Alzheimer's disease (AD), the most common form of dementia, affects more than 40 million people worldwide and is characterized by the decline of cognitive abilities and progressive memory loss (1). To date, there is still no cure available, and the current pharmacotherapeutic strategies do not target the underlying molecular processes of the disease (2, 3).

To establish new treatments for AD, both preclinical models of the disease as well as reliable in vivo biomarkers are essential. Clinical biomarkers include measurement of Abeta42 and (phosphorylated) tau in cerebrospinal fluid, and also MRI and molecular imaging with positron emission tomography (PET). PET is a functional, non-invasive imaging technique that allows in vivo monitoring of pathological and physiological processes (4). ¹⁸F-Fluorodeoxyglucose (FDG)–PET and ¹⁸F-Florbetaben–PET are clinically established biomarkers for the diagnosis of AD capturing two key pathological features of the disease (5, 6). FDG–PET can visualize cerebral glucose metabolism that reflects regional neuronal and synaptic activity and can therefore be used to assess synaptic dysfunctions and neuron loss in the brain (7). ¹⁸F-Florbetaben–PET detects cerebral amyloid load (8). Successful back-translating clinical biomarkers and molecular imaging methods of AD, including PET, is very valuable for the evaluation of new therapeutic strategies and to increase the quality of preclinical studies. However, while PET is an established tool in the assessment of AD patients, its role in preclinical studies using AD mouse models remains unclear.

Several mouse models of AD are available so far showing different pathological hallmarks. Thereby AD models that express mutations in the amyloid precursor protein (APP) and/or presenilin (PS) genes have been widely used to study AD pathology, especially with regard to the Abeta pathology. A well-established and commonly used mouse model is the 5XFAD model, as these mice carry five APP and PS1 mutations that lead to an age-dependent aggressive amyloid plaque pathology, neuronal dysfunction, and neuron loss as well as behavioral deficits (9, 10).

A recent study of our group showed that both ¹⁸F-FDG- and ¹⁸F-Florbetaben-PET are suitable tools for the assessment of AD pathologies in male 5XFAD mice in vivo, postulating its broader use in longitudinal preclinical therapy studies (11). However, only data on male 5XFAD mice have been published so far whereas possible sex differences in ¹⁸F-FDG and ¹⁸F-Florbetaben uptake have not been studied (12–14).

The aim of this study was to evaluate the suitability of ¹⁸F-FDG– and ¹⁸F-Florbetaben–PET in female 5XFAD mice and to assess possible sex differences.

Alzheimer's disease is one of the most challenging diseases of the century. To investigate new therapies, longitudinal evaluation of their effects in vivo is essential and relies heavily on disease-specific biomarkers. Biomarkers, including molecular imaging with ¹⁸F-FDG- and amyloid-PET, have shown their value in the clinical diagnosis of AD (21). However, only a limited number of preclinical studies with PET are available, even though modern small animal PET scanners are able to detect the same molecular pathologies that are seen in humans. Both, ¹⁸F-FDG– and amyloid–PET can detect early changes of the disease, making them valuable targets for in vivo imaging of disease progression and efficacy of new therapeutic strategies. Advances in small animal PET have enabled imaging in AD mouse models, allowing longitudinal follow-up studies (22, 23) and a better translation of findings from bench to bedside.

Neuronal dysfunctions can be detected using ¹⁸F-FDG–PET as ¹⁸F-FDG uptake in neurons is mainly determined by synaptic activity (24). AD patients typically show a pattern of decreased ¹⁸F-FDG uptake in the posterior cingulate cortex proceeding to posterior temporal and parietal cortex areas and eventually to the frontal lobe (25). However, the use of ¹⁸F-FDG as a preclinical biomarker to study AD processes in animal models has been arguable, as inconsistent results with hyper-, hypo-, and normal metabolism have been described in transgenic AD mouse models (12, 26–28).

The 5XFAD mouse model of AD is a widely used model of amyloidosis with five AD-linked mutations. 5XFAD mice show a massive plaque pathology, intraneuronal Abeta, robust microgliosis, and inflammatory processes as well as synaptic and neuronal loss and memory deficits (9, 10, 15, 29, 30). Unbiased stereology revealed significant neuron loss in the subiculum by 9 months and neuron layer 5 of the cortex by 12 months in female 5XFAD mice (15, 29). The robust appearance of histological, molecular, and behavioral AD hallmarks makes the 5XFAD model popular in biomedical research, and the “Alzheimer's Disease Preclinical Efficacy Database” (https://alzped.nia.nih.gov/) shows that ~10% of all AD studies that work with animal models use this strain. Therefore, it is essential that reliable biomarkers are available to monitor the success of a possible therapy in 5XFAD mice. As 5XFAD mice recapitulate major features of AD, ¹⁸F-FDG– and amyloid-PET should theoretically be useful in vivo biomarkers for 5XFAD animals. However, only a few PET studies with inconsistent findings, which largely focus on male mice, have been performed in 5XFAD mice (11, 13, 31, 32). The current study, which is the first preclinical study using PET/MRI with different tracers in female 5XFAD mice, showed distinct reduced ¹⁸F-FDG uptake in the whole brain as well as in all the nine analyzed brain regions including the hippocampus, cortex, thalamus, and hypothalamus. To our knowledge, only one other study analyzed cerebral glucose metabolism in female 5XFAD mice (33). Son et al. focused on the hippocampus and frontal lobe and described a regional hypometabolism in these two brain regions in 9.5-month-old female 5XFAD mice. In line with these findings, Xiao et al. (34) demonstrated decreased ¹⁸F-FDG uptake in the hippocampus, cerebral cortex, and olfactory bulb of 6-month-old male 5XFAD mice. Similarly, DeBay et al. (32) confirmed a hypometabolism in the cerebral cortex, hippocampus, and basal ganglia in 5-month-old male 5XFAD mice. In addition, Macdonald et al. (13) detected lower ¹⁸F-FDG uptake in the whole brain of 13-month-old male 5XFAD mice. In contrast, a study by Rojas et al. (31) showed contradictory findings with increased ¹⁸F-FDG uptake in the whole brain of 11-month-old 5XFAD mice, without stating the sex, using the cerebellum as the reference region. As discussed before (11), this normalization technique seems to heavily influence study results. In contrast to human glucose metabolism in the cerebellum (which is relatively preserved in disease progression), metabolism in the cerebellum of transgenic mouse models can also be influenced, possibly resulting in higher uptake values (27, 35–37). Studies in other AD mouse models using normalized cerebral FDG uptake to the cerebellum also showed higher ¹⁸F-FDG uptake in the brain (27, 36, 37). This factor could explain the results by Rojas et al. (31) in 5XFAD mice.

To our knowledge, no studies on sex differences in ¹⁸F-FDG-PET in 5XFAD mice or other transgenic mouse models of AD are available so far. We could previously demonstrate a significant reduction in glucose metabolism in 7- and 12-month-old male 5XFAD mice in several cortical areas even before the onset of memory defecits (11). In this study, we show for the first time that the hypometabolism in 5XFAD mice is gender-dependent and more pronounced in female mice. Our results demonstrated a lower glucose metabolism in the whole brain as well as in the hippocampus, thalamus, cerebellum, and midbrain of 7-month-old female 5XFAD mice compared with age-matched male 5XFAD mice.

Although the reason is still unknown, AD has a gender-specific epidemiological profile, disproportionately affecting women both in prevalence and in severity (38, 39). A number of female transgenic AD mouse models also display an earlier onset of AD pathologies as well as a more severe pathology than their male counterparts (40–42). Moreover, behavior deficits are often more severe in female mice (43, 44). Sex-based differences in 5XFAD mice have also been demonstrated in several studies ranging from differences in odor detection and motor impairment to working memory deficits (45–47). A gene ontology analysis of the hippocampi of 4-month-old 5XFAD mice detected sex-specific patterns with female 5XFAD mice, which showed more differentially expressed genes compared with males including higher levels of human APP and PS1 mRNA expression. Interestingly, the majority of molecular changes that were more severely affected in female 5XFAD mice were associated with the immune system (48).

Another pathological hallmark of AD that can be detected by PET is cerebral amyloid depositions. Several PET-tracers that are able to visualize amyloid burden have been developed and are used in clinical routine including ¹¹C-labeled Pittsburgh Compound-B (¹¹C-PIB), ¹⁸F-Flutemetamol, ¹⁸F-Florbetapir, and ¹⁸F-Florbetaben (21, 49–52). Amyloid–PET has also been demonstrated as a valuable tool in preclinical studies and can be used to measure therapeutic outcomes via amyloid load in vivo (53–56). The presence of a distinctive plaque pathology in 5XFAD mice has been well-described in vitro with extracellular amyloid plaques beginning at 2 months in the fifth layer of the cortex and progressively increasing with age and spreading to the cortex, hippocampus, and subiculum (9, 15, 57). However, only a few studies on the use of amyloid–PET in 5XFAD mice are available so far, lacking data on female 5XFAD mice. Available studies show higher cerebral uptake of several different amyloid tracers (¹¹C-PIB, ¹⁸F-Florbetapir,¹⁸F-FC119S, and ¹⁸F-Florbetaben) compared with WT mice. Rojas and colleagues reported a higher uptake of the amyloid radiotracers ¹¹C-PIB and ¹⁸F-Florbetapir in 11-month-old 5XFAD mice, whereas Frost et al. showed elevated uptake of ¹⁸F-Florbetapir in 14-month-old mice, both without stating the sex (31, 58). In addition, Oh et al. demonstrated elevated levels of ¹⁸F-FC119S in the cortex, hippocampus, and thalamus of 5.5-month-old male 5XFAD mice (14). We previously showed an increased ¹⁸F-Florbetaben tracer uptake in male 5XFAD mice (11). Here we could confirm that ¹⁸F-Florbetaben is also a valuable tracer for female 5XFAD as our findings demonstrate increased cerebral uptake of ¹⁸F-Florbetaben in 7-month-old female 5XFAD mice.

In contrast to ¹⁸F-FDG uptake, ¹⁸F-Florbetaben–PET did not show significant sex-differences between male and female 5XFAD mice. This is in line with the original manuscript characterizing the 5XFAD model, as Oakley et al. only noted a trend toward greater plaque deposition in young female AD animals relative to males of the same age, but this trend declined with age (9). In contrast, several studies demonstrated higher levels of APP and Aβ42 as well as an increased plaque pathology in female 5XFAD mice (48, 59, 60). In line with our findings, Biechele and colleagues also detected no sex differences in ¹⁸F-Florbetaben uptake in App^NL−G−F mice (61).

Limitations of our study include possible partial volume effect due to analysis of relatively small VOIs. However, this effect might have been avoided as the analyzed volumes were above the suggested threshold in small animal scanners of 9 mm³ [the smallest VOI (amygdala) was 10 mm³] (62–64). Furthermore, there is no standard for small animal brain imaging which can result in high inter-study variations, especially for FDG studies. Therefore, to avoid inconsistencies between different labs, standardization of imaging protocols including fasting protocols, equipment, and animal handling would be beneficial (12, 65). In addition, it might be interesting to study additional age groups for female 5XFAD mice in the future. However, we decided to focus on 7-month-old mice as female 5XFAD mice show a robust Abeta pathology as well as severe memory deficits at this age. Furthermore, this age is often used as an endpoint in treatment studies in 5XFAD mice (66–71). Thus, our present findings underline that PET imaging with ¹⁸F-FDG- and ¹⁸F-Florbetaben could provide a valuable and robust preclinical evaluation tool of therapeutic strategies modulating the AD pathology in 5XFAD mice.

Our results support the feasibility of small animal PET imaging with ¹⁸F-FDG- and ¹⁸F-Florbetaben in the 5XFAD mouse model of AD in both male and female animals. Moreover, our findings highlight the need to account for sex differences in studies working with 5XFAD mice.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Röverskamp 5, 26203 Oldenburg, Germany and Landesamt für Gesundheit und Soziales LAGeSo Darwinstr. 15, 10589 Berlin, Germany.

TF, CI, and NB performed experiments. CB and YB designed the project, performed experiments, analyzed data, and wrote the manuscript. All authors contributed to revising the manuscript and approved the final version.

This work was supported by the Alzheimer Stiftung Göttingen to CB and YB, and in part by the Deutsche Forschungsgemeinschaft (DFG) for PET/MRI use (INST 335/454-1FUGG). We acknowledge support by the Open Access Publication Funds of the Göttingen University.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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