(416b) Brain Imaging Probes Elicit Microglial Inflammatory Responses and Induce Cellular Morphological Changes

Microglia are the resident immune cells of the brain that number in the billions, and are central in maintaining the homeostasis of the neuronal environment through states of activation and quiescence.¹ Microglia mediate inflammatory responses to foreign and pathogenic material, which can elicit downstream neurotoxicity and neurodegeneration. However, despite the broad-scale implementation of fluorescent probes to query neuron function, little attention has been paid to probe interactions with, and effects on the brain microglia. Probes such as calcium dyes,² synthetic nanosensors,³ and voltage probes,⁴ are utilized ubiquitously in neuroscience to study brain structure and neurochemical activity. Herein, we demonstrate the effects of several brain chemistry probes on a SIM-A9 mouse microglial cell line, with particular emphasis on synthetic nanoparticles utilized to image brain chemistry. We show that single-walled carbon nanotube (SWNT)-based dopamine nanosensors are uptaken by, but are not cytotoxic to, SIM-A9 mouse microglia. The activity of SIM-A9 mouse microglia is next quantified with quantitative PCR and with an immune proteomics screen to monitor mRNA and protein expression and secretion levels of common inflammatory cytokine biomarkers. We show that SIM-A9 co-incubation with dopamine nanosensors induces a slight increase in expression of inflammatory cytokines TNF-α, iNOS, IL-1β, and IL-6. The observed nanosensor-induced cytokine upregulation is significantly lower than that caused by lipopolysaccharide stimulation. Live-cell imaging results further reveal that SIM-A9 interactions with SWNT nanosensors elicit a significant change in SIM-A9 cell morphology towards an elongated and ramified state, an in vitro indicator of a quiescent rather than inflammatory microglial state that halts cell proliferation. These contrasting effects indicate that nanosensors affect multiple microglial signaling pathways, possibly through a non-canonical immune response. We also discuss how common passivation techniques, namely PEGylation, can mitigate microglial inflammatory responses to brain imaging probes. PEGylated nanosensors show lower uptake into SIM-A9 cells, a reduced inflammatory response, and partial mitigation of SIM-A9 morphological change. Our results motivate greater attention to the effects of ubiquitously utilized neuro-imaging probes on the brain microglia.

References

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