An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy in neuroscience.

Introduction: Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited. Methods: T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images. Results: Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR. Discussion: The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging.

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye.

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

Transplantation and Noninvasive Longitudinal In Vivo Imaging of Parathyroid Cells: A Proof-of-Concept Study.

The parathyroid cell is a vital regulator of extracellular calcium levels, operating through the secretion of parathyroid hormone (PTH). Despite its importance, the regulation of PTH secretion remains complex and not fully understood, representing a unique interplay between extracellular and intracellular calcium, and hormone secretion. One significant challenge in parathyroid research has been the difficulty in maintaining cells ex vivo for in-depth cellular investigations. To address this issue, we introduce a novel platform for parathyroid cell transplantation and noninvasive in vivo imaging using the anterior chamber of the eye as a transplantation site. We found that parathyroid adenoma tissue transplanted into the mouse eye engrafted onto the iris, became vascularized, and retained cellular composition. Transplanted animals exhibited elevated PTH levels, indicating a functional graft. With in vivo confocal microscopy, we were able to repetitively monitor parathyroid graft morphology and vascularization. In summary, there is a pressing need for new methods to study complex cellular processes in parathyroid cells. Our study provides a novel approach for noninvasive in vivo investigations that can be applied to understand parathyroid physiology and pathology under physiological and pathological conditions. This innovative strategy can deepen our knowledge on parathyroid function and disease.

Ruxolitinib: A new hope for ventilator-induced diaphragm dysfunction.

AIM: Mechanical ventilation (MV) results in diminished diaphragm size and strength, termed ventilator-induced diaphragm dysfunction (VIDD). VID increases dependence, prolongs weaning, and increases discharge mortality rates. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway is implicated in VIDD, upregulated following MV. JAK/STAT inhibition alleviates chronic muscle wasting conditions. This study aimed to explore the therapeutic potential of Ruxolitinib, an FDA approved JAK1/2 inhibitor (JI) for the treatment of VIDD. METHODS: Rats were subjected to 5 days controlled MV (CMV) with and without daily Ruxolitinib gavage. Muscle fiber size and function were assessed. RNAseq, mitochondrial morphology, respirometry, and mass spectrometry were determined. RESULTS: CMV significantly reduced diaphragm size and specific force by 45% (p < 0.01), associated with a two-fold P-STAT3 upregulation (p < 0.001). CMV disrupted mitochondrial content and reduced the oxygen consumption rate (p < 0.01). Expression of the motor protein myosin was unaffected, however CMV alters myosin function via post-translational modifications (PTMs). Daily administration of JI increased animal survival (40% vs. 87%; p < 0.05), restricted P-STAT3 (p < 0.001), and preserved diaphragm size and specific force. JI was associated with preserved mitochondrial content and respiratory function (p < 0.01), and the reversal or augmentation of myosin deamidation PTMs of the rod and head region. CONCLUSION: JI preserved diaphragm function, leading to increased survival in an experimental model of VIDD. Functional enhancement was associated with maintenance of mitochondrial content and respiration and the reversal of ventilator-induced PTMs of myosin. These results demonstrate the potential of repurposing Ruxolitinib for treatment of VIDD.

The anterior chamber of the eye technology and its anatomical, optical, and immunological bases.

The anterior chamber of the eye (ACE) is distinct in its anatomy, optics, and immunology. This guarantees that the eye perceives visual information in the context of physiology even when encountering adverse incidents like inflammation. In addition, this endows the ACE with the special nursery bed iris enriched in vasculatures and nerves. The ACE constitutes a confined space enclosing an oxygen/nutrient-rich, immune-privileged, and less stressful milieu as well as an optically transparent medium. Therefore, aside from visual perception, the ACE unexpectedly serves as an excellent transplantation site for different body parts and a unique platform for noninvasive, longitudinal, and intravital microimaging of different grafts. On the basis of these merits, the ACE technology has evolved from the prototypical through the conventional to the advanced version. Studies using this technology as a versatile biomedical research platform have led to a diverse range of basic knowledge and in-depth understanding of a variety of cells, tissues, and organs as well as artificial biomaterials, pharmaceuticals, and abiotic substances. Remarkably, the technology turns in vivo dynamic imaging of the morphological characteristics, organotypic features, developmental fates, and specific functions of intracameral grafts into reality under physiological and pathological conditions. Here we review the anatomical, optical, and immunological bases as well as technical details of the ACE technology. Moreover, we discuss major achievements obtained and potential prospective avenues for this technology.

Intraocular liver spheroids for non-invasive high-resolution in vivo monitoring of liver cell function.

Longitudinal monitoring of liver function in vivo is hindered by the lack of high-resolution non-invasive imaging techniques. Using the anterior chamber of the mouse eye as a transplantation site, we have established a platform for longitudinal in vivo imaging of liver spheroids at cellular resolution. Transplanted liver spheroids engraft on the iris, become vascularized and innervated, retain hepatocyte-specific and liver-like features and can be studied by in vivo confocal microscopy. Employing fluorescent probes administered intravenously or spheroids formed from reporter mice, we showcase the potential use of this platform for monitoring hepatocyte cell cycle activity, bile secretion and lipoprotein uptake. Moreover, we show that hepatic lipid accumulation during diet-induced hepatosteatosis is mirrored in intraocular in vivo grafts. Here, we show a new technology which provides a crucial and unique tool to study liver physiology and disease progression in pre-clinical and basic research.

3D-Printed Biohybrid Microstructures Enable Transplantation and Vascularization of Microtissues in the Anterior Chamber of the Eye.

Hybridizing biological cells with man-made sensors enable the detection of a wide range of weak physiological responses with high specificity. The anterior chamber of the eye (ACE) is an ideal transplantation site due to its ocular immune privilege and optical transparency, which enable superior noninvasive longitudinal analyses of cells and microtissues. Engraftment of biohybrid microstructures in the ACE may, however, be affected by the pupillary response and dynamics. Here, sutureless transplantation of biohybrid microstructures, 3D printed in IP-Visio photoresin, containing a precisely localized pancreatic islet to the ACE of mice is presented. The biohybrid microstructures allow mechanical fixation in the ACE, independent of iris dynamics. After transplantation, islets in the microstructures successfully sustain their functionality for over 20 weeks and become vascularized despite physical separation from the vessel source (iris) and immersion in a low-viscous liquid (aqueous humor) with continuous circulation and clearance. This approach opens new perspectives in biohybrid microtissue transplantation in the ACE, advancing monitoring of microtissue-host interactions, disease modeling, treatment outcomes, and vascularization in engineered tissues.

Sustained heterologous gene expression in pancreatic islet organoids using adeno-associated virus serotype 8.

Genetic modification of pancreatic islet organoids, assembled in vitro prior to transplantation is an emerging alternative to direct in vivo genetic manipulations for a number of clinical and research applications. We have previously shown that dispersion of islet cells followed by re-aggregation into islet organoids, or pseudoislets, allows for efficient transduction with viral vectors, while maintaining physiological functions of native islets. Among viruses currently used for genetic manipulations, adeno-associated viruses (AAVs) have the most attractive safety profile making them suitable for gene therapy applications. Studies reporting on pseudoislet transduction with AAVs are, however, lacking. Here, we have characterized in detail the performance of AAV serotype 8 in transduction of islet cells during pseudoislet formation in comparison with human adenovirus type 5 (AdV5). We have assessed such parameters as transduction efficiency, expression kinetics, and endocrine cell tropism of AAV8 alone or in combination with AdV5. Data provided within our study may serve as a reference point for future functional studies using AAVs for gene transfer to islet cell organoids and will facilitate further development of engineered pseudoislets of superior quality suitable for clinical transplantation.

Multiple Inositol Polyphosphate Phosphatase Compartmentalization Separates Inositol Phosphate Metabolism from Inositol Lipid Signaling.

Multiple inositol polyphosphate phosphatase (MINPP1) is an enigmatic enzyme that is responsible for the metabolism of inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6 pentakisphosphate (Ins(1,3,4,5,6)P5 in mammalian cells, despite being restricted to the confines of the ER. The reason for this compartmentalization is unclear. In our previous studies in the insulin-secreting HIT cell line, we expressed MINPP1 in the cytosol to artificially reduce the concentration of these higher inositol phosphates. Undocumented at the time, we noted cytosolic MINPP1 expression reduced cell growth. We were struck by the similarities in substrate preference between a number of different enzymes that are able to metabolize both inositol phosphates and lipids, notably IPMK and PTEN. MINPP1 was first characterized as a phosphatase that could remove the 3-phosphate from inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). This molecule shares strong structural homology with the major product of the growth-promoting Phosphatidyl 3-kinase (PI3K), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and PTEN can degrade both this lipid and Ins(1,3,4,5)P4. Because of this similar substrate preference, we postulated that the cytosolic version of MINPP1 (cyt-MINPP1) may not only attack inositol polyphosphates but also PtdIns(3,4,5)P3, a key signal in mitogenesis. Our experiments show that expression of cyt-MINPP1 in HIT cells lowers the concentration of PtdIns(3,4,5)P3. We conclude this reflects a direct effect of MINPP1 upon the lipid because cyt-MINPP1 actively dephosphorylates synthetic, di(C4:0)PtdIns(3,4,5)P3 in vitro. These data illustrate the importance of MINPP1's confinement to the ER whereby important aspects of inositol phosphate metabolism and inositol lipid signaling can be separately regulated and give one important clarification for MINPP1's ER seclusion.

In Vivo CaV3 Channel Inhibition Promotes Maturation of Glucose-Dependent Ca2+ Signaling in Human iPSC-Islets.

CaV3 channels are ontogenetically downregulated with the maturation of certain electrically excitable cells, including pancreatic ß cells. Abnormally exaggerated CaV3 channels drive the dedifferentiation of mature ß cells. This led us to question whether excessive CaV3 channels, retained mistakenly in engineered human-induced pluripotent stem cell-derived islet (hiPSC-islet) cells, act as an obstacle to hiPSC-islet maturation. We addressed this question by using the anterior chamber of the eye (ACE) of immunodeficient mice as a site for recapitulation of in vivo hiPSC-islet maturation in combination with intravitreal drug infusion, intravital microimaging, measurements of cytoplasmic-free Ca2+ concentration ([Ca2+]i) and patch clamp analysis. We observed that the ACE is well suited for recapitulation, observation and intervention of hiPSC-islet maturation. Intriguingly, intraocular hiPSC-islet grafts, retrieved intact following intravitreal infusion of the CaV3 channel blocker NNC55-0396, exhibited decreased basal [Ca2+]i levels and increased glucose-stimulated [Ca2+]i responses. Insulin-expressing cells of these islet grafts indeed expressed the NNC55-0396 target CaV3 channels. Intraocular hiPSC-islets underwent satisfactory engraftment, vascularization and light scattering without being influenced by the intravitreally infused NNC55-0396. These data demonstrate that inhibiting CaV3 channels facilitates the maturation of glucose-activated Ca2+ signaling in hiPSC-islets, supporting the notion that excessive CaV3 channels as a developmental error impede the maturation of engineered hiPSC-islet insulin-expressing cells.

Longitudinal monitoring of pancreatic islet damage in streptozotocin-treated mice with optical coherence microscopy.

Pancreatic islets regulate glucose homeostasis in the body, and their dysfunction is closely related to diabetes. Islet transplantation into the anterior chamber of the eye (ACE) was recently developed for both in vivo islet study and diabetes treatment. Optical coherence microscopy (OCM) was previously used to monitor ACE transplanted islets in non-obese diabetic (NOD) mice for detecting autoimmune attack. In this study, OCM was applied to streptozotocin (STZ)-induced diabetic mouse models for the early detection of islet damage. A custom extended-focus OCM (xfOCM) was used to image islet grafts in the ACE longitudinally during STZ-induced beta cell destruction together with conventional bright-field (BF) imaging and invasive glucose level measurement. xfOCM detected local structural changes and vascular degradation during the islet damage which was confirmed by confocal imaging of extracted islet grafts. xfOCM detection of islet damage was more sensitive than BF imaging and glucose measurement. Longitudinal xfOCM images of islet grafts were quantitatively analyzed. All these results showed that xfOCM could be used as a non-invasive and sensitive monitoring method for the early detection of deficient islet grafts in the ACE with potential applications to human subjects.

Novel aspects of intra-islet communication: Primary cilia and filopodia.

Pancreatic islets are micro-organs composed of a mixture of endocrine and non-endocrine cells, where the former secrete hormones and peptides necessary for metabolic homeostasis. Through vasculature and innervation the cells within the islets are in communication with the rest of the body, while they interact with each other through juxtacrine, paracrine and autocrine signals, resulting in fine-tuned sensing and response to stimuli. In this context, cellular protrusion in islet cells, such as primary cilia and filopodia, have gained attention as potential signaling hubs. During the last decade, several pieces of evidence have shown how the primary cilium is required for islet vascularization, function and homeostasis. These findings have been possible thanks to the development of ciliary/basal body specific knockout models and technological advances in microscopy, which allow longitudinal monitoring of engrafted islets transplanted in the anterior chamber of the eye in living animals. Using this technique in combination with optogenetics, new potential paracrine interactions have been suggested. For example, reshaping and active movement of filopodia-like protrusions of δ-cells were visualized in vivo, suggesting a continuous cell remodeling to increase intercellular contacts. In this review, we discuss these recent discoveries regarding primary cilia and filopodia and their role in islet homeostasis and intercellular islet communication.

Treatment of the metabolic syndrome by siRNA targeting apolipoprotein CIII.

Apolipoprotein CIII (apoCIII) is increased in obesity-induced insulin resistance and type-2 diabetes. Emerging evidences support the advantages of small interfering RNAs (siRNAs) to target disease-causing genes. The aim of this study was to develop siRNAs for in vivo silencing of apoCIII and investigate if this results in metabolic improvements comparable to what we have seen using antisense oligonucelotides against apoCIII. Twenty-four siRNAs were synthesized and tested in a dual luciferase reporter assay. The eight best were selected, based on knockdown at 20 nM, and of these, two were selected based on IC50 values. In vivo experiments were performed in ob/ob mice, an obese animal model for diabetes. To determine the dose-dependency, efficacy, duration of effect and therapeutic dose we used a short protocol giving the apoCIII-siRNA mix for three days. To evaluate long-term metabolic effects mice were treated for three days, every second week for eight weeks. The siRNA mix effectively and selectively reduced expression of apoCIII in liver in vivo. Treatment had to be repeated every two weeks to maintain a suppression of apoCIII. The reduction of apoCIII resulted in increased LPL activity, lower triglycerides, reduced liver fat, ceased weight gain, enhanced insulin sensitivity, and improved glucose homeostasis. No off-target or side effects were observed during the eight-week treatment period. These results suggest that in vivo silencing of apoCIII with siRNA, is a promising approach with the potential to be used in the battle against obesity-induced metabolic disorders.

Uncommon Transplantation Sites: Transplantation of Islets and Islet Organoids in the Anterior Chamber of the Eye of Rodents and Monkeys.

The anterior chamber of the eye is a highly vascularized and innervated location that is also particularly rich in oxygen and immune privileged. This uncommon transplantation site offers unique possibilities for the observation of the transplanted material as well as for local pharmacological intervention. Transplantation of islets and islet organoids to the anterior chamber of the eye of mice and monkeys facilitates a multitude of new approaches for research into islet physiology and pathophysiology and for the treatment of diabetes. We now present a short overview of the experimental possibilities and describe an updated protocol for transplantation of islets and islet organoids into mice and monkeys.

ATF5 is a regulator of ER stress and ß-cell apoptosis in different mouse models of genetic- and diet-induced obesity and diabetes mellitus.

Endoplasmic reticulum (ER) stress is closely associated with type 2 diabetes (T2D). Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element binding protein (CREB) family whose levels are increased upon stress in pancreatic islets from mice. Intriguingly, ATF5 deficiency has been shown to contribute to increased ER stress and apoptosis in mouse islet micro-organs. We hypothesized that either deficiency or overexpression of ATF5 is equally deleterious for pancreatic islets in terms of ER stress and apoptosis. To test this, we used a number of in vitro and in vivo models whereby ATF5 levels were overexpressed. We also determined the regulation of ATF5 in the context of metabolic derangements by using various mouse models of obesity and T2D. Our in vitro results show that ATF5 overexpression promoted palmitic acid (PA)-induced lipotoxic apoptosis. In vivo, global ATF5 overexpression in mice was lethal and pancreas-specific ATF5 overexpressing mice exhibit increased ß-cell apoptosis. Interestingly, ATF5 is downregulated in all mouse models of severe obesity and T2D used in the current study. In conclusion, a tight control on ATF5 levels might be considered when developing novel agents targeting ATF5 for prevention and treatment of metabolic diseases.

Getting them through the door: Social and behavioral determinants of uptake and engagement in an obesity intervention.

Using data from a large-scale screening program (N = 19634), we aimed to prospectively identify factors predicting uptake (i.e. acceptance of the invitation) and engagement (i.e. participation in at least two sessions) in a multi-component-intensive-behavioral-intervention for obesity-management (MBIOM) intervention targeting adolescents (n = 2862; 12-14 years; BMI ≥90th percentile). Approximately one third of adolescents most in need of weight management declined the initial invitation to enter the MBIOM. Poor diet, sedentary behavior, and parental education predicted willingness to enter and stay in the intervention, however measured body mass index did not matter. Perceived family support, instead of initial motivation, facilitated engagement. Our results provide new insights on the importance of regional socio-geographical factors including trust in local authorities.

Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells.

Mitochondrial dynamics and bioenergetics are central to glucose-stimulated insulin secretion by pancreatic beta cells. Previously, we demonstrated that a disturbance in glucose-invoked fission impairs insulin secretion by compromising glucose catabolism. Here, we investigated whether the overexpression of mitochondrial fission regulator Drp1 in MIN6 cells can improve or rescue insulin secretion. Although Drp1 overexpression slightly improves the triggering mechanism of insulin secretion of the Drp1-knockdown cells and has no adverse effects on mitochondrial metabolism in wildtype MIN6 cells, the constitutive presence of Drp1 unexpectedly impairs insulin content, which leads to a reduction in the absolute values of secreted insulin. Coherent with previous studies in Drp1-overexpressing muscle cells, we found that the upregulation of ER stress-related genes (BiP, Chop, and Hsp60) possibly impacts insulin production in MIN6 cells. Collectively, we confirm the important role of Drp1 for the energy-coupling of insulin secretion but unravel off-targets effects by Drp1 overexpression on insulin content that warrant caution when manipulating Drp1 in disease therapy.

Mitochondrial impairment and intracellular reactive oxygen species alter primary cilia morphology.

Primary cilia have recently emerged as cellular signaling organelles. Their homeostasis and function require a high amount of energy. However, how energy depletion and mitochondria impairment affect cilia have barely been addressed. We first studied the spatial relationship between a mitochondria subset in proximity to the cilium in vitro, finding similar mitochondrial activity measured as mitochondrial membrane potential compared with the cellular network. Next, using common primary cilia cell models and inhibitors of mitochondrial energy production, we found alterations in cilia number and/or length due to energy depletion and mitochondrial reactive oxygen species (ROS) overproduction. Finally, by using a mouse model of type 2 diabetes mellitus, we provided in vivo evidence that cilia morphology is impaired in diabetic nephropathy, which is characterized by ROS overproduction and impaired mitochondrial metabolism. In conclusion, we showed that energy imbalance and mitochondrial ROS affect cilia morphology and number, indicating that conditions characterized by mitochondria and radicals imbalances might lead to ciliary impairment.

Biodegradable and Antioxidant DNA Hydrogel as a Cytokine Delivery System for Diabetic Wound Healing.

Impaired diabetic wound healing is associated with the persistence of chronic inflammation and excessive oxidative stress, which has become one of the most serious clinical challenges. Wound dressings with anti-inflammatory and reactive oxygen species (ROS)-scavenging properties are desirable for diabetic wound treatment. In this study, a shape-adaptable, biodegradable, biocompatible, antioxidant, and immunomodulatory interleukin-33 (IL-33)-cytogel is developed by encapsulating IL-33 into physically cross-linked DNA hydrogels and used as wound dressings to promote diabetic wound healing. The porous microstructures and biodegradable properties of the IL-33-cytogel ensure the local sustained-release of IL-33 in the wound area, where the sustained-release of IL-33 is maintained for at least 7 days. IL-33-cytogel can induce local accumulation of group 2 innate lymphoid cells (ILC2s) and regulatory T cells (Tregs), as well as M1-to-M2 transition at the wound sites. Additionally, the antioxidant and biocompatible characteristics of DNA hydrogels promote the scavenging of intracellular ROS without affecting cell viability. As a result, local inflammation in the diabetic wound area is resolved upon IL-33-cytogel treatment, which is accompanied by improved granulation tissue regeneration and accelerated wound closure. This study demonstrates a promising strategy in tissue engineering and regenerative medicine by incorporating DNA hydrogels and cytokine immunotherapy for promoting diabetic wound healing.

Quantitative 3D OPT and LSFM datasets of pancreata from mice with streptozotocin-induced diabetes.

Mouse models for streptozotocin (STZ) induced diabetes probably represent the most widely used systems for preclinical diabetes research, owing to the compound's toxic effect on pancreatic ß-cells. However, a comprehensive view of pancreatic ß-cell mass distribution subject to STZ administration is lacking. Previous assessments have largely relied on the extrapolation of stereological sections, which provide limited 3D-spatial and quantitative information. This data descriptor presents multiple ex vivo tomographic optical image datasets of the full ß-cell mass distribution in mice subject to single high and multiple low doses of STZ administration, and in glycaemia recovered mice. The data further include information about structural features, such as individual islet ß-cell volumes, spatial coordinates, and shape as well as signal intensities for both insulin and GLUT2. Together, they provide the most comprehensive anatomical record of the effects of STZ administration on the islet of Langerhans in mice. As such, this data descriptor may serve as reference material to facilitate the planning, use and (re)interpretation of this widely used disease model.