bromochloroacetic-acid and Eye-Burns

To access the feasibility of using cultivated oral mucosal epithelial cell transplantation (COMET) for the management of severe corneal burn.. COMET was performed to promote re-epithelialization in two eyes with acute alkaline burn and one eye with chronic alkaline burn, and to reconstruct the ocular surface in two eyes with chronic thermal burn. Autologous oral mucosal epithelial cells obtained from biopsy were cultivated on amniotic membrane. Immunoconfocal microscopy for keratins and progenitor cell markers was performed to characterize the cultivated epithelial sheet. Following transplantation, the clinical outcome and possible complications were documented. The patients were followed for an averaged 29.6+/-3.6 (range: 26-34) months.. Cultivated oral mucosal epithelial sheet expressed keratin 3, 13, and progenitor cell markers p63, p75, and ABCG2. After COMET, all the corneas became less inflamed, and the corneal surface was completely re-epithelialized in 6.0+/-3.2 (range: 3-10) days in all but one patients. Microperforation occurred in one patient, and a small persistent epithelial defect developed in another. Both were solved uneventfully. In all patients, superficial corneal blood vessels invariably developed, and to further improve vision, conjunctivo-limbal autografting (N=3) and/or penetrating keratoplasty (N=3) were performed subsequently. The vision of all patients showed substantial improvement after additional surgeries.. This study showed the potential of COMET to promote re-epithelialization and reduce inflammation in acute corneal burn, and to reconstruct the corneal surface in chronic burn. COMET may, therefore, be considered an alternative treatment for severe corneal burn.

Topics: Adolescent; Adult; Amnion; Biomarkers; Burns, Chemical; Cell Culture Techniques; Cell Transplantation; Cells, Cultured; Corneal Injuries; Epithelial Cells; Epithelium, Corneal; Eye Burns; Feasibility Studies; Humans; Keratins; Male; Middle Aged; Mouth Mucosa; Tissue Engineering; Transplantation, Autologous; Young Adult

To investigate the expression of p63 and cytokeratins throughout the course of producing a cultivated autograft of limbal epithelial cells.. A 75 year old male with a severe alkali burn to his right eye received two cultivated autografts of limbal epithelial cells on amniotic membrane followed by a corneal allograft. Immunostaining for p63 and cytokeratins was performed during ex vivo expansion with 3T3 fibroblasts, following subcultivation on amniotic membrane, and on the excised corneal button.. Cultures grown in the presence of 3T3 fibroblasts or on amniotic membrane displayed positive staining for keratins 14 and 19, and p63, but poor staining for keratin 3 (K3). The excised corneal button possessed a stratified epithelium of K3 positive cells residing on amniotic membrane.. Our results document for the first time the co-expression of cytokeratins 14 and 19 with p63 in a cultivated limbal graft. These data support the conclusion that cultivated grafts of limbal epithelium contain predominantly undifferentiated cells with the potential to regenerate a normal corneal epithelium.

Topics: Aged; Burns, Chemical; Cells, Cultured; Corneal Transplantation; DNA-Binding Proteins; Epithelial Cells; Epithelium, Corneal; Eye Burns; Genes, Tumor Suppressor; Humans; Keratin-14; Keratin-3; Keratins; Limbus Corneae; Male; Phenotype; Phosphoproteins; Stem Cell Transplantation; Trans-Activators; Transcription Factors; Transplantation, Autologous; Tumor Suppressor Proteins

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.. Interventional case report.. 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.. 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.. 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.

Topics: Amnion; Biomarkers; Burns, Chemical; Connexin 43; Corneal Diseases; Epithelial Cells; Epithelium, Corneal; Eye Burns; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunophenotyping; Integrin alpha3beta1; Integrin alpha6beta4; Keratins; Keratoplasty, Penetrating; Laminin; Limbus Corneae; Middle Aged; Mucin 5AC; Mucins; Stem Cell Transplantation; Stem Cells; Tissue Preservation; Visual Acuity

Keratocytes synthesize keratan-sulfate proteoglycans (KSPG), lumican and keratocan, to develop and maintain proper collagen interfibrillar spacing and fibril diameter characteristics of the transparent cornea. The purposes of this study are to compare the expression patterns of KSPGs and keratin 12 (K12) respectively by corneal keratocytes and epithelial cells after three different types of injuries; partial and total epithelial debridement and alkali burn.. Corneas of 8-12 week old C57Bl/6J or FVBN mice were wounded by partial epithelial (2 mm in diameter) and total epithelial debridement, and alkali burn (0.1 M NaOH, 30 s) and were allowed to heal for various periods of time, from 1 to 84 days. The corneas were then subjected to light microscopy, in situ and Northern hybridization and RT-PCR for examining the expression of K12 and KSPG in the corneal epithelium and stroma, respectively. Immunohistochemistry with anti-alpha-smooth muscle actin (alpha-SMA) was used to identify myofibroblasts in the stroma of injured cornea.. In 2-3 days, partial epithelial denuded corneas were resurfaced by corneal epithelium positive for K12, and stromal edema caused by debridement disappeared. Total epithelial debridement wounded corneas were resurfaced by conjunctival epithelial cells in 2 weeks. Stromal edema in the total epithelial debridement corneas began to subside after 6 weeks. Corneal epithelial cells resurfaced alkali burned corneas within 3-5 days. In situ and Northern hybridization showed a decrease in keratocan and lumican expression at 6 weeks and increased at 12 weeks post-injury in all wound types. Alpha-SMA positive myofibroblasts in the cornea were detected via immunostaining at the time point when KSPG expression was lowest, 6 weeks post-injury.. The results suggest keratocan and lumican are down-regulated during wound healing at 6 weeks and returned to higher levels at 12 weeks post-injury; implicating that the cells repopulating the injured corneal stroma regained the characteristic function of keratocytes independent of the wound types. However, complete epithelial removal results in irreversible loss of K12 expression.

Topics: Animals; Blotting, Northern; Burns, Chemical; Chondroitin Sulfate Proteoglycans; Cornea; Corneal Injuries; Corneal Stroma; Down-Regulation; Epithelium, Corneal; Eye Burns; Eye Injuries; Eye Proteins; Fibroblasts; Immunoenzyme Techniques; In Situ Hybridization; Keratan Sulfate; Keratin-12; Keratins; Lumican; Mice; Mice, Inbred C57BL; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Hydroxide; Wound Healing

To investigate the proliferation and differentiation of cultured corneal stem cells and determine the effect of corneal stem cells cultured on amniotic membranes on the limbal area for treating corneal burns.. The proliferation and differentiation of corneal stem cells in vitro had been examined using colony-forming efficiency and immunohistochemistry. The stem cells had been cultured on amniotic membranes and transplanted to the limbal area for treating corneal burns.. Corneal stem cells had a high proliferation capacity in primary and first passage, cytokeratin 3 was not expressed in primary culture but partly in first passage. The stem cells could proliferate to form cell layer on an amniotic membrane. When transplanted, stem cells could survive on limbus. After transplantation, ocular inflammation resolved, the cornea re-epithelialized, the stromal opacity reduced, the superficial neovascularity was lessened and the conjunctival fornix re-established.. Ocular surface conditions could be improved by allograft of corneal stem cells cultured on amniotic membranes.

Topics: Alkalies; Amnion; Animals; Burns, Chemical; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Transplantation; Cells, Cultured; Epithelium, Corneal; Eye Burns; Graft Survival; Humans; Keratins; Limbus Corneae; Rabbits; Stem Cells; Treatment Outcome

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.

Topics: Animals; Autoradiography; Burns, Chemical; Cells, Cultured; Cornea; Corneal Injuries; Disease Models, Animal; DNA Probes; Epithelium; Eye Burns; Female; Gene Expression; In Situ Hybridization; Keratins; Male; Rabbits; RNA, Messenger; Sodium Hydroxide; Wound Healing

Limbal basal epithelium is thought to possess corneal epithelial stem cells that are the ultimate source of corneal epithelial proliferation and differentiation during corneal epithelial wound healing. Destruction of the limbal epithelium results in corneal conjunctivalization and vascularization, suggesting that the limbal epithelium also may be a barrier between corneal and conjunctival epithelia. In this experiment, a total corneal epithelial debridement using combined n-heptanol and mechanical scraping was created immediately (one-step) or 5 weeks (two-step) after 15 or 30 sec n-heptanol treatment at the limbus. All defects healed in 1-2 weeks. The severity of corneal vascularization, as judged by external photography, followed the ascending order of 30-sec two-step and 15-sec two-step less than 15-sec one-step less than 30-sec one-step (P less than 0.005). Immunofluorescence studies using monoclonal antibodies AM-3 and AE-5 showed mixed expression of corneal and conjunctival epithelial phenotypes on the corneal surface in the one-step subgroups. By contrast, the two-step subgroups had a normal corneal epithelial phenotype. Impression cytology was used to map goblet-cell distribution on the perilimbal corneal surface. The specimens taken from superior, temporal, and inferior bulbar areas were evaluated by a scoring system at different times. The extent of goblet cells invading onto the corneal surface also followed the same ascending order (P = 0.005). A transient goblet-cell surge was noted, and the extent was related to the extent of corneal vascularization. It is thus evident that in vivo n-heptanol treatment for different durations can result in different extents of corneal conjunctivalization and vascularization. The authors concluded that the capability of the remaining limbal basal epithelium to recover its original full-thickness stratified layers determines the strength of the limbal barrier.

Topics: Alcohols; Animals; Antibodies, Monoclonal; Conjunctiva; Cornea; Epithelium; Eye Burns; Female; Fluorescent Antibody Technique; Heptanol; Keratins; Male; Mucins; Neovascularization, Pathologic; Rabbits; Sclera; Wound Healing

In alkali burned rabbit corneas activities of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase and acid beta-galactosidase were studied histochemically in various time intervals after the traumatization. The technic with semipermeable membranes was employed. Within four days after the injury enzyme activities in the traumatized area were almost lacking. The corresponding activities in the unaffected part of the cornea were within the norm. On the 7th day enzyme activities were on an increase (but still subnormal) in the traumatized area. This area was surrounded by a zone of keratocytes with high levels of enzyme activities. This was particularly remarkable in keratocytes subjacent to the epithelium. The activation of all enzymes studied was present in the basal layer of the epithelium and in the endothelium as well. On the 14th day enzyme activities in the traumatized area were nearly restored and on the 32nd day they could not be distinguished from the normal cornea. Beta-galactosidase displayed a relatively maximal increase in the activity of all enzymes investigated.

Topics: Acetylglucosaminidase; Animals; Burns, Chemical; Cornea; Corneal Injuries; Endothelium; Epithelial Cells; Epithelium; Eye Burns; Galactosidases; Glucuronidase; Glycoside Hydrolases; Keratins; Lysosomes; Rabbits; Sodium Hydroxide; Time Factors; Wound Healing