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Feng-Yi Yang1, Ming-Che Teng1, Maggie Lu2, Hsiang-Fa Liang2, Yan-Ru Lee1, Chueh-Chuan Yen3, Muh-Lii Liang4,5, Tai-Tong Wong51Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 2Drug Delivery Laboratory, Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, 3Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital and National Yang-Ming University School of Medicine, Taipei, 4Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, 5Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, TaiwanBackground: High-dose tissue-specific delivery of therapeutic agents would be a valuable clinical strategy. We have previously shown that repeated transcranial focused ultrasound is able to increase the delivery of Evans blue significantly into brain tissue. The present study shows that repeated pulsed high-intensity focused ultrasound (HIFU) can be used to deliver high-dose atherosclerotic plaque-specific peptide-1 (AP-1)-conjugated liposomes selectively to brain tumors.Methods: Firefly luciferase (Fluc)-labeled human GBM8401 glioma cells were implanted into NOD-scid mice. AP-1-conjugated liposomal doxorubicin or liposomal doxorubicin alone was administered followed by pulsed HIFU and the doxorubicin concentration in the treated brains quantified by fluorometer. Growth of the labeled glioma cells was monitored through noninvasive bioluminescence imaging and finally the brain tissue was histologically examined after sacrifice.Results: Compared with the control group, the animals treated with 5 mg/kg injections of AP-1 liposomal doxorubicin or untargeted liposomal doxorubicin followed by repeated pulsed HIFU not only showed significantly enhanced accumulation of drug at the sonicated tumor site but also a significantly elevated tumor-to-normal brain drug ratio (P < 0.001). Combining repeated pulsed HIFU with AP-1 liposomal doxorubicin or untargeted liposomal doxorubicin has similar antitumor effects.Conclusion: This study demonstrates that targeted or untargeted liposomal doxorubicin, followed by repeated pulsed HIFU, is a promising high-dose chemotherapy method that allows the desired brain tumor region to be targeted specifically.Keywords: repeated focused ultrasound, interleukin-4 receptor, blood-brain barrier, brain tumor, target drug delivery

Exposure to anthropogenic aerosols has been associated with a variety of adverse health effects, increased morbidity, and premature death. Although cigarette smoke poses one of the most significant public health threats, the cellular toxicity of particulate matter contained in cigarette smoke has not been systematically interrogated in a size-segregated manner. In this study, we employed a refined particle size classification to collect cigarette aerosols, enabling a comprehensive assessment and comparison of the impacts exerted by cigarette aerosol extract (CAE) on SH-SY5Y, HEK293T, and A549 cells. Exposure to CAE reduced cell viability in a dose-dependent manner, with organic components having a greater impact and SH-SY5Y cells displaying lower tolerance compared to HEK293T and A549 cells. Moreover, CAE was found to cause increased oxidative stress, mitochondrial dysfunction, and increased levels of apoptosis, pyroptosis, and autophagy, leading to increased cell death. Furthermore, we found that rutin, a phytocompound with antioxidant potential, could reduce intracellular reactive oxygen species and protect against CAE-triggered cell death. These findings underscore the therapeutic potential of antioxidant drugs in mitigating the adverse effects of cigarette aerosol exposure for better public health outcomes.

Although cigarette aerosol exposure is associated with various adverse health issues, its impact on Parkinson's disease (PD) remains elusive. Here, we investigated the effect of cigarette aerosol extract (CAE) on SH-SY5Y cells for the first time, both with and without α-synuclein (α-Syn) overexpression. We found that α-Syn aggravates CAE-induced cell death, oxidative stress, and mitochondrial dysfunction. Fluorescence cross-correlation spectroscopy (FCCS) revealed a dual distribution of α-Syn within the cells, with homogeneous regions indicative of monomeric α-Syn and punctated regions, suggesting the formation of oligomers. Moreover, we observed colocalization of α-Syn oligomers with lysosomes along with a reduction in autophagy activity. These findings suggest that α-Syn overexpression exacerbates CAE-induced intracellular cytotoxicity, mitochondrial dysfunction, and autophagy dysregulation, leading to elevated cell mortality. Our findings provide new insights into the pathogenic mechanisms linking exposure to cigarette aerosols with neurodegenerative diseases.

Background: Danshensu is a bioactive constituent of Salvia miltiorrhiza, a plant commonly used in traditional Chinese medicine. In this study, we investigated the pharmacological efficacy of sodium danshensu, or named salvianic acid A sodium (SAS) on ultraviolet B (UVB)-mediated corneal inflammatory injury in mice., Methods: Albino mice were divided into one blank control group, and three UVB radiation groups, i.e. SAS-untreated group, and prophylactic treatment groups with SAS at 1 and 10 mg/kg via oral administration. The structure integrity and inflammatory changes of cornea were assessed by surface evaluation of smoothness, topographic distortion, opacity, lissamine green staining, and histologic tissue staining. The inflammatory cytokines was measured by bead-based ELISA assays., Results: Prophylactic treatment of SAS significantly inhibited pathologic changes, improved tissue structural integrity, and reduced inflammatory injury in the cornea after UVB exposure. Dosing with SAS treatment attenuated the incidence rate of leukocyte influx by inhibit increase of interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α. Treatment with 10 mg/kg SAS was more effective in preventing the onset of corneal damage than that with 1 mg/kg SAS., Conclusions: These results indicate that SAS exhibit the pharmacological efficacy on corneal protection through its inhibition of UVB induced photodamage and subsequently inflammatory injury in vivo.

Aim: To evaluate and compare the diagnostic capabilities of peripapillary retinal nerve fiber layer (p-RNFL) parameters of Spectralis optical coherence tomography (OCT) versus Stratus OCT to detect glaucoma in patients with high myopia., Methods: This is a retrospective, cross-sectional study. Sixty highly myopic eyes of 60 patients were enrolled, with 30 eyes in the glaucoma group and 30 eyes in the control group. All eyes received peripapillary imaging of the optic disc using Stratus and Spectralis OCT. Areas under the receiver operating characteristic curve (AUROC) and the sensitivity at specificity of >80% and >95% for p-RNFL parameters obtained using the two devices to diagnose glaucoma were analysed and compared., Results: In Spectralis OCT, p-RNFL thickness parameters with the largest AUROC were the temporal-inferior sector (0.974) and the inferior quadrant (0.951), whereas in Stratus OCT, the best parameters were the 7-o'clock sector (0.918) and the inferior quadrant (0.918). Compared to the Stratus OCT parameters, the Spectralis OCT parameters demonstrated generally higher AUROC; however, the difference was not statistically significant., Conclusion: The best p-RNFL parameters for diagnosing glaucoma in patients with high myopia were the temporal-inferior sector on Spectralis OCT and the 7-o'clock sector on Stratus OCT. There were no significant differences between the AUROCs for Spectralis OCT and Stratus OCT, which suggest that the glaucoma diagnostic capabilities of these two devices in patients with high myopia are similar.

The purpose of this study was to investigate the spectrum of organisms causing endophthalmitis after cataract surgery in a tertiary medical center in Taiwan and the antibiotic susceptibilities. This was a retrospective case series study. Patients with endophthalmitis after cataract surgery from January 2004 to July 2015 were reviewed. The outcome measures included the identification of isolates, antibiotic susceptibilities, and final visual outcomes. Twenty-one organisms were isolated from 19 cases. The most common organisms were Enterococcus in 38.1 %, especially Enterococcus faecalis, followed by Staphylococcus epidermidis in 28.6 % and Staphylococcus aureus in 9.5 %. All of the Gram-positive isolates tested were susceptible to vancomycin (100 %), and ceftazidime and amikacin were susceptible for Gram-negative organisms. The Gram-positive organisms remain to be the predominant cause of postoperative endophthalmitis, and Enterococcus species has had an increasing incidence. Vancomycin is still the most powerful antibiotic for Gram-positive organisms, while ceftazidime and amikacin are effective for Gram-negative bacteria.

Context: Only a few studies have assessed intraocular pressure (IOP) changes during the water drinking test (WDT) in patients with primary angle-closure glaucoma (PACG)., Aims: The aim of this study is to investigate IOP changes during WDT in patients with PACG versus primary open-angle glaucoma (POAG)., Settings and Design: This was a prospective and single tertiary center study., Materials and Methods: PACG and POAG patients (n = 15 each) without prior glaucoma surgery were enrolled and subjected to WDT, wherein they consumed an amount of water proportional to their body weight within 10 min. IOP was measured at baseline and every 15 min for 1 h after water intake., Statistical Analysis Used: Intergroup comparisons were performed using Mann-Whitney U-test for continuous variables and Chi-square test for categorical variables. Wilcoxon signed-ranks test was used for comparisons of IOP before and after water intake in the two groups. Regression analysis was used to determine factors associated with IOP fluctuations during WDT., Results: IOP changes over 1 h after water intake showed no significant differences between groups. The mean maximum fluctuation from baseline was 3.61 ± 2.49 and 3.79 ± 1.91 mmHg, respectively, in the PACG and POAG groups. The mean peak IOP was 19.17 ± 4.32 and 19.87 ± 3.44 mmHg in the PACG and PAOG groups, respectively. The axial length and anterior chamber depth showed no correlations with IOP fluctuations., Conclusions: We found similar IOP fluctuation curves and peak IOP values in both PACG and POAG patients subjected to WDT. These findings suggest that WDT is a useful test to induce IOP peaks in both POAG and PACG patients.

Purpose: The aim was to investigate the visual outcomes of treatment with spectacles for bilateral high refractive amblyopia in children three to eight years of age., Methods: Children with previously untreated bilateral refractive amblyopia were enrolled. Bilateral high refractive amblyopia was defined as visual acuity (VA) being worse than 6/9 in both eyes in the presence of 5.00 D or more of hyperopia, 5.00 D or more of myopia and 2.00 D or more of astigmatism. Full myopic and astigmatic refractive errors were corrected, and the hyperopic refractive errors were corrected within 1.00 D of the full correction. All children received visual assessments at four-weekly intervals. VA, Worth four-dot test and Randot preschool stereotest were assessed at baseline and every four weeks for two years., Results: Twenty-eight children with previously untreated bilateral high refractive amblyopia were enrolled. The mean VA at baseline was 0.39 ± 0.24 logMAR and it significantly improved to 0.21, 0.14, 0.11, 0.05 and 0.0 logMAR at four, eight, 12, 24 weeks and 18 months, respectively (all p = 0.001). The mean stereoacuity (SA) was 1,143 ± 617 arcsec at baseline and it significantly improved to 701, 532, 429, 211 and 98 arcsec at four, eight, 12, 24 weeks and 18 months, respectively (all p = 0.001). The time interval for VA achieving 6/6 was significantly shorter in the eyes of low spherical equivalent (SE) (-2.00 D < SE < +2.00 D) than in those of high SE (SE > +2.00 D) (3.33 ± 2.75 months versus 8.11 ± 4.56 months, p = 0.0005). All subjects had normal fusion on Worth four-dot test at baseline and all follow-up visits., Conclusion: Refractive correction with good spectacles compliance improves VA and SA in young children with bilateral high refractive amblyopia. Patients with greater amounts of refractive error will achieve resolution of amblyopia with a longer time., (© 2016 Optometry Australia.)

Purpose: To compare the diagnostic abilities of spectral-domain optical coherence tomography (SD-OCT; Spectralis OCT) and time-domain OCT (TD-OCT; Stratus OCT). Changes in macular parameters in highly myopic eyes of glaucoma patients and highly myopic eyes of glaucoma suspects were evaluated and compared., Methods: We collected data from 72 highly myopic eyes (spherical equivalent, ≤-6.0D). Forty-one eyes had perimetric glaucoma and 31 eyes were suspected to have glaucoma (control group). All eyes underwent SD-OCT and TD-OCT imaging. Area under the receiver operating characteristic (AUROC) curve and sensitivity were examined on macular volume and thickness parameters at a fixed specificity and compared between groups., Results: The highest TD-OCT AUROC curves were found using outer inferior sector macular thickness (AUROC curve, 0.911) and volume (AUROC curve, 0.909). The highest SD-OCT AUROC curves were found using outer inferior region thickness (AUROC curve, 0.836) and volume (AUROC curve, 0.834). The difference between the two imaging modalities was not statistically significant (thickness, p = 0.141; volume, p = 0.138). The sensitivity of TD-OCT macular outer inferior average thickness was highest and was 88.2%, with a specificity of 80.4%. The sensitivity of TD-OCT average volume measurements in this same region was 76.5%, with a specificity of 91.3%. The SD-OCT average thickness measurements also had the highest sensitivity in this region, which was 78.6%, with a specificity of 82.1%. The SD-OCT volume measurements had a sensitivity of 67.9%, with a specificity of 92.3%., Conclusions: Both SD-OCT and TD-OCT measurements of outer inferior macular thickness and volume can differentiate between eyes of glaucoma patients and glaucoma suspects with high myopia. These independent predictors all had good sensitivity. Based on our results, SD-OCT and TD-OCT have similar diagnostic abilities. These parameters may provide useful additional data in highly myopic eyes to complement standard glaucoma diagnosis tools.

Purpose: Excessive exposure to sunlight may be a risk factor for ocular diseases and reduced visual performance. This study was designed to examine the ability of an ultraviolet (UV)-blocking spectacle lens to prevent visual acuity decline and ocular surface disorders in a mouse model of UVB-induced photokeratitis., Methods: Mice were divided into 4 groups (10 mice per group): (1) a blank control group (no exposure to UV radiation), (2) a UVB/no lens group (mice exposed to UVB rays, but without lens protection), (3) a UVB/UV400 group (mice exposed to UVB rays and protected using the CR-39™ spectacle lens [UV400 coating]), and (4) a UVB/photochromic group (mice exposed to UVB rays and protected using the CR-39™ spectacle lens [photochromic coating]). We investigated UVB-induced changes in visual acuity and in corneal smoothness, opacity, and lissamine green staining. We also evaluated the correlation between visual acuity decline and changes to the corneal surface parameters. Tissue sections were prepared and stained immunohistochemically to evaluate the structural integrity of the cornea and conjunctiva., Results: In blank controls, the cornea remained undamaged, whereas in UVB-exposed mice, the corneal surface was disrupted; this disruption significantly correlated with a concomitant decline in visual acuity. Both the UVB/UV400 and UVB/photochromic groups had sharper visual acuity and a healthier corneal surface than the UVB/no lens group. Eyes in both protected groups also showed better corneal and conjunctival structural integrity than unprotected eyes. Furthermore, there were fewer apoptotic cells and less polymorphonuclear leukocyte infiltration in corneas protected by the spectacle lenses., Conclusions: The model established herein reliably determines the protective effect of UV-blocking ophthalmic biomaterials, because the in vivo protection against UV-induced ocular damage and visual acuity decline was easily defined.

Background: The aim was to assess the macular parameters of glaucoma in highly myopic subjects., Methods: In a retrospective study, we enrolled 72 highly myopic eyes (spherical equivalent up to -6.00 D), of which 31 had early to moderate glaucoma and 41 formed a control group. The diagnosis of early to moderate glaucoma was based on glaucomatous visual-field defects with a mean deviation better than -12 dB. All patients underwent imaging with Stratus optical coherence tomography. We subsequently performed analysis for macular volume and thickness parameters between two groups., Results: Using Stratus optical coherence tomography, we found significantly lower values in both macular thickness and volume over outer superior, outer nasal, outer inferior, inner temporal and inner inferior regions in highly myopic eyes with early to moderate glaucoma than in the control group. The macular outer inferior average thickness, macular outer inferior average volume and total average macular volume on Stratus optical coherence tomography had a best combination of sensitivity and specificity (85.4 and 74.2 per cent, respectively). The outer inferior average volume and outer inferior average thickness parameters showed areas under the receiver operating characteristic curve value of 0.85 (95% CI 0.76 to 0.94), which were best for discriminating between early to moderate glaucoma and the control group., Conclusion: Using Stratus optical coherence tomography, the outer inferior macular volume is the only independent predictor for detecting early to moderate glaucoma in high myopia. The macular outer inferior thickness and volume parameters showed good sensitivity, specificity and area under the receiver operating characteristic curve. These parameters may provide a complementary glaucomatous diagnosis in highly myopic eyes., (© 2014 The Authors. Clinical and Experimental Optometry © 2014 Optometrists Association Australia.)

Purpose: To compare postoperative outcomes and assess factors associated with intraocular pressure (IOP) reduction after phacotrabeculectomy in patients with primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG)., Methods: This is a retrospective study of patients who underwent phacotrabeculectomy between 2010 and 2013. Factors including age, gender, visual field (VF), the number of glaucoma medications used, biometric changes, IOP, and surgical success rates were compared between groups., Results: There were 27 PACG and 34 POAG patients. The PACG group had a greater mean IOP reduction after phacotrabeculectomy compared to the POAG group (5.5 ± 7.9 mmHg versus 2.0 ± 4.2 mmHg; p = 0.03). However, the final mean IOP was similar between the two groups (PACG: 12.2 ± 4.8 mmHg, POAG: 12.3 ± 3.1 mmHg; p = 0.92). Phacotrabeculectomy resulted in a mean decrease in axial length (AL) of 0.16 ± 0.15 mm in PACG and 0.16 ± 0.11 mm in POAG ( p = 0.96), and an increase in anterior chamber depth (ACD) of 1.41 ± 0.91 mm in PACG, and 0.87 ± 0.86 mm in POAG ( p = 0.04). At 2 years follow-up, the cumulative success rate of phacotrabeculectomy was 74% in PACG and 62% in POAG. Multivariate analysis found that early glaucoma stage, greater postoperative increase in ACD, and high preoperative IOP were factors associated with greater IOP reduction., Conclusion: Postoperative success rates and mean IOP on the final visit after phacotrabeculectomy were similar between the PACG and POAG groups. Factors associated with IOP reduction were greater postoperative increase in ACD, and high preoperative IOP., Competing Interests: Conflicts of interest: None of the contributing authors declare any conflicts of interest.

The clinical application of chemotherapy to brain tumors has been severely limited because the blood-brain barrier (BBB) often prevents therapeutic levels from being achieved. Here we show that pulsed HIFU and human atherosclerotic plaque-specific peptide-1 (AP-1)-conjugated liposomes containing doxorubicin (AP-1 Lipo-Dox) act synergistically in an experimental brain tumor model. We developed an intracranial brain-tumor model in NOD-scid mice using human brain glioblastoma multiforme (GBM) 8401 cells. Pulsed HIFU was used to transcranially disrupt the BBB in these mouse brains by delivering ultrasound waves in the presence of microbubbles. Prior to each sonication, AP-1 Lipo-Dox or unconjugated Lipo-Dox was administered intravenously, and the concentration in the brains was quantified by fluorometer. Compared to control animals treated with injections of AP-1 Lipo-Dox or unconjugated Lipo-Dox, animals receiving the drug followed by pulsed HIFU exhibited enhanced accumulation of the drug in tumor cells. Drug injection with sonication increased the tumor-to-normal brain doxorubicin ratio of the target tumors by about twofold compared with the control tumors. Moreover, the tumor-to-normal brain ratio was highest after the injection of AP-1 Lipo-Dox with sonication. Combining sonication with AP-1 Lipo-Dox also significantly inhibited tumor growth compared with chemotherapy alone. There was a modest but significant increase in the median survival time in mice treated with AP-1 Lipo-Dox followed by pulsed HIFU, compared to those treated with AP-1 Lipo-Dox without sonication. The use of AP-1-conjugated liposomes carrying cytotoxic agents followed by pulsed HIFU represents a feasible approach for enhanced targeted drug delivery in brain tumor therapies., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Background: Intraocular pressure (IOP) measurements are affected by the central cornea thickness (CCT). The conventional method for CCT measurement is ultrasonic pachymetry. However, noncontact procedures lower the risk of infection and corneal damage. In this study, we compared the CCT measured by Orbscan II, SP3000P, and ultrasonic pachymetry in patients with glaucoma or glaucoma suspect., Methods: The CCT of 208 eyes (46 eyes with glaucoma suspect, 42 with primary angle-closure glaucoma, and 120 with primary open-angle glaucoma) was measured using Orbscan II, SP3000P, and ultrasonic pachymetry. We compared the linear correlation of the CCT between each mode., Results: The mean CCT measured by Orbscan II (563.63 ± 35.867 µm) was larger than with the other two devices. There were significant linear correlations between measurements with ultrasonic pachymetry and Orbscan II (Pearson correlation coefficient (r) = 0.793, p < 0.001), ultrasonic pachymetry and SP3000P (r = 0.890, p < 0.001), and Orbscan II and SP3000P (r = 0.803, p < 0.001). We divided the participants into 3 groups on the basis of the CCT measured with ultrasonic pachymetry: ≤ 500 µm, > 500 µm to ≤ 578 µm, and > 578 µm. There was no significant linear correlation between ultrasonic pachymetry and Orbscan II in the thin group. But, in the intermediate and thick CCT groups, there were significant linear correlations between each of the three devices., Conclusion: We showed good linear correlations of CCT measurements between each of 3 devices, especially in the intermediate and thickest CCTs. These results will be helpful in predicting the relationship between IOP and CCT for the diagnosis and screening of glaucoma; even we used optic systems.

Background/purpose: To investigate the clinical manifestations, disease activity and prognosis in different types of vasculitis., Methods: The charts of pediatric patients with vasculitis diagnosed from December 1997 to December 2007 were retrospectively reviewed. The first clinical manifestations and laboratory results were recorded at the time of diagnosis, and outcome evaluations with history of flare-ups were analyzed., Results: A total of 508 vasculitis patients were included in this study, of whom 124 had Henoch-Schönlein purpura (HSP), and 351 had Kawasaki disease (KD). Hematuria was observed in 79% of recurrent HSP patients at the time of diagnosis, and was associated with an increased risk of relapse (p = 0.000). In Kawasaki disease, the clinical symptoms with erythematous changes in Bacille Calmette-Guérin scars and coronary artery dilatation were more prominent in patients younger than 1 year old, and lymphadenopathy was more common in patients older than 1 year old (p = 0.001). The risk of coronary dilatation was significant in the patients with an initial presentation of thrombocytosis, and greater in patients younger than 1 year old (p = 0.027). Thrombocytopenia was more prominent in vasculitis-associated autoimmune diseases. Marked lymphocytosis with increased C-reactive protein levels was significantly noted in urticarial vasculitis patients compared with HSP patients in multivariate logistic regression analysis., Conclusion: Vasculitis disease activity and prognosis were associated with initial laboratory results and clinical manifestations. Further large-scale clinical trials are warranted to validate these findings., (Copyright © 2011. Published by Elsevier B.V.)

The goal of this study was to evaluate the pharmacokinetics of targeted and untargeted (111)In-doxorubicin liposomes after these have been intravenously administrated to tumor-bearing mice in the presence of blood-brain barrier disruption (BBB-D) induced by focused ultrasound (FUS). An intracranial brain tumor model in NOD-scid mice using human brain glioblastoma multiforme (GBM) 8401 cells was developed in this study. (111)In-labeled human atherosclerotic plaque-specific peptide-1 (AP-1)-conjugated liposomes containing doxorubicin (Lipo-Dox; AP-1 Lipo-Dox) were used as a microSPECT probe for radioactivity measurements in the GBM-bearing mice. Compared to the control tumors treated with an injection of (111)In-AP-1 Lipo-Dox or (111)In-Lipo-Dox, the animals receiving the drugs followed by FUS exhibited enhanced accumulation of the drug in the brain tumors (p<0.05). Combining sonication with drugs significantly increased the tumor-to-normal brain doxorubicin ratio of the target tumors compared to the control tumors. The tumor-to-normal brain ratio was highest after the injection of (111)In-AP-1 Lipo-Dox with sonication. The (111)In-liposomes micro-SPECT/CT should be able to provide important information about the optimum therapeutic window for the chemotherapy of brain tumors using sonication.

Purpose: Ultraviolet B (UVB) irradiation activates nuclear factor-kappa B (NF-κB) and inducible nitric oxide synthase (iNOS) in the cornea, resulting in inflammatory responses and malondialdehyde (MDA) accumulation. This study aims to determine the effect of zerumbone, a potent NF-κB inhibitor and inflammation modulators, on UVB-induced corneal damages in a mouse model., Methods: Fifty female imprinting control region (ICR) mice were randomly divided into five groups. The mice were anaesthetized with their ocular surfaces exposed to UVB light (0.72J/cm(2)/daily), followed by daily dietary zerumbone supplements at 0, 1, 10, and 100 mg/kg of bodyweight. Mice without zerumbone supplements were used as treatment controls and mice without UVB irradiation as blank controls. Corneal surface damages were graded according to smoothness, opacity, and the extent of lissamine green staining. Histopathological changes were also examined, along with the expression of NF-κB, iNOS, and tumor necrosis factor-α (TNF-α). MDA accumulation and the levels of two antioxidant enzymes, glutathione (GSH) and GSH reductase (GR) were also examined., Results: UVB irradiation caused significant damages to cornea, including sustained inflammation, apparent corneal ulcer, and severe epithelial exfoliation, leading to thinning of corneal epithelial layer, and infiltration of polymorphonuclear leukocytes. NF-κB expression was highly activated with nuclear translocation. The expression of iNOS and TNF-α were increased. MDA accumulation was also increased in both the corneal epithelial layer and the stroma. With dietary zerumbone, corneal damages were ameliorated in a dose-dependent manner. NF-κB activation and its nuclear translocation were blocked with decreased expression of iNOS and TNF-α. Infiltration of polymorphonuclear leukocytes was also blocked by dietary zerumbone. Besides, MDA accumulation was reduced with concomitant increase of GSH and GR levels., Conclusions: Dietary zerumbone prevents UVB-induced corneal damages by inhibition of NF-κB, iNOS, and TNF-α, with concomitant reduction of MDA accumulation and increase of GSH and GR levels in the mouse model. Results of this study suggest that dietary zerumbone may be used as a prophylactic agent against UVB-induced photokeratitis.

Purpose: To investigate the preventive effect of dietary zerumbone against UVB-induced cataractogenesis., Methods: A total of 50 six-week-old female ICR mice were split into five groups (each contained 10 mice) and exposed to UVB (0.72 J/cm(2)/daily) at noon for 7 days, except for the blank control group. The mice with UVB exposure were fed with zerumbone as a dietary supplement at 0, 1, 10, and 100 mg/kg of bodyweight, respectively, starting from one day before UVB exposure. On day 7, at 4 h after UVB exposure, all mice were subjected to cataract examination and lens opacity scoring, in correlation with levels of MDA (malondialdehyde), GSH (glutathione), GR (GSH reductase), GPx (glutathione peroxidase), and SOD (superoxide dismutase) in the lens., Results: Dietary zerumbone at 100 mg/kg after UVB exposure was effective in decreasing lens opacity scores (p<0.001) and to reduce MDA (p<0.001), while GSH and GR levels were significantly increased (both p<0.001) in the lens. SOD was also increased with dietary zerumbone at 100 mg/kg (p=0.115), whereas GPx (p=0.171) levels were lower as compared with those without zerumbone after UVB exposure., Conclusions: These results suggest that zerumbone may protect against UVB-induced cataractogensis through reducing lipid peroxides and enhancing the endogenous antioxidant GSH level and GR activity.

Unlabelled: This study evaluated the pharmacokinetics of (99m)Tc-diethylenetriamine pentaacetate acid ((99m)Tc-DTPA) after intravenous administration in healthy and F98 glioma-bearing F344 rats in the presence of blood-brain barrier disruption (BBB-D) induced by focused ultrasound (FUS). The pharmacokinetics of the healthy and tumor-containing brains after BBB-D were compared to identify the optimal time period for combined treatment., Methods: Healthy and F98 glioma-bearing rats were injected intravenously with Evans blue (EB) and (99m)Tc-DTPA; these treatments took place with or without BBB-D induced by transcranial FUS of 1 hemisphere of the brain. The permeability of the BBB was quantified by EB extravasation. Twelve rats were scanned for 2 h to estimate uptake of (99m)Tc radioactivity with respect to time for the pharmacokinetic analysis. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed to examine tissue damage., Results: The accumulations of EB and (99m)Tc-DTPA in normal brains or brains with a tumor were significantly elevated after the intravenous injection when BBB-D was induced. The disruption-to-nondisruption ratio of the brains and the tumor-to-ipsilateral brain ratio of the tumors in terms of radioactivity reached a peak at 45 and 60 min, respectively. EB injection followed by sonication showed that there was an increase of about 2-fold in the tumor-to-ipsilateral brain EB ratio of the target tumors (7.36), compared with the control tumors (3.73). TUNEL staining showed no significant differences between the sonicated tumors and control tumors., Conclusion: This study demonstrates that (99m)Tc-DTPA micro-SPECT/CT can be used for the pharmacokinetic analysis of BBB-D induced by FUS. This method should be able to provide important information that will help with establishing an optimal treatment protocol for drug administration after FUS-induced BBB-D in clinical brain disease therapy.

Purpose: Hyperhomocysteinemia is known to cause degeneration of retinal ganglion cells, but its influence on photoreceptors remains largely unknown. In particular, the role of homocysteine-thiolactone (Hcy-T)--the physiologic metabolite of homocysteine that has been proven to be more cytotoxic than homocysteine itself--as a factor that causes retinopathy, has not been defined. This study aimed to investigate the toxic effects of excessive Hcy-T in a mouse model., Methods: A total of 60 six-week-old female ICR mice were used in this study. The mice were divided into 3 experimental groups and 2 control groups. The mice in the experimental groups were subjected to intravitreal injections of Hcy-T to reach final estimated intravitreal concentrations at 5, 25, and 200 μM, respectively. Mice without injection (blank) and with 0.9 NaCl injections (sham injection) were used as controls. The mice with 200 μM Hcy-T were sacrificed at days 7, 15, 45, and 90 after injection and the mice with 5 or 25 μM Hcy-T were sacrificed at day 90, with the controls sacrificed at day 15 or 90 for comparison. Semi-quantitative dot-blot analysis was performed for confirmation of retinal homocysteinylation. The mouse retinas were evaluated microscopically, with the thickness of total and specific retinal layers determined. Immunohistochemical analysis was performed and the labeled cells were quantified to determine the effects of excessive Hcy-T on specific retinal cells., Results: Dose-dependent retinal homocysteinylation after Hcy-T injection was confirmed. The homocysteinylation was localized in the outer and inner segments of photoreceptors and the ganglion cell layer (GCL). Retinal cell degenerations were found in the GCL, inner nuclear layer, and outer nuclear layer at day 90 after 200 µM Hcy-T injection. Significant thickness reduction was found in the total retina, outer nuclear layer, and the outer and inner segment layers. A trend of thickness reduction was also found in the GCL and inner nuclear layer, although this was not statistically significant. The rhodopsin⁺ photoreceptors and the calbindin⁺ horizontal cells were significantly reduced at day 15, and were nearly ablated at day 90 after 200 μM Hcy-T injection (p<0.001 for both day 15 and day 90), which was not seen in the sham injection controls. The Chx-10⁺ or the Islet-1⁺ bipolar cells and the Pax-6⁺ amacrine cells were severely misarranged at day 90, but no significant reduction was found for both cell types. The GFAP⁺ Müller cells were activated at day 15, but were not significantly increased at day 90 after the injection., Conclusions: Excessive retinal homocysteinylation by Hcy-T, a condition of hyperhomocysteinemia, could lead to degeneration of photoreceptors, which might lead to retinopathies associated with severe hyperhomocysteinemia or diabetes mellitus.

Purpose: Longitudinal follow-up of peripapillary retinal nerve fiber layer (RNFL) thickness after an episode of acute primary angle closure (APAC) using Stratus optical coherence tomography (OCT)., Methods: Seventeen patients who had experienced a single unilateral APAC episode (intraocular pressure, >50 mm Hg) were enrolled. The average and superior, temporal, inferior, and nasal quadrant RNFL thicknesses of the affected and fellow eyes at 1, 4, and 12 weeks after remission were compared by using StratusOCT. The relationship between average RNFL thickness and interval of follow-up were evaluated with regression analysis., Results: The mean duration of the APAC episode was 13.8 hours (range, 3-40). Comparison of the average and four quadrant RNFL thicknesses in the affected eyes longitudinally showed significant differences between 1 and 4, and 1 and 12 weeks, but not between 4 and 12 weeks. The average and four-quadrant RNFL thicknesses for the affected eyes were greater than the analogous values for fellow eyes at 1 week. In contrast, the inferior- and superior-quadrant RNFL thicknesses for the affected eyes were lower at 4 and 12 weeks, whereas the average and nasal quadrant values for the affected eyes were lower than those in fellow eyes at 12 weeks. Average RNFL thickness for the affected eyes was correlated with the interval of follow-up by using inverse regression analysis (P < 0.001; R(2) = 0.60). Controlling for duration of APAC episode, the interval of follow-up on RNFL thickness reduction remained significant (P < 0.001, r = -0.69)., Conclusions: This study demonstrated an initial increase in diffuse RNFL thickness after a single APAC episode, followed by a subsequent decrease.

Background: To find the correlations and differences between the nerve fiber layer parameters of a GDx (polarimetric retinal nerve fiber analyzer) in single high quality images and those in the mean image from three high quality images., Methods: The results of scanning laser polarimetry were selected from 63 eyes of 63 patients (31 male and 32 female). Complete ophthalmic examinations included slit-lamp biomicroscopy, dilated pupil for vitreous and ocular fundus examination, applanation tonometry, and subjective or objective refraction. There were no significant ophthalmic disorders other than glaucoma and mild lens opacity. Nerve fiber layer thickness of each eye was measured with GDx and at least three high quality images (passing the software's quality criteria) were obtained. The best of these three high quality images was selected as a single high quality image (Group One). The mean image (Group Two) was calculated from the same three high quality images., Results: We found that there was a high correlation in all nerve fiber layer parameters between Group One and Group Two. There was no significant difference in the seven relative values of nerve fiber layer parameters (Number, Ellipse Modulation, Symmetry, Superior Ratio, Inferior Ratio, Superior/Nasal and Maximal Modulation) between Group One and Group Two. Group One had significantly higher values compared with Group Two in the other seven absolute values of nerve fiber layer parameters (Average Thickness, Ellipse Average, Superior Average, Inferior Average, Superior Integral, Superior Maximal and Inferior Maximal)., Conclusion: It is reasonable to take a mean image from three good quality images from a cooperative patient. If only one high quality image can be obtained in repeated acquisition of GDx, the seven relative values of nerve fiber layer parameters in this high quality image can be used as a base-line image for detecting retinal nerve fiber layer defects and for determining changes of retinal nerve fiber layer thickness in sequential images of GDx.

Background: The objective of this study was to evaluate the incidence, predisposing surgery, management and final visual outcome of postoperative endophthalmitis over a 14-year period at one institute., Methods: This retrospective study reviewed all intraocular operations performed between 1 January 1991 and 31 August 2004 at Chang Gung Memorial Hospital, Kaohsiung Medical Center, to determine the incidence of nosocomial postoperative endophthalmitis and the characteristics of patients who developed this condition., Results: The overall incidence of postoperative endophthalmitis after intraocular surgery was 0.19% (56 out of 30,219). Postoperative endophthalmitis developed in 56 eyes in 56 patients during the study period. The condition developed after cataract surgery in 46 eyes, after penetrating keratoplasty in 6 eyes, after filtering surgery in 2 eyes, after secondary intraocular lens implant in 1 eye, and after vitrectomy in 1 eye. Postoperative endophthalmitis was culture-positive in 31 cases (55%). The most frequent organism isolated was coagulase-negative Staphylococcus. Factors associated with better visual acuity outcomes included low virulence of isolated pathogen, initial visual acuity of counting fingers or better, and history of cataract surgery compared with other intraocular surgery., Conclusion: The overall incidence of endophthalmitis after intraocular surgery was 0.19%. The results of this 14-year review from a local medical center may serve as a source of comparison for other centers and future studies.

Background: To determine the etiology and morphological features of cataracts in a myopia-endemic area of Taiwan., Methods: This retrospective case-controlled study comprised 332 patients (440 eyes) who underwent cataract-extraction surgery, 163 patients (220 eyes) constituting the presenile cataract study group, and 169 patients (220 eyes) constituting the senile cataract control group. Each group was divided into idiopathic, diabetic, and highly myopic subgroups. Age, gender, ocular and systemic comorbidities, and the types of cataract featured were recorded and analyzed. For the statistical analysis, chi 2 test was used., Results: The mean ages of study subjects were 45.5 +/- 7.6 years for the presenile and 68.7 +/- 8.0 years for the senile cataract groups. We also observed a greater number of cataracts in highly myopic eyes of the presenile group (17.27%) compared to the senile group (0.90%) (p < 0.0001). The distribution of cataract types for the 3 presenile cataract subgroups revealed that nuclear sclerosis contributed most to presenile cataracts in highly myopic patients (p = 0.001), and correspondingly, the posterior subcapsular opacity contributed most in the idiopathic and diabetic cataract subgroups (p = 0.001)., Conclusions: We observed a greater number of cataracts from highly myopic eyes in the presenile group than in the senile group in southern Taiwan, a myopia-endemic area. Nuclear sclerosis contributed most to the presenile cataracts in the highly myopic subgroup.

Background: To detect differences in retinal nerve fiber layer (RNFL) measurements in absolute, advanced and early glaucoma with scanning laser polarimetry (The Nerve Fiber Analyzer GDx), and to assess the usefulness and limitations of this technique for longitudinal follow-up of glaucoma patients., Methods: This is a prospective, cross-sectional study. Twenty-one eyes of 21 patients with absolute glaucoma, twenty-six eyes of 26 patients with advanced glaucoma and twenty-four eyes of 24 patients with early glaucoma were imaged using scanning laser polarimetry. The twelve standard GDx measurement parameters were compared using ANOVA (analysis of variance) and the Tukey test., Results: No significant differences were demonstrated for any of the twelve GDx measurement parameters between absolute and advanced glaucoma cases. There were significant differences for some GDx parameters, including the GDx number (p < 0.0001) superior ratio (p < 0.0001), inferior ratio (p < 0.0001), superior/nasal ratio (p < 0.0001), maximum modulation (p < 0.0001), ellipse modulation (p < 0.0001) and inferior average (p = 0.001) between early and advanced glaucoma, and, between early and absolute glaucoma. Significant differences were demonstrated for the superior average (p = 0.01) parameter between early and absolute glaucoma, but not between early and advanced glaucoma., Conclusions: For follow-up of glaucoma progression, RNFL measurements using scanning laser polarimetry are more useful in the early stage than in the advanced stage.

Aims: The aim of this study was to assess the risk of postoperative endophthalmitis associated with different prophylactic protocols, using povidone-iodine disinfection of the skin and conjunctiva in patients undergoing cataract surgery over an 8-year period at Chang Gung Memorial Hospital (Kaohsiung, Taiwan)., Method: This was a retrospective, comparative, case-controlled study. Data were collected on patients who received different prophylactic disinfection protocols prior to extracapsular cataract extraction with intraocular lens implantation between January 1992 and January 2000. Possible risk factors for the occurrence of postoperative endophthalmitis were analyzed., Results: Medical charts for a total of 10,614 extracapsular cataract extraction surgeries performed during the 8-year period were reviewed, and 12 eyes that developed postoperative endophthalmitis were identified and analyzed. Another 120 eyes matched for age and gender of the patient were randomly selected as controls. Skin preparation with 5% povidone-iodine was associated with an increased risk of postoperative infection, compared to that of 10% povidoneiodine. On the conjunctiva, a lack of 5% povidone-iodine disinfection was associated with an increased risk of postoperative infection, compared to 5% povidone-iodine disinfection., Conclusions: Preoperative skin disinfection with 10% povidone-iodine and conjunctival disinfection with 5% povidone-iodine significantly reduced the relative risk of postoperative endophthalmitis.

A 40-year-old male patient suffered from traumatic optic neuropathy in his right eye. Scanning laser polarimetry was arranged at 2 weeks, 9 weeks, 13 weeks, 24 weeks, and 34 weeks after the trauma. Manual or automated visual field testing was also arranged at 1 week, 5 weeks, 16 weeks, and 28 weeks correspondingly. The data revealed using scanning laser polarimetry (program GDx, version 1.0.05; Laser Diagnostic Technologies, San Diego, Calif, USA) were nearly normal at 2 weeks after trauma, but lower visual field loss was revealed using visual field testing within 2 weeks after the trauma. The superior hump of the GDx deviated from normal at about 9 weeks and some GDx parameters (the Number, Superior/Nasal, Ellipse Modulation (Ellipse Mod.), Maximal Modulation (Max. Mod.), Symmetry, Superior Ratio (Super. Ratio)) became worse later in the series. We propose that visual field defects might be present before retinal nerve fiber layer loss. In this case, scanning laser polarimetry for evaluating the severity of traumatic optic neuropathy was limited especially within 2 weeks after the trauma.

Background: To investigate the risk factors and probability of blindness in patients treated for glaucoma., Methods: The study design was a retrospective, hospital-based, clinical chart, review study. Medical records were reviewed from patients seen between January 2003 and December 2003 at the Kaohsiung Chang Gung Memorial Hospital eye clinic, who had been diagnosed with glaucoma in 1986 or later and who had been treated for at least 2 years for glaucoma., Results: A total of 186 charts were reviewed, which included 66 patients who were blind in at least one eye from glaucoma on presentation. A total of 172 patients and 290 eyes were followed-up for a mean duration of 10.6 +/- 4.67 years. Twenty seven patients and 31 eyes developed blindness from glaucoma during follow-up. The Kaplan-Meier survival estimate at 16 years was 28.6% for glaucoma-related blindness in at least one eye. A worse visual field on presentation, older age, and poor compliance during therapy were significantly associated with the development of blindness. Glaucoma type, a gender difference, systemic disease, greater intraocular pressure fluctuation in the last year of therapy and blindness in one eye on presentation did not show a significant relationship with the rate of development of blindness., Conclusion: Blindness from treated glaucoma is considerable. Our results gave a 28.6% probability of blindness at 16 years in at least one eye. An older age, poor compliance and a worse visual field on presentation were significant risk factors.

We describe a 36-year-old female who suffered from presenile cataract (nuclear sclerosis and posterior subcapsular opacity, with more-severe disease in the right eye than in the left) and report the use of some ophthalmologic examinations to evaluate the anatomic structures and visual functions of both eyes. Slit-lamp biomicroscopy showed increasing horizontal and vertical corneal diameters (14.0/13.5 mm) and iridodonesis. The pupils could not be fully dilated, and the lenses exhibited cataractous changes. The postlimbal depths were 1.772 mm (OD) and 1.690 mm (OS) (normal value, < 0.20 mm). The results of gonioscopy, specular microscopy, Goldmann visual field, and intraocular pressure evaluation were all within normal limits. Because the patient had bilateral megalocornea in the absence of glaucoma, a very deep anterior chamber depth, secondary effects of iridodonesis, and changes to cataractous lenses, anterior megalophthalmos was diagnosed. This disease is rare. Secondary effects such as iridodonesis, miosis, atrophy of the iris stroma lens subluxation, and occurrence of cataractous lens should be kept in mind.

Background: The purpose of this study was to evaluate the complications of a sub-Tenon injection of triamcinolone acetonide in patients with uveitis., Methods: We retrospectively reviewed the records of uveitis cases treated from 1999 to 2003 in Kaohsiung Chang Gung Memorial Hospital. Thirty patients (45 eyes), who received a sub-Tenon injection of 0.5 ml of a triamcinolone acetonide suspension for uveitis control, were included, and 16 (26 eyes) of these patients underwent intraocular pressure (IOP) monitoring for over 3 months after the procedure., Results: The incidence of ocular penetration was 1.6% and of rapidly progressive cataracts was 13.3%. The incidence of ocular hypertension (>21 mmHg) was 76.9% (20/26). The ocular hypertension in most cases was controlled with topical medications, with recovery of normal IOP. Long-term topical antiglaucoma medication was given for 3 eyes, and another 3 eyes (11.5%) received a trabeculectomy performed by glaucoma specialists. The incidence of glaucoma during the follow-up period after treatment is 23.1% (6/26)., Conclusions: The 23.1% incidence of glaucoma in this study is higher than expected. Careful pretreatment evaluation and post-treatment monitoring of IOP are mandatory for patients with uveitis receiving a sub-Tenon injection of triamcinolone acetonide.

Purpose: We compare the intermediate-term outcome of initial trabeculectomy with adjunctive mitomycin C use versus initial trabeculectomy alone for juvenile primary open-angle glaucoma., Methods: This retrospective consecutive analysis included 44 eyes from 36 patients with juvenile primary-open angle glaucoma, all of whom underwent either initial trabeculectomy with adjunctive mitomycin C use (15 eyes) or initial trabeculectomy alone without mitomycin C use (29 eyes). We compared the success rate and complications between the two groups in a three-year follow-up period following surgery., Results: Three years subsequent to surgery, the cumulative success probability was 73% for the mitomycin C group and 68% for the control group, there being no real difference between the two groups (p = 0.89). A greater incidence of hypotony maculopathy was found amongst the mitomycin C group than was the case for the control group (20 versus 0%, respectively, p = 0.034). A lower intraocular pressure amongst the mitomycin C group was noted as compared with the control group (10.8 +/- 3.0 versus 13.3 +/- 3.8 mm Hg, respectively, p = 0.017) amongst the successfully treated patients., Conclusions: Despite the lower intraocular pressure level for the successfully treated patients from the trabeculectomy with mitomycin C group, and a greater incidence of resultant hypotony maculopathy for this group as compared with the trabeculectomy alone group, there appeared to be no significant difference in the cumulative success probability for this group as compared with the trabeculectomy alone group. Therefore, we caution against the use of an initial trabeculectomy with mitomycin C for juvenile primary open-angle glaucoma., (Copyright 2003 S. Karger AG, Basel)

Background: This study aimed to describe the clinical manifestations and surgical outcomes of subluxated lens., Methods: Reviewing charts from January 1991 to June 2000, we studied 110 patients (123 eyes) who had lens subluxation at Chang Gung Memorial Hospital, Kaohsiung. For 21 eyes, patients accepted conservative treatment, and 102 eyes underwent surgery (8 eyes had Marfan syndrome; 18 eyes had idiopathic dislocation; and 76 eyes had experienced trauma). Indications of surgery included reduction in visual acuity, uncorrectable refractive error, and secondary glaucoma., Results: There were 81 male and 29 female patients. Their mean age was 47.6 years (range, 2-76 years). The mean post-operative follow-up period was 22.24 months (range, 2-118 months). In spite of the different lens statuses, similar visual results were found between the surgical treatment group and the nonsurgery group. In the surgery group, 47 eyes (46.1%) achieved best corrected visual acuity (BCVA) of 20/40 or better. A total of 80 eyes (78.4%) achieved an improvement (log MAR change >0.3) in BCVA. The major postoperative complications included glaucoma (11 eyes), retinal detachment (9 eyes) and intraocular lens dislocation into the vitreous cavity (3 eyes)., Conclusions: Results indicate that the presence of a subluxated lens alone is not an absolute indication for surgical removal. The lens status had no significant influence on surgical outcome. Most cases can achieve an improvement in BCVA after appropriate management.

This study was aimed at investigating the impact of antibiotic pulse treatment (APT) of penicillin-G and streptomycin sulfate at a concentration of 1000 mg/l each on the regeneration ability of leaf and shoot-base explants and homogenized sporophytes of Platycerium bifurcatum. The reaction of the plant to APT, as measured by changes in sporophyte regeneration and morphology, was considered to be a stress response. Cell's susceptibility to APT was graded as: tissue homogenized prior to treatment>tissue homogenized after treatment>leaf and shoot-base explants. Biphasic inhibition in sporophyte regeneration was observed in homogenized sporophytes; a sharper decrease occurred with 20-60 min of the APT, followed by a slower decline for up to 180 min. No significant impact on the regeneration ability of leaf and shoot-base explants was found. Morphologically, APT caused leaf cleavage, mesophyll damage and hyperhydricity in some sporophytes. Except for the hyperhydric sporophytes, regenerants were able to continue their growth in soil, and no carry-over effect was found in the next generation.

A rapid and reliable micropropagation method was established for Spathoglottis plicata. Nodal and leaf explants dissected from 8-month-old pot-grown seedlings were cultured on charcoal-amended Murashige and Skoog medium supplemented with 16 combinations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA) at concentrations of 0.54-10.74 µM. Regeneration of protocorm-like bodies (PLBs) and subsequent plantlet development were observed from 98.5% of the nodal explants. Only 6.5% of leaf explants and occasionally some root segments (dissected from regenerated plantlets) were able to produce PLBs and then plantlets. The optimum plant growth regulator (PGR) combination for maximal PLB regeneration was 5.37 µM NAA and 0.44 µM BA. The best combination of PGR for plantlet development was 2.69-10.74 µM NAA and 8.88 µM BA. The NAA to BA ratios for maximal PLB induction and plantlet development were 12.2 and 0.3-1.2, respectively. Regenerated PLBs and plantlets, when cut into pieces of less than 1 mm and subcultured onto the above media, regenerated new PLBs and plantlets in another 3 months.

A simple suspension culture system of Platycerium bifurcatum was developed where sporophytes could be regenerated directly from leaf cells or indirectly through an aposporous gametophyte stage under the same culture conditions. Single cells and aggregates of up to 100 cells developed aposporous gametophytes which later gave rise to sporophytes. Such gametophytes started apogamy when they were mostly less than 0.7 mm in length, bearing only rhizoids. In most cases, only one sporophyte was regenerated from one gametophyte. Aggregates of 500-1000 or more cells, on the other hand, regenerated sporophytes directly. Intercellular interaction was considered to be the physiological cause, and the separation of leaf cells to a certain degree drove the cells to embark on different regeneration paths. It is suggested that the possible existence of a threshold size of cell aggregates separates the two regeneration patterns.