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Protocol for Co-culture of Microglia with Axons.

ABSTRACT: Microglia, the resident immune cells of the central nervous system, accumulate along axons and support neuronal survival during the early postnatal period. Although methods have been developed to isolate microglia or neurons, the procedures for co-culture of these cells can inadvertently affect the interactions between them. Here, we describe a protocol to investigate the accumulation of microglia toward neuronal axons using axon isolation culture devices. This approach is useful for modeling neuron-glia associations. For complete details on the use and execution of this protocol, please refer to Ueno et al. (2013) and Fujita et al. (2020).

SUBMITTER: Fujita Y

PROVIDER: S-EPMC7756915 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Protocol for Co-culture of Microglia with Axons.

Fujita Yuki Y Yamashita Toshihide T

STAR protocols 20200915 3

Microglia, the resident immune cells of the central nervous system, accumulate along axons and support neuronal survival during the early postnatal period. Although methods have been developed to isolate microglia or neurons, the procedures for co-culture of these cells can inadvertently affect the interactions between them. Here, we describe a protocol to investigate the accumulation of microglia toward neuronal axons using axon isolation culture devices. This approach is useful for modeling ne ...[more]

PMID: 33377007

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Project description:BackgroundHepatic encephalopathy (HE) is a neurological complication resulting from acute or chronic liver disease. Hyperammonemia leading to astrocyte swelling and cerebral edema in combination with neuroinflammation including microglia activation, mainly contribute to the pathogenesis of HE. However, little is known about microglia and their inflammatory response, as well as their influence on astrocytic channels and astrocyte swelling under hyperammonemia.ObjectiveTo investigate the effects of ammonia on the microglial activation and morphology in different set-ups of an in vitro astrocyte-microglia co-culture model. Further, potential effects on glial viability, connexin 43 (Cx43) and aquaporin 4 (AQP4) expression were tested.MethodsPrimary rat glial co-cultures of astrocytes containing 5% (M5, representing "physiological" conditions) or 30% (M30, representing "pathological" conditions) of microglia were incubated with 3 mM, 5 mM, 10 mM and 20 mM ammonium chloride (NH4Cl) for 6 h and 24 h in order to mimic the conditions of HE. An MTT assay was performed to measure the viability, proliferation and cytotoxicity of cells. The microglial phenotypes were analyzed by immunocytochemistry. The expression of Cx43 and AQP4 were quantified by immunoblot analysis.ResultsA significant reduction of glial viability was observed in M30 co-cultures after incubation with 20 mM NH4Cl for 6 h, whereas in M5 co-cultures the viability remained unchanged. Microglial activation was detected by immunocytochemistry after incubation with 3 mM, 5 mM and 10 mM NH4Cl for 6 h and 24 h in M5 as well as in M30 co-cultures. The Cx43 expression was slightly increased in M30 co-cultures after 6 h incubation with 5 mM NH4Cl. Also, the AQP4 expression was slightly increased only in M5 co-cultures treated with 10 mM NH4Cl for 6 h. Under the other conditions, Cx43 and AQP4 expression was not affected by NH4Cl.ConclusionsThe novel aspect of our study was the significant microglial activation and decrease of viability after NH4Cl incubation in different set-ups of an in vitro astrocyte-microglia co-culture model, contributing to better understanding of pathophysiological mechanisms of HE. Hyperammonemia led to limited effects on Cx43 and AQP4 expression, the relevance of these minimal changes should be viewed with caution.

Project description:BackgroundAlzheimer disease (AD) is a heterogenous and multifactorial disease, and its pathology is partly driven by microglia and their activated phenotype. Brain organoids (BOs) are gaining prominence as a relevant model of the human brain for the study of AD; however, BOs are commonly devoid of microglia. To overcome this limitation, current protocols incorporate microglia through either (1) co-culture (BO co-culture), or (2) molecular manipulation at critical windows of BO development to have microglia arise innately (BO innate cultures). It is currently unclear whether the microglia incorporated into BOs by either of these two protocols differ in function.MethodsAt in vitro day 90, BO innate cultures and BO-co-cultures were challenged with the AD-related β-amyloid peptide (Aβ) for up to 72 h. After Aβ challenge, BOs were collected for immunoblotting. Immunoblots compared immunodensity and protein banding of Aβ and ionized calcium-binding adapter molecule 1 (IBA1, a marker of microglial activation) in BOs. The translational potential of these observations was supported using 56 human cortical samples from neurocognitively normal donors and patients with early-onset AD and late-onset AD. Statistical analyses were conducted using the Kruskal-Wallis test, a two-way ANOVA, or a simple linear regression, and where applicable, followed by Dunn's or Sidak's test.ResultsWe show that BO co-cultures promote Aβ oligomerization as early as 24 h and this coincides with a significant increase in IBA1 levels. In contrast, the Aβs do not oligomerize in BO innate cultures and the IBA1 response was modest and only emerged after 48 h. In human cortical samples, we found IBA1 levels correlated with age at onset, age at death, and the putative diagnostic Aβ(1-42)/Aβ(1-40) ratio (particularly in their oligomeric forms) in a sex-dependent manner.ConclusionsOur unique observations suggest that BOs with innate microglia model the response of a healthy brain to Aβ, and by extension the initial stages of Aβ challenge. It would be impossible to model these early stages of pathogenesis in BOs where microglia are already compromised, such as those with microglia incorporated by co-culture.

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Protocol for Co-culture of Microglia with Axons.

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Protocol for Co-culture of Microglia with Axons.

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