Presynaptic density determined by SV2A PET is closely associated with postsynaptic metabotropic glutamate receptor 5 availability and independent of amyloid pathology in early cognitive impairment.

INTRODUCTION: Metabotropic glutamate receptor 5 (mGluR5) is involved in regulating integrative brain function and synaptic transmission. Aberrant mGluR5 signaling and relevant synaptic failure play a key role in the pathophysiological mechanism of Alzheimer's disease (AD). METHODS: Ten cognitively impaired (CI) individuals and 10 healthy controls (HCs) underwent [18F]SynVesT-1 and [18F]PSS232 positron emission tomography (PET)/magnetic resonance to assess synaptic density and mGluR5 availability. The associations between mGluR5 availability and synaptic density were examined. A mediation analysis was performed to investigate the possible mediating effects of mGluR5 availability and synaptic loss on the relationship between amyloid deposition and cognition. RESULTS: CI patients exhibited lower mGluR5 availability and synaptic density in the medial temporal lobe than HCs. Regional synaptic density was closely associated with regional mGluR5 availability. mGluR5 availability and synaptic loss partially mediated the relationship between amyloid deposition and cognition. CONCLUSIONS: Reductions in mGluR5 availability and synaptic density exhibit similar spatial patterns in AD and are closely linked. HIGHLIGHTS: Cognitively impaired patients exhibited lower mGluR5 availability and synaptic density in the medial temporal lobe than HCs. Reductions in mGluR5 availability and synaptic density exhibit similar spatial patterns in AD. Regional synaptic density was closely associated with regional mGluR5 availability. mGluR5 availability and synaptic loss partially mediated the relationship between amyloid deposition and global cognition. With further research, modulating mGluR5 availability might be a potential therapeutic strategy for improving synaptic function in AD.

Relationship Between Reactive Astrocytes, by [18F]SMBT-1 Imaging, with Amyloid-Beta, Tau, Glucose Metabolism, and TSPO in Mouse Models of Alzheimer's Disease.

Reactive astrocytes play an important role in the development of Alzheimer's disease (AD). Here, we aimed to investigate the temporospatial relationships among monoamine oxidase-B, tau and amyloid-ß (Aß), translocator protein, and glucose metabolism by using multitracer imaging in AD transgenic mouse models. Positron emission tomography (PET) imaging with [18F]SMBT-1 (monoamine oxidase-B), [18F]florbetapir (Aß), [18F]PM-PBB3 (tau), [18F]fluorodeoxyglucose (FDG), and [18F]DPA-714 (translocator protein) was carried out in 5- and 10-month-old APP/PS1, 11-month-old 3×Tg mice, and aged-matched wild-type mice. The brain regional referenced standard uptake value (SUVR) was computed with the cerebellum as the reference region. Immunofluorescence staining was performed on mouse brain tissue slices. [18F]SMBT-1 and [18F]florbetapir SUVRs were greater in the cortex and hippocampus of 10-month-old APP/PS1 mice than in those of 5-month-old APP/PS1 mice and wild-type mice. No significant difference in the regional [18F]FDG or [18F]DPA-714 SUVRs was observed in the brains of 5- or 10-month-old APP/PS1 mice or wild-type mice. No significant difference in the SUVRs of any tracer was observed between 11-month-old 3×Tg mice and age-matched wild-type mice. A positive correlation between the SUVRs of [18F]florbetapir and [18F]DPA-714 in the cortex and hippocampus was observed among the transgenic mice. Immunostaining validated the distribution of MAO-B and limited Aß and tau pathology in 11-month-old 3×Tg mice; and Aß deposits in brain tissue from 10-month-old APP/PS1 mice. In summary, these findings provide in vivo evidence that an increase in astrocyte [18F]SMBT-1 accompanies Aß accumulation in APP/PS1 models of AD amyloidosis.

Noninvasive Visualization of Amyloid-Beta Deposits in Alzheimer's Amyloidosis Mice via Fluorescence Molecular Tomography Using Contrast Agent.

Alzheimer's disease is pathologically featured by the accumulation of amyloid-beta (Aß) plaque and neurofibrillary tangles. Compared to small animal positron emission tomography, optical imaging features nonionizing radiation, low cost, and logistic convenience. Optical detection of Aß deposits is typically implemented by 2D macroscopic imaging and various microscopic techniques assisted with Aß-targeted contrast agents. Here, we introduce fluorescence molecular tomography (FMT), a macroscopic 3D fluorescence imaging technique, convenient for in vivo longitudinal monitoring of the animal brain without the involvement of cranial window opening operation. This chapter aims to provide the protocols for FMT in vivo imaging of Aß deposits in the brain of rodent model of Alzheimer's disease. The materials, stepwise method, notes, limitations of FMT, and emerging opportunities for FMT techniques are presented.

Reduced SV2A and GABAA receptor levels in the brains of type 2 diabetic rats revealed by [18F]SDM-8 and [18F]flumazenil PET.

PURPOSE: Type 2 diabetes mellitus (T2DM) is associated with a greater risk of Alzheimer's disease. Synaptic impairment and protein aggregates have been reported in the brains of T2DM models. Here, we assessed whether neurodegenerative changes in synaptic vesicle 2 A (SV2A), γ-aminobutyric acid type A (GABAA) receptor, amyloid-ß, tau and receptor for advanced glycosylation end product (RAGE) can be detected in vivo in T2DM rats. METHODS: Positron emission tomography (PET) using [18F]SDM-8 (SV2A), [18F]flumazenil (GABAA receptor), [18F]florbetapir (amyloid-ß), [18F]PM-PBB3 (tau), and [18F]FPS-ZM1 (RAGE) was carried out in 12-month-old diabetic Zucker diabetic fatty (ZDF) and SpragueDawley (SD) rats. Immunofluorescence staining, Thioflavin S staining, proteomic profiling and pathway analysis were performed on the brain tissues of ZDF and SD rats. RESULTS: Reduced cortical [18F]SDM-8 uptake and cortical and hippocampal [18F]flumazenil uptake were observed in 12-month-old ZDF rats compared to SD rats. The regional uptake of [18F]florbetapir and [18F]PM-PBB3 was comparable in the brains of 12-month-old ZDF and SD rats. Immunofluorescence staining revealed Thioflavin S-negative, phospho-tau-positive inclusions in the cortex and hypothalamus in the brains of ZDF rats and the absence of amyloid-beta deposits. The level of GABAA receptors was lower in the cortex of ZDF rats than SD rats. Proteomic analysis further demonstrated that, compared with SD rats, synaptic-related proteins and pathways were downregulated in the hippocampus of ZDF rats. CONCLUSION: These findings provide in vivo evidence for regional reductions in SV2A and GABAA receptor levels in the brains of aged T2DM ZDF rats.

NRF2 Deficiency Promotes Ferroptosis of Astrocytes Mediated by Oxidative Stress in Alzheimer's Disease.

Oxidative stress is involved in the pathogenesis of Alzheimer's disease (AD), which is linked to reactive oxygen species (ROS), lipid peroxidation, and neurotoxicity. Emerging evidence suggests a role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a major source of antioxidant response elements in AD. The molecular mechanism of oxidative stress and ferroptosis in astrocytes in AD is not yet fully understood. Here, we aimed to investigate the mechanism by which Nrf2 regulates the ferroptosis of astrocytes in AD. We found decreased expression of Nrf2 and upregulated expression of the ROS marker NADPH oxidase 4 (NOX4) in the frontal cortex from patients with AD and in the cortex of 3×Tg mice compared to wildtype mice. We demonstrated that Nrf2 deficiency led to ferroptosis-dependent oxidative stress-induced ROS with downregulated heme oxygenase-1 and glutathione peroxidase 4 and upregulated cystine glutamate expression. Moreover, Nrf2 deficiency increased lipid peroxidation, DNA oxidation, and mitochondrial fragmentation in mouse astrocytes (mAS, M1800-57). In conclusion, these results suggest that Nrf2 deficiency promotes ferroptosis of astrocytes involving oxidative stress in AD.

Probing Chemical Complexity of Amyloid Plaques in Alzheimer's Disease Mice using Hyperspectral Raman Imaging.

One of the distinctive pathological features of Alzheimer's disease (AD) is the deposition of amyloid plaques within the brain of affected individuals. These plaques have traditionally been investigated using labeling techniques such as immunohistochemical imaging. However, the use of labeling can disrupt the structural integrity of the molecules being analyzed. Hence, it is imperative to employ label-free imaging methods for noninvasive examination of amyloid deposits in their native form, thereby providing more relevant information pertaining to AD. This study presents compelling evidence that label-free and nondestructive confocal Raman imaging is a highly effective approach for the identification and chemical characterization of amyloid plaques within cortical regions of an arcAß mouse model of AD. Furthermore, this investigation elucidates how the spatial correlation of Raman signals can be exploited to identify robust Raman marker bands and discern proteins and lipids from amyloid plaques. Finally, this study uncovers the existence of distinct types of amyloid plaques in the arcAß mouse brain, exhibiting significant disparities in terms of not only shape and size but also molecular composition.

Imaging increased metabolism in the spinal cord in mice after middle cerebral artery occlusion.

Emerging evidence indicates crosstalk between the brain and hematopoietic system following cerebral ischemia. Here, we investigated metabolism and oxygenation in the spleen and spinal cord in a transient middle cerebral artery occlusion (tMCAO) model. Sham-operated and tMCAO mice underwent [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) to assess glucose metabolism. Naïve, sham-operated and tMCAO mice underwent multispectral optoacoustic tomography (MSOT) assisted by quantitative model-based reconstruction and unmixing algorithms for accurate mapping of oxygenation patterns in peripheral tissues at 24 h after reperfusion. We found increased [18F]FDG uptake and reduced MSOT oxygen saturation, indicating hypoxia in the thoracic spinal cord of tMCAO mice compared with sham-operated mice but not in the spleen. Reduced spleen size was observed in tMCAO mice compared with sham-operated mice ex vivo. tMCAO led to an increase in the numbers of mature T cells in femoral bone marrow tissues, concomitant with a stark reduction in these cell subsets in the spleen and peripheral blood. The combination of quantitative PET and MSOT thus enabled observation of hypoxia and increased metabolic activity in the spinal cord of tMCAO mice at 24 h after occlusion compared to sham-operated mice.

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo.

Background: Abnormal alpha-synuclein and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim at visualizing alpha-synuclein inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. Methods: Fluorescently labelled pyrimidoindole-derivative THK-565 was characterized by using recombinant fibrils and brains from 10-11 months old M83 mice, which subsequently underwent in vivo concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging. The in vivo results were verified against structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 Tesla and scanning transmission X-ray microscopy (STXM) of perfused brains. Brain slice immunofluorescence and Prussian blue staining were further performed to validate the detection of alpha-synuclein inclusions and iron deposition in the brain, respectively. Results: THK-565 showed increased fluorescence upon binding to recombinant alpha-synuclein fibrils and alpha-synuclein inclusions in post-mortem brain slices from patients with Parkinson's disease and M83 mice. i.v. administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 minutes post-injection by wide-field fluorescence compared to non-transgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. Conclusion: We demonstrated in vivo mapping of alpha-synuclein by means of non-invasive epifluorescence and vMSOT imaging assisted with a targeted THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

Inorganic phosphate exporter heterozygosity in mice leads to brain vascular calcification, microangiopathy, and microgliosis.

Calcification of the cerebral microvessels in the basal ganglia in the absence of systemic calcium and phosphate imbalance is a hallmark of primary familial brain calcification (PFBC), a rare neurodegenerative disorder. Mutation in genes encoding for sodium-dependent phosphate transporter 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), platelet-derived growth factor B (PDGFB), platelet-derived growth factor receptor beta (PDGFRB), myogenesis regulating glycosidase (MYORG), and junctional adhesion molecule 2 (JAM2) are known to cause PFBC. Loss-of-function mutations in XPR1, the only known inorganic phosphate exporter in metazoans, causing dominantly inherited PFBC was first reported in 2015 but until now no studies in the brain have addressed whether loss of one functional allele leads to pathological alterations in mice, a commonly used organism to model human diseases. Here we show that mice heterozygous for Xpr1 (Xpr1WT/lacZ ) present with reduced inorganic phosphate levels in the cerebrospinal fluid and age- and sex-dependent growth of vascular calcifications in the thalamus. Vascular calcifications are surrounded by vascular basement membrane and are located at arterioles in the smooth muscle layer. Similar to previously characterized PFBC mouse models, vascular calcifications in Xpr1WT/lacZ mice contain bone matrix proteins and are surrounded by reactive astrocytes and microglia. However, microglial activation is not confined to calcified vessels but shows a widespread presence. In addition to vascular calcifications, we observed vessel tortuosity and transmission electron microscopy analysis revealed microangiopathy-endothelial swelling, phenotypic alterations in vascular smooth muscle cells, and thickening of the basement membrane.

Deep optoacoustic localization microangiography of ischemic stroke in mice.

Super-resolution optoacoustic imaging of microvascular structures deep in mammalian tissues has so far been impeded by strong absorption from densely-packed red blood cells. Here we devised 5 µm biocompatible dichloromethane-based microdroplets exhibiting several orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus enabling single-particle detection in vivo. We demonstrate non-invasive three-dimensional microangiography of the mouse brain beyond the acoustic diffraction limit (<20 µm resolution). Blood flow velocity quantification in microvascular networks and light fluence mapping was also accomplished. In mice affected by acute ischemic stroke, the multi-parametric multi-scale observations enabled by super-resolution and spectroscopic optoacoustic imaging revealed significant differences in microvascular density, flow and oxygen saturation in ipsi- and contra-lateral brain hemispheres. Given the sensitivity of optoacoustics to functional, metabolic and molecular events in living tissues, the new approach paves the way for non-invasive microscopic observations with unrivaled resolution, contrast and speed.

Evaluation of novel anti-CEACAM6 antibody-based conjugates for radioimmunotheranostics of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant solid tumor that lacks early diagnostic methods. Recently, targeted immunotherapy and radiotherapy have been integrated with radionuclide-antibody conjugate drugs, which can be used for targeted diagnosis and dynamic imaging of tumors. CEACAM6 is overexpressed in pancreatic tumors and is a potential theranostic target for PDAC. We aimed to develop a novel targeted carrier for theranostics of PDAC and other solid tumors. METHODS: Based on camelid heavy-chain-only antibodies, we developed a CEACAM6-targeting recombinant antibody NY004, and evaluated it as a novel antibody-carrier for imaging and therapy of cancer in tumor models. We labeled NY004 with theranostic nuclides and applied this self-developed antibody platform in diagnostic imaging and antitumor assessment in PDAC models. RESULTS: Through microPET, IHC, and biodistribution assays, targeting and biodistribution of [89Zr]-NY004 in solid tumors including PDAC was examined, and the investigated tumors were all CEACAM6-positive malignancies. We found that NY004 was suitable for use as a drug carrier for radioimmunotheranostics. Our study showed that NY004 was characterized by high targeted uptake and a long retention time in PANC-1 tumors (up to 6 days post-injection), with good specificity and high imaging efficiency. Therapeutic evaluation of the radionuclide-labeled antibody drug [177Lu]-NY004 in PDAC tumor-bearing model revealed that NY004 had high and prolonged uptake in tumors, relatively low non-target organ uptake, and good anti-tumor efficacy. CONCLUSION: As a drug platform for radiotheranostics, CEACAM6-specific antibody NY004 met the requirements of easy-labeling, targeting specificity, and effective persistence in pancreatic adenocarcinoma tissues. KEY POINTS: • [89Zr]-NY004 has good specificity and high imaging efficiency, and is characterized by high tumor-targeting uptake and a long tumor retention time as a PET molecular imaging tracer. • Therapeutic radionuclide-conjugated antibody drug [177Lu]-NY004 has high uptake and prolonged uptake duration in tumors, low non-target organ uptake, and significant tumor-inhibiting efficacy in PDAC model. • The self-developed antibody structure NY004 is a promising drug platform for radioimmunotheranostics of CEACAM6-positive tumors including pancreatic ductal adenocarcinoma.

Monitoring mouse brain perfusion with hybrid magnetic resonance optoacoustic tomography.

Progress in brain research critically depends on the development of next-generation multi-modal imaging tools capable of capturing transient functional events and multiplexed contrasts noninvasively and concurrently, thus enabling a holistic view of dynamic events in vivo. Here we report on a hybrid magnetic resonance and optoacoustic tomography (MROT) system for murine brain imaging, which incorporates an MR-compatible spherical matrix array transducer and fiber-based light illumination into a 9.4 T small animal scanner. An optimized radiofrequency coil has further been devised for whole-brain interrogation. System's utility is showcased by acquiring complementary angiographic and soft tissue anatomical contrast along with simultaneous dual-modality visualization of contrast agent dynamics in vivo.

Aquaporin 4 is differentially increased and dislocated in association with tau and amyloid-beta.

AIMS: Neurovascular-glymphatic dysfunction plays an important role in Alzheimer's disease and has been analysed mainly in relation to amyloid-beta (Aß) pathology. Here, we aim to investigate the neurovascular alterations and mapping of aquaporin 4 (AQP4) distribution and dislocation associated with tau and Aß. MATERIALS AND METHODS: Perfusion, susceptibility weighted imaging and structural magnetic resonance imaging (MRI) were performed in the pR5 mouse model of 4-repeat tau and the arcAß mouse model of amyloidosis. Immunofluorescence staining was performed using antibodies against AQP4, vessel, astroglia, microglia, phospho-tau and Aß in brain tissue slices from pR5, arcAß and non-transgenic mice. KEY FINDINGS: pR5 mice showed regional atrophy, preserved cerebral blood flow, and reduced cerebral vessel density compared to non-transgenic mice, while arcAß mice showed cerebral microbleeds and reduced cerebral vessel density. AQP4 dislocation and peri-tau enrichment in the hippocampus and increased AQP4 levels in the cortex and hippocampus were detected in pR5 mice compared to non-transgenic mice. In comparison, cortical AQP4 dislocation and cortical/hippocampal peri-plaque increases were observed in arcAß mice. Increased expression of reactive astrocytes were detected around the tau inclusions in pR5 mice and Aß plaques in arcAß mice. SIGNIFICANCE: We demonstrated the neurovascular alterations, microgliosis, astrogliosis and increased AQP4 regional expression in pR5 tau and arcAß mice. We observed a divergent region-specific AQP4 dislocation and association with phospho-tau and Aß pathologies.

Coregistered transcranial optoacoustic and magnetic resonance angiography of the human brain.

Imaging modalities capable of visualizing the human brain have led to major advances in neurology and brain research. Multi-spectral optoacoustic tomography (MSOT) has gained importance for studying cerebral function in rodent models due to its unique capability to map changes in multiple hemodynamic parameters and to directly visualize neural activity within the brain. The technique further provides molecular imaging capabilities that can facilitate early disease diagnosis and treatment monitoring. However, transcranial imaging of the human brain is hampered by acoustic attenuation and other distortions introduced by the skull. Here, we demonstrate non-invasive transcranial MSOT angiography of pial veins through the temporal bone of an adult healthy volunteer. Time-of-flight (TOF) magnetic resonance angiography (MRA) and T1-weighted structural magnetic resonance imaging (MRI) were further acquired to facilitate anatomical registration and interpretation. The superior middle cerebral vein in the temporal cortex was identified in the MSOT images, matching its location observed in the TOF-MRA images. These initial results pave the way toward the application of MSOT in clinical brain imaging.

Automatic analysis of skull thickness, scalp-to-cortex distance and association with age and sex in cognitively normal elderly.

Personalized neurostimulation has been a potential treatment for many brain diseases, which requires insights into brain/skull geometry. Here, we developed an open source efficient pipeline BrainCalculator for automatically computing the skull thickness map, scalp-to-cortex distance (SCD), and brain volume based on T 1 -weighted magnetic resonance imaging (MRI) data. We examined the influence of age and sex cross-sectionally in 407 cognitively normal older adults (71.9±8.0 years, 60.2% female) from the ADNI. We demonstrated the compatibility of our pipeline with commonly used preprocessing packages and found that BrainSuite Skullfinder was better suited for such automatic analysis compared to FSL Brain Extraction Tool 2 and SPM12- based unified segmentation using ground truth. We found that the sphenoid bone and temporal bone were thinnest among the skull regions in both females and males. There was no increase in regional minimum skull thickness with age except in the female sphenoid bone. No sex difference in minimum skull thickness or SCD was observed. Positive correlations between age and SCD were observed, faster in females (0.307%/y) than males (0.216%/y) in temporal SCD. A negative correlation was observed between age and whole brain volume computed based on brain surface (females -1.031%/y, males -0.998%/y). In conclusion, we developed an automatic pipeline for MR-based skull thickness map, SCD, and brain volume analysis and demonstrated the sex-dependent association between minimum regional skull thickness, SCD and brain volume with age. This pipeline might be useful for personalized neurostimulation planning.

PET imaging in animal models of Parkinson's disease.

Alpha-synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, are characterized by aberrant accumulation of alpha-synuclein and synaptic dysfunction leading to motor and cognitive deficits. Animal models of alpha-synucleinopathy have greatly facilitated the mechanistic understanding of the disease and the development of therapeutics. Various transgenic, alpha-synuclein fibril-injected, and toxin-injected animal models of Parkinson's disease and multiple system atrophy that recapitulate the disease pathology have been developed and widely used. Recent advances in positron emission tomography have allowed the noninvasive visualization of molecular alterations, underpinning behavioral dysfunctions in the brains of animal models and the longitudinal monitoring of treatment effects. Imaging studies in these disease animal models have employed multi-tracer PET designs to reveal dopaminergic deficits together with other molecular alterations. This review focuses on the development of new positron emission tomography tracers and studies of alpha-synuclein, synaptic vesicle glycoprotein 2A neurotransmitter receptor deficits such as dopaminergic receptor, dopaminergic transporter, serotonergic receptor, vesicular monoamine transporter 2, hypometabolism, neuroinflammation, mitochondrial dysfunction and leucine rich repeat kinase 2 in animal models of Parkinson's disease. The outstanding challenges and emerging applications are outlined, such as investigating the gut-brain-axis by using positron emission tomography in animal models, and provide a future outlook.

Simultaneous Functional Magnetic Resonance and Optoacoustic Imaging of Brain-Wide Sensory Responses in Mice.

Functional magnetic resonance imaging (fMRI) has massively contributed to the understanding of mammalian brain function. However, the origin and interpretation of the blood oxygen level-dependent (BOLD) signals retrieved by fMRI remain highly disputed. This article reports on the development of a fully hybridized system enabling concurrent functional magnetic resonance optoacoustic tomography (MROT) measurements of stimulus-evoked brain-wide sensory responses in mice. The highly complementary angiographic and soft tissue contrasts of both modalities along with simultaneous multi-parametric readings of stimulus-evoked hemodynamic responses are leveraged in order to establish unequivocal links between the various counteracting physiological and metabolic processes in the brain. The results indicate that the BOLD signals are highly correlated, both spatially and temporally, with the total hemoglobin readings resolved with volumetric multi-spectral optoacoustic tomography. Furthermore, the differential oxygenated and deoxygenated hemoglobin optoacoustic readings exhibit superior sensitivity as compared to the BOLD signals when detecting stimulus-evoked hemodynamic responses. The fully hybridized MROT approach greatly expands the neuroimaging toolset to comprehensively study neurovascular and neurometabolic coupling mechanisms and related diseases.