In Vivo Detection of Age- and Disease-Related Increases in Neuroinflammation by 18F-GE180 TSPO MicroPET Imaging in Wild-Type and Alzheimer's Transgenic Mice

Abstract

Alzheimer's disease (AD) is the most common cause of dementia. Neuroinflammation appears to play an important role in AD pathogenesis. Ligands of the 18 kDa translocator protein (TSPO), a marker for activated microglia, have been used as positron emission tomography (PET) tracers to reflect neuroinflammation in humans and mouse models. Here, we used the novel TSPO-targeted PET tracer (18)F-GE180 (flutriciclamide) to investigate differences in neuroinflammation between young and old WT and APP/PS1dE9 transgenic (Tg) mice. In vivo PET scans revealed an overt age-dependent elevation in whole-brain uptake of (18)F-GE180 in both WT and Tg mice, and a significant increase in whole-brain uptake of (18)F-GE180 (peak-uptake and retention) in old Tg mice compared with young Tg mice and all WT mice. Similarly, the (18)F-GE180 binding potential in hippocampus was highest to lowest in old Tg > old WT > young Tg > young WT mice using MRI coregistration. Ex vivo PET and autoradiography analysis further confirmed our in vivo PET results: enhanced uptake and specific binding (SUV75%) of (18)F-GE180 in hippocampus and cortex was highest in old Tg mice followed by old WT, young Tg, and finally young WT mice. (18)F-GE180 specificity was confirmed by an in vivo cold tracer competition study. We also examined (18)F-GE180 metabolites in 4-month-old WT mice and found that, although total radioactivity declined over 2 h, of the remaining radioactivity, ∼90% was due to parent (18)F-GE180. In conclusion, (18)F-GE180 PET scans may be useful for longitudinal monitoring of neuroinflammation during AD progression and treatment.

Significance statement: Microglial activation, a player in Alzheimer's disease (AD) pathogenesis, is thought to reflect neuroinflammation. Using in vivo microPET imaging with a novel TSPO radioligand, (18)F-GE180, we detected significantly enhanced neuroinflammation during normal aging in WT mice and in response to AD-associated pathology in APP/PS1dE9 Tg mice, an AD mouse model. Increased uptake and specific binding of (18)F-GE180 in whole brain and hippocampus were confirmed by ex vivo PET and autoradiography. The binding specificity and stability of (18)F-GE180 was further confirmed by a cold tracer competition study and a metabolite study, respectively. Therefore, (18)F-GE180 PET imaging may be useful for longitudinal monitoring of neuroinflammation during AD progression and treatment and may also be useful for other neurodegenerative diseases.

Keywords: Alzheimer's disease; GE180 PET tracer; TSPO; microglia; neuroinflammation.

Copyright © 2015 the authors 0270-6474/15/3515717-15$15.00/0.

Figures

Figure 1.

Absence of TSPO IR in thalamus in APP/PS1dE9 Tg mice. Immunohistochemical staining with an anti-Aβ antibody R1282, an anti-TSPO monoclonal antibody, and an anti-Iba-1 antibody (marker for microglia/macrophage) was performed on frozen brain sections from each of four 26-month-old APP/PS1dE9 Tg mice. Representative images showing antibody IR in mouse cortex, hippocampus, cerebellum, striatum, and thalamus are displayed. Although plaque-associated Iba-1 IR was observed in all five brain regions, plaque-associated TSPO IR was observed in cortex, hippocampus, cerebellum, and striatum, but not in thalamus. Therefore, we chose the thalamus as the reference region for our study. Scale bar, 50 μm.

Figure 2.

TSPO IR colocalizes with microglia, especially those associated with Aβ deposits in APP/PS1dE9 Tg mouse and human AD brain. Double IF showed that TSPO IR was closely associated with plaques stained by an anti-Aβ 6E10 (A), an anti-Aβ42 antibody (B), an anti-Aβ40 antibody (C), and thioflavin S (D),and was colocalized with the microglia markers CD45 (E) and CD68 (F), but rarely with the astrocyte marker GFAP (G) in the hippocampus of a 26-month-old APP/PS1dE9 Tg mouse. Similarly, double IF on human AD brain sections indicated that TSPO IR was associated with plaques stained by an anti-Aβ 3A1 (H) and an anti-Aβ42 antibody (I) and was colocalized with the glia markers HLA-DR (J) and, to some degree, GFAP (K). Immunoreactivity of triple IF staining in 26-month-old APP/PS1dE9 Tg mice with anti-CD86 (L), anti-TSPO (M), and 6E10 (N), as well as the merged image (O), showed that TSPO IR colocalized with CD86 IR, a pro-inflammatory phenotypic marker, in microglia that were associated with Aβ plaques. In addition, triple labeling of CD206 (P), TSPO (Q), and 6E10 (R), as well as the merged image (S), indicated that TSPO IR also colocalized with CD206, an anti-inflammatory phenotypic marker, in activated microglia associated with plaques (but not those further away; data not shown). DAPI (blue) was used to indicate the cell nucleus.

Figure 3.

Whole-brain and hippocampal-specific uptake of 18F-GE180 PET tracer in 4- and 26-month-old WT and APP/PS1dE9 Tg mice. A, No significant differences in mouse body weight were found between the groups. B, Mouse brains (without cerebellum) were removed and weighed 2 h after tracer injection. No significant differences in mouse brain weight were found between groups. C, To quantify the whole-brain uptake of GE180 during the 2 h dynamic PET scan, the VOI was drawn manually on the PET images for each mouse. No significant differences in VOI volume were observed between groups. n = 6 per group. D, E, The sagittal views (D) and time–radioactivity curve (E) of 18F-GE180 uptake in young WT, old WT, young APP/PS1dE9 Tg, and old APP/PS1dE9 Tg mouse whole brains scanned by in vivo PET dynamic acquisition for 2 h. F, Hippocampal VOI was identified by PET/MRI image infusion (a) and the coronal views of 18F-GE180 uptake in brains of young WT (b), old WT (c), young APP/PS1dE9 Tg (d), and old APP/PS1dE9 Tg (e) mice scanned by in vivo PET with coregistration of an MRI anatomical template are shown. G, Time–radioactivity curves in thalamus (reference region) for all groups were obtained from 2 h dynamic PET scans. H, Time–radioactivity curves of relative hippocampal uptake (normalized to the thalamus) were calculated for all groups. Two-way ANOVA with post hoc Bonferroni's test were applied for statistical analysis (data shown in Results). n = 6 per group.

Figure 4.

18F-GE180 PET and AR signal were correlated with TSPO IR in hippocampus in 4- and 26-month-old WT and APP/PS1dE9 Tg mice. Hippocampal TSPO IR (% ROI; black bars) in 4- and 26-month-old WT and APP/PS1dE9 Tg mice was quantified by BioQuant software using % ROI (2 planes per mouse, n = 4–6 mice per group). The hippocampal in vivo PET signal during the 60–90 min period of dynamic acquisition (retention, A), ex vivo PET (B), or ex vivo AR (C) was also calculated, respectively, for each group (n = 6) and correlated with TSPO IR. ***p < 0.001, one-way ANOVA with post hoc Bonferroni's test.

Figure 5.

Age- and AD pathology-associated increases in 18F-GE180 uptake in old WT and old APP/PS1dE9 Tg hippocampus (HC) and cortex (CTX) by ex vivo PET analysis. A–D, Representative ex vivo PET images of 1 mm brain slabs of young WT (A), old WT (B), young APP/PS1dE9 Tg (C) and old APP/PS1dE9 Tg (D) mouse brain obtained from 10 min static PET scans. E, Quantification of 18F-GE180 radioactivity in slab 8 was performed in a VOI containing both HC and CTX. F, Uptake of 18F-GE180 in slab 9 was quantified in a VOI containing both HC and CTX. *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA with post hoc Bonferroni's test. n = 6 per group.

Figure 6.

Increased 18F-GE180 uptake in aged WT and aged APP/PS1dE9 Tg mouse brains from ex vivo AR analysis. A, Representative ex vivo AR images are shown for young and old WT, as well as young and old APP/PS1dE9 Tg mice. B, C, For quantitative analysis, a combined hippocampal and cortical ROI was drawn on brain slabs 8 and 9 and a reference region in thalamus was drawn (small white square) on brain slab 8 (B). No difference was found in ROI size between groups (C). D, E, 18F-GE180 radioactivity in HC + CTX ROI in slabs 8 (D) and 9 (E) was normalized by the radioactivity in the thalamus ROI and the ratios (relative radioactivity) were compared between groups. F, G, Representative histogram is shown of the ROI in slab 9 from an old WT mouse (F) and an old APP/PS1dE9 Tg mouse (G) that were processed using ImageJ. H, I, Values of SUV75% in ROI in slabs 8 (H) and 9 (I) of all mice were calculated. *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA with post hoc Bonferroni's test. n = 6 per group.

Figure 7.

Biodistribution of 18F-GE180 tracer in organs or tissues. Organs or tissues were collected at 2 h after tracer injection and radioactivity was measured with a WIZARD2-2480 automatic gamma counter with decay correction. n = 3 in the old WT mice group and old APP/PS1dE9 Tg mice group; n = 4 in young WT mice group.

Figure 8.

Pre-injection of unlabeled GE180 inhibits the binding of 18F-GE180 in mouse brain. A, Sagittal in vivo PET images are shown of 13-month-old WT and APP/PS1dE9 Tg mice that received a pre-injection of saline or unlabeled GE180 (cold) tracer, followed by an injection of 18F-GE180 (hot) tracer. B, Two-hour dynamic in vivo18F-GE180 PET whole-brain time-activity curves of WT and APP/PS1dE9 Tg mice were generated after pre-injection with either saline or cold tracer. The time frames used for image reconstruction were as follows: 1 min × 8 + 2 min × 6 + 10 min × 10. n = 1 per group. C, Representative ex vivo PET images of brain slabs from the WT and APP/PS1dE9 Tg mice in B are shown. D, Quantification of 18F-GE180 uptake in the combined HC and CTX ROI in slabs 8 and 9 by ex vivo PET analysis revealed higher uptake in the APP/PS1dE9 Tg mice. n = 2 per group. E, Representative ex vivo AR images of brain slabs from the WT and APP/PS1dE9 Tg mice in B and C are shown. F, Quantification of 18F-GE180 uptake in the combined HC and CTX ROI in slabs 8 and 9 by ex vivo AR analysis confirmed our in vivo and ex vivo PET findings of increased uptake in APP/PS1dE9 Tg mice compared with WT mice. The data were normalized by the radioactivity in thalamus. n = 2 per group.

Figure 9.

Changes in total metabolites and GE180 in brain, kidney, and liver during the 2 h after tracer injection. Representative HPLC plots for the radioactivity of GE180 and its metabolites in samples of brain (A), kidney (B), liver (C), and plasma (D) collected over the 120 min after tracer injection are shown. E–G, Levels of 18F-GE180 and total metabolites in mouse brains (E), liver (F), and kidney (G) were calculated for samples taken 10, 30, 60, and 120 min after tracer injection. Data quantification was based on the HPLC integration. H, The total radioactivity in mouse plasma, brain, kidney, and liver was reduced over 120 min; however, of the remaining radioactivity, a large portion was parent GE180 in brain, kidney, and blood (as shown in E–G). n = 3/sample/time point.