The effects of topical sodium-hyaluronan on the stromal and endothelial healing were examined in the repair process of experimental corneal alkali wounds. Corneal alkali wounds were produced in one eye of each rabbit by applying a 5.5 mm round filter paper soaked in 1N NaOH onto the central cornea for 60 seconds. Then the eyes were treated topically with either 1% Na-HA(the treatment group)or a phosphate buffered saline(PBS)(the con-trol group)4 times per day for 3 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The stromal healing was assessed by counting polymorphonuclear leukocytes(PMNs)and keratocytes in the central and marginal wounds areas. The stroma treated with Na-HA had less PMNs than that of the control group during the early healing period. The defect area of the endothelium was significantly smaller in the Na-HA treated group than in the control corneas. The present findings indicate that topically applied 1%Na-HA affects stromal and endothelial healing during the early repair process after corneal alkali wounds.
Alkalies* ; Cornea ; Endothelium ; Trypan Blue ; Wounds and Injuries*

PURPOSE: To recognize the anatomical positions of the superior oblique muscle in enucleated eyes using trypan blue. METHODS: Twenty-two surgically-enucleated eyes of 11 bodies were studied. The shortest distance from the nasal insertion of superior rectus to the anterior end of the superior oblique tendon, the distance from the temporal insertion of superior rectus to the anterior end of the superior oblique insertion, and the greatest width of superior oblique tendon insertion were measured by caliper 3 consecutive times. The average values in each of the above 3 points were calculated, and values prior to and after trypan blue staining were compared. RESULTS: Prior to staining with trypan blue, the average distance from the nasal insertion of superior rectus to the anterior end of the superior oblique tendon was 4.97 mm and the average distance from the temporal insertion of superior rectus to the anterior end of the superior oblique insertion was 7.57 mm; after staining with trypan blue, the average values were 5.09 mm and 7.65 mm, respectively. There was no statistically meaningful difference in values prior to and after staining (p > 0.05). Prior to staining, the average value of the greatest width of the superior oblique tendon was 10.32 mm, and after staining with trypan blue, the average value increased to 10.76 mm. There was a statistically meaningful difference between the values (p = 0.02). CONCLUSIONS: Trypan blue staining helped to recognize the location and the width of the superior oblique tendon more precisely.
Diminazene ; Eye ; Muscles ; Tendons ; Trypan Blue

BACKGROUND: The viability of cord blood is an important measure of product quality. Trypan blue (TB) stain is the most commonly and conveniently used method to measure the viability of the cord blood. Recently, cytometric analysis using 7-Aminoactinomycin D (7-AAD) was introduced. Staining with 7-AAD is more sensitive in detecting cellular damage than staining with TB. In addition to this, 7-AAD allows specific measurement of the viability of total nucleated cells (TNC), mononuclear cells (MNC) and CD34+ cells. In this study, we compared the viability of TNC between the TB and 7-AAD method, as well as analyzing the viability of each cell population. METHODS: From February to July 2010, 102 cord blood units were collected and assessed for the viability of TNC by the TB and 7-AAD methods. The viability of mononuclear cells (MNC) and CD34+ cells was assessed by 7-AAD method. RESULTS: The TB and 7-AAD methods were used to assess the viability of TNC, which was 90.1+/-5.7% and 68.4+/-8.0%, respectively. The viability of MNC and CD34+ cells measured by the 7-AAD method was 91.8+/-4.3% and 93.4+/-5.1%, respectively. CONCLUSION: The TNC viability of 7-AAD method was significantly lower than that of TB method. In 7-AAD method, the viabilities of MNC and CD34+ cells were significantly higher than that of TNC. As those are important prognostic factors and measures for successful engraftment after the transplantation, the measurement of the viabilities of MNC and CD34+ cells by 7-AAD method would be helpful to the quality control of the cord blood product.
Cell Survival ; Cryopreservation ; Dactinomycin ; Diminazene ; Fetal Blood ; Quality Control ; Transplants ; Trypan Blue ; Umbilical Cord

PURPOSE: Fas/Fas ligand system plays an important role in modulating keratocyte apoptosis induced by mechanical corneal epithelial injury. It is also hypothesized that keratocyte apoptosis could be involved in the pathogenesis of keratoconus. The purpose of this study is to evaluate apoptosis in the first passage cultured corneal stromal fibroblasts of normal and keratoconus cornea treated with Fas-stimulating antibody. METHODS: Apoptotic cell death was evaluated by trypan blue exclusion assay in the first passage cultured corneal stromal fibroblasts of keratoconus treated with Fas-stimulating antibody. The experiment was performed in comparison with normal corneal stromal fibroblasts as a control. RESULTS: Apoptotic cells were found in the first passage cultured stromal fibroblasts of normal and keratoconus cornea treated with Fas-stimulating antibody. Apoptotic reaction of cultured stromal fibroblasts of keratoconus cornea treated with Fas-stimulating antibody was stronger than that of normal cultured corneal stromal fibroblasts treated with Fas-stimulating antibody. Apoptosis did not occur in cultured stromal fibroblasts of normal and keratoconus cornea treated with normal mouse control IgM. In Hoechst staining of cell suspension including apoptotic cells, characteristic findings such as cell shrinkage and chromatin condensation were observed. CONCLUSIONS: This study showed the differential features of cell death in cultured corneal stromal fibroblasts of keratoconus compared with normal control. Thus, keratocyte apoptosis induced by Fas/Fas ligand system could be an important factor in the pathogenesis of keratoconus.
Animals ; Apoptosis* ; Cell Death ; Chromatin ; Cornea ; Fas Ligand Protein ; Fibroblasts* ; Immunoglobulin M ; Keratoconus* ; Mice ; Trypan Blue

PURPOSE: To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. MATERIALS AND METHODS: Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. RESULTS: Number of fibroblast was significantly more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. CONCLUSION: rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing.
Cell Cycle ; Cell Line* ; Epidermal Growth Factor* ; Fibroblasts* ; Humans* ; S Phase ; Skin ; Trypan Blue ; Wound Healing

BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis. METHODS: To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated. RESULTS: In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced. CONCLUSIONS: Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.
Apoptosis ; Caspases* ; Catalytic Domain ; Cell Death* ; Cisplatin ; HEK293 Cells ; HeLa Cells ; Humans* ; Ribonucleoproteins ; Telomerase* ; Trypan Blue

BACKGROUND: The quality of cord blood largely depends on cell viability. Viability assessments using trypan blue or 7-aminoactinomycin (7-AAD) staining, which are commonly used methods, may not reflect early apoptosis of cord blood cells. We aimed to investigate early apoptosis in cord blood cells following elapsed time after collection using double staining with annexin V and 7-AAD and to compare the result with that of viability evaluation using trypan blue or 7-AAD staining. METHODS: Umbilical cord blood samples were obtained from 30 pregnant women at the time of delivery between July 2012 and March 2013. Viability of cord blood cells was determined at 0 (T0), 24, and 48 hr after collection by using trypan blue exclusion assay, 7-AAD staining, and 7-AAD/annexin V staining. RESULTS: Viabilities defined by 7-AAD/annexin V staining at T0, 24, and 48 hr after collection were respectively as follows: total nucleated cells, 92.8+/-4.5%, 78.4+/-7.8%, and 65.5+/-8.1%; mononuclear cells, 94.4+/-1.7%, 90.8+/-4.2%, and 84.2+/-6.7%; and CD34-positive cells, 92.4+/-3.0%, 90.7+/-4.7%, and 89.3+/-7.0%. The viability using trypan blue was more than 90% until 48 hr after collection. CONCLUSIONS: The mean viability of total nucleated cells using 7-AAD/annexin V staining decreased to less than 80% at 24 hr after collection; however, the viability of CD34-positive cells was more than 85% until 48 hr. Our study's data will provide useful information for the assessing the quality of cord blood products.
Annexin A5 ; Apoptosis ; Cell Survival* ; Female ; Fetal Blood* ; Humans ; Methods ; Pregnant Women ; Trypan Blue ; Umbilical Cord*

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells (1.09 x 10(5) cells/ml). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR: 64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% (3.0 x 10(4) cells/ml) or 20% KSR (4.8 x 10(4) cells/ml) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.
Animals ; Cell Proliferation ; Connective Tissue ; Diminazene ; Extracellular Matrix ; Fibroblasts ; Humans ; Trypan Blue

Autolysis, which is brought about by the release of lysosomal hydrolytic enzymes, has been prevented in cornea with the use of lysosomal membrane stabilizers, such as corticosteroid. So, we performed this study to see whether corticosteroid could reduce endothelial damage in stored corneas, or not. The experiment was performed on 15 albino rabbits which were killed by intravenous air injection. 40 microgram of hydrocortisone sodium succinate was injected into the anterior chamber of the enucleated eye. The whole eye ball was stored in the moist chamber at 4 degrees C for 24 hours, 48 hours, or 72 hours, then the cornea was removed and stained with trypan blue. Unstained endothelial cells were counted with light microscope to determine the density of viable endothelial cells. Same procedures were done on the contralateral eye with injecting normal saline into the anterior chamber instead of hydrocortisone as a control. The density of viable endothelial cells in the steroid group was higher than that in the control group by 1.75%, 14.39%, and 27.40% in 24 hours, 48 hours, and 72 hours storage, respectively.
Anterior Chamber ; Autolysis ; Cornea* ; Endothelial Cells* ; Hydrocortisone ; Membranes ; Rabbits ; Sodium ; Succinic Acid ; Trypan Blue

Autolysis, which is brought about by the release of lysosomal hydrolytic enzymes, has been prevented in cornea with the use of lysosomal membrane stabilizers, such as corticosteroid. So, we performed this study to see whether corticosteroid could reduce endothelial damage in stored corneas, or not. The experiment was performed on 15 albino rabbits which were killed by intravenous air injection. 40 microgram of hydrocortisone sodium succinate was injected into the anterior chamber of the enucleated eye. The whole eye ball was stored in the moist chamber at 4 degrees C for 24 hours, 48 hours, or 72 hours, then the cornea was removed and stained with trypan blue. Unstained endothelial cells were counted with light microscope to determine the density of viable endothelial cells. Same procedures were done on the contralateral eye with injecting normal saline into the anterior chamber instead of hydrocortisone as a control. The density of viable endothelial cells in the steroid group was higher than that in the control group by 1.75%, 14.39%, and 27.40% in 24 hours, 48 hours, and 72 hours storage, respectively.
Anterior Chamber ; Autolysis ; Cornea* ; Endothelial Cells* ; Hydrocortisone ; Membranes ; Rabbits ; Sodium ; Succinic Acid ; Trypan Blue