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Arch Ophthalmol. Author manuscript; available in PMC Jun 19, 2008.
Published in final edited form as:
PMCID: PMC2431172
NIHMSID: NIHMS51181

Naltrexone Prevents Exuberant Granulation Tissue and Neovascularization in the Rat Cornea

Ian S. Zagon, M.S., Ph.D., Matthew S. Klocek, M.S., James W. Griffith, D.V.M., Joseph W. Sassani, M.D., M.H.A., András M. Komáromy, Dr. Med. Vet. Ph.D., and Patricia J. McLaughlin, M.S., D.Ed.

Abstract

Objective

To determine whether topical application of naltrexone (NTX) prevents exuberant granulation tissue formation with neovascularization in diabetic cornea.

Methods

Diabetes (DB) was induced with streptozotocin. A 5 mm corneal abrasion at 9 or 11 weeks was treated topically for 7 days (4X daily) with NTX or sterile vehicle (SV).

Results

Within 2–5 days after re-epithelialization, DB SV rats 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 DB NTX or Normal SV groups. DB SV rats with corneal lesions re-epithelialized more slowly than DB SV animals without such complications, but no difference in blood glucose levels were noted.

Conclusions

Using a minimally invasive model in diabetic rats, topical NTX normalizes corneal wound healing and prevents neovascularization.

Clinical Relevance

Direct application of NTX may serve as an important strategy for facilitating corneal healing and inhibiting corneal neovascularization.

INTRODUCTION

Corneal neovascularization (CNV) has been estimated to affect 1.4 million Americans annually.1 CNV is associated with a wide variety of diseases including contact lens wear, surgical and non-surgical trauma, alkali and other chemical burns, and infection.13, CNV may be helpful in combating infections, assisting in corneal healing, and arresting autoimmune corneal melting.3,4 Nevertheless, such vascularization can lead to corneal scarring, edema, lipid deposition, and inflammation that may significantly compromise corneal transparency and result in severe visual impairment, including blindness.13 The etiology of corneal neovascularization is unclear, though the association of stromal edema proximal to the limbus has been proposed as necessary to allow blood vessels into the usually compact stroma.13,5 Treatment modalities include surgery, topical corticosteroids, angiostatic steroids, non-steroidal anti-inflammatory agents, and natural inhibitors of angiogenesis.13 However, treatments of ocular neovascularization may have limitations such as recurrence, adverse side effects, and ineligibility for therapy.1 Thus, models of ocular neovascularization, as well as treatment regimens for corneal lesions with neovascularization, are needed.

In previous investigations we have reported that topical treatment with the opioid antagonist naltrexone hydrochloride (NTX) facilitates corneal re-epithelialization in normal and diabetic corneas.69 Subsequent preliminary histological examination has revealed that a subset of re-epithelialized poorly controlled diabetic rat corneas exhibit exuberant granulation tissue formation with stromal neovascularization. These observations have led to the present study which examines the hypothesis that topical application of the opioid antagonist NTX prevents granulation tissue formation and accompanying neovascularization in a unique model of delayed wound healing in rats with Type 1 diabetes.

METHODS

ANIMALS AND INDUCTION OF DIABETES

Male Sprague-Dawley rats (~175 g) were obtained from Charles River Laboratories (Wilmington, MA) and housed under standard laboratory conditions. All investigations conformed to the regulations of the Association for Research in Vision and Ophthalmology, National Institutes of Health, and the guidelines of the Institutional Animal Care and Use Committee of The College of Medicine at The Pennsylvania State University.

Type 1 diabetes was induced according to Havel et al.10,11 An intraperitoneal (i.p.) injection of 40 mg/kg streptozotocin (STZ) (Sigma, St. Louis, MO) in ice-cold 0.5 mol/l citrate buffer (pH 4.5) was administered. A second dose of STZ (40 mg/kg) was injected 24 hours later. This regimen produced insulin-deficient diabetes (DB) in 100% of the animals within 48 to 72 hours; this group consisted of 65 rats. Animals not receiving STZ but injected with citrate buffer were considered Normal; this group consisted of 26 rats.

Blood glucose levels were monitored from the tail vein using a True Track Smart System glucometer (Home Diagnostics, Inc., Ft. Lauderdale, FL) immediately prior to receiving STZ, and at 1, 4, and 8 weeks after STZ administration. Glucose levels of >400 mg/dl were considered to be the minimum blood glucose level compatible with a stable non-toxic diabetic state.12

CORNEAL ABRASIONS

The procedures for epithelial debridement and observation of repair followed those reported earlier.68 In brief, animals were anesthetized with a mixture of ketamine (70 mg/kg), xylazine (7 mg/kg) and acepromazine (10 mg/kg). Eyes were examined under a dissecting microscope (SZ-ET; Olympus, Tokyo, Japan). If judged to be disease-free, a 5 mm diameter circle was outlined in the center of the cornea with a disposable dermatological skin punch (Acuderm, Ft. Lauderdale, FL). The encircled corneal epithelium was removed with a No. 15 Bard-Parker scalpel blade. Care was taken not to injure the underlying basement membrane and subjacent corneal tissue.6 Epithelial defects were created between 0730 and 0830 hours or 1600 and 1700 hours; these time points were chosen because previous studies showed no differences in the labeling index between morning and afternoon.14 Any animal that experienced bleeding in the course of mechanical abrasion, was not included in the study. Only one eye was abraded at a time in each animal. The right eye was abraded on the 9th week following injection of STZ. Two weeks later, following closure of the initial epithelial defect, the left eye was abraded.

TOPICAL ADMINISTRATION OF NALTREXONE

NTX (Sigma, Indianapolis, IN) was prepared at concentrations of 10−4 or 10−5 M in Vigamox (moxifloxacin hydrochloride ophthalmic solution, Alcon, Inc., Ft. Worth, TX). Compounds were given as a single drop using the commercial applicator bottle to the central cornea of the injured eye, with the lower eyelid held away from the eye to avoid overflow. Eyedrops were administered to unanaesthetized animals at 0730, 1030, 1330, and 1630 hours for 7 consecutive days. Diabetic rats were randomly assigned to receive either NTX or vehicle, whereas Normal animals received vehicle only.

SLIT LAMP OBSERVATIONS OF CORNEA

All animals were examined with a hand-held slit lamp (Zeiss HSO 10 Hand Slit Lamp, Dublin, CA) to document overall corneal morphology and pathology, particularly the progression of corneal lesions. Corneas were observed with the slit lamp every day after debridement for the first 7 days, and every 2nd day for 3 weeks. Rats were placed in an isoflurane chamber for 60 seconds, and evaluation with the slit lamp was conducted before and after dilation of each eye. Corneal lesions were graded subjectively as 1, 2, and 3 depending on whether they extended no more than 25%, 50%, or 75%, respectively, of the corneal surface area.

PHOTOGRAPHY

Animals were anesthetized in a Plexiglas chamber attached to an isofluorane vaporizer, and the residual epithelial defect was stained with topical fluorescein (Fluor-I-Strip, Ayerst Laboratories. Philadelphia, PA). Rat eyes were viewed using an Olympus dissecting microscope with a tungsten light source and a gelatin Wratten #47 filter, and photographed with a Sony CCD camera at 1.5X magnification. Photographs were taken immediately after epithelial debridement (0 hours) and 16, 24, 32, and 40 hours later. No animal was photographed at intervals less than 12 hours in order to prevent disruption of the healing process. The area of defect was determined using Optimas software (Meyer Instruments, Inc., Houston, TX), and was calculated as the percentage of the original epithelial defect.

HISTOLOGY

Animals were euthanized at 2, 3, 4, or 21 days following debridement by i.p. injection of 100 mg/kg sodium pentobarbital, decapitated; eyes were enucleated, and placed in formalin for 24 hours and prepared for paraffin embedding. Vertical sections (8 μm) that included the corneal surface, limbus, and conjunctiva were processed using hematoxylin and eosin, Masson’s Trichrome, Brown-Hopps, or von Kossa staining protocols.

DATA ANALYSIS

Body weights and glucose measurements were analyzed by the Student’s two-tailed t-test. The area of residual defect was analyzed at each time point using one-way ANOVA, with subsequent analysis by Newman-Keuls tests. The incidence of corneal lesions was analyzed by Chi-Square tests.

RESULTS

INDUCTION OF DIABETES

All rats weighed 174 ± 2 g at the time of STZ injections (Fig. 1A). Normal rats gained approximately 301 g over the course of 8 weeks. Rats in the DB group were comparable in body weight to Normal animals until 2 weeks after injection of STZ. At this time, the DB group had a 15% reduction (p<0.001) in body weight relative to Normal animals. DB rats weighed significantly less (approximately 25–35%) than Normal rats beginning on week 4 and throughout the course of the study.

FIG. 1
Body weights (A) and glucose levels (B) of rats rendered DB with STZ and untreated animals receiving citrate buffer (Normal). (A) Body weights were recorded at the time of STZ injection (week 0), and every 2 weeks thereafter. (B) Blood glucose levels ...

Baseline glucose readings were 139 ± 8 mg/dl for all rats, and these values were consistent in the Normal group throughout the study (Fig. 1B). Rats receiving STZ became hyperglycemic within 5 days, and had glucose levels greater than 450 mg/dl throughout the duration of experimentation.

SLIT LAMP OBSERVATIONS OF CORNEA

Within 2 to 5 days after re-epithelialization, 41% of the 29 DB SV animals exhibited corneas with exuberant granulation tissue and blood vessels extending from the limbus to the corneal lesion (Fig. 2).;. No additional DB SV animals exhibited corneal lesions after this period. However, those animals with corneal lesions often had changes in severity of this complication (Table 1). None of the animals in the Normal SV, or in the DB group receiving either 10−4 or 10−5 M NTX, presented with corneal lesions over the course of the study. The incidence of corneal lesions in the DB SV rats differed significantly (p<0.001) from that recorded for the Normal SV and DB NTX groups.

Fig. 2
Photographs of corneas from Normal and DB rats treated topically with sterile vehicle (SV). Grades 1, 2, and 3 indicate corneal lesions that extended no more than 25%, 50%, or 75%, respectively, of the corneal surface area. Bar = 2 mm.
TABLE 1
Severity of corneal lesions with neovascularization for DB SV animals

HISTOLOGY

Examination of the cornea of rats in all groups revealed a complete epithelial layer by 2 to 3 days after debridement. However, within 4 to 7 days after mechanical abrasion, animals in the DB-SV group were identified with corneal lesions, segmented leukocytes present in clefts in some areas of the stroma, and numerous spindle cell nuclei and melanin pigment. Edema in the stroma was observed in these animals. Rats in the DB-SV group with corneal lesions exhibited capillaries containing red blood cells in the stroma, particularly at the margins of the cornea. Moreover, in some of these specimens the epithelial layer appeared to be detached from the corneal surface.

Histological examination of sections of cornea from DB SV rats collected 3 weeks after debridement were characterized by cellular and vascular indications of inflammation and edema (Fig. 3). Focal necrosis and loss of surface epithelium with segmented leukocytes and macrophages were visible within the subjacent stroma. The inflammatory cells and capillary proliferation disrupted the normally transparent lamellar pattern of collagen within the stroma (see Trichome stained specimen in Fig. 3) cells. There was focal degeneration and mineralization of the subepithelial basement membrane that was confirmed positive for calcium with the von Kossa stain (data not shown). Examination of sections stained with the Brown and Hopps tissue gram stain revealed an absence of bacteria associated with the corneal lesions. Animals in the Normal SV group, as well as rats in the DB group receiving NTX at concentrations of 10−4 and 10−5 M, did not exhibit any histological abnormalities.

Fig. 3
Photomicrographs of sections of the central cornea, taken 21 days after wounding, from rats in the Normal SV (A, B), DB NTX (C,D) and DB SV with a corneal lesion (E,F) groups. Sections were stained with hematoxylin and eosin (A, C) or Masson’s ...

CORNEAL RE-EPITHELIALIZATION

The 5-mm trephine demarcated the entire corneal region of the rat eye but did not encroach on the limbus or conjunctiva (Fig. 4A). Wound healing occurred in a manner consistent with previous studies on normal rat, rabbit, and human, as well as diabetic rat.9,13,15. The initial area of the abrasion ranged from 19.3 mm2 to 25.6 mm2, and corresponded to corneal injuries of 4.9 to 5.7 mm diameter. No differences in the size of the initial abrasions were noted between groups.

FIG. 4
A. Representative photographs of rat eyes stained with fluorescein immediately (0 hours) and at 16, 24, 32, or 40 hours following a 5 mm corneal abrasion. DB animals were topically treated with sterile vehicle (SV) and the photographs are subdivided into ...

DB SV animals (with or without corneal lesions) had corneal epithelial defect measurements that indicated a significant retardation from the Normal SV group at 32 (p<0.05) and 40 (p<0.001) hours, and from the DB groups subjected to 10−4 M or 10−5 M NTX treatment at 16 (p<0.05), 24 (p<0.001), 32 (p<0.01), and 40 (p<0.001) hours (data not shown). The DB SV animals with corneal lesions had greater epithelial defects than those DB SV rats without corneal lesions at 16 and 40 hours (Fig. 4A, B). DB animals receiving NTX at concentrations of either 10−4 M or 10−5 M did not differ from each other in corneal re-epithelialization at any time point (data were collapsed for comparison). However, the DB animals receiving NTX had smaller defects than Normal SV animals at 24 hours, but were comparable at 16, 32, and 40 hours.

BLOOD GLUCOSE LEVELS

Blood glucose levels in the DB SV group that exhibited corneal lesions (564 ± 18; n = 12) did not differ from those of rats in the DB SV group without corneal lesions (524 ± 19; n = 17) at 1, 4, and 8 weeks.

COMMENT

The present study reveals that over 40% of poorly controlled DB rats with corneal abrasions exhibit exuberant granulation tissue formation with neovascularization as determined by slit lamp microscopy and confirmed with histopathology. This abnormality following re-epithelialization of the cornea, with indications at the histological level recorded as early as 2 days after debridement. These complications involved inflammation, edema, mineralization, and neovascularization, but did not appear to include an infectious process.

Our data show that these complications following corneal epithelial repair can be completely prevented by topical treatment with either 10−4 or 10−5 M NTX. Interestingly, DB animals with corneal lesions were found to be correlated with significantly slower rates of re-epithelialization than detected in DB rats without corneal lesions. These results would suggest that the appearance of granulation tissue with neovascularization may be related to an increased susceptibility for damage in these DB corneas brought on by delays in repair of injury. In this regard, it is well-known that there is an irregular thickening and multilamination of the epithelial basement membrane in DB humans and animals.13,14 It may be conjectured that the subset of DB animals displaying more exaggerated delays in re-epithelialization than others have a problem with the integrity of the basement membrane. However, we did find that the blood glucose levels in both groups were similar so that it can be concluded that the magnitude of hyperglycemia was not an issue in this regard. Presumably, the exuberant granulation tissue disrupts the repaired corneal epithelium as the excessive granulation tissue protrudes above the level of the surrounding epithelium. Given the edema in the stroma and the appearance of new blood vessels from the limbus, our observations support the hypothesis of Cogan5 that stromal edema occurring near the limbus may be necessary to allow blood vessels into the corneal stroma. Thus, for the first time, we have defined a model for a pathologic response to complications in re-epithelialization that involve exuberant granulation tissue formation and neovascularization. Moreover, we have demonstrated a treatment modality using NTX that aborts this process.

The mechanism(s) concerning NTX’s capacity to attenuate neovascularization in the repair of the abraded cornea of diabetic rats is unclear. One possibility is that NTX directly diminishes the proliferation of these blood vessels. However, it is known that 5 μg of NTX placed on a methycellulose disk stimulates angiogenesis (blood vessel number and length) compared to vehicle controls in a chick chorioallantoic membrane preparation.16,17 Moreover, NTX at a dosage that induced a continuous opioid receptor blockade (i.e., 30 mg/kg, daily administration) has been reported to elevate DNA synthesis in the intima and media, and to increase intimal thickness and reduce luminal area, in the carotid artery that was denuded with balloon catherization in comparison to vehicle exposed control rats.18 These data are consistent with findings that sustained opioid receptor blockade with NTX or other opioid antagonists such as naloxone results in enhanced DNA synthesis in tissues undergoing development, cellular renewal, or repair, as well as in neoplasia.19 Thus, the evidence does not appear to support the hypothesis that NTX has a direct inhibitory effect on neovascularization. Another possible mechanism regarding NTX treatment and diminished neovascularization is based on the observation that the cornea of the DB SV rats heals slower than that in the Normal SV group.6,7 In the current study, when the DB SV group is subdivided into those with and without corneal lesions, the DB SV group with corneal lesions exhibited even more retarded re-epithelialization than DB SV rats without corneal lesions. This finding suggests that the pace of repair of corneal defects is more highly associated to the appearance of neovascularization, with a greater risk of neovascularization in animals with the slowest rates of re-epithelization. This hypothesis is consonant with the observation that DB animals exposed to topical NTX have an accelerated rate of corneal wound healing that compares to the Normal SV animals, and these DB NTX rats did not display neovascularization. Thus, it could be conjectured that the mechanism for the promotion of neovascularization in the cornea of DB animals may be factors and events occurring in the extended time period of re-epithelialization. If this is the case, then a test of this hypothesis would be to examine the repercussions of a drug or mechanical induced retardation in corneal repair in DB rats, which would be predicted to increase the incidence of neovascularization. Finally, it is known that NTX treatment restores corneal sensitivity in DB rats.7 In this manner, it may optimize the homeostatic milieu and thereby facilitates healing by re-establishing the blink reflex and tear production.

The complications following corneal re-epithelialization in rats and documented herein, has not been reported in humans. One factor that may contribute to the development of the corneal lesions we observed is the severity in degree and duration of hyperglycemia (blood sugars in these rats routinely were greater than 450 mg/dl for more than 8 weeks). Moreover, the human has a Bowman’s membrane that is absent in rats. If a corneal abrasion in a rat can be equated to a superficial corneal ulcer in the human, the lack of this anatomic barrier (i.e., Bowman’s membrane) in the rat could make their corneas more susceptible to inflammatory mediators accompanying re-epithelialization. Further contributing to the complications following corneal epithelial repair in these poorly controlled DB animals may be the presence of decreased corneal sensitivity that we have previously reported and which can be remedied by topical treatment with NTX.15 Decreased corneal sensitivity would be expected to decrease tear production and/or blink reflex, thereby subjecting the newly repaired cornea to an adverse environment.

The healthy human cornea is free from blood vessels, but neovascularization is a common clinical problem seen as a response to chronic hypoxia or various inflammatory stimuli such as bacterial keratitis, alkali burns, and graft rejection.13 The severity of clinical disorders involving corneal neovascularization, which untreated may lead to pronounced visual impairment, emphasizes the importance of broadening our base of knowledge in this field. The fact that the model we describe herein is occurring on the ocular surface makes these lesions very accessible for defining related structural, biochemical, and physiological events, particularly neovascularization. Additionally, such a model provides the unique opportunity to delineate the impact of various treatment regimens. Therefore, for the first time we have characterized a unique model of exuberant granulation tissue with neovascularization that accompanies complications of corneal re-epithelialization that can be tested for toxicity and efficacy of therapeutic modalities.

Acknowledgments

Supported in part by NIH grants EY16666 and K12 EY015398

Footnotes

This article was originally published in Archives of Ophthalmology: http://archopht.ama-assn.org/cgi/content/full/126/4/501

References

1. Lee P, Wang CC, Adamis AP. Ocular neovascularization: An epidemiological review. Survey Ophthalmol. 1998;43:245–269. [PubMed]
2. Chang J-H, Gabison EE, Kato T, Azar DT. Corneal neovascularization. Opin Ophthalmol. 2001;12:242–249. [PubMed]
3. Carmichael TR. Corneal angiogenesis. In: Tombran-Tink J, Barnstable CJ, editors. Ocular Angiogenesis. Chap 3. Totowa, NJ: Humana Press; 2006. pp. 45–71.
4. Dana MR, Streilein JW. Loss and restoration of immune privilege in eyes with corneal neovascularization. Invest Ophthalmol Vis Sci. 1996;37:2485–2494. [PubMed]
5. Cogan DG. Vascularization of the cornea. Its experimental induction by small lesions and a new theory of its pathogenesis. Arch Ophthalmol. 1949;41:406–416. [PubMed]
6. Zagon IS, Jenkins JB, Sassani JW, Wylie JD, Ruth TB, Frey JL, Lang CM, McLaughlin PJ. Naltrexone, an opioid antagonist, facilitates reepithelialization of the cornea in diabetic rat. Diabetes. 2002;51:3055–3062. [PubMed]
7. Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Topically applied naltrexone restores corneal reepithelialization in diabetic rats. J Ocular Pharmacol Therapeutics. 2007;23:89–102. [PubMed]
8. Zagon IS, Sassani JW, McLaughlin PJ. Re-epithelialization of the rat cornea is accelerated by blockade of opioid receptors. Brain Res. 1998;798:254–260. [PubMed]
9. Zagon IS, Sassani JW, McLaughlin PJ. Re-epithelialization of the human cornea is regulated by endogenous opioids. Invest Ophthalmol Vis Sc. 2000;41:73–81. [PubMed]
10. Havel PJ, Hahn TM, Sindelar DK, Baskin DG, Dallman MF, Weigle DS, Schwartz MW. Effects of streptozotocin-induced diabetes and insulin treatment on the hypothalamic melanocortin system and muscle uncoupling protein 3 expression in rats. Diabetes. 2000;49:244–252. [PubMed]
11. Ahren B, Stern JS, Gingerich RL, Curry DL, Havel PJ. Glucagon secretory responses to hypoglycemia, adrenaline, and carbachol in streptozotocin diabetic rats. Acta Physiol Scand. 1995;155:215–221. [PubMed]
12. Nakamura M, Sato N, Chikama T-I, Hasegawa Y, Nishida T. Fibronectin facilitates corneal epithelial wound healing in diabetic rats. Exp Eye Res. 1997;64:355–359. [PubMed]
13. Rehany U, Ishii Y, Lahav M, Rumelt S. Ultrastructural changes in corneas of diabetic patients. Cornea. 2000;19:534–538. [PubMed]
14. Taylor HR, Kimsey RA. Corneal epithelial basement membrane changes in diabetes. Invest Ophthalmol Vis Sci. 1981;20:548–553. [PubMed]
15. Zagon IS, Klocek MS, Sassani JW, Mauger DT, McLaughlin PJ. Corneal safety of topically applied naltrexone. J Ocular Pharmacol Therapeutics. 2006;22:377–387. [PubMed]
16. Blebea J, Mazo JE, Kihara TK, Vu J-H, McLaughlin PJ, Atnip RG, Zagon IS. Opioid growth factor modulates angiogenesis. J Vascular Surg. 2000;32:364–373. [PubMed]
17. Blebea J, Vu J-H, Assadnia S, McLaughlin PJ, Atnip RG, Zagon IS. Differential effects of vascular growth factors on arterial and venous angiogenesis. J Vascular Surg. 2002;35:532–538. [PubMed]
18. Zagon IS, Essis FM, Verderame MF, Healy DA, Atnip RG, McLaughlin PJ. Opioid growth factor inhibits intimal hyperplasia in balloon-injured rat carotid artery. J Vascular Surg 2003. 2003;37:636–643. [PubMed]
19. Zagon IS, Verderame MF, McLaughlin PJ. The biology of the opioid growth factor receptor (OGFr) Brain Res Res. 2002;38:351–376. [PubMed]
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