OBJECTIVE: To investigate the relationship among mossy fiber axon sprouting(MFS), synaptic reorganization, and the alteration of expression of Eph A5 and ephrin A3 in the dentate gyrus in rats with pilocarpine-induced chronic temporal lobe epilepsy. METHODS: Mossy fiber sprouting and synaptic formation in rats were observed by Neo-Timm staining, after the acute status epilepticus and chronic spontaneous temporal lobe epilepsy induced by lithium-chloride and pilocarpine. In situ hybridization was used to detect ephrin A3 mRNA and an immunohistochemical staining was applied to determine Eph A5 protein. RESULTS: In entorhinal cortex, only Eph A5 mRNA and protein expressed, which significantly decreased on Day 7 after pilocarpine induced status epilepticus(P<0.01),and resumed to normal levels on Day 30 (P>0.05). In the dentate granule cells, ephrin A3 mRNA reduced obviously on Day 7 after pilocarpine-induced status epilepticus (P<0.01), and returned to normal levels on Day 30 (P>0.05). CONCLUSION The down-regulation of Eph A5 mRNA and protein in entorhinal cortex and dentate gyrus, and ephrin A3 mRNA in dentate gyrus after status epilepticus may be part of the endogenous molecular mechanism of mossy fiber sprouting to the inner molecular layer of dentate gyrus.
Animals ; Axons ; physiology ; Chromosome Pairing ; physiology ; Dentate Gyrus ; physiopathology ; Down-Regulation ; Ephrin-A3 ; biosynthesis ; Epilepsy, Temporal Lobe ; chemically induced ; metabolism ; physiopathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Receptor, EphA5 ; biosynthesis ; Status Epilepticus ; chemically induced ; metabolism ; physiopathology

BACKGROUNDIn a previous study, we demonstrated that ephrin-A2 and -A3 negatively regulate the growth of neural progenitor cells in the central nervous system. Adult mice deficient in ephrin-A2 and -A3 (A2(-/-)A3(-/-)) displayed active ongoing neurogenesis throughout the brain, and mice deficient in ephrin-A3 alone showed increased proliferation of ciliary epithelium derived retinal stem cells. This study aimed to detect that the increase in proliferation and neurogenic potential of Müller cells is influenced by the absence of ephrin-A2 and -A3.

METHODSWe assessed the retinal and Müller cell expression of ephrin-As and their receptor and neural progenitor cell markers by immunostaining and real-time PCR. We cultured purified primary Müller cells derived from wild-type and A2(-/-)A3(-/-) mice in a defined culture medium that enables trans-differentiation of Müller cells into retinal neurons. To evaluate proliferating Müller cells in vivo, we injected 5'-ethylnyl-2'-deoxiuridine (EdU) intraperitoneally to adult mice.

RESULTSExpression of ephrin-A2/A3 and their receptor EphA4 were detected in the retinas of adult mice, with EphA4 expression particularly enriched in Müller cells. Müller cells of A2(-/-)A3(-/-) mice exhibited significantly elevated expression of retinal progenitor cell markers, Pax6 and Chx10, when compared with those from wild-type mice. Moreover, a higher percentage of Müller cells of A2(-/-)A3(-/-) mice trans-differentiated and became recoverin+ and β-III-tublin+ in the culture than those from wild type mice. Strikingly, an increased number of EdU+ retinal cells was detected in the retinas of adult A2(-/-)A3(-/-) mice as compared with wild-type mice.

CONCLUSIONSEphrin-A2 and -A3 are negative regulators of the proliferative and neurogenic potentials of Müller cells. Manipulating ephrin-A signaling may thus represent a novel strategy for stimulating neuroregeneration from endogenous progenitors to participate in retinal repair in case of disease or damage.

Animals ; Cell Differentiation ; genetics ; physiology ; Ephrin-A2 ; genetics ; metabolism ; Ephrin-A3 ; genetics ; metabolism ; Fluorescent Antibody Technique ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Real-Time Polymerase Chain Reaction ; Receptor, EphA4 ; genetics ; metabolism ; Retina ; cytology ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism