Abstract
BackgroundAcute CNS damage is commonly studied using rat and mouse models, but increasingly, molecular analysis is finding species differences that might affect the ability to translate findings to humans. Microglia can undergo complex molecular and functional changes, often studied by in vitro responses to discrete activating stimuli. There is considerable evidence that pro-inflammatory (M1) activation can exacerbate tissue damage, while anti-inflammatory (M2) states help resolve inflammation and promote tissue repair. However, in assessing potential therapeutic targets for controlling inflammation, it is crucial to determine whether rat and mouse microglia respond the same.MethodsPrimary microglia from Sprague-Dawley rats and C57BL/6 mice were cultured, then stimulated with interferon-γ + tumor necrosis factor-α (I + T; M1 activation), interleukin (IL)-4 (M2a, alternative activation), or IL-10 (M2c, acquired deactivation). To profile their activation responses, NanoString was used to monitor messenger RNA (mRNA) expression of numerous pro- and anti-inflammatory mediators, microglial markers, immunomodulators, and other molecules. Western analysis was used to measure selected proteins. Two potential targets for controlling inflammation—inward- and outward-rectifier K+ channels (Kir2.1, Kv1.3)—were examined (mRNA, currents) and specific channel blockers were applied to determine their contributions to microglial migration in the different activation states.ResultsPro-inflammatory molecules increased after I + T treatment but there were several qualitative and quantitative differences between the species (e.g., iNOS and nitric oxide, COX-2). Several molecules commonly associated with an M2a state differed between species or they were induced in additional activation states (e.g., CD206, ARG1). Resting levels and/or responses of several microglial markers (Iba1, CD11b, CD68) differed with the activation state, species, or both. Transcripts for several Kir2 and Kv1 family members were detected in both species. However, the current amplitudes (mainly Kir2.1 and Kv1.3) depended on activation state and species. Treatment-induced changes in morphology and migratory capacity were similar between the species (migration reduced by I + T, increased by IL-4 or IL-10). In both species, Kir2.1 block reduced migration and Kv1.3 block increased it, regardless of activation state; thus, these channels might affect microglial migration to damage sites.ConclusionsCaution is recommended in generalizing molecular and functional responses of microglia to activating stimuli between species.
Highlights
Acute CNS damage is commonly studied using rat and mouse models, but increasingly, molecular analysis is finding species differences that might affect the ability to translate findings to humans
Inflammatory profiling of rat and mouse microglia The terminology for microglial activation is evolving; for clarity, activation states are denoted by the stimulus used, as follows
Anti-inflammatory and “alternative” activation genes and receptors We examined several genes known to be upregulated by IL-4 in mouse microglia: arginase 1 (ARG1), “found in inflammatory zone” 1 (FIZZ1), Mannose receptor (MRC1)/CD206, CCL22, CD163, and peroxisome proliferator-activated receptor gamma (PPAR-γ) [57]
Summary
Acute CNS damage is commonly studied using rat and mouse models, but increasingly, molecular analysis is finding species differences that might affect the ability to translate findings to humans. Microglia can undergo complex molecular and functional changes, often studied by in vitro responses to discrete activating stimuli. Immune responses of mice and humans are increasingly being compared [5,6,7], and it is crucial to determine the similarities and differences between the commonly used rodent species. Changes in activation states are expected to affect functional outcomes, including the capacity of microglia to produce immune mediators, migrate, proliferate, and phagocytose dead cells and debris. To elucidate responses to stimuli that can skew microglia toward a particular activation state, molecular profiles and cell functions are normally assessed in vitro
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