Microglia induce neurogenesis by stimulating PI3K/AKT intracellular signaling in vitro

doi:10.21203/rs.2.11343/v2

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Microglia induce neurogenesis by stimulating PI3K/AKT intracellular signaling in vitro

https://doi.org/10.21203/rs.2.11343/v2

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Background: Emerging evidence suggests that microglia can support neuronal survival, synapse development, and neurogenesis in classic neurogenic niches. Little is known about the ability of microglia to regulate the cortical environment and stimulate cortical neurogenesis outside classic neurogenic niches. We used an in vitro co-culture model system to investigate the hypothesis that microglia respond to soluble signals from cortical cells, particularly following injury, to alter the cortical environment and promote cortical cell proliferation, differentiation, and maintain cortical cell survival via activation of specific cortical intracellular signaling pathways.

Results: Analyses of cell proliferation, apoptosis, protein expression, and intracellular signaling pathway activation were performed on uninjured and injured cortical cells in co-culture with EOC2 microglia. EOC2 microglia in co-culture enhanced cortical cell viability and proliferation of uninjured and injured cortical cells. Co-culture of injured cortical cells with EOC2 microglial cells significantly reduced cortical cell apoptosis. Microglial co-culture significantly increased Nestin+ and a-internexin+ cells within and outside of the injury site. NeuN+ cells increased in injured cortical cultures with microglia. Multiplex ELISA assays showed decreased levels of inflammatory cytokines in conditioned media from injured cortical cell and microglial co-culture. RTPCR analysis of microglial mRNA was performed. EOC2 microglial co-culture environment increased AKT phosphorylation in cortical cells particularly following injury. Inhibition of AKT phosphorylation in cortical cells blocked the microglial-enhanced cortical cell viability and expression of neurogenic markers in vitro.

Conclusion: The in vitro model system presented here allows for assessment of the effect of microglial-derived soluble signals, independent of cell-cell contact, on cortical cell viability, proliferation, and stages of differentiation during homeostasis or following injury. These data suggest that microglia downregulate inflammatory cytokine production following activation by acute cortical injury to enhance proliferation of new cells capable of neurogenesis. Inhibition of AKT signaling in cortical cells blocks the enhanced proliferation and expression of neurogenic markers in cortical cells. Increasing our understanding of the mechanisms that drive cortical neurogenesis stimulated by microglial cells during homeostasis and following injury will provide insight into the potential mechanisms of neuroprotective role of immune activity in the central nervous system (CNS).

Cellular & Molecular Neuroscience

microglia

neurogenesis

PI3K/AKT

Microglia are resident immunocompetent and phagocytic cells of the central nervous system (CNS) and comprise anywhere from 5–12% of cortical cells [1, 2]. During homeostasis, microglia survey the local CNS environment to communicate with neighboring glia and neurons through membrane bound and soluble signals [3, 4]. Emerging evidence suggests that, given specific activator(s), microglia function to support neuronal proliferation, differentiation, synaptic function, and survival [4,5]. Microglia contribute to synaptic development by refining axonal branching and pruning synaptic connections through phagocytic activity [6–9]. Additionally, specific microglial-derived cytokines, growth factors, and cell associated proteins play an important role in the modification and function of both excitatory and inhibitory synaptic connections in the CNS [10–12].

During homeostasis and following injury, microglia support neurogenesis in the classic neural stem cell niches in the dentate gyrus of the hippocampus and subventricular and subgranular zones of the lateral ventricle [13–23]. Further, in vivo studies have shown that specifically stimulated microglia, macrophages, and infiltrating T cells protect neuronal axons from secondary degeneration following injury, degrade inhibitory proteins that restrict neuronal survival and regrowth, reduce pro-inflammatory cytokine production, and induce growth factor and neurotrophin production [24–25]. Production of anti-inflammatory mediators and neurotrophic factors by microglia are likely to be dependent on the nature and duration of the stimulus as well as the severity of injury to which microglia respond [26, 27]. For example, microglia stimulated by damage signals from human peripheral nerve increase BDNF and GDNF secretion; upregulate the expression of migratory cytoskeletal proteins; upregulate proteolytic and debris clearing enzymes; and, enhance both STAT protein expression and NFκB gene transcription [28]. Microglial proliferation and increased release of transforming growth factor—(TGF-) are correlated with neural stem cell proliferation in the adult dentate gyrus [13]. Secretion of insulin growth factor 1 (IGF–1) from microglia following status epilepticus in the adult dentate gyrus stimulates neurogenesis via activation of the p42/44 MAPK pathway [30]. Other work suggests that injury to adult CA1 neurons of the dentate gyrus stimulates IGF–1 release from microglia and astrocytes promoting neuronal survival via AKT phosphorylation and decreased MAPK phosphorylation [31] or via both AKT and MAPK phosphorylation [32]. Increased AKT phosphorylation via PI3K signaling is important for neurogenesis in classic stem cell niches as well as the cortex [31–38]. Recent studies have shown that microglial-derived FGF and EGF growth factor and IL–10/IL–13 cytokine secretion promote the proliferation and differentiation of adult neural stem cells in vitro [39]. While an ever-growing body of work supports the role of microglial soluble signals in proper neurogenesis and plasticity, neuroinflammation caused by microglia activity is also linked to neurodevelopmental and neurodegenerative diseases [40–42]. For example, the reduction in neuroinflammatory cytokines such as TNF-α, IFN-ϒ, MIP–1α and RANTES/CCL5, IL–1α, and IL–1β suppresses apoptosis and enhances neurogenesis [29, 43]. Recent work demonstrates that during development and during inflammatory states associated with degeneration or injury, primary microglia express complex patterns of gene expression resulting functional diverse phenotypes in similar brain regions [43,44]. Taken together, a complex combination of microglial-derived soluble environmental cues with neurogenic or neuroinflammatory functions likely work in combination to promote or restrict neurogenesis and survival during development, survival and into maturity, and repair following injury [7, 18, 39–46].

Outside the classic neural stem cell niches, CNS stem cells have the potential to generate neurons and glial cells following ischemia or traumatic injury [43–4835, 47–50]. Microglial-derived soluble cues are potentially important for local endogenous neurogenesis, migration, and neuronal survival [51]. Interestingly, microglial invasion of the cortical plate overlaps with peak periods of cortical neurogenesis [1–3, 52]. After invasion, microglia remain as morphologically and functionally dynamic cells within the environment of the cortex [2–3, 46, 49]. Microglial-derived cytokines may promote neurogenesis [by supporting progenitor cell survival and mitosis [53–56]. Given the diverse functional roles that microglia can play in the cortex, the development of an in vitro model system to evaluate the neurogenic potential of a uniform population of microglia would be useful. We Consequently, we hypothesize that microglia are able to stimulate localized neurogenesis of cells throughout the cortex during homeostasis or following injury by activating specific intracellular signaling pathways required for neuronal survival and differentiation in cortical cells. Little is known about the mechanisms by which homeostatic microglia or activated microglia responding to injury influence neurogenesis and survival outside of hippocampal and lateral ventricular neurogenic niches.

In this study we present an in vitro model system for investigating the underlying mechanisms by which microglial respond to cortical cues to regulate neuronal differentiation and survival during homeostasis and following acute mechanical injury. Our in vitro system utilizes utilized a microgliaEOC2 microglial cell line suspended above primary rat cortical cells that are were injured or left uninjured as control. Primary cortical cells are were isolated from Sprague-Dawley rat embryos at embryonic day 16–18 because of the high yield of cortical cells that can differentiate into neurons in vitrothe differentiation of these cortical cells has been well characterized in vitro [4957]. Neurogenesis or the differentiation of new neurons in primary cortical cell culture can be determined by evaluating the progressive expression of neurogenic proteins such as Nestin, GFAP, α-internexin, and NeuN [58,59]. Nestin expression has long been known to be upregulated in neural progenitor cells and cortical radial glia [60]. Several studies also report that Nestin protein is expressed in early differentiating neurons in rodents and humans [61–66]. Recently, Nestin has been shown to be co-expressed with doublecortin in immature neurons of the cortex [67,68]. GFAP expression is associated with early stages of neurogenesis and is co-expressed with Nestin in cells of the neurogenic niche, such as neural stem cells, and following hypoxia in the cortical parenchyma [69]. GFAP is also expressed in populations of mature astrocytes and can be used as a marker for these glial cells. Later in neurogenesis, cytoskeletal elements such as α-internexin and neurofilament are upregulated and highly expressed as neurons begin to mature [70–72]. Alpha-internexin is expressed in postmitotic immature neurons as they commense differentiation and can be found co-localized with neurofilament triplets in mature neurons in the CNS [72]. The neuronal splicing regulator, NeuN, is expressed in both post-mitotic immature and mature cortical neurons [73]. We utilized the well characterized stages of neurogenesis in primary cortical cells to assess the effects of microglia during homeostasis and cortical injury on cortical cell viability, proliferation, differentiation, and intracellular signaling.

Our data suggest that EOC2 microglial-derived soluble cues promote cortical cell viability and enhance proliferation of cortical cells. Microglial- derived cues reduced apoptosis of primary cortical cells following acute mechanical injury in vitro. Significantly increased expression of neurogenic markers Nestin and α-internexin was seen within the site of injury where proliferation was enhanced. Expression of the mature neuronal marker NeuN increased in injured cortical cells outside the injury site when co-cultured with microgliaEOC2 microglial cells. MicrogliaEOC2 microglial cells responding to acute injury downregulated their expression of pro-inflammatory cytokines. AKT phosphorylation was increased in cortical cells co-cultured with microgliaEOC2 microglial cells. Inhibition of AKT phosphorylation reduces reduced the enhanced expression of neurogenic markers in cortical cell and microglial co-cultures. These results show that this co-culture in vitro system provides a model to evaluate cortical cell responses to microglial derived soluble cues and to investigate the underlying mechanisms of the functional states of microglia in response to cortical signals during homeostasis or following injury. This system may provide valuable insight into those mechanisms that should be evaluated for relevance in vivo and the potential for stimulating neuroimmune interactions that favor recovery after cortical injury.

CThe in vitro system utilized ortical and EOC2 microglial cells cultured on Transwell® inserts suspended directly above uninjured or injured primary cortical cells. These co-cultures co-cultures were established to investigate the effect of microglial responses during states ofto cortical cell homeostatic homeostasis and injury signals from cortical cellsfollowing cortical mechanical injury on cortical cell proliferation, survival, and differentiation.in vitro. Primary cortical cultures were established using methods previously described [4957]. To characterize the cortical cell types at the time of microglial co-culture, immunocytochemical analysis of cortical cell marker expression was performed at two days in vitro (Figure 1). In the control cortical culture, 56.3 ± 0.3% of cells expressed Nestin, 51.3 ± 2.0% expressed α-internexin, 41.7 ± 0.3% of cells expressed the mature neuronal marker TUJI, and 4.3 ± 0.3% expressed glial fibrillary acidic protein (Figure 1D). Only 1.9 ± 0.6% of over 1000 cells counted were immunopositive for the microglial marker CD11b (CD11b+) demonstrating that the culture conditions did not support primary microglial cell proliferation and survival (Figure 1 E; over 300 cells were counted per experiment in three separate experiments; ± represents standard error of the mean (SEM)).

Immediately prior to co-culture with suspended EOC2 microglial cellsmicroglia, cortical cells were left uninjured or were injured using a sterile stylet to disrupt and remove cortical cells from the culture surface [74]plate. Uninjured and iInjured cortical cells were then cultured for two additional days with or without microgliaEOC2 microglial cells on Transwell® inserts. Two days following injury in cortical cultures without EOC2 microglia, the site of injury (indicated by the dashed white line) was observable and few neurofilament immunopositive (NF+) cells or processes were found in the injury site (Figure 2A, B; black scale bar represents 100 µm). In cortical cultures with EOC2 microglia, the site of injury (indicated by the dashed white line) was associated with increased cell density and increased neurofilament expression at the site of injury (Figure 2C, D). MicrogliaEOC2 microglial cells used for co-culture experiments are CD11b+ (Figure 2 E, F; white scale bar represents 50 µm).

Cortical cell viability following injury and co-culture with microglial was measured using 3-(4,5-Dimethylthiazol–2-yl)–2,5-Diphenyltetrazolium Bromide (MTT) colorimetric assays that measure metabolic activity in living cells. Injured cortical cultures without microgliaEOC2 microglial cells and uninjured cortical cultures with and without microgliaEOC2 microglial cells were also assessed using the MTT assay. Quantification of optical density (O. D.) of three separate MTT assays performed in triplicate shows that in the absence of cortical cell injury, microglial-derived soluble cues significantly enhanced cortical cell mitochondrial activity by a mean difference of 0.28 ± 0.03 O. D. units (*p<0.05, ± represents SEM, n = 3) as compared to uninjured cortical cells cultured in control media alone (Figure 2G). Co-culture of injured cortical cells with microgliaEOC2 microglial cells significantly increased mitochondrial activity by a mean difference of 0.39 ± 0.03 O. D. units (*p<0.05, ± represents SEM, n = 3) when compared to uninjured conditions without microgliaEOC2 microglial cells and by 0.42 ± 0.02 O. D. units (**p<0.01, ± represents SEM, n = 3) when compared to injured cortical cells co-cultured without microgliaEOC2 microglial cells (Figure 2G). Following injury, metabolic activity as a measure of cell viability was not significantly different from MTT activity in uninjured control conditions (Figure 2G, p>0.05, n = 3).

Immunocytochemistry assays specific for measuring cell proliferation and survival were used to examine the response of primary cortical cells to co-culture with soluble signals from microgliaEOC2 microglial cells. Cell proliferation was measured by incorporation of a modified, fluorescently labeled thymidine analogue EdU into newly synthesized DNA. Large field confocal image analysis of uninjured cortical cells without microgliaEOC2 microglial cells showed the presence of EdU+ cells demonstrating that these cultures have at least a limited number of dividing cells upon isolation from the cortex (Figure 3A). In the presence of microgliaEOC2 microglial cells, the number of EdU+ cells increased in uninjured cortical cell culture (Figure 3B). Mechanical injury of cortical cells striped away cortical cells as indicated by dashed white lines (Figure 3C-D). Without microgliaEOC2 microglial cells, few EdU+ cells were in the damaged area (Figure 3C). When injured cortical cells were co-cultured with microgliaEOC2 microglial cells, an increase in proliferating EdU+ cells was seen throughout the culture and within the damaged area (Figure 3D). Full magnification of the boxed area within the injured site and EdU+ cells (Figure 3D) is shown in Figure 3E. Quantification of proliferating cells in uninjured cortical culture without microgliaEOC2 microglial cells showed that 45.7 ± 5.0% of the cells were EdU+. In the presence of microgliaEOC2 microglial cells, the average percent of EdU+ cells increased to 74.3 ± 5.6%. This 28.6 ± 7.5% increase in EdU+ cells in the presence of microgliaEOC2 microglial cells was significant (Figure 3E, *p< 0.05, ± represents SEM, n = 3). Following injury, the percent of EdU+ cells in cortical cultures without microgliaEOC2 microglial cells was 47.2 ± 9.3% and was not significantly different from the control, uninjured cortical cells cultured without microgliaEOC2 microglial cells (Figure 3F, p>0.05, ± represents SEM, n = 3,). When cultured with microgliaEOC2 microglial cells, the number of proliferating EdU+ cells in injured cortical cultures increased 38.5 ± 6.0% to 84.3 ± 3.3% compared to uninjured control cells (Figure 3F, **p<0.01, ± represents SEM, n = 3). The difference in the percent of proliferating cells between uninjured and injured cortical cells co-cultured with microglia did not reach significance (p>0.05).

To evaluate the effect of microgliaEOC2 microglial cells on cell survival, Click-iT® fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. Figure 4 illustrates TUNEL+ immunocytochemistry observed using large field confocal imaging of uninjured and injured cortical cultures in the presence or absence of microgliaEOC2 microglial cells. In the absence of microgliaEOC2 microglial cells, very few TUNEL+ cells were present in uninjured cortical cell cultures (Figure 4A). Following injury, cortical cells in the absence of microgliaEOC2 microglial cells, showed increased TUNEL expression particularly in the area of damage (Figure 4C). When injured cortical cells were co-cultured with microgliaEOC2 microglial cells, a decrease in TUNEL+ cells were observed both within the injured area and throughout the cell culture (Figure 4D). Full magnification of the boxed area within the injury site clearly revealed the presence of TUNEL+ cells (Figure 4E). Quantification of TUNEL staining in uninjured cortical cultures without microgliaEOC2 microglial cells shows that 1.72 ± 0.2% of the cells were TUNEL+. Co-culture of microgliaEOC2 microglial cells with uninjured cortical cells did not significantly alter the percent of TUNEL+ cells (3.44 ± 0.6%, ± represents SEM, n = 3, p>0.05, Figure 4F). Following injury, the percent of TUNEL+ cells in cortical cultures without microgliaEOC2 microglial cells significantly increased to 30.1 ± 4.9% (± represents SEM, n = 3, *p<0.05, Figure 4F). When cultured with microgliaEOC2 microglial cells, the number of TUNEL+ cells in injured cortical cultures decreased to 5.6 ± 1.2% (± represents SEM, n = 3, Figure 4F). The reduction of TUNEL staining by 24.6 ± 4.8% in injured cortical cells co-cultured with microglia was highly significant (**p<0.01, ± represents SEM, n = 3, Figure 4F). The percent of TUNEL+ cells in uninjured cortical cultures in the presence of microgliaEOC2 microglial cells was not significantly different from the percent of TUNEL+ cells observed in uninjured cortical cultures used as control (p>0.05, n = 3, Figure 4F).

Since EdU expression was increased and TUNEL expression was reduced in injured cortical cells co-cultured with EOC2 microglial cells, we sought to determine whether the proliferating cells were expressing neurogenic protein markers. Progressive expression of proteins such as Nestin, glial fibrillary acidic protein (GFAP), and α-internexin, and NeuN is indicative of stages of neurogenesis [5158–731]. Quantification of the expression of Nestin+, glial fibrillary acidic protein (GFAP)+, α-internexin+, and NeuN+ in cortical cells following injury and exposure to EOC2 microglial cells was determined using immunocytochemical and western blot analyses.

In order to address whether microglial derived soluble signaling cues influence the number of cells expressing Nestin, GFAP, α-internexin, and NeuN, co-culture experiments were established where cortical cells were injured or left uninjured and then cultured with EOC2 microglia. After 2DIV, cortical co-cultures were fixed and immunostained for Nestin, GFAP, α-internexin, and NeuN. Nuclei were identified with DAPI (blue immunofluorescence). In all experiments at least 100 cells were counted per field from three separate culture fields for each condition. The percent of immunopositive cells per field was determined by dividing the number of immunopositive cells by the total number of DAPI stained cells per field. The average percent of immunopositive cells was determined for three biological replicates and compared to controls. Two-way ANOVA with multiple comparisons followed by Tukey’s multiple comparisons test was used to determine significance of these data. The presence of EOC2 microglial soluble cues significantly increased the percent of Nestin immunopositive (Nestin+) cells in uninjured control cortical cultures by ~23% (Figure 5A, ***p<0.001) as compared to control, uninjured cortical cultures without microglia. Microglial soluble cues increased the percent of Nestin immunopositive cells within the site of injury by more than 45% and by approximately 38% outside the site of injury as compared to control, cortical cultures without EOC2 microglia. These differences were highly significant (Figure 5A,****p<0.0001). Injury of confluent cortical cultures without EOC2 microglial soluble cues resulted in a significant loss of over 20% of Nestin+ cells within the injury site and no significant change in the percent of Nestin+ cells outside the injury site (Figure 5A, p>0.05). Following injury, co-culture with EOC2 microglial soluble cues significantly increased the percent of Nestin+ cells at the site of injury by over 22% and outside the site of injury by approximately 15% (Figure 5A, ***p<0.001 and *p<0.05). In all culture conditions, the percent of GFAP immunopositive cells that increased in the uninjured and injured cortical cultures with EOC2 microglia was not significantly different from that of control, uninjured cortical cells without microglial co-culture (Figure 5B, p>0.05). The expression of α-internexin, an intermediate filament expressed in post-mitotic and mature neurons, was significantly increased in cortical cultures when in the presence of EOC2 microglial soluble cues (Figure 5C). The percent of uninjured cortical cells expressing α-internexin increased nearly 17% in the presence of microglial-derived soluble cues as compared to controls (Figure 5C, *p<0.05). Injury of cortical cultures in the absence of EO2 microglia decreased the percent of α-internexin immunopositive (α-internexin+) cells by nearly 32% (Figure 5C, ****p<0.0001). Co-culture of injured cortical cells with microglial-derived soluble cues significantly increased the percent of α-internexin+ cells by nearly 73% within the site of injury to 88.8 ± 4.3% and over 74% outside the site of injury to 89.8 ± 4.1% (Figure 5C, ***p<0.001 and ****p<0.0001, respectively). The percent of NeuN immunopositive (NeuN+) cells in uninjured cortical cultures in the presence of EOC2 microglia increased by approximately 5% as compared to uninjured cortical cultures without microglia (Figure 5D, *p>0.05). Injury of cortical cells significantly decreased the percent of NeuN+ cells by nearly 20% at the site of injury as compared to uninjured controls in the absence or presence of microglia (Figure 5D, ***p<0.001 and **p<0.01, respectively). Outside the site of injury, the percent of NeuN+ cells in cultures without microglia was not significantly different from uninjured control conditions (Figure 5D, *p<0.05) and was approximately 20% higher than the percent of NeuN+ cells seen within the injury site (Figure 5D, ***p<0.001). The presence of EOC2 microglia increased the percent of NeuN+ cells outside the site of injury by approximately 19% to 48.5 ± 1.1% as compared to sites outside of injury without EOC2 microglial in co-culture (Figure 5D, **p<0.01). The increase in NeuN+ cells in the presence of microglia following injury is of interest since expression of neurogenic markers should lead to neuronal differentiation as indicated by NeuN expression. Higher power images of NeuN immunoreactivity show NeuN+ cells at the site of injury and outside the side of injury (Figure 1S). An increase in the number of total cells was seen in cortical cultures in the presence of EOC2 microglial-derived soluble cues as compared to those cortical cultures alone (Figure 1SA,B). Several cells within the injury site show low NeuN immunoreactivity and did not meet the criteria for positive immunoreactivity (Figure 1SB).

Relative immunofluorescence units (RFU) of immunocytochemistry experiments were measured to assess the level of neurogenic protein expression. RFU for each labeled primary and secondary antibody conjugate directed against a neurogenic protein was calculated by measuring pixel intensity for each fluorochrome in images acquired with the same exposure settings for all experimental conditions. For each protein, the immunofluorescence of at least 100 cells per field from three separate culture fields were averaged and compared to controls. These experiments were performed using cortical cultures from three biological replicates (Figure 6). Following injury, cortical cells were cultured in the presence of EOC2 microglial cells or in media alone. Immunofluorescent images for Nestin and GFAP or α-internexin and GFAP, or NeuN and GFAP showed increased expression of these early neurogenic and neuronal markersproteins in injured areas when co-cultured with microgliaEOC2 microglial cells as compared to media alone. Nestin and GFAP expression was significantly enhanced in injured neuronal cultures co-cultured with microgliaEOC2 microglial cells as compared to cortical cells cultured alone (Figures 5A6A). Cells immunopositive for both Nestin (green) and GFAP (red) are indicated by yellow immunofluorescence. Nestin immunofluorescence increased 7.4 ± 0.3fold in injured areas of cortical cells co-cultured with EOC2 microglia as compared to Nestin immunofluorescence in injured cortical cultures without EOC2 microglial co-culture(± represents SEM, n = 3, ****p<0.0001, Figure 6B). A 4.0 ± 0.2fold increase in GFAP immunofluorescence was observed in injured cortical cultures co-cultured with microgliaEOC2 microglial cells as compared to controls (± represents SEM, n = 3 ***p<0.001, Figure 5B6B). Expression of α-internexin, significantly increased 16.7 ± 0.8fold in injured cortical cultures when exposed to microglial-derived soluble cues as compared to injured controls alone (± represents SEM, n = 3, ****p <0.0001, Figure 5B6B). NeuN immunofluorescence was 0.4 ± 0.1fold higher in injured cortical cultures with microgliaEOC2 microglial cells than in control cultures without microglia at 2 DIV (± represents SEM, n = 3 **p<0.051 Figure 5B6B).

Western blot analysis was used to assess protein expression of neurogenic markers in injured as well as uninjured cortical cultures (Figure 6A7A). Nestin protein expression in uninjured and injured cortical cultures increased 1.7 ± 0.1fold and 1.5 ± 0.1fold respectively following co-culture with microgliaEOC2 microglial cells as compared to expression in uninjured control cultures alone (± represents SEM, n = 3, ****p<0.0001, ***p <0.001, Figure 6B7B). No significant change in Nestin expression was observed in cortical cultures that were injured and not co-cultured with microgliaEOC2 microglial cells (p>0.05, n = 3, Figure 6B7B). A 1.9 ± 0.5fold increase in α-internexin expression was observed in uninjured cortical cultures co-cultured with microgliaEOC2 microglial cells as compared to control, uninjured neurons cultured alone (± represents SEM, n = 3, **p <0.01, Figure 6B7B). Injured cortical cells co-cultured with microgliaEOC2 microglial cells exhibited a 2.4 ± 0.4fold increase in α-internexin expression compared to control, uninjured neurons cultured without microgliaEOC2 microglial cells (± represents SEM, n = 3**p <0.01, Figure 6B7B). No significant change in Nestin expression was observed in cortical cultures that were injured and not co-cultured with microgliaEOC2 microglial cells (p>0.05, n = 3, Figure 6B7B). GFAP, a protein expressed during neurogenesis and in mature astrocytes, levels increased 1.6 ± 0.1fold in uninjured cortical cells co-cultured with microgliaEOC2 microglial cells and 1.9 ± 0.2fold in injured cortical cells co-cultured with microgliaEOC2 microglial cells as compared to control uninjured cortical cells (± represents SEM, n = 3, ***p <0.001, Figure 6B7B). Expression of GFAP also significantly increased ~1.6 fold in injured cortical cells without microglial co-culture (***p<0.001, n = 3, Figure 6B7B). Injury of cortical cells followed by 2 DIV without microglial co-culture reduced NeuN expression significantly by 0.25 ± 0.1fold as compared to uninjured control cortical cultures (± represents SEM, n = 3, *p <0.05, Figure 6B). NeuN protein expression in injured cortical cells co-cultured with microgliaEOC2 microglial cells was not significantly different that NeuN expression in control conditions as determined by western blot analysis of total protein collected from cortical cultures (± represents SEM, n = 3, p>0.05, Figure 6B7B).

In order to begin to examine changes in the cytokine environment of cortical cells in the presence of EOC2 microglia, multiplex ELISA assays were used to determine the presence of well-characterized microglial-derived cytokines in microglial-conditioned media following injury of co-cultured cortical cells and then compared to the cytokine levels in microglial-conditioned media following co-culture with uninjured cortical cells. Analysis of three separate assays performed in triplicate showed that the concentration of several cytokines was significantly different from the levels observed in microgliaEOC2 microglial cells-conditioned media when suspended above uninjured cortical cells (Figure 78). The concentration of cytokines measured in media collected from uninjured cortical cell and microglial co-culture was used as the baseline, normalized, and set equal to one (Figure 7A). When compared to control cytokine concentrations, MCP–1 concentration increased 22.0 ± 0.02% above control levels while IFN- and TNF- expression concentration decreased to 41.3 ± 0.07% and 73.5 ± 0.08% below control levels, respectively (± represents SEM, n = 3, *p <0.05, Figure 7A8A). Concentrations of MIP–1 and RANTES decreased by ~20% in media from injured cortical and microglial co-cultures compared to media from uninjured cortical and microglial co-cultures but these decreases were not significant (p>0.05, Figure 7B8B). IL–1, IL–1, IL–2, IL–4, IL–6, and GM-CSF were either undetectable or not not significantly different in conditioned media from uninjured and injured cortical and microglial co-cultures (data not shownFigure 2S). IL–1, IL–1, IL–2, IL–4 levels were undetectable in conditioned media from uninjured and injured cortical cultures with and without microglia. IL–6 was measured to be 3.5 ± 14.4 pg/ml (± is SD) in uninjured cortical cell and microglia conditioned media. The large standard deviation does not allow for accurate interpretation of IL–6 in this co-culture system. GMCSF was also detected in uninjured and injured cortical cell and microglia co-culture media. GMCSF concentration was 18.2 ± 13.2 pg/ml (± is SD) and 14.1 ± 16.3 pg/ml (± is SD) in uninjured cortical cell and injured cortical microglial co-culture media respectively (Figure 2S). The large standard deviation does not allow for accurate interpretation of GMCSF in this co-culture system (Figure 2S).

To begin to investigate whether the differences in cytokine concentrations are due to changes in microglial cytokine expression, RT-CPR was used to compare cytokine mRNA levels in of microgliaEOC2 microglial cells following co-culture with injured or uninjuredco-cultured with injured neurons cortical cellsto mRNA levels in control microglia co-cultured with uninjured neurons. Since EOC2 microglial cells are physically separated from cortical cells by Transwell permeable supports, RNA can be specifically isolated from EOC2 microglial cells upon removal of the Transwell from the cortical culture. MicrogliaEOC2 microglial cells in co-culture with injured cortical neurons, demonstrated decreased mRNA expression as compared to controls (indicated by the dashed line) for IFN- (decreased by 22.2±10.2%), MCP–1 (decreased by 79.7 ± 2.9%), MIP–1α (decreased by 60.2 ± 6.7%) TNF- (decreased by 97.6 ± 4.1%) and RANTES (decreased by 62.5 ±11.6%) (± is SEM, Figure 7B8B). Decreased expression of MCP–1 mRNA in microgliaEOC2 microglial cells suggests that the increase in MCP–1 protein levels was not microglial derived (Figure 8B). Given that microglial cells, neurons, and astroglial cells can secrete a variety of soluble factors, studies directed at identifying neurogenic microglial, neuronal, or glial soluble signals are being addressed using this in vitro system.

Several signaling pathways are activated by soluble signaling molecules during neurogenesis [29, 57–58] and may also underlie microglial-enhanced neurogenesis observed in our co-culture system. To begin to investigate possible signaling pathways important for microglial-enhanced neurogenesis, injured and uninjured cortical cells in co-culture with and without microgliaEOC2 microglial cells were treated with inhibitors for intracellular signaling pathways. We then used the MTT viability assay to screen for those inhibitors that blocked microglial-enhanced viability of cortical cells. Microglial co-culture increased viability of uninjured and injured cortical cultures as compared to cortical cultures alone as shown previously (***p <0.001, see supplemental data, Figure 2SA3SA-D). Inhibitors for MEK (PD98059), p38 MAPK (SKF86002), PKCα/βI/βII/γ (GF109203X), and Janus Kinase 2 protein (AG490) did not block the increased metabolic activity and viability of cortical cells co-cultured with microgliaEOC2 microglial cells. AG490 and GF109203X at 40 µm did significantly influence viability of cortical cultures but these affects were not specific for microglial-enhanced viability (Figure 2S3S). LY294002, an inhibitor of PI3K, and downstream AKT phosphorylation, specifically reduced microglial-enhanced cortical cell viability by ~50% or 0.52 ± 0.02 and 0.51 ± 0.02 optical density (O. D.) units at 10 and 40 µm respectively as compared to untreated, uninjured cortical cells co-cultured with microgliaEOC2 microglial cells (± represents SEM, n = 3, ****p <0.0001, Figure 8A9A). Following injury, 10 and 40 µm LY294002 treatment significantly reduced microglial-enhanced cortical cell viability by ~73% or 0.73 ± 0.02 and 0.74 ± 0.02 O. D. units (± represents SEM, n = 3, ****p <0.0001, Figure 8A9A). Treatment of uninjured or injured cortical cells in the absence of microgliaEOC2 microglial cells with LY294002 did not significantly affect metabolic activity as measured by MTT (± represents SEM, n = 3, p >0.05, Figure 8A9A). Western blot analysis of cortical cells confirmed that the presence of microgliaEOC2 microglial cells in suspension above cortical cultures increased phosphorylation of AKT, a PI3K target, in cortical cells (Figure 8B9B). Analysis of western blots showed that the presence of microgliaEOC2 microglial cells in uninjured cortical cultures increased AKT phosphorylation 3.6 ± 1.0fold as compared to uninjured cortical cells alone (± represents SEM, n = 3, *p <0.05, Figure 8C9C). Injury alone did not significantly increase (~0.8 fold) phosphorylation of AKT as compared to control levels (Figure 8C9C, p>0.05). Following injury and co-culture with microgliaEOC2 microglial cells, AKT phosphorylation increased 5.0 ± 1.0fold as compared to injured cortical cells alone (± represents SEM, n = 3, **p <0.01, Figure 8C9C). This increase was also significantly different from AKT phosphorylation levels measured in injured cortical cells without microglial co-culture (4.2 ± 1.0fold increase, *p<0.05, ± represents SEM, n = 3, Figure 8C9C). These experiments suggest that phosphorylation of AKT may be necessary for microglial-enhanced expression of specific neurogenic proteins.

To further investigate the necessity of the AKT phosphorylation for microglial-enhanced neurogenesisexpression of neurogenic proteins, immunocytochemical analysis of neurogenic protein expression was assessed in injured and uninjured cortical cells co-cultured with microgliaEOC2 microglial cells in the presence of 40 m LY294002. Uninjured cortical cells incubated with LY294002 but notbut without microgliaEOC2 microglial cells served as the control and baseline for the normalization of protein expression. Incubation of cortical cultures with LY294002 completely blocked the increase in AKT phosphorylation and reduced neurogenic protein expression seen in cortical cells when cultured with EOC2 microglial cells (Figure 9D). LY294002 reduced Nestin expression by 0.44 ± 0.3fold in uninjured cortical cells co-cultured with EOC2 microglial cells as compared to controls (**p<0.01, n = 3, Figure 9E). LY294004 treatment reduced Nestin expression by 0.26 ± 0.0fold in injured cortical cells co-cultured with EOC2 microglial cells as compared to uninjured controls (***p<0.001, n = 3, Figure 8E9E) and by 0.36 ± 0.1fold in injured cortical cells cultured without microgliaEOC2 microglial cells (**p<0.01, n = 3, Figure 8E9E). Expression of the neuronal intermediate filament α-internexin was significantly reduced by 0.59 ± 0.1fold (****p<0.0001) in injured cortical cells co-cultured with microgliaEOC2 microglial cells when compared with uninjured controls and by 0.40 ± 0.1fold (**p<0.01) when compared with injured cortical cells not cultured with microgliaEOC2 microglial cells (Figure 8E9E, n = 3). Additionally, microglial-enhanced expression of GFAP in uninjured and injured cortical cells was significantly reduced following treatment with LY294002 (Figure 8D 9D and 8E9E). GFAP expression was reduced by 0.76 ± 0.0fold as compared to uninjured controls and 0.85 ± 0.1fold as compared to injured cortical cells without microglial co-culture (****p<0.0001, n = 3, Figure 8E9E).

Immunocytochemical analysis was also used to evaluate the effect of blocking PI3K activity and AKT phosphorylation (Figure 910). Co-cultures were established as previously described and cortical cells were incubated with LY294002. After co-culture, cells were fixed and evaluated for the expression of the neurogenic markers- Nestin, -internexin, and GFAP (Figure 9A10A) since the expression of these markers was enhanced in our co-culture system. DAPI immunofluorescence was used to identify nuclei (Figure 9A10A). Immunocytochemical analysis confirmed western blot data showing that LY294002 treatment and inhibition of AKT phosphorylation reduced Nestin, α-internexin, and GFAP expression in injured cortical cells co-cultured with microgliaEOC2 microglial cells (Figure 9A10A). Quantification of neurogenic protein expression was determined by calculating RFU for each labeled primary and secondary antibody conjugate directed against neurogenic proteins in images acquired with the same exposure settings for all experimental conditions as described previously. Evaluation of RFU for Nestin expression showed that co-culture with microgliaEOC2 microglial cells significantly enhanced Nestin in uninjured or injured cortical cultures as compared to Nestin expression in cortical cultures alone (*p<0.05, Figure 9B10B). Application of 40 m LY294002 reduced Nestin expression in co-cultures of microgliaEOC2 microglial cells and uninjured cortical cells by 8.5 ± 2.6 RFU or ~44% (***p<0.001, ± is SEM, n = 3, Figure 9B10B). In co-cultures of microgliaEOC2 microglial cells and injured cortical cells, 40 m LY294002 reduced Nestin expression by 17.3 ± 3.9 RFU or ~77% (***p<0.001, ± is SEM, n = 3, Figure 9B10B). LY294002 application did not significantly affect Nestin expression in cortical cells that were not cultured with microgliaEOC2 microglial cells (p>0.05, n = 3, Figure 9B10B). Expression of the neurofilament -internexin was similarly reduced by inhibiting the PI3K pathway by 12.0 ± 1.9 RFU or ~40 % (***p<0.001, n = 3) in injured neuronal co-cultures with microgliaEOC2 microglial cells (Figure 9C10C). GFAP expression was also significantly reduced to 26.4% of control. LY294002 did not significantly change protein expression in uninjured or injured neurons cultured in control media alone (p>0.05, Figure 9D10D). These data underscore the importance and necessity of AKT phosphorylation via PI3K activity for microglial-enhanced neurogenesis in this in vitro system.

This study examines the ability of EOC2 microglial cells to support the viability, proliferation, neurogenesis, and survival of primary cortical cells outside of the classic neurogenic niche during homeostasis and following mechanical injury. Stem cell progenitors outside of the classic neurogenic niches have the capacity for neurogenesis during normal homeostasis and following injury or disease given exposure to the proper combination of neurogenic cues [478, 55–5648–50]. Cortical microglial cells are a potential source of neurogenic signaling molecules [4, 39–42, 51–5645–47]. Microglia have been shown to contribute to neuronal synapse development, survival, and neurogenesis in neurogenic niches of the CNS during development and following injury [5–6, 8, 10–23, 45–4751–56]. Neurogenic potential of microglial-derived cues may be controlled by the mechanism and duration of activation since infection, trauma, and disease also stimulate pro-inflammatory responses from microglia that can lead to neurotoxicity and neurodegeneration [26–27, 43, 75,7657–58]. Several in vitro studies suggest that microglial-conditioned media is neurotoxic depending on the mechanism of microglial activation [43, 57–5944–46, 75, 76]. Recent characterization of primary microglia using single cell sequencing as well as other methods shows that microglia are highly diverse and that subpopulations of functional microglia are present within specific brain regions and in responses to particular environmental cues [45,46]. When investigating primary microglia, it is important to consider their functional diversity and sensitivity to environmental conditions. Of note, even the conditions of microglial isolation from the cortex can result in unique microglia phenotypes and functions in vitro [46]. Because of the diversity of primary microglia, this study presents an in vitro model system using the EOC2 microglial cell line and primary cortical cells in co-culture to begin to examine the neurogenic potential of microglial soluble cues during cortical cell homeostasis and following cortical cell injury. Suspension of microglia on Transwell® cell culture inserts directly above cortical cells allows for evaluation of how the cortical environment is influenced by microglial-derived soluble cues. The methods of isolating primary cortical cells utilized in this in vitro system have been well characterized [52] and our data show that these cells express primarily neurogenic and neuronal cell markers with evidence of less than 5% astrocytic protein expression and less than 2% microglial protein expression (Figure 1F). Using this system where EOC2 microglia and primary cortical cells are not in direct contact also allows for protein and RNA to be collected independently from either cell population. This reductionist in vitro system allows for preliminary questions to be asked about microglial neurogenic potential in the cortex.

Results from the experiments presented here show that soluble cues from microgliaEOC2 microglial cells responding to injury signals from primary cortical cells enhanced proliferation, suppressed apoptosis, and promoted the expression of neurogenic and mature neuronal markers in the primary cortical cultures. Immunocytochemical analysis and MTT assays of cortical cells in co-culture with suspended EOC2 microglial cells following injury showed that microgliaEOC2 microglial cells enhanced viability of neurofilament immunopositive cells and increased extension of neurofilament (heavy and light) immunopositive processes into injured areas of cortical cultures (Figure 2). Neurofilament is expressed in neuronal progenitor cells and mature neurons. Neurofilament expression is associated with structural maturation of neurons and axonal function as light, medium, and heavy neurofilaments assemble [64,65,7069, 71]. Our immunocytochemical observations show that microgliaEOC2 microglial cells responding to injury increase heavy and light neurofilament expression in viable cortical cells in vitro. Neurodegeneration and inflammatory responses of microglia are often associated with reduced neurofilament expression, disrupted axon development, and demyelination [102] . although the role of microglia-derived inflammatory response in axon degeneration and regeneration in the visual system has recently been called into question [64]. Given our current understanding, increased expression of neurofilament in these in vitro studies suggests a reduction in the release of proinflammatory soluble signals released from EOC2 microglia and/or cortical cells in the microglial co-culture environment.

To more specifically address how microgliaEOC2 microglial cells during homeostasis and during responses to cortical injury influence cortical cell differentiation, the in vitro system was used to assess cell proliferation, apoptosis, and expression of neurogenic markers in uninjured and injured co-culture conditions. Immunocytochemical analyses revealed that co-culturing uninjured or injured cortical cells with microgliaEOC2 microglial cells significantly increased the percent of EdU positive cells in cortical cultures as compared to controls (Figure 3). EdU positive, or proliferating cells were observed in the area of damagewithin the injury site and throughout the culture (Figure 3). Particularly striking is the proliferation of cortical cells within the injury site. Enhanced proliferation is not seen within the injury site injured cortical cultures without microglia (Figure 3). Microglial responses in the subgranular and subventricular zones can influence the number of mature neurons that are generated by regulating neuronal stem cell and neuronal progenitor cell proliferation and differentiation throughout life [4–5, 10–11, 21, 39, 445]. In the subgranular zone and subventricular zones the microglial responses are dependent upon location and specific developmental time points [4–5, 46–4751–52]. Enhancement of cortical cell proliferation outside of the neurogenic niche as shown using this in vitro system suggests that the soluble environment of cortical cells as directed by the presence of EOC2 microglia is able to significantly support cortical cell proliferation during homeostasis and following injury (Figure 3).

Apoptosis as measured by TUNEL staining showed that co-culture with microgliaEOC2 microglial cells significantly reduced apoptosis in cortical cells following injury (Figure 4). Microglia can increase or reduce apoptosis in neurogenic zones and this response is dependent upon the combination of proinflammatory cytokines, growth factors, and phagocytic activity of microglia [5, 22–23, 26–27, 29]. Recently it has been shown that neuroblasts can be recruited from the subventricular zone of hippocampus to sites of injury in the cortex by microglial-derived specific cues suggesting that microglia responding to cortical damage may be activated, at least at specific times following injury, to attract differentiating neuronal progenitors into cortical tissue [6477]. Chemical depletion of microglia in acute injury in vivo models proposes microglia are not involved in axon regeneration [77]. It could be reasoned that microglia may play a role in specific phases of recovery after damage, such as cellular proliferation, and not others, such as axonal outgrowth. These studies Further studies suggest microglia may also act locally to stimulate neurogenesis of responsive cortical cells at noncanonical neurogenic regions [4847, 65, 6951–56, 79]. We propose that this in vitro model system is useful to investigate whether microglial soluble signals following cortical injury are have neurogenic potential outside of previously investigated subventricular and subgranular zones. Further this in vitro system allows for analysis of neurogenic protein expression and activation of intracellular signaling pathways mediated by microglia in cortical cells.

Microglial-conditioned media increased expression of neurofilament immunopositive cells and processes following cortical injury (Figure 2) so further investigation of expression of neurogenic markers, Nestin, -internexin, GFAP, and NeuN was warranted (Figures 5–7). Quantification of the percent of cortical cells expressing Nestin, -internexin, GFAP, and NeuN as well as the relative amount of Nestin, -internexin, GFAP, and NeuN protein expression showed significant differences in neurogenic protein expression when uninjured and injured cortical cells are co-cultured with EOC2 microglia. Nestin expression alone or Nestin and GFAP expression are characteristic protein expression patterns of early, primary neurogenic stem cells and progenitors in classic neurogenic regions [2,44,65,6660–63, 80–81].

Nestin immunopositive progenitors can eventually give rise to intermediate progenitors that produce immature neurons or neuron-committed progenitors [60–68, 80–8165–66]. These microglial-derived soluble cues may also enhance gliogenesis. [67–70]. Nestin and GFAP expression are associated with the proliferation of migratory astrocyte progenitors following trauma in the neurogenic niche of the subependymal zone [55–56, 82–8353, 68]. Either directly or by stimulating nearby cells, the modification of microglial-derived Enhancement of Nestin expression in injured cortical cultures following exposure to microglial-derived soluble cues at homeostasis and more so in response to injury, presents a potential mechanism by which local signaling maymay help shape a local environment that can stimulate the process of neurogenesisthe Nestin+ cell proliferation that is important for generation of new neurons and glia following injury in the cortex outside of classic neurogenic regions [69]. The expression of -internexin, a Type IV neuronal intermediate filament protein occurs during later stages of neuronal differentiation and axon development [54, 669–710]. Immunocytochemical and western blot results showed that -internexin expression was significantly increased in injured neurons following co-culture with microgliaEOC2 microglial cells (Figures 5–6). Microglial co-culture enhances increases the percent of cortical cells expressing neurogenic proteins (Figure 5). For Nestin and -internexin the percent of immunopositive cells present in injured co-cultures suggests that microglial responses to injury allows for proliferation of Nestin+ and -internexin+ cells at the site of injury (Figure 5A,B). Increased Nestin+ and -internexin+ cells were seen outside of the injury site as well. The over two-fold increase in expression of -internexin in injured cortical cultures exposed to microglial-conditioned media suggests that proliferating cells may be neuronal progenitors (Figures 6–7). Quantification of western blot experiments also showed a significant increase in -internexin in injured cortical cultures exposed to microglial-conditioned media as compared to uninjured controls (Figure 7). GFAP + immunoreactivity increases showing the same response to microglia in co-culture but the increases in GFAP+ cells did not reach significance (Figure 5B). These data suggest that GFAP expression is transient as is seen in cortical neurogenesis.The over two-fold increase in expression of -internexin in injured cortical cultures exposed to microglial-conditioned media suggests that proliferating cells were neuronal progenitors (Figures 5–6). Neuronal progenitor cells expressing -internexin were clearly distinct from GFAP expressing cells. Quantification of western blot experiments also showed a significant increase in -internexin in injured cortical cultures exposed to microglial-conditioned media as compared to uninjured controls (Figure 6). Immunocytochemical analysis of GFAP immunoreactivity in injured cortical cultures co-cultured with microgliaEOC2 microglial cells showed an increase in GFAP expressing cells with the morphology of mature astrocytes as compared to injured cortical neurons alone (Figure 55–6). Western blot analysis confirmed a significant increase in GFAP protein in injured cortical cells cultured with microgliaEOC2 microglial cells (Figure 67). It is possible likely that microglial responding to injury stimulate neurogenesis and GFAP expression in progenitor cells as well as some mature astrocytes. As these cells differentiate and increase in size the protein expression is likely to increase as seen in our western blot analysis shown in Figure 7. Microglial soluble signals have been shown to stimulate astrocyte differentiation from progenitor cells [7155–56].

In order to determine whether microgliaEOC2 microglial cells were able to promote differentiation of mature neurons and enhance the survival of existing neurons following injury we used immunofluorescent and western blot analysis to visualize NeuN expression. NeuN is a marker for mature neurons [7352, 55]. The percent of NeuN+ cells increased in injured cortical cell and microglial co-cultures (Figure 5D). Enhanced NeuN immunofluorescence was observed in injured cortical cultures exposed to microglial-conditioned media (Figures 56, 1S). During injury NeuN+ cells were removed from the culture dish at the site of injury (Figure 5, 6). Outside the site of injury NeuN+ cells are found more extensively when the cortical cells are co-cultured with microglia. The presence of NeuN positive cells in injured areas may represent microglial-directed neuronal differentiation of newly generated neuronal progenitor cells. Few of these cells are present in injured areas when neurons are cultured in control media. Western blot analysis showed an increase in NeuN expression when uninjured or injured neurons were exposed to microglial- conditioned media but this increase did not reach significance when compared with NeuN expression in cortical cultures without microgliaEOC2 microglial cells (Figure 6A7A-B). There are several possible explanations for this result. For western blot analysis, protein was isolated from the entire neuronal culture which included injured as well as uninjured regions and would be less specific for detecting proliferation and maturation in injured areas alone. Maturation of neuronal progenitors takes time up to one week in culture [57–58]. and given theGiven the expression pattern neurogenic markers observed in these experiments, microglial soluble cues are likely promoting proliferation of cortical cells at the site of injury and observing cortical cells moving into the we are likely observing primarily early and later stages of neurogenesis. Long-term experiments are underway to determine whether maturation of mature neurons results from effector microglial-enhanced neurogenesis. Our supplemental data showing increased NeuN immunofluorescence in the area of injury following cortical co-culture with microgliaEOC2 microglial cells, suggests that microglial-enhanced neurogenesis and differentiation of mature neurons is occurring (Figure 1S, 2S). Taken together, these data suggest that microglial soluble signals released following co-culture with cortical cells during homeostasis and more so during activation by cortical injury promote the proliferation and survival of neurogenic cells. and that these soluble signals can give rise to mature neurons and astrocytes necessary for cortical repair and normal function.

While continued studies are necessary to identify the specific injury signals that stimulate EOC2 microglial-associated neurogenic properties, our results showed the early neurogenic potential of cortical cells when exposed to soluble signals present in an environment modified by co-culture with microglial-derived soluble signals. Multiplex ELISA assays of microglial-conditioned media from co-culture experiments with uninjured and injured cortical cells revealed that the expression of several microglial-derived cytokines significantly changed following microglia stimulation by neuronal injury. Specifically, multiplex ELISA data showed significant upregulation of MCP–1/CCL2 and downregulation of IFN-, MIP–1, TNF- and RANTES (Figure 87A). IL–1, IL–1, IL–2, IL–4, IL–6, and GM-CSF were detected in our co-culture conditions, however, the concentration of these cytokines was not significantly different in EOC2 microglial co-culture with uninjured or injured neurons and are unlikely contributors to microglial-enhanced neurogenesis observed in this study (data not shown). Upregulation of MCP–1/CCL2 is interesting since MCP–1/CCL2 is associated with inflammation as well as subventricular zone and neocortical neurogenesis and neurogenic migration [72–7585–90]. MCP–1/CCL2 is expressed by microglia, neurons, neural stem cells and astrocytes [85, 8976]. Our data suggest that the increase in MCP–1 protein is unlikely to be microglial-derived since MCP–1 mRNA levels are lower in EOC2 microglia responding to injury than in control microglia (Figure 87B). MCP–1 may be secreted from the increased number of Nestin immunopositive cells as pluripotent cells have been shown to secrete MCP–1 [76,7785–87, 90] and may contribute to the activation of microglia [88] and microglia-derived neurogenesis [52].. Work focused on investigating this possibility is currently underway. Decreased levels of IFN- and other inflammatory cytokines following co-culture of microglia with injured neurons may also favor neurogenesis in neuronal cultures [54, 9149]. Inflammatory cytokines are known to act at specific concentrations and in certain combinations to regulate neurogenesis and neuronal survival [29]. More specifically, low levels of IFN-, rather than high levels produced during responses to inflammatory mediators, have been shown to stimulate both neurogenesis and oligodendrogenesis [20, 29, 7054, 7856]. TNF-, while primarily associated with inflammatory responses associated with neurotoxicity [29], has varied effects on neurogenesis and can stimulate neurons to secrete CCL2 [8973]. Most recent studies show that suppression of TNF-α enhances neurogenesis [29, 18,29,43,7579]. Similarly, MIP–1 and RANTES have diverse roles in the CNS. Various studies have shown that MIP–1, RANTES, and other ligands for CCR5 receptors on neurons contribute to pro-inflammatory neurotoxicity [890]. However, these ligands may also play an important role in the development and migration of neurons [29, 4650,890]. Interestingly, secretion of pro-inflammatory cytokines by microglia such as IL–1ß, IL–6, inducible nitric oxide synthase (iNOS), and TNF- are suppressed by RANTES signaling [4751, 8193]. These results suggest that RANTES may function to regulate microglia effector function and contribute to the neurotrophic and neurogenic properties of microglia. Microglia can also secrete growth factors and neurotrophins [39, 61]. Neurotrophin release may be enhanced when microglia respond to cortical injury [82,8394,95]. Additional ELISA and RT-PCR analyses are required to determine whether neurotrophins or other soluble signals, such as prokineticins, contribute to the microglial-enhanced neurogenesis presented in this in vitro system [7764]. We suggest, as have others [18] that microglial effects on neuronal survival, proliferation, and differentiation is largely dependent upon the entire composition of the mixture of soluble signals that are released by microglia and cortical neuronal and/or glial cells in response to stimulation.

Microglial-derived cues have been shown to activate both MAPK and PI3K/AKT pathways while stimulating neurogenesis of cultured progenitor cells and in neurogenic niches [301–38]. For example, IGF–1 secreted by microglia in the hippocampus stimulates neuronal differentiation and can activate MAPK and PI3K/AKT intracellular signaling but only PI3K/AKT signaling is necessary for neuronal differentiation and survival [30–32]. IGF–1 in combination with other growth factors such as TGF-β stimulate neurogenesis and promote cortical cell survival and proliferation via PI3K-p110α [37]. Expression of Nestin during neurogenesis has been shown to require the PI3K pathway but not the MAPK pathway [8066]. This study adds to this growing body of work suggesting that the process of neurogenesis requires the activation of PI3K/AKT intracellular signaling. Our results showed that AKT phosphorylation was increased in cortical cultures following exposure to EOC2 microglial-conditioned media (Figure 98). Application of the PI3K/AKT inhibitor, LY294002, blocks microglial-enhanced neuronal survival and proliferation following injury (Figure 8) and the expression of neurogenic markers (Figure 109). Inhibition of other intracellular signaling pathways associated with viability, proliferation, and neurogenesis such as MEK [9684], p38MAPK [8462], PKCα/βI/βII/γ [85–8797–99], and Janus Kinase 2 protein [100–10188, 89] did not block the increased metabolic activity and viability of cortical cells co-cultured with EOC2 microglia (see supplemental Figure 2S3S).

Taken together, our data provide an enticing view of the dynamic and multifunctional role of microglia in the cortex. Microglia responding to cortical cues, particularly following injury, may stimulate local neurogenesis and may impact cortical function and potential repair after injury. Downregulation of pro-inflammatory cytokines release from microglia could allow for increased proliferation, reduced apoptosis and increased neurogenesis [5,29]. Continued investigations are underway to better dissect the complex milieu of neurogenic soluble signals released by microglia or other secretory cells in the presence of microglia. Our results suggest that in combination the microglial-derived soluble cuesthe presence of microglia enhances neurogenesis by stimulating the PI3K/AKT signaling pathway in cortical cells. While other intracellular signaling pathways are likely also stimulated, the inhibition of the PI3K/AKT pathway and not other pathways previously implicated in neurogenesis such as MEK, p38 MAPK, PKCα/βI/βII/γ and Janus Kinase 2 blocked EOC2 microglial-enhanced neurogenesis. Further elucidation of the intracellular mechanisms regulating neurogenic function of microglia is essential for understanding the intrinsic neuroprotective role of immune activity in the CNS and may aid in the development of methodologies to promote such activity during neurodegenerative disease or following traumatic injury. The in vitro model system presented here provides an experimental tool to investigate the mechanisms of microglial responses to cortical injury outside of the neurogenic niche.

Here we present an in vitro model system allowing for the assessment of microglial-derived soluble signals on cortical cell viability, proliferation, and differentiation during homeostasis or following cortical injury. Using this model system intracellular signaling pathways are readily investigated in cortical cells and/or microglia. These studies show that cortical injury activates neurogenic properties in EOC2 microglia that involve at least the reduction in pro-inflammatory cytokine gene expression and cytokine release. Microglial-derived or microglial-driven soluble signals produced during homeostasis and following activation by acute cortical injury enhance neurogenesis by upregulating AKT signaling in cortical cells. Increasing our understanding of the mechanisms that drive cortical neurogenesis stimulated by microglia during homeostasis and following injury will provide insight into neuroprotective role of immune activity in the CNS.

Isolation of rat cortical cells

Female timed-pregnant Sprague Dawley rats (200–250 g) were purchased from the Charles River Laboratories (USA). Use of animals was performed in strict accordance with the Institutional Animal Care and Use committee guidelines as approved by the IACUC committee at Creighton University (protocol #0793). Timed Sprague Dawley dams were housed for up to 3 days in Creighton’s Animal Resource Faculty that is AALAC accredited. Ad libitum food and water and normal 24h light dark schedules were followed. Rat cortical cultures were established as described by Meberg and Miller [5749]. Briefly, Sprague-Dawley dams were euthanized by CO₂ asphyxiation. For, CO₂ asphyxiation dam were placed in a clear chamber with CO₂ delivery at 20% of chamber volume per minute. After 1 min of cessation of all respiratory movement, toe pinch tests were performed to determine a lack of reflexive responses and a thoracotomy was used to ensure death. E16-E18 rat embryos were removed from their placental sacks and immediately decapitated. The brains were removed and the cerebral cortices were dissected from day 15–16 embryonic Sprague-Dawley rats (Sasco, Wilmington, MA), mechanically dissociated in Ca²⁺/Mg²⁺-free Hank’s balanced salt solution, with 0.035% sodium bicarbonate and 1mM pyruvate (pH 7.4) following 15 min digestion with 2.5% trypsin. Trypsin was neutralized with Dulbecco’s Modified Eagles Media (DMEM: Hyclone, Thermofisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum and the cell suspension was washed three times and resuspended with neurobasal media supplemented with B–27® and penicillin/streptomycin (Thermofisher Scientific, Waltham, MA). Cells were then plated onto poly-D-lysine coated plates and coverslips (Sigma, St. Louis, MO) at density of 1.5 x 10⁶ cells/well in 6-well plates and 5 x 10⁵ cells/well in 24-well plates and were maintained at 37 ºC in 5% CO₂inneurobasal supplementedmedia. Each cortical culture from 1 pregnant dam was considered to be a biological replicate because embryonic brain tissue is used for co-culture, immunocytochemistry, Multiplex ELISA, RTPCR and western blot experiments all performed in triplicate. In total, 24 Sprague Dawley rats were used for these data.

Cultivation of microglia

EOC 2 microglia isolated from brain tissue of Mus musculus were purchased from American Type Culture Collection (ATCC CRL–2467; Manassas, VA) and were maintained in DMEM (Hyclone, Thermofisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum, 1% l-glutamine, 1% penicillin/streptomycin, and 20% LADMAC conditioned media. Cells were grown in 100-mmtissue culture dishes at 37 ºC in 5% CO₂ and allowed to reach 80% confluency before the cells were passed. LADMAC conditioned media was collected from Mus musculus bone marrow derived LADMAC cells (ATCC CRL–2420; Manassas, VA) 5–7 days after initial plating of cells at 1 x 10⁵ in Eagle’s Minimal Essential Media (MEM; Hyclone) supplemented with 10% FBS, 1% l-glutamine, 0.1mM nonessential amino acids, 1.0 mM sodium pyruvate, and 1% penicillin/streptomycin. LADMAC conditioned media was collected, filter sterilized, and frozen until needed as a media supplement for microglia. LADMAC conditioned media was used to provide colony-stimulating factor 1 (CSF–1) to the microglial cultures as outlined by the ATCC culture instructions for EOC microglial cells (ATCC CRL–2420; Manassas, VA).

Neuronal—Microglial Co-Cultures

Primary cortical cells were cultured for 48 hours then either injured by mechanical transection using a sterile stylet or left uninjured [7450]. Briefly, mechanical transection using a sterile stylet involves the application of the sterile stylet tip directly to the cortical cell culture. Pressure is placed on the stylet while dragging the stylet tip across the cortical cell culture to form parallel sites of injury in the cortical culture. Microglia were pre-seeded directly onto 6-well permeable Transwells® at 5 x 10⁵ cells/well or onto 24-well Transwells® at 4 x 10⁴ cells/well (Corning,Tewksbury, MA) and cultured for 24 hours before being suspended above cortical cells using Transwells® in the co-culture model system. Microglia seeded onto Transwells® and injured or uninjured (control) primary cortical cells were co-cultured for an additional 48 hours in unsupplemented neurobasal media prior to cellular assays. For immuncytochemical analysis of EOC2 microglia only, EOC2 microglia were cultured on glass coverslips and placed into Transwells® and then removed for fixing and immunostaining (see below).

Immunocytochemistry

Primary cortical cultures were plated onto sterile poly-D-lysine coated coverslips. Microglia were plated onto sterile glass coverslips suspended above cortical cultures in Transwells®. Following co-culture experiments, cortical cells and microglia were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed with 1X PBS. Cells were permeabilized with 0.2% Triton X–100 in PBS for 10 minutes, washed, and blocked for 1 hour in PBS, 0.2% BSA, and 0.2% Triton X–100. Primary antibodies were applied and incubated overnight at 4 ºC in PBS, 0.2% BSA, 0.2% Triton X–100. Primary antibodies were purchased from RMD Millipore Sigma (Darmstadt, Germany) and included: mouse anti-Nestin (1:200, Millipore Cat# AB5922, RRID:AB_91107), rabbit anti-GFAP (1:400, Millipore Cat# AB5541, RRID:AB_177521), mouse anti-α internexin (1:100, Millipore Cat# AB5354, RRID:AB_91800), mouse anti-TUJI/beta tubulin III (1:200, Millipore Cat# MAB1637, RRID:AB_2210524)and mouse anti-NeuN (1:50, Millipore cat#MAB377l, RRID:AB_2298772).Primary antibody directed against neurofilament was a mouse monoclonal neurofilament antibody, p-NF-H (7H11) (1:200, Santa Cruz Biotechnologies, Cat# sc–20015, RRID: AB_670161). To confirm microglial characteristics, microglial cells were immunostained with rabbit anti-mouse CD11b conjugated to Alexa 488 (2 µg/100 µl, Caltag Laboratories, Burlingame, CA). Secondary antibodies were applied for 1 hour at a concentration of 1:500 for goat anti-rabbit IgG (H+L) rhodamine conjugate and goat anti-mouse IgG (H+L) fluorescein conjugate (Pierce, Rockford, IL). Nuclei were visualized using a DAPI stain (300 mmol, MP Biomedicals, Santa Ana, CA). Qualitative and quantitative analysis of immunocytochemistry was performed by acquiring images with a Leica DMI4000B inverted microscope with a cooled CCD camera (Q Imaging, Surrey, BC) and fluorescent capabilities. For quantification of the percent of cells expressing neurogenic markers was determined by counting the number of immunopositive cells for each marker and dividing that number by the total number of cells counted in the field. Experiments were performed in triplicate with at least 300 cells counted manually per experiment for each condition. For quantification of relative fluorescence intensity units associated with the immunocytochemistry experiments localizing protein expression in cortical cell cultures.

In all experiments, iImages were analyzed with Volocity (PerkinElmer,USA) and ImageQuant (GE Healthcare, USA) software were used for image analysis and presentation. ImageProPlus software (MediaCybernetics, Rockville, MD). For image data, 3 field views of at least 100 cells from 3 separate experiments were analyzed for each condition.

Measurement of Cell Viability

Viability of cortical cells and microglia were measured by metabolism of thiazolyl blue, 3-[4,5-dimethylthiazol–2yl]–2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich). Injured and uninjured cortical cells cultured with and without microglia were incubated with 100 µl MTT in 1 ml of media for one hour. Media was removed and cells were dissolved in 300 µl dimethylsulfoxide (DMSO) and aliquoted to 100 µl/well in 96-well plates. Absorbance was read at 540–590nm on an ELISA plate reader. Three experiments were performed in triplicate.

Measurement of Cortical Cell Proliferation

Cell proliferation was measured using Click-iT® EdU Alexa Fluor 647 according to the manufacturer’s instructions (C10340, Thermo Fisher Scientific, Waltham, MA). Briefly, Click-iT® EdU Alexa Fluor 647 is a modified thymidine analogue EdU (5-ethynyl–2’-deoxyuridine, a nucleoside analog of thymidine) that is incorporated into newly synthesized DNA. The EdU is fluorescently labeled with a photostable Alexa Fluor® dye during the click reaction. Briefly, uninjured and injured cortical cells co-cultured with and without microglia for 2 DIV (days in vitro) were fixed in 3.7% formaldehyde in PBS for 15 min at RT. Fixed cells were washed twice with 1 ml of 3% BSA in PBS. Cells were permeabilized in 0.5% Triton®x–100 for 20 min at RT, washed and 1X Click-iT® EdU reaction cocktail was added for 30 min at RT. The reaction cocktail was removed, cells were washed in 3% BSA and PBS, counterstained with DAPI, mounted and imaged for analysis. Imaging was performed using IBIF Leica TCS SP8 MP Confocal Microscope at 20x magnification. Experiments were performed in triplicate with at least 300 cells counted manually per experiment for each condition. Volocity (PerkinElmer,USA) and ImageQuant (GE Healthcare, USA) software were used for image analysis and presentation.

Measurement of Cortical Cell Apoptosis

Apoptosis of cortical cultures was measured following co-culture with microglia for 2 DIV (days in vitro) using a Click-iT® TUNEL Alexa Fluor 488 imaging assay (C10245, Thermo Fisher Scientific, Waltham, MA). Manufacturer’s instructions were followed. Briefly, injured and uninjured cortical cells cultured with or without microglia were fixed with 4% PFA in PBS for15 min then permeabilized with 0.25% Triton-X® 100 for 20 min. Each condition was incubated with 100 µl of TdT reaction buffer for 10 min at RT then removed. Cells were incubated in 100 µl TdT reaction cocktail for one hr at 37 ^oC. Cells were washed twice in 3% BSA in PBS for 2 min then incubated with Click-iT® reaction buffer with additive for 30 min at RT protected from light. Cells were rinsed and counterstained with DAPI, mounted, and cover slipped for analysis. Imaging was performed using IBIF Leica TCS SP8 MP Confocal Microscope at 20x magnification. Experiments were performed in triplicate with at least 300 cells counted manually per experiment for each condition. Volocity (PerkinElmer,USA) and ImageQuant (GE Healthcare, USA) software were used for image analysis and presentation.

ELISA Analysis

Conditioned media was collected from uninjured and injured neuronal and microglia co-cultures from three separate experiments and cytokine expression was determined by Q-Plex™ mouse cytokine –Inflammation multiplex assay. Concentrations of mouse microglia-derived cytokines MCP–1, IFN-, MIP–1, TNF, RANTES, IL–1, IL–1, IL–2, IL–4, IL–3, IL–6, IL–10, IL–12, 1L–17 and GM-CSF were evaluated by Quansys Biosciences (#110449MS, Logan, UT). Cytokine concentrations in media collected from uninjured cortical cells cultured with microglia were used as the reference and control for these experiments. In order to use cytokine concentrations from uninjured cortical and microglial co-cultures as our control condition, each cytokine concentration measured in the uninjured cortical cell and microglia co-culture condition was normalized and set equal to one. Cytokine concentrations in media collected from injured cortical cells co-cultured with microglia were measured, normalized, and expressed as the percent change in cytokine concentration as compared to uninjured control concentrations for that cytokine. Multiplex ELISA assays were run in triplicate in three biological replicate experiments. Significance of the percent change from control was determined using student T test with Bonferroni correction. The percent change in cytokine concentration was considered significant if p<0.05, error bars represent the standard error of the mean of the percent change. Cytokine levels that were not consistently detected in either condition or were not significantly different in control and experimental conditions are not shown.

RT-PCR Analysis

For real-time PCR analysis of cytokines and mature miRNAs, total RNA of EOC2 microglial cultured on Transwell permeable inserts that were physically separated from cortical cells was extracted using the mirVana miRNA Isolation kit (Ambion). An amount of 200 ng total RNA was reverse-transcribed using the Invitrogen™ NCode™ miRNA First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Comparative real-time PCR was performed using the Invitrogen™ SYBR GreenER™ qPCR SuperMix Universal (Thermo Fisher Scientific) on the Bio-Rad CFX96 Touch™ Real-Time PCR Detection System. Primers were purchased from QIAGEN (Ccl3, Ccl5, Ifn, Mcpt1, Tnfα, Gapdh). Normalization was performed using Gapdh. Relative expression was calculated using the comparative Ct (ΔΔCt) method.

Western Blots

Following co-culture, protein collected separately from cortical cells or microglia was assessed using western blot analysis. Cortical cells or microglia were lysed with 500 µl lysis buffer (10x lysis buffer, Cat#9803, Cell Signaling, Danvers, MA), supplemented with 0.1M PMSF (Cat # 36978, Thermo Fisher Scientific, Waltham, MA), and HALT™ protease and phosphatase inhibitor diluted to 1X (Cat#78446, Thermo Fisher Scientific, Waltham, MA) per 3 wells of the 6-well plates or per 3 Transwells®.Lysates were spun 10,000 RPM for 10 minutes at 4 °C. Lysate supernatant were collected and heated at 95 °C for 5 minutes with 4X sample buffer plus 10mM DTT. Denatured protein samples were separated by SDS-PAGE gel electrophoresis on 10% TGS gels. Proteins were transferred to PVDF membranes in Tris-glycine transfer buffer. After transfer, membranes were blocked using BSA Blocking Buffer™ in TBS(Cat#37520, Thermo Fisher Scientific, Waltham, MA) for 1 hour and then incubated with primary antibody diluted in BSA Blocking Buffer™ in TBS overnight at 4 °C. Primary antibodies include rabbit anti-phospho-p44/42 MAPK (1:1000, 1:2000, Cell Signaling Technology Cat# 4376, RRID:AB_331772), rabbit anti-pan AKT (1:1000, Cell Signaling Technology Cat# 4691, RRID:AB_915783) rabbit anti-phospho-AKT (1:100, Cell Signaling Technology Cat# 9270, RRID:AB_329824). Following washing in Tris Buffered Saline with 0.1% Tween® 20 and BSA blocking buffer™, appropriate secondary antibodies (anti-rat IgG, HRP-linked antibody,1:1000, Cell Signaling Technology, Danvers, MA) were applied for detection. Membranes were developed using chemiluminescence SuperSignal™ ELISA Pico Chemiluminescent Substrate (Cat#37069, Thermo Fisher Scientific, Waltham, MA) following manufacturer’s instructions. Statistical analyses involved semi-quantitative measurements of chemiluminescence using BioRad ChemiDoc QRS (Hercules, CA) imaging system and software. Total protein loading was assessed by detecting GAPDH in each sample. Three separate experiments were performed for measurements of protein expression by densitometry.

Assays for Intracellular Signal Transduction

Primary cortical and microglial co-cultures were established as described above. Stock solutions of kinase inhibitors in DMSO were prepared at stock concentrations recommended by the manufacturer. Stocks were stored at –20^oC and diluted into cell culture media prior to use. Four hours prior to injury and co-culture with microglia, signaling pathway inhibitors were added at concentrations of 0 µM, 10 µM, or 40 µM to the cortical cultures. Inhibitors tested were the following: MAPK inhibitor PD98059 (Cat#9900S), PI3K/AKT inhibitor LY294002 (Cat#9901S), PKC and Glycogen synthase kinase–3 inhibitor GF109203X (Cat#984150), Janus kinase 2 inhibitor AG490 (Cat#14704S). All inhibitors were purchased from Cell Signaling Technologies (Danvers, MA). Uninjured and injured cortical cells that were not cultured with microglia were used as controls. For control experiments, DMSO vehicle diluted in culture media was used in the experiments. After 48 hours, cells were analyzed using MTT assays (see above) or fixed with 4% PFA in PBS to observe expression for neurogenic markers, Nestin, α-internexin, and GFAP using immunocytochemical methods as described above. To quantify imaging data, 3 field views of at least 100 cells from 3 separate experiments were analyzed for each condition.

Immunoprecipitation for AKT/pAKT Analysis

Immunoprecipitation for AKT and pAKT was used to increase specificity and detection of AKT protein in cellular lysates. Cellular cultures were lysed as described above and each condition was split into two aliquots (200 µl each). Primary antibodies AKT (pan) (C67E7) rabbit mAB (Cell Signaling Technology Cat# 4691, RRID:AB_915783) and Phospho- AKT (Thr308) rabbit mAB (Cell Signaling Technology Cat#9275, RRID:AB_329828) were added at 1:50 for each sample and rotated overnight at 4 °C. A 50% slurry of EZview Red Protein A Affinity Gel Beads (Cat#P6486, EDM Millipore Sigma, Darmstadt, Germany) were added at 1:10 for each sample and rotated for 1 hour at 4 °C. Cells were centrifuged at 8,200g for 1 minute and washed with lysis buffer 3 times. Samples were heated at 95 °C for 5 minutes with 25 µl 3X sample buffer. Samples were run on 4–20% gradient SDS-polyacrylamide gels (Cat#4561096, BioRad, Hercules, CA) using SDS-PAGE and then transferred to PVDF membrane. After transfer, membranes were blocked using BSA Blocking Buffer™ in TBS for 1 hour and then gently rocked with the primary AKT or pAKT antibody at 1:1000 in BSA Blocking Buffer™ in TBS overnight at 4°C. Blots were washed and incubated in secondary anti-rabbit HRP conjugated antibody for 1 hour at RT. Membranes were developed using chemiluminescence as described above. Three separate experiments were performed. Statistical analysis involved analysis of densitometric images acquired with BioRad ChemiDoc QRS imaging system and software (BioRad,Hercules CA) were performed as described above.

Statistical Analysis

Data are expressed as mean values and error bars represent standard error of the mean (SEM). Student T test with Bonferroni’s correction or one- way ANOVA followed by Tukey-Kramer post hoc tests were performed where appropriate. For determination of significant differences between percents and for multiple comparisons between culture conditions, two-way ANOVA followed by Tukey-Kramer multiple analyses post hoc tests were used. Values of p<0.05 were considered to be significant. All statistical analyses were performed with Graphpad Prism 6 8 (La Jolla, CA).

AKT: Protein Kinase B, PI3K, phosphatidylinositol 3-kinase, E: embryonic,TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling, NeuN, Fox–3, Rbfox3: Hexaribomucleotide binding protein 3; RT-PCR: Reverse transcriptase polymerase chain reaction, MCP–1: monocyte chemoattractant protein 1, CCL2:chemokine ligand 2 or monocyte chemotactic protein 1, IFN-,: Interferon gamma, MIP–1,: macrophage inflammatory protein 1 alpha, TNF: tumor necrosis factor alpha, RANTES (CCL5): regulation on activation, normal T cell expressed and secreted, chemokine ligand 5, CNS: central nervous system, BDNF: brain derived neurotrophic factor, GDNF: glial derived neurotrophic factor, STAT: signal transducer and activation of transcription, NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells, TGF-: transforming growth factor beta, IGF–1: Insulin growth factor, MAPK: mitogen activated protein kinase, CA1: cornu Ammon 1 of the hippocampus, FGF: Fibroblast growth factor, EGF: epidermal growth factor, IL1–13: Interleukins 1–13, GFAP: glial fibrillary acidic protein, TUJI: neuron specific class III beta tubulin, SEM: standard error of the mean, NF: neurofilament, MTT: 3-(4,5-Dimethylthiazol–2-yl)–2,5-Diphenyltetrazolium Bromide, O. D. optical density, EdU, 5’-ethynyl–2’-deoxyridine, DNA: deoxyribonucleic acid, RFU: relative immunofluorescence units, DAPI: 4’,6-diamino–2-phenylindole, DIV: days in vitro, ELISA: enzyme-linked immunosorbent assay, GM-CSF: granulocyte macrophage colony stimulating factor, mRNA: Messenger ribonucleic acid, MEK: mitogen-activated protein kinase kinase, p38 MAPK: p38 mitogen-activated protein kinase, PKC: protein kinase C, DMEM: Dulbecco’s Modified Eagles Media, FBS: fetal bovine serum, DMSO, dimethylsulfoxide, PBS: phosphate buffered saline, Gapdh: glyceraldehyde 3-phosphate dehydrogenase, ΔΔCt: delta-delta cycle threshold

Ethics Approval and consent to participate: Not applicable for humans since there are no human subjects or samples in this study. Use of animals was performed in strict accordance with the Institutional Animal Care and Use committee guidelines as approved by the IACUC committee at Creighton University (protocol #0793).

Consent for publication: Not applicable.

Availability of data and material: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests: The authors declare that they have no competing interests

Funding: This publication was made possible by Creighton University College of Arts and Sciences Faculty Summer Fellowship and Grant Number P20 RR16469 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. Publication costs are shared between these funding mechanisms and Creighton University.

Authors contributions: KC contributed to the design and implementation of the co-culture system, immunocytochemistry and intracellular signaling pathway analysis. KC was also a major contributor to the writing of this manuscript. NWM contributed to co-culture implementation, RT-PCR and analysis and was a significant contributor to the writing of this manuscript. ERW contributed to co-culture experimental design and immunocytochemistry analysis. AE contributed to ELISA experimental design, implementation and analysis. JC contributed to immunocytochemistry and western blot analysis. MB contributed to data analysis and to review and revision of this manuscript. XMC contributed to experimental design and to the writing of this manuscript. AS was responsible for conceptualization of the experiments, experimental design, data analysis, and writing of the manuscript. All authors read and approved the final manuscript.

Acknowledgements: NA

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Microglia induce neurogenesis by stimulating PI3K/AKT intracellular signaling in vitro

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Abstract

Figures

Background

Results

Discussion

Conclusions

Materials and Methods

Isolation of rat cortical cells

Cultivation of microglia

Neuronal—Microglial Co-Cultures

Immunocytochemistry

Measurement of Cell Viability

Measurement of Cortical Cell Proliferation

Measurement of Cortical Cell Apoptosis

ELISA Analysis

RT-PCR Analysis

Western Blots

Assays for Intracellular Signal Transduction

Immunoprecipitation for AKT/pAKT Analysis

Statistical Analysis

List of Abbreviations

Declarations

References

Supplementary Files

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