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

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

BACKGROUND: Trichomonas vaginalis is a pathogenic protozoa infecting human genitourinary tract. Metronidazole is currently the drug of choice to treat T. vaginalis infection. However, because of the side effects and the occurrence of resistant strains of metronidazole, it is needed to investigate alternatives. METHODS: The antiprotozoal effect of aquatic extract from Sophora flavescens on the growth and fine structure of T. vaginalis was examined by using trypan blue exclusion assay and electron microscopy. RESULTS: One hour after the addition of 4 mg/mL extract and half hour after the addition of 5 mg/mL showed antiprotozoal effect. One to two hours after the addition of 3 mg/mL extract, the movement of flagella and axostyle had disappeared, but death of the cells had not occurred until two hours after the addition. The fine structure of the cytoplasm was also changed half an hour to two hours after addition. The number of polyribosome decreased when that of single ribosomes in the cytoplasm increased. CONCLUSION: These results indicated that S. flavescens had the antiprotozoal effect on T. vaginalis by inhibition of cell multiplication as well as an impairment of protein synthesis.
Cell Proliferation ; Cytoplasm ; Flagella ; Humans ; Metronidazole ; Microscopy, Electron ; Polyribosomes ; Ribosomes ; Sophora* ; Trichomonas vaginalis* ; Trichomonas* ; Trypan Blue

PURPOSE: To evaluate the effects of trypan blue (TB) on the survival of cultured human trabecular meshwork cells (HTMCs). METHODS: Primarily cultured HTMCs were exposed to 0.05, 0.10 or 0.50% TB for 1, 5 or 30 min. Cellular survival was assessed using the MTT assay and degree of apoptosis was analyzed with flow cytometry using annexin-V/propidium iodide double staining. RESULTS: Long-term exposure or high concentration of TB decreased the survival of HTMCs (p < 0.05). In flow cytometric analysis, exposure to 0.50% TB for 30 min increased the degree of apoptosis (p < 0.05). Commercial TB decreased cell survival after exposure for 5 min and increased the degree of apoptosis after exposure for 30 min (p < 0.05). CONCLUSIONS: TB may cause cellular damage of cultured HTMCs and apoptosis could be the underlying mechanism. In TB-assisted cataract surgery, TB should be used for the shortest time possible and removed completely.
Apoptosis ; Cataract ; Cell Survival ; Flow Cytometry ; Humans ; Trabecular Meshwork* ; Trypan Blue*

PURPOSE: To evaluate the effect of mitomycin-C (MMC) on osteotomy site as an adjunctive therapy for dacryocystorhinostomy, the effect of MMC on cultured human osteoblasts was tested. METHODS: Cultured osteoblasts which was obtained from the human iliac crest, were treated with four different concentrations of MMC (0 mg/ml, 0.2 mg/ml, 0.02 mg/ml, 0.002 mg/ml) and cultured for 24hours. To observe the effect of exposed time dependency, cells were treated with MMC during 5, 30minutes, and 24hours and washed and changed with fresh osteogenic media, and then cultured for 24 hours. The effect of fibroblast growth factor (FGF) and transforming growth factor-beta(TGF-beta) on the MMC-treated cells was evaluated. Cell viability was measured using trypan blue staining method and MTT assay. RESULTS: As compared with control group, the lowest growth rate of osteoblasts was 6.8% in 0.2 mg/ml MMC-treated cells. There were no significant differences in the growth rate between 5 minutes and 30 minutes MMC treatment groups, but in case of 24 hours treatment group with MMC (0.2 mg/ml) the growth rate was suppressed to 77.5% of control group with statistical significance. Both growth factors had promotive effect on the growth of in 0.02 mg/ml MMC-treated osteoblasts, but not in 0.2 mg/ml MMC-treated cells. CONCLUSIONS: Osteoblasts which were treated for longer time and with higher concentration of MMC showed more suppression in growth rate. These results suggest that intraoperative application of MMC during dacryocystirhinostomy could have a positive effect of mucosal ostium with suppression of osteoblasts proliferation.
Cell Survival ; Dacryocystorhinostomy ; Fibroblast Growth Factors ; Humans* ; Intercellular Signaling Peptides and Proteins ; Mitomycin* ; Osteoblasts* ; Osteotomy ; Trypan Blue

BACKGROUND: Adipose tissue damage of cryopreserved fat after autologous fat transfer is inevitable in several processes of re-transplantation. This study aims to compare and analyze the survivability of adipocytes after thawing fat cryopreserved at -20degrees C by using thawing methods used in clinics. METHODS: The survival rates of adipocytes in the following thawing groups were measured: natural thawing at 25degrees C for 15 minutes; natural thawing at 25degrees C for 5 minutes, followed by rapid thawing at 37degrees C in a water bath for 5 minutes; and rapid thawing at 37degrees C for 10 minutes in a water bath. The survival rates of adipocytes were assessed by measuring the volume of the fat layer in the top layers separated after centrifugation, counting the number of live adipocytes after staining with trypan blue, and measuring the activity of mitochondria in the adipocytes. RESULTS: In the group with rapid thawing for 10 minutes in a water bath, it was observed that the cell count of live adipocytes and the activity of the adipocyte mitochondria were significantly higher than in the other two groups (P<0.05). The volume of the fat layer separated by centrifugation was also measured to be higher, which was, however, not statistically significant. CONCLUSIONS: It was shown that the survival rate of adipocytes was higher when the frozen fat tissue was thawed rapidly at 37degrees C. It can thus be concluded that if fats thawed with this method are re-transplanted, the survival rate of cryopreserved fats in transplantation will be improved, and thus, the effect of autologous fat transfer will increase.
Adipocytes ; Adipose Tissue ; Autografts ; Baths ; Cell Count ; Centrifugation ; Cryopreservation ; Fats ; Mitochondria ; Survival Rate ; Trypan Blue ; Water

The effects of topical dexamethasone on the endothelial healing and the change of aqueous composition were examined in the repair process of experimental corneal alkali wounds. Corneal alkali wounds were induced, then the eyes were treated topically with either 0.1%dexamethasone or abalanced salt solution[BSS]4 times per day for 8 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The concentrations of ascorbic acid, glucose, and the ions, Na, K , Ca2 and Mg2 , were measured in the aqueous humor. Endothelial healing in control corneas showed a biphasic pattern of healing:an initial short-term healing for the first week and then a late long-term healing following a secondary endothelial breakdown. Topical administration of 0.1%dexamethasone deterred endothelial healing during the early period and prevented secondary endothelial breakdown. Total repair process of endothelium was accelerated by the dexamethasone treatment. Among the various components of the aqueous humor examined, ascorbic acid seemed most sensitive to change caused by the alkali injury and dexametha-sone treatment. The present data indicate that dexamethasone may have a therapeutic potential in the management of endothelial healing after corneal alkali injury.
Administration, Topical ; Alkalies* ; Aqueous Humor ; Ascorbic Acid ; Cornea ; Dexamethasone ; Endothelium ; Glucose ; Ions ; Trypan Blue ; Wounds and Injuries*

BACKGROUND: 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed (CFSE) is the fluorescent labelling agent of living cells and used to trace the cells in vivo after transplatnation of various cells. The CFSE labelled cells can maintain fluorescence for up to 7 days after labelling. The MC3T3-E1 cell line (MC3T3) has been used for many studies about osteoblast, which is well known as a mouse preosteoblast. So the CFSE would be used to trace the transplanted MC3T3. However there are few reports about CFSE labelling of MC3T3. This study is aimed to know about adequate concenturation and incubation time of CFSE to MC3T3. MATERIALS AND METHODS: The MC3T3 was incubated in a humidified atmosphere of 95% air with 5% CO2 at 37 degrees C using alpha-minimal essential medium (alpha-MEM) containing10% FBS and gentamycin. Ten mM CFSE solution in dimethylsulphoxide (DMSO: 1%) was diluted with phosphate buffered saline (PBS) and final concentration of culture medium was, respectively, 5, 10, 15, 20, 25 and 30 microM. Then the MC3T3 was incubated with CFSE in a humidified atmosphere of 95% air with 5% CO2 at 37 degrees C for 5, 10, 15, 20, 25, 30, 35, 40 and 45 minutes in each concentration. The fluorescence of CFSE labelled cells was analysed with a inverted fluorescence microscope. The duration of cell labelling was also studied. Trypan blue dye exclusion test was done for cell viability. RESULTS: For concentration between 5 and 10 microM, CFSE did not significantly label the MC3T3 in vitro. The destruction of MC3T3 was observed at the concentration of 20 microM. In the concentration of 15 microM., the best labelling was obtained at an incubation period between 15 and 30 minutes. The MC3T3 labelled with an incubation period of 15 minutes at 15 microM. was still fluorescent 7 days after CFSE labelling. The mean cell viability was 95.93%. CONCLUSION: These results suggests an incubation period of 15 minutes at 15 microM. of CFSE provides best labelling of MC3T3 in vitro.
Animals ; Atmosphere ; Cell Line* ; Cell Survival ; Dimethyl Sulfoxide ; Fluorescence ; Gentamicins ; Mice ; Osteoblasts ; Trypan Blue