Persistent Gliosis Interferes with Neurogenesis in Organotypic Hippocampal Slice Cultures

Abstract

Neurogenesis in the adult hippocampus has become an intensively investigated research topic, as it is essential for proper hippocampal function and considered to bear therapeutic potential for the replacement of pathologically lost neurons. On the other hand, neurogenesis itself is frequently affected by CNS insults. To identify processes leading to the disturbance of neurogenesis, we made use of organotypic hippocampal slice cultures (OHSC), which, for unknown reasons, lose their neurogenic potential during cultivation. In the present study, we show by BrdU/Prox1 double-immunostaining that the generation of new granule cells drops by 90% during the first week of cultivation. Monitoring neurogenesis dynamically in OHSC from POMC-eGFP mice, in which immature granule cells are endogenously labeled, revealed a gradual decay of the eGFP signal, reaching 10% of initial values within 7 days of cultivation. Accordingly, reverse transcription quantitative polymerase chain reaction analysis showed the downregulation of the neurogenesis-related genes doublecortin and Hes5, a crucial target of the stem cell-maintaining Notch signaling pathway. In parallel, we demonstrate a strong and long-lasting activation of astrocytes and microglial cells, both, morphologically and on the level of gene expression. Enhancement of astroglial activation by treating OHSC with ciliary neurotrophic factor accelerated the loss of neurogenesis, whereas treatment with indomethacin or an antagonist of the purinergic P2Y12 receptor exhibited potent protective effects on the neurogenic outcome. Therefore, we conclude that OHSC rapidly lose their neurogenic capacity due to persistent inflammatory processes taking place after the slice preparation. As inflammation is also considered to affect neurogenesis in many CNS pathologies, OHSC appear as a useful tool to study this interplay and its molecular basis. Furthermore, we propose that modification of glial activation might bear the therapeutic potential of enabling neurogenesis under neuropathological conditions.

Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation

“…Similarities in cellular composition and architecture between the in vitro organotypic brain slice cultures and the in vivo setting offer advantages to model complex brain physiology and pathophysiology even though a detailed characterization of the cellular and molecular features of the brain slice culture system was lacking as it pertains to microglia properties in this experimental system. These presumed similarities prompted many studies using variants of the brain slice culture platform to study pathogenic mechanisms involved in stroke, epilepsy, neurodegenerative proteinopathies, and responses to specific neuroinflammatory stimuli (Bernardino, 2005 ; Vinet et al, 2012 ; Ziemka-Nałecz et al, 2013 ; Olajide et al, 2014 ; Hellwig et al, 2015 ; Gerlach et al, 2016 ; Masuch et al, 2016a , b ; Bhatia et al, 2017 ; Saliba et al, 2017 ; Yousif et al, 2018 ; Araki et al, 2019 ; Croft et al, 2019a ; Grabiec et al, 2019 ; Richter et al, 2019 ; Sheppard et al, 2019 ). Revisiting these studies in light of our results may reveal new insights into the role of microglia in these settings.…”

Time-lapse imaging reveals highly dynamic structural maturation of postnatally born dentate granule cells in organotypic entorhino-hippocampal slice cultures

“…At DIV 28, DCX expression was limited to fewer cells that were located in the SGZ (Figs 1 and 2), thus revealing neurogenesis in postnatal OTCs. Previous studies have reported a substantial decrease in neurogenesis in OTCs during the first week of cultivation4546. Since DCX expression can persist up to 3 weeks in individual GCs21224849, DCX-positive cells at DIV 28 may represent GCs that were generated at early time points but were still immature and continued to express DCX at the time of fixation.…”

“…showed primarily symmetrical division of neuron-committed cells from neural progenitors at DIV 2, thus revealing ongoing neurogenesis at least during the first days in culture 44 . However, with maturation of the cultures the generation of newborn cells subsides 45 46 , making it difficult to use older, i.e. more mature cultures to study adult neurogenesis at later time points.…”

“…Since DCX expression can persist up to 3 weeks in individual GCs 21 22 48 49 , DCX-positive cells at DIV 28 may represent GCs that were generated at early time points but were still immature and continued to express DCX at the time of fixation. Alternatively, since neurogenesis does not cease completely in OTCs 24 25 45 46 , it could also be possible that a low number of GCs was generated at a later period. As in our cultures the number of DCX-expressing cells was much lower at the end of 4 weeks, our results reflect previous findings showing that the rate of neurogenesis in OTCs declines over time.…”

A Modular Assembly of Spinal Cord–Like Tissue Allows Targeted Tissue Repair in the Transected Spinal Cord

“…However, it is critical to predifferentiate NSCs into OPCs before applying CNTF so as to avoid astrocytic differentiation of NSCs as triggered by CNTF effect 22. Our previous studies focused on using NT‐3 and its receptor TrkC gene modification technique to promote neuronal differentiation of NSCs with synaptic transmission capability 11, 23.…”