| How to protect survival of RGCs and promote axonal regeneration after ON injury has become a focus all over the world. Now, it has been proved that lens injury can promote survival of RGCs and axonal regeneration. The induction of lens injury results in release of lentogenic factors which promote survival of RGCs and stimulate axonal regeneration throughout the optic pathway, and arrive at the thalamus at last. But, what're on earth the lentogenic factors? It has not been answered until today. Leon found that there were a lot of macrophages in the retina after lens injury, and thought that activated macrophages played a great role. However, there's time discrepancy between macrophage infiltration and axonal regeneration, and axonal regeneration can not be explained completely by the effect of macrophage.Lens is a tissue contenting the highest protein, and composed of 66% water and 33% protein. Soluble crystallins(α,β,γ)make up 90%～95% of the total protein of the lens. As self-antigens, crystallins can activate macrophages and promote their infiltration in local tissue. And as small heat shock proteins, alpha-A and alpha-B, two alpha-crystallin subunit of α-crystallin, can inhibit the activation of caspase-3, and promote the survival of retinal pigment epithelial cells, photoreceptors and lens epithelium cells.Whether crystallins can promote the survival of RGCs or not?Purpose: To establish culture method and evaluation technique of retinal ganglion cells in Long Evans rats in vitro. Study the effect of crystallins on survival and growth of rat RGCs in vitro, and discuss the relation between crystallins and activated macrophages about their effect on rat RGCs in vitro. Thereby, ques a new method for the treatment of optic nerve injury and provide experimental basis on the regeneration of optic nerve injury.Methods: Crystallins were extracted from Long Evans rats, and culture media of macrophages were prepared by culturing enterocoelia macrophages of Long Evans rats for 24h. RGCs in Long Evans rats were cultured in DMEM, DMEM containing crystallins, and DMEM containing culture media of macrophages activated by DMEM or crystallins. The growth regularity and survival time of RGCs in vitro was observed under phase-contrast microscope. The numbers and length of the longest processes of RGCs with processes and cells activity were measured when RGCs were cultured for 1d, 3d, and 5d. Results: 1. Survival time of RGCs in vitro: RGCs died out after cultured for 5d to 6d in DMEM, 6d to 7d in DMEM containing culture media of macrophages activated by DMEM, and 7d to 8d in DMEM containing culture media of macrophages activated by crystallins. However, RGCs could survived for 12d to 14d in DMEM containing crystallins, and the survival time of RGCs was longer significantly than other three groups(p<0.05).2. The numbers of RGCs with processes: Whether RGCs had been cultured for 1d ,3d or 5d, the numbers of RGCs with processes of crystallines group, and the two groups of culture media of macrophages activated by DMEM or crystallins were more than DMEM group(p<0.01). And contrasted to the two groups of culture media of macrophages activated by DMEM or crystallins, crystallines could make more RGCs with processes survive(p<0.01). However, there were no difference between the two groups of culture media of macrophage activated by DMEM and crystallins.3. The length of the longest processes of RGCs: When RGCs had been cultured for 1d or 3d, the length of the longest processes of RGCs of crystallins group, and the two groups of culture media of macrophages activated by DMEM or crystallins were longer than DMEM group(p<0.01). After cultured for 5d, the three experimental groups could still elongate the length of the longest processes of RGCs, but the group of culture media of macrophages activated by DMEM was less effective than other two experimental groups(p<0.05). As the same as the numbers of RGCs with processes, there was no difference between the two groups of culture media of macrophages activated by DMEM a...
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