Regulation of Tenon's capsule fibroblast cell proliferation by the opioid growth factor and the opioid growth factor receptor axis.

PURPOSE: Glaucoma filtration surgery often fails because of the fibrotic reaction from Tenon's capsule fibroblasts (TCFs). This study examined whether the interaction of the opioid growth factor (OGF) [Met(5)]-enkephalin with its receptor (OGFr) is a regulator of TCF proliferation. METHODS: The presence of OGF and its receptor (OGFr) was determined in rabbit TCFs (RTCFs) by immunocytochemistry. The kinetics of OGFr were established in receptor binding assays. The ability of OGF to inhibit RTCF proliferation was assessed with dose-response, receptor mediation, and reversibility studies. Dependence on OGF and OGFr was ascertained by antibody neutralization and siRNA studies, respectively. The mechanism of action of the OGF-OGFr axis on survival (apoptosis, necrosis) and DNA synthesis of RTCFs was elucidated. RESULTS: OGF and OGFr were detected in RTCF cells, and specific and saturable binding to OGFr was recorded. Exogenous OGF had a dose-dependent, reversible, and receptor-mediated inhibitory effect on cell proliferation. Endogenous OGF was found to be constitutively produced and tonically active in cell replication, with neutralization of this peptide causing acceleration of cell proliferation. The silencing of OGFr by using siRNA technology stimulated cell replication, validating OGFr's integral role. The mechanism of OGF-OGFr action was not related to cell survival, but rather to DNA synthesis-specifically, the cyclin-dependent kinase inhibitory pathway. Knockdown of p16 or p21 eliminated OGF's inhibitory effect on growth. CONCLUSIONS: The OGF-OGFr system is a native biological regulator of cell proliferation in RTCFs and may offer a means of improving the success of glaucoma filtration surgery in a safe and nontoxic manner.

Diabetic keratopathy and treatment by modulation of the opioid growth factor (OGF)-OGF receptor (OGFr) axis with naltrexone: a review.

The opioid growth factor (OGF)-OGF receptors (OGFr) axis plays an important role in the homeostasis and re-epithelialization of the mammalian cornea. This tonically active growth regulatory inhibitory pathway is involved in cell replication, and the endogenous neuropeptide OGF targets cyclin-dependent kinase inhibitors, p16 and/or p21. Blockade of OGF-OGFr interfacing by systemic or topical administration of opioid antagonists such as naltrexone (NTX) results in accelerated DNA synthesis, cell replication, and tissue repair. Molecular manipulation of OGFr using sense constructs delayed corneal re-epithelialization, whereas antisense constructs accelerated repair of the corneal surface. Corneal keratopathy, a significant complication of diabetes mellitus, is manifested by delays in corneal re-epithelialization following surgery, injury, or disease. Tissue culture studies have shown that addition of NTX stimulates DNA synthesis and explant outgrowth of rabbit corneal epithelium, whereas OGF depresses DNA synthesis and explant outgrowth in a receptor-mediated manner. NTX accelerated corneal re-epithelialization in organ cultures of human and rabbit cornea. Systemic application of NTX to the abraded corneas of rats, and topical administration of NTX to the injured rabbit ocular surface, increased re-epithelialization. Systemic injections or topical administration of NTX facilitates re-epithelialization of the cornea in diabetic rats. Given the vital role of the corneal epithelium in maintaining vision, the frequency of corneal complications related to diabetes (diabetic keratopathy), and the problems occurring in diabetic individuals postoperatively (e.g., vitrectomy), and that conventional therapies such as artificial tears and bandage contact lenses often fail, topical application of NTX merits clinical consideration.

Dry eye reversal and corneal sensation restoration with topical naltrexone in diabetes mellitus.

OBJECTIVE: To determine if topical application of naltrexone hydrochloride (NTX), an opioid antagonist, restores tear production and corneal sensation in rats with diabetes mellitus. METHODS: Type 1 diabetes was induced with streptozotocin in rats. Tear production was measured by the Schirmer test, and corneal sensitivity, by an esthesiometer. Eye drops of 10(-5)M NTX or sterile vehicle were administered either once only or 4 times a day for 1 or 5 days; a single drop of insulin (1 U) was given once only. RESULTS: Dry eye and corneal insensitivity were detected in the diabetic rats beginning 5 weeks after streptozotocin injection. One drop of NTX or 4 times a day for 1 or 5 days reestablished tear production and corneal sensitivity within 1 hour of administration. The reversal of dry eye lasted for up to 2 to 3 days depending on drug regimen, but restitution of corneal sensation lasted for 4 to 7 days. Topical application of 1 eye drop of insulin restored corneal sensitivity within 1 hour and lasted for at least 2 days. In contrast, 1 eye drop of insulin did not increase tear production at 1, 24, or 48 hours compared with diabetic animals receiving sterile vehicle. CONCLUSION: Topical treatment with NTX normalizes tear production and corneal sensitivity in type 1 diabetic rats. CLINICAL RELEVANCE: Topical application of NTX to the ocular surface may serve as an important strategy for treating dry eye and corneal anesthesia in diabetes. Its effect, if any, in other forms of decreased corneal sensitivity and/or dry eye should be investigated.

Naltrexone and insulin are independently effective but not additive in accelerating corneal epithelial healing in type I diabetic rats.

Patients with diabetes are at increased risk for developing corneal disorders, termed diabetic keratopathy. Treatments for diabetic keratopathy are limited. Preclinical studies have demonstrated that topical administration of either naltrexone (NTX) or insulin (INS) accelerates corneal re-epithelialization in type I diabetic rats. This study determined whether the combination of NTX and INS would have additive effect(s) on the re-epithelialization of corneal abrasions in diabetic male Sprague-Dawley rats beyond either agent alone. Type 1 diabetes (DB) (glucose levels>400mg/dl) was induced with streptozotocin; glycemic levels were not controlled with INS. Eight weeks after induction of diabetes, a 5mm diameter circular abrasion was created in the center of the cornea in one eye of each rat. Eye drops (0.05ml) of INS [1U ( approximately 6nM)] and NTX (10(-5)M) in Vigamox were administered separately 4 times daily for 7 days (NTX/INS); DB control rats received drops of sterile vehicle (DB SV) 4 times daily. Two other groups of rats were given only NTX (DB NTX) or only INS (DB INS). Re-epithelialization was monitored by fluorescein staining, and images were recorded with a CCD camera. Areal measurements were made using Optimas software, and the percentage of epithelial defect over a 40h period was calculated. Twenty-four hour after formation of an abrasion ( approximately 21.7+/-0.4mm(2)area), corneal wounds in DB rats treated with NTX, INS, or NTX/INS were significantly smaller (p<0.001) than those in DB SV rats, with reductions in the size of the defect ranging from 24 to 84%. DB rats treated with NTX or INS alone also were observed to have reductions in wound size of 22 and 29%, respectively, from subjects in the DB SV group at 16h. At 16h both the DB NTX and DB INS groups had defects that were 13 and 27%, respectively, smaller than those for the DB NTX/INS group, and at 40h the DB INS animals had 78% smaller corneal wounds than in the DB NTX/INS group. Therefore, the DB NTX/INS group exhibited some slight delays in wound repair compared to the DB NTX and DB INS groups. Topical application of NTX and/or INS to the cornea had no effect on non-invasive measures that included ocular morphology, intraocular pressure, or corneal thickness. These data demonstrate that although NTX or INS accelerates wound healing, concomitant application of NTX and INS to corneal abrasions in diabetic animals does not have an additive effect on re-epithelialization.

Prevention of exuberant granulation tissue and neovascularization in the rat cornea by naltrexone.

OBJECTIVE: To determine whether topical application of naltrexone prevents exuberant granulation tissue formation with neovascularization in diabetic rat corneas. METHODS: Diabetes was induced with streptozotocin. A 5-mm corneal abrasion at 9 or 11 weeks was treated topically for 7 days (4 times daily) with naltrexone or a sterile vehicle. RESULTS: Within 2 to 5 days after reepithelialization, diabetic rats given the sterile vehicle had a 41% incidence of corneal lesions represented by exuberant granulation tissue with corneal neovascularization extending from the limbus. These lesions exhibited edema, cellular and vascular inflammation, and disruption of stromal lamella by fibrovascular tissue and calcium mineralization, but infection was not detected. No corneal lesions were recorded in the diabetic group treated with naltrexone or the control group given the sterile vehicle. Diabetic rats with corneal lesions given the sterile vehicle reepithelialized more slowly than diabetic rats given the sterile vehicle without such lesions, but no difference in blood glucose levels were noted. CONCLUSIONS: Using a minimally invasive model in diabetic rats, topical naltrexone normalizes corneal wound healing and prevents neovascularization. CLINICAL RELEVANCE: Direct application of naltrexone may serve as an important strategy for facilitating corneal healing and inhibiting corneal neovascularization.

Use of topical insulin to normalize corneal epithelial healing in diabetes mellitus.

OBJECTIVE: To determine whether topical application of insulin normalizes delayed corneal wound healing in rats with diabetes mellitus (DB). METHODS: Diabetes mellitus was induced with streptozocin. A 5-mm corneal abrasion at 9 or 11 weeks was treated topically for 7 days (4 times daily) with 1, 2, or 5 U of insulin or with sterile vehicle (SV). RESULTS: Residual corneal epithelial defects of rats with DB receiving SV (hereafter called DB SV rats or animals) were approximately 35% larger than in healthy animals receiving SV (hereafter called healthy SV rats or animals). Rats with DB receiving topical insulin had wounds ranging from 19% to 60% smaller than DB SV rats, corresponding to wound sizes in healthy SV rats. Topical insulin had no effect on reepithelialization of corneal wounds in healthy SV rats. Insulin did not affect corneal thickness, ocular pressure, or serum glucose level. The corneal sensitivity of DB SV rats was markedly reduced from healthy SV rats, but rats with DB given insulin had corneal sensitivity values comparable to the healthy SV group. DNA synthesis was decreased in DB SV corneal epithelium but was comparable to that in healthy SV rats after they received insulin; apoptosis and necrosis levels were similar in all groups. CONCLUSION: Topical insulin normalizes corneal reepithelialization in diabetic rats. CLINICAL RELEVANCE: Direct application of insulin may serve as an important strategy for treating diabetic keratopathy.

Topically applied naltrexone restores corneal reepithelialization in diabetic rats.

PURPOSE: Complications of diabetes include an increased risk of delayed corneal epithelial wound healing. This study examined the efficacy of naltrexone (NTX), an opioid antagonist, by topical application in facilitating the repair of such lesions. METHODS: NTX, in concentrations of 10(-4), 10(-5), or 10(-6) M, was administered topically 4 times per day for 7 days to the abraded eye of type 1 diabetic rats (DB), DB animals receiving insulin and normoglycemic (DB-IN), and nondiabetic (Normal) rats, beginning 8 weeks following the onset of diabetes. RESULTS: In comparison to DB animals receiving sterile vehicle (SV), DB-NTX rats had 25%-83% smaller defects, a reduction of 24 h (approximately 33%) in closure time, and 20%-42% faster healing rates. DB rats receiving 10(-4) and 10(-5) M NTX were equal to, or surpassed, Normal animals in all parameters of wound closure. NTX also enhanced wound repair in DB-IN and Normal animals relative to their respective SV group. No adverse effects of NTX administration on a variety of noninvasive or invasive measures were discerned. CONCLUSIONS: Topically applied NTX is not only feasible, but also effective over a one hundredfold dosage for accelerating corneal wound healing in diabetic subjects.

Corneal safety of topically applied naltrexone.

PURPOSE: Naltrexone (NTX), an opioid antagonist, accelerates wound healing of corneal epithelium in normal and diabetic animals. This study examined the safety of NTX by topical application in the cornea. METHODS: NTX in concentrations of 10(-3), 10(-4), 10(-5), 10(-6), or 10(-7) M was administered topically 4 times/daily for 7 d to the eye of Type 1 diabetic rats (glucose levels >400 mg/dL) (DB), DB animals receiving insulin to maintain normoglycemia (DB-IN), and normal (nondiabetic) (Normal) subjects beginning 8 weeks after onset of diabetes. RESULTS: No differences in intraocular pressures, corneal thickness, endothelial cell number, or epithelial apoptosis, necrosis, or organization were observed between DB, DB-IN, and Normal groups with and without treatment with NTX. The DB group had a twofold decrease in corneal sensitivity from the Normal and DB-IN groups prior to NTX treatment but were comparable to the Normal and DB-IN groups for at least 2 weeks after chronic exposure to 10(-3) to 10(-7) M NTX was terminated. No differences between Normal and DB-IN groups were noted. CONCLUSIONS: Topical application of NTX over a 10,000-fold range of dosage had no overt toxicity for the parameters studied, indicating that efficacy studies for the use of NTX in corneal wound healing are warranted.