BV2 Cells: Key Microglial Models in Neuroinflammation

Microglial cells are the primary immune responders in the central nervous system, playing a crucial role in neuroinflammation. Studying their behavior under pathological conditions is essential for understanding neurodegenerative diseases and developing therapeutic strategies. However, primary microglia have limitations, including variability between samples and ethical concerns regarding human-derived cells.

BV2 cells, an immortalized murine microglial cell line, offer a widely used alternative for investigating neuroinflammatory mechanisms. Their reproducibility and ease of culture make them valuable for studying microglial activation and interactions with other neural components.

Structural And Molecular Characteristics

BV2 cells exhibit morphological and molecular features similar to primary microglia, making them a widely accepted model for studying microglial function. Under basal conditions, they display a small, amoeboid shape with short, retracted processes, differing from the highly ramified morphology of resting primary microglia. This structural difference stems from their immortalized nature, which alters cytoskeletal dynamics and baseline activation states. Despite this, BV2 cells extend processes and adopt a more complex morphology in response to environmental cues, reflecting their plasticity.

At the molecular level, BV2 cells express microglia-associated markers, including ionized calcium-binding adapter molecule 1 (Iba1), which is involved in actin cytoskeleton remodeling, and CD11b, a component of the complement receptor 3 complex that mediates adhesion and phagocytosis. Additionally, they maintain expression of purinergic receptors such as P2X4 and P2X7, which are implicated in ATP-mediated signaling and cellular responses to stress.

Gene expression profiling has revealed transcriptional similarities to primary microglia, particularly in pathways linked to metabolism and stress responses. However, genes associated with homeostatic microglial functions, such as Tmem119 and Sall1, are downregulated in BV2 cells. This suggests they may not fully replicate all aspects of microglial physiology, particularly in maintaining a quiescent state. Nonetheless, their ability to upregulate inflammatory and stress-related genes in response to stimuli makes them a reliable model for studying microglial activation.

Role In Neuroinflammatory Pathways

BV2 cells serve as a tool for investigating molecular and signaling cascades that drive neuroinflammation. They detect and respond to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) through pattern recognition receptors such as Toll-like receptors (TLRs). TLR4, in particular, is well characterized, as its activation by lipopolysaccharide (LPS) induces pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). The expression levels of these cytokines in BV2 cells closely mirror those in primary microglia.

Beyond cytokine release, BV2 cells activate intracellular signaling cascades regulating neuroinflammation. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway drives inflammatory gene expression, while mitogen-activated protein kinase (MAPK) signaling amplifies inflammation through extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 MAPK. Blocking NF-κB or MAPK signaling significantly reduces inflammatory mediator expression, highlighting their relevance in microglial activation.

Oxidative stress is another key aspect of neuroinflammation that BV2 cells help elucidate. Upon activation, they generate reactive oxygen species (ROS) and nitric oxide (NO) through inducible nitric oxide synthase (iNOS) upregulation. Excessive ROS and NO production contribute to neuronal damage, playing a role in neurodegenerative diseases such as Alzheimer’s and Parkinson’s. BV2 cells exposed to LPS or amyloid-beta (Aβ) peptides exhibit increased oxidative stress, making them a useful system for studying the interplay between inflammation and oxidative damage.

Techniques To Induce Activation

BV2 cells can be activated through chemical, environmental, and genetic approaches. LPS is one of the most commonly used agents, engaging TLR4 and triggering intracellular signaling. The concentration and duration of LPS exposure influence the activation profile, with low doses (10–100 ng/mL) inducing a moderate response and higher concentrations (above 500 ng/mL) leading to robust transcriptional and functional changes. Activation is assessed using nitric oxide production assays, enzyme-linked immunosorbent assays (ELISA) for cytokine release, and quantitative PCR for inflammatory gene expression.

Interferon-gamma (IFN-γ) is another potent activator, either alone or in combination with LPS. IFN-γ drives BV2 cells toward a pro-inflammatory phenotype by upregulating major histocompatibility complex (MHC) class II expression and increasing inflammatory mediator production. Unlike LPS, which signals through TLR4, IFN-γ activates the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, promoting immune surveillance and antigen presentation. Co-treatment with LPS and IFN-γ amplifies cytokine release and oxidative stress markers.

Environmental factors such as hypoxia and oxidative stress also activate BV2 cells. Hypoxic conditions (1–5% O₂) mimic ischemic stroke and neurodegenerative states, leading to hypoxia-inducible factor 1-alpha (HIF-1α) upregulation, which regulates metabolic adaptation and inflammation. Similarly, hydrogen peroxide (H₂O₂) induces oxidative stress, triggering mitochondrial dysfunction and stress-related signaling pathways.

Genetic manipulation strategies, including CRISPR-Cas9 and RNA interference (RNAi), allow precise control over BV2 cell activation. Silencing NF-κB subunits reduces the inflammatory response to LPS, while overexpressing pro-inflammatory transcription factors enhances cytokine production. These tools provide targeted insights into therapeutic interventions.

Phenotypic Markers

BV2 cells express phenotypic markers that define their identity and functional state. These markers are assessed through immunocytochemistry, flow cytometry, and gene expression analysis. Ionized calcium-binding adapter molecule 1 (Iba1) is consistently expressed, reflecting their microglial lineage and role in cytoskeletal remodeling.

CD11b, a component of the complement receptor 3 complex, increases following activation and indicates enhanced phagocytic capacity. CD68, a lysosomal-associated glycoprotein, also marks phagocytic activity, with elevated levels corresponding to an activated state.

Surface markers such as CD40 and CD86 provide further insights into activation. CD40, a co-stimulatory molecule, is upregulated in response to inflammatory cues, while CD86, associated with antigen presentation, increases during heightened activation. These markers help delineate functional shifts under different conditions.

Conditioned Media Investigations

BV2-conditioned media contains cytokines, chemokines, and bioactive molecules that influence surrounding neural and immune components. This approach has been instrumental in studying neuroinflammatory cascades and microglia-mediated neuronal damage or protection. Pro-inflammatory treatments such as LPS or IFN-γ lead to high concentrations of TNF-α, IL-6, and NO, all of which modulate neuronal survival and synaptic function.

Beyond inflammatory mediators, BV2-conditioned media contains extracellular vesicles (EVs), including exosomes and microvesicles, which play a role in intercellular communication. These vesicles carry proteins, lipids, and microRNAs that influence neuronal signaling and glial crosstalk. Exosomes from resting BV2 cells support neuronal viability, while those from LPS-stimulated cells contain inflammatory miRNAs that exacerbate neurodegeneration. Manipulating BV2-conditioned media provides a controlled system for assessing therapeutic interventions targeting microglial-secreted factors.

Interactions With Other Cells

BV2 cells actively interact with neurons, astrocytes, and other glial populations, shaping the inflammatory landscape of the central nervous system. Their bidirectional communication with neurons influences synaptic plasticity, neurotransmitter balance, and survival. In response to neuronal distress signals such as ATP release or misfolded protein aggregates, BV2 cells undergo morphological and functional changes that can either facilitate repair or contribute to neurotoxicity. Pro-inflammatory phenotypes often lead to excitotoxicity through excessive glutamate release and oxidative stress.

Interactions with astrocytes further modulate BV2 behavior. Astrocyte-derived cytokines and metabolic factors influence microglial responses, with transforming growth factor-beta (TGF-β) suppressing inflammatory gene expression and promoting a homeostatic phenotype. Conversely, reactive astrocytes release interleukin-1 beta (IL-1β) and complement proteins that enhance BV2-mediated neuroinflammation. Studying BV2 cells in multicellular systems captures the complexity of neuroinflammatory signaling.