Studying stress in BV2 microglial cells: from model development to translational perspectives

Chronic stress is a major risk factor for neuropsychiatric disorders. At the brain level, stressors elicit activation of the HPA axis and consequent glucocorticoid (GC) release. While acute GC signalling promotes adaptive responses through the balance between mineralocorticoid (MR) and glucocorticoid (GR) receptors, chronic exposure leads to a dysregulated GR-driven state, consistently implicated in such conditions.

Microglia, highly responsive to stress-related cues, act as mediators of the stress-induced neurobiological changes underlying neuropsychiatric disorders. However, most cellular models of chronic stress focus primarily on neurons and, to a lesser extent, astrocytes.

To address this gap and model the impact of chronic exposure to stress on microglial function in vitro, the BV2 murine microglial cell line was treated for 96h with increasing concentrations of corticosterone (CORT), the principal GC in rodents, to mimic prolonged signalling. All results were expressed as a percentage of the control group, which was set to 100%. Differences between untreated controls and treatment groups, as well as among the several concentrations, were analysed with GraphPad Prism software using one-way ANOVA followed by Tukey’s post hoc test for all pairwise comparisons.

CORT treatment induced an increase in GR nuclear expression, measured through Western Blot, with respect to controls following a concentration-dependent trend and reaching significance from 0.50μM onwards (CORT 0.50μM: +250%, p=0.0218; CORT 1μM: +271%, p=0.0128; CORT 5μM: +306%, p=0.0052). Its ratio with MR nuclear expression displayed a similar modulation (CORT 0.50μM: +369%, p=0.0669; CORT 1μM: +603%, p=0.0023; CORT 5μM: +607%, p=0.0022), indicating an imbalance of the ‘MR-GR switch’ towards GR, a GC profile consistent with chronic stress. This was coupled with an impairment of its FKBP5-mediated negative feedback mechanism, indicated by no change in the protein expression of its mediator compared to the control group (CORT 0.05μM: -6%; CORT 0.25μM: -21%; CORT 0.50μM: -8%; CORT 1μM: +1%; CORT 5μM: +10%; p>0.9 for all).

In line with existing literature, these data support the model’s validity and broader applicability for investigating stress-driven alterations across cellular pathways.

While GC signalling showed a concentration-dependent response across all tested concentrations, CORT induced a non-linear modulation of anti-inflammatory arginase-1 protein expression, with significant differences observed between concentrations, implying a shift in microglial response across increasing stress levels and suggesting that the capacity to counteract the challenge may be reduced beyond a certain threshold (CORT 1 μM vs 0.50 μM: −56%, p = 0.0457).

A similar pattern was observed for the antioxidant response mediated by catalase, whose activity, measured by a well-established enzymatic assay based on hydrogen peroxide decomposition, significantly decreased only at higher concentrations relative to controls (CORT 0.05μM: -22%, p=0.7994; CORT 0.25μM: -13%, p=0.9697; CORT 0.50μM: -21%, p=0.8766; CORT 1μM: -76%, p=0.0165; CORT 5μM: -77%, p=0.0090).

In light of these findings, this model may offer valuable insights into stress-related molecular changes in biological processes beyond direct GC signalling, such as inflammation and redox balance, as well as serve as a platform to evaluate potential therapies. Furthermore, it could complement existing in vivo and ex vivo systems to improve our understanding of such alterations in humans.

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