Longitudinal Imaging Research Articles

Ultrasonographic technique and appearance of the coelomic organs in crocodilians

IntroductionCrocodilians have significant ecological, conservational, and economic roles. They are also commonly raised for commercial purposes and kept as zoological specimens. Although ultrasonography has been used in zoological contexts for health assessments of crocodilians, published studies on a detailed ultrasonography protocol and ultrasonographic anatomy are lacking. This study aimed to establish a standardized ultrasonography protocol and pictorial reference of the ultrasonographic appearances of the coelomic organs of crocodilians.MethodsA total of 7 crocodilians comprising 4 different species were included in this study. The crocodilians were manually restrained and underwent a non-contrasted and contrasted computed tomography (CT) scan, followed by an ultrasonography (USG) examination. Ultrasound fusion imaging technique enabled greater confidence in establishing a clear organ localization and correlation between modalities by visualizing the same anatomy from the same view angle.ResultsThe heart, caudal vena cava, liver, fat body (steatotheca), spleen, stomach, duodenal loops, pancreas, kidneys, testes, ovaries and cloaca were visualized in all species. Longitudinal and transverse images of the coelomic structures were acquired when possible. The ultrasonographic characteristics of the coelomic organs, including transducer positioning, acoustic window and approach, shape, size, marginations, and echo pattern were documented.DiscussionThe findings of this study provided a useful ultrasonographic protocol and anatomical reference of the coelomic organs in crocodilians. Invaluable insights into the practicality and adequacy of ultrasonography in evaluating the coelomic structures of crocodilians as part of health assessment and disease diagnosis were also discussed.

Gestational and early postnatal protein malnutrition disrupts neurodevelopment in rhesus macaques.

Adequate nutrition during gestation is critical for fetal development, and deficits in protein are associated with neurological and behavioral impairments in offspring placing a significant burden on global health. Fetal and neonatal longitudinal magnetic resonance assessments of brain development spanning mid-gestation to 11months of age were conducted in rhesus macaque (Macaca mulatta) (n= 22; 9 females) generated from an established nonhuman primate model of gestational protein reduction to ascertain the neurodevelopmental effects of reduced maternal protein intake. Structural abnormalities were identified in two reduced diet groups, in addition to age-dependent whole-brain volume deficits in the most severely reduced (50% vs. 33% reduction) protein cohort, primarily restricted to gray matter structures; i.e. cortical/subcortical gray matter and the cerebellum. Diffusion-weighted imaging revealed widespread postnatal reductions in white matter fractional anisotropy, concentrated in the corpus callosum for both reduced protein levels relative to control diet. Despite extensive neurodevelopmental alterations detectable by longitudinal imaging, early behavioral assessments conducted at 1month revealed minor perturbations. These results highlight differential impacts of reduced maternal and infant protein intake on gray and white matter formation and organization, with potential implications for early motor development.

Longitudinal Magnetic Resonance Imaging in Asymptomatic C9orf72 Mutation Carriers Distinguishes Phenoconverters to Amyotrophic Lateral Sclerosis or Amyotrophic Lateral Sclerosis With Frontotemporal Dementia.

We prospectively studied asymptomatic C9orf72 mutation carriers, identifying those developing amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). We enrolled 56 asymptomatic family members (AFM) with a C9orf72 mutation (AFM C9+), 132 non-carriers (AFM C9-), and 359 population-based controls. Using 3 T magnetic resonance imaging, we measured cortical thickness, gyrification, and subcortical volumes longitudinally. Linear mixed-effects models on non-converting AFM C9+ scans (n = 107) created a reference for these measurements, establishing individual atrophy patterns. Atrophy patterns from presymptomatic phenoconverters (n = 10 scans) served as a template for group comparisons and similarity assessments. Similarity with phenoconverters was quantified using Dice similarity coefficient (DSC) for cortical and Kullback-Leibler similarity (KLS) for subcortical measures. Using longitudinal similarity assessments, we predicted when participants would reach the average similarity level of phenoconverters at their first post-onset scan. Five AFM C9+ converted to ALS or ALS-FTD. Up to 6 years before symptoms, these phenoconverters exhibited significant atrophy in frontal, temporal, parietal, and cingulate cortex, along with smaller thalamus, hippocampus, and amygdala compared to other AFM C9+. Some non-converted AFM C9+ had high DSC and KLS, approaching values of phenoconverters, whereas others, along with AFM C9- and controls, had lower values. At age 80, we predicted 27.9% (95% confidence interval, 13.2-40.1%) of AFM C9+ and no AFM C9- would reach the same DSC as phenoconverters. Distinctive atrophy patterns are visible years before symptom onset on presymptomatic scans of phenoconverters. Combining baseline and follow-up similarity measures may serve as a promising imaging biomarker for identifying those at risk of ALS or ALS-FTD. ANN NEUROL 2024.

EyeLiner: A Deep Learning Pipeline for Longitudinal Image Registration using Fundus Landmarks

ObjectiveDetecting and measuring changes in longitudinal fundus imaging is key to monitoring disease progression in chronic ophthalmic diseases, such as glaucoma and macular degeneration. Clinicians assess changes in disease status by either independently reviewing or manually juxtaposing longitudinally acquired color fundus photos (CFPs). Distinguishing variations in image acquisition due to camera orientation, zoom, and exposure from true disease-related changes can be challenging. This makes manual image evaluation variable and subjective, potentially impacting clinical decision making. We introduce our deep learning pipeline, “EyeLiner,” for registering, or aligning, two-dimensional CFPs. Improved alignment of longitudinal image pairs may compensate for differences that are due to camera orientation, while preserving pathological changes. DesignEyeLiner registers a “moving” image to a “fixed” image using a deep learning-based keypoint matching algorithm. ParticipantsWe evaluate EyeLiner on three longitudinal datasets: Fundus Image REgistration (FIRE), Sequential Fundus for Glaucoma Forecast (SIGF), and our internal glaucoma dataset from the Colorado Ophthalmology Research Information System (CORIS). MethodsAnatomical keypoints along the retinal blood vessels are detected from the moving and fixed images using a convolutional neural network and subsequently matched using a transformer-based algorithm. Finally, transformation parameters are learned using the corresponding keypoints. Main Outcome MeasuresWe computed the mean distance (MD) between manually annotated keypoints from the fixed and the registered moving image. For comparison to existing state-of-the-art retinal registration approaches, we used the mean area under the curve (mAUC) metric introduced in the FIRE dataset study. ResultsEyeLiner effectively aligns longitudinal image pairs from FIRE, SIGF and CORIS, as qualitatively evaluated through registration checkerboards and flicker animations. Quantitative results show that the MD decreased for this model after alignment from 321.32 to 3.74 pixels for FIRE, 9.86 to 2.03 pixels for CORIS, and 25.23 to 5.94 pixels for SIGF. We also obtain an AUC of 0.85, 0.94 and 0.84 on FIRE, CORIS and SIGF respectively, beating the current state-of-the-art SuperRetina (AUCFIRE=0.76, AUCCORIS=0.83, AUCSIGF=0.74). ConclusionsOur pipeline demonstrates improved alignment of image pairs in comparison to the current state-of-the-art methods on three separate datasets. We envision that this method will enable clinicians to align image pairs, and better visualize changes in disease over time.

Longitudinal imaging of therapeutic enzyme expression after gene therapy for Fabry disease using positron emission tomography and the radiotracer [18F]AGAL

Longitudinal, non-invasive, in vivo monitoring of therapeutic gene expression is an unmet need for gene therapy (GT). Positron Emission Tomography (PET) radiotracers designed to bind to therapeutic proteins may provide a sensitive imaging platform to guide treatment response and dose optimization in GT. Herein, we evaluated a novel PET tracer ([18F]AGAL) for targeting alpha galactosidase A (α-GalA), an enzyme deficient in Fabry disease. Gla knockout mice were subjected to either GT with an adeno-associated virus encoding the human a-Gal A (AAVGLA) or recombinant α-GalA for enzyme replacement studies. PET imaging, ex vivo autoradiography, biochemical analyses and radiation dosimetry were performed. [18F]AGAL exhibited pH-dependent binding to a-GalA, suggesting recognition of the active enzyme residing within the acidified lysosomes. Imaging studies in the Fabry mouse model showed quick renal clearance with high radioactive uptake in the heart at 6 weeks that was sustained for 26 weeks after a single administration of AAVGLA, indicating effective and durable transgene expression from GT. Good concordance was achieved between in vivo PET imaging and ex vivo quantification of α-GalA levels in biofluids and tissues. Biodistribution and dosimetry in non-human primate showed acceptable radiation exposure for multiple injections demonstrating its potential for translation to clinical trial use.

Deep learning model for diagnosis of thyroid nodules with size less than 1 cm: A multicenter, retrospective study

ObjectiveTo develop a ultrasound images based dual-channel deep learning model to achieve accurate early diagnosis of thyroid nodules less than 1 cm. MethodsA dual-channel deep learning model called thyroid nodule transformer network (TNT-Net) was proposed. The model has two input channels for transverse and longitudinal ultrasound images of thyroid nodules, respectively. A total of 9649 nodules from 8455 patients across five hospitals were retrospectively collected. The data were divided into a training set (8453 nodules, 7369 patients), an internal test set (565 nodules, 512 patients), and an external test set (631 nodules, 574 patients). ResultsTNT-Net achieved an area under the curve (AUC) of 0.953 (95 % confidence interval (CI): 0.934, 0.969) on the internal test set and 0.941 (95 % CI: 0.921, 0.957) on the external test set, significantly outperforming traditional deep convolutional neural network models and single-channel swin transformer model, whose AUCs ranged from 0.800 (95 % CI: 0.759, 0.837) to 0.856 (95 % CI: 0.819, 0.881). Furthermore, feature heatmap visualization showed that TNT-Net could extract richer and more energetic malignant nodule patterns. ConclusionThe proposed TNT-Net model significantly improved the recognition capability for thyroid nodules with size less than 1 cm. This model has the potential to reduce overdiagnosis and overtreatment of such nodules, providing essential support for precise management of thyroid nodules while complementing fine-needle aspiration biopsy.

Quantitative micro-CT-derived biomarkers elucidate age-related lung fibrosis in elder mice

BackgroundIdiopathic Pulmonary Fibrosis (IPF), prevalently affecting individuals over 60 years of age, has been mainly studied in young mouse models. The limited efficacy of current treatments underscores the need for animal models that better mimic an aged patient population. We addressed this by inducing pulmonary fibrosis in aged mice, using longitudinal micro-CT imaging as primary readout, with special attention to animal welfare.MethodsA double bleomycin dose was administered to 18–24 months-old male C57Bl/6j mice to induce pulmonary fibrosis. Bleomycin dosage was reduced to as low as 75% compared to that commonly administered to young (8–12 weeks-old) mice, resulting in long-term lung fibrosis without mortality, complying with animal welfare guidelines. After fibrosis induction, animals received Nintedanib once-daily for two weeks and longitudinally monitored by micro-CT, which provided structural and functional biomarkers, followed by post-mortem histological analysis as terminal endpoint.ResultsCompared to young mice, aged animals displayed increased volume, reduced tissue density and function, and marked inflammation. This increased vulnerability imposed a bleomycin dosage reduction to the lowest tested level (2.5 µg/mouse), inducing a milder, yet persistent, fibrosis, while preserving animal welfare. Nintedanib treatment reduced fibrotic lesions and improved pulmonary function.ConclusionsOur data identify a downsized bleomycin treatment that allows to achieve the best trade-off between fibrosis induction and animal welfare, a requirement for antifibrotic drug testing in aged lungs. Nintedanib displayed significant efficacy in this lower-severity disease model, suggesting potential patient stratification strategies. Lung pathology was quantitatively assessed by micro-CT, pointing to the value of longitudinal endpoints in clinical trials.

Development of a nomogram-based model incorporating radiomic features from follow-up longitudinal lung CT images to distinguish invasive adenocarcinoma from benign lesions: a retrospective study

PurposeTo develop and validate a radiomic model for differentiating pulmonary invasive adenocarcinomas from benign lesions based on follow-up longitudinal CT images.MethodsThis is a retrospective study including 336 patients (161 with invasive adenocarcinomas and 175 with benign lesions) who underwent baseline (T0) and follow-up (T1) CT scans from January 2016 to June 2022. The patients were randomized in a 7:3 ratio into training and test sets. Radiomic features were extracted from lesion volumes of interest on longitudinal CT images at T0 and T1. Differences in radiomic features between T1 and T0 were defined as delta-radiomic features. Logistic regression was used to build models based on clinicoradiological (CR), T0, T1, and delta radiomic features and compute signatures. Finally, a nomogram based on the CR, T0, T1 and delta signatures was constructed. Model performance was evaluated for calibration, discrimination, and clinical utility.ResultsThe T1 radiomic model was superior to the other independent models. In the training set, it had an area under the curve (AUC) of 0.858), superior to the CR model (AUC 0.694), the T0 radiomic model (AUC 0.825), and the delta radiomic model (AUC 0.734). In the test set, it had an AUC of 0.817, again outperforming the CR model (AUC 0.578), the T0 radiomic model (AUC 0.789), and the delta radiomic model (AUC 0.647). The nomogram incorporating the CR, T0, T1 and delta signatures showed the best predictive performance in both the training (AUC: 0.906) and test sets (AUC: 0.856), and it exhibited excellent fit with calibration curves. Decision curve analysis provided additional validation of the clinical utility of the nomogram.ConclusionA nomogram utilizing radiomic features extracted from longitudinal CT images can enhance the discriminative capability between pulmonary invasive adenocarcinomas and benign lesions.

Synthesis and Evaluation of 68Ga- and 177Lu-Labeled [Pro14]bombesin(8-14) Derivatives for Detection and Radioligand Therapy of Gastrin-Releasing Peptide Receptor-Expressing Cancer.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in a variety of cancers and represents a promising target for diagnosis and therapy. However, the extremely high accumulation in the pancreas observed for most of the clinically evaluated GRPR-targeted radiopharmaceuticals could limit their applications. In this study, we synthesized one GRPR antagonist (ProBOMB5) and two GRPR agonists (LW02056 and LW02057) by replacing the 4-thiazolidinecarboxylic acid (Thz14) residue in our previously reported GRPR-targeted tracers with Pro14. The 68Ga and 177Lu labeling were conducted in HEPES (2 M, pH 5.0) buffer and acetate (0.1 M, pH 4.5) buffer, respectively, and the radiolabeled products were obtained in a 24-57% decay-corrected radiochemical yield and >92% radiochemical purity. The binding affinities (Ki) of Ga-ProBOMB5, Ga-LW02056, Ga-LW02057, and Lu-ProBOMB5 were measured via in vitro competition binding assays and were 12.2 ± 1.89, 14.7 ± 4.81, 13.8 ± 2.24, and 13.6 ± 0.25 nM, respectively. The PET imaging and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice at 1 h post injection. [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 enabled clear tumor visualization in PET images. The tumor uptake values of [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 were 12.4 ± 1.35, 8.93 ± 1.96, and 7.64 ± 0.55%ID/g, respectively, and their average pancreas uptake values were minimal (0.60-1.37%ID/g). Longitudinal SPECT imaging and ex vivo biodistribution studies were also conducted for [177Lu]Lu-ProBOMB5 and clinically validated [177Lu]Lu-RM2. Despite comparable tumor uptake at 1 h post injection ([177Lu]Lu-ProBOMB5:8.09 ± 1.70%ID/g; [177Lu]Lu-RM2:7.73 ± 0.96%ID/g), a faster clearance from PC-3 tumor xenografts was observed for [177Lu]Lu-ProBOMB5, leading to a lower radiation-absorbed dose delivered to tumors. Our data demonstrate that [68Ga]Ga-ProBOMB5 is a promising tracer for clinical translation for detecting GRPR-expressing tumor lesions. However, further optimizations are needed for [177Lu]Lu-ProBOMB5 to prolong tumor retention for therapeutic applications.

A novel framework for elucidating the effect of mechanical loading on the geometry of ovariectomized mouse tibiae using principal component analysis.

Murine models are used to test the effect of anti-osteoporosis treatments as they replicate some of the bone phenotypes observed in osteoporotic (OP) patients. The effect of disease and treatment is typically described as changes in bone geometry and microstructure over time. Conventional assessment of geometric changes relies on morphometric scalar parameters. However, being correlated with each other, these parameters do not describe separate fractions of variations and offer only a moderate insight into temporal changes. The current study proposes a novel image-based framework that employs deformable image registration on in vivo longitudinal images of bones and Principal Component Analysis (PCA) for improved quantification of geometric effects of OP treatments. This PCA-based model and a novel post-processing of score changes provide orthogonal modes of shape variations temporally induced by a course of treatment (specifically in vivo mechanical loading). Errors associated with the proposed framework are rigorously quantified and it is shown that the accuracy of deformable image registration in capturing the bone shapes (∼1 voxel = 10.4μm) is of the same order of magnitude as the relevant state-of-the-art evaluation studies. Applying the framework to longitudinal image data from the midshaft section of ovariectomized mouse tibia, two mutually orthogonal mode shapes are reliably identified to be an effect of treatment. The mode shapes captured changes of the tibia geometry due to the treatment at the anterior crest (maximum of 0.103mm) and across the tibia midshaft section and the posterior (0.030mm) and medial (0.024mm) aspects. These changes agree with those reported previously but are now described in a compact fashion, as a vector field of displacements on the bone surface. The proposed framework enables a more detailed investigation of the effect of disease and treatment on bones in preclinical studies and boosts the precision of such assessments.

Hearing loss and its relation to longitudinal changes in white matter microstructure in older adults: The Rotterdam Study

Hearing loss is considered a potentially modifiable risk factor for dementia. The sensory deprivation theory postulates that hearing loss adversely affects cognition in older adults through structural brain changes, but longitudinal studies are scarce. To find evidence for a possible detrimental effect of hearing loss on white matter microstructure, we carried out a longitudinal study in the population-based Rotterdam Study. A total of 1877 participants with a median age at baseline of 56.4 years (IQR: [52.2–60.0]) underwent audiometry and had longitudinal diffusion imaging data available with a mean follow-up of 4.0 years. A lower level of hearing acuity was associated with worse white matter microstructure in the left uncinate fasciculus and superior longitudinal fasciculus at baseline. Poorer hearing acuity was also associated with faster microstructural deterioration over time in the left superior longitudinal fasciculus. The strongest effects were observed for low-frequency hearing thresholds, while the high-frequency thresholds showed the weakest associations. These results suggest that hearing loss may contribute to the age-related decline in brain structure, consistent with the sensory deprivation theory.

Patterns of Brain Maturation in Autism and Their Molecular Associations

In the neurotypical brain, regions develop in coordinated patterns, providing a fundamental scaffold for brain function and behavior. Whether altered patterns contribute to clinical profiles in neurodevelopmental conditions, including autism, remains unclear. To examine if, in autism, brain regions develop differently in relation to each other and how these differences are associated with molecular/genomic mechanisms and symptomatology. This study was an analysis of one the largest deep-phenotyped, case-control, longitudinal (2 assessments separated by approximately 12-24 months) structural magnetic resonance imaging and cognitive-behavioral autism datasets (EU-AIMS Longitudinal European Autism Project [LEAP]; study dates, February 2014-November 2017) and an out-of-sample validation in the Brain Development Imaging Study (BrainMapASD) independent cohort. Analyses were performed during the 2022 to 2023 period. This multicenter study included autistic and neurotypical children, adolescents, and adults. Autistic participants were included if they had an existing autism diagnosis (DSM-IV/International Statistical Classification of Diseases and Related Health Problems, Tenth Revision or DSM-5 criteria). Autistic participants with co-occurring psychiatric conditions (except psychosis/bipolar disorder) and those taking regular medications were included. Neuroanatomy of neurotypical and autistic participants. Intraindividual changes in surface area and cortical thickness over time, analyzed via surface-based morphometrics. A total of 386 individuals in the LEAP cohort (6-31 years at first visit; 214 autistic individuals, mean [SD] age, 17.3 [5.4] years; 154 male [72.0%] and 172 neurotypical individuals, mean [SD] age, 16.35 [5.7] years; 108 male [62.8%]) and 146 individuals in the BrainMapASD cohort (11-18 years at first visit; 49 autistic individuals, mean [SD] age, 14.31 [2.4] years; 42 male [85.7%] and 97 neurotypical individuals, mean [SD] age, 14.10 [2.5] years; 58 male [59.8%]). Maturational between-group differences in cortical thickness and surface area were established that were mostly driven by sensorimotor regions (eg, across features, absolute loadings for early visual cortex ranged from 0.07 to 0.11, whereas absolute loadings for dorsolateral prefrontal cortex ranged from 0.005 to 0.06). Neurodevelopmental differences were transcriptomically enriched for genes expressed in several cell types and during various neurodevelopmental stages, and autism candidate genes (eg, downregulated genes in autism, including those regulating synaptic transmission; enrichment odds ratio =3.7; P =2.6 × -10). A more neurotypical, less autismlike maturational profile was associated with fewer social difficulties and more typical sensory processing (false discovery rate P <.05; Pearson r ≥0.17). Results were replicated in the independently collected BrainMapASD cohort. Results of this case-control study suggest that the coordinated development of brain regions was altered in autism, involved a complex interplay of temporally sensitive molecular mechanisms, and may be associated with both lower-order (eg, sensory) and higher-order (eg, social) clinical features of autism. Thus, examining maturational patterns may provide an analytic framework to study the neurobiological origins of clinical profiles in neurodevelopmental/mental health conditions.

TdCoxSNN: Time-dependent Cox survival neural network for continuous-time dynamic prediction

Abstract The aim of dynamic prediction is to provide individualized risk predictions over time, which are updated as new data become available. In pursuit of constructing a dynamic prediction model for a progressive eye disorder, age-related macular degeneration (AMD), we propose a time-dependent Cox survival neural network (tdCoxSNN) to predict its progression using longitudinal fundus images. tdCoxSNN builds upon the time-dependent Cox model by utilizing a neural network to capture the nonlinear effect of time-dependent covariates on the survival outcome. Moreover, by concurrently integrating a convolutional neural network with the survival network, tdCoxSNN can directly take longitudinal images as input. We evaluate and compare our proposed method with joint modelling and landmarking approaches through extensive simulations. We applied the proposed approach to two real datasets. One is a large AMD study, the Age-Related Eye Disease Study, in which more than 50,000 fundus images were captured over a period of 12 years for more than 4,000 participants. Another is a public dataset of the primary biliary cirrhosis disease, where multiple laboratory tests were longitudinally collected to predict the time-to-liver transplant. Our approach demonstrates commendable predictive performance in both simulation studies and the analysis of the two real datasets.

Real-Time Imaging Assessment of Stress-Induced Vascular Inflammation Using Heartbeat-Synchronized Motion Compensation.

Chronic mental stress accelerates atherosclerosis through complicated neuroimmune pathways, needing for advanced imaging techniques to delineate underlying cellular mechanisms. While histopathology, ex vivo imaging, and snapshots of in vivo images offer promising evidence, they lack the ability to capture real-time visualization of blood cell dynamics within pulsatile arteries in longitudinal studies. An electrically tunable lens was implemented in intravital optical microscopy, synchronizing the focal plane with heartbeats to follow artery movements. ApoE-/- mice underwent 2 weeks of restraint stress before baseline imaging followed by 2 weeks of stress exposure in the longitudinal imaging, while nonstressed mice remained undisturbed. The progression of vascular inflammation was assessed in the carotid arteries through intravital imaging and histological analyses. A 4-fold reduction of motion artifact, assessed by interframe SD, and an effective temporal resolution of 25.2 Hz were achieved in beating murine carotid arteries. Longitudinal intravital imaging showed chronic stress led to a 6.09-fold (P=0.017) increase in myeloid cell infiltration compared with nonstressed mice. After 3 weeks, we observed that chronic stress intensified vascular inflammation, increasing adhered myeloid cells by 2.45-fold (P=0.031), while no significant changes were noted in nonstressed mice. Microcirculation imaging revealed increased circulating, rolling, and adhered cells in stressed mice's venules. Histological analysis of the carotid arteries confirmed the in vivo findings that stress augmented plaque area, myeloid cell and macrophage accumulation, and necrotic core volume while reducing fibrous cap thickness indicating accelerated plaque formation. We visualized the 3-dimensional structure of the carotid artery and 4-dimensional dynamics of the venules in the cremaster muscle. Dynamic focusing motion compensation intravital microscopy enabled subcellular resolution in vivo imaging of blood cell dynamics in beating arteries under chronic restraint stress in real time. This novel technique emphasizes the importance of advanced in vivo imaging for understanding cardiovascular disease.

Imaging the large-scale and cellular response to focal traumatic brain injury in mouse neocortex.

Traumatic brain injury (TBI) affects neural function at the local injury site and also at distant, connected brain areas. However, the real-time neural dynamics in response to injury and subsequent effects on sensory processing and behavior are not fully resolved, especially across a range of spatial scales. We used in vivo calcium imaging in awake, head-restrained male and female mice to measure large-scale and cellular resolution neuronal activation, respectively, in response to a mild/moderate TBI induced by focal controlled cortical impact (CCI) injury of the motor cortex (M1). Widefield imaging revealed an immediate CCI-induced activation at the injury site, followed by a massive slow wave of calcium signal activation that traveled across the majority of the dorsal cortex within approximately 30 s. Correspondingly, two-photon calcium imaging in primary somatosensory cortex (S1) found strong activation of neuropil and neuronal populations during the CCI-induced traveling wave. A depression of calcium signals followed the wave, during which we observed atypical activity of a sparse population of S1 neurons. Longitudinal imaging in the hours and days after CCI revealed increases in the area of whisker-evoked sensory maps at early time points, in parallel to decreases in cortical functional connectivity and behavioral measures. Neural and behavioral changes mostly recovered over hours to days in our M1-TBI model, with a more lasting decrease in the number of active S1 neurons. Our results in unanesthetized mice describe novel spatial and temporal neural adaptations that occur at cortical sites remote to a focal brain injury.

Ultrasonography for non-invasive sex identification and reproductive assessment in Nile tilapia (Oreochromis niloticus).

Sex identification and the selection of monosex male tilapia are crucial for tilapia aquaculture. In this study, we evaluated the application of ultrasonography as an alternative procedure for sex identification and reproductive assessment in Nile tilapia (Oreochromis niloticus). Ultrasonography was performed on 23 fish weighing 232-1,281 g to capture longitudinal and transverse images of the ovaries and testes. Female tilapia were identified by the presence of numerous small egg granules and gray or light gray ovarian tissue and male tilapia by the homogeneous echogenicity and uniform gray tubular appearance of the testes. These distinct echogenic patterns allowed for accurate differentiation between the female and male fish. Ultrasonography achieved a 95% accuracy in sex identification, with minimal stress to the fish during the procedure, compared to manual sex sorting, which demonstrated an accuracy of 87%. Furthermore, the method facilitated the assessment of the reproductive status of the fish, including the maturation stages and fecundity potential. The use of ultrasonography offers significant advantages over traditional methods, such as animal welfare enhancements and improved breeding program efficiency. Our findings support the integration of ultrasonography into aquaculture practices and will promote sustainable and humane fish farming while optimizing reproductive management and productivity.

Longitudinal Imaging of the Foveal Cone Mosaic in CNGA3-Associated Achromatopsia.

The purpose of this study was to assess the natural history of the foveal cone mosaic in CNGA3-associated achromatopsia (ACHM). Thirteen eyes from 10 genetically confirmed patients underwent longitudinal imaging with optical coherence tomography (OCT) and non-confocal split detection adaptive optics scanning light ophthalmoscopy (AOSLO). OCT scans assessed outer nuclear layer (ONL) thickness, foveal ellipsoid zone (EZ) disruption, and foveal hypoplasia. AOSLO images were analyzed to calculate peak foveal cone density (PCD) and mean inter-cell distance (ICD) between cones. Mixed effects models were used to analyze the rate of annual change of PCD and ICD. Mean (±SD) age at visits was 29 ± 10 years, with a follow-up of 2.6 ± 1 years. There was no change in ONL thickness, degree of EZ disruption, or foveal hypoplasia over the follow-up period. We also observed a stable foveal cone mosaic using AOSLO imaging, with no significant change in PCD or ICD. Mean PCD was 15,346 cones/mm² at the mean age of 29 years old (cf. 64,000-324,000 cones/mm² in previously reported healthy controls), with a mean rate of change of -117.79 cones/mm² (0.8%) per year, P = 0.130. Mean ICD at the mean age was 13.82 µm, with a rate of change of 0.17 µm per year, P = 0.83. CNGA3-associated ACHM displays stable foveal cone structure over time with a similar rate of change to CNGB3-associated ACHM (2% decline per year). The stable PCD, small cohort, and large variability within the cohort means significant age associations were not detected.

Revealing real-time 3D in vivo pathogen dynamics in plants by label-free optical coherence tomography

Microscopic imaging for studying plant-pathogen interactions is limited by its reliance on invasive histological techniques, like clearing and staining, or, for in vivo imaging, on complicated generation of transgenic pathogens. We present real-time 3D in vivo visualization of pathogen dynamics with label-free optical coherence tomography. Based on intrinsic signal fluctuations as tissue contrast we image filamentous pathogens and a nematode in vivo in 3D in plant tissue. We analyze 3D images of lettuce downy mildew infection (Bremia lactucae) to obtain hyphal volume and length in three different lettuce genotypes with different resistance levels showing the ability for precise (micro) phenotyping and quantification of the infection level. In addition, we demonstrate in vivo longitudinal imaging of the growth of individual pathogen (sub)structures with functional contrast on the pathogen micro-activity revealing pathogen vitality thereby opening a window on the underlying molecular processes.

Letrozole-Based Near-Infrared Dynamic Imaging Targeting Ductal-Vascular RhoJ From Pancreatic Intraepithelial Neoplasia to Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) relies heavily on neoangiogenesis for its progression, making early detection crucial. Here, LTZi-MHI148 (Letrozole inhibitor bonding with MHI-148 dye), a near-infrared (NIR) fluorescent agent is developed, to target RhoJ (Ras Homolog Family Member J), a protein expressed in neonatal vasculature, for both imaging and therapy of early PDAC. This agent is synthesized by conjugating Letrozole with MHI-148, exhibiting excellent NIR characteristics and photostability. In vitro studies showed that LTZi-MHI148 selectively accumulated within pancreatic cancer cells through Organic Anion Transporting Polypeptide (OATP) transporters and bound to cytoplasmic RhoJ. In vivo, the probe effectively targeted neoangiogenesis and Pancreatic Intraepithelial Neoplasias (PanINs) in various PDAC models, including the orthotopic, ectopic, spontaneous, and tamoxifen-induced tumors. Notably, LTZi-MHI148 detected preneoplastic PanIN lesions with Overexpressed RhoJ and active neoangiogenesis in both spontaneous and tamoxifen-induced PDAC murine models. Longitudinal imaging studies revealed that RhoJ-targeted neoangiogenesis tracks lesion progression, highlighting LTZi-MHI148's utility in monitoring disease progression. Furthermore, multiple LTZi-MHI148 administrations attenuated PanINs to PDAC progression, suggesting its potential as a therapeutic intervention. These findings underscore the translational potential of LTZi-MHI148 for the early detection and targeted therapy of PDAC, utilizing NIR-I/II imaging to monitor RhoJ overexpression in precancerous ductal neoplasia associated with neoangiogenesis.

A 3D imaging system for dosimetry in FLASH radiotherapy

This study examines the feasibility of reconstructing the spatial dose distribution delivered by a 9 MeV electron beam within a plastic scintillator block using optical tomography. The system is designed for use in 3D-dosimetry for clinical applications in quality assurance and beam characterization, especially with FLASH-beam sources. The proposed detector reconstructs the 3D dose distribution map employing a monolith with 10 cm side, imaged in four different projections by scientific cameras. The 3D dose distribution simulated with Geant4 was reconstructed using a maximum likelihood expectation maximization algorithm applied to the lateral projection, resulting in a longitudinal image with a standard deviation of 0.2 Gy. Furthermore, a preliminary experimental test has been performed.