In vivo imaging of the neurovascular unit in CNS disease
The neurovascular unit—comprised of glia, pericytes, neurons and cerebrovasculature— is a dynamic interface that ensures physiological central nervous system (CNS) functioning. In disease dynamic remodeling of the neurovascular interface triggers a cascade of responses that determine the extent of CNS degeneration and repair. The dynamics of these processes can be adequately captured by imaging in vivo, which allows the study of cellular responses to environmental stimuli and cell-cell interactions in the living brain in real time. This perspective focuses on intravital imaging studies of the neurovascular unit in stroke, multiple sclerosis (MS) and Alzheimer disease (AD) models and discusses their potential for identifying novel therapeutic targets.
In vivo dynamics of innate immune sentinels in the CNS
The innate immune system is comprised of cellular sentinels that often serve as the first responders to injury and invading pathogens. Our basic understanding of innate immunity is derived from research conducted in peripheral lymphoid tissues. However, it is now recognized that most non-lymphoid tissues throughout the body are equipped with specialized innate immune cells that are uniquely adapted to the niches in which they reside. The central nervous system (CNS) is a particularly interesting compartment because it contains a population of post-mitotic cells (neurons) that are intolerant of robust, cytopathic inflammatory responses observed in many peripheral tissues. Thus, evolutionary adaptations have fitted the CNS with a unique array of innate immune sentinels that facilitate the development of local inflammatory responses but attempt to do so in a manner that preserves the integrity of its post-mitotic residents. Interestingly, studies have even suggested that CNS resident innate immune cells contribute to the homeostasis of this compartment and promote neural activity. In this review we discuss recent advances in our understanding of CNS innate immune sentinels and how novel imaging approaches such as intravital two-photon laser scanning microscopy (TPLSM) have shed light on these cells during states of health and disease.
Novel in vivo imaging techniques for the liver microvasculature
Intravital microscopy is a valuable tool in studying the liver, a complex organ with a unique sinusoidal microcirculation and both metabolic and immune functions. The liver is also subject to a large variety of diseases including viral, bacterial and parasitic infections. We developed novel recording techniques to visualize dynamic events in the hepatic microvasculature without the need of fluorescent markers. In combination with cellular and molecular probes, reporter mice and Plasmodium as a hepatotropic model microorganism, we demonstrate the power of these techniques in monitoring the development of the malaria parasite and the response of the hepatic microenvironment to infection.
Motion compensation using a suctioning stabilizer for intravital microscopy
Motion artifacts continue to present a major challenge to single cell imaging in cardiothoracic organs such as the beating heart, blood vessels or lung. In this study, we present a new water-immersion suctioning stabilizer that enables minimally invasive intravital fluorescence microscopy using water-based stick objectives. The stabilizer works by reducing major motion excursions and can be used in conjunction with both prospective or retrospective gating approaches. We show that the new approach offers cellular resolution in the beating murine heart without perturbing normal physiology. In addition, because this technique allows multiple areas to be easily probed, it offers the opportunity for wide area coverage at high resolution.
In vivo assessment of the pulmonary microcirculation in elastase-induced emphysema using probe-based confocal fluorescence microscopy
Introduction: The alveolar capillary bed, which appears essential for the maintenance of alveolar septa, is altered in pulmonary emphysema. Until recently, techniques that allow its analysis in vivo in spontaneously breathing conditions were lacking. Fibered confocal fluorescence microscopy (FCFM) is a new technique that enables distal lung microstructures imaging in vivo. FCFM can be coupled with I.V fluorescein injection to image the pulmonary capillary network. The aim of this study was to assess the lung microcirculation in vivo using FCFM and I.V fluorescein in rats with experimental emphysema.
Results: In vivo pulmonary microcirculation imaging was possible in 7/7 elastase animals and in 6/7 controls. Using FCFM, intercapillary distances and alveolar facets diameters were found significantly higher in the elastase group compared with controls (49.5 vs. 41.8 µm p < 0.001, and 118.5 vs. 95.1 µm p < 0.001, respectively). Ex vivo mean interwall distance (MIWD) was correlated with the alveolar facets diameters measured in vivo (rs = 0.65 ; p = 0.016).
Methods: 14 Sprague-Dawley rats were assigned to intratracheal instillation of porcine pancreatic elastase (n = 7) or saline (n = 7). The subpleural microcirculation was assessed using FCFM in spontaneously breathing rats, through a 2mm thoracic window using a continuous aspiration system, after I.V. injection of fluorescein-dextran. FCFM sequences were recorded and the image analysis was performed separately by two observers, blindly to the animal group. Fluorescence intensity (FI), maximal intercapillary distances, and alveolar facets diameters measured with FCFM were compared between groups, and to ex vivo lung morphometric measurements (MIWD).
Conclusion: FCFM allows the quantitative assessment of the microcirculation alterations due to emphysema in vivo.
In vivo tracking of hematopoietic cells in the retina of chimeric mice with a scanning laser ophthalmoscope
We examine the effect of bone marrow transplantation (BMT) on retinal cell turnover by performing simultaneous cell tracking of native microglia and engrafting donor bone marrow-derived cell (BMDC) populations in the retinae of live mice using a custom-built multi-color confocal scanning laser ophthalmoscope (SLO) specifically developed for murine retinal imaging. CX3CR1GFP/+ mice whose retinal microglia express the green fluorescent protein (GFP) were exposed to a lethal dose of gamma radiation and subsequently rescued with bone marrow cells from universal DsRed donor mice. Over a time course of four months after the irradiation and BMT, progressive loss of GFP+ microglia was accompanied by delayed engraftment of DsRed+ BMDC. Morphologic examination revealed that the remaining GFP+ microglia were ramified, while engrafting DsRed+ cells exhibited both ramification and dendriform shape. Leukocyte endothelial interaction, normally absent in healthy retinal vasculature, was observed even after three months, indicating sustained inflammation long after the radiation exposure. Fluorescein angiography demonstrated that the blood-retina barrier is compromised early after irradiation. In vivo imaging provides a powerful means to study dynamic cellular processes over a broad range of timescales from seconds to months that have previously not been accessible by ex vivo analysis.