Research progress of HDACs and associated inhibitors on regulation of acute lung injury / 中国药理学通报

Acute lung injury ( ALI) and its most extreme form a-cute respiratory distress syndrome ( ARDS) are lung diseases with high morbidity and mortality. There is no effective therapeutic intervention until now for its complicated pathophysiologi-cal processes and sophisticated regulatory mechanism. Histone deacetylases (HDACs) are a family of proteins with deacetylase activity. Studies have shown that HDACs are involved in the pathophysiological processes of ALI/ARDS, including inflammatory responses,endothelial permeability,oxidative stresses,alveolar fluid clearance and lung tissue repairment. Simultaneously, the use of HDACs inhibitors (HDACIs) can interfere with ALI/ ARDS progression. In this review we describe and summarize the pathophysiological processes and the underlying mechanisms in ALI/ARDS regulated by HDACs and HDACIs in detail, in order to provide the basis for the clinical application of HDACs-targe- ted agents and indicate directions for future study.

La vitamina C, implicaciones terapéuticas en el paciente con quemaduras graves / Vitamin C, therapeutic implications in the patient with serious burns / Vitamina C, implicações terapêuticas no paciente com queimaduras graves

Resumen: La atención a pacientes con quemaduras extensas es compleja, la quemadura condiciona efectos en el sitio de la lesión y a nivel sistémico. A nivel de la microcirculación se presenta respuesta de mediadores químicos inflamatorios y excesiva producción de especies reactivas de oxígeno y nitrógeno, además condiciona disminución de la capacidad antioxidante de vitamina C, por lo que se altera el balance fisiológico de óxido-reducción, dando paso al estado de estrés oxidativo, esto trae como consecuencia un incremento en la inflamación, disfunción endotelial e incremento de la permeabilidad capilar. Uno de los objetivos de la reanimación del paciente quemado es restaurar el volumen intravascular generado por el estado de choque, en el cual se implementan estrategias como el uso de cristaloides, coloides, plasma, terapias dialíticas, uso limitado de opioides y la administración de vitamina C. El objetivo de este trabajo es dar a conocer a la comunidad médica las características físicas y químicas, los mecanismos moleculares de la vitamina C en los que se encuentra implicada en condiciones de quemaduras graves, con la finalidad de la implementación durante la fase de reanimación del quemado.

Abstract: The care of patients with extensive burns is complex, the burn conditions effects at the site of the injury and at the systemic level. At the microcirculation level, there is a response of inflammatory chemical mediators and excessive production of reactive oxygen and nitrogen species, which also causes a decrease in the antioxidant capacity of vitamin C, which is why the physiological balance of oxide-reduction is altered, giving way to the state of oxidative stress, this results in an increase in inflammation, endothelial dysfunction and an increase in capillary permeability. One of the objectives of the resuscitation of the burned patient is to restore the intravascular volume generated by the state of shock, in which strategies such as the use of crystalloids, colloids, plasma, dialysis therapies, limited use of opioids and the administration of vitamins are implemented C. The objective of this work is to make known to the medical community, the physical and chemical characteristics, the molecular mechanisms of vitamin C in which it is involved in severe burn conditions, with the purpose of implementation during the resuscitation phase of burn.

Resumo: O atendimento a pacientes com queimaduras extensas é complexo, a queimadura condiciona efeitos no local da lesão e em nível sistêmico. Ao nível da microcirculação, há uma resposta de mediadores químicos inflamatórios e produção excessiva de espécies reativas de oxigénio e nitrogênio, condiciona também uma diminuição da capacidade antioxidante da vitamina C, que altera o equilíbrio fisiológico de oxidação-redução, dando lugar a o estado de estresse oxidativo, isso resulta em aumento da inflamação, disfunção endotelial e aumento da permeabilidade capilar. Um dos objetivos da ressuscitação do paciente queimado é restaurar o volume intravascular gerado pelo estado de choque, no qual se implementam estratégias como o uso de cristalóides, colóides, plasma, terapias dialíticas, uso limitado de opióides e administração de vitamina C. O objetivo deste trabalho é dar a conhecer à comunidade médica as características físicas e químicas, os mecanismos moleculares da vitamina C em que está envolvida em condições de queimaduras graves, com vista à sua aplicação durante a fase de reanimação do paciente queimado.

Activation of A2b adenosine receptor decreases lipopolysaccharide-induced pulmonary microvascular permeability / 中华危重病急救医学

Objective To explore the role of the A2b adenosine receptor (Adora2b) in lipopolysaccharide (LPS)-induced injury of human pulmonary microvascular endothelial cells (HPMECs), and its mechanism. Methods HPMECs were cultured in vitro. The LPS dose-effect experiment, time-effect experiment and the Adora2b agonist/antagonist intervention experiment were performed respectively. ① Dose-effect and time-effect experiments: HPMECs were stimulated with 1, 10, 100, 1 000 μg/L LPS for 24 hours, or 100 μg/L LPS for 4, 8, 12, 16, 24 hours. Cell viability was measured by cell counting kit-8 (CCK8). The protein and mRNA expressions of Adora2b were determined by Western Blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) respectively. ② Adora2b agonist/antagonist intervention experiment: serum-starved HPMECs were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then incubated with 100 μg/L of LPS for 24 hours. The HPMECs without treatment were served as blank control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. The monolayer permeability of HPMECs was determined by fluorescein isothiocyanate (FITC)-dextran. Cell cycle was analyzed by flow cytometry. The mRNA expressions of VE-cadherin, occludin, vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANGPT1) were determined by RT-PCR. Results ① Dose-effect and time-effect experiments: LPS induced the decreased cell viability of HPMECs in dose and time-dependent manner. Compared with the control group, the protein expression of Adora2b was sharply up-regulated after 100 μg/L or 1 000 μg/L LPS stimulation. Meanwhile, LPS was shown to cause a dose and time-dependent induction of Adora2b transcript level. ② Adora2b agonist/antagonist intervention experiments: compared with the control group, the monolayer permeability of HPMECs was rapidly enhanced after LPS treatment, and lower cell viability and proliferation, as well as the expression of cell junction and angiogenic factors were downregulated. Compared with LPS group, 0.1, 1, 10 μmol/L BAY 60-6583 pretreatment could decrease the endothelial cell barrier leakage in a dose-dependent manner [Pd: (203.06±15.24)%, (164.15± 17.82)%, (125.69±10.38)% vs. (218.53±12.05)%], and promote cell proliferation of HPMECs [the proportion of S and G2 phases: (24.36±1.40)%, (32.37±0.95)%, (40.05±2.99)% vs. (18.83±0.73)%]. Pretreatment of 10 μmol/L BAY60-6583 also upregulated the mRNA expressions of cell junction and angiogenic factors [VE-cadherin (2-ΔΔCt):2.17±0.23 vs. 0.56±0.10, occludin (2-ΔΔCt): 5.32±0.28 vs. 0.48±0.11, VEGF (2-ΔΔCt): 4.44±0.34 vs. 0.58±0.09, ANGPT-1 (2-ΔΔCt): 5.98±0.73 vs. 0.66±0.10, all P < 0.01]. PSB1115 pretreatment aggravated injury of microvascular endothelial cells after LPS incubation, with lower cell viability, slower proliferation and less expression of VEGF and ANGPT1. There was no influence of BAY 60-6583 or PSB1115 single treatment on cell viability, cell cycle and the expression of angiogenic factors in HPMECs. Conclusions In vitro studies of cultured HPMECs exposed to LPS are identified as dose and time-dependent induction of Adora2b transcript and corresponding protein induction. Activation of Adora2b attenuates LPS-induced pulmonary microvascular endothelial cell barrier enhancement by regulating intercellular junction and promoting angiogenesis, suggesting Adora2b as potential therapeutic target in the treatment of LPS-induced forms of acute lung injury.

Effects of MRP4 gene on lipopolysaccharide-induced endothelial permeability and the cytoskeleton of endothelial cells changes / 中华急诊医学杂志

Objective To investigate the effects of multidrug resistance-associated protein 4 (MRP4) on the cytoskeleton and cellular permeability of rat pulmonary microvascular endothelial cells (PMVECs) induced by lipopolysaccharide (LPS).Methods PMVECs were cultured for 3 to 6 generations were randomly divided into 4 groups:control group,LPS group,Ad-shMRP4 group (adenoviral expression of a short-hairpin RNA directed against MRP4),Ad-shRNA group.The infection rate of cells was detected by fluorescence microscope observation.The level of MRP4 was assayed by Western botting.Monolayer permeability was determined by the Transwell assay.The morphological characteristic and distribution of Factin was measured by laser confocal fluorescence microscope.Results Compared with control group,the expression of MRP4 protein was up-regulated (P ＜ 0.05) and the significant increase in the permeability of endothelial cells (2 h,6 h,12 h and 24 h respectively:0.28 ±0.02 vs.0.41 ±0.04,0.32 ±0.02,0.30 ±0.01 vs.0.53±0.04,0.39±0.03,0.33 ±0.04 vs.1.12±0.17,0.70 ±0.07,0.32±0.03 vs.0.79 ± 0.02,0.57 ± 0.05,P ＜ 0.05),the F-actin was remodeled,and the stress fibers were formed in LPS group and Ad-shMRP4 group.However,compared with LPS group,the expression of MRP4 protein was down-regulated (P ＜ 0.05) and the markedly decrease in the permeability of endothelial ceils (2 h,6 h,12 h and 24 h respectively:0.41 ± 0.04 vs.0.32 ± 0.02,0.53 ± 0.04 vs.0.39 ± 0.03,1.12 ± 0.17 vs.0.70 ± 0.07,0.79 ± 0.02 vs.0.57 ± 0.05,P ＜ 0.05) was found,and the remodeling of F-actin,and the formation of stress fibers were observed in Ad-shMRP4 group.Conclusions Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton,thus playing an important role in the protection of endothelial cell barrier.

Effect of multidrug resistant associated protein 4 overexpression on lipopolysaccharide-induced vascular endothelial hyperpermeability and its mechanism / 中华危重病急救医学

Objective To investigate the effect of multidrug resistance protein 4 (MRP4) overexpression on lipopolysaccharide (LPS)-induced vascular endothelial hyperpermeability of rat pulmonary micro-vascular endothelial cells (PMVECs) and its molecule mechanism. Methods Three to six passages of PMVECs were cultured in vitro, and they were divided into three groups: the cells in LPS group were only challenged by LPS 10 μg/mL after being cultured in serum-free medium for 24 hours; the cells in Ad-shRNA and Ad-MRP4 groups were infected with the empty virus control or recombinant adenovirus expressing MRP4 for 2 hours, and then were cultured in serum-free medium for 24 hours followed by stimulation of LPS 10 μg/mL. Endothelial permeability was assayed by the Transwell chamber models at 2, 6, 12, and 24 hours after LPS stimulation. Intracellular cyclic adenosine monophosphate (cAMP) levels were detected by enzyme-linked immunosorbent assay (ELISA). The morphological characteristics and distribution of F-actin was determined by laser confocal fluorescence microscope. The protein expressions of MRP4,β-catenin, vascular endothelium-cadherin (VE-cad) and ZO-1 were measured by Western Blot. Results ① After LPS stimulation, endothelium permeability and intracellular cAMP levels in PMVECs were significantly increased, peaked at 12 hours, and then decreased after 24 hours. Compared with LPS group and Ad-shRNA group, PMVECs of Ad-MRP4 group were exhibited a significant increase in endothelial permeability [12-hour permeability (A value):1.88±0.06 vs. 1.12±0.17, 1.10±0.18] and a significant decrease in intracellular cAMP level [12-hour cAMP (μg/L):2.39±0.02 vs. 2.97±0.01, 3.00±0.02, all P 0.05).② Under laser confocal fluorescence microscope, after LPS stimulation, the stress fiber formation was induced in three groups. But there were pronounced irregular aggregation of fiber in PMVECs of Ad-MRP4 group. ③ Furthermore, compared with LPS group and Ad-shRNA group, protein expression of MRP4 in Ad-MRP4 group was dramatically increased (gray value: 0.76±0.03 vs. 0.44±0.02, 0.43±0.02, both P 0.05). Conclusion MRP4 overexpression can decrease intracellular cAMP levels, reduce intercellular junction protein expression, and then exaggerate LPS-induced vascular endothelial hyperpermeability.

Effects of MRP4 gene on lipopolysaccharide-induced endothelial permeability and the cytoskeleton of endothelial cells changes / 中华急诊医学杂志

Objective To investigate the effects of multidrug resistance-associated protein 4 (MRP4) on the cytoskeleton and cellular permeability of rat pulmonary microvascular endothelial cells (PMVECs) induced by lipopolysaccharide (LPS).Methods PMVECs were cultured for 3 to 6 generations were randomly divided into 4 groups:control group,LPS group,Ad-shMRP4 group (adenoviral expression of a short-hairpin RNA directed against MRP4),Ad-shRNA group.The infection rate of cells was detected by fluorescence microscope observation.The level of MRP4 was assayed by Western botting.Monolayer permeability was determined by the Transwell assay.The morphological characteristic and distribution of Factin was measured by laser confocal fluorescence microscope.Results Compared with control group,the expression of MRP4 protein was up-regulated (P ＜ 0.05) and the significant increase in the permeability of endothelial cells (2 h,6 h,12 h and 24 h respectively:0.28 ±0.02 vs.0.41 ±0.04,0.32 ±0.02,0.30 ±0.01 vs.0.53±0.04,0.39±0.03,0.33 ±0.04 vs.1.12±0.17,0.70 ±0.07,0.32±0.03 vs.0.79 ± 0.02,0.57 ± 0.05,P ＜ 0.05),the F-actin was remodeled,and the stress fibers were formed in LPS group and Ad-shMRP4 group.However,compared with LPS group,the expression of MRP4 protein was down-regulated (P ＜ 0.05) and the markedly decrease in the permeability of endothelial ceils (2 h,6 h,12 h and 24 h respectively:0.41 ± 0.04 vs.0.32 ± 0.02,0.53 ± 0.04 vs.0.39 ± 0.03,1.12 ± 0.17 vs.0.70 ± 0.07,0.79 ± 0.02 vs.0.57 ± 0.05,P ＜ 0.05) was found,and the remodeling of F-actin,and the formation of stress fibers were observed in Ad-shMRP4 group.Conclusions Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton,thus playing an important role in the protection of endothelial cell barrier.

Relationship between endothelial damage and p120-catenin in paraquat intoxication and the protective effect of mangiferin / 中华危重病急救医学

Objective To investigate the relationship between endothelial damage and p120-catenin (p120-ctn) in a model of paraquat intoxication,and the modulatory effect of mangiferin on p120-ctn.Methods Human umbilical vein endothelial cells (HUVECs) were cultured in two compartment spreading apparatus in vitro.The endothelial cells were divided into three groups:control group (cultured in DMEM with 10％ fetal bovine serum),paraquat group (paraquat was added to the medium with final concentration of 0.05 μmol/L) and mangiferin group (cultured in medium with addition of paraquat for 30 minutes,then mangiferin was added in a final concentration of 20 μmol/L).The cellular permeability at 6,12,24,48,72 hours after culture in the three groups was measured.The expressions of p120-ctn 1A,p120-ctn 3A mRNA and p120-ctn protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot analysis.The distribution of p120-ctn protein was observed by immunofluorescence.Results Compared with control group,cellular permeability in paraquat and mangiferin groups were increased with prolongation of time,and peaked at 72 hours [(29.86 ± 3.98)％,(24.39 ± 2.79)％ vs.(11.71 ± 1.67)％,both P＜0.05].The cellular permeability was significantly lower in mangiferin group than that in paraquat group at different time points (all P＜0.05).At 6 hours after intoxication,the expressions of p120-ctn 1A,p 120-ctn 3A mRNA (gray value) and p 120-ctn protein (gray value) were significantly lower in paraquat group than those in control group (p120-ctn 1A mRNA:0.150 ± 0.024 vs.0.433 ± 0.024,p120-ctn 3A mRNA:0.316 ± 0.043 vs.0.701 ±0.020,p120-ctn protein:0.485 ±0.031 vs.0.763 ±0.038,all P＜0.01).The expressions of p120-ctn 1A,p120-ctn 3A mRNA and p120-ctn protein were significantly higher in mangiferin group than those in paraquat group from 6 hours on (p120-ctn 1A mRNA:0.281 ± 0.021 vs.0.150 ± 0.024,p120-ctn 3A mRNA:0.602 ± 0.042 vs.0.316 ± 0.043,p120-ctn protein:0.675 ± 0.031 vs.0.485 ± 0.031,all P＜0.01),and they were gradually increased with prolongation of time,and peaked at 72 hours (p120-ctn 1A mRNA:1.376 ±0.128 vs.0.150 ± 0.024,p120-ctn 3A mRNA:1.251 ± 0.059 vs.0.316 ± 0.043,p120-ctn protein:0.844 ± 0.050 vs.0.485 ± 0.031,all P＜ 0.01).Under upright fluorescence microscope,p120-ctn was mainly distributed in the cell membrane in control group,with a slight expression in cytoplasm,and no expression in the nuclei.With prolongation of time,p120-ctn expression in the cell membrane was gradually decreased in paraquat group,while it was increased in the cytoplasm and nuclei,with blurring of cell membrane and widening of cellular gap.p120-ctn expression was improved on the cell membrane in mangiferin group at corresponding time points,with decreased in expression in nuclei and cytoplasm.Conclusion The p120-ctn protein plays an important role in the enhancement of endothelial permeability in paraquat intoxication,and mangiferin may attenuate endothelial injury in paraquat intoxication possibly through modulation of p 120-ctn protein.

Expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury / 中华急诊医学杂志

Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury (ALI) and explore the function of Slit2 and Robo4 in ALI.Methods Forty-eight Sprague-Dawley rats were randomly (random number) divided into control group (n =24) and ALl group (n =24).ALI model was reproduced by cecum ligation and puncture (CLP).The control group only experienced a simulated operation without CLP.Both groups were further divided into 3 subgroups with 8 rats in each subgroup:12 h,24 h,and 48 h subgroups.artery blood gas analysis,lung tissue wet/dry weight (W/D) ratio,lung histopathologic changes,pulmonary microvascular permeability were observed.The serum tumor nocrosis factor-α (TNF-α) was measured with enzyme linked immunosorbent assay (ELISA).The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR).The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry.Date were analyzed by one-way ANOVA with SPSS version 13.0 software.Statistical significance was established at a P value of less than 0.05.Results Compared with the control group,in ALI rats at different time points,partial pressure of oxygen in arterial blood (PaO2) decreased significantly,lung W/D weight ratio and pulmonary microvascular permeability,the serum TNF-α increased significantly (all P ＜ 0.05),histopathology of lung revealed signs of injury.The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [(0.56±0.13) vs.(0.87±0.05),F=41.39,P＜0.05,(0.42±0.10) vs.(0.85±0.07),F=93.54,P＜0.05,(0.26±0.08) vs.(0.89 ±0.09),F=227.05,P＜0.05].but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [(0.86±0.07) vs.(0.83±0.05),F=0.695,P＞0.05,(0.82±0.05) vs.(0.89±0.08),F=2.061,P ＞ 0.05,(0.86 ± 0.08) vs.(0.86 ± 0.05),F =0.035,P ＞ 0.05].Immunohistochemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells,while lung epithelial cell nuclei and endochylema.Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells.Compared with the control group,expression of Slit2 protein in lung tissue in ALI group decreased markedly [(0.37 ± 0.05) vs.(0.45 ± 0.07),F =6.82,P ＜ 0.05,(0.32±0.06) vs.(0.47±0.09),F=23.54,P＜0.05,(0.28±0.07) vs.(0.46±0.06),F=28.01,P ＜ 0.05].As good as RT-PCR,there were no significant difference in expression of Robo4 protein in lung tissue between two groups [(0.53±0.04) vs.(0.52±0.05),F=0.155,P＞0.05,(0.53± 0.09) vs.(0.50±0.05),F=0.498,P＞0.05,(0.55±0.06) vs.(0.56±0.07),F=0.073,P ＞ 0.05].Conclusions Lung tissues of control group rats express Slit2 and Robo4.The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALI caused by CLP may be associated with the occurrence of ALI.

Endothelial Cell Products as a Key Player in Hypoxia-Induced Nerve Cell Injury after Stroke

OBJECTIVE: Activated endothelial cells mediate the cascade of reactions in response to hypoxia for adaptation to the stress. It has been suggested that hypoxia, by itself, without reperfusion, can activate the endothelial cells and initiate complex responses. In this study, we investigated whether hypoxia-induced endothelial products alter the endothelial permeability and have a direct cytotoxic effect on nerve cells. METHODS: Hypoxic condition of primary human umbilical vein endothelial cells(HUVEC) was induced by CoCl2 treatment in culture medium. Cell growth was evaluated by 3,4,5-dimethyl thiazole-3,5-diphenyl tetrazolium bromide (MTT) assay. Hypoxia-induced products (IL-1beta, TGF-beta1, IFN-gamma, TNF-alpha, IL-10, IL-6, IL-8, MCP-1 and VEGF) were assessed by enzyme-linked immunosorbent assay. Endothelial permeability was evaluated by Western blotting. RESULTS: Prolonged hypoxia caused endothelial cells to secrete IL-6, IL-8, MCP-1 and VEGF. However, the levels of IL-1, IL-10, TNF-alpha, TGF-beta, IFN-gamma and nitric oxide remained unchanged over 48 h hypoxia. Hypoxic exposure to endothelial cells induced the time-dependent down regulation of the expression of cadherin and catenin protein. The conditioned medium taken from hypoxic HUVECs had the cytotoxic effect selectively on neuroblastoma cells, but not on astroglioma cells. CONCLUSION: These results suggest the possibility that endothelial cell derived cytokines or other secreted products with the increased endothelial permeability might directly contribute to nerve cell injury followed by hypoxia.

Endothelial Function of Cornea Preserved in Korean Corneal Storage Media

PURPOSE: To evaluate the endothelial function of cornea preserved in newly developing korean corneal storage media (CS002, CS003) by estimating the permeability of corneal endothelium and the change of corneal thickness. METHODS: The cornea were divided into six experimental groups - fresh group immediately after enucleation, 4degrees Cmoist chamber group preserved for 24 hours and 48 hours, Optisol & CS002 group for 1, 3, 5, and 7 days, and Likorol & CS003 group for 7, 10, and 14 days after enucleation, and then corneal endothelial permeability(Pac) was measured using carboxyfluorescein solution. Corneal thickness was measured using pachymeter(fine focus adjustment) of the specular microscope. RESULTS: Corneal endothelial Pac (x1 0(- 4) cm/min) was 3.64+/-0.33 in fresh group, 4.79+/-0.28 in 4degrees Cmoist chamber group for 24 hours. Each endothelial Pac of CS002 group at 5 and 7 days was 5.81+/-0.55 and 5.65+/-0.58, which were different with 4degrees Cmoist chamber preservation group for 24 hours(p<0.05) but not different with Optisol groups at same days. Each endothelial Pac of CS003 group at 7, 10, and 14 days was 4.34+/-0.34, 4.66+/-0.59, and 4.66+/-0.27, which were not different from those of Likorol. Each corneal thickness of CS002 and Optisol group at 7days was 417.80+/-19.37 mu m and 421.00+/-19.75mu m, which were resemble increment. Corneal thickness was 426.75+/-22.43mu m in CS003 group and 476.00+/- 40.08mu m in Likorol group at 7days. There was statistical difference between the two group(P<0.05), and this difference was sustained for 14days (P<0.05). CONCLUSION: There was no difference in the effect on corneal endothelial permeability between korean corneal storage media such as CS002 and CS003, and that of previous corneal storage media such as Optisol and Likorol. Corneal thickness of cornea preserved in korean corneal storage media was thinner than that of Likorol.

The Change of Corneal Endothelial Permeability and Corneal Thickness after Topical Steroid Treatment

Changes of corneal endothelial permeability and corneal thickness after instillation of topical steroid was evaluated. In five rabbits, 0.1%dexamethasone eye solution was dropped into right eyes, and in another five rabbits, 1%prednisolone acetate eye solution was dropped into right eyes, and each left eyes were used as a control with dropping of Tears Naturale(r)II (Alcon-couvreur, Belgium). After dropping eyedrops for seven days, corneal endothelial permeability and corneal thickness were measured. Mean corneal thickness changed from 371.4 micrometer to 334.2 micrometer after dropping eyedrops in 0.1%dexamethasone group, and from 375.8 micrometer to 347.8 micrometer in control group where no statistical difference was noted between the two groups. Corneal endothelial permeability was 3.58x10(-4)cm/min in 0.1%dexamethasone group, and 3.54x10(-4) cm/min in control group(p>0.05). Mean corneal thickness changed from 347.4 micrometer to 323.8 micrometer after dropping eyedrops in 1% prednisolone acetate group, and from 342.4 micrometer to 335.6 micrometer in control group. There was also no statistical difference between the two groups. Corneal endothelial permeability was 4.08x10(-4)cm/min in 1%prednisolone acetate group, and 4.26x10(-4) cm/min in control group(p>0.05). In conclusion, topical application of 0.1%dexamethasone or 1%prednisolone acetate for a short period may have no effect on corneal endothelial permeability and thickness.

Protective Effect of Hyaluronic Acid on the Corneal Endothelial Function Against Free-Radical Damage

PURPOSE: To investigate the protective effects of hyaluronic acid with glutathione and ascorbic acid on corneal endothelial function against free-radical damage. METHODS: bovine corneal endothelial(BCEN) cells were treated with a flux of chemically generated superoxide anion produced by the combination of 1 mM hypoxanthine and 0.06 U/ml xanthine oxidase(HX-XO) for 10 minutes, and rabbit corneas were mounted in the dual-chamber specular microscope and perfused with bicarbonate Ringer(BR) solution for one hour and their endothelial surface was exposed to HX-XO for five minutes, and then perfused with glutathione, hyaluronic acid, or ascorbic acid in BR solution for three hours. BCEN cells was observed using MTT assay and rabbit corneal thickness was measured every 15 minutes and corneal swelling rates were calculated by linear regression analysis. Also, corneal endothelial permeability was measured using carboxyfluorescein and fluorometer. RESULTS: MTT assay showed less cytotoxicity in the cells treated with glutathione, hyaluronic acid, or ascorbic acid compared to HX-XO alone. Glutathione, hyaluronic acid, or ascorbic acid reduced the rabbit corneal swelling caused by HX-XO. Corneal endothelial permeability(Pac) increased in corneas perfused with HX-XO(7.88 x 10 cm/min) while those with BR had Pac of 4.54 x 10 cm/min. Following treatment with glutathione, hyaluronic acid, or ascorbic acid, Pac decreased to 4.96, 6.81, and 5.25 respectively(p<0.05). CONCLUSIONS: In conclusion, these data suggest that hyaluronic acid scavenges HX-XO-generated oxyradicals as well as glutathione and less likely ascorbic acid.

The Effect of Taurine on Corneal Endothelial Damage by Free-radicals

PURPOSE: To investigate the protective effect of taurine on the corneal endothelial damage by oxidative stress. METHODS: Rabbit corneas were mounted in the dual-chambered specular microscope and perfused with bicarbonate-Ringer solution(BR) for 1 hour, and endothelial surface was treated with hypoxanthine-xanthine oxidase(HX-XO) for 5 minutes, and perfused with BR for 3 hours in control group, while perfused with 0.5, 1.0, 2.0 mM taurine in BR in test group. Corneal thickness was measured every 15 minutes and corneal swelling rates were calculated by linear regression analysis. Also, corneal permeability was measured using carboxyfluorescein and fluorometer. Using bovine corneal endothelial cells, MTT assay was done. RESULTS: On MTT assay, cytotoxicity of HX-XO group was 47.69% while those treated with 0.5, 1.0, 2.0 mM taurine were 36.22%, 29.73%, 24.90%, respectively(P<0.05). 0.5, 1.0 and 2.0 mM taurine group (12.88, 10.75 and 8.95 um/hr, respectively) reduced the HX-XO-induced corneal swelling rate(20.08 um/hr)(P<0.05). Corneal endothelial permeability(Pac) showed 7.96 x 10(-4) cm/min in corneas perfused with HX-HO. Also, each taurine solutions markedly reduced Pac(7.00+/-0.29 x 10(-4), 6.51+/-0.25 x 10(-4) and 5.37+/-1.41 x 10(-4) cm/min, respectively)(P<0.05). CONCLUSIONS: The results of this study showed that taurine may prevent hydrogen peroxide-induced corneal endothelial damage.

The Effect of Mitomycin-C on the Function of Corneal Endothelial Cells in Rabbit

The purpose of this study is to investigate the toxicity of mitomycin-C[MMC]to the corneal endothelial cells, which is medical adjunct to pterygium and glaucoma surgery.Rabbit corneas were mounted in the in-vitro dual-chambered specular microscope.Corneal endothelium was perfused with the glutathione-bicarbonate-Ringer[GBR]solution for one hour, then, perfused with 0.02%, 0.01%, and 0.005%MMC in GBR solution in experimental groups, and with GBR solution only in control group.Corneal thickness was measured every 15 minutes during perfusion and corneal swelling rate was calculated.Corneal endothelial permeability was also measured in another experiment.In MMC-mixed group, the early swelling rate decreased due to osmolarity of MMC, but after removal of MMC, the swelling rate increased compared to that of the control group.The pattern of increase was not a linear form, but a secondary curve with the plateau. In 0.02%and 0.01%MMC group, corneas swelled significantly, but not in 0.005%group.Corneal endothelial permeability was 4.21 +/-0.50 x10-4cm/min at 0.005%MMC, 4.10 +/-0.93 x10-4cm/min in control, and 4.25 +/-0.48 x10-4cm/min at 0.01% MMC, 3.73 +/-0.73 x10-4cm/min in control. No significant changes in permeability was observed.In conclusion, MMC of 0.01% or higher exposed to corneal endothelial cells induced corneal swelling of which mechanism was thought to be due to inhibition of Na/K-ATPase by MMC.

The Effect of Fluorescein and Indocyanine Green on Corneal Endothelial Function

Both sodium fluorescein[FL] and indocyanine green[ICG] were used for fundus angiography. Recently, these were also used during cataract surgery for enhancement of capsular visualization in white mature or hypermature cataract. So, ICG and FL may influence corneal endothelial function if left in anterior chamber. To evaluate the effect of intracameral FL or ICG on corneal endothelial function, rabbit corneas were isolated &mounted in the in-vitro specular microscope for endothelial perfusion. Experimental corneas were perfused with different concentrations of FL or ICG. Control corneas were perfused with glutathione-bicarbonate Ringer solution[GBR]. Corneal thickness was measured every 15 minutes during the perfusion and corneal swelling rates were calculated. Corneal endothelial permeability[Pa c] was measured according to the method of Watsky et al. The corneas perfused with FL, 2.5% deswelled probably due to high osmolarity. Swelling rates of corneas perfused with 1% and 0.5% FL did not differ significantly from control[p>0.05]. The corneas perfused with 0.01%, 0.005%, 0.002%, and 0.001% ICG swelled significantly[p0.05]. Pa c in corneas perfused with ICG, 0.005% increased markedly compared to control while corneas perfused with FL, 1% showed decreased permeability. The results of this study showed that FL did not affect endothelial function of rabbit cornea in relatively high concentrations while ICG affected endothelial function even in lower concentrations. With respect to clinical use of intracameral ICG, close attention must be paid.

Effect of Dorzolamide on Corneal Endothelium

The purpose of this study was to investigate the effect of 2% dorzolamide, a topical carbonic anhydrase inhibitor, on corneal endothelium. Corneal endothelial permeability, central corneal thickness, central corneal endothelial cell density and intraocular pressure were measured in both eyes before(baseline) and after the use of 2% dorzolamide t.i.d. in one eye for one week. There was significant decrease in intraocular pressure(p=0.04) after the use of 2% dorzolamide t.i.d. for one week, but there were no significant changes in corneal endothelial permeability, central corneal thickness and central corneal endothelial cell density(p>0.05 in all).