ROLE OF REACTIVE GLIA IN MODULATION OF NEURO- AND SYNAPTOGENESIS IN VITRO

DOI: https://doi.org/10.29296/24999490-2021-06-10

A.V. Vinogradova, O.P. Tuchina Immanuel Kant Baltic Federal University, Laboratory for Synthetic biology, School of Life Sciences, ul. Universitetskaya, 2, Kaliningrad, 236041, Russian Federation E-mail: [email protected]

Introduction. Activation of glial cells by lipopolysaccharide leads to the release of proinflammatory cytokines and the development of neuroinflammation, which, in turn, affects the processes of neuro- and synaptogenesis. However, the specific mechanisms of neuroglial and astrocytemicroglial interactions in these processes remain the subject of discussion. Aim of the study. Our aim was to study the role of microglia in the acquisition of a reactive phenotype by astrocytes in vitro, and the effect of lipopolysaccharide and astrocyte-conditioned medium on the neuro- and synaptogenesis of hippocampal neurons in vitro by immunocytochemistry methods. Methods. The study was carried out on glial and neuronal cell cultures from rat hippocampus. Lipopolysaccharide was added to mixed glial culture and pure astrocyte culture at concentrations of 1 and 10 μg/ml for 6, 12 and 24 hours. The cells were then fixed, stained for astrocytic marker GFAP. Lipopolysaccharide at a concentration of 10 μg/ml at 6 and 12 hours, as well as an astrocyte-conditioned medium were added to the culture of hippocampal neurons which were later fixed and stained for the marker of cell proliferation Ki67, presynaptic (Syn1) and postsynaptic (PSD95) proteins. Results. The number of GFAP increased in mixed glial cultures after 6 and 12 h of incubation with lipopolysaccharide; in astrocyte culture, GFAP on the contrary, decreased. The addition of lipopolysaccharide and astrocyte-conditioned medium to neurons led to a decrease in cell proliferation and an increase in the amount of postsynaptic protein PSD95, while the amount of Syn1 changed only in case when lipopolysaccharide was added directly to neurons. Conclusion. In our work it was determined that astrocytes acquire a characteristic reactive phenotype only in the presence of microglial cells in vitro, as evidenced by an increase in the amount of GFAP. Astrocytes incubated with lipopolysaccharide are capable of modulating neuronal proliferation and synaptogenesis processes in vitro.
Keywords: 
hippocampus, neurogenesis, lipopolysaccharide, neuroinflammation
Список литературы: 
  1. Ben Haim L., Rowitch D.H. Functional diversity of astrocytes in neural circuit regulation. Nature Reviews Neuroscience. 2016; 18 (1): 31–41. https://doi.org/10.1038/nrn.2016.159.
  2. Tuchina O.P., Adamovskaja S.S. Regional'naja geterogennost' astrotsitov v otnoshenii ekspressii glial'nogo fibrilljarnogo kislogo belka i sintetazy glutamina in vitro. Sovremennye problemy nauki i obrazovanija. 2020; 2: 158–62. https://doi.org/10.17513/spno.29751 [Tuchina O.P., Adamovskaya S.S. Regional heterogeneity of astrocytes in respect to glial fibrillary acidic protein and glutamine synthetase in vitro. Modern problems of science and education. 2020; 2: 158–62. https://doi.org/10.17513/spno.29751 (in Russian)].
  3. Franco R., Fernández-Suárez D. Alternatively activated microglia and macrophages in the central nervous system. Progress in Neurobiology. 2015; 131: 65–86. https://doi.org/10.1016/j.pneurobio.2015.05.003.
  4. Tuchina O.P. Nejro-immunnye vzaimodejstvija v holinergicheskom protivovospalitel'nom puti. Geny i kletki. 2020; XV (1): 23–8. https://doi.org/10.23868/202003003 [Tuchina O.P. Neuro-immune interactions in cholinergic anti-inflammatory pathway. Genes and Cells. 2020; XV (1): 23–8. https://doi.org/10.23868/202003003 (in Russian)].
  5. Banks W.A., Robinson S.M. Minimal penetration of lipopolysaccharide across the murine blood-brain barrier. Brain Behav Immun. 2009; 24 (1): 102–9. https://doi.org/10.1016/j.bbi.2009.09.001.
  6. Patlaj N.I., Sotnikov E.B., Kurilova E.A., Tuchina O.P. Rannie izmenenija reaktivnogo profilja glial'nyh kletok gippokampa myshi v otvet na lipopolisaharid. Sovremennye problemy nauki i obrazovanija. 2020; 6: 136–9. https://doi.org/10.17513/spno.30310 [Patlay N.I., Sotnikov E.B., Kurilova E.A., Tuchina O.P. Early changes in the reactive profile of mouse hippocampal glial cells in response to lipopolysaccharide. Modern problems of science and education. 2020; 6: 136–9. https://doi.org/10.17513/spno.30310 (in Russian)].
  7. Sotnikov E.B., Patlaj N.I., Nikolaeva A.Ju., Tuchina O.P. Ekspressija glial'nyh markerov, tsitokinov i markerov nejrogeneza v gippokampe myshi pri starenii i v otvet na lipopolisaharid. Molekuljarnaja meditsina. 2021; 19 (3): 38–43. https://doi.org/10.29296/24999490-2021-03-06 [Sotnikov E.B., Patlay N.I., Nikolaeva A.Y., Tuchina O.P. Expression of glial markers, cytokines and markers of neurogenesis in the mouse hippocampus during aging and in response to lipopolysaccharide. Molekulyarnaya meditsina 2021; 19 (3): 38–43. https:// doi.org/10.29296/24999490-2021-03-06 (in Russian)].
  8. Patlaj N.I., Sotnikov E.B., Tuchina O.P. Rol' mikroglial'nyh tsitokinov v moduljatsii nejrogeneza vo vzroslom mozge. Mezhdunarodnyj zhurnal prikladnyh i fundamental'nyh issledovanij. 2020; 5: 15–23. https://doi.org/10.17513/mjpfi.13062 [Patlay N.I., Sotnikov E.B., Tuchina O.P. The role of microglial cytokines in the modulation of neurogenesis in the adult brain. International journal of applied and fundamental studies. 2020; 5: 15–23. https://doi.org/10.17513/mjpfi.13062 (in Russian)].
  9. Liddelow S.A., Guttenplan K.A., Clarke L.E., Bennett F.C., Bohlen C.J., Schirmer L., Bennett M.L., Münch A.E., Chung W.-S., Peterson T.C., Wilton D.K., Frouin A., Napier B.A., Panicker N., Kumar M., Buckwalter M.S., Rowitch D.H., Dawson V.L., Dawson T.M., Stevens B., Barres B.A. Neurotoxic reactive astrocytes are induced by activated microglia. Nature. 2017; 541: 481–7. https://doi.org/10.1038/nature21029.
  10. You L.-H., Yan C.-Z., Zheng B.-J., Ci Y.-Z., Chang S.-Y., Yu P., Gao G.-F., Li H.-Y., Dong T.-Y., Chang Y.-Z. Astrocyte hepcidin is a key factor in LPS-induced neuronal apoptosis. Cell Death Dis. 2017; 8: e2676. https://doi.org/10.1038/cddis.2017.93.
  11. Tamashiro T.T., Dalgard C.L., Byrnes K.R. Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue. J. Vis Exp. 2012; 2: e3814. https://doi.org/10.3791/3814.
  12. Beaudoin G.M.J., Lee S.-H., Singh D., Yuan Y., Ng Y.-G., Reichardt L.F., Arikkath J. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc. 2012; 7: 1741–54. https://doi.org/10.1038/nprot.2012.099.
  13. Middeldorp J., Hol E.M. GFAP in health and disease. Progress in Neurobiology. 2011; 421–43. https://doi.org/10.1016/j.pneurobio.2011.01.005.
  14. Leow-Dyke S., Allen C., Denes A., Nilsson O., Maysami S., Bowie A.G., Rothwell N.J., Pinteaux E. Neuronal Toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro. J. Neuroinflammation. 2012; 9: 230–5. https://doi.org/10.1186/1742-2094-9-230.