Intravital multiphoton microscopy as a novel tool in the field of immunopharmacology

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Abstract

Intravital microscopy with multiphoton excitation is a recently developed optical imaging technique for deep tissue imaging without fixation or sectioning, which permits examination of fundamental concepts regarding the dynamic nature of cells under physiological and pathological conditions in living animals. This novel technique also offers exciting opportunities for pharmacological research by providing new platforms for the study of cellular dynamics in response to drugs in vivo. Moreover, fluorescent chemical probes for functional or molecular analysis in single cells in vivo play important roles in pharmacology. For example, we have recently revealed the pharmacodynamic actions of different biological agents for the treatment of rheumatoid arthritis (RA) in vivo by directly visualizing drug-induced cellular behaviors and functions of osteoclasts on bone surfaces. This review focuses on the principles and advantages of intravital imaging for the dissection of pharmacological mechanisms, and discusses how such imaging can contribute to the drug development process, introducing recent trials that evaluated the in vivo pharmacological effects of various agents.

Introduction

Many advances have been made in molecular and cellular biology, drug discovery, and their clinical applications over the past two decades. The pathogenesis of acquired diseases, such as cancer, inflammatory diseases, infection, and bone diseases, and their responses to therapeutics, are highly dynamic processes. However, most recent basic research in this area has been based on the analysis of static samples, such as tissue sections or cell suspensions, and thus does not necessarily reflect the natural dynamics of biological processes in the native microenvironment in vivo.

Intravital imaging is a recently developed technique for the observation of living, intact biological tissues in experimental animals. By capturing images continuously, intravital imaging allows longitudinal and direct visualization of cellular behaviors in space and time, which enables analysis of the characteristics of cell migration (Germain, Robey, & Cahalan, 2012), motility (Kikuta et al., 2013), proliferation (Sakaue-Sawano et al., 2008), death (Mempel et al., 2006), and division (Lefrançais et al., 2017), as well as cell–cell interactions (Mempel, Henrickson, & Von Andrian, 2004), in complex tissue microenvironments. Fluorescent labeling of target cells, immobilization of fields of view, and quantitative analysis of imaging data are often difficult, but necessary steps for intravital imaging. Multiphoton excitation microscopy (MPEM) has provided support in this research area. In this system, a multiphoton laser induces focal excitation of fluorophores by simultaneous stimulation by multiple photons. MPEM has several advantages over the use of conventional fluorescence and confocal microscopy for the observation of intact deep tissue, including reduced phototoxicity, photobleaching, and autofluorescence. The near-infrared femtosecond lasers employed by MPEM provide these advantages because the laser beam is concentrated in a small region (Fig. 1). MPEM can also detect second harmonic signals; collagen is the strongest source of such signals in animal tissue due to its unique triple helix structure (Cox & Kable, 2006). Synergistic multicolor imaging using multiple excitation lasers and an effective spectral unmixing algorithm enable multiplex fluorescent intravital imaging (Rakhymzhan et al., 2017).

Therapeutic approaches for immunological diseases have changed markedly over the last several years, and a diverse range of novel drugs, including biological agents and molecular targeted drugs, has emerged. These drugs are generally highly effective and have revolutionized clinical practice for rheumatic and allergic diseases, although their actual pharmacological actions have not been elucidated fully. The International Union of Pharmacology (IUPHAR) has issued guidelines regarding immunopharmacology for these new therapeutics (Ishii, 2017; Tiligada et al., 2015). Several key technologies are involved in the promotion of immunopharmacology, with intravital MPEM representing the chief such technology. Real pharmacological actions in vivo would be best analyzed by direct visualization of live tissues and organs in intact tissue. Using MPEM, our group has investigated the dynamic processes of bone metabolism under physiological and pathological conditions, and responses to therapeutics. This review discusses the principles and applications of intravital imaging using MPEM, the methods used for analysis of the obtained images, and their contributions to medical science and pharmacology. We also describe recent advances in imaging analysis of bone metabolism and how they have contributed to the in vivo pharmacological evaluation of some bone protecting agents in the contexts of RA and osteoporosis.

Section snippets

Advantages of intravital multiphoton microscopy for pharmacological research

While recent genome-wide studies have allowed rapid identification of large quantities of target molecules for pharmaceuticals, drug discovery and development have become complex processes with high costs and labor requirements because of their high degrees of uncertainty and high rates of failure (Hon & Lee, 2017). For example, some molecular targeted drugs, including small molecules and monoclonal antibodies (mAbs), have off-target effects, involving the binding of drugs to proteins other

Intravital multiphoton imaging of living bone tissue

Bone is a dynamic tissue that undergoes continuous building and degradation throughout life. During bone remodeling, homeostasis is regulated by endogenous systems to match bone formation by osteoblasts with the amount of resorption by osteoclasts. Osteoblasts and osteoclasts are considered to communicate with each other to meet these needs, and this local cell–cell communication system, named “coupling,” is mediated by various soluble and membrane-bound factors (Sims & Martin, 2014; Sims &

Evaluation of biological agents for rheumatoid arthritis

Intravital imaging of osteoclasts is not only an essential tool for analysis of their migration, differentiation, and function, but would also contribute to the discovery and development of drugs for the treatment of osteoclast-mediated bone diseases.

RA is a common autoimmune disease characterized by chronic inflammation and destruction of synovial joints. In addition to joint stiffness, tenderness, and swelling, articular bone erosion is a central clinical feature of RA. Prolonged inflammation

Evaluation of osteogenic agents for osteoporosis

Osteoporosis is characterized by low bone mineral density and poor bone microarchitecture, associated with high fracture risk. This disease is caused by excessive bone resorption without an equivalent increase in bone formation. The most common therapeutic strategies for osteoporosis have traditionally involved the use of antiresorptive drugs, primarily bisphosphonates; some osteogenic agents, such as parathyroid hormone (PTH) and romosozumab, a specific inhibitor of sclerostin, have also been

Summary and future perspectives

While recent advances in molecular biology have accelerated the discovery of bioactive molecules, pharmacological research faces serious concerns regarding labor requirements and costs because of the complex and unpredictable effects of these molecules in living organisms. Intravital imaging provides unbiased spatiotemporal information on living tissues in the native microenvironment, which has revealed much more complexity than previously thought. It can also provide information regarding

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported in part by CREST, Japan Science and Technology Agency; Grants-in-Aid for Scientific Research (A) from the Japan Society for the Promotion of Science (JSPS to MI); grants from the Uehara Memorial Foundation (to MI); from the Kanae Foundation for the Promotion of Medical Sciences (to MI); from Mochida Memorial Foundation (to MI); and from the Takeda Science Foundation (to MI).

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