Synergistic Efficacy Of Pigment Epithelium-derived Factor And Macrophage-conditioned Medium Or Lens Injury Promote Retinal Ganglion Cell Survival And Axon Regeneration

BackgroundRetinal ganglion cells (RGCs) are responsible for transmission of visual signals to the brain. Disease and trauma in central nervous system (CNS) often lead to the neuronal death and loss of the functional connections. After optic nerve injury in mature mammals, RGCs are unable to regenerate their axons. The strategies aimed at repairing the injured axons involve stimulation of both neuronal survival and axon regeneration. Pigment epithelium-derived factor (PEDF) is a neurotrophic, neuroprotective and antiangiogenic protein that can protect neurons from a variety of the damages. And it is also an anti-inflammatory factor for neurons. However, if the lens is injured at the time of nerve injury, many RGCs will survive and regenerate their axons. Lens injury could induce macrophages to infiltrate, and the factors secreted by macrophages could stimulate RGCs to regenerate their axons. Macrophage activation seems to play a key role in the axon regeneration, because injection of Zymosan stimulated macrophages in the absence of lens injury and promoted RGCs to regenerate their axons.PurposeWe aimed to explore whether or not combination of PEDF with macrophage- conditioned medium (MCM) or lens injury could enhance their effect on the RGCs survival and axon regeneration in the optic nerve of adult rats. Methods1. Cell culture: RGC cells from SD rat borne within 24h and peritoneal cavity macrophages of SD rats were cultured in this study. Their morphous were observed and they were identified by immunohistochemistry. Their survival curve was obtained through MTT method. Their survival pattern was revealed.2. Cell study: (1) Cultured RGCs were incubated with Zymosan vesicles with or without serum. The numbers of the survival macrophages and of the vesicles engulfed in the macrophages were counted under a microscope. (2) MCM was collected and the effects of combination of PEDF with MCM on the number of the survival cultured RGC cells and the lengths of RGC cell axon regeneration were assessed.3. Animal studies: (1) FluoroGold was injected into the superior colliculus of mature SD rats and immature rat borne 2-week, and RGCs were retrogradely labelled by application of FG. The disposition pattern and the number of RGCs were viewed. The optic nerve crush model and lens injury model were established. The effect of combination of PEDF with lens injury on the number of the survival RGC cells, the length of RGC cell axon regeneration and the expression of GAP-43 mRNA were assessed.Results1. We successfully cultured RGCs of SD rat. GAP-43-positive RGCs were seen. The 71.4 % cells were survived on day 4. The majority of the cells were dead on day 6, and 46.3 % cells were survived. Only 7.1%cells were survived on day 8. We also successfully cultured SD rat peritoneal cavity macrophages. CD-68-positive cells were seen. The cells could survive about for about 2 weeks, and more than the half of cells were dead on day 8.2. When the cultured RGCs were incubated with Zymosan vesicles for 2 days, the numbers of the survival cells cultured with serum were more than the control (P<0.01). The number of the survival cells cultured with MCM was 31.4±3.7 (P<0.05) on day 3, and the number of the survival cells (34.7±4.2; P<0.01) cultured with combination of PEDF with MCM increased significantly when compared to the control (27.6±2.2). The number of cells cultured with combination of PEDF with MCM increased even more than the cells cultured with MCM (P<0.05). The axon length of RGCs cultured with MCM was 57.6±3.7μm on day 3, and the axon length of RGCs (88.6±9.5μm ) cultured with combination of PEDF with MCM was significantly longer than the control (41.8±5.4μm, P<0.01). The axon length of RGCs cultured with combination of PEDF with MCM was longer than the cells cultured with MCM (P<0.01).3. The retrogradely labelled RGCs model was successfully made. The density of RGCs in retina was 2219±113 RGC/mm2. The density of RGCs survived in the rat with intravitreal injection of PEDF plus lens injury strongly increased after optic nerve injury. The combination of PEDF with lens injury protected more number of RGCs compared with independent administration of PEDF or lens injury (1411±84 RGC/mm2, P < 0.01). A single lens injury could promote the axon regeneration of RGCs into the distal optic nerve. This approach led to a dramatic increase in the axon length of RGCs after combination of intravitreal injection of PEDF with lens injury, and the axon length was longer than that of RGCs in rat with a single lens injury (P<0.01). The quantitative analysis showed that a dramatic increase in the expression of GAP-43 mRNA after combination of intravitreal injection of PEDF with lens injury, higher than injection with PEDF or lens injury only (P < 0.01).Conclusion1. The serum could enhance the phagocytosis of macrophages.2. A single application of PEDF couldn't increase RGCs survival and axon regeneration; MCM could enhance RGCs survival and axon regeneration. And the combination PEDF with MCM had a synergistic efficacy on either RGCs survival or axon regeneration.3. A single intravitreal injection of PEDF protein or lens injury could potentiate RGCs survival, and combination PEDF with lens injury had a synergistic action on RGCs survival. Lens injury could enhance axon regeneration, and combination PEDF with lens injury had a synergistic action on it. A single intravitreal injection of PEDF protein or lens injury could increase the expression of GAP-43 mRNA, and combination PEDF with lens injury had a synergistic action on the expression.Innovation1. In this study, we first reportd retrogradely labelled RGCs of immature rat.2. The effect of PEDF on axon regeneration of cultured RGCs and the effect of PEDF on axon regeneration of RGCs after the optic nerve injury were first explored in our study.3. Whether or not combination PEDF with MCM or lens injury had a synergistic action on either RGCs survival or axon regeneration was first investigated in our study.