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Deposition of α-synuclein fibrils is implicated in Parkinson's disease (PD) and dementia with Lewy bodies (DLB), while in vivo detection of α-synuclein pathologies in these illnesses has been challenging. Here, we have developed a small-molecule ligand, C05-05, for visualizing α-synuclein deposits in the brains of living subjects. In vivo optical and positron emission tomography (PET) imaging of mouse and marmoset models demonstrated that C05-05 captured a dynamic propagation of fibrillogenesis along neural pathways, followed by disruptions of these structures. High-affinity binding of 18 F-C05-05 to α-synuclein aggregates in human brain tissues was also proven by in vitro assays. Notably, PET-detectable 18 F-C05-05 signals were intensified in the midbrains of PD and DLB patients as compared with healthy controls, providing the first demonstration of visualizing α-synuclein pathologies in these illnesses. Collectively, we propose a new imaging technology offering neuropathology-based translational assessments of PD and allied disorders toward diagnostic and therapeutic research and development., Competing Interests: Declaration of interests M.O., M.-R.Z., and M. Higuchi filed a patent on compounds related to the present report (2019-034997, PCT/JP2020/002607)., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Significance: Two-photon optogenetics and simultaneous calcium imaging can be used to visualize the response of surrounding neurons with respect to the activity of an optically stimulated target neuron, providing a direct method to assess neuronal connectivity., Aim: We aim to develop a two-photon optogenetics-based method for evaluating neuronal connectivity, compare it to the existing indirect resting-state synchrony method, and investigate the application of the method to brain pathophysiology., Approach: C1V1-mScarlet was introduced into GCaMP6s-expressing transgenic mice with an adeno-associated virus. Optical stimulation of a single target neuron and simultaneous calcium imaging of the target and surrounding cells were performed. Neuronal connectivity was evaluated from the correlation between the fluorescence intensity of the target and surrounding cells., Results: The neuronal connectivity in the living brain was evaluated using two-photon optogenetics. However, resting-state synchrony was not always consistent with two-photon optogenetics-based connectivity. Comparison with neuronal synchrony measured during sensory stimulation suggested that the disagreement was due to external sensory input. Two-photon optogenetics-based connectivity significantly decreased in the common carotid artery occlusion model, whereas there was no significant change in the control group., Conclusions: We successfully developed a direct method to evaluate neuronal connectivity in the living brain using two-photon optogenetics. The technique was successful in detecting connectivity impairment in hypoperfusion model mice., (© 2024 The Authors.)

Sarcopenia is a common skeletal muscle disease in older people. Lower limb muscle strength is a good predictive value for sarcopenia; however, little is known about its genetic components. Here, we conducted a genome-wide association study (GWAS) for knee extension strength in a total of 3452 Japanese aged 60 years or older from two independent cohorts. We identified a significant locus, rs10749438 which is an intronic variant in TACC2 (transforming acidic coiled-coil-containing 2) (P = 4.2 × 10 -8 ). TACC2, encoding a cytoskeleton-related protein, is highly expressed in skeletal muscle, and is reported as a target of myotonic dystrophy 1-associated splicing alterations. These suggest that changes in TACC2 expression are associated with variations in muscle strength in older people. The association was consistently observed in young and middle-aged subjects. Our findings would shed light on genetic components of lower limb muscle strength and indicate TACC2 as a potential therapeutic target for sarcopenia., (© 2024. The Author(s).)

We herein describe our experience with patients who had been diagnosed with BRCA1/2 pathogenic variants and metastatic breast cancer. Three patients who experienced postoperative recurrences had received chemotherapy before recurrence, while an additional patient with stage Ⅳ disease at diagnosis required chemotherapy before olaparib administration. Prior anthracycline and/or taxane-based therapies needed prior to administration of poly(adenosine diphosphate ribose) polymerase inhibitors might still be controversial in terms of patient benefits.

Background and objective The treatment for symptomatic meniscus-deficient knees with cartilage defects remains challenging on account of insufficient meniscal substitutes. One solution for this might involve combining meniscal allograft transplantation (MAT) and cartilage repair. In this study, we aimed to analyze the effectiveness and safety of MAT concomitant with cartilage repair for symptomatic lateral meniscus-deficient knees in a setting with limited availability of meniscal transplants in Japan. Methods Nine patients who underwent MAT concomitant with osteochondral transplantation (five) and/or autologous chondrocyte implantations (seven) were followed up for at least two years (mean: 51.2 months, range: 24-84 months). Their demographic data and other characteristics were as follows - mean age: 51.7 years, range: 36-67 years; men/women: 4/5; cause: trauma/discoid meniscus: 8/1; cartilage defect size: mean: 6.7 cm 2 /knee, range: 1.0-11.3. The effectiveness and safety were evaluated clinically by using the Lysholm Knee Scoring Scale (LKSS) and Japanese Orthopaedic Association (JOA) knee score, physical examination, X-rays, and MRI preoperatively and at one, 12, and 24 months after the implantation. Differences between the variables were analyzed using the Friedman test and Scheffe's multiple comparisons. Results The median LKSS and JOA scores significantly improved from 70 points (range: 21-80) and 35 (25-45) preoperatively to 86.5 (65-98) and 87.5 (80-95) at 24 months after surgery, respectively (p<0.001, p=0.0013). The range of motion (ROM), femorotibial angle, and the lateral joint space showed no significant changes. However, lateral meniscal extrusions (LMEs) increased by 3.0 mm (range: 0-6.3 mm) at one month postoperatively and remained unchanged until two years postoperatively. Treatment failure occurred in one case, which was revised by total knee arthroplasty (TKA) at 18 months postoperatively. Additional surgeries were needed in some cases: lateral meniscal tear (three cases), contracture (two cases), and patellar instability (one case). However, neither infection nor allergic reaction was observed in the blood exams. Conclusions Although MAT concomitant with cartilage repair showed good clinical outcomes, half of the cases needed additional surgeries. Based on our findings, this technique should be adopted only in select cases and performed by a handful of highly experienced surgeons., Competing Interests: The authors have declared financial relationships, which are detailed in the next section., (Copyright © 2023, Uchio et al.)

Ossification of the posterior longitudinal ligament of the spine (OPLL) is an intractable disease leading to severe neurological deficits. Its etiology and pathogenesis are primarily unknown. The relationship between OPLL and comorbidities, especially type 2 diabetes (T2D) and high body mass index (BMI), has been the focus of attention; however, no trait has been proven to have a causal relationship. We conducted a meta-analysis of genome-wide association studies (GWASs) using 22,016 Japanese individuals and identified 14 significant loci, 8 of which were previously unreported. We then conducted a gene-based association analysis and a transcriptome-wide Mendelian randomization approach and identified three candidate genes for each. Partitioning heritability enrichment analyses observed significant enrichment of the polygenic signals in the active enhancers of the connective/bone cell group, especially H3K27ac in chondrogenic differentiation cells, as well as the immune/hematopoietic cell group. Single-cell RNA sequencing of Achilles tendon cells from a mouse Achilles tendon ossification model confirmed the expression of genes in GWAS and post-GWAS analyses in mesenchymal and immune cells. Genetic correlations with 96 complex traits showed positive correlations with T2D and BMI and a negative correlation with cerebral aneurysm. Mendelian randomization analysis demonstrated a significant causal effect of increased BMI and high bone mineral density on OPLL. We evaluated the clinical images in detail and classified OPLL into cervical, thoracic, and the other types. GWAS subanalyses identified subtype-specific signals. A polygenic risk score for BMI demonstrated that the effect of BMI was particularly strong in thoracic OPLL. Our study provides genetic insight into the etiology and pathogenesis of OPLL and is expected to serve as a basis for future treatment development., Competing Interests: YK, MT, MN, NO, HS, XL, TE, SI, KK, TK, SK, MK, HS, TT, TM, HI, TY, MK, HN, KA, YT, KT, SI, KT, KH, YI, YK, SM, HN, KM, AS, SF, TK, KW, TT, KK, SK, MT, KY, HT, HL, SN, KO, YM, AO, MM, NI, CT, SI No competing interests declared, YK Consulting fees from Medacta International, MY representatives of Japanese Organization of the Study for Ossification of Spinal Ligament, (© 2023, Koike, Takahata, Nakajima et al.)

Brain capillaries play a crucial role in maintaining cellular viability and thus preventing neurodegeneration. The aim of this study was to characterize the brain capillary morphology at rest and during neural activation based on a big data analysis from three-dimensional microangiography. Neurovascular responses were measured using a genetic calcium sensor expressed in neurons and microangiography with two-photon microscopy, while neural acivity was modulated by stimulation of contralateral whiskers or by a seizure evoked by kainic acid. For whisker stimulation, 84% of the capillary sites showed no detectable diameter change. The remaining 10% and 6% were dilated and constricted, respectively. Significant differences were observed for capillaries in the diameter at rest between the locations of dilation and constriction. Even the seizures resulted in 44% of the capillaries having no detectable change in diameter, while 56% of the capillaries dilated. The extent of dilation was dependent on the diameter at rest. In conclusion, big data analysis on brain capillary morphology has identified at least two types of capillary states: capillaries with diameters that are relatively large at rest and stable over time regardless of neural activity and capillaries whose diameters are relatively small at rest and vary according to neural activity.

In Alzheimer's disease (AD), network hyperexcitability is frequently observed and associated with subsequent cognitive impairment. Dysfunction of inhibitory interneurons (INs) is thought to be one of the key biological mechanisms of hyperexcitability. However, it is still unknown how INs are functionally affected in tau pathology, which is a major pathology in AD. To clarify this, we evaluated the neuronal activity of cortical INs in 6-month-old rTg4510 mice, a model of tauopathy. Calcium imaging with mDlx enhancer-driven labeling revealed that neuronal activity in INs was decreased in rTg4510 mice. In the patch clamp recording, the firing properties of fast-spiking INs were altered so as to reduce their activity in rTg4510 mice. In parallel with microglial activation, perineuronal nets around parvalbumin-positive INs were partially disrupted in rTg4510 mice. Taken together, our data indicate that the excitability of cortical fast-spiking INs is decreased, possibly because of the disruption of perineuronal nets., Competing Interests: The authors declare the following interests: T.K., K.S., and M.Y. are employees of Sumitomo Pharma Co., Ltd. Moreover, this study was supported by Sumitomo Pharma Co., Ltd. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript., (© 2023 The Author(s).)

Introduction: As the movement of water in the brain is known to be involved in neural activity and various brain pathologies, the ability to assess water dynamics in the brain will be important for the understanding of brain function and the diagnosis and treatment of brain diseases. Aquaporin-4 (AQP4) is a membrane channel protein that is highly expressed in brain astrocytes and is important for the movement of water molecules in the brain., Methods: In this study, we investigated the contribution of AQP4 to brain water dynamics by administering deuterium-labeled water (D 2 O) intraperitoneally to wild-type and AQP4 knockout (AQP4-ko) mice that had undergone surgical occlusion of the middle cerebral artery (MCA). Water dynamics in the infarct region and on either side of the anterior cerebral artery (ACA) was monitored with proton-density-weighted imaging (PDWI) performed on a 7T animal MRI., Results: D 2 O caused a negative signal change quickly after administration. The AQP4-ko mice showed a delay of the time-to-minimum in both the contralateral and ipsilateral ACA regions compared to wild-type mice. Also, only the AQP4- ko mice showed a delay of the time-to-minimum in the ipsilateral ACA region compared to the contralateral side. In only the wild-type mice, the signal minimum in the ipsilateral ACA region was higher than that in the contralateral ACA region. In the infarct region, the signal attenuation was slower for the AQP4-ko mice in comparison to the wild-type mice., Discussion: These results suggest that AQP4 loss affects water dynamics in the ACA region not only in the infarct region. Dynamic PDWI after D 2 O administration may be a useful tool for showing the effects of AQP4 in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Urushihata, Takuwa, Takahashi, Kershaw, Shibata, Nitta, Tachibana, Yasui, Higuchi and Obata.)

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer's disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770 + mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770 + mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (App NL-F/NL-F ) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770 + :App NL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood-brain barrier upon administration of anti-Aβ antibodies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

To assess if magnetic resonance spectroscopy (MRS)-measured Glutamate (Glu) and GABA reflect excitatory and inhibitory neural activities, respectively, we conducted MRS measurements along with two-photon mesoscopic imaging of calcium signals in excitatory and inhibitory neurons of living, unanesthetized mice. For monitoring stimulus-driven activations of a brain region, MRS signals and mesoscopic neural activities were measured during two consecutive sessions of 15-min prolonged sensory stimulations. In the first session, putative excitatory neuronal activities were increased, while inhibitory neuronal activities remained at the baseline level. In the second half, while excitatory neuronal activities remained elevated, inhibitory neuronal activities were significantly enhanced. We assessed regional neurochemical statuses by measuring MRS signals, which were overall in accordance with the neural activities, and neuronal activities and neurochemical statuses in a mouse model of Dravet syndrome under resting condition. Mesoscopic assessments showed that activities of inhibitory neurons in the cortex were diminished relative to wild-type mice in contrast to spared activities of excitatory neurons. Consistent with these observations, the Dravet model exhibited lower concentrations of GABA than wild-type controls. Collectively, the current investigations demonstrate that MRS-measured Glu and GABA can reflect spontaneous and stimulated activities of neurons producing and releasing these neurotransmitters in an awake condition.

Positron emission tomography (PET) allows biomolecular tracking but PET monitoring of brain networks has been hampered by a lack of suitable reporters. Here, we take advantage of bacterial dihydrofolate reductase, ecDHFR, and its unique antagonist, TMP, to facilitate in vivo imaging in the brain. Peripheral administration of radiofluorinated and fluorescent TMP analogs enabled PET and intravital microscopy, respectively, of neuronal ecDHFR expression in mice. This technique can be used to the visualize neuronal circuit activity elicited by chemogenetic manipulation in the mouse hippocampus. Notably, ecDHFR-PET allows mapping of neuronal projections in non-human primate brains, demonstrating the applicability of ecDHFR-based tracking technologies for network monitoring. Finally, we demonstrate the utility of TMP analogs for PET studies of turnover and self-assembly of proteins tagged with ecDHFR mutants. These results establish opportunities for a broad spectrum of previously unattainable PET analyses of mammalian brain circuits at the molecular level., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Fluctuations of neuronal activities in the brain may underlie relatively slow components of neurofunctional alterations, which can be modulated by food intake and related systemic metabolic statuses. Glutamatergic neurotransmission plays a major role in the regulation of excitatory tones in the central nervous system, although just how dietary elements contribute to the tuning of this system remains elusive. Here, we provide the first demonstration by bimodal positron emission tomography (PET) and magnetic resonance spectroscopy (MRS) that metabotropic glutamate receptor subtype 5 (mGluR5) ligand binding and glutamate levels in human brains are dynamically altered in a manner dependent on food intake and consequent changes in plasma glucose levels. The brain-wide modulations of central mGluR5 ligand binding and glutamate levels and profound neuronal activations following systemic glucose administration were further proven by PET, MRS, and intravital two-photon microscopy, respectively, in living rodents. The present findings consistently support the notion that food-associated glucose intake is mechanistically linked to glutamatergic tones in the brain, which are translationally accessible in vivo by bimodal PET and MRS measurements in both clinical and non-clinical settings.

Protein malnutrition is epidemiologically suggested as a potential risk factor for senile dementia, although molecular mechanisms linking dietary proteins and amino acids to neurodegeneration remain unknown. Here, we show that a low-protein diet resulted in down-regulated expression of synaptic components and a modest acceleration of brain atrophy in mice modeling neurodegenerative tauopathies. Notably, these abnormal phenotypes were robustly rescued by the administration of seven selected essential amino acids. The up-regulation of inflammation-associated gene expression and progressive brain atrophy in the tauopathy model were profoundly suppressed by treatment with these essential amino acids without modifications of tau depositions. Moreover, the levels of kynurenine, an initiator of a pathway inducing neuroinflammatory gliosis and neurotoxicity in the brain, were lowered by treatment through inhibition of kynurenine uptake in the brain. Our findings highlight the importance of specific amino acids as systemic mediators of brain homeostasis against neurodegenerative processes.

Background: Aquaporin-4 is a membrane channel protein that is highly expressed in brain astrocytes and facilitates the transport of water molecules. It has been suggested that suppression of aquaporin-4 function may be an effective treatment for reducing cellular edema after cerebral infarction. It is therefore important to develop clinically applicable measurement systems to evaluate and better understand the effects of aquaporin-4 suppression on the living body., Methods: Animal models of focal cerebral ischemia were created by surgically occluding the middle cerebral artery of wild-type and aquaporin-4 knockout mice, after which multi-b-value multi-diffusion-time diffusion-weighted imaging measurements were performed. Data were analyzed with both the apparent diffusion coefficient (ADC) model and a compartmental water-exchange model., Results: ADCs were estimated for five different b value ranges. The ADC of aquaporin-4 knockout mice in the contralateral region was significantly higher than that of wild-type mice for each range. In contrast, aquaporin-4 knockout mice had significantly lower ADC than wild-type mice in ischemic tissue for each b-value range. Genotype-dependent differences in the ADC were particularly significant for the lowest ranges in normal tissue and for the highest ranges in ischemic tissue. The ADCs measured at different diffusion times were significantly different for both genotypes. Fitting of the water-exchange model to the ischemic region data found that the water-exchange time in aquaporin-4 knockout mice was approximately 2.5 times longer than that in wild-type mice., Conclusions: Multi-b-value multi-diffusion-time diffusion-weighted imaging may be useful for in vivo research and clinical diagnosis of aquaporin-4-related diseases., (© 2021. The Author(s).)

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

Objective: Positron emission tomography (PET) has been used to investigate changes in the concentration of endogenous neurotransmitters. Recently, this technique has been applied to the imaging of serotonin 2A receptors using [ 18 F]altanserin. In these measurements, a reduction in binding potential (BP) suggests an increase in endogenous serotonin levels caused by pharmacological or cognitive stimulations, and the sensitivity of BP reduction depends on the characteristics of [ 18 F]altanserin. In this study, we evaluated an analytical method for estimating the changes in endogenous serotonin levels based on PET scans with [ 18 F]altanserin at baseline and stimulated states and validated it using simulations and small animal PET studies., Methods: First, in the simulations, the time-activity curves at baseline and the stimulated states were generated using an extended compartment model including the competition for the receptors between the administered [ 18 F]altanserin and endogenous serotonin. In the stimulated state, the magnitude and onset of the endogenous serotonin elevation were altered to varying degrees. In these time-activity curves, BP was estimated using the simplified reference tissue model (SRTM), and the reduction in BP was evaluated by comparison with that of the baseline state. Next, the proposed method was applied to mouse PET studies. Endogenous serotonin levels were elevated by treatment with selective serotonin reuptake inhibitors (SSRIs), and PET studies were performed twice, once with and once without treatment. In both scans, BP was estimated using the SRTM with the cerebellum as a reference region, and the reduction in BP after SSRI treatment was evaluated., Results: In the simulations, the BP estimate of the stimulated state was smaller than that of the baseline state, and their reduction was related to the amount of change in the serotonin concentration. BP reduction was also affected by the onset of serotonin elevation. In the mouse studies, the BP of the cerebral cortex decreased in the scans with SSRI treatment., Conclusions: The reduction in BP estimated using the SRTM from [ 18 F]altanserin-PET studies at baseline and in stimulated states can detect changes in the binding conditions of serotonin 2A receptors. This may be useful for investigating the elevation of endogenous serotonin levels caused by stimulations., (© 2021. The Japanese Society of Nuclear Medicine.)

Objective: Quantification of angiographic images with two-photon laser scanning fluorescence microscopy (2PLSM) relies on proper segmentation of the vascular images. However, the images contain inhomogeneities in the signal-to-noise ratio (SNR) arising from regional effects of light scattering and absorption. The present study developed a semiautomated quantification method for volume images of 2PLSM angiography by adjusting the binarization threshold according to local SNR along the vessel centerlines., Methods: A phantom model made with fluorescent microbeads was used to incorporate a region-dependent binarization threshold., Results: The recommended SNR for imaging was found to be 4.2-10.6 that provide the true size of imaged objects if the binarization threshold was fixed at 50% of SNR. However, angiographic images in the mouse cortex showed variable SNR up to 45 over the depths. To minimize the errors caused by variable SNR and a spatial extent of the imaged objects in an axial direction, the microvascular networks were three-dimensionally reconstructed based on the cross-sectional diameters measured along the vessel centerline from the XY-plane images with adapted binarization threshold. The arterial volume was relatively constant over depths of 0-500 µm, and the capillary volume (1.7% relative to the scanned volume) showed the larger volumes than the artery (0.8%) and vein (0.6%)., Conclusions: The present methods allow consistent segmentation of microvasculature by adapting the local inhomogeneity in the SNR, which will be useful for quantitative comparison of the microvascular networks, such as under disease conditions where SNR in the 2PLSM images varies over space and time., (© 2021 John Wiley & Sons Ltd.)

A panel of radiochemicals has enabled in vivo positron emission tomography (PET) of tau pathologies in Alzheimer's disease (AD), although sensitive detection of frontotemporal lobar degeneration (FTLD) tau inclusions has been unsuccessful. Here, we generated an imaging probe, PM-PBB3, for capturing diverse tau deposits. In vitro assays demonstrated the reactivity of this compound with tau pathologies in AD and FTLD. We could also utilize PM-PBB3 for optical/PET imaging of a living murine tauopathy model. A subsequent clinical PET study revealed increased binding of 18 F-PM-PBB3 in diseased patients, reflecting cortical-dominant AD and subcortical-dominant progressive supranuclear palsy (PSP) tau topologies. Notably, the in vivo reactivity of 18 F-PM-PBB3 with FTLD tau inclusion was strongly supported by neuropathological examinations of brains derived from Pick's disease, PSP, and corticobasal degeneration patients who underwent PET scans. Finally, visual inspection of 18 F-PM-PBB3-PET images was indicated to facilitate individually based identification of diverse clinical phenotypes of FTLD on a neuropathological basis., Competing Interests: Declaration of Interests H. Shimada, M.-R.Z., T.S., and M.H. hold patents on compounds related to the present report (JP 5422782/EP 12 884 742.3/CA2894994/HK1208672)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The present study describes methodological aspects of image analysis for angiographic image data with long-term two-photon microscopy acquired for the investigation of dynamic changes in the three-dimensional (3D) network structure of the capillaries (less than 8 μm in diameter) in the mouse cerebral cortex. Volume images of the identical capillaries over different periods of days up to 32 days were compared for adaptation under either chronic hypoxia (8-9% O 2 ) or hyperoxia (40-50% O 2 ). We observed that the median diameters of measured capillaries were 5.8, 8.4, 9.0, and 8.4 μm at 0, 1, 2, and 3 weeks during exposure to hypoxia, respectively (N = 1, n = 2193 pairs at day 0), and 5.4, 5.7, 5.4, 6.0, and 6.1 μm measured weekly up to 32 days under hyperoxia (N = 1, n = 1025 pairs at day 0). In accordance with these changes in capillary diameters, tissue space was also observed to change in a depth-dependent manner under hypoxia, but not hyperoxia. The present methods provide us with a method to quantitatively determine three-dimensional vascular and tissue morphology with the aid of a computer-assisted graphical user interface, which facilitates morphometric analysis of the cerebral microvasculature and its correlation with the adaptation of brain cells imaged simultaneously with the microvasculature.

Cerebral capillaries respond to changes in neural activity to maintain regional balances between energy demand and supply. However, the quantitative aspects of the capillary diameter responses and their contribution to oxygen supply to tissue remain incompletely understood. The purpose of the present study is to check if the diameters measured from large-scale angiographic image data of two-photon laser scanning fluorescent microscopy (2PLSM) are correctly determined with a custom-written MATLAB software and to investigate how the measurement errors can be reduced, such as at the junction areas of capillaries. As a result, nearly 17% of the measured locations appeared to be outliers of the automated diameter measurements, in particular arising from the junction areas where three capillary segments merged. We observed that about two-thirds of the outliers originated from the measured locations within 6 μm from the branching point. The results indicate that the capillary locations in the junction areas cause non-negligible errors in the automated diameter measurements. Considering the common site of the outliers, the present study identified that the areas within 6 μm from the branch point could be separately measured from the diameter analysis, and careful manual inspection with reference to the original images for these transition areas around the branch point is further recommended.

Digital breast tomosynthesis (DBT) is an emerging imaging tool for both the screening and diagnosis of breast cancers. However, the use of DBT in diagnosis of calcifications remains ambiguous. In this study, we investigated DBT findings that might help differentiate between benign and malignant calcifications. We enrolled 256 subjects and evaluated 303 breasts with grouped or segmental calcifications. All imaging examinations were performed using two-directional full-field digital mammography (FFDM) and DBT. We divided subjects into two groups, namely, "dense" and "fatty," based on the quantity of breast tissue and evaluated whether the growth of cancer causes increased the density overlapping with calcifications. Increased overlapping density was significantly associated with malignant calcifications (p < 0.001), and the identification of increased density was more accurate using DBT than using FFDM. Furthermore, we used DBT to evaluate whether segmental calcifications were continuous or discontinuous. Significantly more malignant than benign calcifications were associated with a continuous distribution (p = 0.035). Increased density overlapping with grouped calcifications was significantly associated with invasive cancers (p = 0.017). The findings of this study suggest that DBT improves the ability to differentiate between benign and malignant calcifications.

The chemogenetic technology designer receptors exclusively activated by designer drugs (DREADDs) afford remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities and potential off-target effects of CNO represent areas for improvement. Here, we provide a new high-affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 μg per kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 μg per kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents a potent, selective, metabolically stable and fast-acting DREADD agonist with utility in both mice and nonhuman primates for a variety of applications.

Objective: We examined the therapeutic effect of adding of pertuzumab in combination with trastuzumab as neoadjuvant chemotherapy for HER2-positive breast cancer., Participants: Overall, 27 patients with HER2-positive breast cancer who had completed neoadjuvant chemotherapy and undergone surgery between January 2014 and January 2020 were included in the study. We performed a retrospective analysis of the relationship between HER2 gene amplification and the therapeutic effect of pertuzumab in this group. For the anti-HER2 treatment, only trastuzumab was administered to all patients until December 2018, and then a combination of trastuzumab and pertuzumab was administered to 4 patients from January 2019., Results: The case distribution based on histological therapeutic effect was: 4 patients with Grade 1, 12 patients with Grade 2, and 11 patients with Grade 3. Patients with immunohistochemistry scores of 3+ and high signal ratios of HER2/ CEP17 in FISH testing tended to have better therapeutic outcomes than other patients., Discussion: Outcomes similar to those observed in this study were reported in other cases where pertuzumab was used for anti-HER2 treatment. We expected that the therapeutic effects of pertuzumab would be better in patients with high HER2 expression. However, further research is required to understand the effects of pertuzumab in patients with cT1c that were not included in the Neo Sphere trial. The therapeutic effects of adding pertuzumab to the treatment in patients with cT1c and lymph node-positive breast cancer were indicated by comparison with our cases.