Protein tyrosine phosphatase 1B regulates the activity of retinal pigment epithelial cells.

PURPOSE: To determine whether protein tyrosine phosphatase 1B (PTP1B) is expressed in rat retinal pigment epithelium (RPE) cells, to evaluate whether inhibition of PTP1B contributes to initiation of RPE cells into an active state, and to investigate the signaling pathways involved in this process. METHODS: Rat retinas were detached by trans-scleral injection of 1.4% sodium hyaluronate into the subretinal space. Immunocytochemistry evaluated the expression of PTP1B in RPE cells located at normal and detached retinas. From the cultured RPE cells treated with TCS-401, cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetracolium bromide assay, and the protein expression levels of cyclin A and cyclin D1 were determined. The effect of TCS-401 on cell differentiation was confirmed by immunostaining for α-smooth muscle actin and by western blot. Cell migration activity and PTP1B signaling mechanism were determined. Migration Assay was used to evaluate cell migration activity. PTP1B signaling mechanism was determined by use of PD98059 and LY294002. RESULTS: PTP1B was expressed in the RPE layer of the normal retina. After retinal detachment, weak immunolabeling of PTP1B was seen in the RPE cells. TCS-401 promoted the proliferation and expression of cyclin A and cyclin D1 in RPE cells. TCS-401 induced RPE cells to differentiate toward better contractility and motility. A migration assay proved that inhibiting PTP1B improved the migratory activity of RPE cells. TCS-401 activated extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) phosphorylation. Pretreatment with PD98059 and LY294002 abolished TCS-401-induced activation of Erk, Akt, cell proliferation, and cell migration. CONCLUSIONS: PTP1B may be involved in regulating the active state of RPE cells. The inhibition of PTP1B promoted the proliferation, myofibroblast differentiation, and migration of RPE cells, and MEK/Erk and PI3K/Akt signaling pathways played important roles in the proliferation and migration process.

[In vitro study on expression of tumor stem cell biomarker and transdifferentiation towards endothelial cells of retinoblastoma cells under hypoxia condition].

OBJECTIVE: To investigate the capability of RB cells that represent characteristics of tumor stem cell and trans-differentiate towards endothelial cells under hypoxia microenvironment, as well as their mechanism. METHODS: Experimental research. RB cell line Y79 was cultured in hypoxia environment with or without rapamycin treatment. Morphological changes, expression of neuron specific enolase (NES) and ATP binding cassette transporters G2 (ABCG2), and expression of HIF-1α protein and mRNA were detected. After been induced toward endothelial cells, expression of CD31 and vWF, up-taking of Dil-acLDL, vasculogenic mimicry (VM) formation in 3D culture and expression of HIF-1α,EphA2 and PI3K were detected. The ANOVA test was performed to compare the differences among groups, and SNK-q test was performed to further comparison. RESULTS: Y79 cells in normal oxygen group were single or agminated suspending cells. In hypoxia group, part of Y79 cells became adherent. No adherent cells were observed in rapamycin fore-treated group. Positive rates of NES and ABCG2 had significant difference among these three groups (FNES = 698.45, FABCG2 = 864.48, all P < 0.01). Compared with normal group [(98.2 ± 2.5)%, (2.1 ± 2.1)%],NES positive rate [(35.1 ± 3.4)%] was significantly reduced and ABCG2 positive rate [(67.4 ± 3.6)%] was significantly enhanced in hypoxia group (q = 46.11, 50.89; both P < 0.01), no significant changes were observed in rapamycin fore-treated group(P > 0.05). Expressions of HIF-1α protein and mRNA showed significant difference among these three groups (Fprotein = 314.85, FmRNA = 132.01, all P < 0.01). Compared with normal oxygen group (0.165 ± 0.056,0.927 ± 0.715) , HIF-1α protein (1.094 ± 0.077) and mRNA (6.408 ± 0.686) were significantly increased in hypoxia group(q = 31.81, 18.40; both P < 0.01), HIF-1α mRNA (7.219 ± 0.591) was significantly increased but no increased protein expression (0.218 ± 0.061) was observed in rapamycin fore-treated group. After transdifferentiation induction,Y79 cell in hypoxia group expressed CD31 and vWF, acquired the abilities of acLDL up-taking and VM formation. Compared with normal group(0.327 ± 0.108, 0.194 ± 0.033, 0.402 ± 0.068), expression of HIF-1α,EphA2 and PI3K protein (1.440 ± 0.089,0.377 ± 0.056,0.762 ± 0.090) was significant increased in hypoxia group(q = 8.72-23.00, all P < 0.01). CONCLUSIONS: Under hypoxic condition, part of RB cells can express tumor stem cell markers, transdifferentiate towards endothelial cells and acquire part of endothelial cell function and VM formation capability. HIF-1α through EphA2/PI3K pathway may play an important role in VM formation.

Structural measurements and vessel density of spectral-domain optic coherence tomography in early, moderate, and severe primary angle-closure glaucoma.

AIM: To compare the macular ganglion cell-inner plexiform layer (GCIPL) thickness, retinal nerve fiber layer (RNFL) thickness, optic nerve head (ONH) parameters, and retinal vessel density (VD) measured by spectral-domain optical coherence tomography (SD-OCT) and analyze the correlations between them in the early, moderate, severe primary angle-closure glaucoma (PACG) and normal eyes. METHODS: Totally 70 PACG eyes and 20 normal eyes were recruited for this retrospective analysis. PACG eyes were further separated into early, moderate, or severe PACG eyes using the Enhanced Glaucoma Staging System (GSS2). The GCIPL thickness, RNFL thickness, ONH parameters, and retinal VD were measured by SD-OCT, differences among the groups and correlations within the same group were calculated. RESULTS: The inferior and superotemporal sectors of the GCIPL thickness, rim area of ONH, average and inferior sector of the retinal VD were significantly reduced (all P<0.05) in the early PACG eyes compared to the normal and the optic disc area, cup to disc ratio (C/D), and cup volume were significantly higher (all P<0.05); but the RNFL was not significant changes in early and moderate PACG. In severe group, the GCIPL and RNFL thickness were obvious thinning with retinal VD were decreasing as well as C/D and cup volume increasing than other three groups (all P<0.01). In the early PACG subgroup, there were significant positive correlations between retinal VD and GCIPL thickness (except superonasal and inferonasal sectors, r=0.573 to 0.641, all P<0.05), superior sectors of RNFL thickness (r=0.055, P=0.049). More obvious significant positive correlations were existed in moderate PACG eyes between retinal VD and superior sectors of RNFL thickness (r=0.650, P=0.022), and temporal sectors of RNFL thickness (r=0.740, P=0.006). In the severe PACG eyes, neither GCIPL nor RNFL thickness was associated with retinal VD. CONCLUSION: The ONH damage and retinal VD loss appears earlier than RNFL thickness loss in PACG eyes. As the PACG disease progressed from the early to the moderate stage, the correlations between the retinal VD and RNFL thickness increases.

[The expression and meanning of Skp2, p27, PTEN proteins in non-Hodgkin B-cell lymphoma of ocular adnexa].

OBJECTIVE: To investigate the expression and the correlation of protein of S-phase kinase association protein 2(Skp2), p27 and phosphatases phosphatase and tensin homologue (PTEN) in non-Hodgkin B-cell lymphoma (NHL)of ocular adnexa. METHODS: Experimental study. Paraffin sections were collected from patients suffering from B cell NHL (n = 30) in Qingdao Affiliated Hospital from January 1995 to January 2011 and lymphadenosis (n = 10) of ocular adnexa as the control group. The expression of Skp2, p27 and PTEN proteins were detected by immunohistochemistry and the positive expression rate between lymphomas and lymphadenosis was compared by χ(2) inspection. Spearman rank correlation was used to estimate the relationships among three proteins in ocular lymphoma. Pathologic grade were evaluated as independent factor of clinical information. RESULTS: The expression of three proteins were related to pathologic type of tumors, but not related to age, gender, pathogenetic locations. In non-Hodgkin B-cell lymphoma of ocular adnexa, the expression of Skp2 (76.7%, 23/30) was higher than that in control group (0/10) (χ(2) = 19.79, P = 0.001), while the expression of p27 (73.3%, 22/30) and PTEN (56.7%, 17/30) was lower than that in the control group (10/10 and 10/10) (χ(2) = 8.37, P = 0.039;χ(2) = 13.48, P = 0.004). With the increase of pathologic grade, Skp2 labeling frequency increase gradually, while p27 and PTEN labeling frequency decrease gradually;there was a negative correlation between p27 and Skp2 in MALT (r = -0.134, P < 0.05) while there was a positive correlation between p27 and PTEN (r = 0.828, P < 0.05). The relationship between Skp2 and PTEN was also negative (r = -0.883, P < 0.05). CONCLUSIONS: The higher expression of Skp2 and lower expression of p27 and PTEN may play a role in the tumorigenesis and different pathologic type of the B cell NHL of ocular adnexa. The expressions of three proteins correlate with each other in mucosa-associated lymphoid tissue.

NADPH oxidase 2 plays a protective role in experimental Aspergillus fumigatus keratitis in mice through killing fungi and limiting the degree of inflammation.

AIM: To explore whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) is expressed in fungal keratitis in mice and investigate its role in this disease. METHODS: NOX2 expression was detected in C57BL/6 mice. After testing the inhibitory effect of diphenyleneiodonium chloride (DPI) on NOX2, its impact on clinical performance, myeloperoxidase levels, the number of colonies forming units, the level of H3, the generation of reactive oxygen species (ROS) and the release of cytokines [NF-κB, interleukin-17A (IL-17A), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), Nrf2, IL-10, and TGF-ß] were compared. A one-way ANOVA and an unpaired, two-tailed Student's t-test was used to determine the statistical significance. RESULTS: NOX2 expression was significantly increased after Aspergillus fumigatus injection in corneas and that this increase could be reduced by treatment with DPI. DPI treatment produced more severe inflammation and resulted in higher clinical scores, more neutrophils infiltration, a weakened ability to clear fungi, the release of fewer ROS and the formation of neutrophil extracellular traps. Treatment with DPI increased the expression of the proinflammatory cytokines NF-κB, IL-17A, IL-6, and TNF-α and decreased the expression of the anti-inflammatory cytokines Nrf2, IL-10 and TGF-ß compared to their expression levels without DPI treatment. CONCLUSION: NOX2 plays an important role against Aspergillus fumigatus in the mouse cornea through killing fungi and limiting the degree of inflammation.

[Mechanism of the immune response to keratomycosis].

Immune system played an important role in recognition and elimination of fungal pathogens. The balance between host factors and fungal pathogens can influence the prognosis of disease. As located on the surface of the eyeball. It was easy for cornea to be attracted by pathogens when traumatic injury occur. Moreover, cornea had characters of transparency and vascularization, which keep it on immune privilege situation relatively. The progress in the study of the mechanism of immunity participating in the keratomycosis and ways of fungus escaped from the attract of immune system was reviewed.

[Expression of substance P in experimental fungal keratitis].

OBJECTIVE: To detect the change process of substance P (SP)expression and the difference between fungus and bacterial keratitis and explore effects of SP on the damage and repair process of keratitis model of Wistar rats. METHODS: Wistar rats were divided randomly as blank controlled group, fungal keratitis group and bacterial keratitis group. Inoculate fusarium and staphylococcus into cornea of Wistar rats to make animal keratitis model. Choose the right eye as experimental eye and the left eye as control. After the model was created successfully, 25 rats were executed randomly at 1, 5, 8, 10 and 14 days after the surgery. Detailed record and hematoxylin-eosin dye were done to each group to examine the process of ulcer. Expression of SP were detected through immunohistochemistry and reverse transcription polymerase chain reaction. Use variance analysis, T test and Q test to evaluate the results. RESULTS: One day after infection, the cornea of experimental group showed ulcer with disordered shallow matrix layer collagenous fibre arrangement and neutral granular cell infiltration; after 5 days, ulcer worsened and granular cells infiltrated through the whole layer; after 8 days, ulcer shrinked and fibroblast began to increase with new vessels formed; after 10 days, new vessels began to decrease; after 14 days, the matrix level textile fiber assumes scar type restructuring. The expression of SP increased in endothelium cells, inflammatory cells, fibroblast cells and endothelium of new vessels 1 d after surgery (absorbance for fusarium and staphylococcus group were 0.3313 ± 0.0133 and 0.3995 ± 0.0191 respectively; mRNA were 0.4525 ± 0.0170 and 0.5532 ± 0.0258), peaked at 8 d (for fusarium and staphylococcus group were 0.5525 ± 0.0171 and 0.7050 ± 0.0119 respectively; mRNA were 0.5975 ± 0.0221 and 0.7150 ± 0.0238), began to decrease at 10 d (for fusarium and staphylococcus group were 0.1533 ± 0.0176 and 0.3125 ± 0.0170 respectively; mRNA were 0.2416 ± 0.0082 and 0.4835 ± 0.0082) and dropped to lower than normal at 14 d (for fusarium and staphylococcus group were 0.1150 ± 0.0128 and 0.1675 ± 0.0126 respectively; mRNA were 0.1275 ± 0.0126 and 0.2325 ± 0.0171), which had significant difference (the absorbance of SP: for fusarium group, F = 832.24, q = 35.3675, 12.9044, 27.3621, 34.6506, 22.4632, 62.7296, 70.0182, 40.2664, 47.5550, 7.2886, P < 0.01;for staphylococcus group F = 636.17, q = 38.7494, 11.4245, 10.8298, 28.6082, 27.3249, 49.5791, 67.3575, 22.2543, 40.0327, 17.7784, P < 0.01. mRNA expression: for fusarium group F = 658.60, q = 18.9941, 9.5132, 27.4719, 42.0007, 9.4809, 46.4661, 60.9948, 36.9852, 51.5139, 14.5287, P < 0.01; for staphylococcus group F = 335.13, q = 16.9266, 4.1677, 7.1081, 32.4724, 12.7589, 24.0347, 49.3990, 11.2759, 36.6402, 25.3643, P < 0.01). Two experimental groups showed similar changes as time changes, the bacterial group showed more SP expression than fungal group, which had significant difference (absorbance of SP t = 6.5493, 7.3867, 16.0505, 14.5479, 6.5360, P < 0.05; mRNA expression t = 7.2878, 9.5232, 8.8149, 43.6256, 11.1269, P < 0.05). Controlled eyes showed increased SP expression at 1 d (absorbance was 0.2840 ± 0.0212; mRNA was 0.3950 ± 0.0129) and dropped to normal at 5 d (absorbance was 0.2125 ± 0.0174; mRNA was 0.3321 ± 0.0041). There was significant difference between bacterial group, fungal group and controlled eyes (absorbance of SP: compared with fusarium group t = 4.2261, 18.3314, 35.5163, 5.3609, 13.4826; compared with staphylococcus group t = 9.0508, 25.6639, 63.4924, 7.4828, 7.1301, P < 0.05. mRNA expression: compared with fusarium group t = 6.0249, 31.9158, 26.0413, 9.1550, 19.1741; compared with staphylococcus group t = 12.2636, 53.4404, 36.8727, 15.8687, 8.2939, P < 0.05). CONCLUSION: SP possibly participated in the early time damage and the later period repair process in fungus keratitis and its lower expression level possibly participated in the mechanism of lighter ache in fungus keratitis.

[Clinical observation of visual quality after implantation of toric intraocular lens].

OBJECTIVE: To observe the visual quality after implantation of Acrysof toric intraocular lens (IOL) in cataract patients. METHODS: Eighty eyes (60 patients) had implantation of Acrysof toric IOL in our hospital between Oct. 2009 and Sep. 2010. The patients were divided into four groups according to the toric models (T3, T4 and T5). The uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), residual cylinders, contrast sensitivity, high order aberration and IOL axis were evaluated preoperatively and 3 months postoperatively. Sixty-five eyes (45 patients) had implantation of Acrysof Natural IOL. RESULTS: The UCVA improved in the toric groups three months postoperatively. The UCVA of group T5b was lower than that of the other three groups. The UCVA of toric group was (0.19 ± 0.14) which was better than that of the control group (t = 4.04, P < 0.05). The residual cylinder was (1.69 ± 0.68) D in group T5b, which was statistically different from the other three groups. The residual cylinder of the toric group was (0.51 ± 0.78) D, which was much lower than the control group (t = 2.54, P < 0.05). The difference of contrast sensitivity between the four toric groups was not statistically significant. The low-order aberration and coma of group T5b was higher than that of the other three groups. The difference of contrast sensitivity between the toric group and the control group was not statistically significant. The low-order aberration and coma of the toric group were lower than those of the control group. The IOLs in 67 eyes (83.75%) rotated less than 5 degrees and all IOLs rotated less than 10 degrees. CONCLUSION: Implantation of the AcrySof toric IOL proves to be an effective, safe, and stable method of managing corneal astigmatism in cataract patients and provides good visual quality.

[Research of factors related to invasion and metastasis in choroidal melanoma].

OBJECTIVE: To investigate the expression of factors related to invasion and metastasis in choroidal melanoma and to determine their relationships with malignant features. METHODS: The expression of Connexin43 (Cx43), epithelial cadherin (E-cadherin), phosphatidylinositol 3-kinase (PI3K) and connective tissue growth factor (CTGF) in choroidal melanoma and nevi were detected by immunohistochemistry, and the correlation between these factors and clinicopathological features were analyzed. RESULTS: Positive rates of Cx43, E-cadherin, PI3K and CTGF were 74.07% (20/27), 44.4% (12/27), 74.07% (20/27) and 66.67% (18/27) in choroidal melanoma tissues, respectively; and 33.33% (5/15), 86.67% (13/15), 33.33% (5/15) and 20.00% (3/15) in the nevi tissues, respectively. There were significant differences in the expression of these markers between the two groups (χ(2) = 5.060, P = 0.024; χ(2) = 5.490, P = 0.019; χ(2) = 5.060, P = 0.024; χ(2) = 6.637, P = 0.010). The expression rates of Cx43 protein were 40% (4/10), 88.89% (8/9) and 100% (8/8) in spindle, mixed and epithelioid cell type, respectively. The expression of these data was related to histological type (χ(2) = 9.874, P = 0.007). The expression rates of PI3K protein were 42.86% (3/7), 75% (9/12) and 100% (8/8), in small, medium and large tumors, respectively, and their expression were co-related to the tumor size (χ(2) = 6.357, P = 0.042). Positive rates of Cx43, E-cadherin, PI3K and CTGF were 50% (6/12), 83.33% (10/12), 50% (6/12) and 41.66% (5/12), respectively, in choroidal melanoma tissues without sclera invasion and were 93.33% (14/15), 40% (6/15), 93.33% (14/15) and 86.67% (13/15), respectively, in choroidal melanoma tissues with violation involved the sclera. There were significant differences of the expression of these markers between the two groups (χ(2) = 4.457, P = 0.016; χ(2) = 3.546, P = 0.028; χ(2) = 4.457, P = 0.016; χ(2) = 4.218, P = 0.019). CONCLUSION: Increased expression of Cx43, PI3K and CTGF and decreased expression of E-cadherin are involved in the processes of invasion and metastasis of choroidal melanoma.

[Substance P and corneal wound healing].

Cornea is one of the sites that have many nerve fiber terminals which play critical roles in maintaining normal cornea functions and the wound healing of cornea. SP is one of the neurotransmitters released by cornea nerve fiber and is very important in signal transduction and feedback control of the cornea. The relationship between SP and cornea wound healing has been discussed.

Anti-inflammatory effects of astaxanthin against fungal keratitis.

AIM: To characterize effect of astaxanthin (ASX) in Aspergillus fumigatus (A. fumigatus) induced keratitis in mouse model. METHODS: In vivo, fungal keratitis mouse model was established in C57BL/6 mice using A. fumigatus, followed by ASX or dimethyl sulfoxide (DMSO) treatment. Clinical responses were evaluated by clinical score and myeloperoxidase (MPO) assay. Inflammatory cytokines were assessed by reverse-transcription polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, and enzyme-linked immuno sorbent assay (ELISA). RESULTS: In animal model, ASX improved corneal transparency and clinical response, suppressed the expression of inflammatory cytokine like IL-1ß, TNF-α, and HMGB-1. Neutrophil levels have been shown to decrease in ASX-treated cornea by immunofluorescence and MPO. TLR2 and TLR4 levels were lower in ASX-treated group than DMSO-treated. CONCLUSION: ASX can suppress inflammatory response and reduce inflammatory cytokine production in mice model with A. fumigatus keratitis.

Expression and role of aryl hydrocarbon receptor in Aspergillus fumigatus keratitis.

AIM: To observe the expression and role of aryl hydrocarbon receptor (AhR) in the immune response of mouse cornea infected with Aspergillus fumigatus (A. fumigatus). METHODS: Murine models of A. fumigatus keratitis were established by scraping the central epithelium of mouse cornea, daubing A. fumigatus on the cornea and covering with a contact lens. The mice were randomly divided into the control group and the A. fumigatus-infected (A.F.) group for 1, 3 and 5d respectively, which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection. In this study, immunofluorescence staining was used to detect the expression and localization of AhR in mouse corneas, and the mRNA and protein of AhR were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. In addition, mouse peritoneal macrophages were stimulated by A. fumigatus with or without the pretreatment of AhR antagonist CH223191 and AhR agonist FICZ, and the tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and Arg-1 mRNA were detected by RT-PCR. RESULTS: According to the results of the slit light photography, it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3rd day after the infection of A. fumigatus. Contrasted with the control group, the expression of AhR in the corneal epithelial cells infected with A. fumigatus was significantly increased detected by immunofluorescence staining. AhR mainly expressed in the nucleus and cytoplasm of corneal epithelial cells. Consistent with the transcriptional level of AhR mRNA, the expression level of AhR protein reached the peak on the 3rd day after infection which was detected by Western blot. Furthermore, RT-PCR showed that CH223191 up-regulated the expression of TNF-α and iNOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages; inversely, FICZ reduced the expression of TNF-α and iNOS while elevated the expression of IL-10 and Arg-1. CONCLUSION: AhR is involved in the pathogenesis of A. fumigatus keratitis and induced immune protection in anti-A. fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.

Regulation of LOX-1 on adhesion molecules and neutrophil infiltration in mouse Aspergillus fumigatus keratitis.

AIM: To determine whether lectin-like ox-LDL receptor (LOX-1) regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus (A. fumigatus) keratitis of C57BL/6 mice. METHODS: C57BL/6 mice were pretreated with a neutralizing antibody to LOX-1 (5 µg/5 µL) or control nonspecific IgG (5 µg/5 µL), LOX-1 inhibitor Poly-I (2 µg/5 µL) or PBS by subconjunctival injection. Fungal keratitis (FK) mouse models of C57BL/6 mice were established by scraping corneal central epithelium, smearing A. fumigatus on the corneal surface and covering the eye with contact lenses. The corneal response to infection was assessed via clinical score. The mRNA levels of the adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin were tested in control and infected corneas by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of ICAM-1 were evaluated by immunofluorescence (IF) and Western blot. Neutrophils were extracted from the abdominal cavity of C57BL/6 mice followed by pretreatment using antibody to LOX-1 (10 µg/mL) or control nonspecific IgG (10 µg/mL), the Poly-I (4 µg/mL) or PBS. The cells were then stimulated with A. fumigatus and tested mRNA and protein levels of lymphocyte function-associated antigen-1 (LFA-1) using RT-PCR and Western blot. IF and myeloperoxidase (MPO) assays were used to assess neutrophil infiltration in mice corneas. RESULTS: Pretreatment of LOX-1 antibody or the Poly-I reduced the degree of inflammation of cornea and decreased the clinical FK score compared with pretreatment of IgG or PBS (both P<0.01). And these pretreatment also displayed an obvious decline in the mRNA levels of ICAM-1, VCAM-1, P-selectin, E-selectin and LFA-1 expression compared with control groups (all P<0.01). Furthermore, pretreated with LOX-1 antibody or Poly-I, the protein levels of ICAM-1 and LFA-1 also decreased compared with control groups (all P<0.05). Neutrophil infiltration in the cornea was significantly reduced after pretreatment of LOX-1 antibody or Poly-I compared with control groups by IF and MPO assays (both P<0.01). CONCLUSION: Inhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A. fumigatus infected corneas of C57BL/6 mice.

The anti-inflammatory regulation of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis.

AIM: To analyze the impact of calcitonin gene-related peptide (CGRP) in mouse keratitis after Aspergillus fumigatus (A. fumigatus) infection. METHODS: C57BL/6 mice were treated subconjunctivally with different concentrations of exogenous CGRP, and BALB/c mice were treated with CGRP8-37 (a CGRP antagonist) before corneas were infected with A. fumigatus. The cornea was assessed under the slit-lamp and the clinical score was recorded. The mRNA levels of IL-1ß, TNF-α, IL-6, and MIP-2 were detected by quantitative real-time polymerase chain reaction (PCR), while the protein level of IL-1ß was determined by Western blotting. In vitro, RAW264.7 cells were used to investigate NLRP3 and IL-1ß expression induced by A. fumigatus after the pretreatment of exogenous CGRP or CGRP8-37. Cytokines expression in RAW264.7 cells was evaluated by real-time PCR and Western blotting. RESULTS: Using exogenous CGRP resulted in down-regulated synthesis of IL-1ß and MIP-2 stimulated by A. fumigatus in C57BL/6 mice keratitis, and the synthesis of IL-1ß, MIP-2 and IL-6 was up-regulated in BALB/c mice corneas after the pretreatment with CGRP8-37. Pretreatment with exogenous CGRP and CGRP8-37 did not influence TNF-α mRNA levels either in BALB/c or C57BL/6 mice keratitis. The levels of NLRP3 and IL-1ß were both reduced in A. fumigatus stimulated-macrophages after treatment with exogenous CGRP. And CGRP8-37 pretreatment would increase NLRP3 and IL-1ß levels. CONCLUSION: CGRP may alleviate the inflammatory reaction in mice keratitis after infection with A. fumigatus. The anti-inflammatory effect may be related to the inhibition of NLRP3 expression by CGRP.

Changes in corneal innervation and pain responses in fungal keratitis.

AIM: To characterize changes in the cornea nerve and pain responses in fungal keratitis (FK). METHODS: A retrospective analysis of in vivo confocal microscopy images of 11 FK corneas was performed, and the results were compared with those for 11 normal corneas. Subbasal corneal nerves were analyzed for total nerve number, main nerve trunk number, branching patterns and tortuosity. C57BL/6 mice were infected with Aspergillus fumigatus. Disease severity was determined through clinical scoring and slit lamp photography. Corneas were harvested at 1, 3, 5, and 7d post infection (p.i.) and assessed for ß III tubulin. Corneal mechanical sensitivity thresholds were detected by von Frey test. ß-endorphin (ß-EP) and µ receptor protein expression was detected through Western blotting. RESULTS: Total nerve number, main nerve trunk number, and nerve branching were significantly lower in FK patients than in controls, but tortuosity was not significantly different. In infected mice, subbasal nerve density decreased from 1d p.i., reaching a minimum at 5d p.i. Clinical scores rose at 1d p.i., peaked at 3d p.i., and decreased at 5d p.i. Mechanical sensitivity thresholds showed the same trends. ß-EP and µ receptor protein expression increased after infection. CONCLUSION: Corneal nerve density is lower in FK patients and Aspergillus fumigatus-infected mice than in controls. Pain sensitivity decreases with postinfection corneal ulcer aggravation. ß-EP and µ receptor proteins are both upregulated in infected mouse corneas.

High-mobility group box1 as an amplifier of immune response and target for treatment in Aspergillus fumigatus keratitis.

AIM: To determine the roles of high-mobility group box1 (HMGB1) in pro-inflammation, host immune response and its potential target for treatment in Aspergillus fumigatus (A.fumigatus) keratitis. METHODS: Expression of HMGB1 was tested in C57BL/6 normal and infected corneas. Dual immunostaining tested co-expression of HMGB1 with TLR4 or LOX-1. C57BL/6 mice were pretreated with Box A or PBS and then infected. Clinical scores, polymerase chain reaction, ELISA, and MPO assay were used to assess the disease response. Flow cytometry were used to test the effect of Box A on reactive oxygen species (ROS) expression after A.fumigatus stimulation in polymorphonuclear neutrophilic leukocytes (PMN). C57BL/6 peritoneal macrophages were pretreated with Box B before A.fumigatus stimulation, and MIP-2, IL-1ß, TNF-α, HMGB1 and LOX-1 were measured. Macrophages were pretreated with Box B or Box B combined with Poly(I) (an inhibitor of LOX-1) before stimulating with A.fumigatus, and MIP-2, IL-1ß, TNF-α, LOX-1, p38-MAPK, p-p38-MAPK were measured. RESULTS: HMGB1 levels were elevated in C57BL/6 mice after infection. HMGB1 co-expressed with TLR4, and LOX-1 in infiltrated cells. Box A vs PBS treated C57BL/6 mice had lower clinical scores and down-regulated corneal HMGB1, MIP-2, IL-1ß expression and neutrophil influx. Box B treatment amplified expression of MIP-2, IL-1ß, TNF-α, HMGB1 and LOX-1 that induced by A.fumigatus in macrophage. Compared to the treatment of Box B only, the protein expression of IL-1ß, TNF-α showed inhibition of Box B combined with Poly(I), which also reduced the A.fumigatus-evoked protein level of LOX-1 and phosphorylation level of p38-MAPK. The production of A.fumigatus-stimulated ROS was significantly declined after Box A pretreatment in PMN. CONCLUSION: Blocking HMGB1 reduces the disease response in C57BL/6 mice. HMGB1 can amplify the host immune response through p38-MAPK, and is a target for treatment of A.fumigatus keratitis.

Clinical effects of three types of silicone intubations in repairing lacerations of canaliculus.

OBJECTIVE: To evaluate the clinical effects of one-passage, double-passage and circular canalicular intubations in repairing lacerations of canaliculus. METHODS: A total of 109 eyes in 109 cases of canalicular laceration were repaired with three types of silicone intubations, among which 23 with one-passage canalicular intubation, 51 with double-passage canalicular intubation, and 35 with circular canalicular intubation. The average follow-up period was 12-15 months. RESULTS: The wound/junction of the lacrimal canaliculi was ruptured in 5 cases (9.80%) of the double-passage group, 3 cases (8.57%) of the circular group, and 8 cases (34.78%) of the one-passage group. The rupture incidence of the one-passage group was significantly higher than that of the other two groups (X(2) equal to 9.416, P less than 0.01). During the intubation, canaliculitis was observed in 12 cases (23.53%) of the double-passage group, while only 3 cases (8.57%) in the circular group and 8 cases (34.78%) in the one-passage group. The circular group had significantly lower incidence of canaliculitis than the other two groups (X(2) equal to 6.095, P less than 0.05). After extubation 6 months after laceration repair, the lacrimal passage remained patent with canalicular irrigation in 46 cases (90.20%) in the double-passage group, 30 cases (85.71%) in the circular group and 15 cases (65.22%) in the one-passage group. Six months after surgery, the canalicular patency in the one-passage group was significantly lower than that of the other two groups (X(2) equal to 7.390, P less than 0.05). CONCLUSIONS: Circular canalicular intubation is more stable and has less surgical complications than the double-passage and one-passage canalicular intubations. It is also more effective clinically 12-15 months after laceration surgery.

Efficiency and therapeutic effect of modified pigtail probe in anastomosing lacerated lacrimal canaliculus.

OBJECTIVE: To investigate the necessity of modification to the traditional pigtail probe and evaluate its efficiency and therapeutic effect in searching the nasal cut ends and anastomosing the lacerated lacrimal canaliculus. METHODS: Eighty-seven patients (including 87 eyes) suffering from canalicular laceration were randomized into two groups: 41 patients treated with traditional pigtail probes (Group A) and 46 with modified pigtail probes (Group B). During the reconstruction of the lacerated canaliculi, the traditional pigtail probe and the modified pigtail probe were used respectively to seek for the nasal cut ends of lacerated lacrimal canaliculi. Peripherally inserted central catheter (PICCTM) silicone tube with diameter of 0.95 mm was intubated as a stent for 4-6 months. The surgical outcomes were retrospectively analyzed after stent removal. RESULTS: In Group B, the primary success rate of searching the nasal cut ends of lacerated lacrimal canaliculi was 93.48% (43/46) and the final success rate was 97.83% (45/46). No false passage formed in Group B. Statistical significance was found between Group A and Group B as the primary success rates of searching the nasal cut ends (X(2) equal to 10.522, P less than 0.01) and the false passage forming rates were concerned (X(2)) equal to 4.704, P less than 0.05), whereas no significance was found between the two groups as the final success rates were concerned (X(2) equal to 0.007, P larger than 0.05). The mean time of searching the nasal cut ends of lacerated lacrimal canaliculi in Group B was (5.02+/-0.73) minutes and the mean time of operation was (33.90+/-4.84) minutes, and both were significantly shorter than those of Group A (t(1) equal to 9.779, t(2) equal to 10.700, P less than 0.01). The cure rate of Group B was 95.65%, though higher than that of Group A, no statistical significance was found (Z equal to -0.007, P larger than 0.05). Totally, 2 patients (2.30%) were found to be absent of common canaliculus and underwent bicanalicular nasal intubation in the two groups. CONCLUSIONS: Pigtail probes are efficient and convenient apparatus for searching the nasal cut ends of the lacerated lacrimal canaliculi in the reconstruction of canalicular laceration. Necessary or proper modifications to the pigtail probes can minimize the risk of iatrogenic damages or complications and enhance the efficiency and therapeutic effect of canalicular repair.

Cloning, expression and functional analyses of human platelet-derived growth factor-B chain peptide for wound repair of cat corneal endothelial cells.

OBJECTIVE: To investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application. METHODS: Total RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively. RESULTS: PDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells. CONCLUSIONS: The construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.

[The effect of rapamycin liposome gutta inhibiting rat corneal neovascularization].

OBJECTIVE: To explore the inhibiting effect of rapamycin (RAPA) on corneal neovascularization (CNV) of rats and the functional mechanism. METHODS: A design group was adopted. 102 Wistar rats were divided into four groups at random, including rapamycin liposome treated group (24 rats), the rapamycin solved in bean oil treated group (24 rats), blank liposome treated group (24 rats), blank treated group (24 rats) and normal control group (6 rats). All right eyes of 96 rats were induced by alkali cauterization. Rapamycin liposome were prepared by thin film hydration and the major factors were studied by the method of orthogonal design. After alkali burn, cauterized rats were observed by slitlamp biomicroscope every day. On the 1st, 4th, 7th, 14th days after operation, the expression of HIF-1alpha and VEGF were examined by immunohistochemical method and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The analysis of variance and q test groups for analysis were adopted to analyze the results. RESULTS: (1) The bodies of RAPA liposome were intact kinds of spheres, the average diameter was 145.2 nm, and the envelopment rate was 90.02%. (2) After the burn of 14 d, CNV area of B, C, D and E group were (28.289 +/- 0.703), (28.005 +/- 0.801), (20.002 +/- 1.005) and (22.300 +/- 0.853) mm(2) (F = 159.62, P < 0.05). The CNV of both the rapamycin liposome treated group and the rapamycin solved in bean oil group grew slowly and smaller than that of blank liposome treated group and blank treated group (q = 47.80, 46.20, 34.60, 32.90;P = 0.00). While the rapamycin liposome treated group changed more obviously than the rapamycin solved in bean oil group (q = 13.20, P = 0.00). After alkali burn, the expression of HIF-1alpha and VEGF increased dramatically, meanwhile the expression of HIF-1alpha and VEGF were significantly decreased by RAPA. CONCLUSIONS: Liposome body is an excellent medicine carrier for the RAPA. RAPA can obviously suppress the growth of CNV. The possibly mechanism is weakening VEGF expression by inhibiting the transcription factor HIF-1alpha.