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3-Dimentional model of dorsal root ganglia explants in Matrigel for visualization of neural cell migration, axon guidance and branchingтезисы доклада
- Авторы: Semina E.V., Rubina K.A., Rysenkova K.D., Sysoeva V.Yu, Tkachuk V.A.
- Сборник: 21st Biennial Meeting of the International Society of Developmental Neuroscience Conference. ISDN 2016 Abstract Book
- Тезисы
- Год издания: 2016
- Первая страница: P2.005
- Аннотация: Isolation, culturing and immunofluorescent (IF) staining of axons and migrated nerve cells in 3-dimentional ex vivo culture of mice dorsal root ganglion (DRG) explants in Matrigel (MG) can be used to evaluate axonal growth and neural cell migration in a 3-dimentional culture. The methodology also allows to assess the impact of different potential pharmacological agents on the growth and branching of neurites as well as to test the effect of these substances on cell migration - basic processes, reflecting regeneration of nerve tissue. To evaluate the rate of neurites elongation we used darkfield light microscopy; cell migration, axonal direction and branching were assessed using IF labeling and confocal imaging. For experiments, mice were lethal anesthetized and DRG were isolated under aseptic conditions using Olympus SZX16 stereo microscope, placed in chambered borosilicate coverglass (Lab-Tek), and covered with the drop of MG. The samples were cultured in sterile humidified incubator. Imaging was performed using light microscopy (Zeiss Axoivert 2000) at low magnification within 2 weeks. Image processing (neurites length, number of migrated cells) was obtained with MetaMorph software (Meta Imaging Series). For staining samples were fixed, permeabilized, treated for blocking of non-specific binding and subsequently incubated with 1st (NF200 for mature axon) and 2nd (AlexaFluor 594) antibodies, nuclei counterstained by DAPI. Images were acquired by confocal laser scanning microscopy (TCS SP5, Leica) equipped with a Plan-Apo x10, 1.40 NA objective and 543-Argon laser. We tested the impact of urokinase (uPA at 10 ng/ml) and blocking of urokinase receptor (anti-uPAR, 25 mkg/ml) on the directed growth and branching of axons, spontaneous cell migration and the rate cell migration from DRG to MG. uPA administration significantly increased both, neurite outgrowth and cell migration. Thus, uPA stimulates axonal elongation (1.5 times longer compared to control, p<0.05) and activates spontaneous cell migration (2 times compared to control, p<0.05). uPAR is important for regulation of axon growth, branching and branches organization. Also blocking of uPAR attenuates cell migration from explants to Matrigel.
- Добавил в систему: Семина Екатерина Владимировна