Effects of isoflurane anaesthesia depth and duration on renal function measured with [99mTc]Tc-mercaptoacetyltriglycine SPECT in mice.

BACKGROUND: The influence of anaesthetic depth and the potential influence of different anaesthetic beds and thus different handling procedures were investigated in 86 severe combined immunodeficient (SCID) mice using semi-stationary dynamic single photon emission computed tomography (SPECT) for kidney scintigraphy. Therefore, isoflurane concentrations were adjusted using respiratory rate for low (80-90 breath/min) and deep anaesthesia (40-45 breath/min). At low anaesthesia, we additionally tested the influence of single bed versus 3-mouse bed hotel; the hotel mice were anaesthetized consecutively at ~ 30, 20, and 10 min before tracer injections for positions 1, 2, and 3, respectively. Intravenous [99mTc]Tc-MAG3 injection of ~ 28 MBq was performed after SPECT start. Time-activity curves were used to calculate time-to-peak (Tmax), T50 (50% clearance) and T25 (75% clearance). RESULTS: Low and deep anaesthesia corresponded to median isoflurane concentrations of 1.3% and 1.5%, respectively, with no significant differences in heart rate (p = 0.74). Low anaesthesia resulted in shorter aortic blood clearance half-life (p = 0.091) and increased relative renal tracer influx rate (p = 0.018). A tendency toward earlier Tmax occurred under low anaesthesia (p = 0.063) with no differences in T50 (p = 0.40) and T25 (p = 0.24). Variance increased with deep anaesthesia. Compared to single mouse scans, hotel mice in position 1 showed a delayed Tmax, T50, and T25 (p < 0.05 each). Furthermore, hotel mice in position 1 showed delayed Tmax versus position 3, and delayed T50 and T25 versus position 2 and 3 (p < 0.05 each). No difference occurred between single bed and positions 2 (p = 1.0) and 3 (p = 1.0). CONCLUSIONS: Deep anaesthesia and prolonged low anaesthesia should be avoided during renal scintigraphy because they result in prolonged blood clearance half-life, delayed renal influx and/or later Tmax. Vice versa, low anaesthesia with high respiratory rates of 80-90 rpm and short duration (≤ 20 min) should be preferred to obtain representative data with low variance.

Chronic exposure to a synthetic cannabinoid alters cerebral brain metabolism and causes long-lasting behavioral deficits in adult mice.

In recent years, there has been growing evidence that cannabinoids have promising medicinal and pharmacological effects. However, the growing interest in medical cannabis highlights the need to better understand brain alterations linking phytocannabinoids or synthetic cannabinoids to clinical and behavioral phenotypes. Therefore, the aim of this study was to investigate the effects of long-term WIN 55,212-2 treatment-with and without prolonged abstinence-on cerebral metabolism and memory function in healthy wildtype mice. Adult C57BI/6J mice were divided into two treatment groups to study the acute effects of WIN 55,212-2 treatment as well the effects of WIN 55,212-2 treatment after an extended washout phase. We could demonstrate that 3 mg/kg WIN 55,212-2 treatment in early adulthood leads to a hypometabolism in several brain regions including the hippocampus, cerebellum, amygdala and midbrain, even after prolonged abstinence. Furthermore, prolonged acute WIN 55,212-2 treatment in 6-months-old mice reduced the glucose metabolism in the hippocampus and midbrain. In addition, Win 55,212-2 treatment during adulthood lead to spatial memory and recognition memory deficits without affecting anxiety behavior. Overall we could demonstrate that treatment with the synthetic CB1/CB2 receptor aganist Win 55,212-2 during adulthood causes persistent memory deficits, especially when mice were treated in early adulthood. Our findings highlight the risks of prolonged WIN 55,212-2 use and provide new insights into the mechanisms underlying the effects of chronic cannabinoid exposure on the brain and behavior.

Monitoring orthodontic tooth movement in rats after piezocision by bone scintigraphy.

AIM: Piezocision, corticocision of mineralized tissue by ultrasound showed promising results in accelerating tooth movement induced by orthodontic appliances although the biologic effects of this procedure are not well-understood so far. The aim of this study was to investigate the impact of piezocision on bone remodeling in rats by bone SPECT imaging. MATERIAL AND METHODS: Ten male Wistar rats underwent surgical placement of orthodontic appliances on each side of the maxilla followed by piezocision on one side only. Each rat underwent 99mTc-MDP bone SPECT/CT imaging before surgery (T0), and 2 (T1) and 4 weeks (T2) after surgery. Bone uptake is expressed as median [IQR] min-max in percentage of the injected activity per ml computed from the 10 voxels with the highest uptake (%IAmax10/ml). RESULTS: Pooled data regardless of the piezocision showed a significant increase in bone uptake from T0 (3.2 [2.8-3.9] 2.6-4.9) to T1 (4.4 [3.8-4.6] 3.4-4.8; p = 0.001). Thereafter, the uptake decreased to T2 (3.8 [3.1-4.4] 2.8-4.8; p = 0.116). No significant differences in bone uptake were found between the maxilla sides without and with piezocision: T1: without (4.3 [3.8-4.5] 3.4-4.8) vs. with (4.5 [3.7-4.6] 3.5-4.7; p=0.285), T2: without (4.0 [3.1-4.5] 2.8-4.8) vs. with (3.7 [3.0-4.4] 2.8-4.8; p=0.062). CONCLUSION: 99mTc-MDP bone SPECT imaging in rats was able to reproduce changes in bone uptake in the maxilla after placement of orthodontic appliances inducing measurable tooth movement. An additional effect of piezocision on bone remodeling in terms of bone uptake was not detectable which is probably due to the pronounced and significant effects induced by the orthodontic appliances per se, which may mask the potential effects of additional piezocision.

Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy.

PURPOSE: Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies. METHODS: FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule-based FAP-targeting agent. RESULTS: Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of 68Ga-FAP-2286, 111In-FAP-2286, and 177Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. 177Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46. CONCLUSION: In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of 68Ga-FAP-2286 for imaging and 177Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.

Search strategy analysis of Tg4-42 Alzheimer Mice in the Morris Water Maze reveals early spatial navigation deficits.

Spatial disorientation is one of the earliest symptoms in Alzheimer's disease and allocentric deficits can already be detected in the asymptomatic preclinical stages of the disease. The Morris Water Maze (MWM) is used to study spatial learning in rodent models. Here we investigated the spatial memory of female 3, 7 and 12 month-old Alzheimer Tg4-42 mice in comparison to wild-type control animals. Conventional behavior analysis of escape latencies and quadrant preference revealed spatial memory and reference memory deficits in female 7 and 12 month-old Tg4-42 mice. In contrast, conventional analysis of the MWM indicated an intact spatial memory in 3 month-old Tg4-42 mice. However, a detailed analysis of the swimming strategies demonstrated allocentric-specific memory deficits in 3 month-old Tg4-42 mice before the onset of severe memory deficits. Furthermore, we could show that the spatial reference memory deficits in aged Tg4-42 animals are caused by the lack of allocentric and spatial strategies. Analyzing search strategies in the MWM allows to differentiate between hippocampus-dependent allocentric and hippocampus-independent egocentric search strategies. The spatial navigation impairments in young Tg4-42 mice are well in line with the hypometabolism and synaptic deficits in the hippocampus. Therefore, analyzing search strategies in the Tg4-42 model can be a powerful tool for preclinical drug testing and identifying early therapeutic successes.

Discovery of a novel pseudo ß-hairpin structure of N-truncated amyloid-ß for use as a vaccine against Alzheimer's disease.

One of the hallmarks of Alzheimer's disease (AD) are deposits of amyloid-beta (Aß) protein in amyloid plaques in the brain. The Aß peptide exists in several forms, including full-length Aß1-42 and Aß1-40 - and the N-truncated species, pyroglutamate Aß3-42 and Aß4-42, which appear to play a major role in neurodegeneration. We previously identified a murine antibody (TAP01), which binds specifically to soluble, non-plaque N-truncated Aß species. By solving crystal structures for TAP01 family antibodies bound to pyroglutamate Aß3-14, we identified a novel pseudo ß-hairpin structure in the N-terminal region of Aß and show that this underpins its unique binding properties. We engineered a stabilised cyclic form of Aß1-14 (N-Truncated Amyloid Peptide AntibodieS; the 'TAPAS' vaccine) and showed that this adopts the same 3-dimensional conformation as the native sequence when bound to TAP01. Active immunisation of two mouse models of AD with the TAPAS vaccine led to a striking reduction in amyloid-plaque formation, a rescue of brain glucose metabolism, a stabilisation in neuron loss, and a rescue of memory deficiencies. Treating both models with the humanised version of the TAP01 antibody had similar positive effects. Here we report the discovery of a unique conformational epitope in the N-terminal region of Aß, which offers new routes for active and passive immunisation against AD.

Quantitative Brain Positron Emission Tomography in Female 5XFAD Alzheimer Mice: Pathological Features and Sex-Specific Alterations.

Successful back-translating clinical biomarkers and molecular imaging methods of Alzheimer's disease (AD), including positron emission tomography (PET), are very valuable for the evaluation of new therapeutic strategies and increase the quality of preclinical studies. 18F-Fluorodeoxyglucose (FDG)-PET and 18F-Florbetaben-PET are clinically established biomarkers capturing two key pathological features of AD. However, the suitability of 18F-FDG- and amyloid-PET in the widely used 5XFAD mouse model of AD is still unclear. Furthermore, only data on male 5XFAD mice have been published so far, whereas studies in female mice and possible sex differences in 18F-FDG and 18F-Florbetaben uptake are missing. The aim of this study was to evaluate the suitability of 18F-FDG- and 18F-Florbetaben-PET in 7-month-old female 5XFAD and to assess possible sex differences between male and female 5XFAD mice. We could demonstrate that female 5XFAD mice showed a significant reduction in brain glucose metabolism and increased cerebral amyloid deposition compared with wild type animals, in accordance with the pathology seen in AD patients. Furthermore, we showed for the first time that the hypometabolism in 5XFAD mice is gender-dependent and more pronounced in female mice. Therefore, these results support the feasibility of small animal PET imaging with 18F-FDG- and 18F-Florbetaben in 5XFAD mice in both, male and female animals. Moreover, our findings highlight the need to account for sex differences in studies working with 5XFAD mice.

Impact of piezocision on orthodontic tooth movement.

OBJECTIVE: This study investigated the impact of a single piezocision in the maxillary alveolar process on the speed of tooth movement. The null hypothesis was that the speed of tooth movement will be equal with and without piezocision. METHODS: All maxillary molars on one side were moved against the combined incisors in 10 ten-week-old male Wistar rats. Under general anesthesia, a force of 25 cN was applied on either side using a Sentalloy closed coil spring. After placing the orthodontic appliance, vertical corticision was performed using a piezotome under local anesthesia, 2 mm mesial from the mesial root of the first molar on a randomly selected side; the other side served as the control. At the beginning of the treatment, and 2 and 4 weeks later, skull micro-computed tomography was performed. After image reconstruction, the distance between the mesial root of the first molar and the incisive canal, and the length of the mesial root of the first maxillary molar were measured. Moreover, the root resorption score was determined as described by Lu et al. RESULTS: Significantly higher speed of tooth movement was observed on the corticision side; thus, the null hypothesis was rejected. The loss of root length and root resorption score were significantly more pronounced after piezocision than before. A strong correlation was observed between the speed of tooth movement and root resorption on the surgical side, but the control side only showed a weak correlation. CONCLUSIONS: Piezocision accelerates orthodontic tooth movement and causes increased root resorption.

Long-circulating XTEN864-annexin A5 fusion protein for phosphatidylserine-related therapeutic applications.

Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE-/- mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug.

Radionuclide, magnetic resonance and computed tomography imaging in European round back slugs (Arionidae) and leopard slugs (Limacidae).

Other than in animal models of human disease, little functional imaging has been performed in most of the animal world. The aim of this study was to explore the functional anatomy of the European round back slug (Arionidae) and leopard slug (Limacidae) and to establish an imaging protocol for comparative species study. Radionuclide images with single photon emission computed tomography (SPECT) and positron emission tomography (PET) were obtained after injections of standard clinical radiopharmaceuticals 99mtechnetium dicarboxypropane diphosphonate (bone scintigraphy), 99mtechnetium mercaptoacetyltriglycine (kidney function), 99mtechnetium diethylenetriaminepentaacetic acid (kidney function), 99mtechnetium pertechnetate (mediated by the sodium-iodide symporter), 99mtechnetium sestamibi (cardiac scintigraphy) or 18F-fluoro-deoxyglucose (glucose metabolism) in combination with magnetic resonance imaging (MRI) and computed tomography (CT) for uptake anatomic definition. Images were compared with anatomic drawings for the Arionidae species. Additionally, organ uptake data was determined for a description of slug functional anatomy in comparison to human tracer biodistribution patterns identifying the heart, the open circulatory anatomy, calcified shell remnant, renal structure (nephridium), liver (digestive gland) and intestine. The results show the detailed functional anatomy of Arionidae and Limacidae, and describe an in vivo whole-body imaging procedure for invertebrate species.

SPECT/CT Imaging, Biodistribution and Radiation Dosimetry of a 177Lu-DOTA-Integrin αvß6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin αvß6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin αvß6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin αvß6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC. METHODS: The expression of integrin αvß6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model. RESULTS: RT-qPCR and immunofluorescence results showed high expression of integrin αvß6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin αvß6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 ± 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 ± 2.5 %IA/g, declining much more rapidly than in tumors. CONCLUSION: 177Lu-DOTA-integrin αvß6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

Right-ventricular dysfunction in HFpEF is linked to altered cardiomyocyte Ca2+ homeostasis and myofilament sensitivity.

AIMS: Heart failure with preserved ejection fraction (HFpEF) is frequently (30%) associated with right ventricular (RV) dysfunction, which increases morbidity and mortality in these patients. Yet cellular mechanisms of RV remodelling and RV dysfunction in HFpEF are not well understood. Here, we evaluated RV cardiomyocyte function in a rat model of metabolically induced HFpEF. METHODS AND RESULTS: Heart failure with preserved ejection fraction-prone animals (ZSF-1 obese) and control rats (Wistar Kyoto) were fed a high-caloric diet for 13 weeks. Haemodynamic characterization by echocardiography and invasive catheterization was performed at 22 and 23 weeks of age, respectively. After sacrifice, organ morphometry, RV histology, isolated RV cardiomyocyte function, and calcium (Ca2+ ) transients were assessed. ZSF-1 obese rats showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV diastolic dysfunction (including increased LV end-diastolic pressures and E/e' ratio), and preserved LV ejection fraction. ZSF-1 obese animals developed RV dilatation (50% increased end-diastolic area) and mildly impaired RV ejection fraction (42%) with evidence of RV hypertrophy. In isolated RV cardiomyocytes from ZSF-1 obese rats, cell shortening amplitude was preserved, but cytosolic Ca2+ transient amplitude was reduced. In addition, augmentation of cytosolic Ca2+ release with increased stimulation frequency was lost in ZSF-1 obese rats. Myofilament sensitivity was increased, while contractile kinetics were largely unaffected in intact isolated RV cardiomyocytes from ZSF-1 obese rats. Western blot analysis revealed significantly increased phosphorylation of cardiac myosin-binding protein C (Ser282 cMyBP-C) but no change in phosphorylation of troponin I (Ser23, 24 TnI) in RV myocardium from ZSF-1 obese rats. CONCLUSIONS: Right ventricular dysfunction in obese ZSF-1 rats with HFpEF is associated with intrinsic RV cardiomyocyte remodelling including reduced cytosolic Ca2+ amplitudes, loss of frequency-dependent augmentation of Ca2+ release, and increased myofilament Ca2+ sensitivity.

Accurate Monte Carlo Modeling of Small-Animal Multi-Pinhole SPECT for Non-Standard Multi-Isotope Applications.

Recent advances in preclinical SPECT instrumentation enable non-standard multi-isotope acquisitions at the edge of physical feasibility to improve efficiency of pharmaceutical research. Due to the variety of applications, optimization of imaging hardware, acquisition protocols and reconstruction algorithms is a central and recurring task. For this purpose, we developed a Monte Carlo simulation model of a preclinical state-of-the-art multi-pinhole SPECT system, the NanoSPECT/CTPLUS, with emphasis on high accuracy for multi-isotope experiments operating near the system range limits. The GATE/ GEANT4 model included an accurate description of multi-pinhole collimators and all substructures of the detector back compartment. The readout electronics was modeled with a variety of signal processors partially extended to incorporate non-simplified measured response functions. The final model was able to predict energy spectra, planar images and tomographic reconstructions with high accuracy for both standard and non-standard multi-isotope experiments. Complex activity distributions could be reproduced for a wide range of noise levels and different modes of angular undersampling. Using the example of a dual-isotope triple-tracer experiment, the model has proven to be a powerful tool for protocol optimization and quantitative image correction at the performance range limits of multi-isotope multi-pinhole SPECT.

Nuclear Medicine Imaging Procedures in Oncology.

Nuclear medicine radionuclide imaging is a quantitative imaging modality based on radioisotope-labeled tracers which emit radiation in the form of photons used for image reconstruction. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the two noninvasive tomographic three-dimensional radionuclide imaging procedures for both clinical and preclinical settings. In this review on nuclear medicine imaging procedures in oncology, a variety of standard SPECT and PET tracers including radioiodine, 18Fluorine fluorodeoxyglucose (18F-FDG), and 68Gallium-labeled small proteins like Prostate Specific Membrane Antigen (PSMA) or somatostatin analogues and their application as targeted molecular imaging probes for improved tumor diagnosis and tumor phenotype characterization are described. Absolute and semiquantitative approaches for calculation of tracer uptake in tumors during the course of disease and during treatment allow further insight into tumor biology, and the combination of SPECT and PET with anatomical imaging procedures like computed tomography (CT) or magnetic resonance imaging (MRI) by hybrid SPECT/CT, PET/CT, and PET/MRI scanners provides both anatomical information and tumor functional characterization within one imaging session. With the recent establishment of novel molecular radiolabeled probes for specific tumor diagnosis, prognosis, and treatment monitoring, nuclear medicine has been able to establish itself as a distinct imaging modality with increased sensitivity and specificity.

Systematic Identification of MACC1-Driven Metabolic Networks in Colorectal Cancer.

MACC1 is a prognostic and predictive metastasis biomarker for more than 20 solid cancer entities. However, its role in cancer metabolism is not sufficiently explored. Here, we report on how MACC1 impacts the use of glucose, glutamine, lactate, pyruvate and fatty acids and show the comprehensive analysis of MACC1-driven metabolic networks. We analyzed concentration-dependent changes in nutrient use, nutrient depletion, metabolic tracing employing 13C-labeled substrates, and in vivo studies. We found that MACC1 permits numerous effects on cancer metabolism. Most of those effects increased nutrient uptake. Furthermore, MACC1 alters metabolic pathways by affecting metabolite production or turnover from metabolic substrates. MACC1 supports use of glucose, glutamine and pyruvate via their increased depletion or altered distribution within metabolic pathways. In summary, we demonstrate that MACC1 is an important regulator of metabolism in cancer cells.

Dual SGLT-1 and SGLT-2 inhibition improves left atrial dysfunction in HFpEF.

BACKGROUND: Sodium-glucose linked transporter type 2 (SGLT-2) inhibition has been shown to reduce cardiovascular mortality in heart failure independently of glycemic control and prevents the onset of atrial arrhythmias, a common co-morbidity in heart failure with preserved ejection fraction (HFpEF). The mechanism behind these effects is not fully understood, and it remains unclear if they could be further enhanced by additional SGLT-1 inhibition. We investigated the effects of chronic treatment with the dual SGLT-1&2 inhibitor sotagliflozin on left atrial (LA) remodeling and cellular arrhythmogenesis (i.e. atrial cardiomyopathy) in a metabolic syndrome-related rat model of HFpEF. METHODS: 17 week-old ZSF-1 obese rats, a metabolic syndrome-related model of HFpEF, and wild type rats (Wistar Kyoto), were fed 30 mg/kg/d sotagliflozin for 6 weeks. At 23 weeks, LA were imaged in-vivo by echocardiography. In-vitro, Ca2+ transients (CaT; electrically stimulated, caffeine-induced) and spontaneous Ca2+ release were recorded by ratiometric microscopy using Ca2+-sensitive fluorescent dyes (Fura-2) during various experimental protocols. Mitochondrial structure (dye: Mitotracker), Ca2+ buffer capacity (dye: Rhod-2), mitochondrial depolarization (dye: TMRE) and production of reactive oxygen species (dye: H2DCF) were visualized by confocal microscopy. Statistical analysis was performed with 2-way analysis of variance followed by post-hoc Bonferroni and student's t-test, as applicable. RESULTS: Sotagliflozin ameliorated LA enlargement in HFpEF in-vivo. In-vitro, LA cardiomyocytes in HFpEF showed an increased incidence and amplitude of arrhythmic spontaneous Ca2+ release events (SCaEs). Sotagliflozin significantly reduced the magnitude of SCaEs, while their frequency was unaffected. Sotagliflozin lowered diastolic [Ca2+] of CaT at baseline and in response to glucose influx, possibly related to a ~ 50% increase of sodium sodium-calcium exchanger (NCX) forward-mode activity. Sotagliflozin prevented mitochondrial swelling and enhanced mitochondrial Ca2+ buffer capacity in HFpEF. Sotagliflozin improved mitochondrial fission and reactive oxygen species (ROS) production during glucose starvation and averted Ca2+ accumulation upon glycolytic inhibition. CONCLUSION: The SGLT-1&2 inhibitor sotagliflozin ameliorated LA remodeling in metabolic HFpEF. It also improved distinct features of Ca2+-mediated cellular arrhythmogenesis in-vitro (i.e. magnitude of SCaEs, mitochondrial Ca2+ buffer capacity, diastolic Ca2+ accumulation, NCX activity). The safety and efficacy of combined SGLT-1&2 inhibition for the treatment and/or prevention of atrial cardiomyopathy associated arrhythmias should be further evaluated in clinical trials.

Multimodal Imaging of 2-Cycle PRRT with 177Lu-DOTA-JR11 and 177Lu-DOTATOC in an Orthotopic Neuroendocrine Xenograft Tumor Mouse Model.

Peptide receptor radionuclide therapy (PRRT) using radiolabeled somatostatin receptor (SSTR) analogs is a common approach in advanced neuroendocrine neoplasms. Recently, SSTR antagonists have shown promising results for imaging and therapy due to a higher number of binding sites than in commonly used agonists. We evaluated PRRT with SSTR agonist 177Lu-DOTATOC and antagonist 177Lu-DOTA-JR11 longitudinally in an orthotopic murine pancreatic neuroendocrine neoplasm model expressing human SSTR2. Morphologic and metabolic changes during treatment were assessed using multimodal imaging, including hybrid PET/MRI and SPECT/CT. Methods: In vitro radioligand binding and internalization assays and cell-cycle analysis were performed. SSTR2-transfected BON cells (BON-SSTR2) were used for in vivo experiments. Tumor-bearing mice received 2 intravenous injections of 100 µL of saline, 30 MBq of 177Lu-DOTATOC, or 20 MBq of 177Lu-DOTA-JR11 with an interval of 3 wk. Weekly T2-weighted MRI was performed for tumor monitoring. Viability of the tumor tissue was assessed by 18F-FDG PET/MRI once after PRRT. Tumor and kidney uptake of the respective radiopharmaceuticals was measured 24 h after injection by SPECT/CT. Results: Compared with 177Lu-DOTATOC, 177Lu-DOTA-JR11 treatment resulted in an increased accumulation of cells in G2/M phase. Animals treated with the SSTR antagonist showed a significant reduction in tumor size (P < 0.001) and an increased median survival (207 d; interquartile range [IQR], 132-228) compared with 177Lu-DOTATOC (126 d; IQR, 118-129). SPECT/CT revealed a 4-fold higher median tumor uptake for the antagonist and a 3-fold higher tumor-to-kidney ratio in the first treatment cycle. During the second therapy cycle, tumor uptake of 177Lu-DOTATOC was significantly lower (P = 0.01) whereas 177Lu-DOTA-JR11 uptake remained stable. Imaging of tumor morphology indicated comparatively larger necrotic fractions for 177Lu-DOTA-JR11 despite further tumor growth. These results were confirmed by 18F-FDG PET, revealing the least amount of viable tumor tissue in 177Lu-DOTA-JR11-treated animals, at 6.2% (IQR, 2%-23%). Conclusion:177Lu-DOTA-JR11 showed a higher tumor-to-kidney ratio and a more pronounced cytotoxic effect than did 177Lu-DOTATOC. Additionally, tumor uptake was more stable over the course of 2 treatment cycles.

A Cyanine-Bridged Somatostatin Hybrid Probe for Multimodal SSTR2 Imaging in Vitro and in Vivo: Synthesis and Evaluation.

Multimodal imaging probes have attracted the interest of ongoing research, for example, for the surgical removal of tumors. Modular synthesis approaches allow the construction of hybrid probes consisting of a radiotracer, a fluorophore and a targeting unit. We present the synthesis of a new asymmetric bifunctional cyanine dye that can be used as a structural and functional linker for the construction of such hybrid probes. 68 Ga-DOTATATE, a well-characterized radiopeptide targeting the overexpressed somatostatin receptor subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our cyanine dye, thus providing additional information along with the data obtained from the radiotracer. We tested the SSTR2-targeting and imaging properties of the resulting probe 68 Ga-DOTA-ICC-TATE in vitro and in a tumor xenograft mouse model. Despite the close proximity between dye and pharmacophore, we observed a high binding affinity towards SSTR2 as well as elevated uptake in SSTR2-overexpressing tumors in the positron emission tomography (PET) scan and histological examination.

In vivo Imaging With 18F-FDG- and 18F-Florbetaben-PET/MRI Detects Pathological Changes in the Brain of the Commonly Used 5XFAD Mouse Model of Alzheimer's Disease.

Imaging biomarkers of Alzheimer's disease (AD) that are able to detect molecular changes in vivo and transgenic animal models mimicking AD pathologies are essential for the evaluation of new therapeutic strategies. Positron-emission tomography (PET) using either 18F-Fluorodeoxyglucose (18F-FDG) or amyloid-tracers is a well-established, non-invasive tool in the clinical diagnostics of AD assessing two major pathological hallmarks. 18F-FDG-PET is able to detect early changes in cerebral glucose metabolism and amyloid-PET shows cerebral amyloid load. However, the suitability of 18F-FDG- and amyloid-PET in the widely used 5XFAD mouse model of AD is unclear as only a few studies on the use of PET biomarkers are available showing some conflicting results. The aim of this study was the evaluation of 18F-FDG-PET and amyloid-PET in 5XFAD mice in comparison to neurological deficits and neuropathological changes. Seven- and 12-month-old male 5XFAD mice showed a significant reduction in brain glucose metabolism in 18F-FDG-PET and amyloid-PET with 18F-Florbetaben demonstrated an increased cerebral amyloid deposition (n = 4-6 per group). Deficits in spatial reference memory were detected in 12-month-old 5XFAD mice in the Morris Water Maze (n = 10-12 per group). Furthermore, an increased plaque load and gliosis could be proven immunohistochemically in 5XFAD mice (n = 4-6 per group). PET biomarkers 18F-FDG and 18F-Florbetaben detected cerebral hypometabolism and increased plaque load even before the onset of severe memory deficits. Therefore, the 5XFAD mouse model of AD is well-suited for in vivo monitoring of AD pathologies and longitudinal testing of new therapeutic approaches.

18F-sodium fluoride bone deposition quantitation with PET in Mice: Variation with age, sex, and circadian rhythm.

AIM: The aim of this study was to establish a data base for normal 18F-sodium fluoride (18F-NaF) bone uptake as a function of age, sex and circadian rhythm in mice. METHODS: In 12 female (F) and 12 male (M) C57BL/6N mice PET images were acquired 90 min after intravenous injection of 20 MBq 18F-NaF for 30 minutes. Each mouse was imaged in follow-up studies at 1, 3, 6, 13 and 21 months of age. In order to assess for physiologic changes related to circadian rhythm, animals were imaged during light (sleep phase) as well as during night conditions (awake phase). Bone uptake is described as the median percentage of the injected activity (%IA) and in relation to bone volume (%IA/ml). RESULTS: A significant smaller bone volume was found in F (1.79 ml) compared to M (1.99 ml; p < 0.001). In sex-pooled data, highest bone uptake occurred at an age of 1 month (61.1 %IA, 44.5 %IA/ml) with a significant reduction (p < 0.001) at age 3 months (43.6 %IA, 23.6 %IA/ml), followed by an increase between 13 (47.3 %IA, 24.5 %IA/ml) and 21 months (52.2 %IA, 28.1 %IA/ml). F had a significantly higher total uptake (F 48.2 %IA, M 43.8 %IA; p = 0.026) as well as a higher uptake per ml bone tissue (F 27.0 %IA/ml; M 22.4 %IA/ml; p < 0.001). A significant impact of circadian rhythm was only found for F at ages of 3 and 6 months with a higher uptake during the sleep phase. CONCLUSION: Circadian rhythm had a significant impact on uptake only in F of 3 and 6 months. Regarding sex, F showed generally higher uptake rates than M. The highest uptake values were observed during bone growth at age 1 month in both sexes, a second uptake peak occurred in elderly F. Designing future bone uptake studies with M, attention must be paid to age only, while in F circadian rhythm and age must be taken into account.