Illuminating characteristic patterns of inflammatory dermatoses: A comprehensive dual-imaging approach using Optical coherence tomography and Line-field confocal optical coherence tomography.

BACKGROUND: Inflammatory skin diseases, such as psoriasis, atopic eczema, and contact dermatitis pose diagnostic challenges due to their diverse clinical presentations and the need for rapid and precise diagnostic assessment. OBJECTIVE: While recent studies described non-invasive imaging devices such as Optical coherence tomography and Line-field confocal OCT (LC-OCT) as possible techniques to enable real-time visualization of pathological features, a standardized analysis and validation has not yet been performed. METHODS: One hundred forty lesions from patients diagnosed with atopic eczema (57), psoriasis (50), and contact dermatitis (33) were imaged using OCT and LC-OCT. Statistical analysis was employed to assess the significance of their characteristic morphologic features. Additionally, a decision tree algorithm based on Gini's coefficient calculations was developed to identify key attributes and criteria for accurately classifying the disease groups. RESULTS: Descriptive statistics revealed distinct morphologic features in eczema, psoriasis, and contact dermatitis lesions. Multivariate logistic regression demonstrated the significance of these features, providing a robust differentiation between the three inflammatory conditions. The decision tree algorithm further enhanced classification accuracy by identifying optimal attributes for disease discrimination, highlighting specific morphologic criteria as crucial for rapid diagnosis in the clinical setting. CONCLUSION: The combined approach of descriptive statistics, multivariate logistic regression, and a decision tree algorithm provides a thorough understanding of the unique aspects associated with each inflammatory skin disease. This research offers a practical framework for lesion classification, enhancing the interpretability of imaging results for clinicians.

Line-field confocal optical coherence tomography: Characteristic hints for the diagnosis of scarring alopecia due to lupus erythematodes: A preliminary study.

INTRODUCTION: Lupus erythematosus (LE) is an inflammatory autoimmune disease, that can affect the skin to varying degree. In particular, discoid LE (DLE) and the rare form of lupus panniculitis/profundus are associated with scarring alopecia. The heterogeneity of the clinical, dermatoscopic, and histologic presentation poses a major challenge to the clinician in the diagnosis and differential diagnosis of other forms of scarring alopecia. OBJECTIVE: While noninvasive imaging techniques using optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) have proven to be helpful in the diagnosis of scarring alopecia in the context of LE, this study aimed to investigate line-field confocal OCT (LC-OCT) to identify characteristic features of cicatricial alopecia in LE. METHODS: Fifteen patients with cicatricial alopecia in LE were included and the most affected/inflamed areas of the scalp were prospectively examined. In analogy to histopathology and previously reported criteria in RCM, all images were evaluated according to seven established criteria and underwent descriptive analyses. RESULTS: LC-OCT revealed characteristic features of cicatricial alopecia, such as lymphocytic interface dermatitis (14/15; 93.3%) and basal cell vacuolization (13/15; 86.7%). The most impressive feature was the occurrence of prominent hyperreflective fibers in 14/15 patients (93.3%). CONCLUSION: LC-OCT imaging can noninvasively detect morphologic criteria such as lymphocytic and vacuolar interface dermatitis of cicatricial alopecia due to LE. In particular, the presence of hyperreflective collagen fibers appears to be a characteristic easily recognizable feature that may facilitate differential diagnosis with other forms of cicatricial alopecia. Further studies are mandatory to differentiate other forms of scarring alopecia.

Line-field confocal optical coherence tomography, a novel non-invasive tool for the diagnosis of onychomycosis.

BACKGROUND AND OBJECTIVES: Onychomycosis is common and important to distinguish from other nail diseases. Rapid and accurate diagnosis is necessary for optimal patient treatment and outcome. Non-invasive diagnostic tools have increasing potential for nail diseases including onychomycosis. This study evaluated line-field confocal optical coherence tomography (LC-OCT) as a rapid non-invasive tool for diagnosing onychomycosis as compared to confocal laser scanning microscopy (CLSM), optical coherence tomography (OCT), and conventional methods. PATIENTS AND METHODS: In this prospective study 86 patients with clinically suspected onychomycosis and 14 controls were examined using LC-OCT, OCT, and CLSM. KOH-preparation, fungal culture, PCR, and histopathology were used as comparative conventional methods. RESULTS: LC-OCT had the highest sensitivity and negative predictive value of all methods used, closely followed by PCR and OCT. Specificity and positive predictive value of LC-OCT were as high as with CLSM, while OCT scored much lower. The gold standard technique, fungal culture, showed the lowest sensitivity and negative predictive value. Only PCR and culture allowed species differentiation. CONCLUSIONS: LC-OCT enables quick and non-invasive detection of onychomycosis, with advantages over CLSM and OCT, and similar diagnostic accuracy to PCR but lacking species differentiation. For accurate nail examination, LC-OCT requires well-trained and experienced operators.

The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk for several neurodegenerative diseases including Alzheimer's disease and frontotemporal dementia (FTD). Homozygous TREM2 missense mutations, such as p.T66M, lead to the FTD-like syndrome, but how they cause pathology is unknown. Using CRISPR/Cas9 genome editing, we generated a knock-in mouse model for the disease-associated Trem2 p.T66M mutation. Consistent with a loss-of-function mutation, we observe an intracellular accumulation of immature mutant Trem2 and reduced generation of soluble Trem2 similar to patients with the homozygous p.T66M mutation. Trem2 p.T66M knock-in mice show delayed resolution of inflammation upon in vivo lipopolysaccharide stimulation and cultured macrophages display significantly reduced phagocytic activity. Immunohistochemistry together with in vivo TSPO small animal positron emission tomography (µPET) demonstrates an age-dependent reduction in microglial activity. Surprisingly, perfusion magnetic resonance imaging and FDG-µPET imaging reveal a significant reduction in cerebral blood flow and brain glucose metabolism. Thus, we demonstrate that a TREM2 loss-of-function mutation causes brain-wide metabolic alterations pointing toward a possible function of microglia in regulating brain glucose metabolism.

Longitudinal TSPO expression in tau transgenic P301S mice predicts increased tau accumulation and deteriorated spatial learning.

BACKGROUND: P301S tau transgenic mice show age-dependent accumulation of neurofibrillary tangles in the brainstem, hippocampus, and neocortex, leading to neuronal loss and cognitive deterioration. However, there is hitherto only sparse documentation of the role of neuroinflammation in tau mouse models. Thus, we analyzed longitudinal microglial activation by small animal 18 kDa translocator protein positron-emission-tomography (TSPO µPET) imaging in vivo, in conjunction with terminal assessment of tau pathology, spatial learning, and cerebral glucose metabolism. METHODS: Transgenic P301S (n = 33) and wild-type (n = 18) female mice were imaged by 18F-GE-180 TSPO µPET at the ages of 1.9, 3.9, and 6.4 months. We conducted behavioral testing in the Morris water maze, 18F-fluordesoxyglucose (18F-FDG) µPET, and AT8 tau immunohistochemistry at 6.3-6.7 months. Terminal microglial immunohistochemistry served for validation of TSPO µPET results in vivo, applying target regions in the brainstem, cortex, cerebellum, and hippocampus. We compared the results with our historical data in amyloid-ß mouse models. RESULTS: TSPO expression in all target regions of P301S mice increased exponentially from 1.9 to 6.4 months, leading to significant differences in the contrasts with wild-type mice at 6.4 months (+ 11-23%, all p < 0.001), but the apparent microgliosis proceeded more slowly than in our experience in amyloid-ß mouse models. Spatial learning and glucose metabolism of AT8-positive P301S mice were significantly impaired at 6.3-6.5 months compared to the wild-type group. Longitudinal increases in TSPO expression predicted greater tau accumulation and lesser spatial learning performance at 6.3-6.7 months. CONCLUSIONS: Monitoring of TSPO expression as a surrogate of microglial activation in P301S tau transgenic mice by µPET indicates a delayed time course when compared to amyloid-ß mouse models. Detrimental associations of microglial activation with outcome parameters are opposite to earlier data in amyloid-ß mouse models. The contribution of microglial response to pathology accompanying amyloid-ß and tau over-expression merits further investigation.

Coupling between physiological TSPO expression in brain and myocardium allows stabilization of late-phase cerebral [18F]GE180 PET quantification.

OBJECTIVES: PET imaging of the 18 kDa translocator protein (TSPO), a biomarker of microglial activity, receives growing interest in clinical and preclinical applications of neuroinflammatory and neurodegenerative brain diseases. In globally affected brains, intra-cerebral pseudo reference regions are not feasible. Consequently, many brain-independent approaches have been attempted, including SUV analysis and normalization to muscle- or heart uptake, aiming to stabilize quantitative analysis. In this study, we systematically compared different image normalization methods for static late phase TSPO-PET imaging of rodent brain. METHODS: We first obtained gamma counter measurements for gold standard quantitation of [18F]GE180 uptake in brain of C57Bl/6 mice (N = 10) after PET, aiming to identify factors contributing significantly to the quantitative results. Subsequently, data from a large cohort of C57Bl/6 mice (N = 79) were compiled to precisely determine the weighted influence and variance attributable these factors by regression analysis. Scan-rescan variability and agreement with histology were used to validate the tested normalization methods in an Alzheimer's disease (AD) mouse model with pathologically increased TSPO expression (PS2APP; N = 24). Longitudinal data from AD model mice (N = 10) scanned at four different ages were used to challenge and validate the different normalization methods in a practical application. RESULTS: Gamma counter results revealed that injected dose, body weight and PET-measured radioactivity concentration in the ventral myocardium all significantly accounted for [18F]GE180 activity in the brain. Skeletal muscle activity had high test-retest variance in this PET only application and was therefore pursued no further. Regression analysis of the large scale evaluation showed that scaling to injected dose or SUV analysis accounted for little variance in brain activity (R2 < 0.5), but inclusion of myocardial activity together with injected dose and body weight in the regression model accounted for most of the variance in brain uptake (R2 = 0.94). Scan-rescan stability, correlation with histology and applicability for longitudinal examination in the disease model were also significantly improved by inclusion of myocadial uptake in the quantitative model. CONCLUSION: Cerebral and myocardial TSPO expression are highly coupled under physiological conditions. Myocardial uptake has great potential for stabilization of static late phase [18F]GE180 quantification in brain in the absence of a valid intra-cerebral pseudo-reference region.

Microglial response to increasing amyloid load saturates with aging: a longitudinal dual tracer in vivo µPET-study.

BACKGROUND: Causal associations between microglia activation and ß-amyloid (Aß) accumulation during the progression of Alzheimer's disease (AD) remain a matter of controversy. Therefore, we used longitudinal dual tracer in vivo small animal positron emission tomography (µPET) imaging to resolve the progression of the association between Aß deposition and microglial responses during aging of an Aß mouse model. METHODS: APP-SL70 mice (N = 17; baseline age 3.2-8.5 months) and age-matched C57Bl/6 controls (wildtype (wt)) were investigated longitudinally for 6 months using Aß (18F-florbetaben) and 18 kDa translocator protein (TSPO) µPET (18F-GE180). Changes in cortical binding were transformed to Z-scores relative to wt mice, and microglial activation relative to amyloidosis was defined as the Z-score difference (TSPO-Aß). Using 3D immunohistochemistry for activated microglia (Iba-1) and histology for fibrillary Aß (methoxy-X04), we measure microglial brain fraction relative to plaque size and the distance from plaque margins. RESULTS: Aß-PET binding increased exponentially as a function of age in APP-SL70 mice, whereas TSPO binding had an inverse U-shape growth function. Longitudinal Z-score differences declined with aging, suggesting that microglial response declined relative to increasing amyloidosis in aging APP-SL70 mice. Microglial brain volume fraction was inversely related to adjacent plaque size, while the proximity to Aß plaques increased with age. CONCLUSIONS: Microglial activity decreases relative to ongoing amyloidosis with aging in APP-SL70 mice. The plaque-associated microglial brain fraction saturated and correlated negatively with increasing plaque size with aging.

Ex Vivo Confocal Laser Scanning Microscopy in Rare Skin Diseases.

While ex vivo confocal laser scanning microscopy has previously demonstrated its utility in most common skin diseases, its use in the assessment of dermatological entities with lower incidence remains unexplored in most cases. We therefore aimed to evaluate the diagnostic efficacy of some rare skin tumors as well as a few inflammatory skin diseases, that have not yet been studied in ex vivo confocal laser scanning microscopy. A total of 50 tissue samples comprising 10 healthy controls, 10 basal cell carcinoma, 10 squamous cell carcinoma, and 20 rare skin conditions were imaged using the newest generation ex vivo confocal microscopy (Vivascope 2500 M-G4, Vivascope GmbH, Munich, Germany). Three blinded investigators were asked to identify characteristic features of rare skin disorders and distinguish them from more common skin diseases in the ex vivo confocal microscopy images. Our findings present the capability of ex vivo confocal microscopy to display distinctive morphologic patterns in common and rare skin diseases. As might be expected, we found a strong correlation between imaging experience and diagnostic accuracy. While the imaging inexperienced dermatohistopathologist reached 60% concordance, the imaging-trained dermatologist obtained 88% agreement with dermatohistopathology. The imaging-trained dermatohistopathologist achieved concordance up to 92% with gold-standard dermatohistopathology. This study highlights the potential of ex vivo confocal laser scanning microscopy as a promising adjunct to conventional dermatohistopathology for the early and precise identification of rare dermatological disorders.

Innovation in Actinic Keratosis Assessment: Artificial Intelligence-Based Approach to LC-OCT PRO Score Evaluation.

Actinic keratosis (AK) is a common skin cancer in situ that can progress to invasive SCC. Line-field confocal optical coherence tomography (LC-OCT) has emerged as a non-invasive imaging technique that can aid in diagnosis. Recently, machine-learning algorithms have been developed that can automatically assess the PRO score of AKs based on the dermo-epidermal junction's (DEJ's) protrusion on LC-OCT images. A dataset of 19.898 LC-OCT images from 80 histologically confirmed AK lesions was used to test the performance of a previous validated artificial intelligence (AI)-based LC-OCT assessment algorithm. AI-based PRO score assessment was compared to the imaging experts' visual score. Additionally, undulation of the DEJ, the number of protrusions detected within the image, and the maximum depth of the protrusions were computed. Our results show that AI-automated PRO grading is highly comparable to the visual score, with an agreement of 71.3% for the lesions evaluated. Furthermore, this AI-based assessment was significantly faster than the regular visual PRO score assessment. The results confirm our previous findings of the pilot study in a larger cohort that the AI-based grading of LC-OCT images is a reliable and fast tool to optimize the efficiency of visual PRO score grading. This technology has the potential to improve the accuracy and speed of AK diagnosis and may lead to better clinical outcomes for patients.

Chronic PPARγ Stimulation Shifts Amyloidosis to Higher Fibrillarity but Improves Cognition.

We undertook longitudinal ß-amyloid positron emission tomography (Aß-PET) imaging as a translational tool for monitoring of chronic treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone in Aß model mice. We thus tested the hypothesis this treatment would rescue from increases of the Aß-PET signal while promoting spatial learning and preservation of synaptic density. Here, we investigated longitudinally for 5 months PS2APP mice (N = 23; baseline age: 8 months) and App NL-G-F mice (N = 37; baseline age: 5 months) using Aß-PET. Groups of mice were treated with pioglitazone or vehicle during the follow-up interval. We tested spatial memory performance and confirmed terminal PET findings by immunohistochemical and biochemistry analyses. Surprisingly, Aß-PET and immunohistochemistry revealed a shift toward higher fibrillary composition of Aß-plaques during upon chronic pioglitazone treatment. Nonetheless, synaptic density and spatial learning were improved in transgenic mice with pioglitazone treatment, in association with the increased plaque fibrillarity. These translational data suggest that a shift toward higher plaque fibrillarity protects cognitive function and brain integrity. Increases in the Aß-PET signal upon immunomodulatory treatments targeting Aß aggregation can thus be protective.

Pre-therapeutic microglia activation and sex determine therapy effects of chronic immunomodulation.

Modulation of the innate immune system is emerging as a promising therapeutic strategy against Alzheimer's disease (AD). However, determinants of a beneficial therapeutic effect are ill-understood. Thus, we investigated the potential of 18 kDa translocator protein positron-emission-tomography (TSPO-PET) for assessment of microglial activation in mouse brain before and during chronic immunomodulation. Methods: Serial TSPO-PET was performed during five months of chronic microglia modulation by stimulation of the peroxisome proliferator-activated receptor (PPAR)-γ with pioglitazone in two different mouse models of AD (PS2APP, AppNL-G-F ). Using mixed statistical models on longitudinal TSPO-PET data, we tested for effects of therapy and sex on treatment response. We tested correlations of baseline with longitudinal measures of TSPO-PET, and correlations between PET results with spatial learning performance and ß-amyloid accumulation of individual mice. Immunohistochemistry was used to determine the molecular source of the TSPO-PET signal. Results: Pioglitazone-treated female PS2APP and AppNL-G-F mice showed attenuation of the longitudinal increases in TSPO-PET signal when compared to vehicle controls, whereas treated male AppNL-G-F mice showed the opposite effect. Baseline TSPO-PET strongly predicted changes in microglial activation in treated mice (R = -0.874, p < 0.0001) but not in vehicle controls (R = -0.356, p = 0.081). Reduced TSPO-PET signal upon pharmacological treatment was associated with better spatial learning despite higher fibrillar ß-amyloid accumulation. Immunohistochemistry confirmed activated microglia to be the source of the TSPO-PET signal (R = 0.952, p < 0.0001). Conclusion: TSPO-PET represents a sensitive biomarker for monitoring of immunomodulation and closely reflects activated microglia. Sex and pre-therapeutic assessment of baseline microglial activation predict individual immunomodulation effects and may serve for responder stratification.

Microglial activation states drive glucose uptake and FDG-PET alterations in neurodegenerative diseases.

2-Deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) is widely used to study cerebral glucose metabolism. Here, we investigated whether the FDG-PET signal is directly influenced by microglial glucose uptake in mouse models and patients with neurodegenerative diseases. Using a recently developed approach for cell sorting after FDG injection, we found that, at cellular resolution, microglia displayed higher glucose uptake than neurons and astrocytes. Alterations in microglial glucose uptake were responsible for both the FDG-PET signal decrease in Trem2-deficient mice and the FDG-PET signal increase in mouse models for amyloidosis. Thus, opposite microglial activation states determine the differential FDG uptake. Consistently, 12 patients with Alzheimer's disease and 21 patients with four-repeat tauopathies also exhibited a positive association between glucose uptake and microglial activity as determined by 18F-GE-180 18-kDa translocator protein PET (TSPO-PET) in preserved brain regions, indicating that the cerebral glucose uptake in humans is also strongly influenced by microglial activity. Our findings suggest that microglia activation states are responsible for FDG-PET signal alterations in patients with neurodegenerative diseases and mouse models for amyloidosis. Microglial activation states should therefore be considered when performing FDG-PET.

Asymmetry of Fibrillar Plaque Burden in Amyloid Mouse Models.

Asymmetries of amyloid-ß (Aß) burden are well known in Alzheimer disease (AD) but did not receive attention in Aß mouse models of Alzheimer disease. Therefore, we investigated Aß asymmetries in Aß mouse models examined by Aß small-animal PET and tested if such asymmetries have an association with microglial activation. Methods: We analyzed 523 cross-sectional Aß PET scans of 5 different Aß mouse models (APP/PS1, PS2APP, APP-SL70, AppNL-G-F , and APPswe) together with 136 18-kDa translocator protein (TSPO) PET scans for microglial activation. The asymmetry index (AI) was calculated between tracer uptake in both hemispheres. AIs of Aß PET were analyzed in correlation with TSPO PET AIs. Extrapolated required sample sizes were compared between analyses of single and combined hemispheres. Results: Relevant asymmetries of Aß deposition were identified in at least 30% of all investigated mice. There was a significant correlation between AIs of Aß PET and TSPO PET in 4 investigated Aß mouse models (APP/PS1: R = 0.593, P = 0.001; PS2APP: R = 0.485, P = 0.019; APP-SL70: R = 0.410, P = 0.037; AppNL-G-F : R = 0.385, P = 0.002). Asymmetry was associated with higher variance of tracer uptake in single hemispheres, leading to higher required sample sizes. Conclusion: Asymmetry of fibrillar plaque neuropathology occurs frequently in Aß mouse models and acts as a potential confounder in experimental designs. Concomitant asymmetry of microglial activation indicates a neuroinflammatory component to hemispheric predominance of fibrillary amyloidosis.

Expression of Translocator Protein and [18F]-GE180 Ligand Uptake in Multiple Sclerosis Animal Models.

Positron emission tomography (PET) ligands targeting the translocator protein (TSPO) represent promising tools to visualize neuroinflammation in multiple sclerosis (MS). Although it is known that TSPO is expressed in the outer mitochondria membrane, its cellular localization in the central nervous system under physiological and pathological conditions is not entirely clear. The purpose of this study was to assess the feasibility of utilizing PET imaging with the TSPO tracer, [18F]-GE180, to detect histopathological changes during experimental demyelination, and to determine which cell types express TSPO. C57BL/6 mice were fed with cuprizone for up to 5 weeks to induce demyelination. Groups of mice were investigated by [18F]-GE180 PET imaging at week 5. Recruitment of peripheral immune cells was triggered by combining cuprizone intoxication with MOG35⁻55 immunization (i.e., Cup/EAE). Immunofluorescence double-labelling and transgene mice were used to determine which cell types express TSPO. [18F]-GE180-PET reliably detected the cuprizone-induced pathology in various white and grey matter regions, including the corpus callosum, cortex, hippocampus, thalamus and caudoputamen. Cuprizone-induced demyelination was paralleled by an increase in TSPO expression, glia activation and axonal injury. Most of the microglia and around one-third of the astrocytes expressed TSPO. TSPO expression induction was more severe in the white matter corpus callosum compared to the grey matter cortex. Although mitochondria accumulate at sites of focal axonal injury, these mitochondria do not express TSPO. In Cup/EAE mice, both microglia and recruited monocytes contribute to the TSPO expressing cell populations. These findings support the notion that TSPO is a valuable marker for the in vivo visualization and quantification of neuropathological changes in the MS brain. The pathological substrate of an increase in TSPO-ligand binding might be diverse including microglia activation, peripheral monocyte recruitment, or astrocytosis, but not axonal injury.

Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism.

Microglia adopt numerous fates with homeostatic microglia (HM) and a microglial neurodegenerative phenotype (MGnD) representing two opposite ends. A number of variants in genes selectively expressed in microglia are associated with an increased risk for neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD). Among these genes are progranulin (GRN) and the triggering receptor expressed on myeloid cells 2 (TREM2). Both cause neurodegeneration by mechanisms involving loss of function. We have now isolated microglia from Grn-/- mice and compared their transcriptomes to those of Trem2-/-mice Surprisingly, while loss of Trem2 enhances the expression of genes associated with a homeostatic state, microglia derived from Grn-/- mice showed a reciprocal activation of the MGnD molecular signature and suppression of gene characteristic for HM The opposite mRNA expression profiles are associated with divergent functional phenotypes. Although loss of TREM2 and progranulin resulted in opposite activation states and functional phenotypes of microglia, FDG (fluoro-2-deoxy-d-glucose)-µPET of brain revealed reduced glucose metabolism in both conditions, suggesting that opposite microglial phenotypes result in similar wide spread brain dysfunction.

Longitudinal PET Monitoring of Amyloidosis and Microglial Activation in a Second-Generation Amyloid-ß Mouse Model.

Nonphysiologic overexpression of amyloid-ß (Aß) precursor protein in common transgenic Aß mouse models of Alzheimer disease likely hampers their translational potential. The novel AppNL-G-F mouse incorporates a mutated knock-in, potentially presenting an improved model of Alzheimer disease for Aß-targeting treatment trials. We aimed to establish serial small-animal PET of amyloidosis and neuroinflammation in AppNL-G-F mice as a tool for therapy monitoring. Methods:AppNL-G-F mice (20 homozygous and 21 heterogeneous) and 12 age-matched wild-type mice were investigated longitudinally from 2.5 to 10 mo of age with 18F-florbetaben Aß PET and 18F-GE-180 18-kDa translocator protein (TSPO) PET. Voxelwise analysis of SUV ratio images was performed using statistical parametric mapping. All mice underwent a Morris water maze test of spatial learning after their final scan. Quantification of fibrillar Aß and activated microglia by immunohistochemistry and biochemistry served for validation of the PET results. Results: The periaqueductal gray emerged as a suitable pseudo reference tissue for both tracers. Homozygous AppNL-G-F mice had a rising SUV ratio in cortex and hippocampus for Aß (+9.1%, +3.8%) and TSPO (+19.8%, +14.2%) PET from 2.5 to 10 mo of age (all P < 0.05), whereas heterozygous AppNL-G-F mice did not show significant changes with age. Significant voxelwise clusters of Aß deposition and microglial activation in homozygous mice appeared at 5 mo of age. Immunohistochemical and biochemical findings correlated strongly with the PET data. Water maze escape latency was significantly elevated in homozygous AppNL-G-F mice compared with wild-type at 10 mo of age and was associated with high TSPO binding. Conclusion: Longitudinal PET in AppNL-G-F knock-in mice enables monitoring of amyloidogenesis and neuroinflammation in homozygous mice but is insensitive to minor changes in heterozygous animals. The combination of PET with behavioral tasks in AppNL-G-F treatment trials is poised to provide important insights in preclinical drug development.