0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Laboratory Sciences |

Effect of Topical Ginkgo biloba Extract on Steroid-Induced Changes in the Trabecular Meshwork and Intraocular Pressure FREE

Li-Yun Jia, PhD; Ling Sun, BSc; Dorothy S. P. Fan, FRCS; Dennis S. C. Lam, MD; Chi Pui Pang, DPhil; Gary H. F. Yam, PhD
[+] Author Affiliations

Author Affiliations: Department of Ophthalmology and Visual Sciences, Chinese University of Hong Kong, Hong Kong (Drs Jia, Fan, Lam, Pang, and Yam and Ms Sun); Joint Shantou International Eye Center of Shantou University, Shantou, China (Ms Sun and Drs Lam and Pang).


Arch Ophthalmol. 2008;126(12):1700-1706. doi:10.1001/archophthalmol.2008.512.
Text Size: A A A
Published online

Objective  To study the effects of Ginkgo biloba extract (GBE) on dexamethasone (DEX)–induced ocular hypertension.

Methods  Rabbits aged 7 weeks received topical TobraDEX (Alcon Labs, Hünenberg, Switzerland) and/or 5 μg of GBE four times daily for 14 days. Intraocular pressure (IOP) was recorded every 3 days. After enucleation, trabecular meshwork (TM) cellularity and extracellular matrix deposition were graded. The effect of GBE on apoptosis and expression of myocilin and cell stress–related genes in DEX-treated human TM cells were studied by immunofluorescence, Western blotting, and quantitative polymerase chain reaction.

Results  Ginkgo biloba extract suppressed DEX-induced IOP elevation in rabbits. It reduced the DEX-associated accumulation of extracellular materials within the cribriform layers of the TM and achieved better TM cellularity. In cultured human TM cells, GBE substantially attenuated anti–Fas ligand–induced apoptosis and reduced DEX-induced myocilin expression. Ginkgo biloba extract modulated the expression of αB-crystallin and heat-shock proteins 70 and 90α but not other stress-related genes. Furthermore, changes associated with DEX were found less in GBE-treated or GBE-primed TM cells.

Conclusion  We showed that GBE, a nontoxic, antiapoptotic, herbal compound significantly suppressed steroid-induced IOP elevation in rabbits and it seems to prevent the adverse effects of DEX on TM cells.

Clinical Relevance  Ginkgo biloba extract could be a therapeutic agent or dietary supplement to prevent steroid-induced ocular hypertension.

Figures in this Article

Topical glucocorticoid is known as an effective treatment of ocular inflammation. Dexamethasone (DEX) inhibits cytokine and chemokine activity and activates neutrophils in the inflammatory process.1,2 However, undesirable induction of ocular hypertension leading to glaucoma may occur in some steroid-responsive subjects. Studies have shown that DEX influences trabecular meshwork (TM) activities, including turnover of cytoskeletal and extracellular matrix proteins, cell movement, adhesion, and morphology.35 Trabecular meshwork, a reticulated network of cell-lined extracellular matrix at the junction of the cornea and iris, regulates aqueous humor outflow.6 Improper functioning of TM could lead to the development of intraocular pressure (IOP)–related glaucoma. In patients with steroid-induced glaucoma, thickened trabeculae and accumulation of abnormal basement membrane–like material in the form of fingerprint-like whorls can be observed.611 Trabecular meshwork cells synthesize and release glycosaminoglycans, glycoproteins, and fibrillar materials12 and are active as phagocytes.13 Thus, the TM maintains the structural and functional integrity of aqueous drainage pathway. Disturbance of the phagocytic capacity of TM could be a pathogenic factor in IOP-associated glaucoma. Myocilin (MYOC) expression was also higher in eyes treated with corticosteroids.14,15 In vitro DEX treatment of human TM cells resulted in a time-dependent induction of MYOC.6,16,17 Aggregated glaucoma-associated mutant MYOC was retained in the endoplasmic reticulum and formed Russell bodies.18 This induced unfolded protein responses, resulting in apoptosis mostly in a caspase-dependent manner. Accordingly, fewer TM cells were documented in patients with galucoma.8,19 The reduced cleaning activity of the TM could lead to impediment of aqueous outflow, subsequently raising IOP.6,20

Gingko biloba extract (GBE), a traditional herbal medicine, is recommended for ophthalmic therapy owing to its cytoprotective, antiapoptotic, and antioxidative properties.2123 Clinical studies have shown that it may be useful in treating visual field damage associated with chronic cerebrovascular insufficiency, glaucomatous damage, macular degeneration, and diabetes mellitus.24 It also alleviates retinal impairment caused by ischemic-reperfusion injury.25 In rabbit corneal epithelial cells and human lens epithelial cells, GBE reduced alloxan-mediated and light-mediated stress, respectively.22 In this study, we investigated the effect of GBE on ocular hypertension caused by topical DEX in rabbits.

TOPICAL DEX AND GBE IN RABBITS

The experimental procedure followed the Declaration of Helsinki and Association for Research in Vision and Ophthalmology animal statement and was approved by the Animal Ethics Committee of the Chinese University of Hong Kong. Thirty male New Zealand albino rabbits aged 7 weeks were randomly chosen for the following topical treatments: group 1 (n = 7), balanced salt solution (BSS); group 2 (n = 8), TobraDEX (0.1% dexamethasone and 0.3% tobramycin) (Alcon, Hünenberg, Switzerland); group 3 (n = 8), 100 μg/mL of GBE; and group 4 (n = 7), TobraDEX and GBE. One drop was applied to the right eye 4 times daily for 14 days. For combined DEX and GBE, DEX was given 20 minutes before GBE to ensure complete drug delivery.

Intraocular pressure was recorded every 3 days with Tono-Pen XL (Solan Ophthalmic, Jacksonville, Florida) by a masked technician (W.L.). The mean (standard deviation) of 5 consecutive readings were calculated and compared by Mann-Whitney U test. A P value less than .05 was considered significant. After the rabbits were killed, enucleated eyes were cut along the nasal-temporal line, fixed in 10% neutral buffered formalin, and paraffin embedded. Ten consecutive sections were stained with hematoxylin-eosin or periodic acid-Schiff (PAS) reagents. Results were examined by 2 masked pathologists (L.-Y.J. and Ta Li Liu, FRCS[Glasg]). Cellularity of TM was graded by counting the nuclei in the TM and categorized as 0, less than 5% less nuclei than the controls; 1, 5% to 20% less; or 2, more than 20% less. Thickening of the TM was graded by impressive judgment as 0, no difference from the controls; 1, mild TM thickening; or 2, marked TM thickening.

DRUG TREATMENTS

Human TM cell lines5 were propagated.26 The cells contained negligible amounts of endogenous MYOC (data not shown). Cells of early passages were grown to confluence before treatment with 100nM DEX (Sigma, St Louis, Missouri) for 5 to 7 days with or without 100 μg/mL of GBE in medium containing 15% charcoal-dextran–treated fetal bovine serum (Hyclone, South Logan, Utah). Ginkgo Plus extract equivalent, providing 24% Ginkgo biloba flavoglycosides and 6% terpene lactones analogous to EGb761, was obtained from VitalHealth Product (Hong Kong, China). The concentration of GBE was optimized to have minimal effect on TM cell growth.

APOPTOSIS

Trabecular network cells were transfected with pFLAG-MYOCWT,27which contained human full-length wild-type MYOC cDNA cloned in mammalian expression vector p3xFLAG-myc-CMV-25 (Sigma). The cells were incubated with 0.2-μg/mL polyclonal anti-Fas antibody (Santa Cruz Biotech Inc, Santa Cruz, California) with or without GBE (0-100 μg/mL) for 48 hours. Cells were fixed and stained28 with monoclonal anti-FLAG (fludarabine, ara-C [cytarabine], and G-CSF [filgrastim]) antibody (Sigma) followed by red-X–conjugated goat antimouse IgG secondary antibody (Invitrogen, Carlsbad, California) and 4,6-diamidino-2-phenylindole (DAPI) (Sigma). Cells immunoreactive for FLAG were examined for nuclear fragmentation. Trabecular meshwork cell apoptosis was also examined by In Situ Cell Death Detection kit (Roche, Basel, Switzerland). In triplicate experiments, 10 random images (×40 objective) were analyzed. Cells treated with 10−8M camptothecin (Sigma) served as a positive control and IgG as a negative control.

WESTERN BLOTTING

Cells were lysed in ice-cold radioimmunoprecipitation assay buffer (Upstate Biotech [Millipore], Billerica, Massachusetts) with protease inhibitor (Roche). Soluble lysate was analyzed with affinity-purified polyclonal antihuman MYOC,27 anti-FLAG, anti–caspase-3 (Santa Cruz Biotech Inc) or anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, Texas) antibodies, followed by the appropriate horseradish peroxidase–conjugated immunoglobulin antibody (Santa Cruz Biotech Inc) and enhanced chemiluminescence (ECL) (Amersham Biosciences, Little Chalfont, England). Band intensities were quantified by Quantity One Image Analysis (BioRad, Hercules, California). Experiments were triplicated and intensities were represented as mean (standard deviation).

QUANTITATIVE POLYMERASE CHAIN REACTION

Total RNA was extracted by RNeasy Kit (Qiagen, Valencia, California) and on-column RNase-free DNase digestion (Qiagen). After reverse transcription, complementary DNA were amplified for αB-crystallin (CRY), interleukin 6 (IL-6), metallothionein (MET), Copper/zinc superoxide dismutase (SOD), heat shock protein 70 (Hsp70), heat-shock protein 90α (Hsp90α), β-actin (eTable) with QuantiTect SYBR Green PCR kit (Qiagen). Target gene expression relative to β-actin was analyzed by 2-ΔΔCT method.29

EFFECT OF GBE AND DEX ON IOP IN RABBITS

The mean (SD) baseline IOP of the rabbits was 10.6 (1.2) mm Hg (Figure 1A). The rabbits demonstrated IOP changes after topical TobraDEX. In preliminary experiments, we compared the marketed TobraDEX with self-prepared 0.1% DEX in Tears Naturale II (Alcon). Both reagents showed similar efficiency of IOP induction in rabbits. No complications or discomfort were observed with TobraDEX. To eliminate the risk of infection, we used TobraDEX. Three rabbits (1 in group 2 and 2 in group 4) died of natural causes unrelated to the topical treatments. Intraocular pressure was monitored throughout treatment. The DEX-treated rabbits (n = 7) showed higher IOPs than the BSS controls (n = 7). The increment range was 3 to 5.5 mm Hg (Figure 1B) and was significant (P = .04) when compared with the BSS controls. In contrast, when rabbits were given TobraDEX followed by GBE (n = 5), mild changes in mean IOP (<2 mm Hg) were observed (Figure 1A and B). By Mann-Whitney U test, the difference between DEX only and combined DEX and GBE treatment was significant at the examination intervals (P = .04). Rabbits with topical GBE only (n = 8) did not show distinct IOP changes when compared with pretreatment values (Figure 1A and B). Light microscopy showed no gross changes in anterior tissues (including cornea, conjunctiva, and eyelids) after topical DEX and/or GBE treatment. In the TM, PAS staining showed increased extracellular materials within the cribriform layers after DEX treatment (Figure 1D, H, and L) compared with the controls (Figure 1C, G, and K). After the combined DEX and GBE treatment, less extracellular matrix material was noted (Figure 1E, I, and M). No distinct alteration of the TM was found after GBE application (Figure 1F and J, and N). Moreover, DEX resulted in an alteration of cellularity, suggesting structural changes that would affect aqueous humor outflow. While strict counts were not conducted, impression by ocular pathologists revealed mild TM thickening with more PAS-positive material and fewer cells in the TM regions of rabbits treated with DEX (Tables 1 and 2; Figure 1H). These changes were fewer in rabbits who had received topical combined DEX and GBE (Figure 1I). Alone, GBE produced no observable changes (Figure 1J), similar to the controls (Figure 1G).

Place holder to copy figure label and caption
Figure 1.

Effect of Ginkgo biloba extract (GBE) on intraocular pressure (IOP) and trabecular meshwork (TM) histology in rabbits. A, Mean IOP and standard deviations during treatments. B, Average IOP fluctuations and standard deviations. Group 1 is the control group (n = 7); group 2 was treated with 0.1% dexamethasone [DEX] and 0.3% tobramycin (TrobaDEX; Alcon Labs, Hünenberg, Switzerland) (n = 7); group 3, TrobaDEX and GBE (n = 5); and group 4, GBE (n = 8). Using the t test, the IOP difference between TobraDEX-treated and TobraDEX and GBE–treated cells was significant (*P < .1; **P < .05). C-J, Trabecular meshwork at low (C-F) and high (G-J) magnifications. K-N, Periodic acid–Schiff staining in the TM indicates deposition of extracellular materials (arrowheads). Scale bars, 0.1 mm (C-F, K-N) and 0.05 mm (G-J).

Graphic Jump Location
Table Graphic Jump LocationTable 1. Grading of Cell Loss in TM Region After Topical TobraDEXa and/or GBE Treatmentsb
Table Graphic Jump LocationTable 2. Grading of Trabecula Thickening After Topical TobraDEXa and/or GBE Treatmentsb
REDUCTION OF DEX-INDUCED MYOC EXPRESSION IN CULTURED HUMAN TM CELLS BY GBE

Trabecular meshwork cells at confluence showed more MYOC staining after 5-day treatment with 100nM DEX (DEX5 group) (Figure 2B) when compared with untreated cells (Figure 2A). This was corroborated by Western blotting (Figure 2E). Densitometry indicated a significant increase in MYOC expression in DEX5 cells, about 10-fold more than in the control cells (P = .01, t test; n = 3) (Figure 2E). This MYOC induction was similar to that of the 10-day DEX treatment (data not shown). During DEX withdrawal, the elevated MYOC level was maintained for 5 days and then reduced gradually (Figure 2F).

Place holder to copy figure label and caption
Figure 2.

Effect of Ginkgo biloba extract (GBE) on myocilin (MYOC) expression. A-D, Immunofluorescence of MYOC. A, Controls; B, Rabbits treated with 100nM dexamethasone for 5 days (DEX5); C, 100nM DEX for 5 days followed by 100 μg/mL of GBE for 5 days (DEX5→GBE5); D, 100 μg/mL of GBE for 5 days followed by 100nM DEX for 5 days (GBE5→DEX5). E, Western blot analysis of MYOC and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in trabecular meshwork (TM) samples treated with 100nM DEX and/or 100 μg/mL of GBE. F, Western blot analysis of MYOC and GAPDH in TM samples with 100nM DEX for 5 days and measured for 10 days after DEX removal. GBE5+DEX5→DEX5 indicates cells pretreated with combined GBE and DEX for 5 days before DEX incubation; scale bars, 10 μm; * P < .05.

Graphic Jump Location

The high MYOC expression level was reduced by more than 60% when cells were incubated with 100 μg/mL of GBE for another 5 days (DEX5→GBE5 group) (Figure 2E). This dosage of GBE was optimized to have minimal effect on cell proliferation. Using immunofluorescence (Figure 2C), weak MYOC staining was noted in the DEX5→GBE5 cells. Moreover, we preincubated TM cells with 100 μg/mL of GBE for 5 days prior to DEX (GBE5→DEX5 group). A weak band of immunoreactive MYOC was observed that had the intensity of about one-fifth that of the DEX5 cells (P = .02, t test; n = 3) (Figure 2D and E). Low MYOC expression was similarly found in cells pretreated with combined GBE and DEX for 5 days before DEX incubation (GBE5+DEX5→DEX5 group) (P = .02, t test; n = 3) (Figure 2E).

ATTENUATION OF ANTI-Fas–INDUCED APOPTOSIS OF MYOC-TRANSFECTED TM CELLS BY GBE

Trabecular meshwork cells were transfected for FLAG-tagged MYOC. Anti-Fas treatment substantially increased the number of apoptotic cells, as revealed by the frequent appearance of fragmented nuclei by the combined immunofluorescence for FLAG and DAPI. An approximately 3-fold increase in the apoptosis rate was found in MYOC-expressing cells after anti-Fas incubation compared with nontransfected cells (Figure 3A). Subsequent GBE lowered the apoptosis rate in a dose-dependent manner. Incubation with 100 μg/mL of GBE for 48 hours resulted in a 75% decrease in apoptosis, approaching the level without anti-Fas. Lower GBE concentrations had lesser preventive effects against apoptosis. Similar results were obtained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) analysis. Incubation of anti–Fas-treated cells with 100 μg/mL of GBE for 2 days resulted in a 3-fold decrease in TUNEL-positive cells from a mean (SD) of 43.6% (10.7%) to 12.6% (3.7%). Controls (6.7% [1.6%]) and GBE-treated cells (8.7% [2.4%]) had similar mean (SD) apoptosis rates. Moreover, anti-Fas–induced activation of caspase-3 was greatly suppressed by GBE (Figure 3B). The active caspase-3 at about 17 kDa was found in MYOC-expressing cells after anti-Fas treatment as well as in camptothecin-treated cells, which served as positive controls. The 30-kDa precursor form is shown in Figure 3B. When anti-Fas treatment was given with 100 μg/mL of GBE incubation for 24 hours, less active caspase-3 was present. Band densitometry of caspase-3 revealed a significant decrease in caspase-3 activation after GBE treatment in anti-Fas–treated MYOC-expressing cells (P = .01, paired t test; n = 3). Cells treated with Ginkgo biloba extract alone had very low levels of active caspase-3.

Place holder to copy figure label and caption
Figure 3.

Effect of Ginkgo biloba extract (GBE) on trabecular meshwork (TM) cell apoptosis. A, Percentage of cells with fragmented nuclei. B, Western blotting of caspase-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in TM samples. Camptothecin (10nM)–treated cells served as positive controls for apoptosis. C, Band densitometry of B. ** indicates GBE-induced reduction of active caspase-3; * pro–caspase-3.

Graphic Jump Location
SAFETY OF GBE FOR TM CELLS

Ginkgo biloba extract in concentrations from 10 to 1000 μg/mL were administered to TM cells. Growth kinetics were determined and the mean of triplicate measurements showed that cells treated with GBE in concentrations of less than 100 μg/mL had similar growth rates to untreated cells (cell doubling with 10 μg/mL of GBE, 40.3 hours; 100 μg/mL GBE, 41.5 hours; 1000 μg/mL GBE, 60 hours; controls, 38.1 hours). Similar results were observed for CHO-K1 cells (data not shown).

We analyzed the expression of genes associated with stress responses (oxidative, heat-shock, and metal-induced) in TM cells treated with 100nM DEX followed by 100 μg/mL of GBE and cells treated only with 100 μg/mL of GBE. A 5-day DEX treatment was chosen, as we aimed to examine early gene induction. First, for 5 days, cells treated with DEX upregulated MYOC expression (7.7-fold more than untreated cells) (Figure 4); this elevated level was maintained after DEX withdrawal for 2 days (Figure 2F). Subsequent GBE treatment gradually reduced MYOC expression by 10% after 60 minutes and 50% after 120 minutes. For cells treated with GBE only, steady-state RNA levels of stress-related genes (CRY, IL-6, MET, and SOD) were not different from those of the controls (Figure 4). Quantitative polymerase chain reaction revealed no significant alteration. However, a 5-day DEX treatment induced an approximately 3-fold increase in CRY expression (Figure 4). Additional incubation with GBE for 60 minutes further increased CRY RNA levels, which decreased after 120 minutes of incubation with GBE. The levels of IL-6, MET, and SOD RNA were not significantly altered by treatment with DEX and/or GBE (Figure 4; eFigure). Moreover, Hsp70 and Hsp90α were upregulated after incubation with DEX and/or GBE (Figure 4).

Place holder to copy figure label and caption
Figure 4.

Stress-response gene expression. Quantitative polymerase chain reaction analysis of CRY, IL-6, MET, SOD, hsp70, hsp90α, and MYOC. Gene expression was normalized with that of β-actin at the respective time point. Tests were performed in triplicate. GBE indicates Ginkgo biloba extract; DEX, dexamethasone.

Graphic Jump Location

This study demonstrated the potential effect of GBE on DEX-induced ocular hypertension. We showed with a rabbit model that GBE suppressed IOP elevation caused by topical DEX. Ginkgo biloba extract exhibited antisteroid action by reducing DEX-induced MYOC expression in cultured human TM cells. We further showed that GBE caused MYOC-expressing cells to be less prone to apoptosis induced by anti–Fas ligand method. Ginkgo biloba extract at our application dosage had no apparent toxicity for TM cells. It moderately upregulated heat-shock responses and molecular chaperones, but did not affect other stress-related genes. This suggests that GBE has the potential to treat IOP-associated glaucoma. In addition, priming TM cells with GBE before treatment with DEX greatly reduced MYOC expression, indicating that either dietary GBE or supplemental GBE in eye drops could reduce the risk of developing steroid-induced ocular hypertension.

Medicinal use of GBE can be traced back 5000 years in China. Today this herbal compound is one of the most widely sold herbal dietary supplements in the United States and Europe. It is known to have antiapoptotic, antioxidative, antiplatelet aggregation, antitumor, antiaging, and nitric oxide inhibitory effects on various cell types.23,3032 The standard extract of GBE, EGb761, is commonly prescribed as an herbal remedy for peripheral vascular, cardiovascular, and cerebrovascular diseases.33,34 It reduces memory loss and symptoms of cognitive disorders, including Alzheimer disease35 and dementias.36 Recently, the antiinflammatory effect of GBE relieved asthma in patients.37 It is proposed to be a natural therapeutic agent for glaucoma, in particular the non–pressure-dependent type.38 Dietary intake of GBE improved the intrinsic glutathione level of retinal glial cells in older guinea pigs.39 In a phase 1 trial, orally administered GBE enhanced ocular blood flow.40 This shows the neuroprotective role of GBE in glaucomatous neuropathy. In this study, we established a novel feature of GBE, ie, the antisteroid effect, in suppressing DEX-induced IOP elevation. From various clinical and animal studies, steroids, in particular DEX, are known to upregulate MYOC expression in a time course similar to the induction of ocular hypertension.16,17 Elevated IOP due to perturbation or blockage of aqueous humor flow along the TM and Schlemm canal9 is a strong risk factor for glaucoma. Sustained high IOP causes progressive damage to retinal ganglion cells, causing loss of bilateral vision. In a rat model of chronic IOP-associated glaucoma, GBE was shown to promote the survival of retinal ganglion cells.41

In this study, GBE was effective in relieving IOP elevation induced by DEX in rabbits aged 7 weeks. Ginkgobiloba extract (5 μg) was topically applied 20 minutes after DEX and the procedure was performed 4 times daily. Measurement of IOP showed that combined DEX and GBE caused much milder IOP fluctuation than DEX only. Increased extracellular materials within the TM layers were observed after DEX treatment was reduced. This indicates the action of GBE in the maintenance of a clear and open outflow channel for the aqueous humor. Moreover, GBE treatment was associated with less apparent cell loss in the TM region.

The antisteroid effect of GBE could be mediated by downregulating DEX-induced MYOC expression in TM cells. Studies have shown that MYOC in TM cells was stimulated by stress, such as hydrogen peroxide and mechanical stretches,42,43 elevated IOP in organ culture of anterior segment,44 or steroids.16 Adding to this list, GBE suppresses MYOC expression in the context of the elevated level induced by steroids. Thus, our results demonstrated a potential antisteroid capability of GBE in human TM cells. Ginkgo biloba (10 to 100 μg/mL) was not cytotoxic and did not affect cell growth. The cause of the slight lengthening of cell-doubling time after 1000 μg/mL of GBE treatment needs further investigation. In addition, GBE showed a protective effect against DEX as evidenced by the low MYOC expression in cells preincubated with GBE. Either priming TM cells with GBE or combined DEX and GBE resulted in a reduced effect of DEX on MYOC induction by about 80%. This result suggests that GBE is a suitable priming agent to suppress DEX-induced MYOC expression and could be envisaged as an ocular nutrient supplement.23,45

Trabecular meshwork cells are stimulated to undergo apoptosis via the Fas:Fas ligand pathway as first demonstrated by Agarwal et al.46 Moreover, low MYOC expression was an advantage toward better TM cell survival.47 In our experiments, immortalized TM cells instead of primary TM culture were used to provide a minimal background of spontaneous apoptosis. When transfected with FLAG-tagged MYOC, our TM culture was prone to apoptosis when induced by anti-Fas. Dexamethasone-induced apoptosis was also observed in another TM cell line48 and in chondrocytes.49Ginkgo biloba extract, with its suppressive action of DEX-induced MYOC expression, maintained a low apoptosis rate for TM cells when exposed to anti-Fas. This was well demonstrated by the low percentage of cells with fragmented nuclei and cells that were TUNEL positive. We further illustrated that a possible mechanism for GBE reduction of TM cell death could be associated with activation of αB-crystallin, Hsp70, and Hsp90α, which are molecular and heat-shock chaperones. Also, αB-crystallin protects TM cells from damage due to oxidative stress and mechanical stretching.50,51 Heat-shock protein 70 promiscuously recognizes substrate proteins with nonnative conformation and, via interaction with Hsp90, promotes protein folding.52 Their upregulation by GBE would be expected to protect TM cells from MYOC induced by DEX. Moreover, GBE did not cause other stress-related changes like that of IL-6 (a marker for inflammation or occurrence of reactive oxygen species), metallothionein (induced by metals, glucocorticoids, inflammatory, and oxidative stress53), and copper/zinc superoxide dismutase (induced by oxidative stress54). Similar antiapoptotic capability of GBE was observed in other cell types.21,32,55

Loss of TM cells was reported in glaucoma patients.8,19 This could reduce the phagocytic capability along the TM and the Schlemm canal, leading to accumulation of extracellular substances that result in blockage of outflow pathways and IOP elevation.13,18,56 In this study, GBE showed a potential antisteroid ability to reduce DEX-induced MYOC expression and an antiapoptotic effect to maintain the TM cell population. Similar effects could be obtained either by priming cells before DEX or rescuing DEX-treated cells. This could allow better TM function. Hence, GBE could be envisaged as a potential drug for treating IOP-associated glaucoma or it could be used as a food supplement to reduce the risk of developing steroid-induced glaucoma.

Correspondence: Gary H. F. Yam, PhD, Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, 147K Argyle St, Kowloon, Hong Kong, China (gary_yam@cuhk.edu.hk).

Submitted for Publication: January 7, 2008; final revision received June 20, 2008; accepted June 28, 2008.

Author Contributions: Li-Yun Jia, PhD, and Ling Sun, BSc contributed equally to the study.

Financial Disclosure: None reported.

Funding/Support: This study was supported by a Block Grant from The Chinese University of Hong Kong.

Additional Contributions: We thank T.D. Nguyen, MD, for providing human TM cells, which were originally established by the late J.R. Polansky, MD, and we are grateful to W.Y. Li, MD, for intraocular pressure measurements.

Goulding  NJEuzger  HSButt  SKPerretti  M Novel pathways for glucocorticoid effects on neutrophils in chronic inflammation. Inflamm Res 1998;47 ((suppl 3)) S158- S165
PubMed Link to Article
Adcock  IMCaramori  G Cross-talk between pro-inflammatory transcription factors and glucocorticoids. Immunol Cell Biol 2001;79 (4) 376- 384
PubMed Link to Article
Wordinger  RJClark  AF Effects of glucocorticoids on the trabecular meshwork. Prog Retin Eye Res 1999;18 (5) 629- 667
PubMed Link to Article
Ishibashi  TTakagi  YMori  K  et al.  cDNA microarray analysis of gene expression changes induced by dexamethasone in cultured human trabecular meshwork cells. Invest Ophthalmol Vis Sci 2002;43 (12) 3691- 3697
PubMed
Leung  YFTam  POLee  WS  et al.  The dual role of dexamethasone on anti-inflammation and outflow resistance demonstrated in cultured human trabecular meshwork cells. Mol Vis 2003;9425- 439
PubMed
Tamm  ER Myocilin and glaucoma. Prog Retin Eye Res 2002;21 (4) 395- 428
PubMed Link to Article
Lütjen-Drecoll  EShimizu  TRohrbach  MRohen  JW Quantitative analysis of ‘plaque material’ in the inner- and outer wall of Schlemm's canal in normal- and glaucomatous eyes. Exp Eye Res 1986;42 (5) 443- 455
PubMed Link to Article
Rohen  JWLütjen-Drecoll  EFlügel  CMeyer  MGrierson  I Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma. Exp Eye Res 1993;56 (6) 683- 692
PubMed Link to Article
Johnson  DGottanka  JFlügel  CHoffmann  FFuta  RLütjen-Drecoll  E Ultrastructural changes in the trabecular meshwork of human eyes treated with corticosteroids. Arch Ophthalmol 1997;115 (3) 375- 383
PubMed Link to Article
Ueda  JWentz-Hunter  KYue  BY Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes. Invest Ophthalmol Vis Sci 2002;43 (4) 1068- 1076
PubMed
Cracknell  KPGrierson  IHogg  PAppleton  PPfeiffer  N Latanoprost-induced iris darkening. Exp Eye Res 2003;77 (6) 721- 730
PubMed Link to Article
Acott  TSWestcott  MPasso  MSvan Buskirk  EM Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye. Invest Ophthalmol Vis Sci 1985;26 (10) 1320- 1329
PubMed
Buller  CJohnson  DHTschumper  RC Human trabecular meshwork phagocytosis. Invest Ophthalmol Vis Sci 1990;31 (10) 2156- 2163
PubMed
Lütjen-Drecoll  EMay  CAPolansky  JRJohnson  DHBloemendal  HNguyen  TD Localization of the stress proteins alpha B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork. Invest Ophthalmol Vis Sci 1998;39 (3) 517- 525
PubMed
Clark  AFKawase  KEnglish-Wright  S  et al.  Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues. Invest Ophthalmol Vis Sci 2001;42 (8) 1769- 1780
PubMed
Nguyen  TDChen  PHuang  WD  et al.  Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J Biol Chem 1998;273 (11) 6341- 6350
PubMed Link to Article
Shepard  ARJacobson  NFingert  JH  et al.  Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 2001;42 (13) 3173- 3181
PubMed
Yam  GHFGaplovska-Kysela  KZuber  CRoth  J Aggregated myocilin induces russell bodies and causes apoptosis. Am J Pathol 2007;170 (1) 100- 109
PubMed Link to Article
Alvarado  JMurphy  CJuster  R Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals. Ophthalmology 1984;91 (6) 564- 579
PubMed Link to Article
Tan  JCPeters  DMKaufman  PL Recent developments in understanding the pathophysiology of elevated intraocular pressure. Curr Opin Ophthalmol 2006;17 (2) 168- 174
PubMed
Ritch  R Potential role for Ginkgo biloba extract in the treatment of glaucoma. Med Hypotheses 2000;54 (2) 221- 235
PubMed Link to Article
Thiagarajan  GChandani  SRao  SH  et al.  Molecular and cellular assessment of gingko biloba extract as a possible ophthalmic drug. Exp Eye Res 2002;75 (4) 421- 430
PubMed Link to Article
Ritch  R Complementary therapy for the treatment of glaucoma: a perspective. Ophthalmol Clin North Am 2005;18 (4) 597- 609
PubMed
Clostre  F Ginkgo biloba extract (EGb 761): state of knowledge in the dawn of the year 2000 [in French]. Ann Pharm Fr 1999;57 ((suppl 1)) 1S8- 1S88
PubMed
Kim  SYKwak  JSShin  JPLee  SH The protection of the retina from ischemic injury by the free radical scavenger EGb 761 and zinc in the cat retina. Ophthalmologica 1998;212 (4) 268- 274
PubMed Link to Article
Polansky  JRWeinreb  RNBaxter  JDAlvarado  J Human trabecular cells I: establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci 1979;18 (10) 1043- 1049
PubMed
Yam  GHFGaplovska-Kysela  KZuber  CRoth  J Sodium 4-phenylbutyrate acts as chemical chaperone on misfolded myocilin to rescue cells from endoplasmic reticulum stress and apoptosis. Invest Ophthalmol Vis Sci 2007;48 (4) 1683- 1690
PubMed Link to Article
Yam  GHFZuber  ChRoth  J A synthetic chaperone corrects the trafficking defect and disease phenotype in a protein misfolding disorder. FASEB J 2005;19 (1) 12- 18
PubMed Link to Article
Livak  KJSchmittgen  TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25 (4) 402- 408
PubMed Link to Article
Chen  LWWang  YQWei  LCShi  MChan  YS Chinese herbs and herbal extracts for neuroprotection of dopaminergic neurons and potential therapeutic treatment of Parkinson's disease. CNS Neurol Disord Drug Targets 2007;6 (4) 273- 281
PubMed Link to Article
Takuma  KHoshina  YArai  S  et al.  Ginkgo biloba extract EGb 761 attenuates hippocampal neuronal loss and cognitive dysfunction resulting from chronic restraint stress in ovariectomized rats. Neuroscience 2007;149 (2) 256- 262
PubMed Link to Article
Eckert  AKeil  UKressmann  S  et al.  Effects of EGb761 Gingko biloba extract on mitochondrial functions and oxidative stress. Pharmacopsychiatry 2003;36 ((suppl 1)) S15- S23
PubMed Link to Article
Sierpina  VSWollschlaeger  BBlumenthal  M Ginkgo bilobaAm Fam Physician 2003;68 (5) 923- 926
PubMed
Zhou  WChai  HLin  PH  et al.  Clinical use and molecular mechanisms of action of extract of Ginkgo biloba leaves in cardiovascular diseases. Cardiovasc Drug Rev 2004;22 (4) 309- 319
PubMed Link to Article
Maurer  KIhl  RDierks  TFrolich  L Clinical efficacy of Ginkgo biloba special extract EGb 761 in dementia of the Alzheimer type. J Psychiatr Res 1997;31 (6) 645- 655
PubMed Link to Article
Le Bars  PLKatz  MMBerman  N  et al.  A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia: North American EGb Study Group. JAMA 1997;278 (16) 1327- 1332
PubMed Link to Article
Tang  YXu  YXiong  S  et al.  The effect of Ginkgo biloba extract on the expression of PKCalpha in the inflammatory cells and the level of IL-5 in induced sputum of asthmatic patients. J Huazhong Univ Sci Technolog Med Sci 2007;27 (4) 375- 380
PubMed Link to Article
Mozaffarieh  MFlammer  J A novel perspective on natural therapeutic approaches in glaucoma therapy. Expert Opin Emerg Drugs 2007;12 (2) 195- 198
PubMed Link to Article
Paasche  GGartner  UGermer  AGrosche  JReichenbach  A Mitochondria of retinal Muller (glial) cells: the effects of aging and of application of free radical scavengers. Ophthalmic Res 2000;32 (5) 229- 236
PubMed Link to Article
Chung  HSHarris  AKristinsson  JK  et al.  Ginkgo biloba extract increases ocular blood flow velocity. J Ocul Pharmacol Ther 1999;15 (3) 233- 240
PubMed Link to Article
Hirooka  KTokuda  MMiyamoto  O  et al.  The Ginkgo biloba extract (EGb 761) provides a neuroprotective effect on retinal ganglion cells in a rat model of chronic glaucoma. Curr Eye Res 2004;28 (3) 153- 157
PubMed Link to Article
Polansky  JRFauss  DJChen  P  et al.  Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Ophthalmologica 1997;211 (3) 126- 139
PubMed Link to Article
Tamm  ERRussell  PEpstein  DLJohnson  DHPiatigorsky  J Modulation of myocilin/TIGR expression in human trabecular meshwork. Invest Ophthalmol Vis Sci 1999;40 (11) 2577- 2582
PubMed
Borrás  T Gene expression in the trabecular meshwork and the influence of intraocular pressure. Prog Retin Eye Res 2003;22 (4) 435- 463
PubMed Link to Article
Bartlett  HEperjesi  F An ideal ocular nutritional supplement? Ophthalmic Physiol Opt 2004;24 (4) 339- 349
PubMed Link to Article
Agarwal  RTalati  MLambert  W  et al.  Fas-activated apoptosis and apoptosis mediators in human trabecular meshwork cells. Exp Eye Res 1999;68 (5) 583- 590
PubMed Link to Article
Wentz-Hunter  KShen  XOkazaki  KTanihara  HYue  BY Overexpression of myocilin in cultured human trabecular meshwork cells. Exp Cell Res 2004;297 (1) 39- 48
PubMed Link to Article
Gu  YZeng  SQiu  PPeng  DYan  G Apoptosis of bovine trabecular meshwork cells induced by dexamethasone [in Chinese]. Zhonghua Yan Ke Za Zhi 2002;38 (5) 302- 304
PubMed
Chrysis  DZaman  FChagin  ASTakigawa  MSavendahl  L Dexamethasone induces apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt-phosphatidylinositol 3′-kinase signaling pathway. Endocrinology 2005;146 (3) 1391- 1397
PubMed Link to Article
Tamm  ERRussell  PJohnson  DHPiatigorsky  J Human and monkey trabecular meshwork accumulate alpha B-crystallin in response to heat shock and oxidative stress. Invest Ophthalmol Vis Sci 1996;37 (12) 2402- 2413
PubMed
Ganea  E Chaperone-like activity of alpha-crystallin and other small heat-shock proteins. Curr Protein Pept Sci 2001;2 (3) 205- 225
PubMed Link to Article
Young  JCAgashe  VRSiegers  KHartl  FU Pathways of chaperone-mediated protein folding in the cytosol. Nat Rev Mol Cell Biol 2004;5 (10) 781- 791
PubMed Link to Article
Haq  FMahoney  MKoropatnich  J Signaling events for metallothionein induction. Mutat Res 2003;533 (1-2) 211- 226
PubMed Link to Article
Maier  CMChan  PH Role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist 2002;8 (4) 323- 334
PubMed Link to Article
Sastre  JLloret  ABorras  C  et al.  Ginkgo biloba extract EGb761 protects against mitochondrial aging in the brain and in the liver. Cell Mol Biol (Noisy-le-grand) 2002;48 (6) 685- 692
PubMed
Shirato  SMurphy  CGBloom  E  et al.  Kinetics of phagocytosis in trabecular meshwork cells. Invest Ophthalmol Vis Sci 1989;30 (12) 2499- 2511
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

Effect of Ginkgo biloba extract (GBE) on intraocular pressure (IOP) and trabecular meshwork (TM) histology in rabbits. A, Mean IOP and standard deviations during treatments. B, Average IOP fluctuations and standard deviations. Group 1 is the control group (n = 7); group 2 was treated with 0.1% dexamethasone [DEX] and 0.3% tobramycin (TrobaDEX; Alcon Labs, Hünenberg, Switzerland) (n = 7); group 3, TrobaDEX and GBE (n = 5); and group 4, GBE (n = 8). Using the t test, the IOP difference between TobraDEX-treated and TobraDEX and GBE–treated cells was significant (*P < .1; **P < .05). C-J, Trabecular meshwork at low (C-F) and high (G-J) magnifications. K-N, Periodic acid–Schiff staining in the TM indicates deposition of extracellular materials (arrowheads). Scale bars, 0.1 mm (C-F, K-N) and 0.05 mm (G-J).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Effect of Ginkgo biloba extract (GBE) on myocilin (MYOC) expression. A-D, Immunofluorescence of MYOC. A, Controls; B, Rabbits treated with 100nM dexamethasone for 5 days (DEX5); C, 100nM DEX for 5 days followed by 100 μg/mL of GBE for 5 days (DEX5→GBE5); D, 100 μg/mL of GBE for 5 days followed by 100nM DEX for 5 days (GBE5→DEX5). E, Western blot analysis of MYOC and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in trabecular meshwork (TM) samples treated with 100nM DEX and/or 100 μg/mL of GBE. F, Western blot analysis of MYOC and GAPDH in TM samples with 100nM DEX for 5 days and measured for 10 days after DEX removal. GBE5+DEX5→DEX5 indicates cells pretreated with combined GBE and DEX for 5 days before DEX incubation; scale bars, 10 μm; * P < .05.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Effect of Ginkgo biloba extract (GBE) on trabecular meshwork (TM) cell apoptosis. A, Percentage of cells with fragmented nuclei. B, Western blotting of caspase-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in TM samples. Camptothecin (10nM)–treated cells served as positive controls for apoptosis. C, Band densitometry of B. ** indicates GBE-induced reduction of active caspase-3; * pro–caspase-3.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Stress-response gene expression. Quantitative polymerase chain reaction analysis of CRY, IL-6, MET, SOD, hsp70, hsp90α, and MYOC. Gene expression was normalized with that of β-actin at the respective time point. Tests were performed in triplicate. GBE indicates Ginkgo biloba extract; DEX, dexamethasone.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Grading of Cell Loss in TM Region After Topical TobraDEXa and/or GBE Treatmentsb
Table Graphic Jump LocationTable 2. Grading of Trabecula Thickening After Topical TobraDEXa and/or GBE Treatmentsb

References

Goulding  NJEuzger  HSButt  SKPerretti  M Novel pathways for glucocorticoid effects on neutrophils in chronic inflammation. Inflamm Res 1998;47 ((suppl 3)) S158- S165
PubMed Link to Article
Adcock  IMCaramori  G Cross-talk between pro-inflammatory transcription factors and glucocorticoids. Immunol Cell Biol 2001;79 (4) 376- 384
PubMed Link to Article
Wordinger  RJClark  AF Effects of glucocorticoids on the trabecular meshwork. Prog Retin Eye Res 1999;18 (5) 629- 667
PubMed Link to Article
Ishibashi  TTakagi  YMori  K  et al.  cDNA microarray analysis of gene expression changes induced by dexamethasone in cultured human trabecular meshwork cells. Invest Ophthalmol Vis Sci 2002;43 (12) 3691- 3697
PubMed
Leung  YFTam  POLee  WS  et al.  The dual role of dexamethasone on anti-inflammation and outflow resistance demonstrated in cultured human trabecular meshwork cells. Mol Vis 2003;9425- 439
PubMed
Tamm  ER Myocilin and glaucoma. Prog Retin Eye Res 2002;21 (4) 395- 428
PubMed Link to Article
Lütjen-Drecoll  EShimizu  TRohrbach  MRohen  JW Quantitative analysis of ‘plaque material’ in the inner- and outer wall of Schlemm's canal in normal- and glaucomatous eyes. Exp Eye Res 1986;42 (5) 443- 455
PubMed Link to Article
Rohen  JWLütjen-Drecoll  EFlügel  CMeyer  MGrierson  I Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma. Exp Eye Res 1993;56 (6) 683- 692
PubMed Link to Article
Johnson  DGottanka  JFlügel  CHoffmann  FFuta  RLütjen-Drecoll  E Ultrastructural changes in the trabecular meshwork of human eyes treated with corticosteroids. Arch Ophthalmol 1997;115 (3) 375- 383
PubMed Link to Article
Ueda  JWentz-Hunter  KYue  BY Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes. Invest Ophthalmol Vis Sci 2002;43 (4) 1068- 1076
PubMed
Cracknell  KPGrierson  IHogg  PAppleton  PPfeiffer  N Latanoprost-induced iris darkening. Exp Eye Res 2003;77 (6) 721- 730
PubMed Link to Article
Acott  TSWestcott  MPasso  MSvan Buskirk  EM Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye. Invest Ophthalmol Vis Sci 1985;26 (10) 1320- 1329
PubMed
Buller  CJohnson  DHTschumper  RC Human trabecular meshwork phagocytosis. Invest Ophthalmol Vis Sci 1990;31 (10) 2156- 2163
PubMed
Lütjen-Drecoll  EMay  CAPolansky  JRJohnson  DHBloemendal  HNguyen  TD Localization of the stress proteins alpha B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork. Invest Ophthalmol Vis Sci 1998;39 (3) 517- 525
PubMed
Clark  AFKawase  KEnglish-Wright  S  et al.  Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues. Invest Ophthalmol Vis Sci 2001;42 (8) 1769- 1780
PubMed
Nguyen  TDChen  PHuang  WD  et al.  Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J Biol Chem 1998;273 (11) 6341- 6350
PubMed Link to Article
Shepard  ARJacobson  NFingert  JH  et al.  Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 2001;42 (13) 3173- 3181
PubMed
Yam  GHFGaplovska-Kysela  KZuber  CRoth  J Aggregated myocilin induces russell bodies and causes apoptosis. Am J Pathol 2007;170 (1) 100- 109
PubMed Link to Article
Alvarado  JMurphy  CJuster  R Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals. Ophthalmology 1984;91 (6) 564- 579
PubMed Link to Article
Tan  JCPeters  DMKaufman  PL Recent developments in understanding the pathophysiology of elevated intraocular pressure. Curr Opin Ophthalmol 2006;17 (2) 168- 174
PubMed
Ritch  R Potential role for Ginkgo biloba extract in the treatment of glaucoma. Med Hypotheses 2000;54 (2) 221- 235
PubMed Link to Article
Thiagarajan  GChandani  SRao  SH  et al.  Molecular and cellular assessment of gingko biloba extract as a possible ophthalmic drug. Exp Eye Res 2002;75 (4) 421- 430
PubMed Link to Article
Ritch  R Complementary therapy for the treatment of glaucoma: a perspective. Ophthalmol Clin North Am 2005;18 (4) 597- 609
PubMed
Clostre  F Ginkgo biloba extract (EGb 761): state of knowledge in the dawn of the year 2000 [in French]. Ann Pharm Fr 1999;57 ((suppl 1)) 1S8- 1S88
PubMed
Kim  SYKwak  JSShin  JPLee  SH The protection of the retina from ischemic injury by the free radical scavenger EGb 761 and zinc in the cat retina. Ophthalmologica 1998;212 (4) 268- 274
PubMed Link to Article
Polansky  JRWeinreb  RNBaxter  JDAlvarado  J Human trabecular cells I: establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci 1979;18 (10) 1043- 1049
PubMed
Yam  GHFGaplovska-Kysela  KZuber  CRoth  J Sodium 4-phenylbutyrate acts as chemical chaperone on misfolded myocilin to rescue cells from endoplasmic reticulum stress and apoptosis. Invest Ophthalmol Vis Sci 2007;48 (4) 1683- 1690
PubMed Link to Article
Yam  GHFZuber  ChRoth  J A synthetic chaperone corrects the trafficking defect and disease phenotype in a protein misfolding disorder. FASEB J 2005;19 (1) 12- 18
PubMed Link to Article
Livak  KJSchmittgen  TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25 (4) 402- 408
PubMed Link to Article
Chen  LWWang  YQWei  LCShi  MChan  YS Chinese herbs and herbal extracts for neuroprotection of dopaminergic neurons and potential therapeutic treatment of Parkinson's disease. CNS Neurol Disord Drug Targets 2007;6 (4) 273- 281
PubMed Link to Article
Takuma  KHoshina  YArai  S  et al.  Ginkgo biloba extract EGb 761 attenuates hippocampal neuronal loss and cognitive dysfunction resulting from chronic restraint stress in ovariectomized rats. Neuroscience 2007;149 (2) 256- 262
PubMed Link to Article
Eckert  AKeil  UKressmann  S  et al.  Effects of EGb761 Gingko biloba extract on mitochondrial functions and oxidative stress. Pharmacopsychiatry 2003;36 ((suppl 1)) S15- S23
PubMed Link to Article
Sierpina  VSWollschlaeger  BBlumenthal  M Ginkgo bilobaAm Fam Physician 2003;68 (5) 923- 926
PubMed
Zhou  WChai  HLin  PH  et al.  Clinical use and molecular mechanisms of action of extract of Ginkgo biloba leaves in cardiovascular diseases. Cardiovasc Drug Rev 2004;22 (4) 309- 319
PubMed Link to Article
Maurer  KIhl  RDierks  TFrolich  L Clinical efficacy of Ginkgo biloba special extract EGb 761 in dementia of the Alzheimer type. J Psychiatr Res 1997;31 (6) 645- 655
PubMed Link to Article
Le Bars  PLKatz  MMBerman  N  et al.  A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia: North American EGb Study Group. JAMA 1997;278 (16) 1327- 1332
PubMed Link to Article
Tang  YXu  YXiong  S  et al.  The effect of Ginkgo biloba extract on the expression of PKCalpha in the inflammatory cells and the level of IL-5 in induced sputum of asthmatic patients. J Huazhong Univ Sci Technolog Med Sci 2007;27 (4) 375- 380
PubMed Link to Article
Mozaffarieh  MFlammer  J A novel perspective on natural therapeutic approaches in glaucoma therapy. Expert Opin Emerg Drugs 2007;12 (2) 195- 198
PubMed Link to Article
Paasche  GGartner  UGermer  AGrosche  JReichenbach  A Mitochondria of retinal Muller (glial) cells: the effects of aging and of application of free radical scavengers. Ophthalmic Res 2000;32 (5) 229- 236
PubMed Link to Article
Chung  HSHarris  AKristinsson  JK  et al.  Ginkgo biloba extract increases ocular blood flow velocity. J Ocul Pharmacol Ther 1999;15 (3) 233- 240
PubMed Link to Article
Hirooka  KTokuda  MMiyamoto  O  et al.  The Ginkgo biloba extract (EGb 761) provides a neuroprotective effect on retinal ganglion cells in a rat model of chronic glaucoma. Curr Eye Res 2004;28 (3) 153- 157
PubMed Link to Article
Polansky  JRFauss  DJChen  P  et al.  Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Ophthalmologica 1997;211 (3) 126- 139
PubMed Link to Article
Tamm  ERRussell  PEpstein  DLJohnson  DHPiatigorsky  J Modulation of myocilin/TIGR expression in human trabecular meshwork. Invest Ophthalmol Vis Sci 1999;40 (11) 2577- 2582
PubMed
Borrás  T Gene expression in the trabecular meshwork and the influence of intraocular pressure. Prog Retin Eye Res 2003;22 (4) 435- 463
PubMed Link to Article
Bartlett  HEperjesi  F An ideal ocular nutritional supplement? Ophthalmic Physiol Opt 2004;24 (4) 339- 349
PubMed Link to Article
Agarwal  RTalati  MLambert  W  et al.  Fas-activated apoptosis and apoptosis mediators in human trabecular meshwork cells. Exp Eye Res 1999;68 (5) 583- 590
PubMed Link to Article
Wentz-Hunter  KShen  XOkazaki  KTanihara  HYue  BY Overexpression of myocilin in cultured human trabecular meshwork cells. Exp Cell Res 2004;297 (1) 39- 48
PubMed Link to Article
Gu  YZeng  SQiu  PPeng  DYan  G Apoptosis of bovine trabecular meshwork cells induced by dexamethasone [in Chinese]. Zhonghua Yan Ke Za Zhi 2002;38 (5) 302- 304
PubMed
Chrysis  DZaman  FChagin  ASTakigawa  MSavendahl  L Dexamethasone induces apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt-phosphatidylinositol 3′-kinase signaling pathway. Endocrinology 2005;146 (3) 1391- 1397
PubMed Link to Article
Tamm  ERRussell  PJohnson  DHPiatigorsky  J Human and monkey trabecular meshwork accumulate alpha B-crystallin in response to heat shock and oxidative stress. Invest Ophthalmol Vis Sci 1996;37 (12) 2402- 2413
PubMed
Ganea  E Chaperone-like activity of alpha-crystallin and other small heat-shock proteins. Curr Protein Pept Sci 2001;2 (3) 205- 225
PubMed Link to Article
Young  JCAgashe  VRSiegers  KHartl  FU Pathways of chaperone-mediated protein folding in the cytosol. Nat Rev Mol Cell Biol 2004;5 (10) 781- 791
PubMed Link to Article
Haq  FMahoney  MKoropatnich  J Signaling events for metallothionein induction. Mutat Res 2003;533 (1-2) 211- 226
PubMed Link to Article
Maier  CMChan  PH Role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist 2002;8 (4) 323- 334
PubMed Link to Article
Sastre  JLloret  ABorras  C  et al.  Ginkgo biloba extract EGb761 protects against mitochondrial aging in the brain and in the liver. Cell Mol Biol (Noisy-le-grand) 2002;48 (6) 685- 692
PubMed
Shirato  SMurphy  CGBloom  E  et al.  Kinetics of phagocytosis in trabecular meshwork cells. Invest Ophthalmol Vis Sci 1989;30 (12) 2499- 2511
PubMed

Correspondence

CME
Also Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
Please click the checkbox indicating that you have read the full article in order to submit your answers.
Your answers have been saved for later.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.

Multimedia

Effect of Topical Ginkgo biloba Extract on Steroid-Induced Changes in the Trabecular Meshwork and Intraocular Pressure
Arch Ophthalmol.2008;126(12):1700-1706.eTable and eFigure

eTable and eFigure -Download PDF (73 KB). This file requires Adobe Acrobat®.
Supplemental Content

Some tools below are only available to our subscribers or users with an online account.

1,425 Views
6 Citations
×

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Collections
PubMed Articles
Jobs