Da-Wen Lu

In vitro and in vivo evaluation of an ocular delivery system of 5-fluorouracil microspheres.

Solvent evaporation technique with O/O (oil/oil) emulsion was used to prepare 5-FU (5-fluorouracil) biodegradable microspheres with a polymer of poly(dl-lactide-co-glycolide) combining lecithin as an emulsifier. In vitro drug release was conducted in phosphate buffer with pH 7.4 at 37 degrees C. For in vivo studies, 10 mg of 5-FU microspheres (containing 1 mg of 5-FU) were implanted in the conjunctival area of the rabbit eye. Rabbits were sacrificed at certain time intervals within 7 days after drug application. Samples of aqueous humor and sclera were prepared and analyzed by the high performance liquid chromatography (HPLC) method. The tested 5-FU microsphere contained 10% drug with a mean particle size of 4.4 +/- 0.6 microm. The microsphere had a burst release initially (64.9 +/- 1.5%), followed by a sustained release; the cumulative release at time points of 1, 10 and 21 days were 71.9 +/- 1.9%, 80.1 +/- 2.1% and 89.8 +/- 2.3%, respectively. For in vivo studies, aqueous humor levels showed a peak at the first sampling point (2 hr), then maintained low levels of 5-FU with a range 0.2-1 microg/mL. Scleral levels were 20-80 microg/mL during a 7-day study. Our results indicate that the prepared 5-FU microsphere provided a long-term release for more than 1 week. The preparation showed no irritation and low toxicity (< 100 microg/mL). For application to the eye, it might be potentially useful as a complement drug system in glaucoma filtration surgery.

Immediate Pars Plana Vitrectomy Improves Outcome in Retained Intravitreal Lens Fragments after Phacoemulsification

Purpose: To investigate the clinical features, visual acuity outcomes and the most appropriate intervention time in patients with retained lens fragments managed by pars plana vitrectomy. Methods: This was a retrospective review of the records of 78 patients who underwent pars plana vitrectomy for retained lens fragments at the Tri-Service General Hospital from January 1, 2000, to December 31, 2006. Results: The mean age of the patients was 70 years (range, 24–92 years). There were 40 men (51%) and 38 women (49%). The mean follow-up period after surgery was 13.8 months. Forty-five patients (58%) had vitrectomy within 1 day of phacoemulsification (group A), 22 (28%) within 1 week (group B) and 11 (14%) after more than 1 week (group C). No patients in group A developed complications, and 76% achieved a final visual acuity of 6/12 or better. In group B, all patients had elevated intraocular pressure, and 45% achieved a final visual acuity of 6/12 or better. In group C, all patients presented with corneal edema, moderate or severe uveitis, and elevated intraocular pressure. Of these patients, 27% had cystoid macular edema, 36% developed retinal detachment, and 27% had a final visual acuity of 6/12 or better. Conclusion: Pars plana vitrectomy performed immediately after cataract surgery for retained lens fragments is a viable option and may achieve a better visual outcome, with reduced risk of secondary glaucoma, retinal detachment or cystoid macular edema.

Erythropoietin protects adult retinal ganglion cells against NMDA-, trophic factor withdrawal-, and TNF-α-induced damage.

Purpose This study aimed to evaluate the neuroprotective effect of EPO in the presence of N-methyl-d-aspartate (NMDA)-, trophic factor withdrawal (TFW)-, and tumor necrosis factor-alpha (TNF-α)-induced toxicity on total, small, and large retinal ganglion cells (RGCs). Methods Retinal cells from adult rats were cultured in a medium containing brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), and forskolin. Expression of RGC markers and EPOR was examined using immunocytochemistry. RGCs were classified according to their morphological properties. Cytotoxicity was induced by NMDA, TFW, or TNF-α. RGC survival was assessed by counting thy-1 and neurofilament-l double-positive cells. Results EPO offered dose-dependent (EC50 = 5.7 ng/mL) protection against NMDA toxicity for small RGCs; protection was not significant for large RGCs. Time-course analysis showed that the presence of EPO either before or after NMDA exposure gave effective protection. For both small and large RGCs undergoing trophic factor withdrawal, EPO at concentrations of 1, 10, or 100 ng/mL improved survival. However, EPO had to be administered soon after the onset of injury to provide effective protection. For TNF-α-induced toxicity, survival of small RGCs was seen only for the highest examined concentration (100 ng/mL) of EPO, whereas large RGCs were protected at concentrations of 1, 10, or 100 ng/mL of EPO. Time-course analysis showed that pretreatment with EPO provided protection only for large RGCs; early post-treatment with EPO protected both small and large RGCs. Inhibitors of signal transduction and activators of transcription such as (STAT)-5, mitogen-activated protein kinases (MAPK)/extracellular-regulated kinase (ERK), and phosphatidyl inositol-3 kinase (PI3K)/Akt impaired the protective effect of EPO on RGCs exposed to different insults. Conclusion EPO provided neuroprotection to cultured adult rat RGCs; however, the degree of protection varied with the type of toxic insult, RGC subtype, and timing of EPO treatment.

Glucosamine Modulates TNF-α–Induced ICAM-1 Expression and Function Through O-Linked and N-Linked Glycosylation in Human Retinal Pigment Epithelial Cells

PURPOSE The purpose of this article was to investigate the effects of glucosamine (GlcN) on the TNF-α-induced expression of intercellular adhesion molecule 1 (ICAM-1) and the function of ICAM-1 in ARPE-19 cells in vitro. METHODS We quantified protein levels of TNF-α-induced ICAM-1 in ARPE-19 cells with Western blotting. The effects of GlcN on O-linked glycosylation, and therefore on ICAM-1 expression, were compared after the addition of alloxan, an inhibitor of O-linked N-acetylglucosamine transferase (OGT), or O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase (O-GlcNAcase [OGA]), or after OGT gene overexpression. The effect of GlcN on the N-linked glycosylation of ICAM-1 was evaluated by the change in its molecular mass on Western blotting. The effect of O-linked glycosylation on the nuclear factor κB (NF-κB) signaling pathway was examined using an NF-κB reporter gene assay. The effect of GlcN on ICAM-1 adhesion activity was examined using an ICAM-1 adhesion assay. RESULTS GlcN, PUGNAc, and OGT overexpression inhibited TNF-α-induced ICAM-1 expression and NF-κB activity in ARPE-19 cells. Alloxan increased ICAM-1 expression and NF-κB activity in TNF-α-induced ARPE-19 cells. GlcN and tunicamycin reduced the molecular mass of TNF-α-induced ICAM-1 in ARPE-19 cells. The proteasome inhibitor MG-132 suppressed the GlcN-induced reduction in the molecular mass of TNF-α-induced ICAM-1. GlcN also attenuated the adhesion activity of TNF-α-induced ICAM-1. CONCLUSIONS GlcN inhibits ICAM-1 expression and functions by modulating the O-linked glycosylation of factors involved in NF-κB signaling and by reducing the N-linked glycosylation of TNF-α-induced ICAM-1 in ARPE-19 cells. These effects may contribute to the GlcN-mediated anti-inflammatory effects in the eye.

Angle changes before and after cataract surgery assessed by Fourier-domain anterior segment optical coherence tomography

PURPOSE: To quantify angle changes after phacoemulsification extracapsular cataract extraction using Fourier‐domain anterior segment optical coherence tomography (AS‐OCT). SETTING: Department of Ophthalmology, Tri‐Service General Hospital, National Defense Medical Center, Taiwan. DESIGN: Case series. METHODS: Fourier‐domain AS‐OCT examinations were performed in patients between 15 and 91 years of age 1 week before and 1 week after cataract surgery. The angle, angle‐opening distance at 500 μm, and trabecular–iris space area at 500 μm in the temporal and nasal quadrants were obtained. Patients were then grouped on the basis of age (<60 years or ≥60 years) and the results compared between groups. RESULTS: Eighty‐nine eyes of 80 patients were evaluated. The angle, angle‐opening distance at 500 μm, and trabecular–iris space area at 500 μm increased significantly after cataract surgery in the entire study population (P<.001). There were larger postoperative differences in all 3 parameters in nasal and temporal measurements in patients 60 years or older (all P<.001). CONCLUSIONS: The angle changed significantly after cataract surgery, particularly in the temporal quadrant and its magnitudes. Postoperative angle changes were larger in older patients, which may be related to the increase in lens volume during the aging process. Fourier‐domain AS‐OCT can be used as a rapid and safe tool in addition to diagnostic gonioscopy in assessing anterior chamber angles. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Adrenergic supersensitivity of rabbit choroidal blood vessels after sympathetic denervation.

Purpose. To demonstrate the phenomenon of adrenergic denervation supersensitivity in rabbit choroidal blood vessels after superior cervical ganglionectomy. Methods. Twenty four albino rabbits of both sexes weighing 2–3 kg were randomly separated into two groups. Twelve rabbits received bilateral superior cervical sympathectomy 2 weeks prior to the study (group s). The other 12 rabbits served as controls (group n). Four different concentrations of 0.1 ml phenylephrine, 0.05%, 0.025%, 0.013%, and 0.007% were slowly injected into the vitreous body near the retinal surface in group n and s rabbits (n = 6 in each group). The choroidal blood flow (PF), blood volume (CMBC), and velocity (V) were measured simultaneously by laser Doppler flowmetry (Perimed, PF 4001). Results. The PF showed similar decreases in group n and srabbits after injection of 0.05%, 0.025%, and 0.013% phenylephrine. With 0.007% phenylephrine, the PF remained unchanged in group n rabbits, but decreased significantly in group s rabbits (p = 0.0007). Velocity decreased similarly in both group n and s rabbits except for the 0.007% phenylephrine, concentration in which velocity decreased significantly in group s rabbits (p = 0.0001). There was no statistical difference in CMBC between group n and s rabbits at any of the test concentrations. Conclusions. The difference in PF decrease between group n and s rabbits with 0.007% phenylephrine demonstrated the existence of choroidal blood vessel denervation supersensitivity. The decrease in PF was achieved mainly through a decrease in blood cell velocity.

Trabeculectomy in patients with primary angle-closure glaucoma.

PurposeTo analyze the results of trabeculectomy in patients with primary angle-closure glaucoma. MethodsA retrospective and noncomparative case series analysis was performed on data from Taiwan, from 2001 to 2004. The outcomes of trabeculectomy in eyes with acute primary angle-closure glaucoma attack (AACG) and those with chronic primary angle-closure glaucoma (CACG) were assessed in terms of final intraocular pressure (IOP), changes to visual acuity, and the incidence of complications. ResultsA total of 52 eyes of 52 patients, 15 patients in AACG group and 37 patients in CACG group, were reviewed. The mean follow-up period was 32 months (range, 26-42 mo). In terms of no visual acuity change after surgery, there were significant differences between the CACG and AACG groups (P=0.02, Fisher exact test). In terms of final IOP control, trabeculectomy outcome was significantly worse in patients in the AACG group than those in the CACG group (P<0.01, Fisher exact test). Moreover, the complication of bleb encapsulation appeared more frequently in the AACG group than in the CACG group (P=0.02, Fisher exact test). ConclusionsCompared with CACG, trabeculectomy may not be as good for patients of AACG, because it may lead to worsened visual acuity; it also seems to present a greater failure rate, more complications, and fewer cases of surgical survival.

The changes of vitreous pH values in an acute glaucoma rabbit model.

The changes of vitreous pH values under acute glaucoma status have never been reported. In this study, we measured the changes of vitreous pH values in an acute glaucoma rabbit model. Under ketamine and xylazine anesthesia, the anterior chamber of the right eye of New Zealand white rabbits was cannulated and connected to a polygraph for continuous intraocular pressure (IOP) monitoring and a mini-pump for persistent injection of viscoelastic substance, Healon GV. The measurement of vitreous pH was done by inserting a pH probe via a third opening through the sclera into the vitreous. Different IOP levels were maintained by varied amounts of Healon GV injections into the anterior chamber. The changes of vitreous pH values following the intracameral injections were recorded and evaluated. The changes of vitreous pH values were minimal when IOPs were maintained at lower than 35 mmHg. Vitreous pH values decreased significantly from 7.32 to less than 7.03, when the IOPs were kept at 70 mmHg or higher. However, the decrease of pH values was reversible if IOP was held at 70 mmHg for less than 10 minutes and then reduced to normal level at once. In addition, with MTT viability assay, it was noted that the decrease in vitreous pH was associated with a higher percentage of retinal cell death.

Bilateral Superior Cervical Ganglionectomy Increases Choroidal Blood Flow in the Rabbit

In this study, we evaluate the effect of bilateral superior cervical ganglionectomy on albino rabbit choroidal blood flow (CBF) in changes of perfusion pressure (PP). Twenty albino rabbits of either sex weighing between 2.0 and 3.0 kg were randomly divided into two groups. The experimental group (group S) included 10 rabbits (20 eyes) that received bilateral superior cervical ganglionectomy one week prior to the study. The other 10 rabbits (20 eyes) served as a control (group N): each received the same procedure as group S except that the superior cervical ganglion was preserved. By means of a laser Doppler flowmeter (Perimed PF4001), blood cell flux (PF), velocity (V), and concentration of moving blood cells (CMBC) were recorded simultaneously while intraocular pressure was increased linearly by a syringe pump. Blood pressure and intraocular pressure were continuously monitored. The laser beam focused on the posterior pole away from major retinal vessels. When PP decreased from 75 to 0 mm Hg, in group N, PF, V and CMBC decreased from 100% to 6.94 ± 0.91%, 8.41 ± 0.87%, and 19.38 ± 1.11%; in group S, it was 17.75 ± 2.58%, 16.78 ± 1.48%, and 34.58 ± 4.42%, respectively. Group S poses higher PF, V, and CMBC than group N while PP decreased gradually. These results indicate that the superior cervical ganglion plays a role in CBF regulation. Bilateral sympathectomy led to a higher PF in the group S rabbits, indicating increased CBF. PF, V, and CMBC remained constant until PP <55 mm Hg demonstrated the existence of CBF autoregulation within a limited perfusion range in both groups. This autoregulation did not change after sympathectomy.

Bevacizumab modulates epithelial‐to‐mesenchymal transition in the retinal pigment epithelial cells via connective tissue growth factor up‐regulation

Purpose:  To investigate the effect of bevacizumab treatment on connective tissue growth factor (CTGF) expression and the induction of epithelial‐to‐mesenchymal transition in ARPE‐19 cells and human donor retinal pigment epithelium (HRPE) cells in vitro.