Lipopolysaccharide-induced neuroinflammation disrupts functional connectivity and community structure in primary cortical microtissues

Abstract

Three-dimensional (3D) neural microtissues are a powerful in vitro paradigm for studying brain development and disease under controlled conditions, while maintaining many key attributes of the in vivo environment. Here, we used primary cortical microtissues to study the effects of neuroinflammation on neural microcircuits. We demonstrated the use of a genetically encoded calcium indicator combined with a novel live-imaging platform to record spontaneous calcium transients in microtissues from day 14–34 in vitro. We implemented graph theory analysis of calcium activity to characterize underlying functional connectivity and community structure of microcircuits, which are capable of capturing subtle changes in network dynamics during early disease states. We found that microtissues cultured for 34 days displayed functional remodeling of microcircuits and that community structure strengthened over time. Lipopolysaccharide, a neuroinflammatory agent, significantly increased functional connectivity and disrupted community structure 5–9 days after exposure. These microcircuit-level changes have broad implications for the role of neuroinflammation in functional dysregulation of neural networks.

A 3D in vitro model of the device-tissue interface: functional and structural symptoms of innate neuroinflammation are mitigated by antioxidant ceria nanoparticles

“…Additionally, we found that tis tissue reaction occurs in the absence of an acute stab wound from implantation, suggesting that innate glia may be sensing and responding to chemical or mechanical cues from the implant, as demonstrated elsewhere [62][63][64][65]. Increases in functional connectivity and disruption of community structure in the wire-implanted microtissues were consistent with LPS-induced neuroinflammatory responses both in vitro and in vivo, alluding to the fact that functional and structural changes to neurons and their synaptic behaviors may be mediated by glial dysregulation [49,54]. Remarkably, we found that while symptoms of oxidative tissue damage occurred locally at the implant surface, changes in functional network dynamics affected the whole microtissue, revealing that localized neuroinflammation impacts the surrounding microcircuitry.…”

“…We developed and characterized a 3D in vitro model of the device-tissue interface by utilizing recent advances in primary 3D cortical culturing platforms [49]. Briefly, cortical tissue was collected from postnatal day 1 rats (figure 1(A)) and dissociated into a single cell suspension (figure 1(B)).…”

“…Nonadherent microwell were made with 2% agarose (16 500, Invitrogen) solutions using a custom injection molding system as described in Atherton et al [49]. Briefly, a 2% agarose solution in phosphate buffered saline was heated in the microwave and mixed in 30 s intervals until fully dissolved.…”

“…Creation and maintenance of 3D primary cultured microtissues were performed as described in Atherton et al [49]. In brief, primary cells were isolated from postnatal day 1 rats by cortical dissection in cold Hibernate A medium (HA, Brainbits) with B27 supplement (17504-044 Invitrogen) (HA-B27), followed by chemical dissociation with papain (Brainbits) in hibernate solution (Brainbits) at 30 • C for 30 min with gentle inversion mixing every 5 min.…”

“…AAV transduction of the genetically encoded GCaMP6s calcium reporter and mRuby control tag (AAV1-hSyn1-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA, AddGene) was performed at DIV1 as described in Atherton et al [49]. Briefly, replacement of CCM with 500 µl viral media, made by diluting viral stock in CCM to a concentration of 1 × 10 6 vg ml −1 , was performed at DIV1 and allowed to incubate for two days before replacement with CCM on DIV4.…”