Yih-Jing Lee

Aluminum overload increases oxidative stress in four functional brain areas of neonatal rats

BackgroundHigher aluminum (Al) content in infant formula and its effects on neonatal brain development are a cause for concern. This study aimed to evaluate the distribution and concentration of Al in neonatal rat brain following Al treatment, and oxidative stress in brain tissues induced by Al overload.MethodsPostnatal day 3 (PND 3) rat pups (n =46) received intraperitoneal injection of aluminum chloride (AlCl3), at dosages of 0, 7, and 35 mg/kg body wt (control, low Al (LA), and high Al (HA), respectively), over 14 d.ResultsAluminum concentrations were significantly higher in the hippocampus (751.0 ± 225.8 ng/g v.s. 294.9 ± 180.8 ng/g; p < 0.05), diencephalon (79.6 ± 20.7 ng/g v.s. 20.4 ± 9.6 ng/g; p < 0.05), and cerebellum (144.8 ± 36.2 ng/g v.s. 83.1 ± 15.2 ng/g; p < 0.05) in the HA group compared to the control. The hippocampus, diencephalon, cerebellum, and brain stem of HA animals displayed significantly higher levels of lipid peroxidative products (TBARS) than the same regions in the controls. However, the average superoxide dismutase (SOD) activities in the cerebral cortex, hippocampus, cerebellum, and brain stem were lower in the HA group compared to the control. The HA animals demonstrated increased catalase activity in the diencephalon, and increased glutathione peroxidase (GPx) activity in the cerebral cortex, hippocampus, cerebellum, and brain stem, compared to controls.ConclusionAluminum overload increases oxidative stress (H2O2) in the hippocampus, diencephalon, cerebellum, and brain stem in neonatal rats.

Intraocular Gene Transfer of Ciliary Neurotrophic Factor Rescues Photoreceptor Degeneration in RCS Rats

Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats.

Involvement of SDF1a and STAT3 in granulocyte colony-stimulating factor rescues optic ischemia-induced retinal function loss by mobilizing hematopoietic stem cells.

PURPOSE Granulocyte colony-stimulating factor (G-CSF) has been applied clinically for several years. In this study, we used G-CSF to induce the mobilization of hematopoietic progenitor cells into peripheral blood in an ischemia-induced retinal degeneration model. METHODS Male Sprague-Dawley rats received G-CSF treatment for 5 days following optic ligation. Histologic and functional evaluations were performed and results were compared with those from untreated rats. Real-time PCR, Western blotting, and immunohistochemical analyses were used to evaluate the expression of retinal cell markers and other substances. RESULTS Retinal histology showed that transient optic ligation induced retinal cell loss. Postischemia, animals that received G-CSF treatment had a higher retinal cell survival rate than that of control animals. Analysis of apoptosis showed that retinas from G-CSF-treated animals exhibited fewer apoptotic cells than those from control retinas. Immunoblotting analyses indicated the presence of greater numbers of CD34-, but less chemokine receptor type 4 (CXCR4)-, and stromal cell-derived factor 1 alpha (SDF1α)-positive cells in the G-CSF-treated ischemic retinas than in ischemic retinas without treatment 14 days after ischemia. The ischemic retinas from G-CSF-treated animals displayed upregulated Thy1 and opsin expression compared with the retinas from untreated animals. Electroretinography indicated superior retinal function in animals treated with G-CSF than in untreated animals postischemia, and that STAT3 might play an important role. CONCLUSIONS Our results suggest that G-CSF reduces optic ischemia-induced retinal cell loss, possibly through STAT3-regulated mobilization of hematopoietic progenitor cells to the retina.

Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells.

This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons.

Aluminum alters NMDA receptor 1A and 2A/B expression on neonatal hippocampal neurons in rats

BackgroundHigh aluminum (Al) content in certain infant formula raises the concern of possible Al toxicity on brain development of neonates during their vulnerable period of growing. Results of in vivo study showed that Al content of brain tissues reached to 74 μM when oral intake up to 1110 μM, 10 times of that in the hi-Al infant formula.MethodsUtilizing a cultured neuron cells in vitro model, we have assessed Al influence on neuronal specific gene expression alteration by immunoblot and immunohistochemistry and neural proliferation rate changes by MTT assay.ResultsMicroscopic images showed that the neurite outgrowth of hippocampal neurons increased along with the Al dosages (37, 74 μM Al (AlCl3)). MTT results also indicated that Al increased neural cell viability. On the other hand, the immunocytochemistry staining suggested that the protein expressions of NMDAR 1A and NMDAR 2A/B decreased with the Al dosages (p < 0.05).ConclusionTreated hippocampal neurons with 37 and 74 μM of Al for 14 days increased neural cell viability, but hampered NMDAR 1A and NMDAR 2A/B expressions. It was suggested that Al exposure might alter the development of hippocampal neurons in neonatal rats.

Homocysteine Facilitates Prominent Polygonal Angiogenetic Networks of a Choroidal Capillary Sprouting Model

Purpose To investigate the effects of homocysteine on choroidal angiogenesis, we established an ex vivo choroidal sprouting explant model and examined the potential growth factors for angiogenesis. Methods Choroid fragments with retinal pigment epithelium were isolated from mouse and embedded in Matrigel. Homocysteine at different concentrations were added to the culture mediums. The choroidal explants were observed at different time points, and the total area of choroidal sprouting was measured and analyzed. Results Homocysteine evoked choroidal capillary sprouting by inducing capillary endothelial cell proliferation with pericyte formation and by facilitating polygonal angiogenetic networks. In some cases, vascular lumens were observed in the newly forming capillaries facilitated by homocysteine. The choroidal sprouting effect of homocysteine can only be observed at a certain range of homocysteine concentration, with 1-mM homocysteine exhibiting the most significantly increased choroidal sprouting areas. Isolectin overexpression was noted in the homocysteine-treated group. Possible growth factors for angiogenesis were detected through immunofluorescent staining, which demonstrated the overexpression of platelet-derived growth factor C and angiopoietin 1 in the homocysteine-treated preparations only. In these preparations, platelet-derived growth factor C was highly expressed in the tip cells of sprouting capillaries. Conclusions We therefore conclude that platelet-derived growth factor C and angiopoietin 1 may play key roles in the choroid angiogenesis evoked by homocysteine.

Hyperhomocysteinemia Causes Chorioretinal Angiogenesis with Placental Growth Factor Upregulation

Hyperhomocysteinemia is a risk factor for atherosclerosis, which may also be associated with retinal vascular disease, diabetic retinopathy, retinal vein occlusion, and glaucoma. For this study, we established a hyperhomocysteinemia animal model to explore homocysteine (hcy)-related choroidal angiogenesis and possible related factors. We injected Sprague Dawley (SD) rats with different concentrations of hcy and performed color fundus imaging, fluorescein angiography, image-guided optical coherence tomography, and retinal histology to observe the retinal and choroidal changes. Subsequently, we observed prominent choroidal vasculature with congested and tortuous retinal and choroidal vessels in fundus angiographies of the hyperhomocysteinemia animal model. In the histological study, the choroidal capillaries proliferated in the hcy-treated eyes, mimicking choroidal neovascularization. Disrupted retinal pigment epithelium (RPE), abnormal branching vascular network (BVN), and polyp-like structures were also observed in the hcy-treated eyes. Furthermore, we found that placental growth factor (PlGF), but not vascular epithelial growth factor (VEGF), was the key mediating factor of this phenomenon. Our findings suggest that hyperhomocysteinemia might cause choroidal angiogenesis.

Subretinal Transplantation of Human Amniotic Membrane Mesenchymal Stem Cell Restores Retinal Function Loss on Light-induced Retinopathy

背景目的：以光照誘發視網膜退化病變的動物模式來探討視網膜感光細胞凋亡的變化，並探討視網膜下腔移植羊膜間質幹細胞可能的治療或視網膜保護功效。實驗方法：實驗以SD大鼠為實驗動物，飼養於光照照度5000到7000 lux，光暗循環各12小時的環境下7天，用以誘發視網膜退化動物模式。光照完成後，部分動物進行視網膜下腔移植人類羊膜間質幹細胞，另一部分動物同部位注射生理食鹽水作為對照組，並以視網膜電位圖、視網膜組織切片、以及免疫螢光染色等方式進行不同組別之研究。結果討論：實驗結果顯示，光照一周後視網膜感光細胞有明顯減少（凋亡）的現象，而視網膜電位圖亦顯示視網膜功能已經明顯退化。經過視網膜下腔移植人類羊膜間質幹細胞之後，三至五週後可以見到有標記的移植羊膜間質細胞已經嵌入被移殖動物的視網膜組之中，並且減少感光細胞的凋亡。這些接受移植的動物在視網膜電位圖的表現也比對照組顯示較佳的視網膜功能。藉由免疫螢光染色的技術更進一步發現，這些移植的羊膜間質幹細胞漸漸分化為視網膜的色素上皮細胞（RPE）、感光細胞（photoreceptor）、以及穆勒膠細胞（Muller cells）。結論：研究結果顯示移植人類間質幹細胞可以修復因長期光照造成受損的視網膜感光細胞並於光照後維持視網膜功能，期望研究成果期望可以做為臨床治療視網膜退化疾病之參考。

Suppression of HSP27 Restores Retinal Function and Protects Photoreceptors From Apoptosis in a Light-Induced Retinal Degeneration Animal Model

Purpose We used a light-induced retinal degeneration animal model to investigate possible roles of heat shock protein 27 (HSP27) in retinal/photoreceptor protection. Methods Sprague-Dawley rats were used for the light-induced retinal degeneration animal model. The histology of eye sections was observed for morphologic changes in the retina. Cell apoptosis was examined in each group using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and electroretinography was used to evaluate retinal function. Protein and mRNA expression levels of different retinal cell markers were also detected through immunofluorescence staining, Western blotting, and real-time PCR. Results The thickness of the outer nuclear layer significantly decreased after 7-day light exposure. Moreover, we injected a viral vector for silencing HSP27 expression into the eyes and observed that photoreceptors were better preserved in the HSP27-suppressed (sHSP27) retina 2 weeks after injection. HSP27 suppression also reduced retinal cell apoptosis caused by light exposure. In addition, the loss of retinal function caused by light exposure was reversed on suppressing HSP27 expression. We subsequently found that the expression of the Rho gene and immunofluorescence staining of rhodopsin and arrestin (cell markers for photoreceptors) increased in sHSP27-treated retinas. HSP27 suppression did not affect the survival of ganglion and amacrine cells. Conclusions Retinal cell apoptosis and functional loss were observed after 7-day light exposure. However, in the following 2 weeks after light exposure, HSP27 suppression may initiate a protective effect for retinal cells, particularly photoreceptors, from light-induced retinal degeneration.