Biomedical subjects

Scheffer C G Tseng

Publications and source records attributed to Scheffer C G Tseng.

At least 37 records · Page 2Linked to original sources

PubMed2005

Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing.

PURPOSE: To purify and characterize the glycoprotein lumican, isolated from human amniotic membrane (AM), and to examine its efficacy in treating corneal epithelium debridement. METHODS: An affinity-purified, anti-human lumican antibody-conjugated protein A Sepharose column was used to purify soluble lumican protein from human AM. The purified AM lumican was characterized by two-dimensional and SDS gel electrophoresis, plus Western blot analysis with anti-lumican antibody. The effects of lumican on corneal epithelial wound healing were examined in an organ culture mouse eye model. RESULTS: Lumican was found to be abundantly present in the stroma of human AM. It was extracted from the AM by isotonic, 1 M NaCl, and 4 M guanidine HCl solutions, suggesting that it is present in both the soluble and matrix-bound states. In two-dimensional gel electrophoresis, the 50-kDa human amniotic lumican purified by antibody-conjugated affinity chromatography migrated in a smear between pH 3.0 and 6.0. After endo-beta-galactosidase digestion, it existed as a single core protein at pH 6.0, suggesting that native human amniotic lumican is a glycoprotein with short sugar moiety. Addition of purified human AM lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. In lumican-knockout (lum(-/-)) mice, the effect of human lumican on promoting corneal epithelial wound healing was even more dramatic than in wild-type mice. CONCLUSIONS: The diversified functions of lumican include modulation of epithelial cells in wound healing and serving as an extracellular matrix component. Administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.

Amnion↗

PubMed2005

The heterogeneous murine corneal stromal cell populations in vitro.

PURPOSE: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. METHODS: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. RESULTS: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. CONCLUSIONS: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.

Actins↗

PubMed2005

High prevalence of Demodex in eyelashes with cylindrical dandruff.

PURPOSE: To determine the prevalence of Demodex in eyelashes with cylindrical dandruff (CD). METHODS: A modified sampling and counting method was applied to 55 clinical cases. Patients were divided in to group A (n = 20) with diffuse CD, group B (n = 12) with sporadic CD, and group C (n = 23) with clean lashes or greasy scales, of which the latter was divided into subgroup C1 (n = 15) without lid hygiene and subgroup C2 (n = 8) using daily lid hygiene for the past year. Each patient underwent a routine complete eye examination and modified counts of Demodex. RESULTS: Demodex was found in all group A and B patients (n = 32) with CD, which was significantly higher than the 22% of group C patients (n = 23) without CD (P < 0.001). The Demodex counts were 4.1 +/- 1.0 and 2.0 +/- 1.2 per epilated lash with retained CD, significantly higher than the 0.2 +/- 0.5 and 0.2 +/- 0.4 per lash without retained CD in groups A and B, respectively (each P < 0.001) and than the 0.01 +/- 0.09 and 0.12 +/- 0.41 per lash in subgroups C1 and C2, respectively (each P < 0.001). Demodex was still found in CD fragments left on the lid skin after epilation. Five Demodex brevis mites were found among the 422 Demodex specimens. CONCLUSIONS: The modified sampling and counting method showed that the prior controversy regarding Demodex has resulted from miscounting and confirmed that lashes with CD are pathognomonic for ocular Demodex infestation. Lid hygiene with shampoo reduces Demodex counts but does not eradicate the mites.

Adult↗

PubMed2004

Oxygen permeability of amniotic membrane and actual tear oxygen tension beneath amniotic membrane patch.

PURPOSE: To investigate the oxygen permeability (Dk) and oxygen transmissibility (Dk/t) of amniotic membrane (AM), and tear oxygen tension beneath AM in rabbits. DESIGN: Experimental study. METHOD: The water content of AM was measured to calculate Dk and Dk/t, and compared with those of a therapeutic soft contact lens (TSCL). The tear oxygen tension beneath AM and TSCL was also measured in four male albino rabbits. RESULTS: The average water content of AM was significantly higher than that of TSCL (96.8 +/- 0.8% (mean +/- SD) vs 78%, P =.0006), giving rise to a significantly higher Dk and Dk/t in AM as compared with TSCL (142.8 +/- 4.7 vs 65.3, respectively, P =.0012, and 92.9 +/- 3.0 vs 29.7, respectively, P =.0008). The average tear oxygen tension under AM was also higher than TSCL (94.9 +/- 2.9 vs 59.1 +/- 4.9 mm Hg, P =.0006). CONCLUSIONS: These findings support the rationale of using AM as a superior bandage in treating persistent corneal epithelial defects or ulcers.

Amnion↗

PubMed2004

Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle.

Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells.

Animals↗

PubMed2004

Sequential changes of lipid tear film after the instillation of a single drop of a new emulsion eye drop in dry eye patients.

PURPOSE: The lipid tear film stabilizes the tears by lowering the surface tension and preventing aqueous tear evaporation and may be analyzed by kinetic analysis of the tear interference images. This study investigated changes in the lipid film after application of a new emulsion-based eye drop of Refresh Endura (Allergan, Irvine, CA) in normal subjects and patients with dry eyes. DESIGN: Comparative, nonrandomized interventional study. PARTICIPANTS: Five normal subjects and 10 aqueous tear deficiency (ATD) patients with or without lipid tear deficiency were enrolled prospectively. METHODS: A complete eye examination was performed, including symptom score, tear break-up time, dye staining, and fluorescein clearance test. One eye received a single dose of emulsion eye drop (EED), whereas the other eye received nonpreserved saline as a control. Kinetic analysis of tear interference images taken by DR-1 (Kowa, Japan) was performed before and at various times after instillation of the drops. MAIN OUTCOME MEASURES: Symptoms, pattern, thickness, and spread time of the tear film. RESULTS: Compared with the control, all eyes receiving EED showed rapid restructuring of the preexisting lipid film. In normal persons, restructuring resulted in a thick lipid band across the cornea and a significant increase of the lipid film thickness. For ATD patients, EED application resulted in either a scant, irregular tear film, which became covered by the lipid film, or a modified lipid film changed without forming a band or irregularities. Emulsion eye drops tended to be more comfortable in patients with more severe ATD. Kinetic analysis showed that the mean spread time improved for all patients after EED when compared with baseline or with controls (P<0.05). CONCLUSIONS: Emulsion eye drop produces significant changes in the tear film of normal and dry eye patients. Further investigation with a prolonged period of treatment is warranted to establish whether EED may help improve the spread and thickness of the lipid tear film in moderate and severe dry eyes.

Adult↗

PubMed2004

How does amniotic membrane work?

Transplantation of amniotic membrane as a temporary or permanent graft promotes epithelial wound healing and exerts potent anti-inflammatory and anti-scarring effects on the ocular surface. These actions depend on the killing of allogeneic amniotic cells and preservation of the cytokine-containing matrix during the preparation of the amniotic membrane. This review describes how these actions inherently operate in utero and how amniotic membrane transplantation aims to recreate such a fetal environment to exert these actions by insulating the surgical site from the host environment. These actions also render the amniotic membrane a unique niche capable of expanding both epithelial and mesenchymal progenitor cells ex vivo, while maintaining their normal cell phenotypes. As a result, the amniotic membrane becomes an ideal substrate for engineering different types of ocular surface tissues for transplantation. Further studies investigating the exact molecular mechanism by which the amniotic membrane works will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine.

Journal Article↗

PubMed2004

A case of conjunctiva-cornea intraepithelial neoplasia successfully treated with topical mitomycin C and interferon alfa-2b in cycles.

PURPOSE: To report a case of conjunctiva-cornea intraepithelial neoplasia (CCIN) treated with topical mitomycin C (MMC) and interferon alfa-2b in cycles. METHODS: A 52-year-old woman referred for limbal stem cell deficiency and epithelial defect was found instead to have CCIN. The diagnosis was confirmed by impression cytology and treated in cycles with topical 0.02% MMC for 14 days in the first cycle, 12 days in the second cycle, and 3 days in the third cycle followed by topical interferon alfa-2b 1 x 106 U/mL for 11 days. RESULT: Topical MMC for 2.5 cycles resulted in 95% reduction of CCIN and improvement of 2 lines of vision. Because of intolerance to MMC, she was switched to topical interferon alfa-2b for 11 days, resulting in total resolution of CCIN, which lasted for at least 10 months. CONCLUSIONS: After differentiation from a persistent corneal epithelial defect and limbal stem cell deficiency by dye staining and impression cytology, the patient in this case of CCIN was successfully treated with topical MMC and interferon alfa-2b in cycles.

Administration, Topical↗

PubMed2004

Incidence of microbial infection after amniotic membrane transplantation.

OBJECTIVE: To report the incidence and characteristics of microbial infection following amniotic membrane transplantation (AMT). METHODS: We retrieved the clinical and microbiological records of a total of 326 patients undergoing AMT from January 1994 to February 2001 at Bascom Palmer Eye Institute to determine the incidence and characteristics of post-AMT infections with respect to the interval between AMT and the time of microbial culturing, types of organisms, and clinical outcomes and to correlate these infections with the microbiologic results of AM storage media submitted immediately after AMT and the underlying ocular diagnosis. RESULTS: We subdivided these 326 patients into two groups: 76 patients (from January 1994 to June 1998) used AM prepared from a research laboratory and did not submit AM storage media for culture under an Institutional Review Board-approved protocol, and 250 patients thereafter used AM obtained from a commercial source and routinely submitted AM storage media for culture. A total of 11 culture-positive infections (3.4%) were identified, and among them 7 infections (9.2%) were from the first group, and 4 (1.6%) were from the second group (P = 0.004). All 4 infections (5.2%) occurring within 1 month after AMT were exclusively from the first group (P = 0.003). All AM storage media from the second group were culture negative. Gram-positive organisms were the most frequent isolate (64%). Infections were not correlated with the underlying ocular diagnosis. CONCLUSIONS: AMT is a safe method for ocular surface reconstruction with a very low rate of microbial infections, especially if AM is prepared according to Good Tissue Banking Practice set forth by FDA. Gram-positive isolates seem to be the most frequent isolates in infections after AMT.

Amnion↗

PubMed2004

Characterization of corneal pannus removed from patients with total limbal stem cell deficiency.

PURPOSE: To determine the epithelial lineage of origin in corneal pannus tissue surgically removed from patients with total limbal stem cell (SC) deficiency. METHODS: The lineage of origin of the entire conjunctivalized pannus removed from eight corneas with a diagnosis of total limbal SC deficiency was characterized by anti-keratin (K)-3 and anti-K19 monoclonal antibodies. The protein and mRNA of epithelial outgrowth from segments of five such pannus specimens were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. RESULTS: Cross sections of all eight specimens showed a stratified epithelium with goblet cells expressing mucin (MUC)-5AC, and a stroma showing blood vessels and inflammatory cell infiltrates. Immunostaining showed full-thickness expression of K19 in the entire pannus of all eight specimens. Expression of K3 was negative in seven patients, but was sporadically positive in a patient with Stevens-Johnson syndrome. In culture, all five pannus specimens generated a compact, small epithelial cell outgrowth, and except for one, reached confluence in 2 to 3 weeks. The K3/K12 pair was expressed by extracts of cell outgrowth from the control limbal epithelial explant, but not in all five pannus specimens. A 60-kDa band of DeltaNp63 was expressed in the control specimen and in all five pannus specimens. Cell outgrowth expressed K3 transcript in three, but none showed K12 transcript. CONCLUSIONS: The resultant epithelial phenotype of the pannus tissue was not corneal, as evidenced by the negative staining to cornea-specific K12 mRNA and protein, but was conjunctival, as evidenced by the presence of goblet cells, the weak expression of K3, and the strong expression of K19. The abundant expression of DeltaNp63 in such conjunctiva-derived epithelium in eyes with total limbal SC deficiency raises doubts as to its validity as a limbal SC marker.

Adult↗

PubMed2004

CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-beta1.

PURPOSE: To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-beta1-induced myofibroblast differentiation. METHODS: Collagenase-isolated keratocytes were seeded and subcultured on plastic or amniotic membrane matrix (AM) in DMEM, with or without 10% FBS, in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS) with 10, 100, and 1000 pg/mL TGF-beta1 or in DMEM with 1% FBS and 10 ng/mL TGF-beta1. Protein expression of CD34 and alpha-smooth muscle actin (alpha-SMA) was measured by Western blot and immunostaining. RESULTS: Keratocytes, expressing CD34 in normal human corneas, continued to express CD34 when cultured on AM in serum-containing medium and on plastic in serum-free medium, but expression was lost on plastic in serum-containing medium. In serum-containing medium, expression of CD34, but not alpha-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed alpha-SMA without CD34 when continuously passaged on plastic. Expression of alpha-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas alpha-SMA expression was upregulated when cells were subcultured on plastic. In serum-free medium, CD34 expression was maintained by cells treated with 10 pg/mL TGF-beta1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became alpha-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-beta1, but failed to do so if cultured on AM, even for 7 days. CONCLUSIONS: These findings indicate that CD34 is expressed by human keratocytes in vivo and in vitro. Myofibroblast differentiation promoted by TGF-beta1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-beta signaling.

Actins↗

PubMed2004

Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells.

PURPOSE: To develop a reproducible method for expanding mouse corneal-limbal epithelial cells and determine the role of extracellular Ca(2+) concentration and serum in modulating their growth and differentiation. METHODS: Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4 degrees C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca(2+) concentration to 0.9 mM, with or without 5% FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin. RESULTS: Intact and viable corneal-limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca(2+) but suppressed by 5% FBS in KSFM at passage 0. CONCLUSIONS: Mouse corneal-limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca(2+) concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca(2+) concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.

Animals↗

PubMed2003

Differentiation of lipid tear deficiency dry eye by kinetic analysis of tear interference images.

OBJECTIVE: To use kinetic changes to characterize tear interference images in patients with lipid tear deficiency (LTD) dry eye. METHODS: We used a DR-1 camera to digitize and analyze sequential images of tear interference on the central 8 mm of the cornea at the start of complete blinking in 11 healthy volunteers and 8 patients with LTD and noninflamed meibomian gland dysfunction. MAIN OUTCOME MEASURES: We studied tear lipid spread time and pattern, stability of the lipid after spread, and distribution of thickness in a prospective, case-control study. RESULTS: On complete lid closure, the lipid spread was horizontal in healthy eyes but vertical in LTD (P<.001). Mean +/- SD lipid spread time was 0.36 +/- 0.22 seconds in healthy eyes but 3.54 +/- 1.86 seconds in LTD (P<.001). Conventional DR-1 grading could not distinguish these groups (P =.32). Mean +/- SD lipid film thickness in healthy eyes was 74.5 +/- 6.9 nm, thicker than the 43.8 +/- 10.6 nm in LTD (P<.001), and this result was confirmed by qualification with intensity histogram (P<.001). CONCLUSIONS: Kinetic analysis of the tear interference revealed distinctive differences in the time and pattern of lipid spread and the distribution and stability of resultant lipid thickness between healthy subjects and patients with LTD. This method can be coupled with others for formulating effective therapies for patients with dry eye.

Adult↗

PubMed2003

Detection of rabbit corneal epithelium over amniotic membrane patch by white-light and laser confocal microscopy.

PURPOSE: To report confocal microscopic observation of rabbit corneal epithelium through amniotic membrane patch (AMP). DESIGN: Experimental study. METHOD: Six rabbit eyes were patched by amniotic membrane. Then, white-light and laser confocal microscopic observation was performed. RESULTS: Images of human amniotic epithelium, amniotic basement membrane, and amniotic stroma were obtained using both devices, followed by detection of rabbit corneal epithelium. CONCLUSION: This study indicates that observation of corneal epithelium through AMP is possible in rabbit eyes using both these methods. This implies that both devices might be used clinically to observe the epithelial healing process under an AMP to better determine when it should be removed.

Amnion↗

PubMed2003

Amniotic membrane transplantation for reconstruction of the conjunctival fornices.

PURPOSE: To describe the clinical outcome of amniotic membrane transplantation (AMT) for fornix reconstruction in a variety of ocular surface disorders. DESIGN: Noncomparative interventional case series. PARTICIPANTS: Seventeen eyes in 15 patients with symblepharon. Four eyes had ocular-cicatricial pemphigoid, two eyes had symblepharon after pterygium excision, four eyes had chemical or mechanical trauma, two eyes had strabismus surgery, two eyes (one patient) had Stevens-Johnson syndrome, one eye had toxic epidermal necrolysis, and two eyes (one patient) had chronic allergic conjunctivitis. INTERVENTION: The subconjunctival scar tissue was dissected from the episclera, and the freed conjunctival flap was recessed to the fornix. A layer of amniotic membrane (AM) was applied to cover the exposed episclera. The fornical edge of the membrane was anchored with sutures passing through the full thickness of the lid. MAIN OUTCOME MEASURES: A deep conjunctival fornix, lack of motility restriction. RESULTS: The mean follow-up period was 37 +/- 24 months (range, 9-84 months). Complete fornix reconstruction was demonstrated in 12 of 17 eyes (70.6%), whereas 2 eyes had a partial success, and 3 eyes (3 patients) had recurrence of symblepharon with restricted motility. In eyes that demonstrated partial success or failure, the underlying etiology was either an autoimmune disorder or a recurrent pterygium. The most successful outcome was observed in eyes with symblepharon associated with trauma. CONCLUSIONS: AMT is an effective method of fornix reconstruction for the repair of symblepharon in a variety of ocular surface disorders. Future modifications, including an epithelial cellular component on the AM (conjunctival autograft or ex vivo expanded epithelial stem cells) may improve the outcome of this surgical procedure.

Adult↗

PubMed2003

Phenotypic study of a case receiving a keratolimbal allograft and amniotic membrane for total limbal stem cell deficiency.

PURPOSE: To report the expression pattern of key molecules by the reconstructed corneal epithelium after a keratolimbal allograft (KLAL) and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency. DESIGN: Interventional case report. METHOD: A 50-year-old woman with severe chemical burns in both eyes received an AMT as a temporary patch at the acute stage, and a KLAL with AMT as a graft at the chronic stage for total limbal stem cell deficiency. The corneal button removed during subsequent corneal transplantation was submitted for immunofluorescence staining with monoclonal antibodies against keratin K3, MUC5AC, connexin 43, integrins alpha3beta1 and alpha6beta4, and laminin 5 for comparison with a normal cornea. RESULTS: Histologically, a normal stratified corneal epithelium has five to six cell layers that lay on the thick amniotic membrane basement membrane. The phenotype was of a corneal origin, based on expression of positive keratin K3, negative MUC5AC, and positive connexin 43. Furthermore, intact basement membrane complexes were present, evidenced by positive staining to integrins alpha3beta1 and alpha6beta4 and to laminin 5. CONCLUSIONS: A normal corneal epithelial phenotype with normal basement membrane complexes was restored after a KLAL and AMT in a case with total limbal stem cell deficiency.

Amnion↗

PubMed2003

Amniotic membrane transplantation with conjunctival limbal autograft for total limbal stem cell deficiency.

PURPOSE: To evaluate the outcomes of corneal surface reconstruction with conjunctival limbal autograft when combined with amniotic membrane transplantation on both the donor and recipient eyes. DESIGN: Retrospective, noncomparative, interventional small case series. PARTICIPANTS: Five eyes of five patients with total limbal stem cell deficiency (LSCD) resulting from pseudopemphigoid (n = 1), chemical burns (n = 3), and extensive removal of conjunctival intraepithelial neoplasia (n = 1) were operated on by one surgeon (SCGT). INTERVENTION: After the removal of fibrovascular pannus from the corneal surface, two conjunctival limbal free grafts were harvested from the fellow eyes in all five patients with unilateral LSCD. Amniotic membrane, with the basement membrane side up, was grafted onto the defect created at the donor site and onto the recipient corneal and limbal sclera before placement of conjunctival limbal grafts. MAIN OUTCOME MEASURES: Symptomatic relief, improvement in visual acuity, fornix deepening, and rapid healing and restoration of normal cornea and limbus in the recipient and donor eyes were assessed. RESULTS: During the mean follow-up of 22 months (range, 11-48 months), all eyes experienced symptomatic relief. All recipient eyes had a mean improvement in visual acuity of nine lines (range, 7-12). The three eyes with stromal vascularization showed regression, and all recipient eyes had marked improvement in corneal clarity. Three eyes receiving simultaneous symblepharon lysis and fornix reconstruction successfully regained deep, stable fornices. The donor eyes showed rapid healing and restoration of the normal limbal landmark, even in one eye where nearly the entire limbus was removed. CONCLUSIONS: Limbal conjunctival transplantation is an effective procedure for restoring the corneal surface integrity in eyes with total LSCD. The additional use of amniotic membrane may contribute to a higher rate of success in the recipient eye and a lower rate of complications in the donor eye, as well as allow the simultaneous correction of concomitant cicatricial abnormalities.

Adult↗

PubMed2003

Amniotic membrane transplantation for bullous keratopathy in eyes with poor visual potential.

PURPOSE: To evaluate the long-term outcomes of epithelial debridement and amniotic membrane transplantation (AMT) for pain and discomfort relief in patients with symptomatic bullous keratopathy and poor visual potential. SETTING: Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, Florida, USA. METHODS: This retrospective study included 18 eyes (18 patients) with bullous keratopathy presenting with intractable pain or discomfort and poor visual potential. After epithelial debridement, all eyes had AMT with the basement membrane side up. During a mean follow-up of 25.1 months +/- 9.6 (SD) (range 12 to 45 months), pain relief, epithelial healing, and visual changes were analyzed. RESULTS: Pain relief was obtained in 88% of patients. Sixty-six percent of eyes had complete resolution of ocular discomfort starting soon after the first postoperative day. One eye had evisceration for persistent pain 10 months postoperatively. Corneal epithelial healing was complete in all except 1 eye. Remaining complaints included foreign-body sensation (5%), tearing (11%), and photophobia (5%). CONCLUSIONS: Amniotic membrane transplantation was a safe, effective, and long-lasting treatment modality for intractable pain associated with chronic bullous keratopathy in eyes with poor visual potential. It can be an alternative to conjunctival flaps for the long-term management of patients with bullous keratopathy in whom corneal transplantation is not indicated. A comparison of the efficacy of AMT with that of other surgical procedures must be performed.

Adult↗