Background

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), mediated by myelin-specific autoreactive CD4⁺ T cells (International Multiple Sclerosis Genetics et al., 2011). Active experimental autoimmune encephalomyelitis (aEAE) is a well-established animal model of MS that is induced by immunizing mice with myelin oligodendrocyte protein–derived peptide (MOG) and complete Freund's adjuvant (CFA) together with pertussis toxin (PTx). However, where and how pathogenic CD4⁺ T cells enter the CNS from peripheral blood and cause the disease had been unclear, since it was believed that the CNS is protected from immune cells by the blood–brain barrier (Liu et al., 2012). Using a passive transfer model of EAE, in which pathogenic CD4⁺ T cells including autoreactive CD4⁺ T cells from the secondary lymphoid tissues of aEAE mice are transferred to naïve mice, we demonstrated that sensory-sympathetic crosstalk, triggered by gravity stimulation via the soleus muscle, leads to the accumulation of pathogenic immune cells at the dorsal vessels of the fifth lumbar cord (L5) locally, which in turn causes microinflammation, a chronic inflammation that enhances the permeability of specific blood vessel sites by elevated local inflammatory mediators, such as IL-6 and TNF-α (Arima et al., 2012). Importantly, this permeability enables autoreactive CD4⁺ T cells to enter the CNS. We named this specific neuro-immune interaction the gateway reflex and have since demonstrated it can be activated by six types of environmental stimuli: gravity, electricity, pain, stress, light, and inflammation (Arima et al., 2012, 2015 and 2017; Hasebe et al., 2022; Murakami et al., 2019; Stofkova et al., 2019).

Coordinated gold nanoclusters (AuNCs) have an atomically precise molecular structure, ultrasmall size, facile surface modification, and characteristic optical properties (Jin et al., 2016). Some AuNCs have been reported to exhibit unique photoluminescence emission with long lifetimes and large Stokes shift from the visible to near-infrared wavelengths (Chen et al., 2015; Yu et al., 2019), as well as high compatibility and photostability. Thus, they are good candidates as luminophores for long-term imaging, high-sensitivity detection, and target-specific treatment (Luo and Liu, 2022; Palmal and Jana, 2014; Zhang et al., 2022). The usefulness of AuNCs as fluorescent probes has already been shown in imaging, detection, and therapy. Phosphine-coordinated AuNCs represent a luminescent gold cluster family (Konishi et al., 2018; Sugiuchi et al., 2017). Among them, diphosphine-coordinated Au₁₃ nanoclusters with an icosahedral gold core motif are interesting, because of their easy accessibility, robustness, and moderately intense photoluminescence in the near-infrared region (Shichibu and Konishi, 2010; Sugiuchi et al., 2015).

The development of tissue-clearing reagents and protocols, efficient fluorescent labeling, and rapid volumetric imaging by light sheet microscopy has enabled new tissue-clearing methods for the 3D imaging of intact tissues and even entire organisms (Hama et al., 2015; Ueda et al., 2020). Despite tissue-clearing approaches enabling high tissue transparency within a few days using organic chemicals (e.g., benzyl alcohol and benzyl benzoate), concerns about the quenching of fluorescent proteins and safety have remained. In response, Tainaka et al. (2014) developed the hydrophilic unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method, which efficiently removes lipids, pigments, and calcium phosphate to retain the fluorescent signals and enable whole-body imaging with single-cell resolution in mice. Combining light sheet microscopy and CUBIC can visualize rare cells and complex structures in various tissues at the whole organ level.

Chronic microinflammation is a hallmark of many inflammatory diseases, including autoimmune, neurodegenerative, and metabolic syndrome–driven diseases. In such inflammatory diseases with stochastic and proliferative processes, single-cell events ultimately affect the health status of the entire organism. Therefore, a detection system that enables quick identification of the development and progression of microinflammation is desired. However, conventional protocols for identifying inflammation sites are mainly based on 2D histology using inflammatory mediator-specific reporter mice or antibodies, which makes it difficult to accurately identify the sites depending on the tissue sections.

In this protocol, we show a rapid 3D-imaging method to detect microinflammation in aEAE mice using Au₁₃ nanoclusters and CUBIC. Au₁₃ signals leaked out from the blood vessels around the L5, indicating enhanced vascular permeability caused by microinflammation, supporting our previous findings of a vascular gate formed for pathogenic immune cells to enter the CNS in this region (Arima et al., 2012). Thus, our method is useful for studying the stepwise development of microinflammation at the whole organ level in various inflammatory diseases and will also provide future perspectives of Au₁₃ nanocluster-based imaging techniques to study biological events, which are induced by the enhancement of vascular permeability at specific vessel sites.