Inflammatory and immunopathological differences in brains of permissive and non-permissive hosts with Angiostrongylus cantonensis infection can be identified using 18F/FDG/PET-imaging

Abstract

Background: Angiostrongylus cantonensis is a parasite that mainly infects the heart and pulmonary arteries of rats and causes human eosinophilic meningitis or meningoencephalitis in certain geographical areas. Current diagnostic methods include detection of the parasite in cerebrospinal fluid (CSF) and eosinophilic immune examination after lumbar puncture, which may be risky and produce false-positive results. 18F- Fluorodeoxyglucose (FDG), a Positron emission tomography (PET) tracer, has been used to assess different pathological or inflammatory changes in the brains of patients. In this study, we hypothesized that A. cantonensis infection-induced inflammatory and immunomodulatory factors of eosinophils result in localized pathological changes in the brains of non-permissive hosts, which could be analyzed using in vivo 18F-FDG PET imaging.

Methodology/findings: Non-permissive host ICR mice and permissive host SD rats were infected with A. cantonensis, and the effects of the resulting inflammation on 18F-FDG uptake were characterized using PET imaging. We also quantitatively measured the distributed uptake values of different brain regions to build an evaluated imaging model of localized neuropathological damage caused by eosinophilic inflammation. Our results showed that the uptake of 18F-FDG increased in the cerebellum, brainstem, and limbic system of mice at three weeks post-infection, whereas the uptake in the rat brain was not significant. Immunohistochemical staining and western blotting revealed that Iba-1, a microglia-specific marker, significantly increased in the hippocampus and its surrounding area in mice after three weeks of infection, and then became pronounced after four weeks of infection; while YM-1, an eosinophilic chemotactic factor, in the hippocampus and midbrain, increased significantly from two weeks post-infection, sharply escalated after three weeks of infection, and peaked after four weeks of infection. Cytometric bead array (CBA) analysis revealed that the expression of TNF in the serum of mice increased concomitantly with the prolongation of infection duration. Furthermore, IFN-γ and IL-4 in rat serum were significantly higher than in mouse serum at two weeks post-infection, indicating significantly different immune responses in the brains of rats and mice. We suggest that 18F-FDG uptake in the host brain may be attributed to the accumulation of large numbers of immune cells, especially the metabolic burst of activated eosinophils, which are attracted to and induced by activated microglia in the brain.

Conclusions: An in vivo 18F-FDG/PET imaging model can be used to evaluate live neuroinflammatory pathological changes in the brains of A. cantonensis-infected mice and rats.

Fig 1

Images (transaxial view) of fused FDG-PET/CT slices from normal mice (A) and rats (B), or mice and rats infected with A. cantonensis 2–4 weeks after 120 min of ¹⁸F-FDG injection. The color bar indicates standardized uptake value (SUV). The image shown is representative of a typical result. (C) (D) The special brain areas as indicated were circled in the figure for analysis. Data are expressed as mean + standard deviation. **p < 0.01, *** p < 0.001, **** p < 0.0001 when compared with the normal control. ^#p < 0.05, ^###p < 0.001, ^####p < 0.0001 indicate comparisons between two indicated groups.

Fig 2. Histological changes in the brains of normal controls and infected animals at 2–4 weeks.

(A1) H&E staining of brain tissue sections of normal mice and rats and those infected with A. cantonensis after 2–4 weeks is shown. (A2) The magnified image from 2-4-weeks post-infection and normal mice and rats. (B) Quantitative analysis of eosinophil numbers. After 2 weeks of infection, increased numbers of immune and inflammatory cells appeared in the mouse brain between the hippocampus, midbrain, and thalamus. Inflammatory cell infiltration further increased at 4 weeks post-infection. However, inflammatory infiltration between the hippocampus and thalamus of rats was delayed until 4 weeks post-infection.

Fig 3

Immunohistochemical staining for microglial and eosinophilic chemotactic factors in the brains of infected mice and rats (A1) Microglial IHC staining of mouse and rat brain sections at 2–4 weeks post-infection and normal control. (A2) The magnified image from 3- and 4-weeks post-infection, where the arrow indicates microglia-accumulated signals. (A3) Quantitative analysis of IBA-1 signals. (B1) IHC staining for eosinophil chemotactic factor of mouse and rat brain sections at 2–4 weeks post-infection, and normal control. (B2) The magnified image of 3- and 4 weeks post-infection, where the arrow indicates eosinophil-accumulated signals. (B3) Quantitative analysis of YM-1 signals. The values are expressed as means ± SD, **p < 0.01, ****p < 0.0001 when compared with the normal control. ^#p < 0.05, ^####p < 0.0001 indicate comparisons between two indicated groups. Slides were visualized under a microscope (Leica, Bensheim, Germany) at (1) 40× and (2) 100× magnification.

Fig 4. qPCR and western blot analyses of IBA, YM-1, and YM-1+YM-2 in the brain.

(A) Relative expression of IBA-1 and YM-1 in mice and rats. (B) Relative expression of IBA-1 and YM-1+YM-2 in mice and rats. The values are presented as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared with the normal control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 indicate comparisons between two indicated groups.

Fig 5. The cytokine ELISA analysis of mice and rats for IFN-γ, IL-2, IL-4, IL-5, and tumor necrosis factor-alpha (TNF-α).

(A) Mouse and rat cytokine levels. (B) IL-5 levels in mice. The values are presented as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared with the normal control, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001. #p<0.05, ##p<0.01, ###p<0.001 indicate comparisons between two indicated groups.

Fig 6. Summary data map of this study.