| In mature mammals, injured axons of retinal ganglion cells (RGCs) are unable to regenerate and soon undergo cellular apoptosis and death. How to protect the RGCs and promote axonal regeneration after optic nerve injury has always been a focus in neuroscience study. Several lines of evidence have suggested thatα-crystallin can promote axon regeneration after optic nerve injury. Furthermore,α-crystallin can inhibit activation of microglia cells and reduce the expression of TNF-αand iNOS in retinal cells, which leading to lessen the damage of RGCs. On the other hand, cells over-expressing heat shock proteins (HSPs) have shown resistance to stress-induced cell death. However, as a member of the small heat shock protein (sHSP) superfamily, the role ofα-crystallin in optic nerve survival and/or regeneration had not been understood well up to now.ObjectiveTo evaluate the effect of exogenousα-crystallin on optic nerve axons by optic nerve injuried rat model. Then, the expression of endogenousα-crystallin in rat RGCs was detected in vitro and in vivo. Finally, using gene transfection technology, the effect of over-expressingα-crystallin on RGCs apoptosis was investigated in vitro.Methods1. Optic nerve injuried rat model was established and a single intravitreal injection ofα-crystallin was administrated at the time of axotomy. Using microscopy technique and silver staining method, the number of axons was measured by computer image analysis.2. Retinal ganglion cells of Long Evans rats were cultured. The expression of endogenousα-crystallin was disclosed by reverse transcriptase-polymerase chain reaction (RT-PCR) methods and immunohistochemistry. In addition, the expression of endogenousα-crystallin was detected in vivo.3. Full lengthα-crystallin cDNA was subcloned into a pAdTrack-CMV vector. The recombinant pAdTrack-CMV-crystallinαwas transfered into BJ5183-AD-1 cells containing bone plasmid pAdeasy-1 to produce adenovirus vector encodingα-crystallin cDNA. Then the adenovirus vector was transfered into 293 cells by lipofectamine to get recombinant adenovirus.4. Retinal ganglion cells of Long Evans rats were cultured and infected by Ad-crystallinα. The expression ofα-crystallin, caspase-3 and bcl-2 was analyzed and the protective effect of over-expressedα-crystallin on hypoxia-induced apoptosis was evaluated.Results1. Enhanced survival of axotomized axons was observed beyond the crush site after a single intravitreal administration ofα-crystallin at the time of axotomy. Axonal density of the retinal ganglion cell was significantly increased compared with that of the untreated controls until 2 weeks after injection. This effect declined at 4 weeks after injection but survival of axons remained greater than controls.2. Positive staining of endogenousα-crystallin was observed in RGCs in vitro by immunofluorescence method. The level of mRNA increased significantly by RT-PCR method (P<0.05).3. Negative staining of endogenousα-crystallin was found in adult rat retina by immunofluorescence method. However, positive staining of endogenousα-crystallin was detected on RGCs in optic nerve injured rat model.4. The constructed recombinant adenovirus carryingα-crystallin was confirmed by RT-PCR, and its titration value determined was 1. 143×109 ifu/mL.5. Compared with normal group and hypoxia group, the expression ofα-crystallin increased significantly in Ad-crystallinαinfected group induced by hypoxia (P<0.05).6. There was a distinguished difference between Ad-crystallinαinfected group and hypoxia group on the expression of caspase-3 induced by hypoxia (P<0.01). In the Ad-crystallinαinfected group, the expression of caspase-3 in RGCs decreased 59%. Conclusions1. Alpha-Crystallin plays a key role in protecting axons after optic nerve injury. Enhanced survival of axotomized axons was observed after a single intravitreal administration ofα-crystallin at the time of axotomy.2. Expression of endogenousα-crystallin in RGCs was significantly increased after 3-5d cultured, indicating thatα-crystallin may be relative to the survival and proliferation in RGCs.3. Compared with negative expression in normal adult rat, injury-induced expression of endogenousα-crystallin was observed in vivo. It suggested thatα-crystallin might participate in endogenous protective effect of retinal injury.4. Alpha-Crystallin expression was elevated by Ad-crystallinαinfection in vivo. In addition, the expression of caspase-3 after hypoxia stimulation was markedly suppressed inα-crystallin-overexpressing cells. The result indicated thatα-crystallin might protect against hypoxia-induced RGCs apoptosis in part by inhibiting caspase-3 activation.
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