Ratio of size of recipient and donor areas in treatment of vitiligo by autologous cultured melanocyte transplantation.

BACKGROUND: Autologous melanocytes can be expanded in vitro, allowing the treatment of large lesions of vitiligo in one session. Theoretically, this procedure could provide a higher donor/recipient size ratio (DR ratio) compared with that in noncultured cell transplantation (with a DR ratio < 1 : 10). However, the exact DR ratio obtained from this procedure has not been reported. OBJECTIVES: To study whether transplantation of cultured pure melanocytes at a high DR ratio is as efficient as that at a low DR ratio. METHODS: One hundred and two patients with vitiligo were treated by transplantation of cultured pure melanocytes and were divided into two groups: a low DR ratio group, including patients with DR ratio ≤ 1 : 10 (mean 1 : 8, 35 cases) and a high DR ratio group with DR ratio > 1 : 10 (mean 1 : 27, 67 cases). The extent of repigmentation between these two groups was compared. RESULTS: There was no significant difference in repigmentation between the low DR ratio group (mean ± SD 77·4 ± 22·5%) and the high DR ratio group (77·6 ± 24·8%). Multiple regression analysis showed that even after adjustment for age, sex, type of vitiligo and transplanted cell density, there was no significant correlation between the extent of repigmentation and the DR ratio, indicating that patients treated with high DR ratio obtained a satisfactory result and showed no difference from the low DR ratio group. CONCLUSIONS: Various surgical procedures for the treatment of vitiligo which involve melanocyte transplantation or skin grafts have different inherent DR ratios. Transplantation of cultured pure melanocytes is an expensive and complicated procedure; however, it provides the highest DR ratio (> 1 : 10 and up to 1 : 60). Surgeons can select one of these methods for the treatment of vitiligo based on their experience and skill, on the size of lesions, and the availability of laboratory support.

Treatment of vitiligo in children and adolescents by autologous cultured pure melanocytes transplantation with comparison of efficacy to results in adults.

BACKGROUND: Transplantation of autologous cultured pure melanocytes is a well-established procedure for the treatment of refractory and stabilized vitiligo. However, there was no report specifically comparing the efficacy with the regard to defined age groups (children-adolescence-adult). OBJECTIVE: We analysed the efficacy of this procedure in the treatment of vitiligo in children and adolescents and compare it with the results in adults treated during the same period and using identical procedures. METHODS: Melanocytes were isolated from the roof of suction blister, cultured and expanded with Hu16 medium in vitro, and transplanted to laser-denuded receipt area. A total of 12 children (8-12 years), 20 adolescents (13-17 years) and 70 adults with vitiligo were treated using this procedure. RESULTS: The patients obtained satisfactory results (repigmentation of 50% or more) results in children, adolescents and adults were 83.3%, 95.0% and 84.0% respectively. The mean extent of repigmentation in children, adolescents and adults was 80.7%, 78.9% and 76.6% respectively. There was no statistical difference in repigmentation among these three groups. After adjusting for all factors (gender, type of vitiligo, period of stability, location of the lesion or transplanted cell density) individually or totally using multiple regression analysis, age still did not correlate to the extent of repigmentation. CONCLUSIONS: The satisfactory results obtained in the treatment of vitiligo in children and adolescents by transplantation of cultured autologous pure melanocytes are comparable with the results in adults. Therefore, this procedure can be considered in refractory and stable vitiligo in children and adolescents, especially in patients with large vitiliginous lesions.

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.

Studies of human uveal melanocytes in vitro: growth regulation of cultured human uveal melanocytes.

PURPOSE: The authors studied the growth requirements and growth regulation of cultured human adult uveal melanocytes (UM). METHODS: The effect of various mitogens and growth factors on the growth of UM were tested separately or combined on cultured UM in multiwell plates. RESULTS: Basic fibroblast growth factor (bFGF) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulate the growth of UM. Without these agents, the UM did not grow or survive. A cyclic adenosine monophosphate (cAMP) stimulator, such as isobutylmethylxanthine or cholera toxin, stimulated growth in the presence of bFGF. Fetal bovine serum (FBS) is also required for growth. In its absence, UM did not grow, even in the presence of bFGF and cAMP stimulators. Other substances, such as epidermal growth factor, acidic FGF, nerve growth factor, and platelet-derived growth factor had no stimulating effects on the growth of UM. CONCLUSIONS: Three classes of agents are required for the growth of UM in vitro: bFGF or TPA, a cAMP stimulator, and FBS. Adult human UM cultured in medium containing all these agents grew well and could be passaged for many generations.

Isolation and cultivation of human iris pigment epithelium.

There have been very few attempts to isolate and culture human iris pigment epithelium (IPE). Earlier efforts that used whole iris explant methods did not achieve pure cultures of IPE. We have developed methods for separating the IPE from the iris stroma of post-mortem eyes that avoid contamination by other cell types. Three different isolation methods were studied: direct dissection, enzyme digestion, and enzyme-assisted microdissection. The latter method yielded the best results. After treatment with enzyme solution, the IPE was easily separated from the stroma under the stereomicroscope and subsequently cultured with supplemented F12 medium. With this method, approximately 2.3 x 10(5) cells were isolated from each iris with an average viability of 90.2%. IPE cells isolated from 19 of 24 eyes grew to confluence in primary culture. The IPE could be maintained in pure culture for many generations over several months with up to 20 population doublings. Cultured IPE demonstrated cytokeratin and S-100 protein by immunocytochemistry. Some of these cells also displayed desmin, indicating origin from the anterior IPE. Cultured IPE cells retained most of the characteristics of IPE in vivo, such as apical/basal polarization, microvilli, and many cell junctions. Gradual dilution of pigment occurred in the dividing IPE cells, suggesting an inability to produce melanin in vitro. A subpopulation of the IPE cells contained myofilaments by electron microscopy, also indicating a anterior IPE origin. This method provides a source for large numbers of human IPE cells and could be useful in studies of the biology of IPE and the role of IPE in pathogenesis of several eye diseases, most notably exfoliation syndrome and its associated glaucomas.

Studies of human uveal melanocytes in vitro: isolation, purification and cultivation of human uveal melanocytes.

PURPOSE: To develop the methods for isolation and cultivation of human uveal melanocytes (UM) from adult donor eyes. METHODS: After removal of the pigment epithelium, the uvea was pretreated in trypsin solution at 4 degrees C overnight, incubated at 37 degrees C with trypsin for 1 hr, then incubated with collagenase for 3 hr. Released cells were collected each hour during the incubation and cultured with F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, isobutylmethylxanthine and cholera toxin. Contaminant cells were eliminated by adding a selective cytotoxic agent, geneticin, when necessary. RESULTS: These methods provide pure melanocyte cultures with high cell yields, good viability, and rapid growth rates. UM isolated and maintained using these methods can be passaged 23 times for a period of 7 mo for more than 35 population doublings. This is comparable to results obtained with cultured neonatal dermal melanocytes and exceeds results obtained with adult dermal melanocytes cultured in media supplemented with phorbol ester, isobutylmethylxanthine, and cholera toxin. CONCLUSION: A method for isolation and cultivation of UM has been developed that yields satisfactory results. Cultured UM may be useful in in vitro studies of UM physiology and may allow development of in vitro models of the pathogenesis of uveal malignant melanoma.

Distribution of carbonic anhydrase among human photoreceptors.

The distribution of carbonic anhydrase (CA) among human photoreceptors has been a topic of dispute. In our experiments, by modifying an established enzyme histochemical technique, reproducible staining was observed. Of the cones in the peripheral retina, 91% were positive for CA. The CA-negative (CA-) cones were absent within approximately 8 arc min of the foveal center and their density peaked at 2 arc deg. No CA activity was found in the rods. Morphologic differences were noted between the CA-positive (CA+) and CA- cones. Compared to the CA+ cones, the CA- cones had longer and more nearly columnar inner segments, more nearly spherical nuclei, and more generous amounts of perikaryal cytoplasm. In the peripheral retina, the distance between CA+ to CA+ nearest neighbors were larger compared to CA- to CA+ nearest neighbors (P less than 0.0001). The frequency distribution and morphology of the CA- cones suggest that they are the blue-sensitive cones. As such, this study demonstrates a biochemical similarity between blue cones and rods that may provide insight into the function and phylogeny of the blue cones.

Melanogenesis by human uveal melanocytes in vitro.

PURPOSE: To study melanogenesis by cultured human uveal melanocytes, and the relationship between melanin production by uveal melanocytes in vitro with the degree of iris pigmentation in vivo. METHODS: Melanin content, melanin production, and tyrosinase activity of cultured uveal melanocytes derived from eyes of various iris color were measured at different stages of cultivation. RESULTS: Cultured uveal melanocytes maintained a constant level of melanin content, expressed tyrosinase activity, and produced measurable amounts of melanin in vitro. Melanosomes in different stages were seen ultrastructurally. Melanin production correlated directly with the degree of iris pigmentation of the eyes from which the uveal melanocytes were isolated. Tyrosinase activity of cultured uveal melanocytes from black versus white donors was significantly different, but, among white donors, there was no correlation with iris pigmentation or with melanin production in vitro. CONCLUSION: Cultured uveal melanocytes can produce melanin in vitro. Cultured uveal melanocytes isolated from eyes of different iris color maintained their inherent capacity for melanogenesis. Therefore, cultured uveal melanocytes are an excellent model system for studying melanogenesis in uveal melanocytes in vitro.

Topical mitomycin chemotherapy for conjunctival malignant melanoma and primary acquired melanosis with atypia: clinical experience with histopathologic observations.

OBJECTIVES: To clinically evaluate topical mitomycin chemotherapy in patients with diffuse, multifocal, or recurrent primary acquired melanosis with atypia and/or conjunctival malignant melanoma and to histopathologically study ocular tissue samples obtained before and after treatment. METHODS: Chemotherapy with topical mitomycin, 0.04% 4 times daily, was administered for 28 days as the primary and only treatment in 7 patients (after biopsy) and for 7 days as adjuvant therapy to excision and cryotherapy in 5 patients. Mean follow-up was 38 months. Five patients developed subconjunctival recurrences, for which 2 underwent orbital exenteration and 3 were treated conservatively. Histopathologic specimens of conjunctival, adnexal, and ocular tissues obtained before and after chemotherapy were evaluated. RESULTS: Regression of tumor was observed in 11 patients with primary or adjuvant topical mitomycin chemotherapy. One patient with nodular melanoma was resistant to mitomycin chemotherapy. Histopathologic findings included regionally variable conjunctival epithelial atrophy and thinning. Dyskeratosis and focal keratinization in conjunctival epithelium were noted. Epithelial nuclei were occasionally pyknotic in areas of atrophic epithelium. Subepithelial inflammation was present and was most intense in areas with severe atrophy and/or keratosis. Two patients with primary treatment and 2 with adjuvant treatment developed subconjunctival recurrence. In patients with recurrent malignant melanoma, the deeper layers of the lamina propria were involved, with sparing of the epithelium and superficial lamina propria. Transient keratoconjunctivitis was observed in all patients during treatment. In evaluation of the exenteration specimens, corneal, scleral, episcleral, retinal, and anterior structures were within normal limits. CONCLUSIONS: Topical mitomycin chemotherapy was found to induce regression of conjunctival melanoma and primary acquired melanosis with atypia. When mitomycin chemotherapy was used as an adjuvant to excision and cryotherapy, 2 (40%) of 5 patients experienced tumor recurrence at a mean of 4.3 years' follow-up. Our histopathologic findings demonstrated a long-term mitomycin chemotherapy-related effect on the conjunctiva. The degree of chronic atrophy and inflammation was not clinically significant. The pattern of effect and location of recurrent disease suggest that this regimen of topical mitomycin chemotherapy was most effective for superficial tumors. No complications that would preclude use of our dose regimen were noted. Although subconjunctival or orbital recurrences were noted, topical mitomycin chemotherapy warrants further investigation as an alternative treatment for primary acquired melanosis with atypia and conjunctival malignant melanoma. Arch Ophthalmol. 2000;118:885-891

Isolation and culture of iris pigment epithelium from iridectomy specimens of eyes with and without exfoliation syndrome.

OBJECTIVE: To culture iris pigment epithelium (IPE) from surgical iridectomy specimens of eyes with and without exfoliation syndrome. METHODS: The IPE was treated to obtain a single cell suspension. Cells were cultured in Ham F12 nutrient mixture, which was supplemented with 30% fetal bovine serum, 50-micrograms/mL [corrected] gentamicin, and 2-mmol/L glutamine. After confluence, the cells were detached using a 0.125% trypsin-0.01% edetic acid solution, resuspended, diluted, and subcultured. The IPE from primary cultures and subcultures was studied by transmission electron microscopy. Immunocytochemical staining was performed. RESULTS: In the primary cultures of IPE from patients with exfoliation syndrome, curved, cross-banded, fine fibrils (diameter, 10-15 nm; periodicity, 10-14 nm) were found on the cell surface. Thicker fibrils (diameter, 24-48 nm; periodicity, 24-36 nm) were found external to the fine fibrils. Subcultures contained mainly fine fibrils. The IPE cells stained positively with anticytokeratin, S100 protein, and vimentin antibodies. CONCLUSION: Iris pigment epithelium can be successfully cultured from eyes with exfoliation syndrome. Studying the production of exfoliation material in vitro should provide information about the pathogenesis of exfoliation syndrome and about the nature of the exfoliation material. The cultivation of normal IPE from surgical specimens provides a source for the study of the growth regulation and pharmacophysiology of IPE in vitro.

The anatomy of the lateral canthal tendon.

This report documents the anatomy of the lateral canthus using gross dissection, histologic examination, computed tomography, magnetic resonance imaging, and clinical measurement. Lateral canthal dissections of 16 cadaver orbits demonstrated a well-defined attachment of the tarsal plates to the orbital rim, averaging 10.6 mm in length and 10.2 mm in width at their insertion on Whitnall's tubercle, 1.5 mm behind the orbital rim and 9.7 mm inferior to the frontozygomatic suture. Histologic examination showed a band of dense fibrous tissue attached to the tarsal plates, with intermingled muscle fibers from the pretarsal orbicularis oculi muscle. A small pocket of fat was identified posterior to the orbital septum and anterior to the lateral canthal tendon. Clinical measurements of normal adults revealed 2 mm of lateral movement of the canthal angle during abduction, apparently caused by posterior fibrous attachments to the check ligament of the lateral rectus muscle.

Microwave diathermy of the retina and choroid.

Microwaves were used to induce chorioretinal scar formation in normal rabbit eyes. We have developed a directional 6.8-gigahertz microwave applicator with a rectangular aperture. It was designed to mimic the shape and function of a T-shaped scleral depressor. For treatment, the applicator was placed on the conjunctiva over the sclera. Then, indentation was used to visualize probe placement during indirect ophthalmoscopy. Thermocouple-controlled heating was initiated such that conjunctival temperatures in a range of 51 degrees C to 65 degrees C were induced for 10 seconds per treatment. We found that treatment at temperatures of 51 degrees C or 52 degrees C for 10 seconds produced circular areas of acute retinal whitening. From these microwave-induced lesions, there evolved chorioretinal attenuation with and without evidence of retinal pigment epithelial hyperplasia. No evidence of scleral damage was noted at these thermal doses.

Epithelial inclusion cyst of the iris.

We describe the clinical presentation, high-frequency ultrasound biomicroscopic characteristics, and pathologic findings associated with a conjunctival inclusion cyst within the iris. The patient had undergone an uncomplicated extracapsular cataract extraction with posterior chamber intraocular lens insertion 9 months prior to presenting with a progressively enlarging iris mass. A clinical examination revealed a solid-appearing white tumor within the midiris stroma, accompanied by limbal-conjunctival hyperemia and anterior chamber inflammation. Ultrasound biomicroscopy revealed an egg-shaped solid iris stromal tumor that displaced the pigment epithelium. The mass was composed of three concentric layers of different echogenicity: a moderately reflective mantle, a less reflective middle zone, and a hyperreflective core. Within 3 days of initiation of topical corticosteroid therapy (prednisolone acetate, 0.5 mg per drop four times daily), the tumor enlarged and induced a plasmoid aqueous and a hypopyon. Histopathologic study revealed a conjunctival inclusion cyst with evidence of acute and chronic inflammation. We have found that the diagnosis of epithelial inclusion cyst within the iris can be aided by an ultrasound evaluation. This case also suggests that it may be preferable to excise these tumors prior to topical corticosteroid treatment.

Iris and ciliary body melanomas: ultrasound biomicroscopy with histopathologic correlation.

OBJECTIVE: To correlate ultrasound biomicroscopic images of iris and ciliary body melanomas with their histopathologic features. METHODS: Ultrasound biomicroscopy was performed in 3 cases of iris melanoma and in 3 cases of ciliary body melanoma. Cross-sectional ultrasound biomicroscopic images were compared with findings from clinical examination and light microscopy to evaluate associations between their histopathologic, surface, and internal ultrasound characteristics. Unique images of intrastomal and obscured posterior tumor margins were visualized by ultrasound biomicroscopy. RESULTS: Results of this study revealed that ultrasound biomicroscopy offers an accurate method to evaluate tumor shape, reflectivity, and local invasion. Neoplastic tissue had only medium echogenicity. Enlarged vessels were correlated to echolucent spaces in the iris stroma. Anterior tumor margins were found within the iris stroma, within the anterior chamber angle, and on the endothelial surface of the cornea. Posterior tumor extension was noted to encroach onto the lens, into the sclera, and serous peripheral retinal detachments were associated with ciliary body tumors. CONCLUSION: Ultrasound biomicroscopic images correlated well with histopathologic features of anterior uveal melanomas including shape, reflectivity, and local extension. Arch Ophthalmol. 2000;118:1515-1521

Influence of haptic materials on the adherence of staphylococci to intraocular lenses.

A recent case-control study indicated that the insertion of an intraocular lens with polypropylene (Prolene) haptic materials was a significant risk factor for postoperative endophthalmitis (odds ratio = 4.5, P < .01). In the present study, we used quantitative techniques to evaluate adherence of Staphylococcus epidermidis to two intraocular lens types--lenses with polypropylene haptic materials and all-polymethyl methacrylate optic and three-piece all-polymethyl methacrylate lenses--using a quantitative culture method, a radioisotope technique, and scanning electron microscopy. All three methods demonstrated approximately twice as many bacteria adherent to lenses with polypropylene haptic materials as to all-polymethyl methacrylate lenses. Scanning electron microscopy showed preferential bacterial adherence to the polypropylene haptic materials. These data provide a pathogenic mechanism to explain our epidemiologic findings of an increased risk of postoperative endophthalmitis associated with implantation of intraocular lenses with polypropylene haptic materials.

Anterior segment implantation cysts. Ultrasound biomicroscopy with histopathologic correlation.

OBJECTIVE: To correlate the clinical, histopathologic, and ultrasound biomicroscopic characteristics of anterior segment implantation cysts. METHODS: We performed a retrospective review of 7 cases of secondary anterior segment implantation cysts. We reviewed the clinical history, visual acuity, clinical findings, and ultrasound biomicroscopic characteristics in all cases. Histopathologic correlation was possible in 4 cases. RESULTS: Six eyes had been subjected to major trauma prior to cyst formation. Trauma was noted as blunt in 3 eyes and surgical in 3 eyes. The diagnosis was confirmed in 1 eye when conjunctival cells were aspirated on fine needle biopsy. Ultrasound biomicroscopy revealed large (mean +/- SD greatest diameter, 4.7 +/- 0.9 mm) cystic tumors. In 1 patient, a cyst-related indentation of the anterior lens surface was seen. Ultrasonographic evaluations of internal reflectivity revealed thick, moderately reflective cyst walls encapsulating a relatively hypoechoic core. In 3 cases, the cyst contents consisted of variably reflective material. The other 4 were completely sonolucent. Histopathologic correlation showed that the cyst walls were lined with stratified squamous epithelium. The moderately reflective cyst contents were found to be degenerated conjunctival cells with inflammatory foci and cholesterol crystals. The sonolucent regions correlated with inflammatory cells and fluid. CONCLUSIONS: This study demonstrates that implantation cysts are unilateral, large, and thick walled. They may be sonolucent or exhibit variable internal reflectivity. These findings as well as the extent of anterior segment involvement (particularly posterior extension) could be evaluated by ultrasound biomicroscopy prior to surgery.

Congenital intraocular teratoma associated with eyelid coloboma.

PURPOSE: To describe the first case of intraocular teratoma associated with eyelid coloboma and the second reported case of intraocular teratoma. DESIGN: Interventional case report. METHODS: A left intraocular tumor was surgically resected from a 2-day-old female with an associated lower eyelid coloboma. RESULTS: Pathologic evaluation revealed a completely intraocular tumor comprising derivatives of all three germ cell layers giving a diagnosis of intraocular teratoma. The eyelid coloboma was repaired, and a scleral-wrapped hydoxyapatite-integrated orbital implant was placed. CONCLUSION: To our knowledge, this is the second reported instance of teratoma originating within the globe and the only reported case of teratoma associated with eyelid coloboma. Although exceedingly rare, intraocular teratoma should be added to the differential diagnosis of congenital intraocular tumors.

Ultrasound biomicroscopic diagnosis of cyclitic membranes.

PURPOSE: To evaluate the utility of ultrasound biomicroscopy in imaging cyclitic membranes. METHODS: Patients with hypotony and suspected or known cyclitic membrane underwent ultrasound biomicroscopic examination. Histopathology of cyclitic membrane was correlated with ultrasound biomicroscopy in three cases. RESULTS: Six eyes of six patients were enrolled. Mean patient age was 62.2 +/- 18.4 (SD) years. The mean intraocular pressure in the affected eye was 4.3 +/- 3.4 mmHg. Three eyes were pseudophakic and three eyes were aphakic. All eyes had undergone two or more previous intraocular surgeries. Ultrasound biomicroscopy imaged the cyclitic membrane in all six eyes. Histopathology revealed fibroproliferative cyclitic membranes with associated inflammatory cells. CONCLUSION: Ultrasound biomicroscopy is useful in detecting the presence of those cyclitic membranes that may not be identified on clinical examination.

Wound healing following intrastromal photorefractive keratectomy with the Nd:YLF picosecond laser in the cat.

BACKGROUND: We studied the histopathology of the stromal wound healing response in the cat cornea following intrastromal photorefractive keratectomy (IPRK) with the Nd:YLF picosecond laser. METHODS: Intrastromal PRK was performed in the anterior stroma of cat corneas with the Nd:YLF picosecond laser. The cats were sacrificed at predetermined intervals ranging from immediately to 6 months postoperatively. Effects of the laser treatment on the epithelium, Bowman's layer, stroma, and the endothelium were evaluated using light and scanning electron microscopy. No anti-inflammatory agents were used. RESULTS: Intrastromal PRK resulted in no perceptible damage to the corneal epithelium or Bowman's layer either acutely or at 6 months. The corneal stroma showed multiple cavitations immediately after intrastromal PRK, which collapsed over several hours, followed by thinning of the cornea over 2 weeks. At 1 month, the stromal collagen was abnormal with surrounding hypercellularity. The endothelium showed no injury, acutely or at 6 months. No thermal effects on stromal collagen were observed at 6 months, and disruption of the lamellar pattern was not apparent after the cavitation bubbles were reabsorbed. CONCLUSION: Intrastromal PRK can effectively remove stromal tissue without acute damage to the adjacent lamellae, epithelium, or endothelium. There is a transient cellular wound healing response associated with a transient stromal collagen abnormality at 2 weeks to 1 month, which was not apparent 2 months after the procedure.

Effects of melatonin, its precursors and derivatives on the growth of cultured human uveal melanoma cells.

The effects of melatonin, its precursors and derivatives on the growth of cultured human uveal melanoma cells were studied. The melanoma cells were plated into 24-well plates. Melatonin, its 6-hydroxy or 6-chloro derivative, serotonin, tryptophan or kynurenine was added to the medium in concentrations of 0.001 to 1000 nM. After 5 days the cells were detached, counted, and compared with the controls. Melatonin inhibited the growth of uveal melanoma cell lines in a dose-dependent manner (0.1-10 nM). This growth inhibition occurred at concentrations of melatonin (2 nM) found in human aqueous humour. The melatonin derivatives also inhibited the growth of uveal melanoma cells; 6-chloromelatonin was more potent than melatonin and 6-hydroxymelatonin was the least active (6-chloromelatonin > melatonin > 6-hydroxymelatonin). The precursors of melatonin (tryptophan and serotonin) and the abnormal metabolite of tryptophan (kynurenine) did not inhibit the growth of the melanoma cells, indicating that changes to the metabolic processes of melatonin may play a role in the pathogenesis of uveal melanoma.