Altered IGF-I activity and accelerated bone elongation in growth plates precedes excess weight gain in a mouse model of juvenile obesity

Nearly one-third of children in the United States are overweight or obese by their pre-teens. Tall stature and accelerated bone elongation are characteristic features of childhood obesity, which co-occur with conditions such as limb bowing, slipped epiphyses, and fractures. Obese children paradoxically have normal circulating IGF-I, the major growth-stimulating hormone. Here we describe and validate a mouse model of excess dietary fat to examine mechanisms of growth acceleration in obesity. We used in vivo multiphoton imaging and immunostaining to test the hypothesis that high-fat diet increases IGF-I activity and alters growth plate structure before the onset of obesity. We tracked bone and body growth in male and female C57BL/6 mice (N = 114) on high-fat (60% kcal fat) or control (10% kcal fat) diets from weaning (3-weeks) to skeletal maturity (12-weeks). Tibial and tail elongation rates increased after brief (1-2 week) high-fat diet exposure without altering serum IGF-I. Femoral bone density and growth plate size were increased, but growth plates were disorganized in not-yet-obese high-fat diet mice. Multiphoton imaging revealed more IGF-I in the vasculature surrounding growth plates of high-fat diet mice, and increased uptake when vascular levels peaked. High-fat diet growth plates had more activated IGF-I receptors and fewer inhibitory binding proteins, suggesting increased IGF-I bioavailability in growth plates. These results, which parallel pediatric growth patterns, highlight the fundamental role of diet in the earliest stages of developing obesity-related skeletal complications and validate the utility of the model for future studies aimed at determining mechanisms of diet-enhanced bone lengthening.

Self-confocal NIR-II fluorescence microscopy for in vivo imaging

Benefiting from low scatter of NIR-II light in biological tissues and high spatial resolution of confocal microscopy, NIR-II fluorescence confocal microscopy has been developed recently and achieve deep imaging in vivo. However, independence of excitation point and detection point makes this system difficult to be adjusted. New, improved, self-confocal NIR-II fluorescence confocal systems are created in this work. Based on a shared pinhole for excitation light and fluorescence, the system is easy and controlled to be adjusted. The fiber-pinhole confocal system is constructed for cerebrovascular and hepatocellular NIR-II fluorescence intensity imaging. The air-pinhole confocal system is constructed for cerebrovascular NIR-II fluorescence intensity imaging, hepatic NIR-II fluorescence lifetime imaging, and hepatic multiphoton imaging.

Pigmentation Affects Elastic Fiber Patterning and Biomechanical Behavior of the Murine Aortic Valve

The aortic valve (AoV) maintains unidirectional blood distribution from the left ventricle of the heart to the aorta for systemic circulation. The AoV leaflets rely on a precise extracellular matrix microarchitecture of collagen, elastin, and proteoglycans for appropriate biomechanical performance. We have previously demonstrated a relationship between the presence of pigment in the mouse AoV with elastic fiber patterning using multiphoton imaging. Here, we extended those findings using wholemount confocal microscopy revealing that elastic fibers were diminished in the AoV of hypopigmented mice (KitWv and albino) and were disorganized in the AoV of K5-Edn3 transgenic hyperpigmented mice when compared to wild type C57BL/6J mice. We further used atomic force microscopy to measure stiffness differences in the wholemount AoV leaflets of mice with different levels of pigmentation. We show that AoV leaflets of K5-Edn3 had overall higher stiffness (4.42 ± 0.35 kPa) when compared to those from KitWv (2.22 ± 0.21 kPa), albino (2.45 ± 0.16 kPa), and C57BL/6J (3.0 ± 0.16 kPa) mice. Despite the striking elastic fiber phenotype and noted stiffness differences, adult mutant mice were found to have no overt cardiac differences as measured by echocardiography. Our results indicate that pigmentation, but not melanocytes, is required for proper elastic fiber organization in the mouse AoV and dictates its biomechanical properties.

A Novel Assay Allowing Drug Self-Administration, Extinction, and Reinstatement Testing in Head-Restrained Mice

Multiphoton microscopy is one of several new technologies providing unprecedented insight into the activity dynamics and function of neural circuits. Unfortunately, some of these technologies require experimentation in head-restrained animals, limiting the behavioral repertoire that can be integrated and studied. This issue is especially evident in drug addiction research, as no laboratories have coupled multiphoton microscopy with simultaneous intravenous drug self-administration, a behavioral paradigm that has predictive validity for treatment outcomes and abuse liability. Here, we describe a new experimental assay wherein head-restrained mice will press an active lever, but not inactive lever, for intravenous delivery of heroin or cocaine. Similar to freely moving animals, we find that lever pressing is suppressed through daily extinction training and subsequently reinstated through the presentation of relapse-provoking triggers (drug-associative cues, the drug itself, and stressors). Finally, we show that head-restrained mice will show similar patterns of behavior for oral delivery of a sucrose reward, a common control used for drug self-administration experiments. Overall, these data demonstrate the feasibility of combining drug self-administration experiments with technologies that require head-restraint, such as multiphoton imaging. The assay described could be replicated by interested labs with readily available materials to aid in identifying the neural underpinnings of substance use disorder.

New Endothelial Mechanisms in Glomerular (Patho)biology and Proteinuria Development Captured by Intravital Multiphoton Imaging

In the past two decades, intravital imaging using multiphoton microscopy has provided numerous new visual and mechanistic insights into glomerular biology and disease processes including the function of glomerular endothelial cells (GEnC), podocytes, and the development of proteinuria. Although glomerular endothelial injury is known to precede podocyte damage in several renal diseases, the primary role of GEnCs in proteinuria development received much less attention compared to the vast field of podocyte pathobiology. Consequently, our knowledge of GEnC mechanisms in glomerular diseases is still emerging. This review highlights new visual clues on molecular and cellular mechanisms of GEnCs and their crosstalk with podocytes and immune cells that were acquired recently by the application of multiphoton imaging of the intact glomerular microenvironment in various proteinuric disease models. New mechanisms of glomerular tissue remodeling and regeneration are discussed based on results of tracking the fate and function of individual GEnCs using serial intravital multiphoton imaging over several days and weeks. The three main topics of this review include (i) the role of endothelial injury and microthrombi in podocyte detachment and albumin leakage via hemodynamic and mechanical forces, (ii) the alterations of the endothelial surface layer (glycocalyx) and its interactions with circulating immune cells in lupus nephritis, and (iii) the structural and functional remodeling and regeneration of GEnCs in hypertension, diabetes, and other experimental injury conditions. By the comprehensive visual portrayal of GEnCs and the many other contributing glomerular cell types, this review emphasizes the complexity of pathogenic mechanisms that result in proteinuria development.