Glucose-stimulated calcium dynamics in beta cells from C57BL/6J, C57BL/6N, and NMRI mice: A systematic comparison of activation, activity, and deactivation properties in tissue slices

Although mice are a very instrumental model in islet beta cell research, possible phenotypic differences between strains and substrains are largely neglected in the scientific community. In this study, we show important phenotypic differences in beta cell responses to glucose between NMRI, C57BL/6J, and C57BL/6N mice, i.e., the three most commonly used strains. High-resolution multicellular confocal imaging of beta cells in acute pancreas tissue slices was used to measure and quantitatively compare the calcium dynamics in response to a wide range of glucose concentrations. Strain- and substrain-specific features were found in all three phases of beta cell responses to glucose: a shift in the dose-response curve characterizing the delay to activation and deactivation in response to stimulus onset and termination, respectively, and distinct concentration-encoding principles during the plateau phase in terms of frequency, duration, and active time changes with increasing glucose concentrations. Our results underline the significance of carefully choosing and reporting the strain to enable comparison and increase reproducibility, emphasize the importance of analyzing a number of different beta cell physiological parameters characterizing the response to glucose, and provide a valuable standard for future studies on beta cell calcium dynamics in health and disease.

Consecutive-Day Ventricular and Atrial Cardiomyocyte Isolations from the Same Heart: Shifting the Cost–Benefit Balance of Cardiac Primary Cell Research

Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4–8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.

Micro-RNA 92a as a Therapeutic Target for Cardiac Microvascular Dysfunction in Diabetes

Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect.

A Hyaluronic Acid Demilune Scaffold and Polypyrrole-Coated Fibers Carrying Embedded Human Neural Precursor Cells and Curcumin for Surface Capping of Spinal Cord Injuries

Tissue engineering, including cell transplantation and the application of biomaterials and bioactive molecules, represents a promising approach for regeneration following spinal cord injury (SCI). We designed a combinatorial tissue-engineered approach for the minimally invasive treatment of SCI—a hyaluronic acid (HA)-based scaffold containing polypyrrole-coated fibers (PPY) combined with the RAD16-I self-assembling peptide hydrogel (Corning® PuraMatrix™ peptide hydrogel (PM)), human induced neural progenitor cells (iNPCs), and a nanoconjugated form of curcumin (CURC). In vitro cultures demonstrated that PM preserves iNPC viability and the addition of CURC reduces apoptosis and enhances the outgrowth of Nestin-positive neurites from iNPCs, compared to non-embedded iNPCs. The treatment of spinal cord organotypic cultures also demonstrated that CURC enhances cell migration and prompts a neuron-like morphology of embedded iNPCs implanted over the tissue slices. Following sub-acute SCI by traumatic contusion in rats, the implantation of PM-embedded iNPCs and CURC with PPY fibers supported a significant increase in neuro-preservation (as measured by greater βIII-tubulin staining of neuronal fibers) and decrease in the injured area (as measured by the lack of GFAP staining). This combination therapy also restricted platelet-derived growth factor expression, indicating a reduction in fibrotic pericyte invasion. Overall, these findings support PM-embedded iNPCs with CURC placed within an HA demilune scaffold containing PPY fibers as a minimally invasive combination-based alternative to cell transplantation alone.

Label-free imaging of collagen fibers in tissue slices using phase imaging with computational specificity

Evaluating the tissue collagen content in addition to the epithelial morphology has been proven to offer complementary information in histopathology, especially in disease stratification and patient survivability prediction. One imaging modality widely used for this purpose is second harmonic generation microscopy (SHGM), which reports on the nonlinear susceptibility associated with the collagen fibers. Another method is polarization light microscopy (PLM) combined with picrosirius-red (PSR) tissue staining. However, SHGM requires expensive equipment and provides limited throughput, while PLM and PSR staining are not part of the routine pathology workflow. Here, we advance phase imaging with computational specificity (PICS) to computationally infer the collagen distribution of unlabeled tissue, with high specificity. PICS utilizes deep learning to translate quantitative phase images (QPI) into corresponding PSR images with high accuracy and speed. Our results indicate that the distributions of collagen fiber orientation, length, and straightness reported by PICS closely match the ones from ground truth.

On-tissue spatial proteomics integrating MALDI-MS imaging with shotgun proteomics reveals soy consumption-induced biomarkers in a fragile X syndrome mouse model

Soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for unbiased protein biomarker identification in Fragile X syndrome (FXS) in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging (MSI) with label-free proteomics in a mouse model of FXS to map the spatial distribution and quantify the levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1,004 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in the GABAergic system, synaptic transmission, and co-expression network analysis indicated that protein in soy group was significantly associated with metabolism and synapse modules in the Fmr1KO brain. Ultimately, this spatial proteomics work laid the ground for identifying novel therapeutic targets and biomarkers for FXS.

Analysis of vascularization in thyroid gland nodes with superb microvascular imaging (SMI) and CD34 expression histology: a pilot study

Background The Doppler sonography technique known as "superb microvascular imaging" (SMI) is advancing sonographic micro vascularization imaging in various disciplines. In this study, we aimed to determine whether SMI could reliably reproduce the blood flow in thyroid nodes and whether malignancy could be diagnosed, based on vascularization properties. Immunhistochemical staining by CD34 and SMI where used to determine the vascularization of nodes in terms of quantified vascularization parameters gained by computational evaluation. Methods We used image analysis programs to investigate whether the quantitative value for vascularization strength in the thyroid node, measured with SMI, was correlated with the actual degree of vascularization, determined microscopically. We included 16 patients that underwent thyroid resections. We prepared thyroid gland tissue slices for immunohistochemistry and labelled endothelial cells with CD34 to visualize blood vessels microscopically. We used image analysis programs, ImageJ, to quantify SMI Doppler sonographic measurements and CellProfiler to quantify CD34 expression in histological sections. We evaluated the numeric values for diagnostic value in node differentiation. Furthermore, we compared these values to check for correlations. Results Among the 16 nodes studied, three harboured malignant tumours (18.75%): two papillary and one follicular carcinoma. Among the 13 benign lesions (81.25%), four harboured follicular adenomas. Malignant and benign nodes were not significantly different in sonographic (0.88 ± 0.89 vs. 1.13 ± 0.19; p = 0.2790) or immunohistochemical measurements of vascularization strength (0.05 ± 0.05 vs. 0.08 ± 0.06; p = 0.2260). Conclusion We found a positive, significant correlation (r = 0.55588; p = 0.0254) between SMI (quantitative values for vascularization strength) and immunohistochemistry (CD34 staining) evaluations of thyroid nodes.

2D vs 3D morphological analysis of dorsal root ganglia in health and painful neuropathy

Dorsal root ganglia (DRGs) are clusters of sensory neurons that transmit the sensory information from the periphery to the central nervous system, and satellite glial cells (SGCs), their supporting trophic cells. Sensory neurons are pseudounipolar neurons with a heterogeneous neurochemistry reflecting their functional features. DRGs, not protected by the blood brain barrier, are vulnerable to stress and damage of different origin (i.e., toxic, mechanical, metabolic, genetic) that can involve sensory neurons, SGCs or, considering their intimate intercommunication, both cell populations. DRG damage, primary or secondary to nerve damage, produces a sensory peripheral neuropathy, characterized by neurophysiological abnormalities, numbness, paraesthesia and dysesthesia, tingling and burning sensations and neuropathic pain. DRG stress can be morphologically detected by light and electron microscope analysis with alterations in cell size (swelling/atrophy) and in different sub-cellular compartments (i.e., mitochondria, endoplasmic reticulum, and nucleus) of neurons and/or SGCs. In addition, neurochemical changes can be used to portray abnormalities of neurons and SGC. Conventional immunostaining, i.e., immunohistochemical detection of specific molecules in tissue slices can be employed to detect, localize and quantify particular markers of damage in neurons (i.e., nuclear expression ATF3) or SGCs (i.e., increased expression of GFAP), markers of apoptosis (i.e., caspases), markers of mitochondrial suffering and oxidative stress (i.e., 8-OHdG), markers of tissue inflammation (i.e., CD68 for macrophage infiltration), etc. However classical (2D) methods of immunostaining disrupt the overall organization of the DRG, thus resulting in the loss of some crucial information. Whole-mount (3D) methods have been recently developed to investigate DRG morphology and neurochemistry without tissue slicing, giving the opportunity to study the intimate relationship between SGCs and sensory neurons in health and disease. Here, we aim to compare classical (2D) vs whole-mount (3D) approaches to highlight “pros” and “cons” of the two methodologies when analysing neuropathy-induced alterations in DRGs.