IGF-1-Mediated Survival from Induced Death of Human Primary Cultured Retinal Pigment Epithelial Cells Is Mediated by an Akt-Dependent Signaling Pathway.

Degeneration of the human retinal pigmented epithelium (hRPE) is involved in several eye disorders such as age-related macular degeneration (AMD). In this study, we investigated the protective effect of IGF-1 on human primary cultured RPE cells and its underlying mechanism. IGF-1 dose- and time-dependently promoted the survival of RPE cells from serum deprivation. Western blot showed that IGF-1 stimulated the activation of the PI3K/Akt and MAPK pathways in hRPE. Inhibition of the PI3K/Akt pathway by the PI3K-specific inhibitor, LY294002 or inhibition of Akt by Akt-specific inhibitors Akt inhibitor VIII or SN-38, or downregulation Akt with siRNA specific for Akt blocked the effect of IGF-1 on hRPE. In contrast, blockade of the MAPK pathway with a specific inhibitor PD98059 had no effect. Interestingly, vitreous IGF-1 injection reversed the inhibitory effect of light exposure (a dry AMD model) on both a wave and b wave. Immunocytochemistry showed that vitreous IGF-1 injections promoted the survival of RPE cells in rat retina and the expression of RPE65 in RPE cells from light injury. These results indicate that IGF-1 is able to protect hRPE cell from different insults in vivo and in vitro. Further detailed studies may lead the way to a therapeutic intervention for retinal diseases in which cell death is an underlying contributory mechanism.

Effects of different concentrations of tetramethylpyrazine, an active constituent of Chinese herb, on human corneal epithelial cell damaged by hydrogen peroxide.

AIM: To discuss the effects of different concentrations of tetramethylpyrazine (TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell (SDHCEC) induced by hydrogen peroxide. METHODS: We detected the combined effects of TMP with concentrations ranging from 4 mg/mL to 0.03 mg/mL and 800 µM hydrogen peroxide on SDHCEC. The methyl thiazolyl tetrazolium (MTT) assay was processed at 3, 6 and 12h separately while the detection of cell apoptosis at 6h only by flow cytometry. RESULTS: The viability of SDHCEC with 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL TMP joint with 800 µM hydrogen peroxide at 3h and 6h was significantly higher than that with 800 µM hydrogen peroxide only, P<0.05. However, except 0.25 mg/mL, TMP with other concentrations joint with 800 µM hydrogen peroxide at 12h could not significantly inhibit decreased SDHCEC viability induced by 800 µM hydrogen peroxide. At 12h, TMP of 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL could significantly inhibit SDHCEC early apoptosis induced by 800 µM hydrogen peroxide, most remarkable at 0.25 mg/mL TMP, P<0.05. CONCLUSION: Our results suggested that hydrogen peroxide can induce apoptosis related damage to SDHCEC. TMP can protect SDHCEC from the damage, and the protective effects may be associated with its anti-apoptosis mechanism.

Establishment of the mild, moderate and severe dry eye models using three methods in rabbits.

BACKGROUND: Dry eye (DE) is a common eye disease, and appropriate animal models are essential to explore the pathogenesis and therapy of DE. In this study, we aimed to establish rabbit models by three methods. METHODS: In group A, the lacrimal gland, Harderian gland, and nictitating membrane of the left eyes were surgically removed. In group B, the bulbar conjunctiva of the left eyes was burned with 50% trichloroacetic acid. In group C, both methods above were used. The right eyes served as normal controls. The Schirmer I test (SIt), fluorescein staining, and impression cytology were evaluated at baseline and on days 28, 42, and 56. RESULTS: Both the SIt and goblet cell density were significantly lower in operated eyes compared to the control eyes, while the corneal fluorescein staining scores in the operated eyes were significantly higher than in the control eyes on days 28, 42, and 56 (p < 0.05, p < 0.01 or p < 0.001). The SIt and goblet cell densities in groups B and C were significantly lower than group A on days 28, 42, and 56 (p < 0.05, p < 0.01 or p < 0.001). In addition, the corneal fluorescein staining scores in group C were significantly higher than either group A or group B on days 28, 42, and 56, while fluorescein staining scores were higher in group B than group A on days 42 and 56 days (p < 0.05, p < 0.01 or p < 0.001), with mean score 3.8 ± 1.30 (group A), 7.4 ± 1.14 (group B) and 10.8 ± 1.30 (group C) on day 56. CONCLUSIONS: Results suggest that three separate DE models, with mild, moderate, and severe manifestations of DE, could be stably established, in which conjunctival goblet cells took an important role.

Comparison of the Schirmer I test with and without topical anesthesia for diagnosing dry eye.

AIM: To determine the value of Schirmer I test (S I t) without anesthesia and with topical anesthesia for diagnosing dry eye (DE). METHODS: Totally 220 eyes in 110 patients, male (44) and female (66), (39.56±12.67) years old diagnosed with DE were examined. S I t without anesthesia was performed firstly, and 15 minutes later, it was applied again in the same person after topical anesthesia with 0.5% proparacaine hydrochloride eye drops. The wetting strips counted <10mm per 5 minutes were defined positive, while ≤5mm per 5 minutes were defined strong positive. RESULTS: The wetting length in S I t after topical anesthesia was significantly lower than that in S I t without anesthesia (P<0.001). The positive rate and strong positive rate of S I t after topical anesthesia were significantly higher than that of S I t without anesthesia (P<0.001). The positive rate and strong positive rate of S I t without anesthesia and the strong positive rate of S I t after topical anesthesia in patients with aqueous-deficiency dry eye (ADDE) were significantly higher than those in total patients whereas those in patients with evaporative dry eye (EDE) were significantly lower than those in total patients (P<0.001). CONCLUSION: S I t after topical anesthesia with 0.5% proparacaine hydrochloride eye drops is more objective and reliable than that without anesthesia in reflecting the status of DE, and its diagnostic value in patients with ADDE was even higher, making itself a meaningful evidence for the diagnosis and treatment of DE.

Protective effects and mechanism of tetramethylpyrazine against lens opacification induced by sodium selenite in rats.

Tetramethylpyrazine (TMP), extracted from the Chinese herbal medicine Ligusticum wallichii franchat (chuan xiong in Chinese), is a potent anti-free radical and calcium antagonist. Correspondingly, two important hypotheses in the causation of cataracts are free radical toxicity and calcium ion overload. In this study we investigated the effect of TMP on lens opacification induced by sodium selenite in rats, addressing the potential of TMP eye drops to prevent and treat cataracts. Results showed that the extent of lens opacification in the untreated Normal Control group (NC group) was significantly less than that of selenite-injected untreated rats (MC group) on days 3, 5, 7 and 10 (p < 0.001), while TMP treated selenite-injected rats (TMP group) had less lens opacification than the MC group on days 3, 5, 7 and 10 (p < 0.05). Compared with the NC group, the MC group had significantly decreased activity of super-oxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) and significantly elevated malondialdehyde (MDA) and calcium ion content (p < 0.001). Compared with the MC group, the activity of (SOD), (GSH-PX) and (CAT) were significantly higher while (MDA) and calcium ion levels were significantly lower in the TMP group at all time points (p < 0.01). The findings demonstrate that the selenite-induced cataract rat models were successfully built and the TMP eye drops can delay lens opacification induced by sodium selenite in rats. The mechanism by which TMP preserves lens transparency from selenite treated animals is associated with the lenses' ability to maintain normal levels of activity of SOD, GSH-PX and CAT and normal concentrations of MDA and calcium ion.

Ganoderma spore lipid inhibits N-methyl-N-nitrosourea-induced retinal photoreceptor apoptosis in vivo.

The purpose of the present study was to investigate the effect of Ganoderma spores lipid (GSL) on Bax, Bcl-xl and Caspase-3 expression in damaged retina and to address the effect of GSL on photoreceptor cell lesions induced by N-methyl-N-nitrosourea (MNU). Thirty 50-day-old female Sprague-Dawley rats were divided randomly into five groups to detect the dose-response effect of GSL by electroretinogram (ERG) analysis. Four groups received different daily GSL doses (0.5, 1, 2 and 4 g/kg, respectively) and one control group received no treatment. Sixty rats were divided randomly into an untreated MNU model control group and the GSL treatment group. Retina tissue samples were obtained sequentially 0 h before and 1, 3, 7 and 10 d after MNU injection. Expressions of Bax, Bcl-xl and Caspase-3 were detected by RT-PCR and immunofluorescence assays, then photoreceptor cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) signals. GSL had a dose-response effect on retina ERG and reversed retinal photoreceptor damage induced by MNU. RT-PCR analysis demonstrated that transcription levels of Bax, Bcl-xl and Caspase-3 in MNU control group and GSL treatment group were all upregulated on 1 d (p < 0.01) and peaked on 3 d (p < 0.01) after MNU injection compared to before MNU injection. GSL treatment significantly decreased mRNA levels of Bax on 1, 3, 7 and 10 d vs. MNU group (all p < 0.01) and mRNA levels of Caspase-3 on 1, 3, 7 d (p < 0.01) and 10 d (p < 0.05) vs. MNU group. Bcl-xl was clearly upregulation on 1, 3, 7 and 10 d vs. MNU group (all p < 0.01). Expression ratios of Bax/Bcl-xl were raised after MNU injection on 1 and 3 d vs. 0 h before MNU (both p < 0.01), peaked on 3 d, then dropped after GSL treatment on 1, 3, 7 and 10 d vs. MNU group (all p < 0.01). Immunofluorescence assays showed GSL decreased Bax and Caspase-3 positive staining levels in retina and increased Bcl-xl level. TUNEL-positive cells were evoked only in the outer nuclear layer and peaked on 3 d in rats receiving MNU (p < 0.01 vs. 0 h before MNU). GSL administration decreased apoptosis levels significantly, and the apoptotic indexes (AIs) of the GSL group were lower than those of MNU group on 1 and 3 d (both p < 0.01). Taken together, these data suggest that GSL may regulate the expressions of Bax, Bcl-xl and Caspases-3, inhibiting MNU-induced rat photoreceptor cell apoptosis and protecting retinal function.

[Aqueous humor penetration of ligustrazine hydrochloride eye drops after topical administration in rabbit eyes].

PURPOSE: To study the aqueous humor penetration of ligustrazine hydrochloride after ocular administration in rabbits. METHODS: Eighteen rabbits were randomly divided into 6 groups. The aqueous humor was collected at 5, 15, 30, 60, 120 and 180 minutes following topical administration of ligustrazine hydrochloride eye drops and the concentration of ligustrazine hydrochloride was determined by HPLC. Analytical column was Diamonsil C18 stainless steel column (250 mm x 4.6 mm, 5 microm); The mobile phase was methanol: water(62:38); Flow rate was 0.9 ml/min; The detection wavelength was 280 nm. RESULTS: The concentrations of ligustrazine hydrochloride in aqueous humor were (15.785 +/- 2.988) microg/mL, (11.900 +/- 1.743) microg/mL, (8.286 +/- 1.182) microg/mL, (2.745 +/- 0.807) microg/mL, (0.379 +/- 0.091) microg/mL, (0.049 +/- 0.038) microg/mL, respectively, after a time interval of 5 min, 15 min, 30 min, 60 min, 120 min, 180 min. The maximum concentration was 15.785 microg/mL at 5 min, and then the concentration of ligustrazine hydrochloride gradually decreased and declined to a lower point at 180 min. CONCLUSION: The penetration of ligustrazine hydrochloride in aqueous humor after ocular administration was good. The results provided experimental data for the management of eye diseases with ligustrazine hydrochloride by topical administration.

[Pharmacokinetics of puerarin in the aqueous humor and vitreous of rabbit eye following systemic administration].

PURPOSE: To study dynamic changes of puerarin in aqueous humor and vitreous humor of rabbit eye following a single dose intraperitoneal injection. METHODS: Rabbits were randomly divided into 11 groups. Puerarin (80 mg/kg) was given by a single dose intraperitoneal injection. After the administration, the aqueous humor, vitreous were harvested at different time points and the concentration of puerarin was determined by reversed phase high performance liquid chromatography (RP-HPLC). RESULTS: The concentration-time data of puerarin in aqueous humor, vitreous was subject to two-compartment open model. The pharmacokinetic parameters were separately as following. Cmax = 1.61, 0.09 microg/ml. tmax = 1.68, 1.81 h. t1/2alpha = 1.36, 1.05 h. t1/2beta = 19.72, 15.18 h. CL = 2.17, 12.43 L/h. The practical data of the concentration of puerarin in aqueous humor and vitreous humor that were detected was (0.78 +/- 0.29) microg/ml, (0.06 +/- 0.02) microg/ml at 30 min respectively and reach maximum concentration was (2.32 +/- 0.15) microg/ml, (0.12 +/- 0.04) microg/ml at 2 h. Then the concentration of puerarin in aqueous humor and vitreous humor was gradually decreased. The concentration of puerarin was 0.57 microg/ml in the aqueous humor, 0.05 microg/ml in the vitreous humor at 6 h. The concentration of puerarin declined to a lower point 0.03 microg/ml or below in aqueous humor and vitreous humor after 16 h. CONCLUSIONS: Using HPLC method to detect puerarin in the aqueous humor and vitreous humor was particular, accurate and sensitive. The puerarin can penetrate blood-ocular barrier to enter aqueous humor and vitreous humor. The concentration of puerarin in the aqueous humor was obviously more than that in the vitreous humor. The quantitation of puerarin entering vitreous humor was limited. The clearance of the compound were slower and half-life were longer in aqueous humor and vitreous, that can provide principle basis for treating eye diseases with puerarin by systemic administration.

[The toxic effect of N-methyl-N- nitrosourea on retina in rats].

PURPOSE: To observe the toxic effect of N-methyl-N-nitrosourea (MNU) on photoreceptor cells within retina of SD rats. METHODS: At 50 days of age, 100 female rats received a single intraperitoneal injection of MNU at different doses of 50 mg/kg, 60 mg/kg, 70 mg/kg and 80 mg/kg body weight, respectively. Each group had 6 rats and 4 untreated rats were used as normal control. At 24, 48 and 72 hours and 7 days after the administration of MNU, the animals were sacrificed and both eyes were enucleated immediately and processed for histological examination. RESULTS: It was found that all doses of MNU could sequentially damage the central and peripheral retina. The first evidence of retinapathy 24 hours after the application of MNU was pyknosis and disruption of photoreceptor cells nuclei and the disorientation of the photoreceptor outer segments; loss of photoreceptor cell deteriorated significantly at 48 hours or 72 hours; the outer nuclear layer and photoreceptor layer were almost completely lost at 7 days. CONCLUSION: The results demonstrated that MNU could selectively damage the photoreceptor cells in the retina of the rats, which was dose-dependent and time-dependent.