Jie-Xin Deng

The development of blood-retinal barrier during the interaction of astrocytes with vascular wall cells

Astrocytes are intimately involved in the formation and development of retinal vessels. Astrocyte dysfunction is a major cause of blood-retinal barrier injury and other retinal vascular diseases. In this study, the development of the retinal vascular system and the formation of the blood-retinal barrier in mice were investigated using immunofluorescence staining, gelatin-ink perfusion, and transmission electron microscopy. The results showed that the retinal vascular system of mice develops from the optic disc after birth, and radiates out gradually to cover the entire retina, taking the papilla optica as the center. First, the superficial vasculature is formed on the inner retinal layer; then, the vasculature extends into the inner and outer edges of the retinal inner nuclear layer, forming the deep vasculature that is parallel to the superficial vasculature. The blood-retinal barrier is mainly composed of endothelium, basal lamina and the end-feet of astrocytes, which become mature during mouse development. Initially, the naive endothelial cells were immature with few organelles and many microvilli. The basal lamina was uniform in thickness, and the glial end-feet surrounded the outer basal lamina incompletely. In the end, the blood-retinal barrier matures with smooth endothelia connected through tight junctions, relatively thin and even basal lamina, and relatively thin glial cell end-feet. These findings indicate that the development of the vasculature in the retina follows the rules of “center to periphery” and “superficial layer to deep layers”. Its development and maturation are spatially and temporally consistent with the functional performance of retinal neurons and photosensitivity. The blood-retinal barrier gradually becomes mature via the process of interactions between astrocytes and blood vessel cells.

Death Receptor 5 and Neuroproliferation

Tumor necrosis factor-related apoptosis-inducing ligand or Apo2 ligand is a member of the tumor necrosis factor superfamily of cytokines that induces apoptosis upon binding to its death domain-containing transmembrane receptors, death receptors 4 and 5 (DR4, DR5). However, DR5 is also expressed in the developing CNS where it appears to play a role unrelated to apoptosis, and instead may be involved in the regulation of neurogenesis. We report on the distribution of DR5 expression in mouse hippocampus, cerebellum, and rostral migratory stream (RMS) of olfactory bulb from embryonic (E) day 16 (E16) to postnatal (P) day (P180). At E16, DR5-positive cells were distributed widely in embryonic hippocampus with strong immunostaining in the developing dentate gyrus. In newborn hippocampus, DR5-positive cells were predominantly located in proliferative zones, such as dentate gyrus, subventricular zone, and RMS. After postnatal day 7 (P7), the number of DR5-positive cells decreased, and cells with intense fluorescence were primarily restricted to the subgranular layer (SGL), although the granular cell layer showed weak fluorescence. After P30, only few DR5-positive cells were found in SGL, and mature granule cells were negative for DR5 expression. To address whether DR5 expression is a restricted to progenitor cells and newborn neurons, we performed 5-bromo-deoxyuridine labeling. We report that proliferative cells in the SGL selectively express DR5, with lower levels of expression in cells positive for doublecortin, a marker of newborn neurons. In addition, the stem cells in intestine, cerebellum, and RMS were also demonstrated to be DR5-positive. In the meantime, in cerebellum, DR5-positive cells were also positive for glial fibrillary acidic protein, a marker of proliferative Bergmann cells. We conclude that DR5 is selectively expressed by neuroprogenitor cells and newborn neurons, suggesting that the DR5 death receptor is likely to play a key role in neuroproliferation and differentiation.

Neural differentiation and synaptogenesis in retinal development.

To investigate the pattern of neural differentiation and synaptogenesis in the mouse retina, immunolabeling, BrdU assay and transmission electron microscopy were used. We show that the neuroblastic cell layer is the germinal zone for neural differentiation and retinal lamination. Ganglion cells differentiated initially at embryonic day 13 (E13), and at E18 horizontal cells appeared in the neuroblastic cell layer. Neural stem cells in the outer neuroblastic cell layer differentiated into photoreceptor cells as early as postnatal day 0 (P0), and neural stem cells in the inner neuroblastic cell layer differentiated into bipolar cells at P7. Synapses in the retina were mainly located in the outer and inner plexiform layers. At P7, synaptophysin immunostaining appeared in presynaptic terminals in the outer and inner plexiform layers with button-like structures. After P14, presynaptic buttons were concentrated in outer and inner plexiform layers with strong staining. These data indicate that neural differentiation and synaptogenesis in the retina play important roles in the formation of retinal neural circuitry. Our study showed that the period before P14, especially between P0 and P14, represents a critical period during retinal development. Mouse eye opening occurs during that period, suggesting that cell differentiation and synaptic formation lead to the attainment of visual function.

Protective effect of autophagy in neural ischemia and hypoxia: Negative regulation of the Wnt/β-catenin pathway

Autophagy is a highly conserved process of self-digestion to promote cell survival in response to nutrient starvation and other metabolic stresses. However, whether ischemic-hypoxic (IH) injury-induced autophagy acts as a neuroprotective mechanism or leads to neuroinjury is a subject of debate. It is known that autophagy is regulated by signaling pathways, including the mammalian target of rapamycin pathway. However, in neural IH injury, whether other signaling pathways are involved in the regulation of autophagy remains to be fully elucidated. In the present study, using the autophagy agonist (rampycin), autophagy antagonist [3-methyl adenine (3-MA)] and lysosome antagonist (MHY1485), autophagy was intervened with at oxygen-glucose deprivation (OGD) 6 h, in order to elucidate the regulatory mechanisms of autophagy. Using immunocytochemistry and western blot analysis, the expression levels of stress-related proteins, such as hypoxia-inducible factor-1α (HIF-1α) (a key regulator in hypoxia) and cyclooxygenase 2 (COX2; inflammatory indicator), were analyzed. In addition, the upstream proteins (Wnt1 and Wnt3a), downstream proteins (Dvl2, β-catenin) and target proteins (C-myc and cyclin D) in the Wnt/β-catenin signaling pathway were examined by immunocytochemistry and western blot analysis. The present study revealed that autophagy was activated with the upregulation of autophagic flux in IH injury; it was demonstrated that autophagy had a protective role in IH injury. The Wnt/β-catenin pathway was involved in IH injury regulation, and the upstream proteins in the Wnt/β-catenin signaling pathway were upregulated, whereas downstream proteins were downregulated by the activity of autophagy accordingly.

The effects of prenatal alcohol exposure on the developmental retina of mice.

AIMS Our aim is to investigate the effects of prenatal alcohol exposure (PAE) on the development of retinal bipolar and horizontal cells. METHODS The alterations of the retinal bipolar and horizontal cells in P7, P14 and P30 mice were observed after PAE, with immunofluorescent labeling and DiI diolistic assay. RESULTS The retinal development of filial pups was affected by PAE in a dose-dependent and long-term manner. The number of bipolar cells of alcohol groups was significantly lower than that of the control, and the dendritic receptive field of horizontal cells was also significantly smaller than those of the control groups (P < 0.01). CONCLUSION PAE was able to cause retarded development of pup retinal neural cells.

14,15-EET Suppresses Neuronal Apoptosis in Ischemia–Reperfusion Through the Mitochondrial Pathway

Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia–reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia–reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen–glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia–reperfusion by 14,15-EET.

Stress injuries and autophagy in mouse hippocampus after chronic cold exposure

Cold exposure is an external stress factor that causes skin frostbite as well as a variety of diseases. Estrogen might participate in neuroprotection after cold exposure, but its precise mechanism remains unclear. In this study, mice were exposed to 10°C for 7 days and 0–4°C for 30 days to induce a model of chronic cold exposure. Results showed that oxidative stress-related c-fos and cyclooxygenase 2 expressions, MAP1LC3-labeled autophagic cells, Iba1-labeled activated microglia, and interleukin-1β-positive pyramidal cells were increased in the hippocampal CA1 area. Chronic cold exposure markedly elevated the levels of estrogen in the blood and the estrogen receptor, G protein-coupled receptor 30. These results indicate that neuroimmunoreactivity is involved in chronic cold exposure-induced pathological alterations, including oxidative stress, neuronal autophagy, and neuroimmunoreactivity. Moreover, estrogen exerts a neuroprotective effect on cold exposure.

Effects of chronic cold exposure on murine central nervous system.

Recently, cold‐adaptation medicine has gotten more and more attention because of its specific significance to health care, military activities, sports performance, and so on. Although numerous studies have focused on respiratory, immune, and circulatory systems as well as skin damage upon cold exposure, the impacts on central nervous system are not well understood. This study explores the effects of chronic cold exposure on the murine central nervous system. To establish a chronic cold‐exposure animal model, adult male mice from postnatal days 40–50 (P40–50) were housed at 0–4°C for 20 days. During the study period, estrogen receptors were labeled via immunohistochemistry, the dendritic spines of visual cortical pyramidal cells were labeled with DiI diolistic assay, and synaptic ultrastructure was observed by transmission electron microscopy. The results showed that cold exposure could inhibit neural proliferation significantly, with an increase of G‐protein‐coupled receptor 30 (GPR30) expression. Chronic cold exposure could also induce a decrease in the dendritic spines of pyramidal cells in visual cortex, along with a decrease in the number of synaptic formations. The ultrastructure of synapses after cold exposure was observed. It was found that pre‐ and postsynaptic membranes were fused, with a vague synaptic cleft. Furthermore, neuronal cytoplasmic and organelle swellings were also observed, along with microtubule disintegration. In conclusion, chronic cold exposure can cause structural and functional changes in the mouse central nervous system, possibly by direct participation of estrogen and its receptor, GPR30, in response to chronic cold exposure.

Experimental study on the neurotoxic effect of β-amyloid on the cytoskeleton of PC12 cells

The aim of the present study was to establish a cell model of Alzheimers disease (AD) and investigate the neurotoxic effects of β-amyloid (Aβ) on the cytoskeleton. PC12 cells were cultured and treated with Aβ25-35, and cell survival was analyzed with the MTT assay. Cell apoptosis was visualized using 4′,6-diamidino-2-phenylindole staining and the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Immunocytochemistry and phalloidin staining were used to label the cytoskeleton of PC12 cells. Aβ25-35 was found to induce PC12 cell apoptosis in a dose-dependent manner (P<0.05). Moreover, Aβ25-35 also caused dose-dependent disintegration of the cytoskeleton (P<0.05). Therefore, the PC12 cell cytoskeleton was found to be sensitive to Aβ25-35 neurotoxicity. The disintegration of the cytoskeleton is likely an important pathological alteration in AD, and Aβ is a key molecule involved in AD pathogenesis.

The impairment of learning and memory and synaptic loss in mouse after chronic nitrite exposure

The objective of this study is to understand the impairment of learning and memory in mouse after chronic nitrite exposure. The animal model of nitrite exposure in mouse was created with the daily intubation of nitrite in adult healthy male mice for 3 months. Furthermore, the mouses learning and memory abilities were tested with Morris water maze, and the expression of Synaptophysin and γ‐Synuclein was visualized with immunocytochemistry and Western blot. Our results showed that nitrite exposure significantly prolonged the escape latency period (ELP) and decreased the values of the frequency across platform (FAP) as well as the accumulative time in target quadrant (ATITQ) compared to control, in dose‐dependent manner. In addition, after nitrite exposure, synaptophysin (SYN) positive buttons in the visual cortex was reduced, in contrast the increase of γ‐synuclein positive cells. The results above were supported by Western blot as well. We conclude that nitrite exposure could lead to a decline in mices learning and memory. The overexpression of γ‐synuclein contributed to the synaptic loss, which is most likely the cause of learning and memory impairment.