How to optimize neuroinflammation models

This article is based on a poster originally written by MS Heins, S. Hatusupy, MF Olthuis and presented at SfN 2024.

Neuroinflammation contributes to many neurodegenerative diseases. The blood-brain barrier (BBB) maintains an immune-privileged state in the brain, making peripherally produced inflammation unsuitable for the study of neuroinflammation.

Peripheral inflammation can spread to the central nervous system by affecting the integrity of the BBB, transporting inflammatory cells and cytokines from the bloodstream to the brain. A more representative model would show local neuroinflammation in the brain without peripheral effects.

This study explores the best experimental design for a locally produced model of neuroinflammation in adult male C57Bl/6J mice using three stimuli (LPS, BzATP, and LPS+BzATP).

Levels of proinflammatory cytokines were measured in various tissues and fluids, including interstitial fluid collected by push-pull microdialysis or open-flow microperfusion, and blood and brain tissues were analyzed.

The differential distribution and localization of pro-inflammatory cytokines responding to various stimuli were studied.

Methods

Studies were conducted in an AAALAC accredited laboratory in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals (2011) and the standards of European Union Directive 2010/63. The Dutch National Animal Experimentation Commission approved all animal treatments.

A comparison was first made between acute intracerebroventricular (ICV) administration of LPS in anesthetized animals and ICV administration of LPS and/or BzATP after cannulation surgery and recovery in awake, freely moving animals. . Terminal brain tissues were harvested four hours after administration and analyzed using the IL-1β ELISA (R&D Systems).

The timing of LPS and BzATP administration could then be examined. Adult male C57Bl/6J mice were surgically cannulated at the left ICV for administration and right CTX for collection of microdialysate or microperfusate.

After recovery, awake and freely moving animals were processed and microdialysate or microperfusate samples were collected.

All samples obtained were analyzed using IL-1β ELISA (R&D Systems) or a mouse inflammatory panel, particularly for levels of TNF-α, IL-1β, and IL- 6 (MSD). Only data from the most recent trial with optimal timing are presented here.

Tissue levels 4 h after ICV administration

Acute administration of LPS by ICV (blue bars) to anesthetized adult male C57Bl/6J mice, followed by terminal tissue sampling four hours after administration, shows strong elevations of IL-1β throughout the brain , but this is not detected in animals that received vehicle (black bars). ). Blood and CSF IL-1β levels for vehicle-administered animals were below the limit of quantification (LLOQ). LPS-treated rats significantly increased IL-1β levels in CSF (~2,200 pg/mL), but only moderately increased in plasma (~100 pg/mL).

Acute administration of ICV. Image credit: Charles River Laboratories

ICV injection of LPS (blue bars), BzATP (red bars), or LPS + BzATP (purple bars) in freely moving cannulated adult male C57Bl/6J mice followed by terminal tissue sampling four hours later administration, showed a significant increase in IL-1β for both LPS- and LPS+BzATP-administered animals. This was not observed in animals that received BzATP or vehicle (black bars). The effects were stronger in the left and right cortex than in the striatum and hippocampus tissues. Animals receiving vehicle had lower IL-1β levels at LLOQ, while LPS + BzATP-treated mice had a slight increase in plasma (~60 pg/mL).

Free-motion cannulated ICV administration. Image credit: Charles River Laboratories.

Microdialysis after ICV administration

ICV administration of LPS at t = 120 minutes, followed by BzATP at t = 0 minutes (purple squares), increased levels of TNF-α, IL-1β, and IL-6 in the interstitial space cerebral cortex. Animals treated with vehicle (black circles) showed no immediate increase.

After BzATP ICV administration, the levels of TNF-α, IL-1β, and IL-6 were significantly increased compared to vehicle controls.

These results were obtained both in push-pull microdialysates and in open-flow microperfusates; however, push-pull microdialysates (top graphs) appeared less variable than open-flow microperfusates (bottom graph).

IL-1β levels in terminal tissue samples were consistent with interstitial fluid levels and previous tissue collection experiments. Animals that received LPS + BzATP showed a significant increase in IL-1β levels (purple bars), while animals that received vehicle showed no or low levels (black bars).

Optimizing the timing of LPS and BzATP stimulation resulted in elevated levels of the inflammatory cytokines TNF-α, IL-1β, and IL-6 in mouse brain interstitial fluids.

These could be identified by push-pull microdialysis and open-flow microperfusion. Based on these experimental results, push-pull microdialysis is recommended over open-flow microperfusion since the microdialysis results are more consistent.

Topically applied LPS combined with microanalysis reduces peripheral side effects of neuroinflammation and allows the study of potential time-dependent effects of anti-neuroinflammatory drugs.

Image credit: Charles River Laboratories.

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