Reepithelialization of the human cornea is regulated by endogenous opioids.

PURPOSE: To determine the influence of endogenous opioid modulation on reepithelialization of the human cornea. METHODS: Eight-millimeter-diameter epithelial defects were created with a trephine and mechanical scraping in the center of human corneas. Resurfacing was studied in organ culture. The size of the defect, the number of specimens with complete reepithelialization, and rate of closure were evaluated using topical fluorescein and morphometric analysis. The influence of opioid receptor blockade was studied using the potent and long-acting opioid antagonist, naltrexone (NTX; 10(-6) M), and the effects of excess (10(-6) M) opioid growth factor (OGF), [Met5]enkephalin, also were determined. The modulatory activity of NTX and OGF on DNA synthesis was evaluated by monitoring the labeling index (LI) using radioactive thymidine. The presence and location of OGF and its receptor (OGFr) were ascertained by immunocytochemistry 1 hour and 24 hours after abrasion. RESULTS: NTX accelerated the wound-healing process, with 21% to 89% less defect than controls observed from 24 to 96 hours. At 72 hours, 62% of the subjects in the NTX group had complete closure of the corneal defects, in contrast to only 19% of the control specimens. All epithelial abrasions were resurfaced in the NTX group between 96 and 120 hours, whereas all controls were not closed until 168 hours. The rate of healing in the NTX group was 1.06 mm2/h compared to a rate of 0.68 mm2/h in the control group. OGF delayed corneal wound healing, with 24% to 260% more defect recorded than in control specimens at day 7. The healing rate of the OGF group was 0.42 mm2/h compared to 0.82 mm2/h for control subjects. The corneal epithelium adjacent to the wound had an LI that was 152% greater than control specimens, whereas OGF decreased the LI of this region by 75%. OGF and OGFr were detected in the epithelium bordering the damaged region at 1 hour, and both peptide and receptor were noted in the regenerating epithelium at 24 hours. CONCLUSIONS: These results indicate that an endogenous opioid is present and functions as a tonically active, receptor-mediated, negative growth factor during reepithelialization of the abraded human cornea.

Nuclear magnetic resonance study of free and bound water fractions in normal lenses.

Proton NMR relaxation times T1 and T2 were determined for normal lenses excised from sexually mature animals from seven different species. Lenses were immersed in physiological buffer during measurements, and suppression methods were employed to null the buffer signal. This enabled selective analysis of lenticular water. Observed relaxation times were correlated with protein and water content. At 37 degrees C and 1.89 Tesla, single-exponential spin-lattice relaxation was observed, but spin-spin relaxation was found to be double-exponential. It was shown that the short-T2 fraction is proportional to protein concentration; this fraction was attributed to water bound to protein. The long-T2 fraction was attributed to free lenticular water. The amounts of free and bound water thus obtained were used in the spin-lattice relaxation rate equation for rapid exchange in a two-component system to calculate the magnitudes of the two corresponding T1 relaxation components.

Anatomic features of the eye disclosed with nuclear magnetic resonance imaging.

Nuclear magnetic resonance (NMR) imaging of the eye and paraorbital areas was performed in 35 volunteers and in four patients with ocular pathology. Two- and three-dimensional images were performed with saturation recovery (SR), inversion recovery (IR), and spin echo (SE) pulse sequences. Fat was brighter than surrounding tissue on images obtained with all pulse sequences, while muscle and optic nerve were of decreased intensity. The optic chiasm and vitreous were of decreased intensity compared with orbital fat on SR. The lens had even less signal intensity than the vitreous on SR and IR images and blended into the surroundings on SE images. A melanoma of the ciliary body and a lymphoma of the lacrimal gland were identified. In conclusion, NMR images can be used to identify normal and pathologic orbital and eyeball anatomy. Image contrast is provided by high intensity fat, which is interspersed throughout other orbital structures.

Massively invasive diffuse choroidal melanoma.

A 74-year-old woman was found to have increasing proptosis in a blind, painful left eye with neovascular glaucoma. Uveal malignant melanoma with massive orbital involvement was diagnosed, and the patient underwent orbital exenteration, with preoperative and postoperative orbital irradiation. The tumor was a mixed-cell, diffuse uveal malignant melanoma with involvement of the optic nerve adjacent to the line of surgical transection and of the optic nerve sheath. Subsequently, the cerebrospinal fluid cytology disclosed cells consistent with malignant melanoma, despite the absence of neurologic signs or symptoms. Cerebrospinal fluid cytology is essential in such cases, and ultrasonography is of value.

Traumatic intraconal hematic cyst of the orbit.

A 35-year-old man with a history of repeated previous facial injuries presented with acute onset of a right orbital mass less than 2 weeks after blunt trauma to the right eye. Orbital B-scan ultrasonography and computed tomography showed an intraconal cystic lesion. The tumor was excised via lateral orbitotomy. Pathologic evaluation demonstrated a hematic cyst containing fresh blood. The presence of hemosiderin in the cyst wall suggested recent bleeding in a preexisting orbital hemorrhagic lesion. Patients with hematic cysts of the orbit usually present with a subperiosteal mass months to years after trauma.

Phenotypic variation in combined granular-lattice (Avellino) corneal dystrophy.

OBJECTIVE: To describe the phenotypic variation exhibited by members of families with combined granular-lattice (Avellino) corneal dystrophy. SETTING: We examined 40 patients (age range, 12 to 85 years) from six unrelated families with Avellino corneal dystrophy. This included the first individuals to ever be examined near the presumed site of origin in Italy. In addition, one family was the first to trace its origins to Germany rather than to Italy. We studied the phenotypic expression of the disease in the cornea, visual acuities, subjective complaints, complications, treatment, and histologic condition of these individuals. RESULTS: The granular stromal lesions reach their mature quantity and size early in life, and appear as either gray and crumb-shaped deposits or superficial with an annular and planar distribution. The lattice component appears gradually, beginning and maturing later in life. The phenotypic variation within families was found to be substantial. Widely variable proportions of lattice and granular changes were found within single sibships. Visual acuities ranged from 20/20 to 20/400. Recurrent corneal erosions were present but infrequent. Subjective complaints included glare and decreased night vision. Penetrating keratoplasty was required in one individual to restore vision. Histopathologic examination revealed typical amyloid and granular deposits. Granular deposits were found replacing Bowman's membrane and extending to the corneal surface. These deposits probably represent the cause of recurrent erosions. CONCLUSIONS: Combined granular and lattice corneal dystrophy may present with substantial phenotypic variation. The disease can be found in individuals who trace their ancestry to both Italy and Germany, a wider geographic distribution than previously proposed.

Ocular invasion in mucormycosis.

The histopathologic findings in a case of ocular invasion in rhinocerebral mucormycosis are described. The findings of hyphae in the inner sclera and marked involvement of the posterior ciliary arteries suggested an arterial route of ocular invasion by fungus. Only five other cases of rhinocerebral mucormycosis with ocular fungal invasion have been reported to our knowledge. All six patients died from the infection. As a group, these cases suggest that the presence of ocular infiltration by fungus may indicate poor prognosis in rhinocerebral mucormycosis.

The autocrine derivation of the opioid growth factor, [Met5]-enkephalin, in ocular surface epithelium.

Endogenous opioid peptides serve as growth factors in developing, renewing, healing, and neoplastic cells and tissues. A native opioid peptide, [Met5]-enkephalin, termed opioid growth factor (OGF), has been discovered to regulate DNA synthesis in the epithelium of the ocular surface. OGF and its receptor zeta have been localized in both the basal and suprabasal cells of the epithelium. This study examined the hypothesis that OGF is an autocrine growth factor. Using probe for preproenkephalin (PPE) mRNA that encodes OGF, and in situ hybridization techniques, silver grains related to PPE mRNA were detected in both basal and suprabasal cells of the central and peripheral cornea, limbus, and conjunctiva. No distinct regional differences in the presence or location of message, as reflected by the density and distribution of PPE mRNA signal, were noted. These results demonstrate that a growth factor known to serve as a tonic, inhibitory, and receptor-mediated influence on the epithelium of the ocular surface is derived in an autocrine manner, thereby permitting local control of homeostatic cellular replication.

Re-epithelialization of the rabbit cornea is regulated by opioid growth factor.

An endogenous opioid peptide, [Met5]-enkephalin, termed opioid growth factor (OGF) is a tonically active, autocrine-produced inhibitory molecule related to developing, neoplastic, renewing and healing tissues. The present investigation was designed to examine the role of OGF on corneal epithelial wound closure in the rabbit under in vitro and in vivo conditions. A 10-mm diameter epithelial defect was made in the center of the rabbit cornea, and the size of the defect, number of specimens with complete re-epithelialization, and rate of wound closure were evaluated using topical fluorescein and morphometric analysis. In organ culture, the influence of a complete opioid receptor blockade by naltrexone (NTX) showed an acceleration in re-epithelialization compared to controls. The action of excessive agonist (OGF) application revealed that exposure of wounded epithelium to OGF delayed wound closure under in vitro conditions, and did so in a receptor-mediated fashion. The modulatory capability of opioids on wound healing in vivo was explored by examining the effects of opioid peptide-receptor disruption using topical application of NTX, and enhanced healing of the abraded rabbit cornea was noted. The presence and location of OGF and the zeta (zeta) receptor in the normal and injured rabbit corneal epithelium were ascertained by immunocytochemistry, and both OGF and the zeta receptor were detected in basal and suprabasal epithelial cells. These results show that an opioid peptide, OGF, plays a direct role in the repair of injury to the corneal epithelium in the rabbit and acts as a receptor-mediated and constitutively expressed inhibitory molecule.

Re-epithelialization of the rat cornea is accelerated by blockade of opioid receptors.

A native opioid peptide, [Met5]-enkephalin, termed opioid growth factor (OGF), serves as a constitutively expressed and autocrine produced inhibitory molecule related to developing, neoplastic, renewing, and healing tissues. The present study was designed to examine the effects of interfering with opioid-receptor interaction during re-epithelialization of the cornea in the rat using both systemic injections and topical applications of the potent opioid antagonist naltrexone (NTX). A 4 mm diameter epithelial defect was made in the center of the rat cornea. NTX injected twice daily or applied as eyedrops four times daily significantly accelerated re-epithelialization compared to controls. Beginning as early as 8 h after wounding, both the systemic and topical NTX treatment groups had defects that were approximately 10% to 67% smaller than control abrasions at the time points examined. Similarly, the rate of healing for the NTX groups was 4.7- and 2.8-fold greater than controls for systemic and topical paradigms, respectively. The incidence of complete re-epithelialization in animals given systemic administration of NTX was markedly accelerated in comparison to control rats; however, differences in incidence of repair between NTX and control groups receiving topical application were not observed. These results show that native opioid peptides function in wound healing, and exert a tonically inhibitory influence at the receptor level on repair of corneal epithelial injuries.

Cellular dynamics of corneal wound re-epithelialization in the rat. I. Fate of ocular surface epithelial cells synthesizing DNA prior to wounding.

The fate of ocular surface epithelial cells in response to injury of the cornea was examined. Corneal epithelial cells were labeled during DNA synthesis with [3H]thymidine 1 h prior to wounding. A 3-mm diameter epithelial defect was made in the center of the rat cornea, with the basement membrane remaining intact. Within 12 h of abrasion, labeled cells were detected in the regenerating surface. At 18 h, there was a 2.7- and 17-fold increase of labeled basal and suprabasal cells, respectively, in the epithelium adjacent to the wound, and at 24 and 30 h there was an excessive number of cell layers (up to 7) at the margin of the abrasion. Re-epithelialization progressed as a gradient of cell layers that became diminished towards the center of the wound. Completion of layers 1, 2, 3, and 4 were recorded at 24, 30, 36, and 72 h, respectively. No changes in the labeling index of the limbus or conjunctiva were noted. These results suggest that processes of centripetal and vertical migration, as well as events related to cell division, in the uninjured corneal surface are not impeded by wounding of the corneal epithelium. However, wound healing appears to require cells with a basal phenotype, presumably because of this cell type's migratory capability.

Conserved expression of the opioid growth factor, [Met5]enkephalin, and the zeta (zeta) opioid receptor in vertebrate cornea.

In addition to neuromodulation, endogenous opioids serve as growth factors. The naturally occurring opioid peptide, [Met5]enkephalin, termed opioid growth factor (OGF), has been found to be a potent and tonic inhibitor of processes related to growth and renewal, particularly cell proliferation. OGF mediates its actions through the zeta (zeta) opioid receptor. In order to determine if OGF and/or the zeta receptor are present in human corneal epithelium, immunocytochemistry was utilized. Immunoreactivity with regard to OGF and to the zeta receptor could be detected in the cortical cytoplasm of both basal and suprabasal epithelial cells, but was not associated with the cell nucleus. Investigation of the ubiquity of OGF and zeta receptor in the vertebrate cornea showed that both elements are present in a wide variety of classes of the phylum Chordata, including mammalia, aves, reptilia, amphibia, and osteichthyes. These results suggest that an endogenous opioid system related to growth may have originated as early as 300 million years ago, and that the function of this system in cellular renewal and homeostasis is a requirement of the vertebrate corneal epithelium.

Cellular dynamics of corneal wound re-epithelialization in the rat. II. DNA synthesis of the ocular surface epithelium following wounding.

To determine the fate of ocular surface epithelial cells in response to corneal injury, epithelial cells undergoing DNA synthesis were labeled at discrete time points following abrasion. A 3-mm diameter epithelial defect was made in the center of the rat cornea. One hour prior to sacrifice, animals received an intraperitoneal injection of [3H]-thymidine. DNA synthesis in the regenerating epithelium was of low abundance (< 2%). The undamaged corneal epithelium had a labeling index (LI) that was markedly elevated from unwounded specimens in the first 48 h after abrasion. The LIs of suprabasal cells were increased at 24 h and 36 h; these cells were believed to be displaced basal epithelial cells. Basal and suprabasal cells of the limbus and conjunctiva exhibited increases in LIs from unwounded subjects at singular timepoints (within 24 h of injury). These results, using rigorous statistical analysis, show that DNA synthesis is not impeded--but rather often accelerated--following denuding of the corneal epithelium. Re-epithelialization is not dependent on DNA synthesis in the regenerating region, but appears to be related to an increase in DNA synthesis (along with centripetal migration) in the adjacent, undamaged epithelium. The increase in DNA synthesis occurring in the limbus and conjunctiva does not directly supply cells to the regenerating region within the 72 h required for epithelial restitution, but is conjectured to serve in replenishing ocular surface epithelial cells used in the repair process.

Immunoreactive atrial natriuretic peptide in the retina of rats and rabbits.

Immunoreactive atrial natriuretic peptide (ANP) has been detected in retinal tissue by radioimmunoassay, although it is unclear whether it was in retinal cells or represented residual peptide from blood and/or ocular fluids. We report here, using the immunoperoxidase method and a highly specific anti-rat ANP, that the peptide was localized consistently in the outer and inner plexiform layers of retinas of rats and rabbits. This information raises important issues: what are the implications for retinal function and, since blood-borne radioligands do not reveal retinal 'receptors', what is the endogenous target of retinal ANP?

Homeostasis of ocular surface epithelium in the rat is regulated by opioid growth factor.

Endogenous opioid peptides serve as growth factors in developing, renewing, and neoplastic cells and tissues. This study examined the hypothesis that opioids serve to modulate the homeostatic renewal of ocular surface epithelium in the rat. DNA synthesis in the epithelium of the central (CC) and peripheral (PC) cornea, limbus (LM), and conjunctiva (CN) was investigated using adult male rats. Animals received an injection of opioid growth factor (OGF), [Met5]-enkephalin, OGF and naloxone (NAL), NAL alone, naltrexone (NTX), or an equivalent volume of sterile water (CO) and sacrificed 4 h later (i.e. 16:00 h). [3H]thymidine was administered 1 h before sacrifice. With the exception of NTX (20 mg/kg), all compounds were given at 10 mg/kg. Examination of 5 time points over an 18-h period revealed no variation in DNA synthesis within a region of ocular surface basal epithelium (BE). OGF depressed DNA synthesis of the BE by 25, 48, and 50% in the PC, LM, and CN, respectively; little labeling was recorded in the BE of the CC. Exposure to OGF-NAL or NAL alone did not alter DNA synthesis of the BE. Complete blockade of OGF-zeta receptor interaction by administration of the potent opioid antagonist, NTX, increased the number of epithelial cells in the PC, LM, and CN undergoing DNA synthesis by 30 to 72%. The effects of OGF and NTX on DNA synthesis of BE also were observed in an organ culture setting. Utilizing immunocytochemistry, OGF and its receptor zeta were associated with both the basal and the suprabasal cells of the ocular surface epithelium. These results indicate that an endogenous opioid peptide, OGF, and its receptor are present and govern homeostatic cellular renewal processes in ocular surface epithelium. OGF regulates DNA synthesis in a direct manner, and does so by a tonic, inhibitory, and receptor-mediated mechanism.

Cellular dynamics of corneal wound re-epithelialization in the rat. III. Mitotic activity.

The number and distribution of mitotic epithelial cells in the ocular surface during homeostasis and in response to abrasion of the mammalian cornea were determined. Normal rats and those receiving a 3 mm diameter centrally located epithelial defect, received an intraperitoneal injection of colchicine 6 h prior to sacrifice. Mitosis in the basal epithelium during homeostasis was comparable in magnitude across the ocular surface epithelium, with the exception of a few mitotic figures in the midline. Thirty percent of the mitotic figures were in the basal layer (layer 1), and 70% were in layer 2; the cells in layer 2 were often noted to retain connection to the basal lamina by cytoplasmic stalks. Mitosis was rarely noted in the regenerating epithelium. However, summation of M phase cells in both the basal and suprabasal epithelium adjacent to the wound showed increases of 3- and 5-fold at 30 and 36 h after abrasion, respectively, from levels at homeostasis and the time of injury. In striking contrast to homeostatic epithelium, 80% of the mitotic cells were located in layer 1 of the corneal epithelium, with normal distribution observed by 72 h. Mitosis in the limbus and conjunctiva was increased 3-fold at 30 h and 24 h, respectively, from values at homeostasis and the time of debridement. These results, using rigorous statistical analysis and precise topographic assessment, showed that mitosis is not impeded - but rather often accelerated - following denuding of the corneal epithelium and that the spatial distribution of mitotic cells is correlated with wounding. The data revealed that re-epithelialization of the corneal epithelium is not dependent on mitosis in the regenerating epithelium, but rather in the adjacent unwounded epithelium of the cornea, with most cells being located in the basal layer until re-epithelialization is completed. Mitotic cells in the limbus and conjunctiva may be related to replenishment of ocular surface epithelial cells used in the repair process rather than directly supplying the abraded surface.

Epithelial adhesion complexes and organ culture of the human cornea.

The effects of extended organ culture of human cornea on the structural integrity, particularly adhesion complexes, of the epithelium were determined. Human corneas were placed in organ culture using an immersion method. The structure of the cornea prior to culture (0 h) and at 7, 14, and 18 days in culture was evaluated by staining with hematoxylin/eosin, and by ultrastructural analysis that included a morphometric study of the type and number of adhesion complexes. Human corneas prepared immediately (0 h) and those in culture after 7 days showed similar structural organization and anatomical features. In contrast to 0 h specimens, the corneal epithelium at 14 days in culture exhibited signs of deterioration, with increases in cellular contraction, extracellular space, electron density of the cytoplasm, nuclear invaginations, and nucleoplasmic opacity, as well as aggregations of junctional complexes between cells. At 18 days in culture, the ocular surface epithelium was markedly reduced in thickness and consisted of no more than 2-3 cell layers; a distinct basal layer was not detected, and the morphology of the suprabasal and basal layers were similar. The basement membrane was disorganized, and anchoring complexes composed of hemidesmosomes were often absent. The number and type of the anchoring complexes associated with the basal epithelium and Bowman's membrane were comparable until 14 days of age, although the total number of hemidesmosomes per microm of epithelial plasmalemma was subnormal. After 2 weeks in culture, there were 38-72% fewer anchoring complexes and a decrease of 44% in the number of hemidesmosomes/microm of membrane from samples prepared immediately and after 7 days in culture. These results indicate that the structural integrity of human corneal epithelium in organ culture is compromised after 14 days in vitro using an immersion system of tissue culture. Thus, long-term use of cultures to define homeostasis and wound healing of the ocular surface epithelium, which necessitates normal architecture including anchoring complexes between epithelium and Bowman's membrane, may not be appropriate and requires careful monitoring both qualitatively and quantitatively at the electron microscopic level of resolution.

Anophthalmos in an infant with multiple congenital anomalies.

A full-term, 2,828-g male infant who lived five weeks had histologically proven, bilateral, congenital anophthalmos. The infant had multiple congenital anomalies including esophageal atresia, choanal stenosis, tetralogy of Fallot, persistent left superior vena cava, arhinencephaly, retardation of myelination in the brain, cerebellar sclerosis, and dysplasias, as well as other developmental anomalies of the central nervous system. There was no family history of anophthalmos, and, in view of the arhinencephaly, we diagnosed sporadic secondary anophthalmos.

Inverted follicular keratosis.

We reviewed 17 cases of inverted follicular keratosis. The median age of the patients at the time of surgery was 69 years. Follow-up in 14 cases showed no recurrences of inverted follicular keratosis, which is a benign skin lesion, often mistaken clinically and pathologically for a malignancy. Inverted follicular keratosis is characterized histologically by the presence of squamous eddies, acantholysis, acanthosis, and hyperkeratosis.

Cosegregation of X-linked retinitis pigmentosa and hemophilia A.

We examined a family pedigree in which retinitis pigmentosa and hemophilia A were inherited in an X-linked manner. Six female carriers were identified by electroretinography. Results of ophthalmoscopic examination were normal in two women, while four displayed marked variability in phenotypic expression. Six of seven males identified with retinitis pigmentosa had hemophilia A. One asymptomatic boy had a markedly abnormal electroretinogram despite normal ophthalmoscopic examination. Pedigree analysis showed a high recombination rate, which would be expected as these two genes are known to be at opposite arms of the X chromosome.