Too bright for 2 dimensions: recent progress in advanced 3-dimensional microscopy of the kidney.

The kidney is a structurally and functionally complex organ responsible for the control of water, ion, and other solute homeostasis. Moreover, the kidneys excrete metabolic waste products and produce hormones, such as renin and erythropoietin. The functional unit of the kidney is the nephron, which is composed by a serial arrangement of a filter unit called the renal corpuscle and several tubular segments that modulate the filtered fluid by reabsorption and secretion. Within each kidney, thousands of nephrons are closely intermingled and surrounded by an intricate network of blood vessels and various interstitial cell types, including fibroblasts and immune cells. This complex tissue architecture is essential for proper kidney function. In fact, kidney disease is often reflected or even caused by a derangement of the histologic structures. Frequently, kidney histology is studied using microscopic analysis of 2-dimensional tissue sections, which, however, misses important 3-dimensional spatial information. Reconstruction of serial sections tries to overcome this limitation, but is technically challenging, time-consuming, and often inherently linked to sectioning artifacts. In recent years, advances in tissue preparation (e.g., optical clearing) and new light- and electron-microscopic methods have provided novel avenues for 3-dimensional kidney imaging. Combined with novel machine-learning algorithms, these approaches offer unprecedented options for large-scale and automated analysis of kidney structure and function. This review provides a brief overview of these emerging imaging technologies and presents key examples of how these approaches are already used to study the normal and the diseased kidney.

Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases.

Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered low-molecular-weight proteins through receptor-mediated endocytosis and lysosomal processing. Congenital and acquired dysfunctions of the proximal tubule are consistently reflected by the inappropriate loss of solutes including low-molecular-weight proteins in the urine. The zebrafish pronephros shares individual functional segments with the human nephron, including lrp2a/megalin-dependent endocytic transport processes of the proximal tubule. Although the zebrafish has been used as a model organism for toxicological studies and drug discovery, there is no available assay that allows large-scale assessment of proximal tubule function in larval or adult stages. Here we establish a transgenic Tg(lfabp::½vdbp-mCherry) zebrafish line expressing in the liver the N-terminal region of vitamin D-binding protein coupled to the acid-insensitive, red monomeric fluorescent protein mCherry (½vdbp-mCherry). This low-molecular-weight protein construct is secreted into the bloodstream, filtered through the glomerulus, reabsorbed by receptor-mediated endocytosis and processed in the lysosomes of proximal tubule cells of the fish. Thus, our proof-of-concept studies using zebrafish larvae knockout for lrp2a and clcn7 or exposed to known nephrotoxins (gentamicin and cisplatin) demonstrate that this transgenic line is useful to monitor low-molecular-weight proteinuria and lysosomal processing. This represents a powerful new model organism for drug screening and studies of nephrotoxicity.

Rhabdoid cavitated adenocarcinoma in the jejunum, an exceptional case by enteroscopy.

We have read the article published by Abdulkader I et al., which described two cases of a rhabdoid tumor of the small bowel diagnosed by surgery. We present a similar case in the jejunum diagnosed by double balloon enteroscopy (DBE). We present the case of a 64-year-old patient with multifactorial anemia and transfusional requirements and a flat lesion of 2 cm in the colon, which showed undifferentiated adenocarcinoma on histopathological analysis.

Gastric ulcer due to mucormycosis in a critical patient.

We have read the article published by Sánchez-Velázquez P et al., which described a clinical case of gastrointestinal hemorrhage secondary to gastric ulcer due to Mucor. We present a similar clinical case, as an example of one identified by gastroscopy. The case was a 71-year-old female with multiple organ failure secondary to nosocomial pneumonia who required mechanical ventilation, vasoactive drugs, corticosteroids, antibiotherapy and continuous venovenous hemofiltration. Her room was adjacent to a building under construction. The patient had severe upper gastrointestinal bleeding and therefore, an urgent upper gastrointestinal endoscopy was performed. A small amount of blood was identified, as well as a large ulcer without a white base extending from the fundus to the antrum region of the stomach, with bleeding due to rubbing and nodular edges that suggested degeneration.

The rebleeding rate in patients evaluated for obscure gastrointestinal bleeding after negative small bowel findings by device assisted enteroscopy.

BACKGROUND: data on the long-term outcome of patients with obscure gastrointestinal bleeding (OGIB) with positive small bowel findings in capsule endoscopy but negative small bowel findings in device-assisted enteroscopy are scarce. OBJECTIVE: this study aimed to evaluate the rebleeding rate and time to rebleed in patients with no small bowel findings in enteroscopy, after a positive capsule endoscopy in the setting of OGIB. Baseline predictors for rebleeding were assessed. METHODS: a retrospective double-center study was performed, including patients with OGIB with positive findings by capsule endoscopy and negative small bowel findings by enteroscopy. RESULTS: thirty-five patients were included. Rebleeding occurred in 40 % of patients during a median follow-up of 27 months. Further evaluation in patients with a rebleed was performed in 85.7 %, leading to a final diagnosis in 78.6 %. The rebleeding rate increased progressively over time, from 17.2 % at one month to 54.4 % at four years. Overt bleeding at the time of the first episode was a predictor of rebleeding (p = 0.03) according to the multivariate analysis. This was 50 % at one year compared with 21.8 % in patients with occult bleeding on admission. CONCLUSIONS: in obscure gastrointestinal bleeding, long-term follow-up and further evaluation may be considered after a positive capsule endoscopy. Even if there are no small bowel findings by device-assisted enteroscopy. The rebleeding rate in our study was 40 %, mainly in the presence of an overt bleeding on admission.

Recurrent small-bowel bleeding from a Dieulafoy's lesion after combined endoscopic treatment.

INTRODUCTION: Dieulafoy's lesion of the small bowel is an uncommon cause of gastrointestinal (GI) bleeding that often recurs after endoscopic treatment. MATERIAL AND METHODS: we report an observational, descriptive, retrospective, single-center study in 15 patients with small bowel bleeding who were diagnosed with a Dieulafoy's lesion by capsule endoscopy or double-balloon enteroscopy. RESULTS AND CONCLUSIONS: all patients underwent combined endoscopic treatment. During a median follow-up of 33.5 months (range, 2-145), three of the 12 cases that stayed in follow-up (25 %) recurred, all within 48 hours after treatment. Two were successfully re-treated with a repeat endoscopic procedure.

A degenerate Todani Ia choledochal cyst in a patient with pancreas divisum.

Bile duct cysts represent congenital abnormalities associated with biliopancreatic maljunction that may undergo malignant degeneration. We report herein the case of a 72-year-old male patient with cholangitis. MR-cholangiography and abdominal CT revealed a mass at the biliary-pancreatic-duodenal crossroads, extrahepatic biliary dilation up to 38 mm, and pancreas divisum. Gastroscopy found an infiltrative bulbar mucosa with adenocarcinoma in biopsy samples, and extrinsic bulging of the second duodenal portion. Endoscopic ultrasound showed a choledochal cystic dilation with solid contents, and FNA findings were nonspecific. ERCP confirmed an adenomatous papilla at the lower portion of the extrinsic formation, and a large cystic, saccular dilation of extrahepatic bile ducts (Todani Ia). Fistulotomy was required for deep cannulation of the proximal biliary tract, and attention was drawn to extruding polypoid lesions originating in the biliary epithelium, identified in biopsies as adenoma with dysplasia. Finally, a diagnosis was made of advanced adenocarcinoma in choledochal cyst.

Label-free, three-dimensional multiphoton microscopy of the connective tissue in the anterior vaginal wall.

INTRODUCTION AND HYPOTHESIS: Multiphoton microscopy (MPM) is a nonlinear, high-resolution laser scanning technique and a powerful approach for analyzing the spatial architecture within tissues. To demonstrate the potential of this technique for studying the extracellular matrix of the pelvic organs, we aimed to establish protocols for the detection of collagen and elastin in the vagina and to compare the MPM density of these fibers to fibers detected using standard histological methods. METHODS: Samples of the anterior vaginal wall were obtained from nine patients undergoing a hysterectomy or cystocele repair. Samples were shock frozen, fixed with formaldehyde or Thiel's solution, or left untreated. Samples were imaged with MPM to quantify the amount of collagen and elastin via second harmonic generation and autofluorescence, respectively. In six patients, sample sections were also histologically stained and imaged with brightfield microscopy. The density of the fibers was quantified using the StereoInvestigator and Cavalieri software. RESULTS: With MPM, collagen and elastin could be visualized to a depth of 100 µm, and no overlap of signals was detected. The different tissue processing protocols used did not result in significantly different fiber counts after MPM. MPM-based fiber quantifications are comparable to those based on conventional histological stains. However, MPM provided superior resolution, particularly of collagen fibers. CONCLUSIONS: MPM is a robust, rapid, and label-free method that can be used to quantify the collagen and elastin content in thick specimens of the vagina. It is an excellent tool for future three-dimensional studies of the extracellular matrix in patients with pelvic organ prolapse.

A dramatic increase of C1q protein in the CNS during normal aging.

The decline of cognitive function has emerged as one of the greatest health threats of old age. Age-related cognitive decline is caused by an impacted neuronal circuitry, yet the molecular mechanisms responsible are unknown. C1q, the initiating protein of the classical complement cascade and powerful effector of the peripheral immune response, mediates synapse elimination in the developing CNS. Here we show that C1q protein levels dramatically increase in the normal aging mouse and human brain, by as much as 300-fold. This increase was predominantly localized in close proximity to synapses and occurred earliest and most dramatically in certain regions of the brain, including some but not all regions known to be selectively vulnerable in neurodegenerative diseases, i.e., the hippocampus, substantia nigra, and piriform cortex. C1q-deficient mice exhibited enhanced synaptic plasticity in the adult and reorganization of the circuitry in the aging hippocampal dentate gyrus. Moreover, aged C1q-deficient mice exhibited significantly less cognitive and memory decline in certain hippocampus-dependent behavior tests compared with their wild-type littermates. Unlike in the developing CNS, the complement cascade effector C3 was only present at very low levels in the adult and aging brain. In addition, the aging-dependent effect of C1q on the hippocampal circuitry was independent of C3 and unaccompanied by detectable synapse loss, providing evidence for a novel, complement- and synapse elimination-independent role for C1q in CNS aging.

Deletion of Sirt3 does not affect atherosclerosis but accelerates weight gain and impairs rapid metabolic adaptation in LDL receptor knockout mice: implications for cardiovascular risk factor development.

Sirt3 is a mitochondrial NAD(+)-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR(-/-)) and LDLR/Sirt3 double-knockout (Sirt3(-/-)LDLR(-/-)) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR(-/-) mice. Sirt3 deficiency does not affect atherosclerosis in LDLR(-/-) mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.

Enhancement of CA3 hippocampal network activity by activation of group II metabotropic glutamate receptors.

Impaired function or expression of group II metabotropic glutamate receptors (mGluRIIs) is observed in brain disorders such as schizophrenia. This class of receptor is thought to modulate activity of neuronal circuits primarily by inhibiting neurotransmitter release. Here, we characterize a postsynaptic excitatory response mediated by somato-dendritic mGluRIIs in hippocampal CA3 pyramidal cells and in stratum oriens interneurons. The specific mGluRII agonists DCG-IV or LCCG-1 induced an inward current blocked by the mGluRII antagonist LY341495. Experiments with transgenic mice revealed a significant reduction of the inward current in mGluR3(-/-) but not in mGluR2(-/-) mice. The excitatory response was associated with periods of synchronized activity at theta frequency. Furthermore, cholinergically induced network oscillations exhibited decreased frequency when mGluRIIs were blocked. Thus, our data indicate that hippocampal responses are modulated not only by presynaptic mGluRIIs that reduce glutamate release but also by postsynaptic mGluRIIs that depolarize neurons and enhance CA3 network activity.

Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples.

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.

Removal of extracellular human amyloid beta aggregates by extracellular proteases in C. elegans.

The amyloid beta (Aß) plaques found in Alzheimer's disease (AD) patients' brains contain collagens and are embedded extracellularly. Several collagens have been proposed to influence Aß aggregate formation, yet their role in clearance is unknown. To investigate the potential role of collagens in forming and clearance of extracellular aggregates in vivo, we created a transgenic Caenorhabditis elegans strain that expresses and secretes human Aß1-42. This secreted Aß forms aggregates in two distinct places within the extracellular matrix. In a screen for extracellular human Aß aggregation regulators, we identified different collagens to ameliorate or potentiate Aß aggregation. We show that a disintegrin and metalloprotease a disintegrin and metalloprotease 2 (ADM-2), an ortholog of ADAM9, reduces the load of extracellular Aß aggregates. ADM-2 is required and sufficient to remove the extracellular Aß aggregates. Thus, we provide in vivo evidence of collagens essential for aggregate formation and metalloprotease participating in extracellular Aß aggregate removal.

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples.

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

Calsyntenins mediate TGN exit of APP in a kinesin-1-dependent manner.

Kinesin motors are required for the export of membranous cargo from the trans-Golgi network (TGN), yet information about how kinesins are recruited to forming transport intermediates is sparse. Here we show that the Kinesin-1 docking protein calsyntenin-1 localizes to the TGN in vivo and directly and specifically recruits Kinesin-1 to Golgi/TGN membranes as well as to dynamic post-Golgi carriers. Overexpression of various calsyntenin chimeras and kinesin light chain 1 (KLC1) at high levels caused the formation of aberrant membrane stacks at the endoplasmic reticulum (ER) or the Golgi, disrupted overall Golgi structure and blocked exit of calsyntenin from the TGN. Intriguingly, this blockade of calsyntenin exit strongly and selectively impeded TGN exit of amyloid precursor protein (APP). Using live cell microscopy we found that calsyntenins exit the TGN in Kinesin-1-decorated tubular structures which may serve as carriers for calsyntenin-1-mediated post-TGN transport of APP. Abrogation of this pathway via virus-mediated knockdown of calsyntenin-1 expression in primary cultured neurons caused a marked elevation of APP C-terminal fragments. Together, these results indicate a role for calsyntenin-1 in Kinesin-1-dependent TGN exit and post-Golgi transport of APP-containing organelles and further suggest that distinct intracellular routes may exhibit different capacities for proteolytic processing of APP.

Rapid and reversible formation of spine head filopodia in response to muscarinic receptor activation in CA1 pyramidal cells.

A key feature at excitatory synapses is the remodelling of dendritic spines, which in conjunction with receptor trafficking modifies the efficacy of neurotransmission. Here we investigated whether activation of cholinergic receptors, which can modulate synaptic plasticity, also mediates changes in dendritic spine structure. Using confocal time-lapse microscopy in mouse slice cultures we found that brief activation of muscarinic receptors induced the emergence of fine filopodia from spine heads in all CA1 pyramidal cells examined. This response was widespread occurring in 48% of imaged spines, appeared within minutes, was reversible, and was blocked by atropine. Electron microscopic analyses showed that the spine head filopodia (SHFs) extend along the presynaptic bouton. In addition, the decay time of miniature EPSCs was longer after application of the muscarinic acetylcholine receptor agonist methacholine (MCh). Both morphological and electrophysiological changes were reduced by preventing microtubule polymerization with nocodazole. This extension of SHFs during cholinergic receptor activation represents a novel structural form of subspine plasticity that may regulate synaptic properties by fine-tuning interactions between presynaptic boutons and dendritic spines.

Characterization of Deposits in Calcific Tendinitis of the Shoulder: Deposits Are Composed of Large Aggregates of Highly Crystalline, Rod-Like Crystals.

BACKGROUND: In the current literature, deposits in calcific tendinitis are described as amorphous masses of hydroxyapatite with a size in the range of 5 to 20 µm. Theoretically, these are too big to be phagocytized by macrophages and induce an inflammatory reaction. PURPOSE: To better characterize the deposits seen in calcific tendinitis. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Included in the study were 6 patients with a history of at least 1 year of shoulder pain (range, 1-14 years). Shoulder arthroscopy was performed under general anesthesia, and calcium deposits from the supraspinatus tendon and biopsies from the adjacent subacromial bursa were taken. Samples were analyzed by light microscopy and immunostained for macrophages. Scanning electron microscopy and energy-dispersive x-ray (EDX) analysis were used to assess the morphology and chemical composition of the calcific deposits. RESULTS: Light microscopy showed round and bulky calcium deposits partially surrounded by activated CD68-positive macrophages within inflammatory tissue. Some hemosiderin positive mononuclear cells, indicative for (micro-) hemorrhage, were seen. Scanning electron microscopy revealed that the large calcific deposits (1-20 µm) were composed of rod-like structures. These highly crystalline rods had a size of approximately 100 nm in length and 20 nm in width. Chemical composition by EDX analysis showed that crystals were composed of mainly calcium, oxygen, and phosphorus, equaling the chemical composition of hydroxyapatite. CONCLUSION: Deposits in calcific tendinitis of the rotator cuff are not amorphous but composed of highly crystalline structures. Fragmentation of these aggregates and subsequent release of the needle-like nanocrystals might initiate the strong inflammatory reaction often seen in patients with calcifying tendinitis of the rotator cuff.