Expressions of matrix metalloproteinases-1 and -9 and opioid growth factor in rabbit cornea after lamellar keratectomy and treatment with 1% nalbuphine (original) (raw)
Veterinary Ophthalmology, 2012
Purpose To study the effects of topical administration of 1% morphine on corneal analgesia in rabbits submitted to lamellar keratectomy and to assess the expression of matrix metalloproteinase-1, metalloproteinase-2, metalloproteinase-9 (MMPs), type IV collagen, and interleukin-10 (IL-10) during the treatment. Methods Morphine group (MG) received 50 lL of topical 1% morphine four times daily, while the control group received saline instead. Corneal touch threshold (CTT) and the wound area were assessed until corneal healing. Corneal samples were processed for routine histology, immunohistochemistry, zymography, and ELISA. Results Following keratectomy, CTT increased significantly from 6 to 96 h time points. Mean corneal re-epithelization rate and scores of leukocyte infiltration did not differ significantly between treatment groups. Immunolabeling pattern for MMP-1, MMP-9, and type IV collagen was similar in both treatment groups. In the MG, zymography indicated significantly higher levels of active MMP-2 on days 6 and 12; and in the latent MMP-9, on days 3 and 6, and in the active MMP-9, on day 6. Latent MMP-2 and MMP-9, and active MMP-9 decreased to values close to those of healthy corneas on day 12, but levels of active MMP-2 remained significantly elevated in the MG. IL-10 levels measured on days 1-6 were reduced as compared to those of healthy corneal tissue and returned to levels close to those of healthy corneas on day 12. Conclusion Topical morphine promoted corneal analgesia for up to 4 days and did not delay corneal re-epithelization. The re-establishment of MMPs and IL-10 to levels close to baseline values at the end of the study and the expression of type IV collagen in both groups reinforce that, with caution, 1% morphine can be used after lamellar keratectomy in rabbits.
Veterinary Ophthalmology, 2013
Objective To evaluate the effects of agents on corneal re-epithelization and metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) activities in corneas of rats submitted to ulceration. Animals Studied Ninety eight healthy rats. Procedures Corneal ulcers were created using 1N NaOH in their left eye. Eyes were treated every 6 h with 1% ethylenediaminetetraacetic acid (EDTA), 3% chondroitin sulfate (CS), 10% N-acetylcysteine NAc and saline (S) at 6-h intervals. Corneas were stained with fluorescein and photographed at the same time points. Following 20 h and 40-42 h of corneal injury, corneas were processed for scanning electron microscopy (SEM) to quantify microvilli density, and MMPs activities were analyzed using zymography. Results The percentage of wound area and the time in hours for corneal re-epithelization did not differ significantly among treatment groups (P > 0.05). In first and the second moments, latent MMP-2 was significantly elevated in the eyes treated with NAC and CS (P < 0.001). Active MMP-2 did not change significantly among treatment groups in the first moment (P > 0.05); significantly higher activity was observed in the second moment in the eyes treated with CS (P <0.001). In the second moment, latent MMP-9 decreased significantly in eyes treated with EDTA and S (P < 0.01). Microvilli corneal density did not change significantly between healthy subjects and treatment groups (P > 0.05). Conclusion Any of the studied substances did not accelerate corneal re-epithelization and did not add protection to the corneal microvilli. Significant higher levels of active form of MMP-2 in 3% chondroitin sulfate-treated group may indicate that the agent acts as substrate for such enzyme. At the end of the experiment, 1% EDTA was the most efficient agent to inhibit significantly the latent form of MMP-9. However, any of the substances add benefit over saline on reducing the proteolytic activity in the cornea of rats after alkali injury.
Collegium antropologicum, 2008
We aim to find a link between keratokonus (KC) and bullous keratopathy (BK), and extra cellular matrix re-modellation molecules. The activities of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), pro-matrix metalloproteinase-13 (proMMP-13) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) were measured using immunoassay in three human corneal tissue layers (epithelium, stroma and endothelium) supernatants of the patients with KC and BK which underwent the perforative keratoplasty. MPP-2, MMP-9, proMMP-13 and TIMP-1 activity was detected in all samples. The epithelial layer showed significantly higher levels of MMP-9 and proMMP-13 in BK than in KC. Increased levels of MMP-2 (p=0.07) levels were found in bullous keratopathy compared to keratoconus patients. Epithelial TIMP-1 showed no significant difference in activity between KC and BK. All these findings suggest an active degradation of the extra-cellular matrix in epithelial corneal layer in Bullou...
Investigative Ophthalmology & Visual Science, 2013
PURPOSE. Nerve growth factor (NGF) is a neuropeptide essential for the development, survival, growth, and differentiation of corneal cells. Its effects are mediated by both TrkA and p75 receptors. Clinically relevant use of NGF was introduced to treat neurotrophic ulcerations in patients. Herein, we examine the mechanisms by which NGF enhances epithelial wound healing both in vivo and in vitro. METHODS. An animal model using adult hens was implemented for the in vivo experiments. Laser ablation keratectomy was performed and animals were observed for up to 7 days. Epithelial healing was measured with fluorescein. In addition, proliferation was measured using BrdU incorporation and both TrkA and matrix metalloprotease-9 (MMP-9) expression were measured by immunohistochemistry (IHC) and Western blot (WB). In vitro experiments were carried out with telomerase-immortalized human corneal epithelial cells (HCLE). The rate of proliferation was measured using a colorimetric assay and BrdU incorporation. Realtime migration was evaluated with an inverted microscope. MMP-9 expression was evaluated by immunocytochemistry (ICC), WB, zymography, and RT-PCR. Finally, beta-4 integrin (b4) expression was assessed by ICC and WB. RESULTS. Faster epithelial healing was observed in NGF-treated corneas compared with controls (P < 0.01). These corneas showed increased proliferation, TrkA upregulation, and enhanced MMP-9 presence (P < 0.01). In vitro, faster spreading and migration were observed in response to NGF (P < 0.01). Enhanced proliferation, as well as enhanced TrkA and MMP-9 expression, and decreased b4 levels were observed after adding NGF (P < 0.01). CONCLUSIONS. NGF plays a major role during the epithelial healing process by promoting migration, a process that is accelerated by cell spreading. This effect is mediated by both the upregulation of MMP-9 and cleavage of b4 integrin.
Extracellular Matrix and Na + ,K + -ATPase in Human Corneas Following Cataract Surgery
Cornea, 2002
To examine the distribution of extracellular matrix (ECM) and basement membrane (BM) components and of Na + ,K + -ATPase in postcataract surgery (PCS) corneas. These corneas were from patients who never developed pseudophakic or aphakic bullous keratopathy (PBK/ABK) after cataract surgery. PCS corneas were compared with PBK/ABK and Fuchs' dystrophy corneas. Methods. The distribution of PBK/ABK ECM and BM markers and of all three Na + ,K + -ATPase ␣ subunits was studied by immunofluorescence in 10 healthy, 11 PCS, 16 PBK/ABK, and 12 Fuchs' dystrophy corneas. Results. Fibrotic ECM proteins, tenascin-C and fibrillin-1, were found in only 1 of 10 healthy and in 2 of 11 PCS corneas. In contrast, these proteins were expressed in all PBK/ABK and more than half of the Fuchs' dystrophy corneas. BM components in PCS corneas were altered to a greater extent (40-60%), especially fibronectin and laminin-10. A decreased epithelial immunostaining for Na + ,K + -ATPase ␣ subunits was seen in approximately 40% of PCS corneas and in approximately two thirds of PBK/ABK and Fuchs' dystrophy corneas. However, the endothelial staining was normal in all groups. Conclusions. Because tenascin-C and fibrillin-1 were mostly found in diseased but not in PCS corneas, their expression may be related to later, clinical stages of corneal edema development. However, BM components abnormal in PBK/ABK and Fuchs' dystrophy corneas were also altered in PCS corneas without clinical evidence of ocular disease. This may result from subclinical corneal changes resulting from cataract surgery, lens removal, exposure to the intraocular lens, or endothelial cell damage. Alterations of epithelial Na + ,K + -ATPase point to the importance of epithelial changes in the development of corneal edematous diseases.
Cornea, 2002
Purpose. To characterize extracellular matrix (ECM) and nine matrix metalloproteinase (MMP) changes in two corneas that underwent a complicated laser-assisted in situ keratomileusis (LASIK) procedure. Methods. The first patient underwent bilateral LASIK. The flap on the left eye was transected in several locations and placed back. This cornea later developed edema, and the removed flap was analyzed after lamellar keratoplasty. The second patient had a LASIK flap lifted, replaced twice, and then completely removed. The epithelium grew over the stroma, but haze and severe ectasia occurred. After penetrating keratoplasty, the recipient cornea was analyzed. An autopsy cornea from a person who underwent uneventful LASIK and ten normal autopsy corneas served as controls. Corneas were analyzed by immunohistochemistry. Results. Both flap regions in the treated corneas had marked alterations of ECM components and MMPs. Stromal deposits of various ECM proteins, including those normally absent in the central cornea (tenascin-C, fibrillin-1, type XIV collagen), were found. Rare myofibroblasts and inflammatory cells were present. The epithelial basement membrane (BM) was altered in both cases. The most dramatic change was poor or no staining for ␣3-␣6 type IV collagen chains and thrombospondin. The limbal ␣1-␣2 type IV collagen and laminin-2 (␣21␥1) appeared in the central epithelial BM. Other components were altered to a lesser extent. The anterior stroma was positive for MMP-1 and MMP-2, and some MMP-7 was seen in the epithelium. These ECM and MMP patterns were not seen in uneventful LASIK or normal corneas. Conclusions. In the flaps of LASIK-treated corneas, fibrosed areas of anterior stroma had increased levels of MMP-1 and MMP-2 that may have caused loss of specific type IV collagen isoforms in the epithelial BM. These changes may reflect an on-going wound healing process and contribute to the development of ectasia.
Acta Ophthalmologica, 2009
To evaluate the combined effect of intense pulsed light (IPL) therapy and meibomian gland expression on extracellular matrix metalloproteinase-9 (MMP-9) levels and clinical outcomes of moderate and severe meibomian gland dysfunction (MGD) treatment. Methods: This retrospective study was conducted on 45 eyes of 23 patients with moderate and severe MGD. Each eye underwent three IPL sessions and meibomian gland expression at 2-week intervals. In this study the evaluated parameters included tear film break-up time (TBUT), corneal and conjunctival fluorescein staining scores, biomicroscopic examination of lid margins and meibomian glands, ocular surface disease index (OSDI) questionnaire score, and extracellular MMP-9 levels using the immunoassay device before and two weeks after the last treatment session. Linear mixed model and generalized estimating equations model were used to evaluate possible differences. Results: There were significant improvements in TBUT (P < 0.001), SICCA ocular staining score (P = 0.008), Oxford staining score (P = 0.023), lid margin irregularity (P < 0.001 for upper and lower eyelids), lid thickness (P < 0.001 for upper and lower eyelids), meibomian gland plugging (P = 0.010 and P = 0.012 for upper and lower eyelids), meibum color (P = 0.044 and P < 0.001 for upper and lower eyelids), meibum consistency (P < 0.001 for upper and lower eyelids), MGD grade (P < 0.001), and OSDI questionnaire score (P < 0.001). Incidence of positive results for MMP-9 immunoassay significantly decreased from 84.0% to 56.0% (P = 0.031) after treatment. Conclusion: In patients with moderate to severe MGD, three sessions of IPL combined with meibomian gland expression improved objective findings, subjective symptoms, meibomian gland function, and MMP-9 immunoassay results. The results support the combination of IPL and meibomian gland expression for treating moderate to severe MGD.
Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury
The American journal of pathology, 1996
Delayed re-epithelialization of the cornea after injury usually precedes stromal ukceration. Previous findings using a rat thermal injury model suggested that re-epithelialization is impeded by products of resident corneal cells, which destroy adbesive structures at the basement membrane zone. In this study, we provide additional evidencefor this concept. Failure to re-epithelialize was found to correlate with an increase in the amounts of gelatinolytic matrix metalloproteinases present in the rat cornea. One of these gelatinases, gelatinase B, is synthesized by the resident corneal cells, and inhibition of its synthesis correlated with inhibition of basement membrane dissolution. The matrix metalloproteinases collagenase and stromelysin are also synthesized by resident corneal cells in thermaly injured corneas of rabbits, but the timing of bulk enzyme synthesis correlated more closely with deposition of repair tissue in the stroma than with failure to re-epithelialize. Nevertheless, in human corneas with repair defects, gelatinase B and collagenase are synthesized by cells in the basal layer of the epithelium directly adjacent to the basement membrane, suggesting that both couldparticipate in dissolution ofthis structure. Importantly, treatment of thermally injured corneas with a synthetic inhibitor of matrix metalloproteinases significantly improved basement membrane integrity. These data support the concept that over-expression of matrix metalloproteinases by resident corneal cells impedes re-epithelialization after some types ofcorneal injury. (Am J Pathol 1996, 149:1287-1302 Corneal ulceration is a devastating disorder that can cause blindness. Ulcers manifest as a breakdown of the collagenous stromal tissue, a process that was once thought to be a simple physical dissolution described as corneal melting. A major change in our understanding of stromal ulceration occurred when this process was shown to be associated with the secretion of type collagen-degrading enzymes from living cells.' In organ culture, collagenolytic activity was shown to be produced by superficial sections of ulcerating cornea containing the epithelial layer and a small amount of anterior stroma. However, the observation that neutrophil infiltration is a hallmark of stromal ulceration suggested that these cells (with their accumulated stores of hydrolytic enzymes) might provide the more important source of collagenase. Experiments showing that chemical injuries do not ulcerate in animals that have been made neutropenic have provided support for this hypothesis.2