Alpha-crystallin promotes rat retinal neurite growth on myelin substrates in vitro.

BACKGROUND: Myelin-associated molecules are major impediments to axon regeneration after optic nerve injury. Intravitreal injection of α-crystallin can protect axons from optic nerve degeneration after crushing in rats. OBJECTIVES: Our purpose was to investigate whether α-crystallin could counteract the inhibitory effect of myelin and promote neurite growth. METHODS: Newborn rat retinal neurons were cultured on myelin-coated dishes with DMEM containing α-crystallin (10(-4) g/l) or bovine serum albumin. The density of neurons with neurites and the length of the longest neurite of the cells were analyzed on days 1, 3 and 5. RESULTS: Cultures containing α-crystallin had significantly higher neurite-containing cell densities, and the neurites were significantly longer compared with cultures containing bovine serum albumin. These findings indicated that α-crystallin could counteract the effect of myelin inhibitory factors and stimulate neurite growth.

α-Crystallin promotes rat axonal regeneration through regulation of RhoA/rock/cofilin/MLC signaling pathways.

Intravitreal injection of α-crystallin can promote axons from optic nerve regeneration after crushing in rats. We have previously demonstrated that α-crystallin can counteract the effect of myelin inhibitory factors and stimulate neurite growth. And a common crucial signaling event for myelin inhibitory factors is the activation of RhoA. To investigate whether α-crystallin counteracts the inhibitory effect of myelin inhibitory factors through regulation of RhoA/Rock signaling pathway, α-crystallin (10(-4) g/L) was injected into rat vitreous at the time the optic nerve crushed. The RhoA protein activity and the expression of RhoA and Rock were evaluated after 3 days of optic nerve axotomy. Rock downstream effectors, phosphorylated cofilin, and phosphorylated myosin light chain were detected when retinal neurons were cultured for 3 days. Axonal regeneration and neurites growth of cultured cells were observed also. Our results showed that α-crystallin decreased the RhoA protein activity and the phosphorylation of both cofilin and myosin light chain, and promoted the axonal growth. However, the expression of RhoA and Rock was not affected by α-crystallin. These findings indicated that α-crystallin could counteract the effect of myelin inhibitory factors through the regulation of RhoA/Rock signaling pathway.

Distribution and expression of RhoA in rat retina after optic nerve injury.

BACKGROUND: RhoA is a small guanosine triphosphatase which participates in signaling pathways of axonal repellents or inhibitors. However, the distribution and expression of RhoA in the rat retina after optic nerve injury has not been elucidated yet. OBJECTIVES: To study the distribution and expression of RhoA in the rat retina after optic nerve injury. METHODS: Immunohistochemistry was used to determine the distribution of RhoA in rat retina after optic nerve injury. The expression of RhoA was analyzed by Western blot. RESULTS: In normal retina and the retina 1 day after optic nerve injury, RhoA was distributed in the retinal ganglion cell (RGC) layer. Three days after optic nerve injury, it existed in RGCs and the inner plexiform layer. However, 7 days after surgery its immunoreactivity was abundant not only in the RGC and inner plexiform layers but also in the inner nuclear and outer plexiform layers. Western blot analysis showed that the expression of RhoA increased significantly in the retina after optic nerve injury in comparison with normal retina. CONCLUSION: These results indicate that the distribution and expression of RhoA were extended and enhanced after optic nerve injury, and that RhoA plays an important role in optic nerve regeneration.

[Influence of fluorescent protein expression on the proliferation of NIH3T3 cells in vitro].

OBJECTIVE: To investigate the influence of fluorescent protein expression on the proliferation of murine NIH3T3 cells, so as to provide a theoretical basis for cell tracing technology. METHODS: NIH3T3 cells were cultured in vitro, and were randomly divided into control, pLEGFP-N1 (with transfection of pLEGFP-N1 retroviral vector), pEGFP-N1 (with transfection of pEGFP-N1 vector) and pDsRed2-C1 (with transfection of pDsRed2-C1 vector) groups. Then the cells were screened by G418 for 3 weeks. The changes in cell adhesive rate were observed and the population doubling times was determined by growth curve. RESULTS: There was obvious fluorescent protein expression in the transfected NIH3T3 cells after G418 selection, and the highest percentage of labeled NIH3T3 cells was found in pLEGFP-N1 group. The population doubling time in pDsRed2-C1 (40.3+/-0.7 h) , PEGFP-N1 (39.6 +/- 0.6 h) and pLEGFP-N1 (36.5 +/- 0.7 h) groups was evidently longer than that in control (27.9 +/- 0.6 h, P < 0.01), with high adhesive rate in each group. CONCLUSION: The expression of fluorescent protein exhibited some inhibitory effect on the proliferation of NIH3T3 cells in vitro. Since the inhibitory effect by retroviral vector was weaker compared with eukaryotic vector, it should be the first choice for fluorescent protein labeling during cell transplantation.