Exaggerated Increases in Microglia Proliferation, Brain Inflammatory Response and Sickness Behaviour upon Lipopolysaccharide Stimulation in Non-Obese Diabetic Mice

Abstract

The non-obese diabetic (NOD) mouse, an established model for autoimmune diabetes, shows an exaggerated reaction of pancreas macrophages to inflammatory stimuli. NOD mice also display anxiety when immune-stimulated. Chronic mild brain inflammation and a pro-inflammatory microglial activation is critical in psychiatric behaviour.

Objective: To explore brain/microglial activation and behaviour in NOD mice at steady state and after systemic lipopolysaccharide (LPS) injection.

Methods: Affymetrix analysis on purified microglia of pre-diabetic NOD mice (8-10 weeks) and control mice (C57BL/6 and CD1 mice, the parental non-autoimmune strain) at steady state and after systemic LPS (100 μg/kg) administration. Quantitative PCR was performed on the hypothalamus for immune activation markers (IL-1β, IFNγ and TNFα) and growth factors (BDNF and PDGF). Behavioural profiling of NOD, CD1, BALB/c and C57BL/6 mice at steady state was conducted and sickness behaviour/anxiety in NOD and CD1 mice was monitored before and after LPS injection.

Results: Genome analysis revealed cell cycle/cell death and survival aberrancies of NOD microglia, substantiated as higher proliferation on BrdU staining. Inflammation signs were absent. NOD mice had a hyper-reactive response to novel environments with some signs of anxiety. LPS injection induced a higher expression of microglial activation markers, a higher brain pro-inflammatory set point (IFNγ, IDO) and a reduced expression of BDNF and PDGF after immune stimulation in NOD mice. NOD mice displayed exaggerated and prolonged sickness behaviour after LPS administration.

Conclusion: After stimulation with LPS, NOD mice display an increased microglial proliferation and an exaggerated inflammatory brain response with reduced BDNF and PDGF expression and increased sickness behaviour as compared to controls.

Fig. 1

Use of the proliferation marker BrdU revealed a higher percentage of microglia in the DNA-replicating S-phase 3 days after BrdU administration when compared to CD1 controls. Data expressed as mean ± SEM, n = 3 per group. * p < 0.05, *** p < 0.001.

Fig. 2

Investigation of mRNA expression of inflammatory markers in whole-brain tissue and isolated microglia. For whole-brain tissue, qPCR was used and data could be expressed as mean ± SEM (bars), n = 4-6 per group. * p < 0.05, LPS vs. control. ^# p < 0.05, NOD vs CD1. For microglia, Affymetrix analysis data are given of 1 single experiment with pooled microglia. Four hours after LPS administration, significant increases in whole-brain tissue mRNA and microglial expression of TNFα (a), IL-1β (b) and IL-6 (c) were reported in both NOD and CD1 mice. No obvious differences between the NOD and CD1 mice were evident, although NOD always scored slightly higher. Microglial expression followed these patterns by and large, although IL-6 did not show significant increases. Investigation of mRNA expression of IFNγ (d) and IDO (e) in whole-brain tissue revealed a significantly elevated expression in NOD mice following LPS administration when compared to vehicle-treated controls (Veh). These increases were also significantly greater than expression in the CD1 strain following LPS. Investigation of isolated microglia revealed expression of IFNγ that was up-regulated after LPS only in NOD mice. A primer for IDO was not present on the microarray chip used in this study, so no information was available for isolated microglia.

Fig. 3

Investigation of mRNA expression of growth factors in whole-brain tissue and isolated microglia. For whole-brain tissue, qPCR was used and data could be expressed as mean ± SEM (bars), n = 4-6 per group. * p < 0.05, LPS vs. control. ^# p < 0.05, NOD vs. CD1. For microglia, Affymetrix analysis data are given of 1 single experiment with pooled microglia. Investigation of mRNA expression of growth factors in whole-brain tissue revealed different responses to LPS in the NOD and CD1 mice. NOD mice had a significantly reduced expression of BDNF (a) and PDGF (b) in whole-brain tissue following LPS administration. By and large, microglial expression followed this pattern for PDGF. Veh = Vehicle-treated control.

Fig. 4

Behaviour of male NOD (filled bars) in comparison to male CD1, Balb/C and BL6 mice (all open bars) in various paradigms under novel and home cage conditions in the absence of LPS stimulation. NOD mice showed the highest activity in novel situations such as the open-field (a) and modified hole board (b) tests. c This hyper-activity was not present in the home cage environment. NOD mice had a shorter immobility time during initial exposure to the forced-swim test (FST1, d); however, when re-exposed to the FST 24 h later (FST2), the change in the passive stress-coping strategy (i.e. the time immobile in FST2 minus that in FST1) was highest in the NOD mice (e). f In the novelty-induced hypophagia test, an anxiety test independent of locomotor behaviour, NOD mice showed the highest latency to consume palatable liquid (i.e. latency in novel cage minus latency in home cage). g During the series of testing of basal behaviour, all NOD mice were in a pre-diabetic state as shown by the glucose levels determined in the blood drawn after the last test. Data are expressed as mean ± SEM, n = 10/strain; * p < 0.05, ** p < 0.01, *** p > 0.001.

Fig. 5

Comparison of NOD (filled bars) with CD1 mice (open bars) in various paradigms after LPS or saline (control) i.p. injection. In response to LPS, NOD mice showed an exaggerated and prolonged behavioural response to LPS stimulation as indicated by a strong reduction in body weight 24 h after LPS (a) which was independent of food intake (b), a more pronounced reduction in home cage activity 4 h after LPS (c) and a reduction in rearing that was maintained for at 24 h after LPS (d). Data expressed as mean ± SEM, n = 5-6 per group. *** p < 0.001, ** p < 0.01, * p < 0.05, LPS vs. control. ^### p < 0.001, ^## p < 0.01, ^# p < 0.05, NOD vs. CD1.