Neuronal nitric oxide (NO) synthase (nNOS), a Ca2+ dependent enzyme, is expressed by distinct populations of neocortical neurons. Although neuronal NO is well known to contribute to the blood flow increase evoked by neural activity, the relationships between nNOS neurons activity and vascular responses in the awake state remain unclear. We imaged the barrel cortex in awake, head-fixed mice through a chronically implanted cranial window. The Ca2+ indicator GCaMP7f was expressed selectively in nNOS neurons using adenoviral gene transfer in nNOScre mice. Air-puffs directed at the contralateral whiskers or spontaneous motion induced Ca2+ transients in 30.2 ± 2.2% or 51.6 ± 3.3% of nNOS neurons, respectively, and evoked local arteriolar dilation. The greatest dilatation (14.8 ± 1.1%) occurred when whisking and motion occurred simultaneously. Ca2+ transients in individual nNOS neurons and local arteriolar dilation showed various degrees of correlation, which was strongest when the activity of whole nNOS neuron ensemble was examined. We also found that some nNOS neurons became active immediately prior to arteriolar dilation, while others were activated gradually after arteriolar dilatation. Discrete nNOS neuron subsets may contribute either to the initiation or to the maintenance of the vascular response, suggesting a previously unappreciated temporal specificity to the role of NO in neurovascular coupling.

Sensory stimulation evokes a local, vasodilation-mediated blood flow increase to the activated brain region, which is referred to as functional hyperemia. Spontaneous vasomotion is a change in arteriolar diameter that occurs without sensory stimulation, at low frequency (∼0.1 Hz). These vessel diameter changes are a driving force for perivascular soluble waste clearance, the failure of which has been implicated in neurodegenerative disease. Stimulus-evoked vascular reactivity is known to propagate along penetrating arterioles to pial arterioles, but it is unclear whether spontaneous vasomotion propagates similarly. We therefore imaged both stimulus-evoked and spontaneous changes in pial arteriole diameter in awake, head-fixed mice with 2-photon microscopy. By cross-correlating different regions of interest (ROIs) along the length of imaged arterioles, we assessed vasomotion propagation. We found that both during rest and during visual stimulation, one-third of the arterioles showed significant propagation (i.e., a wave), with a median (interquartile range) wave speed of 405 (323) µm/s at rest and 345 (177) µm/s during stimulation. In a second group of mice, with GCaMP expression in their vascular smooth muscle cells, we also found spontaneous propagation of calcium signaling along pial arterioles. In summary, we demonstrate that spontaneous vasomotion propagates along pial arterioles like stimulus-evoked vascular reactivity.

The extraction of fluorophore lifetimes in a biological sample provides useful information about the probe environment that is not readily available from fluorescence intensity alone. Cell membrane potential, pH, concentration of oxygen ([O2]), calcium ([Ca2+]), NADH and other ions and metabolites are all regularly measured by lifetime-based techniques. These measurements provide invaluable knowledge about cell homeostasis, metabolism and communication with the cell environment. Fluorescence lifetime imaging microscopy (FLIM) produces spatial maps with time-correlated single-photon counting (TCSPC) histograms collected and analyzed at each pixel, but traditional TCSPC analysis is often hampered by the low number of photons that can reasonably be collected while maintaining high spatial resolution. More important, traditional analysis fails to employ the spatial linkages within the image. Here, we present a different approach, where we work under the assumption that mixtures of a global set of lifetimes (often only 2 or 3) can describe the entire image. We determine these lifetime components by globally fitting precise decays aggregated over large spatial regions of interest, and then we perform a pixel-by-pixel calculation of decay amplitudes (via simple linear algebra applied to coarser time-windows). This yields accurate amplitude images (Decay Associate Images, DAI) that contain stoichiometric information about the underlying mixtures while retaining single pixel resolution. We collected FLIM data of dye mixtures and bacteria expressing fluorescent proteins with a two-photon microscope system equipped with a commercial single-photon counting card, and we used these data to benchmark the gDAI program.

Two-photon fluorescence lifetime microscopy (2P-FLIM) is a non-invasive optical technique that can obtain cellular metabolism information based on the intrinsic autofluorescence lifetimes of free and enzyme-bound NAD(P)H, which reflect the metabolic state of single cells within the native microenvironment of the living tissue. NAD(P)H 2P-FLIM was initially performed in bone marrow stromal cell (BMSC) cultures established from Col (I) 2.3GFP or OSX-mCherry mouse models, in which osteoblastic lineage cells were labelled with green or red fluorescence protein, respectively. Measurement of the mean NAD(P)H lifetime, τM, demonstrated that osteoblasts in osteogenic media had a progressively increased τM compared to cells in regular media, suggesting that osteoblasts undergoing mineralization had higher NAD+/NAD(P)H ratio and may utilize more oxidative phosphorylation (OxPhos). In vivo NAD(P)H 2P-FLIM was conducted in conjunction with two-photon phosphorescence lifetime microscopy (2P-PLIM) to evaluate cellular metabolism of GFP+ osteoblasts as well as bone tissue oxygen at different locations of the native cranial bone in Col (I) 2.3GFP mice. Our data showed that osteocytes dwelling within lacunae had higher τM than osteoblasts at the bone edge of suture and marrow space. Measurement of pO2 showed poor correlation of pO2 and τM in native bone. However, when NAD(P)H 2P-FLIM was used to examine osteoblast cellular metabolism at the leading edge of the cranial defects during repair in Col (I) 2.3GFP mouse model, a significantly lower τM was recorded, which was associated with lower pO2 at an early stage of healing, indicating an impact of hypoxia on energy metabolism during bone tissue repair. Taken together, our current study demonstrates the feasibility of using non-invasive optical NAD(P)H 2P-FLIM technique to examine cellular energy metabolism at single cell resolution in living animals. Our data further support that both glycolysis and OxPhos are being used in the osteoblasts, with more mature osteoblasts exhibiting higher ratio of NAD+/NAD(P)H, indicating a potential change of energy mode during differentiation. Further experiments utilizing animals with genetic modification of cellular metabolism could enhance our understanding of energy metabolism in various cell types in living bone microenvironment.

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.

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Background: Intravital microscopy is an emerging technique in life science with applications in kidney research. Longitudinal observation of (patho-)physiological processes in living mice is possible in the smallest functional unit of the kidney, a single nephron (sn). In particular, effects on glomerular filtration rate (GFR) - a key parameter of renal function - can be assessed. Methods: After intravenous injection of a freely filtered, non-resorbable, fluorescent dye in C57BL/6 mice, a time series was captured by multiphoton microsopy. Filtration was observed from the glomerular capillaries to the proximal tubule (PT) and the tubular signal intensity shift was analyzed to calculate the snGFR. Results: Previously described methods for snGFR analysis relied on two manually defined measurement points in the PT and the tubular volume was merely estimated in 2D images. We present an extended image processing workflow by adding continuous measurement of intensity along the PT in every frame of the time series using ImageJ. Automatic modelling of actual PT volume in a 3D dataset replaced 2D volume estimation. Subsequent data analysis in R, with a calculation of intensity shifts in every frame and normalization against tubular volume, allowed exact assessment of snGFR by linear regression. Repeated analysis of image data obtained in healthy mice showed a striking increase of reproducibility by reduction of user interaction. Conclusions: These improvements in image processing and data analysis maximize the reliability of a sophisticated intravital microscopy technique for the precise assessment of snGFR, a highly relevant predictor of kidney function.

Intravital microscopy is an imaging technique aimed at the visualization of the dynamics of biological processes in live animals. In the last decade, the development of nonlinear optical microscopy has enormously increased the use of this technique, thus addressing key biological questions in different fields such as immunology, neurobiology and tumor biology. In addition, new upcoming strategies to minimize motion artifacts due to animal respiration and heartbeat have enabled the visualization in real time of biological processes at cellular and subcellular resolution. Recently, intravital microscopy has been applied to analyze different aspect of mucosal immunity in the gut. However, the majority of these studies have been performed on the small intestine. Although crucial aspects of the biology of this organ have been unveiled, the majority of intestinal pathologies in humans occur in the large intestine.Here, we describe a method to surgically expose and stabilize the large intestine in live mice and to perform short-term (up to 2 h) intravital microscopy.

To understand how arousal state impacts cerebral hemodynamics and neurovascular coupling, we monitored neural activity, behavior, and hemodynamic signals in un-anesthetized, head-fixed mice. Mice frequently fell asleep during imaging, and these sleep events were interspersed with periods of wake. During both NREM and REM sleep, mice showed large increases in cerebral blood volume ([HbT]) and arteriole diameter relative to the awake state, two to five times larger than those evoked by sensory stimulation. During NREM, the amplitude of bilateral low-frequency oscillations in [HbT] increased markedly, and coherency between neural activity and hemodynamic signals was higher than the awake resting and REM states. Bilateral correlations in neural activity and [HbT] were highest during NREM, and lowest in the awake state. Hemodynamic signals in the cortex are strongly modulated by arousal state, and changes during sleep are substantially larger than sensory-evoked responses.

Dendritic spines are microscopic protrusions on neurons that house the postsynaptic machinery necessary for neurotransmission between neurons. As such, dendritic spine structure is intimately linked with synaptic function. In pathology, dendritic spine behavior and its contribution to disease are not firmly understood. It is well known that dendritic spines are highly dynamic in vivo. In our recent publication, we used an intravital imaging approach, which permitted us to repeatedly visualize the same neurons located in lamina II, a nociceptive processing region of the spinal cord. Using this imaging platform, we analyzed the intravital dynamics of dendritic spine structure before and after nerve injury-induced pain. This effort revealed a time-dependent relationship between the progressive increase in pain outcome, and a switch in the steady-state fluctuations of dendritic spine structure. Collectively, our in vivo study demonstrates how injury that leads to abnormal pain may also contribute to synapse-associated structural remodeling in nociceptive regions of the spinal cord dorsal horn. By combining our live-imaging approach with measures of neuronal activity, such as with the use of calcium or other voltage-sensitive dyes, we expect to gain a more complete picture of the relationship between dendritic spine structure and nociceptive physiology.