Subconjunctival Tissue Research Articles

Senile ocular amyloidosis in SAM and BALB/c strains of mice

We investigated whether amyloid deposition can affect retinal atrophy in old SAMR1, SAMP1 and BALB/c mice. Immunohistochemistry revealed that old SAMP1 mice showed the deposition of the murine senile amyloid protein fibril, AApoA-II in the subconjunctival tissue, the vessel walls near the chamber angle, and the sheaths of the external ocular muscles and the conjunctival glands, but was never observed in the retina or the choroid. Although the old SAMR1 mice also showed a remarkable loss of retinal photoreceptor and ganglion cells, they never showed any amyloid deposition. The BALB/c strain did not showed any amyloid deposition either. Our data suggest that atrophy of the retina is not related to senile systemic amyloidosis in mice.

APOPTOSIS IN THE CORNEA IN RESPONSE TO EPITHELIAL INJURY: SIGNIFICANCE TO WOUND HEALING AND DRY EYE.

Chronic ocular surface injury is a hallmark of dry eye disease. The extent of objective surface abnormality is highly variable between patients, and roughly correlates with the severity of the disease. These surface changes extend from mild conjunctival rose bengal staining to diffuse conjunctival and corneal rose bengal and fluorescein staining. Previous studies have demonstrated that corneal surface injury triggers keratocyte apoptosis and a subsequent wound healing response in the stroma. However, the majority of patients with mild to moderate surface abnormalities from dry eye have no evidence of stromal haze, unstable refractive error, or other changes that would be expected if significant ongoing stromal wound healing were triggered by the surface changes. Superficial injury to the corneal or conjunctival epithelium probably does not induce stromal wound healing because of the intact barrier function of the epithelium and lack of penetration of proapoptotic cytokines into the corneal stroma and subconjunctival tissues. New data suggest in response to IL-1 or tumor necrosis factor (TNF) alpha is monocyte chemotactic and activating factor (MCAF)18 (also Hong, Liu, and Wilson, unpublished data, 2000). This upregulation of MCAF in response to IL-1 or TNF alpha stimulation was confirmed by RNAse protection assay (mRNA) and western blotting (protein). MCAF has chemotactic effects on monocyte and macrophage cells. This observation has led us to formulate a model to explain factors attracting inflammatory cells into corneal stroma or subconjunctival connective tissue in response to epithelial injury. This hypothesis holds that: 1) Significant injury to the epithelium (mechanical or other) results in the release of IL-1 and TNF alpha from the epithelial cells. 2) If the injury to the epithelium is sufficient to break down the epithelial barrier function, then IL-1 and TNF alpha penetrate into the stroma or subconjunctival tissues. Some types of injury might directly trigger release of IL-1 or TNF alpha from the basal epithelial cells. 3) IL-1 or TNF alpha that penetrates into the subepithelial tissues binds to receptors expressed by keratocytes or conjunctival fibroblasts, and triggers MCAF production. 4) MCAF attracts and activates macrophages. Work is in progress to characterize the effects of MCAF. We also hope to identify other cytokines released by keratocytes or conjunctival fibroblasts that attract T cells and polymorphonuclear cells in response to epithelial injury or other pathophysiological triggers.

The pharmacokinetics and effects of diltiazem in rabbits.

To investigate the effect of diltiazem on wound healing after the creation of conjunctival flaps in rabbit eyes. Also, to investigate the pharmacokinetics of diltiazem in rabbits after subconjunctival and topical administration. For the histopathological study, a limbal-based flap was prepared and diltiazem was injected subconjunctivally for five days after the surgery. The rabbits were euthanised 20 days after surgery. The effectiveness of diltiazem on wound healing was evaluated by histopathological examination and measurement of the thickness of subconjunctival fibrous tissue. For the pharmacokinetic study, diltiazem was applied topically or injected subconjunctivally. Aqueous paracenteses were performed 0.5, 1, 2, 4 hours thereafter. The histopathological study found no difference in thickness of the subconjunctival fibrous tissue in control and diltiazem-treated eyes. No significant toxicity was observed in eyes treated with diltiazem. The peak aqueous concentration was 3.8 +/- 0.4 microg/ml after topical application and 15.3 +/- 1.1 microg/ml after subconjunctival injection. The peak aqueous concentration was achieved 1/2 hours after administration in both cases. Diltiazem did not appear to affect wound healing at the dose tested. Topical and subconjunctival diltiazem successfully penetrated the aqueous humor of rabbit eyes.

Augmentation of filtering blebs with perfluoropropane gas bubble: An experimental and pilot clinical study

ObjectiveTo evaluate the use of perfluoropropane (C3F8) gas bubble in the augmentation of glaucoma-filtering blebs. DesignExperimental animal study and a pilot clinical study. ParticipantsTwenty rabbits in the animal study and 20 humans in the pilot study participated. InterventionTwenty patients underwent trabeculectomy for medically uncontrolled glaucoma. Surgical techniques were similar to those used in standard limbus-based trabeculectomies. The Tenon’s capsule and conjunctival wound were closed separately using continuous sutures. Before the final knots were tied, 0.1 to 0.2 ml of 15%:85%–C3F8:air mixture gas bubble was introduced into the subconjunctival space with a silicone tube attached to a 27-gauge Rycrof cannula. The sutures were immediately knotted on withdrawal of the tube. The patients were reviewed after surgery for the appearance, size, and location of the gas bubble. The bleb morphology and intraocular pressures were also monitored. Main outcome measuresThe parameters measured were intraocular pressure control, Snellen visual acuity, appearance of the bleb in relation to its elevation and degree of inflammation of the subconjunctival tissue, and characteristics of the gas bubble (e.g., size, number, and position). ResultsThe gas bubbles stayed in the conjunctival bleb for an average duration of 17 days (range, 13–27 days). In all cases, the gas bubbles helped to maintain the filtering bleb until their complete resolution. No excessive inflammation was noted in the filtering blebs. The mean preoperative intraocular pressure was 27.3 ± 5.5 millimeters of mercury (mmHg), and the mean postoperative intraocular pressure was 14.5 ± 2.3 mmHg, with only two patients requiring supplementary topical medication. All cases resulted in good filtering blebs with satisfactory intraocular pressures after 16 months of follow-up. ConclusionsSubconjunctival C3F8 gas bubbles, via a “spacer” effect, aid the maintenance of filtering blebs in the early postoperative period. This technique may be useful in augmenting or salvaging blebs at risk of failure.

Immunolocalization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human subconjunctival tissues

Purpose. To evaluate the roles of fibroblast proteins in the remodeling of the subconjunctival connective tissue, we immunohistochemically assessed the expression of matrix metalloproteinases (MMP)−1 and −2, and the tissue inhibitors of matrix metalloproteinases (TIMP)−1 and −2 in cultured human subconjunctival fibroblasts and in normal and healing human subconjunctival connective tissue.Methods. Cultured fibroblasts derived from human subconjunctival connective tissue and surgical specimens of normal and healing conjunctiva were immunostained with monoclonal antibodies directed against human MMPs and TIMPs and examined by light and electron microscopy.Results. In the cultured fibroblasts, MMP-1 and TIMP-1 antibodies stained the cytoplasm in a fine granular pattern, suggesting localization of those proteins in the endoplasmic reticulum (ER) and Golgi apparatus. Antibodies to MMP-2 and TIMP-2 reacted with fibroblast cytoplasm in a granular pattern. Electron microscopy of those fibroblasts revealed MMP-1 and TIMP-1 immunoreactivity in the ER cisternae or on the membrane of the ER. In surgical samples, MMP-1 and TIMP-1 were immunohistochemically detected in healing subconjunctival tissue, but not in conjunctival epithelium or normal subconjunctival tissue.Conclusions. MMPs and TIMPs may be involved in remodeling of subconjunctival connective tissue and in fibroblast population after surgical interventions. These proteins may play a crucial role in the post-operative fibrotic process occurring during scar formation in subconjunctival tissue. Curr. Eye Res. 17: 445–451, 1998.This paper was presented in abstract form by Dr. Kawashima at the Annual Meeting of The Association for Research in Vision and Ophthalmology, May 13, 1997, Fort Lauderdale, FL, USA.

Limbal Autograft Transplantation for Recurrent and Advanced Pterygia

This study was conducted to examine the usefulness of limbal autograft transplantation in the treatment of recurrent and advanced pterygia. Eleven patients with recurrent and 16 with advanced pterygia (a total of 27 pterygia) were treated with limbal autograft transplantation. Once a pterygium had been excised, superior limbal tissue was taken with conjunctival flap and transferred to the excised area. All the grafts were planted promptly, and donor sites were re-epithelialized with no excessive scar tissue formation. Although slight recurrence was noted in 2 eyes (7.4%), subconjunctival tissue invasions were limited to less than 1 mm, and no further surgical interventions were needed. These results indicate that limbal autograft transplantation may be effective for the treatment of recurrent and advanced pterygia.

Fluorescence angiography of the anterior eye segment in severe eye chemical burns

Severe eye burns usually result in extensive necrosis of the conjunctiva and subconjunctival tissue. To reduce the reactive inflammatory response excision of the necrotic tissue is necessary. Between August 1990 and December 1994 fluorescein angiograms of the anterior eye segment were performed in 13 (17 eyes) patients with most severe eye burns. The angiographies were carried out 1-4 days after the accident. Conventional photo angiography was performed using a fundus camera (NF 505-AF Nikon). The angiograms revealed characteristic patterns that provided exact information on the perfusion of burned conjunctival and underlying tissue. It was particularly useful in the detection of clinically not recognisable subconjunctival ischemia. In addition, perfusion of the episcleral vessels and the extent of scleral ischemia could be quantified. According to the angiographic findings subtile excision of the necrotic tissue was performed and vascularized tissue was preserved. Anterior segment angiography is a valuable method in assessing the extent of necrosis of the conjunctiva and underlying tissue and in determining scleral ischemia. It provides a basis for deciding the extent of surgical debridement of necrotic tissue in the acute phase of the burn. The quantification of limbal and scleral ischemia may give useful information for early plastic-reconstructive procedures.

Inhibition by Rapamycin of PDGF- and bFGF-Induced Human Tenon Fibroblast Proliferation in Vitro

Fibrosis of subconjunctival tissues is a major cause of bleb failure following glaucoma filtration surgery. The aim of the present investigation was to demonstrate the effect of Rapamycin, a clinically relevant macrolide antibiotic with potent immunosuppressive properties, on human Tenon fibroblast proliferation induced by platelet-derived growth factor and basic fibroblast growth factor. Primary Tenon fibroblast cultures were derived from patients undergoing trabeculectomies or routine cataract extractions. Rapamycin was added in concentrations of 0.1-100 ng/ml with or without 3-30 ng/ml of porcine platelet-derived growth factor or of human recombinant basic fibroblast growth factor. Two days after treatment, the cells were examined and counted. The results were expressed as the percent of cell growth in treated culture relative to its untreated control. Rapamycin was not cytotoxic at any of the concentrations tested. Inhibition of platelet-derived growth factor-induced Tenon fibroblast proliferation occurred with all doses of Rapamycin, the most marked effect being observed with 30 ng/ml (60% inhibition, p < 0.001). In contrast, optimal inhibition of basic fibroblast growth factor-induced proliferation was only 37% (p < 0.01), achieved with 10 ng/ml of the peptide. Rapamycin potently inhibits platelet-derived growth factor-induced fibroblast proliferation in vitro without any apparent cytotoxicity. It may eventually prove to be a useful adjunct to glaucoma filtration surgery.

Orbital fat prolapse in the dog

Five dogs presented with varying degrees of subconjunctival swelling and enophthalmos. Biopsy revealed the subconjunctival tissue to be fat. Surgical excision in two cases resulted in resolution of the clinical signs and demonstrated that the fat came from the retrobulbar region.

In vitro and in vivo evaluation in rabbits of a controlled release 5-fluorouracil subconjunctival implant based on poly(D,L-lactide-co-glycolide).

To design a controlled release 5-fluorouracil (5-FU) implant to provide prolonged antifibroblast concentrations of 5-FU in the subconjunctival tissues but low concentrations of 5-FU in other ocular tissues. Implants (5 mg; 2.5 mm diameter x 1.2 mm thickness) of 5-FU or 9:1, 8:2, 7:3 5-FU to polymer mass ratios were made by compression. Poly(D,L-lactide-co-glycolide) polymers with 50:50 and 75:25 lactide to glycolide ratios were used. In vitro release characteristics of four types of implants were studied: 5-FU alone (CT), 5-FU/ polymer matrices (MT), coated 5-FU/polymer matrices with a central hole drilled through the matrix and coating (CM1), and with a central hole in the coating (CM2). MT and CM1 (9:1 drug/polymer) were selected for subconjunctival implantation in rabbits. 14C-5-FU levels in various ocular tissues and retrieved pellets were monitored. First-order release was observed from CT, MT and CM1 implants. Zero-order release profiles were observed from CM2 implants. Drug release was retarded by formulating 5-FU in a matrix comprising poly(D,L-lactide-co-glycolide) which in turn could be modified by the lactide/glycolide ratio of the polymer, the drug to polymer ratio, coating, and hole dimensions. First-order release kinetics were observed for MT and CM1 implants in the in vivo study in rabbits. A correlation between in vitro and in vivo release was established which allowed in vivo release to be predicted from in vitro release data. For CM1, therapeutic tissue concentrations of 35.2 micrograms/g (conjunctiva) and 17.7 micrograms/g (sclera) were found at the implantation site up to 200 hours post-implantation. Tracer levels were undetectable in other ocular tissues. The CM1 implant maintained steady antifibroblast levels in target tissues and minimized levels in nontarget tissues.

Antiproliferative activity of a liposomal delivery system of mitoxantrone on rabbit subconjunctival fibroblasts in an ex-vivo model.

Wound healing is the main cause of the failure of filtering surgery in glaucoma. We developed a liposomal delivery system of mitoxantrone (MITX), an anthracyclin derivative, to allow a single adjuvant administration and to lessen ocular side-effects of the drug. In order to evaluate the antiproliferative activity of liposomal MITX, an ex vivo model consisting in the culture of subconjunctival tissue explants from rabbits pretreated with subconjunctival injections of free or liposomal MITX was used. We found that both forms of MITX decreased the growth rate as well as the explant proliferation surfaces 15 days or 1 month after a single administration of the drug in vivo. A morphometric analysis of the cells showed that the surface of the fibroblasts exposed to both forms of MITX was from 10 to 12 times as important as that of the control cells exposed to the empty liposomes and to the control buffer. A radioautographic study showed that more than 95% of the fibroblasts exposed to both forms of MITX were in the G1 phase of the cell cycle, while the control cell population was equally distributed among the different phases of the cell cycle.

Conjunctiva and subconjunctival tissue in primary open-angle glaucoma after long-term topical treatment: an immunohistochemical and ultrastructural study.

Primary open-angle glaucoma is commonly treated with long-term hypotensive medical therapy. When this approach becomes inadequate, therapy proceeds with surgery. The present study investigates morphological changes in the conjunctival and subconjunctival tissues induced by short- and long-term topical medical therapy of primary open-angle glaucoma. Comparisons were made between biopsy specimens from glaucomatous patients, who received specific eyedrop therapy (timolol and pilocarpine) for various periods of time, and control patients with no conjunctival pathology or topical treatment. Histological, immunohistochemical and ultrastructural parameters were investigated. The morphometric analysis of histological sections and immunohistochemistry (anti-fibronectin antibody) in medium- and long-term therapy patients showed: (a) significant increases in the thickness and number of epithelial cell layers; (b) significant increases in the fibroblast density in both subepithelial and deep connective tissue; and (c) a more compact connective tissue, richer in collagen fibers arranged in whirls, with some inflammatory elements. These findings were confirmed by the ultrastructural analysis. In the same patients, the other immunohistochemical parameters investigated (anti-HLA-DR, anti-CD1a, anti-CD4, anti-CD8, anti-IL2 and C3b antibodies) revealed a tendency to chronic inflammation. Following specific surgery, this tendency manifested itself in a diffuse immune response, especially in those patients who underwent medium- and long-term medical therapy. According to these results, antiglaucomatous surgery should be rehabilitated and considered as an alternative to long-term medical therapy in the first-instance treatment of primary open-angle glaucoma.

Biocompatibility of a new semisolid bioerodible poly(ortho ester) intended for the ocular delivery of 5-fluorouracil.

The biocompatibility of a new semisolid, hydrophobic poly(ortho ester) (POE) intended for controlled drug delivery to the eye was evaluated. The polymer was injected subconjunctivally in rabbits, and clinical and histologic examinations were performed 3, 10, 15, and 21 days after injection. Polymers injected as controls were an aqueous gel of sodium hyaluronate (SH), 1% in phosphate buffer, and medical grade silicone oil. After injection, the POE emulsified into small droplets and a focal eosinophilic reaction was noted at 3 days' implantation. At 10 days' implantation, the POE was not identified in the implantation site and the inflammatory reaction had resolved, with fibroblasts being the predominant cell type. At 15 and 21 days, no POE was identified and normal appearing tissue was present in the injection site. Sodium hyaluronate was not inflammatory over the period of the implantations. Silicone oil induced a slight inflammation at 3 days, with the presence of eosinophils and limited necrosis with cellular debris. Silicone oil was present in the implantation site at 3, 10, 15, and 21 days. The inflammatory response to the respective polymers was evaluated in the subconjunctival tissue. The inflammatory reaction was quantified at the implant site, adjacent subconjunctival tissues, and scleral and corneal stroma. The inflammatory cell densities in these respective tissue zones were determined, and the ratio of eosinophils over total inflammatory cells was calculated. POE did not become encapsulated with fibrous tissue, but biodegraded in a short time, indicating its potential for use after glaucoma filtration surgery.

Histopathologic Effects of Mitomycin C After Trabeculectomy in Human Glaucomatous Eyes With Persistent Hypotony

We evaluated the histopathologic findings in seven patients who underwent surgical revision of the filtration site after trabeculectomy with mitomycin C because of persistent hypotonous maculopathy. Light microscopic examination of subconjunctival tissue and sclera demonstrated hypocellularity of fibroblasts and disruption of the normal architecture. Tissue fragments at the margin of the bleb wall demonstrated scarring and contained multiple fibroblasts. Additionally, we investigated the histopathologic changes in an eye obtained from a patient who died one week after a trabeculectomy with mitomycin C. Transmission electron microscopy showed myelin figures, increased melanolipofuscin granules, vacuolated cytoplasm, and disrupted mitochondria of the ciliary body epithelium underlying the site of mitomycin C application. On the basis of these findings, both overfiltration because of tissue disorganization of the filtering bleb and aqueous hyposecretion because of ciliary body toxicity might be involved in the causes of persistent hypotony after mitomycin C trabeculectomy.

Epithelial Ingrowth and Glaucoma Drainage Implants

The authors report four cases of conjunctival epithelial invasion into the fibrous capsule surrounding a Baerveldt glaucoma implant. All cases were associated with concurrent or recent prior scleral buckling surgery. Three patients had postoperative conjunctival wound leaks, one in the immediate postoperative period and the other two at 9 and 17 days postoperatively after release of the tube ligature. In the fourth patient, a fistulous tract developed over the implant after a first-stage insertion. Histopathologic confirmation of subconjunctival epithelialization was obtained in two of these patients. Surgical revision was performed in all patients. Excision and debridement of all epithelium-lined subconjunctival tissues and extensive bipolar cautery were used in the three patients with wound leaks. Implant removal also was performed in one of these. Fistulectomy and bipolar cautery were used in the fourth patient. No recurrent wound leaks or other adverse sequelae were noted. Epithelial invasion of the subconjunctival space and inner bleb wall after implantation of glaucoma drainage devices can lead to breakdown of the conjunctival wound and persistent aqueous leak. Prior or concurrent scleral buckling surgery may predispose to this occurrence. Surgical revision involving epithelial debridement, cautery, and meticulous wound closure, with or without implant removal, has been used successfully in the management of this complication.

Single intraoperative applications of 5-fluorouracil during filtration surgery: early results.

Experimental studies from our laboratory have suggested that single exposures to 5-fluorouracil (5-FU) would have a similar effect to subconjunctival injections of 5-FU and a less permanent effect on subconjunctival fibroblasts than mitomycin C (MMC). Thirty four eyes of 33 glaucoma patients with an increased risk of filtration surgery failure were treated with a single intraoperative 5 minute exposure of sclera and subconjunctival tissues to 5-FU, 25 mg/ml. The follow up period ranged from 3-9 months, during which two high risk eyes failed completely and one required topical beta blockers to control intraocular pressure. No low/moderate risk eyes failed. Single 5 minute intraoperative exposures to 5-FU are convenient, inexpensive, have no significant corneal side effects, and may be a useful adjunctive treatment to optimise the results of glaucoma filtration surgery, particularly in the large group of low/moderate risk patients.

Diode Laser Surgery: Ab Interno and Ab Externo versus Conventional Surgery in Rabbits

Fibroblastic proliferation of subconjunctival tissues remains a primary mechanism of failure in filtration surgery. Minimizing the surgical manipulation of episcleral tissues may reduce scarring. Laser sclerostomy surgery involves minimal tissue dissection, and is gaining attention as a method of potentially improving filter duration in high-risk cases. Twenty-five New Zealand rabbits underwent filtration surgery in one eye, and the fellow eye remained as the unoperated control. Ten rabbits underwent ab externo diode laser sclerostomy surgery, ten underwent ab interno diode sclerostomy surgery, and five had posterior sclerostomy procedures. Filtration failure was defined as a less-than-4-mmHg intraocular pressure (IOP) difference between the operative and control eyes. The mean time to failure for the ab externo, ab interno, and conventional posterior sclerostomy techniques measured 17.4 +/- 11.5, 13.1 +/- 6.7, and 6.0 +/- 3.1 days, respectively. In a comparison of the laser-treated groups with the conventional procedure, the time to failure was significantly longer (P = 0.02) for the ab externo filter. The mean ab interno sclerostomy duration was longer than the posterior lip procedure, but this difference was not statistically significant (P = 0.15). The overall level of IOP reduction was similar in the three groups. These data suggest that diode laser sclerostomy is a feasible technique in rabbits, and the ab externo approach resulted in longer filter duration than the conventional posterior lip procedure in this model.

Effect of Subconjunctival Daunorubicin on Glaucoma Surgery in Rabbits

ABSTRACT Daunorubicin (DNR), an antibiotic-antimetabolite, was used as an adjunct to standardized partialthickness filtration surgery in 21 rabbits to determine its effects on that surgery. Postoperatively, one of the eyes of each rabbit received subconjunctival injections of DNR 25 µg or 50 µg, while the fellow eyes received only diluent, daily for 2 to 14 days. Bleb survival, fistula patency, the thickness of the subconjunctival connective tissue, and postoperative complications were investigated. The filtration blebs lasted significantly longer (P &lt; .005), the rate of fistula closure was significantly lower (P &lt; .005), and the subepithelial connective tissue was much looser and thicker in the experimental eyes than in the control eyes. Corneal toxicity occurred more frequently in the eyes treated with the higher-dose DNR than in those treated with the lower dose and in the control eyes.

Investigation of enzyme activities in severe burns of the anterior eye segment.

In severe burns of the anterior eye segment, including the cornea, limbus and adjacent conjunctiva, ischemia resulted from the necroses. While necrotic conjunctival and subconjunctival tissues may be removed to eliminate the toxic influence, the opaque cornea and ischemic sclera could not be removed. In the surrounding healthy tissues an inflammatory reaction developed, which brought about an infiltration of the damaged tissues by leukocytes and the release of lysosomal marker enzymes. N-Acetylglucosaminidase and cathepsin-D represent a number of other destructive enzymes involved with corneal and corneoscleral ulceration. Initially, their activities were low in the turbid, acellular cornea and increased 3 weeks after the burn. In the surrounding conjunctiva, these enzyme activities were normally higher than in the cornea and increased significantly after the burn. The elevated activities of N-acetylglucosaminidase and cathepsin-D in the conjunctiva and cornea were related clinically to corneal and corneoscleral ulceration.

Prolonged Localized Tissue Effects From 5-Minute Exposures to Fluorouracil and Mitomycin C

Rabbits undergoing full-thickness glaucoma filtering surgery were exposed for 5 minutes to one of three intraoperative treatments: (1) distilled water; (2) fluorouracil, 50 mg/mL; or (3) mitomycin C, 0.4 mg/mL. Tissue samples were taken from the subconjunctival and scleral tissues at the treated area and 90 degrees and 180 degrees from the center of the treated area and the adjacent cornea 2 mm from the limbus, 1 hour, 5 days, and 30 days postoperatively. The biopsy specimens were then placed in tissue culture media and the fibroblast outgrowths measured. Five-minute intraoperative treatments with fluorouracil resulted in a reversible delay of fibroblast outgrowths from treated subconjunctival and scleral tissues of just over 1 week in this model, whereas treatment with mitomycin C, 0.4 mg/mL, resulted in prolonged inhibition of at least 30 days. These effects were localized to the area treated. The many clinical implications of these findings are discussed.