Two-Photon Microscopy for the Study of Tendons.

Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in various biological systems. This technique relies on nonlinear light-matter interactions to detect two distinct signals: the second harmonic generated (SHG) diffusion signal, which facilitates the visualization of collagen fibers and their orientation, and the near-infrared excitation signal for imaging ultraviolet excited autofluorescence. SHG imaging proves especially effective in visualizing collagen fibers due to the non-centrosymmetric crystalline structure of fibrillar collagen I. Given that tendons are matrix-rich tissues with a limited number of cells, their high collagen content makes them ideal candidates for analysis using two-photon microscopy. Consequently, two-photon microscopy offers a valuable means to analyze and characterize collagen abnormalities in tendons. Its application extends to studying tendon development, injuries, healing, and aging, enabling the comprehensive characterization of tendon cells and their interactions with the ECM under various conditions using two-photon microscopy tools. This protocol outlines the use of two-photon microscopy in tendon biology and presents an adapted methodology to achieve effective imaging and characterization of tendon cells during development and after injury. The method allows the utilization of thin microscopic sections to create a comprehensive image of the ECM within tendons and the cells that interact with this matrix. Most notably, the article showcases a technique to generate 3D images using two-photon microscopy in animal models.

Spatial High-resolution Analysis of Gene Expression Levels in Tendons.

In recent years, many protocols have been developed for high-resolution transcriptomics in many different medical and biology fields. However, matrix-rich tissues, and specifically, tendons were left behind due to their low cell number, low RNA amount per cell, and high matrix content, which made them complicated to analyze. One of the recent and most important single-cell tools is the spatial analysis of gene expression levels in tendons. These RNA spatial tools have specifically high importance in tendons to locate specific cells of new and unknown populations, validate single-cell RNA-seq results, and add histological context to the single-cell RNA-seq data. These new methods will enable the analysis of RNA in cells with exceptional sensitivity and the detection of single-molecule RNA targets at the single-cell level, which will help to molecularly characterize tendons and promote tendon research. In this method paper, we will focus on the available methods to analyze spatial gene expression levels on histological sections by using novel in situ hybridization assays to detect target RNA within intact cells at single-cell levels. First, we will focus on how to prepare the tendon tissue for the different available assays and how to amplify target-specific signals without background noise but with high sensitivity and high specificity. Then, the paper will describe specific permeabilization methods, the different probe designs, and the signal amplification strategies currently available. These unique methods of analyzing transcription levels of different genes in single-cell resolution will enable the identification and characterization of the tendon tissue cells in young and aged populations of various animal models and human tendon tissues. This method will also help analyze gene expression levels in other matrix-rich tissues such as bones, cartilage, and ligaments.

Fibromodulin Ablation Exacerbates the Severity of Acute Colitis.

Introduction: Epidemiological studies have associated pigment production with protection against certain human diseases. In contrast to African Americans, European descendants are more likely to suffer from angiogenesis-dependent and inflammatory diseases, such as wet age-related macular degeneration (ARMD) and ulcerative colitis (UC), respectively. Methods: In a mouse model of dextran sulfate sodium (DSS)-induced acute colitis, the effect of fibromodulin (FMOD) depletion was examined on colitis severity. Results: In this study, albino mice that produce high levels of FMOD developed less severe acute colitis compared with mice lacking in FMOD as assessed by clinical symptoms and histopathological changes. FMOD depletion affected the expression of tight junction proteins, contributing to the destruction of the epithelial barrier. Furthermore, this study revealed a stronger inflammatory response after DSS treatment in the absence of FMOD, where FMOD depletion led to an increase in activated T cells, plasmacytoid dendritic cells (pDCs), and type I interferon (IFN) production. Discussion: These findings point to FMOD as a potential biomarker of disease severity in UC among light-skinned individuals of European descent.

A distinct transition from cell growth to physiological homeostasis in the tendon.

Changes in cell proliferation define transitions from tissue growth to physiological homeostasis. In tendons, a highly organized extracellular matrix undergoes significant postnatal expansion to drive growth, but once formed, it appears to undergo little turnover. However, tendon cell activity during growth and homeostatic maintenance is less well defined. Using complementary methods of genetic H2B-GFP pulse-chase labeling and BrdU incorporation in mice, we show significant postnatal tendon cell proliferation, correlating with longitudinal Achilles tendon growth. Around day 21, there is a transition in cell turnover with a significant decline in proliferation. After this time, we find low amounts of homeostatic tendon cell proliferation from 3 to 20 months. These results demonstrate that tendons harbor significant postnatal mitotic activity, and limited, but detectable activity in adult and aged stages. It also points towards the possibility that the adult tendon harbors resident tendon progenitor populations, which would have important therapeutic implications.

A robust method for RNA extraction and purification from a single adult mouse tendon.

BACKGROUND: Mechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts. METHODS: Single Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed. RESULTS: After testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons. DISCUSSION: Our work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples.

Repair after nephron ablation reveals limitations of neonatal neonephrogenesis.

The neonatal mouse kidney retains nephron progenitor cells in a nephrogenic zone for 3 days after birth. We evaluated whether de novo nephrogenesis can be induced postnatally beyond 3 days. Given the long-term implications of nephron number for kidney health, it would be useful to enhance nephrogenesis in the neonate. We induced nephron reduction by cryoinjury with or without contralateral nephrectomy during the neonatal period or after 1 week of age. There was no detectable compensatory de novo nephrogenesis, as determined by glomerular counting and lineage tracing. Contralateral nephrectomy resulted in additional adaptive healing, with little or no fibrosis, but did not also stimulate de novo nephrogenesis. In contrast, injury initiated at 1 week of age led to healing with fibrosis. Thus, despite the presence of progenitor cells and ongoing nephron maturation in the newborn mouse kidney, de novo nephrogenesis is not inducible by acute nephron reduction. This indicates that additional nephron progenitors cannot be recruited after birth despite partial renal ablation providing a reparative stimulus and suggests that nephron number in the mouse is predetermined at birth.

Wnt signaling orients the proximal-distal axis of chick kidney nephrons.

The nephron is the fundamental structural and functional unit of the kidney. Each mature nephron is patterned along a proximal-distal axis, with blood filtered at the proximal end and urine emerging from the distal end. In order to filter the blood and produce urine, specialized structures are formed at specific proximal-distal locations along the nephron, including the glomerulus at the proximal end, the tubule in the middle and the collecting duct at the distal end. The developmental processes that specify these different nephron segments are not fully understood. Wnt ligands, which are expressed in the nephric duct and later in the nascent nephron itself, are well-characterized inducers of nephrons, and are both required and sufficient for initiation of nephron formation from nephrogenic mesenchyme. Here, we present evidence that Wnt signaling also patterns the proximal-distal nephron axis. Using the chick mesonephros as a model system, a Wnt ligand was ectopically expressed in the coelomic lining, thereby introducing a source of Wnt signaling that is at right angles to the endogenous Wnt signal of the nephric duct. Under these conditions, the nephron axis was re-oriented, such that the glomerulus was always located at a position farthest from the Wnt sources. This re-orientation occurred within hours of exposure to ectopic Wnt signaling, and was accompanied initially by a repression of the early glomerular podocyte markers Wt1 and Pod1, followed by their re-emergence at a position distant from the Wnt signals. Activation of the Wnt signaling pathway in mesonephric explant cultures resulted in strong and specific repression of early and late glomerular markers. Finally, cytoplasmic ß-catenin, indicative of active canonical Wnt signaling, was found to be enriched in the distal as compared with the proximal region of the forming nephron. Together, these data indicate that Wnt signaling patterns the proximal-distal axis of the nephron, with glomeruli differentiating in regions of lowest Wnt signaling.

Generation of the podocyte and tubular components of an amniote kidney: timing of specification and a role for Wnt signaling.

Kidneys remove unwanted substances from the body and regulate the internal body environment. These functions are carried out by specialized cells (podocytes) that act as a filtration barrier between the internal milieu and the outside world, and by a series of tubules and ducts that process the filtrate and convey it to the outside. In the kidneys of amniote vertebrates, the filtration (podocyte) and tubular functions are tightly integrated into functional units called nephrons. The specification of the podocyte and tubular components of amniote nephrons is currently not well understood. The present study investigates podocyte and tubule differentiation in the avian mesonephric kidney, and presents several findings that refine our understanding of the initial events of nephron formation. First, well before the first morphological or molecular signs of nephron formation, mesonephric mesenchyme can be separated on the basis of morphology and the expression of the transcription factor Pod1 into dorsal and ventral components, which can independently differentiate in culture along tubule and podocyte pathways, respectively. Second, canonical Wnt signals, which are found in the nephric duct adjacent to the dorsal mesonephric mesenchyme and later in portions of the differentiating nephron, strongly inhibit podocyte but not tubule differentiation, suggesting that Wnt signaling plays an important role in the segmentation of the mesonephric mesenchyme into tubular and glomerular segments. The results are discussed in terms of their broader implications for models of nephron segmentation.

Minute-to-minute urine flow rate variability: a new renal physiology variable.

BACKGROUND: Urine output is a surrogate for tissue perfusion and is typically measured at 1-hour intervals. Because small urine volumes are difficult to measure in urine collection bags, considerable over- or underestimation is common. To overcome these shortcomings, digital urine meters were developed. Because these monitors measure urine volume in 1-minute intervals, they provide minute-to-minute measurements of the urine flow rate (UFR). In a previous study, we observed that the minute-to-minute variability in the UFR disappeared during hypovolemia. The aim of this study was to describe the minute-to-minute variability in the UFR as a new physiological variable and to show its relationship to blood volume depletion. METHODS: Seven adult pigs were used in this study. The UFR, minute-to-minute UFR, mean arterial blood pressure, heart rate, and base excess were measured at euvolemia and during gradual hemorrhaging (10%, 20%, and 30% of estimated blood volume). Variance and wavelet spectral analysis were used to measure the disappearance of the minute-to-minute UFR variability. RESULTS: The UFR decreased from 2.2 ± 0.2 to 1.0 ± 0.1 mL/min after a 10% estimated blood volume loss (±1 SE, n = 7, P = 0.0348). The variance in the minute-to-minute UFR decreased from 1.4 ± 0.3 to 0.4 ± 0.1 mL/min (±1 SE, n = 7, P = 0.046). CONCLUSIONS: The UFR and its minute-to-minute variability decrease during hemorrhaging. The variability in the UFR may be useful as an aid for the diagnosis of hypovolemia.

Flow enhances photosynthesis in marine benthic autotrophs by increasing the efflux of oxygen from the organism to the water.

Worldwide, many marine coastal habitats are facing rapid deterioration due in part to human-driven changes in habitat characteristics, including changes in flow patterns, a factor known to greatly affect primary production in corals, algae, and seagrasses. The effect of flow traditionally is attributed to enhanced influx of nutrients and dissolved inorganic carbon (DIC) across the benthic boundary layer from the water to the organism however, here we report that the organism's photosynthetic response to changes in the flow is nearly instantaneous, and that neither nutrients nor DIC limits this rapid response. Using microelectrodes, dual-pulse amplitude-modulated fluorometry, particle image velocimetry, and real time mass-spectrometry with the common scleractinian coral Favia veroni, the alga Gracilaria cornea, and the seagrass Halophila stipulacea, we show that this augmented photosynthesis is due to flow-driven enhancement of oxygen efflux from the organism to the water, which increases the affinity of the RuBisCO to CO(2). No augmentation of photosynthesis was found in the absence of flow or when flow occurred, but the ambient concentration of oxygen was artificially elevated. We suggest that water motion should be considered a fundamental factor, equivalent to light and nutrients, in determining photosynthesis rates in marine benthic autotrophs.

Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus.

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l(-1)). On the other hand, pH varied greatly, from 8.7+/-0.15 in the gastrolith cavity through 7.6+/-0.2 in muscle to 6.9+/-0.5 in the stomach.

Contribution of Chloroflexus respiration to oxygen cycling in a hypersaline microbial mat from Lake Chiprana, Spain.

In dense stratified systems such as microbial mats, photosynthesis and respiration are coupled due to a tight spatial overlap between oxygen-producing and -consuming microorganisms. We combined microsensors and a membrane inlet mass spectrometer with two independent light sources emitting in the visible (VIS) and near infrared (NIR) regions to study this coupling in more detail. Using this novel approach, we separately quantified the activity of the major players in the oxygen cycle in a hypersaline microbial mat: gross photosynthesis of cyanobacteria, NIR light-dependent respiration of Chloroflexus-like bacteria (CLB) and respiration of aerobic heterotrophs. Illumination by VIS light induced oxygen production in the top approximately 1 mm of the mat. In this zone CLB were found responsible for all respiration, while the contribution of the aerobic heterotrophs was negligible. Additional illumination of the mat with saturating NIR light completely switched off CLB respiration, resulting in zero respiration in the photosynthetically active zone. We demonstrate that microsensor-based quantification of gross and net photosyntheses in dense stratified systems should carefully consider the NIR light-dependent behaviour of CLB and other anoxygenic phototrophic groups.