at P17 when compared to the non-punctured eyes (p<0.001) although there was no effect on retinal ischaemia. PEDF immunreactivity was strongest in the ganglion cells of the retina, and intensity increased in punctured eyes at P13. There was more immunoreactive PEDF in ganglion cells adjacent to retinal venules than arterioles. At P13, retinal PEDF mRNA was also increased in punctured eyes compared to non-punctured controls (p<0.05), although there was no differential at P17. CONCLUSIONS: Penetrating ocular injury suppresses retinal neovascularization and modulates expression of PEDF. These findings have implications for intra-vitreal delivery of angiostatic agents since ocular perforation may provoke an acute, endogenous anti-angiogenic response that should be taken into account.

Fibroblast growth factor 2: roles of regulation of lens cell proliferation and epithelial-mesenchymal transition in response to injury.

PURPOSE: To examine the role of fibroblast growth factor 2 (FGF2) in regulating lens cell proliferation and epithelial-mesenchymal transition (EMT) in response to injury. METHODS: The amount of FGF2 protein was determined in healing, injured rat lenses by enzyme immunoassay. The effects of FGF2 and transforming growth factor beta2 (TGFbeta2) on cell proliferation of alphaTN4 cells (a mouse lens epithelial cell line) were determined. FGF2-knockout mice were used to further examine the role of endogenous FGF2 on injury-induced epithelial cell proliferation and EMT. The anterior lens capsule was injured by a hypodermic needle under both general and topical anesthesia in one eye of 34 fgf2+/+ mice and 42 fgf2-/- mice. At days 2, 5, and 10 post-injury the mice were sacrificed following a 2 h labeling period with bromo-deoxyuridine (BrdU). The number of BrdU-positive cells in each specimen was determined. RESULTS: A capsular break caused a 10 fold increase of FGF2 protein accumulated in rat lens 14 days after injury. Addition of 3.43 ng/ml FGF2 enhanced proliferation of alphaTN4 cells. This occurred in the presence or absence of exogenous TGFbeta2, that has an inhibitory effect on alphaTN4 cell proliferation. Significantly fewer BrdU-labeled cells were found in fgf2-/- mice than in fgf2+/+ mice during healing post-injury. However, lacking FGF2 did not alter the expression patterns of alpha-smooth muscle actin and collagen type I, markers of EMT in lens cells. CONCLUSIONS: Endogenous FGF2 is required for increased cell proliferation but not essential for EMT during the lens response to injury.

A long-term follow-up of metallic intraocular foreign bodies, employing diagnostic x-ray spectrometry.

We describe 10 eyes harboring metallic intraocular foreign bodies (IOFBs) for 9 to 46 years that had undergone diagnostic x-ray spectrometry to measure metal dissolution 9 years before this study. Findings from the present clinical and laboratory examinations, including perimetry, electroretinography, electrooculography, and diagnostic x-ray spectrometry examinations are reported. Deterioration in visual acuity consequent to metal dissolution of the IOFB was found in only one eye. Visual fields and electrophysiologic performances deteriorated in two other eyes for reasons that were probably unrelated to IOFB dissolution. Visual functions remained stable during the follow-up period in seven eyes, although excess copper levels were demonstrated in one of them. Eight of 10 eyes retained visual acuity of 6/24 or better. Diagnostic x-ray spectrometry confirmed siderosis in one patient and ruled out metallosis in the two patients with deteriorated vision for reasons that were unrelated to metallosis. The favorable outcome of this series of patients may influence one to consider a more conservative approach in the decision to remove IOFBs. A careful, life-long follow-up might be considered with periodic electroretinographic and diagnostic x-ray spectrometry examinations; the IOFB should be promptly removed if and when metallosis with damage to visual functions is demonstrated.

High expression of chemokines Gro-alpha (CXCL-1), IL-8 (CXCL-8), and MCP-1 (CCL-2) in inflamed human corneas in vivo.

OBJECTIVE: To investigate in vivo expression of chemokines in normal and inflamed human corneas, to determine whether chemokines are responsible for the recruitment of inflammatory cells. METHODS: In situ hybridization of the CXC chemokines growth-related oncogene-alpha (Gro-alpha) (CXCL-1), interleukin 8 (CXCL-8), macrophage interferon-gamma inducible gene (CXCL-9), and interferon-gamma inducible protein 10 (CXCL-10) and of the CC chemokines macrophage chemoattractant protein 1 (MCP-1) (CCL-2), macrophage inflammatory protein 1alpha (CCL-3), and regulated on activation, normal T-cell expressed and secreted (CCL-5) was performed to localize chemokine messenger RNA. Immunohistochemistry was used to identify the cellular infiltrate within the cornea. Three normal human eyes were compared with eyes enucleated because of chronic inflammation (n = 10), secondary to perforating injuries. RESULTS: In normal corneas, no chemokine expression was detected. In inflamed lesions, a high intensity of signals from Gro-alpha (CXCL-1) and MCP-1 (CCL-2) messenger RNA was observed in limbal epithelium and from Gro-alpha (CXCL-1), interleukin 8 (CXCL-8), and MCP-1 (CCL-2) in corneal stroma. The Gro-alpha (CXCL-1) was the only chemokine expressed by central corneal epithelium. All other examined chemokines were only moderately expressed in limbus and corneal stroma, or barely detectable. CONCLUSIONS: These cytokines are important agents in the cytokine network and contribute to the cell-specific and spatially restricted recruitment of neutrophils and mononuclear cells in acute inflammatory lesions of the human cornea. Clinical Relevance Understanding the role of chemokines in corneal inflammation may lead to the development of a selective receptor blockage of highly expressed chemokines to inhibit the recruitment of leukocyte subsets.

The effect of trauma on the ocular penetration of intravenous ciprofloxacin.

PURPOSE: To study the intraocular pharmacokinetics of intravenously administered ciprofloxacin following eye trauma. METHODS: Twenty-three New Zealand albino rabbits and 12 Yorkshire pigs each received a surgically induced scleral injury to the right eye. Following repair, each rabbit received a single 30-mg intravenous infusion of ciprofloxacin. Each pig received either two 200-mg doses or two 400-mg doses of intravenous ciprofloxacin given 12 hours apart. Vitreous and serum samples were harvested at 0.5, 1, 4, 6, and 12 hours after antibiotic administration in rabbits and 1 hour after the second dose in pigs. Bioassays for ciprofloxacin were performed on each sample, and results were statistically compared by t test. The untraumatized left eye in each animal served as a control. RESULTS: The mean vitreous concentration of ciprofloxacin in traumatized rabbit eyes was 0.37 microgram/ml. This level was sustained above levels in control eyes (0.18 microgram/ml) for at least 4 hours following antibiotic administration. In control eyes, intravitreal levels peaked at 1 hour. Mean vitreous concentrations +/- SD in traumatized pig eyes were 0.091 +/- 0.017 microgram/ml in swine that had received 200-mg doses of ciprofloxacin vs 0.312 +/- 0.153 microgram/ml in swine that had received 400-mg doses (P = .02). Mean vitreous concentrations of ciprofloxacin in control eyes were not affected by increasing dosage. CONCLUSION: In both animal models, experimental surgical trauma increased intravitreal ciprofloxacin delivery. In addition, systemically administered ciprofloxacin achieved intravitreous levels exceeding minimum inhibitory concentrations for common ocular pathogens, suggesting a role for ciprofloxacin in the prophylaxis of posttraumatic endophthalmitis.