Microglia of rat ventral midbrain recovers its resting state over time in vitro: Let microglia rest before work

Cristóvão, Ana Clara; Saavedra, Ana; Fonseca, Carla P.; Campos, Filipa; Duarte, Emília P.; Baltazar, Graça

doi:10.1002/jnr.22219

Cited by 10 publications

(12 citation statements)

References 49 publications

(53 reference statements)

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“…The supernatant enriched with microglia was collected and centrifuged (200g, 5 min). Microglial cell culture purity was quantified after immunolabelling using CD11b antibody (1 : 200; Abcam, Belgium) and found to be above 90% (see details in the electronic supplementary material) in accordance with previous reports that used a similar isolation technique [18,28]. A total of 6 Â 10 4 viable cells ml 21 were seeded on P(TMC-CL) substrates secured at the bottom of a 24-well plate with a silicone o-ring (Epidor, Spain) and cultured in DMEM/F12 medium (Gibco) supplemented with 10% FBS and 1% P/S for 1 or 5 days.…”

supporting

confidence: 80%

The role of the surface on microglia function: implications for central nervous system tissue engineering

Pires

Rocha

Ambrosio

et al. 2015

J. R. Soc. Interface.

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In tissue engineering, it is well accepted that a scaffold surface has a decisive impact on cell behaviour. Here we focused on microglia-the resident immune cells of the central nervous system (CNS)-and on their response to poly(trimethylene carbonate-co-1-caprolactone) (P(TMC-CL)) fibrous and flat surfaces obtained by electrospinning and solvent cast, respectively. This study aims to provide cues for the design of instructive surfaces that can contribute to the challenging process of CNS regeneration. Cell morphology was evidently affected by the substrate, mirroring the surface main features. Cells cultured on flat substrates presented a round shape, while cells with elongated processes were observed on the electrospun fibres. A higher concentration of the pro-inflammatory cytokine tumour necrosis factor-a was detected in culture media from microglia on fibres. Still, astrogliosis is not exacerbated when astrocytes are cultured in the presence of microgliaconditioned media obtained from cultures in contact with either substrate. Furthermore, a significant percentage of microglia was found to participate in the process of myelin phagocytosis, with the formation of multinucleated giant cells being observed only on films. Altogether, the results presented suggest that microglia in contact with the tested substrates may contribute to the regeneration process, putting forward P(TMC-CL) substrates as supporting matrices for nerve regeneration.

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supporting

confidence: 80%

The role of the surface on microglia function: implications for central nervous system tissue engineering

Pires

Rocha

Ambrosio

et al. 2015

J. R. Soc. Interface.

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“…The protocol here described originates microglial cultures that exceeds 97% purity and has been used as a model for activated CNS-resident microglia (Carson et al, 1998; Schmid et al, 2009) and to prepare polarized M1 and M2 phenotypes (Jang et al, 2013). Indeed, it was previously suggested that the isolation process is a sufficient stimulus to induce microglia activation (Cristóvão et al, 2010). There is a high controversy on whether neonatal microglia are less (Moussaud and Draheim, 2010) or more reactive than adult (Christensen et al, 2014) and aged cells (Njie et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, these cells were analyzed either 24–48 h after isolation (Njie et al, 2012; Lai et al, 2013) or following trypsinization when kept in culture for several weeks in the presence of conditioned medium containing increased levels of mitogens (von Bernhardi et al, 2011). Such methods may promote microglia activation and bias the translation of culture findings, since it has been suggested that microglia may need some time in culture to recover its quiescent state (Cristóvão et al, 2010). Moreover, there are no means to isolate degenerating microglia for experimentation (Njie et al, 2012) once only the more resistant ones will survive to the isolation procedure.…”

Section: Introductionmentioning

confidence: 99%

Microglia change from a reactive to an age-like phenotype with the time in culture

Caldeira

Oliveira

Cunha

et al. 2014

Front. Cell. Neurosci.

140

176

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Age-related neurodegenerative diseases have been associated with chronic neuroinflammation and microglia activation. However, cumulative evidence supports that inflammation only occurs at an early stage once microglia change the endogenous characteristics with aging and switch to irresponsive/senescent and dystrophic phenotypes with disease progression. Thus, it will be important to have the means to assess the role of reactive and aged microglia when studying advanced brain neurodegeneration processes and age-associated related disorders. Yet, most studies are done with microglia from neonates since there are no adequate means to isolate degenerating microglia for experimentation. Indeed, only a few studies report microglia isolation from aged animals, using either short-term cultures or high concentrations of mitogens in the medium, which trigger microglia reactivity. The purpose of this study was to develop an experimental process to naturally age microglia after isolation from neonatal mice and to characterize the cultured cells at 2 days in vitro (DIV), 10 DIV, and 16 DIV. We found that 2 DIV (young) microglia had predominant amoeboid morphology and markers of stressed/reactive phenotype. In contrast, 16 DIV (aged) microglia evidenced ramified morphology and increased matrix metalloproteinase (MMP)-2 activation, as well as reduced MMP-9, glutamate release and nuclear factor kappa-B activation, in parallel with decreased expression of Toll-like receptor (TLR)-2 and TLR-4, capacity to migrate and phagocytose. These findings together with the reduced expression of microRNA (miR)-124, and miR-155, decreased autophagy, enhanced senescence associated beta-galactosidase activity and elevated miR-146a expression, are suggestive that 16 DIV cells mainly correspond to irresponsive/senescent microglia. Data indicate that the model represent an opportunity to understand and control microglial aging, as well as to explore strategies to recover microglia surveillance function.

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“…Relatively to microglia, in vitro cell models, either microglial cell lines, or primary microglia isolated from embryonic (Gingras et al, 2007 ) or neonatal animals (Floden and Combs, 2007 ), though largely used (Moussaud and Draheim, 2010 ), fail in mimicking adult behavior cells (Sierra et al, 2007 ). Furthermore, primary cultures of microglia were shown to change their activation profile according with the time in culture (Cristóvão et al, 2010 ). All of these features contribute to data inconsistency.…”

Section: Introductionmentioning

confidence: 99%

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

Caldeira

Cunha

Vaz

et al. 2017

Front. Aging Neurosci.

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Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aβ production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aβ1–42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aβ treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aβ in 2 DIV microglia. Additionally, Aβ downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

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Microglia of rat ventral midbrain recovers its resting state over time in vitro: Let microglia rest before work

Cited by 10 publications

References 49 publications

The role of the surface on microglia function: implications for central nervous system tissue engineering

The role of the surface on microglia function: implications for central nervous system tissue engineering

Microglia change from a reactive to an age-like phenotype with the time in culture

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

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