Inhibition of choroidal neovascularization by lentivirus-mediated PEDF gene transfer in rats.

AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated PEDG gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.

Neuroprotective effect of curcumin against oxidative damage in BV-2 microglia and high intraocular pressure animal model.

PURPOSE: The involvement of local and systemic oxidative stress in intraocular pressure (IOP) elevation and optic nerve damage has been hypothesized in the pathogenesis of glaucoma. In this study, we aim to evaluate the antioxidant effects of curcumin in BV-2 microglia oxidative damage and assess its neuroprotective effects in a chronic high IOP rat model. METHODS: BV-2 microglia cell line was used in an in vitro study and Wistar rats were used in an in vivo study. Cultured BV-2 microglia cells were pretreated with 10, 1, or 0.1 µM curcumin for 1 h, and sustained oxidative stress was induced by subjecting BV-2 microglia to 200 µM hydrogen peroxide (H2O2) for 24 h. MTT assay was used to determine cell viability. Changes of intracellular reactive oxygen species (ROS) and apoptosis were analyzed by flow cytometry. Three episcleral veins were cauterized to induce high IOP in Wistar rats and measured by Tonopen. After 6 weeks of treatment with curcumin (10 mg/kg/day) by intragastric administration, surviving of retinal ganglion cells was quantified. Activation of caspase 3, cytochrome c, BAX, and BCL2 was quantified by Western blotting both in BV-2 microglia and in animal model. Data were analyzed with the GraphPad Prism 5.0 software, and P<0.05 was considered to be statistically significant. RESULTS: The in vitro study showed that when BV-2 microglia was pretreated with curcumin, the cell viability increased and the intracellular ROS and apoptosis significantly decreased. In the in vivo study, chronic mild IOP elevation was induced for 4 weeks. In the curcumin-treated group, curcumin protected rat BV-2 microglia from death significantly. In both H2O2-treated BV-2 microglia and glaucoma models, caspase 3, cytochrome c, and BAX were downregulated and BCL2 was upregulated in the curcumin-treated group. CONCLUSIONS: Curcumin affords neuroprotective effects by inhibiting oxidative damage and could be a new or adjunctive treatment for glaucoma.

[Analysis of component and source of fine particulate matter in sarcoidosis granulomatous cells].

OBJECTIVE: To explore the source of the fine particulate matter (PM(2.5)) in the sarcoidosis granulomatous cell and the relationship between the sarcoidosis and the PM(2.5) in the atmosphere. METHODS: Paraffin-embedded tissues of 50 cases of human sarcoidosis biopsy samples, 10 cases of non-sarcoidosis autopsy lung samples, 18 cases of lung tissues (with granulomatous lesions) of rats exposed to PM(2.5) by bronchial infusion, and the free PM(2.5) sample in the atmosphere were collected. The characteristics of tissues above mentioned were observed under the light microscopy, which stained by HE staining and Warthin-Starry silver staining. The characteristics of the PM(2.5) in the four groups were analyzed using confocal Raman microscopy. The component of the PM(2.5) in the sarcoidosis granuloma was analyzed using transmission electron microscope-energy dispersive X-ray detector (TEM-EDX), and the component of the PM(2.5) in the atmosphere was analyzed with X-ray fluorescence separately. RESULTS: The PM(2.5) in the four groups have the similar Raman spectrum, they share the feature of carbonaceous composition, the element component of PM(2.5) in the human sarcoidosis was the same as PM(2.5) in the atmosphere. CONCLUSION: The study provided the further evidence that the PM(2.5) in the sarcoidosis lesion was from PM(2.5) in the atmosphere, and it should be not excepted that sarcoidosis may be a sensitive individual reaction to the PM(2.5) inhaled from the atmosphere.

[Olfactory neuroblastoma with unusual structures: a clinical pathologic study].

OBJECTIVE: To investigate unusual pathological features of olfactory neuroblastoma (ONB) and its correlation with the clinical prognosis. METHODS: Totally 40 cases of ONB were studied using histology and immunohistochemistry techniques. All the cases of ONB were graded according to Hyams Grading system. RESULTS: ONB consisted of small round tumor cells growing in nests or lobules separated by fibrovascular septa. Characteristically, there were neurofibrillary intercellular matrices and Homer-Wright or Flexner-Wintersteiner rosette formation. The unusual structures including epithelial components such as mucous or squamous cell nests which were found in 45.0% (18/40), and 15.0% (6/40) respectively. In addition, 3 cases showed an in-situ form with invasion of tumor into olfactory epithelium, and there was exogenous papillary proliferation seen in 2 cases. Log-rank survival analysis demonstrated that Hyams Grading had no statistical correlation with the prognosis. The presence of necrosis was correlated with a poor prognosis (P = 0.016) while the presence of mucous cells was correlated with a good prognosis (P = 0.011). CONCLUSIONS: Unusual pathological structures including epithelial structures, in-situ invasion of tumor tissue into the involving olfactory epithelium and exogenous papillary proliferation can be found in ONB, suggesting that ONB may originate from the undifferentiated basal cells of olfactory epithelium, through bipotential differentiation. The presence of tumor necrosis in ONB is a poor prognostic indicator while the presence of mucous cells suggests a good prognosis.

[Histopathology of lung of rats exposed to suspension of PM(2.5) with silver staining].

OBJECTIVE: To observe the histopathological changes of the lung of rats which exposed to the suspension of PM(2.5) and detect the effect of silver staining showing dust particles deposited in the lungs. METHODS: The dissociative PM(2.5) of Beijing city was collected to make suspension. The rats were divided into different groups and exposed to different dosage of PM(2.5) (0.3 mg/0.2 ml per rat, 0.75 mg/0.2 ml per rat, 2 mg/0.2 ml per rat) by intratracheal instillation every week. These rats were sacrificed at 4, 12 weeks and 24 weeks (total dosage: 7.2 mg per rat, 18 mg per rat, 48 mg per rat) after the treatment, and their lungs were sampled. The pathological varieties and the situation of these rats' lungs were observed macroscopically and using hematoxylin and eosin (HE) staining, Warthin-Starry (WS) silver stain, as well as the transmission electron microscope (TEM). The dust particles in these rats' lungs were observed by x-ray spectrum chemical element analysis (X-RSA). The granulomatous lesion in the lungs of the rats was counted, and the deposition degree, integrated optical density (IOD) value and integrated area density (IAD) value of the dust particles deposited in the lungs were measured. The variance, least significance difference, and the unitary linear related and regression were analyzed. RESULTS: The number of the granulomatous lesion in the lungs of the rats became more and more with time. In WS staining the dust particles were dark brown and became clearer. The IOD and IAD value of these dust particles were much higher in WS staining than that in HE staining (P < 0.05). The IOD value of the dust particles was positively correlated with the number of the granulomatous lesion (R = 0.639, P < 0.01). The ultrastructure of the dust particles in the rats' lungs and the dissociative PM(2.5) was basically same in TEM. Their main compositions were similar, by X-RSA, and both of them were silicon. CONCLUSION: The suspension of PM(2.5) could result in the granulomatous lesion in the lung of rats. WS silver staining is a good method to show PM(2.5) phagocytized by macrophage, and is better than HE staining.

[Histomorphological feature of silicotic nodules under Warthin-Starry silver staining and its possible prompt value in the histopathologic examination].

OBJECTIVE: To investigate histomorphological feature of silicotic nodules under Warthin-Starry (WS) silver staining and its value in the histopathological examination. METHODS: Six cases with silicosis obtained by autopsy and 21 cases with sarcoidosis were collected (among which 3 cases were obtained by autopsy and 18 cases were obtained by biopsy). The serial sections of those paraffin embedded samples were applied respectively for (1) hematoxylin and eosin (HE) staining, (2) WS staining, (3) streptomyces avidin-peroxidase (SP) immunohistochemical staining for mouse anti-human CD68 monoclonal antibody, (4) observing under transmission electron microscope (TEM), (5) X-ray spectrum chemical element analysis(X-RSA). The emphasis of observation and analysis were the dust particles in silicotic nodules and granulomas cells (dust cells, epithelioid cells and multinucleated giant cells in the granulomas). The dust particles deposit in the granulomas were graded under the HE and WS staining. RESULTS: Under the HE staining the dust particles deposit degrees were (+++) in cellular silicotic nodules, (+) in the fibrous ones, and (-) in the sarcoid nodules; under the WS staining and the dust particles deposit degrees were (+++) in both silicotic nodules whose dust particles were characteristically black, and (+/++) in sarcoid nodules. The dust particles deposit degrees in silicotic nodules were markedly higher than those in sarcoidosis (P < 0.01). The results of immunohistochemical staining indicated that the expression of CD68 in both cells of silicotic nodules and sarcoid nodules were positive. The positive degrees decreased successively with the content of the dust particles. The dust particles of silicotic nodules could be more readily observed than those of sarcoidosis in size and electronic density under TEM. The results of X-RSA indicated that the main chemical element in both dust particles was silicon. CONCLUSION: WS staining is better than HE staining in showing the dust particles of silicotic nodules, which appear characteristically black, especially in the fibrous ones. Together the TEM observation and X-RSA, the silicotic nodules may be prompted.