Faculty Bibliography

Maternal care, including by non-biological parents, is important for offspring survival^1-8. Oxytocin^1,2,9-15, which is released by the hypothalamic paraventricular nucleus (PVN), is a critical maternal hormone. In mice, oxytocin enables neuroplasticity in the auditory cortex for maternal recognition of pup distress¹⁵. However, it is unclear how initial parental experience promotes hypothalamic signalling and cortical plasticity for reliable maternal care. Here we continuously monitored the behaviour of female virgin mice co-housed with an experienced mother and litter. This documentary approach was synchronized with neural recordings from the virgin PVN, including oxytocin neurons. These cells were activated as virgins were enlisted in maternal care by experienced mothers, who shepherded virgins into the nest and demonstrated pup retrieval. Virgins visually observed maternal retrieval, which activated PVN oxytocin neurons and promoted alloparenting. Thus rodents can acquire maternal behaviour by social transmission, providing a mechanism for adapting the brains of adult caregivers to infant needs via endogenous oxytocin.

Segmentation and mutant classification of high-frequency ultrasound (HFU) mouse embryo brain ventricle (BV) and body images can provide valuable information for developmental biologists. However, manual segmentation and identification of BV and body requires substantial time and expertise. This paper proposes an accurate, efficient and explainable deep learning pipeline for automatic segmentation and classification of the BV and body. For segmentation, a two-stage framework is implemented. The first stage produces a low-resolution segmentation map, which is then used to crop a region of interest (ROI) around the target object and serve as the probability map of the auto-context input for the second-stage fine-resolution refinement network. The segmentation then becomes tractable on high-resolution 3D images without time-consuming sliding windows. The proposed segmentation method significantly reduces inference time (102.36 to 0.09 s/volume≈1000x faster) while maintaining high accuracy comparable to previous sliding-window approaches. Based on the BV and body segmentation map, a volumetric convolutional neural network (CNN) is trained to perform a mutant classification task. Through backpropagating the gradients of the predictions to the input BV and body segmentation map, the trained classifier is found to largely focus on the region where the Engrailed-1 (En1) mutation phenotype is known to manifest itself. This suggests that gradient backpropagation of deep learning classifiers may provide a powerful tool for automatically detecting unknown phenotypes associated with a known genetic mutation.

The rodent heart is frequently used to study human cardiovascular disease (CVD). Although advanced cardiovascular ultrasound imaging methods are available for human clinical practice, application of these techniques to small animals remains limited due to the temporal and spatial-resolution demands. Here, an ultrasound vector-flow workflow is demonstrated that enables visualization and quantification of the complex hemodynamics within the mouse heart. Wild type (WT) and fibroblast growth factor homologous factor 2 (FHF2)-deficient mice (Fhf2KO/Y), which present with hyperthermia-induced ECG abnormalities highly reminiscent of Brugada syndrome, were used as a mouse model of human CVD. An 18-MHz linear array was used to acquire high-speed (30 kHz), plane-wave data of the left ventricle (LV) while increasing core body temperature up to 41.5°C. Hexplex (i.e., six output) processing of the raw data sets produced the output of vector-flow estimates (magnitude and phase); B-mode and color-Doppler images; Doppler spectrograms; and local time histories of vorticity and pericardium motion. Fhf2WT/Y mice had repeatable beat-to-beat cardiac function, including vortex formation during diastole, at all temperatures. In contrast, Fhf2KO/Y mice displayed dyssynchronous contractile motion that disrupted normal inflow vortex formation and impaired LV filling as temperature rose. The hexplex processing approach demonstrates the ability to visualize and quantify the interplay between hemodynamic and mechanical function in a mouse model of human CVD.

The cell cycle is a progression of four distinct phases (G1, S, G2, M), with various cycle proteins being essential in regulating this process. In breast cancer, alterations in the cell cycle and uncontrolled proliferation led to several studies interrogating the relationship between cyclins and their counterpart cyclin-dependent-kinases (CDKs). We aimed to develop a radiolabeled CDK4/6 inhibitor for breast cancer imaging. Our transfluorinated analog ([¹⁸F]-CDKi) was evaluated and validated as a novel PET imaging agent to quantify CDK4/6 expression in ER-positive HER2-negative breast cancer. Methods: [¹⁸F]-CDKi was synthesized and assayed for its inhibitory activities against CDK4/6 kinases. [¹⁸F]-CDKi was prepared with a 2-step automated synthetic strategy that yielded the final product with remarkable purity and molar activity. In vitro and in vivo biologic activity and specificity was assessed in a MCF-7 cell line and in mice bearing MCF-7 breast tumors. Non radioactive Palbociclib (Inbrance, Pfizer®) was used as blocking agent to investigate the binding specificity and selectivity of [¹⁸F]-CDKi. Results: To generate [¹⁸F]-CDKi, we introduced an F-18 prosthetic group (¹⁸F-fluorobenzoic acid, [¹⁸F-FBA]), and transformed palbociclib into a different, PET active functional molecule. [¹⁸F]-CDKi was obtained with an overall radiochemical uncorrected yield of 15% and radiochemical purity > 98 %. The total synthesis time from the start of synthesis to final injectable formulated tracer is 70 minutes. The retention time reported for [¹⁸F]-CDKi and [¹⁹F]-CDKi is 27.4 min as demonstrated by co-injection with [¹⁹F]-CDKi in a HPLC. The first experiments aimed to analyze pharmacokinetics (PK) and in vitro activity of [¹⁸F]-CDKi (figure 5, Blood HL). In vivo blood half-life [t1/2(weighted) = 7.03 minutes], and octanol/water phase partition coefficient (logDO/W = 1.91 ± 0.24) showed a mainly lipophilic behavior. [¹⁸F]-CDKi is stable in vitro and in vivo (>98% at 4h post injection) and maintained its potent targeting affinity to CDK4/6 (~¹³NM for CDK4 and ~15nM for CDK6). Cellular uptake experiments performed in MCF-7 breast cancer cell line (ER-positive/HER2-negative) demonstrated specific uptake with a maximum intracellular concentration of ~65% as early as 10 minutes post incubation. The tracer uptake was reduced to <5% when cells were co-incubated with a molar excess of Palbociclib. In vivo imaging and ex-vivo biodistribution of ER-positive/HER-2 negative MCF-7 breast cancer models showed a ~4%ID/g tumor specific uptake (reduced to ~0.3%ID/g with a 50-fold excess of cold palbociclib). A comprehensive biodistribution analysis revealed also a significantly lower activation of CDK4/6 in non-targeting organs. Conclusion: [¹⁸F]-CDKi represents the first ¹⁸F positron emission tomography (PET) CDK4/6 imaging agent and a promising imaging agent for ER-positive HER2-negative breast cancer.

Vascular changes occur early in the development of obstructive airways disease. However, the vascular remodeling and dysfunction due to World Trade Center-Particulate Matter (WTC-PM) exposure are not well described and are therefore the focus of this investigation. C57Bl/6 female mice oropharyngeally aspirated 200 µg of WTC-PM₅₃ or phosphate-buffered saline (PBS) (controls). 24-hours (24-hrs) and 1-Month (1-M) after exposure, echocardiography, micro-positron emission tomography(µ-PET), collagen quantification, lung metabolomics, assessment of antioxidant potential and soluble-receptor for advanced glycation end products (sRAGE) in bronchoalveolar lavage(BAL) and plasma were performed. 24-hrs post-exposure, there was a significant reduction in (1) Pulmonary artery(PA) flow-velocity and pulmonary ejection time(PET) (2) Pulmonary acceleration time(PAT) and PAT/PET, while (3) Aortic ejection time(AET) and velocity time integral(VTI) were increased, and (4) Aortic acceleration time (AAT)/AET, cardiac output and stroke volume were decreased compared to controls. 1-M post-exposure, there was also significant reduction of right ventricular diameter as right ventricle free wall thickness was increased and an increase in tricuspid E, A peaks and an elevated E/A. The pulmonary and cardiac standard uptake value and volume 1-M post-exposure was significantly elevated after PM-exposure. Similarly, α-smooth muscle actin(α-SMA) expression, aortic collagen deposition was elevated 1-M after PM exposure. In assessment of the metabolome, prominent subpathways included advanced glycation end products (AGEs), phosphatidylcholines, sphingolipids, saturated/unsaturated fatty acids, eicosanoids, and phospholipids. BAL superoxide dismutase(SOD), plasma total-antioxidant capacity activity, and sRAGE (BAL and plasma) were elevated after 24-hrs. PM exposure and associated vascular disease are a global health burden. Our study shows persistent WTC-Cardiorespiratory and Vascular Dysfunction (WTC-CaRVD), inflammatory changes and attenuation of antioxidant potential after PM exposure. Early detection of vascular disease is crucial to preventing cardiovascular deaths and future work will focus on further identification of bioactive therapeutic targets.

Microvascular rarefaction, or the decrease in vascular density, has been described in the cerebrovasculature of aging humans, rats, and, more recently, mice in the presence and absence of age-dependent diseases. Given the wide use of mice in modeling age-dependent human diseases of the cerebrovasculature, visualization, and quantification of the global murine cerebrovasculature is necessary for establishing the baseline changes that occur with aging. To provide in vivo whole-brain imaging of the cerebrovasculature in aging C57BL/6 mice longitudinally, contrast-enhanced magnetic resonance angiography (CE-MRA) was employed using a house-made gadolinium-bearing micellar blood pool agent. Enhancement in the vascular space permitted quantification of the detectable, or apparent, cerebral blood volume (aCBV), which was analyzed over 2 years of aging and compared to histological analysis of the cerebrovascular density. A significant loss in the aCBV was detected by CE-MRA over the aging period. Histological analysis via vessel-probing immunohistochemistry confirmed a significant loss in the cerebrovascular density over the same 2-year aging period, validating the CE-MRA findings. While these techniques use widely different methods of assessment and spatial resolutions, their comparable findings in detected vascular loss corroborate the growing body of literature describing vascular rarefaction aging. These findings suggest that such age-dependent changes can contribute to cerebrovascular and neurodegenerative diseases, which are modeled using wild-type and transgenic laboratory rodents.