Multiphoton Microscopy Research Articles

Looking below the surface - using intravital imaging to decipher inflammatory renal disease and renal cell injury.

Renal function can be perturbed by a range of stimuli that cause cellular injury and inflammation in the kidney. These injurious and inflammatory processes are typically dynamic and progressive, involving the actions of highly migratory cells such as leukocytes and cellular responses that occur over time spans ranging from seconds to weeks. Understanding these dynamic responses has entailed the use of imaging technologies that allow visualisation and capture of events over different time spans, ideally in intact organs in live, experimental animals. The technique that allows this is intravital imaging. Intravital imaging, particularly multiphoton intravital microscopy, has been crucial to the investigation of dynamic physiological and pathophysiological processes in the kidney for many years, driving key developments in our understanding of renal (patho)physiology. This includes the mechanisms of ultrafiltrate generation, the response to acute kidney injury and how inflammatory leukocytes are recruited to and cause injury in the kidney. This Review describes the key studies that have applied intravital imaging to the investigation of models of inflammatory renal disease. The responses examined include those restricted to the glomerulus and the effects of acute kidney injury on the tubulointerstitium. Future innovations and directions in this field of research are also discussed.

Multiphoton imaging-based quantifiable collagen signatures for predicting outcomes in patients with pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) presents a clinical challenge due to its poor prognosis and high mortality rate. Here, we aimed to enhance the prognostic prediction of patients with PDAC by studying collagen features in tumor microenvironment using multiphoton microscopy (MPM) combining with image processing technique. We identified eight distinct tumor-associated collagen signatures (TACS1-8) from multiphoton images of PDAC tissues and developed an optical biomarker, TACS-score, based on the TACS1-8 using ridge regression analysis. Additionally, we also extracted 142 microscopic TACS (M-TACS) from second-harmonic generation (SHG) images and constructed a new robust biomarker, M-TACS-score, using the least absolute shrinkage and selection operator (LASSO) regression analysis. Our statistical results demonstrate that as two new optical biomarkers, TACS- and M-TACS-score, are independent prognostic factors and have good discriminatory ability (high AUC) as well as risk stratification (high HR) comparing with traditional clinical model (combining seven clinical risk factors, age, sex, TNM stage, tumor location and differentiation, perineural and lymph-vascular invasion) in predicting overall survival (OS) of patients with PDAC, highlighting their potential prognostic and predictive value. A combination of label-free multiphoton imaging technique and computer-aided image processing method may offer a novel and promising approach for finding new biomarkers to improve prognosis prediction and thereby tailor treatment strategies more effectively.

Endothelialization of coronary stents after intra-luminal adenoviral VEGF-A gene transfer in a preclinical porcine restenosis model - Studies with optical coherence tomography, angioscopy, multiphoton and scanning electron microscopy.

Coronary stenting operations have become the main option for the treatment of coronary heart disease. Vessel recovery after stenting has emerged as a critical factor in reducing possible complications. In this study, we evaluated the feasibility, safety and efficacy of locally administered intraluminal gene therapy delivered using a specialized infusion balloon catheter. Sixteen pigs received bare metal stents (BMS) in the left circumflex coronary artery and drug-eluting stents (DES) in the right coronary artery. Adenoviral (Ad) gene transfers (dose 1,5e10 pfu) of VEGF-A and LacZ were performed with a ClearWay™ RX infusion balloon catheter on both stents. In vivo imaging included angiography, OCT and angioscopy. Tissue samples were collected for analyses at day 14 and studied using scanning electron microscopy and multiphoton microscopy. AdVEGF-A accelerated re-endothelialization in the BMS compared to the other groups. The highest restenosis was in the BMS AdLacZ group. DES groups had statistically significant reduced maximum stenosis compared to BMS AdLacZ. No major in-stent thrombosis events were detected. Ex vivo imaging showed that intraarterial imaging is not very accurate in the detection of endothelial layer. Biodistribution of the Ad vector and measured safety parameters (off-target tissues and blood tests) did not show any major safety concerns. Adenoviral gene transfer using the ClearWay™ RX catheter was feasible and safe. AdVEGF-A accelerated re-endothelization in BMS. However, DES showed better outcomes in the short-term regarding restenosis and do not benefit from intraluminal AdVEGF-A gene transfer.

Design and characterization of hollow microneedles for localized intrascleral drug delivery of ocular formulations.

Effective drug delivery to the posterior segment of the eye remains a challenge owing to the limitations of conventional methods such as intravitreal injections, which are associated with significant side effects. This study explored the use of hollow microneedles (HMNs) for localized intrascleral drug delivery as a minimally invasive alternative. Stainless steel HMNs with bevel angles of 30°, 45°, 60°, and 75° were fabricated using wire electron discharge machining. The penetration force of these HMNs in ex vivo porcine sclera was assessed using a texture analyser, revealing that the 60° bevel angle required the lowest force (<2N), making it optimal for scleral penetration. To ensure precision in drug delivery, 3D-printed adapters were developed to control the injection angles and volumes. The distribution of a model dye, rhodamine B, was studied via digital imaging, multiphoton microscopy, and confocal microscopy. The results showed that HMNs with a 60° bevel angle could penetrate the sclera to a depth of approximately 450µm at a 45° injection angle, providing enhanced distribution within the scleral layers. This study confirmed that the use of HMNs enables effective and controlled intrascleral drug delivery, resulting in the formation of localized depots with minimal tissue damage. This research demonstrates the potential of HMNs as a promising alternative to traditional ocular drug delivery methods, offering improved bioavailability and the potential to reduce patient discomfort.

Application of Microneedles for High-Molecular-Weight Dextran Penetration Across the Buccal Mucosa

Objectives: This work aimed at investigating the effect of different microneedle-based strategies on the permeation of high-molecular-weight model molecules (fluorescently labeled dextrans (FDs), 70 and 150 kDa) across the buccal mucosa. Methods: Two different approaches were evaluated: (1) stainless steel microneedles (MNs) of 500 µm height used for tissue pre-treatment; and (2) soluble microneedles of different lengths (150, 500, and 800 µm), made of polyvinylpyrrolidone and FDs, prepared using the solvent casting technique. Porcine esophageal epithelium was used as a model for the buccal mucosa. Results: The application of soluble MNs promoted high-molecular-weight dextran transport across pig esophageal epithelium. The transport was proportional to MN length, with a minimum of 500 µm, regardless of the molecular weight of the FDs. The use of solid MNs of the same length to pre-treat the tissue, followed by the application of a solution of the permeant, did not produce the same effect in terms of onset of permeation, which was found to be much slower. Conclusions: The results obtained show that by applying soluble MNs of appropriate length (500 and 800 µm), the transport of high-molecular-weight dextrans (70 and 150 kDa) across and into the mucosal tissue occurs very rapidly. The multiphoton microscopy analysis confirmed the presence of holes in the tissue and the presence of fluorescein-labeled dextrans.

Shallow-angle intracranial cannula for repeated infusion and in vivo imaging with multiphoton microscopy.

Multiphoton microscopy serves as an essential tool for high-resolution imaging of the living mouse brain. To facilitate optical access to the brain during imaging, the cranial window surgery is commonly used. However, this procedure restricts physical access above the imaging area and hinders the direct delivery of imaging agents and drugs. To overcome this limitation, we have developed a cannula delivery system that enables the implantation of a low-profile cannula nearly parallel to the brain surface at angles as shallow as 8 degrees, while maintaining compatibility with multiphoton microscopy. To validate this approach, we perform direct infusion and imaging of various fluorescent cell markers in the brain. Additionally, we successfully demonstrate tracking of degenerating neurons over time in Alzheimer's disease mice using Fluoro-Jade C. Furthermore, we show longitudinal imaging of brain tissue partial pressure of oxygen using a phosphorescent oxygen sensor. Our developed technique should enable a wide range of new longitudinal imaging studies in the mouse brain.

Local characterization of collagen architecture and mechanical properties of tissue engineered atherosclerotic plaque cap analogs.

Many cardiovascular events are triggered by fibrous cap rupture of an atherosclerotic plaque in arteries. However, cap rupture, including the impact of the cap's structural components, is poorly understood. To obtain better mechanistic insights in a biologically and mechanically controlled environment, we previously developed a tissue-engineered fibrous cap model. In the current study, we characterized the (local) structural and mechanical properties of these tissue-engineered cap analogs. Twenty-four collagenous cap analogs were cultured. The analogs were imaged with multiphoton microscopy with second-harmonic generation to obtain local collagen fiber orientation and dispersion. Then, the analogs were mechanically tested under uniaxial tensile loading until failure, and the local deformation (strain) and failure characteristics were analyzed. Our results demonstrated that the tissue-engineered analogs mimic the dominant (circumferential) fiber direction of human plaques. The analogs also exhibited a physiological strain stiffening response, similar to human fibrous plaque caps. Ruptures in the analogs initiated in and propagated towards local high-strain regions. The local strain values at the rupture sites were similar to the ones reported for carotid human fibrous plaque tissue. Finally, the study revealed that the rupture propagation path in the analogs followed the local fiber direction. STATEMENT OF SIGNIFICANCE: Many cardiovascular events are triggered by mechanical rupture of atherosclerotic plaque caps. Yet, cap rupture mechanics is poorly understood. This is mainly due to the scarcity of plaques for high-throughput testing and the structural complexity of plaques. To overcome this, we previously developed tissue-engineered cap analogs. The current study characterizes (local) structural and mechanical properties of these cap analogs. Our findings show that: (1) cap analogs closely mimic human fibrous caps, including fiber orientation and strain stiffening responses; (2) structural and mechanical properties of cap analogs are associated, which provides critical information for understanding plaque rupture; and (3) cap ruptures commonly start in and propagate towards high-strain areas, indicating the potential use of strain measurements for cap rupture risk assessment.

Label-free Detection and Characterization of Metabolism in Fresh and Cryopreserved Macrophages.

Cryopreservation is a widely used technique to preserve biological samples for extended periods of time at low temperatures. Even though it is known to have significant effects on cell viability, its effect on their metabolism remains unexplored. Studying how cryopreservation influences the metabolism of cells is important to guarantee the reliability of samples transported between sites for analysis. Optical metabolic imaging allows for the study of cellular metabolism in a label-free manner by using the autofluorescence properties of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), two metabolic coenzymes. The goal of this research is to study the metabolic changes in macrophages after cryopreservation and compare these results with freshly isolated macrophage samples to evaluate if the metabolic data is retained after cryopreservation. The metabolism of macrophages was analyzed with fluorescence lifetime imaging microscopy (FLIM) using a multiphoton microscope. Monocytes were isolated from human whole blood and separated into two groups. In Group 1, freshly isolated monocytes were differentiated into macrophages using macrophage-colony stimulating factor (M-CSF) over 8 days. In Group 2, isolated monocytes were cryopreserved after separation from whole blood and then thawed and stimulated with M-CSF for 8 days. Single-cell analysis showed there are significant changes in FLIM parameters between the groups suggesting that the metabolic data of macrophages is altered after a cryopreservation and cell thawing cycle. Our research bridges the current gap by studying the metabolic changes of cells after cryopreservation using a non-invasive and label-free imaging technique.

A novel workflow for multi-modal imaging of musculoskeletal tissues.

According to the World Health Organization (WHO) musculoskeletal conditions are a leading contributor to disability worldwide. This fact is often somewhat overlooked, since musculoskeletal conditions are less likely to be associated with mortality. Nonetheless, treatments, therapies and management of these conditions are extremely costly to national healthcare systems. As with all systemic conditions, biomedical imaging of relevant tissues plays a major role in understanding the fundamental biological processes involved in musculoskeletal health. However, the skeletal system with its relatively large proportion of dense, opaque (often mineralised) tissues can often be more challenging to image, and recently important advances have been made in imaging these complex musculoskeletal tissues. Thus, we here describe a novel workflow in which recent advanced imaging techniques have been modified and optimised for use in musculoskeletal tissues (specifically bone and cartilage). This will allow for investigations, of different phases of these tissues, at new and higher resolutions. Furthermore, the process has been designed to fit with the existing and standard processes which are typically used with these samples (i.e. μCT imaging and standard histology). The additional modalities which have been included here are second harmonic generation (SHG) imaging, tissue clearing, specifically the Passive Clear Lipid-exchanged Acrylamide-hybridised Rigid Imaging Tissue hYdrogel (CLARITY) method known as PACT, and then imaging of these tissues with confocal, multiphoton and light-sheet microscopy. This paper serves to introduce a combination of existing new methods and improvements in imaging of musculoskeletal tissues.

Long-term excessive salt consumption alters villous and crypt morphology and the protein expression of uroguanylin, TRPV6 and PMCA1b in the rat small intestine.

Although long-term high dietary sodium consumption often aggravates hypertension and bone loss, sodium in the intestinal lumen has been known to promote absorption of nutrients and other ions, e.g., glucose and calcium. However, whether high-salt diet (HSD) altered mucosal morphology, villous cell turnover and calcium transporter expression remained elusive. Herein, rats were treated with HSD containing 8% wt/wt NaCl for up to 5 months. HSD rats exhibited a marked increase in sodium intake with high fecal and urinary sodium excretion, as compared to the control group treated with normal diet. Intestinal histomorphometry revealed increasing of crypt depth and villous height in 3- and 4-month HSD groups, respectively, consistent with larger mucosal-to-serosal amplification ratio that reflected an increased surface area for nutrient absorption. The signals of Ki-67-positive cells was enhanced in the crypts as visualized by multiphoton fluorescence microscopy, whereas the TUNEL-positive cells were decreased in the villi of HSD, suggesting greater crypt cell proliferation and a reduction of villous cell apoptosis. Confocal microscopy showed higher expression of TRPV6 protein in the villous tip of HSD, while PMCA1 expression was increased in villous tip and crypt areas. The percentage of cells with highly expressed uroguanylin-an endogenous intestinal natriuretic peptide-was significantly higher in HSD group. In conclusion, HSD profoundly changed the intestinal morphology and turnover of epithelial cell, increased the expression of calcium transporters and uroguanylin. Our findings reflect pathophysiological adaptations in the intestine, which might be another target organ for drug discovery against HSD-induced osteopathy in the future.

Differential bone and vessel type formation at superior and dura periosteum during cranial bone defect repair

The cranial mesenchyme, originating from both neural crest and mesoderm, imparts remarkable regional specificity and complexity to postnatal calvarial tissue. While the distinct embryonic origins of the superior and dura periosteum of the cranial parietal bone have been described, the extent of their respective contributions to bone and vessel formation during adult bone defect repair remains superficially explored. Utilizing transgenic mouse models in conjunction with high-resolution multiphoton laser scanning microscopy (MPLSM), we have separately evaluated bone and vessel formation in the superior and dura periosteum before and after injury, as well as following intermittent treatment of recombinant peptide of human parathyroid hormone (rhPTH), Teriparatide. Our results show that new bone formation along the dura surface is three times greater than that along the superior periosteal surface following injury, regardless of Teriparatide treatment. Targeted deletion of PTH receptor PTH1R via SMA-CreER and Col 1a (2.3)-CreER results in selective reduction of bone formation, suggesting different progenitor cell pools in the adult superior and dura periosteum. Consistently, analyses of microvasculature show higher vessel density and better organized arterial-venous vessel network associated with a 10-fold more osteoblast clusters at dura periosteum as compared to superior periosteum. Intermittent rhPTH treatment further enhances the arterial vessel ratio at dura periosteum and type H vessel formation in cortical bone marrow space. Taken together, our study demonstrates a site-dependent coordinated osteogenic and angiogenic response, which is determined by regional osteogenic progenitor pool as well as the coupling blood vessel network at the site of cranial defect repair.

Compact module for video-rate image mosaics in two-photon microscopy.

Biological applications using multiphoton microscopy increasingly seek a larger field of view while maintaining sufficient temporal sampling to observe dynamic biological processes. Multiphoton imaging also requires high numerical aperture microscope objectives to realize efficient non-linear excitation and collection of fluorescence. This combination of low-magnification and high-numerical aperture poses a challenge for system design. To address this, the use of a liquid crystal polarization grating stack is proposed here to temporally sequence through multiple fields of view. This solution pans the native field of view with minimal latency and zero inertial movement of either the microscope or biological sample. Implemented as a simple add-on unit to existing multi-photon microscopes, this device increases the total field size by 4x, covering up to 7.6mm2. Performance constraints and functional demonstration of imaging neural activity are presented.

Characterizing astrocyte-mediated neurovascular coupling by combining optogenetics and biophysical modeling.

Vasoactive signaling from astrocytes is an important contributor to the neurovascular coupling (NVC), which aims at providing energy to neurons during brain activation by increasing blood perfusion in the surrounding vasculature. Pharmacological manipulations have been previously combined with experimental techniques (e.g., transgenic mice, uncaging, and multiphoton microscopy) and stimulation paradigms to isolate in vivo individual pathways of the astrocyte-mediated NVC. Unfortunately, these pathways are highly nonlinear and non-additive. To separate these pathways in a unified framework, we combine a comprehensive biophysical model of vasoactive signaling from astrocytes with a unique optogenetic stimulation method that selectively induces astrocytic Ca2+ signaling in a large population of astrocytes. We also use a sensitivity analysis and an optimization technique to estimate key model parameters. Optogenetically-induced Ca2+ signals in astrocytes cause a cerebral blood flow (CBF) response with two major components. Component-1 was rapid and smaller (ΔCBF∼13%, 18 seconds), while component-2 was slowest and highest (ΔCBF ∼18%, 45 seconds). The proposed biophysical model was adequate in reproducing component-2, which was validated with a pharmacological manipulation. Model's predictions were not in contradiction with previous studies. Finally, we discussed scenarios accounting for the existence of component-1, which once validated might be included in our model.

Compact inverted digital holographic microscope based on common-path configuration

Ongoing advancements in holographic imaging technology have a considerable impact in various fields, specifically life sciences, owing to the 3D and precise visualization of microscopic structures. However, present-day imaging technologies, such as confocal, multiphoton microscopy, and digital holography microscopy, face constraints such as tagging bulky and expensive components. Its functionality requires specific laboratory conditions and relies on relatively complicated alignment mechanics. To address these limitations, we have proposed a compact label-free, easy-to-align imaging solution, which is what we believe to be a state-of-the-art inverted holographic 3D-printed microscopy prototype based on common-path holographic configuration compatible with high numerical aperture objectives. The microscope has several advantages, including simplicity, robustness, high temporal stability, and compatibility with other imaging modalities, and it is standalone. The designed architecture is automated by using the controller and sophisticated designed stages, making it a self-focusing device along all three axes. The cutting-edge prototype presented here influences pioneering light field technology, providing simplification of the optical setup and improvement of robustness as well as real-time monitoring of live organisms and transparent samples. These features enable its applicability for in-field measurements without using an antivibration optical table. The proposed device not only overwhelms the flaws of existing technologies but also opens up new ventures for future in-field life sciences applications, to our knowledge.

Deep learning-based aberration compensation improves contrast and resolution in fluorescence microscopy

Optical aberrations hinder fluorescence microscopy of thick samples, reducing image signal, contrast, and resolution. Here we introduce a deep learning-based strategy for aberration compensation, improving image quality without slowing image acquisition, applying additional dose, or introducing more optics. Our method (i) introduces synthetic aberrations to images acquired on the shallow side of image stacks, making them resemble those acquired deeper into the volume and (ii) trains neural networks to reverse the effect of these aberrations. We use simulations and experiments to show that applying the trained ‘de-aberration’ networks outperforms alternative methods, providing restoration on par with adaptive optics techniques; and subsequently apply the networks to diverse datasets captured with confocal, light-sheet, multi-photon, and super-resolution microscopy. In all cases, the improved quality of the restored data facilitates qualitative image inspection and improves downstream image quantitation, including orientational analysis of blood vessels in mouse tissue and improved membrane and nuclear segmentation in C. elegans embryos.

Implications of compressive loading of the stomach wall: Interplay between mechanical deformation and microstructure.

During gastric surgery, the stomach wall is compressed with clamps and sutures or staple lines. These short- and long-term deformations can severely compromise the integrity of the tissue and make it difficult for the stomach wall to respond and remodel to the new loading conditions. Consequently, serious intra- and postoperative complications such as the formation of leaks during bariatric surgeries, can be associated with these immense tissue deformations. Hence, the study aimed to investigate the effects of compressive loading of the stomach wall in the radial direction. This was done by macroscopic mechanical loading of the stomach wall in each region of the stomach and evaluating the microstructural changes inflicted in the tissue. For this purpose, several imaging techniques were used, i.e., a histological analysis, second-harmonic generation microscopy, and X-ray micro-computed tomography. The combination of these three methods allowed us to investigate the gradual compression of the different stomach layers as well as the local reorientation and deformation of the main microstructural components, e.g., collagen fibers and muscle bundles. Importantly, this study found that the collagen bundles in the stomach wall straighten and reorient toward the circumferential-longitudinal plane and partially fan out with increased radial compressive deformation. The 3D scans of the stomach wall indicated a deterioration of the blood vessels and buckling of the mucosal glands due to compression. Statement of significance Unfortunately, little is known about the load transfer in the stomach wall during gastric surgery and the associated deformations on the macro- and microscale. The present study investigates the structural changes of the stomach wall, its layers and the inherent biological building blocks using histology, multi-photon microscopy, and micro-computed tomography. For the first time, the layer-specific response to stepwise radial compression of the stomach wall was studied, the related collagen fiber parameters were estimated, and a 3D sample structure was visualized. This clinically-oriented study links the structural changes within the wall to the postoperative remodel- ing process and the irreversibly altered gastric motility, thereby underscoring its relevance to the field of biomedical engineering, e.g., the development and improvement of surgical instruments.

Quantitative Histopathology Analysis Based on Label-free Multiphoton Imaging for Breast Cancer Diagnosis and Neoadjuvant Immunotherapy Response Assessment.

Accurate diagnosis and assessment of breast cancer treatment responses are critical challenges in clinical practice, influencing patient treatment strategies and ultimately long-term prognosis. Currently, diagnosing breast cancer and evaluating the efficacy of neoadjuvant immunotherapy (NAIT) primarily rely on pathological identification of tumor cell morphology, count, and arrangement. However, when tumors are small, the tumors and tumor beds are difficult to detect; relying solely on tumor cell identification may lead to false negatives. In this study, we used the label-free multiphoton microscopy (MPM) method to quantitatively analyze breast tissue at the cellular, extracellular, and textural levels, and identified 11 key factors that can effectively distinguish different types of breast diseases. Key factors and clinical data are used to train a two-stage machine learning automatic diagnosis model, MINT, to accurately diagnose breast cancer. The classification capability of MINT was validated in independent cohorts (stage 1 AUC = 0.92; stage 2 AUC = 1.00). Furthermore, we also found that some factors could predict and assess the efficacy of NAIT, demonstrating the potential of label-free MPM in breast cancer diagnosis and treatment. We envision that in the future, label-free MPM can be used to complement stromal and textural information in pathological tissue, benefiting breast cancer diagnosis and neoadjuvant therapy efficacy prediction, thereby assisting clinicians in formulating personalized treatment plans.

Record-high hyperpolarizabilities in atomically precise single metal-doped silver nanoclusters.

Recent developments in optical imaging techniques, particularly multi-photon excitation microscopy that allows studies of biological interactions at a deep cellular level, have motivated intensive research in developing multi-photon absorption fluorophores. Biological tissues are optically transparent in the near-infrared region. Therefore, fluorophores that can absorb light in the near-infrared (NIR) region by multi-photon absorption are particularly useful in bio-imaging. For instance, photoluminescence from ligand-protected gold nanoclusters has drawn extensive research interest in the past decade due to their bright, non-blinking, stable emission and tunability from the blue to the NIR emission. In this work, using the control of single metal doping on silver nanoclusters (Ag25 protected by thiolate SR = 2,4-dimethylbenzenethiol (DMBT) ligand), we aim to explore the effects of metal doping on the (photo)stability and nonlinear optical response of liganded nanoclusters. We study two-photon excited photoluminescence and the second harmonic response upon excitation in the NIR (780-950 nm) range. Particular emphasis is placed on the effect of metal doping on the second-order nonlinear optical scattering properties (first hyperpolarizability, β(2ω)) of Ag25 nanoclusters. In addition, β(2ω) values are one order higher than the one reported for Au25 nanoclusters and represent the largest values ever reported for ligand-protected nanoclusters. Such enhanced hyperpolarizability leads to a strong second harmonic response and renders them attractive targets in bioimaging.

Renal Proximal Tubule Cell-Specific Megalin Deletion Does Not Affect Atherosclerosis But Induces Tubulointerstitial Nephritis in Mice Fed a Western Diet.

Pharmacological inhibition of megalin (also known as LRP2 [low-density lipoprotein receptor-related protein-2]) attenuates atherosclerosis in hypercholesterolemic mice. Since megalin is abundant in renal proximal tubule cells (PTCs), the purpose of this study was to determine whether PTC-specific deletion of megalin reduces hypercholesterolemia-induced atherosclerosis in mice. Female Lrp2 f/f mice were bred with male Ndrg1-Cre ERT2 +/0 mice to develop PTC-LRP2 +/+ and PTC-LRP2 -/- littermates. To study atherosclerosis, all mice were bred to an LDL (low-density lipoprotein) receptor -/- background and fed a Western diet to induce atherosclerosis. PTC-specific megalin deletion did not attenuate atherosclerosis in LDL receptor -/- mice in either sex. Serendipitously, we discovered that PTC-specific megalin deletion led to interstitial infiltration of CD68+ cells and tubular atrophy. The pathology was only evident in male PTC-LRP2 -/- mice fed a Western diet but not in mice fed a normal laboratory diet. Renal pathologies were also observed in male PTC-LRP2 -/- mice in an LDL receptor +/+ background fed the same Western diet, demonstrating that the renal pathologies were dependent on diet and not on hypercholesterolemia. In contrast, female PTC-LRP2 -/- mice had no apparent renal pathologies. In vivo multiphoton microscopy demonstrated that PTC-specific megalin deletion dramatically diminished ALB (albumin) accumulation in PTCs within 10 days of Western diet feeding. RNA-sequencing analyses demonstrated the upregulation of inflammation-related pathways in the kidney. PTC-specific megalin deletion does not affect atherosclerosis but leads to tubulointerstitial nephritis in mice fed a Western diet, with severe pathologies in male mice.

Development of small-molecule fluorescent probes targeting neutrophils via N-formyl peptide receptors.

N-Formyl peptide receptors (FPRs) are membrane receptors that are abundantly expressed in innate immune cells, including neutrophils and platelets, demonstrating potential new targets for immune system regulation and the treatment of inflammatory conditions. We report here the development and bio-physical validation of new FPR imaging agents as effective tools to track FPR distribution, localisation and functions, ultimately helping to establish FPR exact roles and functions in pathological and physiological conditions. The new series of probes feature a small molecule-based FPR address system conjugated to suitable fluorophores, resulting in highly specific FPR agents, including a partial agonist endowed with high affinity (i.e. low/sub-nanomolar potency) on FPR-transfected cells and human neutrophils. Preliminary imaging studies via multiphoton microscopy demonstrate that the probes enable the visualisation of FPRs in live cells, thus representing valid bio-imaging tools for the analysis of FPR-mediated signalling, such as the activation of neutrophils in inflammatory events.