Erythropoietin inhibits gamma-irradiation-induced apoptosis by upregulation of Bcl-2 and decreasing the activation of caspase 3 in human UT-7/erythropoietin cell line.

1. Erythropoietin (EPO) can reverse radiotherapy-induced anaemia by stimulating bone marrow cells to produce erythrocytes. However, there are limited studies that address the mechanisms by which EPO exerts its beneficial effects in radiotherapy-induced anaemia. In the present study, we used a human bone marrow-derived EPO-dependent leukaemia cell line UT-7/EPO that progressed further in erythroid development to evaluate the anti-apoptotic effects of EPO on irradiated human erythroid progenitor. 2. The UT-7/EPO cells exposed to gamma-irradiation were cultured in the presence or absence of EPO at a concentration of 7 U/mL. The cell viability, cell apoptosis and the expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 were examined. 3. The results showed that EPO protected the viability of human UT-7/EPO cells exposed to gamma-irradiation. EPO significantly inhibited gamma-irradiation-induced apoptosis in human UT-7/EPO cells: a significant decrease in the percentage of apoptotic cells was observed (62, 69 and 62% at 24, 48 and 72 h, respectively). Furthermore, EPO significantly increased the expression of Bcl-2 protein and the relative Bcl-2/Bax ratio, and decreased the activation of caspase 3 and formation of the p17 and p12 cleavage in similar conditions. 4. In conclusion, EPO exerts anti-apoptotic effects on irradiated human UT-7/EPO cells through upregulation of Bcl-2 protein and the relative Bcl-2/Bax ratio, and by decreasing the activation of caspase 3. These findings may contribute to our understanding of the beneficial function of EPO in radiotherapy-induced anaemia.

The new targets of ouabain in retinal interneurons of Sprague-Dawley rats.

Ouabain is both a cardiac glycoside used in therapy of congestive heart failure and an endogenous steroid hormone. It specifically binds to Na(+), K(+)-ATPase (NKA) and blocks its activity. Overdose of ouabain induces retinal damage. In different species ouabain-induced retinal degeneration affects different cell types. In fish and rabbit ouabain induces retinal cell death preferentially in the ganglion cell layer and outer photoreceptor segments respectively. In rats, the pattern of NKA expression has been studied with most detail among retinal neurons. In addition, ouabain selectively destroyed some types of neurons in rodents. However, ouabain-sensitive retinal neurons remain unclear in rats. We show here that injection of ouabain into the rat vitreous body induced dramatic cell death in the inner nuclear layer (INL). The cell death was time- and dose-dependent. Ouabain-induced dying cells in the INL were TUNEL-positive. Immunohistochemistry analysis revealed that there was a significant decrease in the number of calbindin D-28K- and syntaxin-1-positive horizontal and amacrine cells in the INL of ouabain-treated rat retinas. Thus our results revealed that the horizontal and amacrine cells are the most sensitive cell types to ouabain in the retina of Sprague-Dawley rat.

Comparison of blood-nerve barrier disruption and matrix metalloprotease-9 expression in injured central and peripheral nerves in mice.

To investigate the involvement of blood-born factors and extracellular proteases in axonal degeneration and regeneration in both PNS and CNS, we directly compared the differences of blood-nerve barrier (BNB) disruption and matrix metalloprotease-9 (MMP-9) induction between the sciatic nerve and optic nerve after crush injury in the same animal. In sciatic nerve, BNB disruption, fibrin(ogen) deposition and MMP-9 expression were observed only in the first week following injury. Neurofilament (NF) immunoreactivity dramatically decreased in the first 2 days, gradually recovered to the normal levels by day 28. In contrast, the immunoglobulin G deposits spanned from 4 h to 28 days in crushed optic nerves. Fibrin(ogen) deposition was only observed in the first 2 days, while MMP-9 induction did not occur until a week after injury but lasted for 3 weeks in the crushed optic nerves. The NF immunoreactivity did not change much until day 7 and almost completely disappeared on day 28. The decrease of NF immunoreactivity coincided with the induction of MMP-9 after optic nerve crush. These results show that BNB disruption and MMP-9 induction are differentially regulated in the PNS and CNS after injuries, and they may contribute to the different regeneration capacities of the two systems.

Sonic hedgehog promotes stem-cell potential of Müller glia in the mammalian retina.

Müller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Müller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Müller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Müller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Müller glia-derived cells. Together, these results provide evidences that Müller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.

Acute photoreceptor degeneration down-regulates melanopsin expression in adult rat retina.

Melanopsin in retinal ganglion cells plays an important role in mammalian circadian systems. Previous studies indicate melanopsin is responsible for circadian photoentrainment independent of classical rods and cones. However, expression of melanopsin in ganglion cells may be regulated by photoreceptors. In this study, we investigated the effects of N-methyl-N-nitrosourea (MNU)-induced acute photoreceptor degeneration on melanopsin mRNA expression and protein distribution in adult rats. Expression of melanopsin was analyzed 0.5, 1, 5, 7, 13 and 28 days after MNU administration by real-time RT-PCR and immunohistochemistry. MNU-induced gradual degeneration of photoreceptors, and by day 7 most of the photoreceptors were lost. The number of ganglion cells did not change significantly at all time points after MNU injection. In contrast, melanopsin mRNA decreased gradually with the loss of photoreceptors, at the same time pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA levels, which co-express with melanopsin in ganglion cells, were not affected by MNU treatment, indicating decrease of melanopsin mRNA levels is not due to ganglion cell damage. Distribution of melanopsin protein in the dendrites of ganglion cells dramatically decreased with the degeneration of photoreceptors, but its expression in the soma persisted for a long time. Our results suggest that intact photoreceptors maintain the expression of melanopsin and its distribution in ganglion cell dendrites.