In situ Microinflammation Detection Using Gold Nanoclusters and a Tissue-clearing Method

Abstract

Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4⁺ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au₁₃ nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.

Keywords: Au13 nanoclusters; CD4+ T cells; CUBIC; Experimental autoimmune encephalomyelitis (EAE); Gateway reflex; Microinflammation; Myelin oligodendrocyte glycoprotein (MOG).

Figure 1.. Overview of procedures for microinflammation detection using Au₁₃ nanoclusters and the CUBIC tissue-clearing method.

Mice were immunized with MOG_35-55/CFA (IFA containing M. tuberculosis H37RA) and underwent PTx injection. On days 16–20, when experimental autoimmune encephalomyelitis (EAE) develops with clinical scores of 2–3, the EAE mice were injected with Au₁₃ nanoclusters, anesthetized after 2 h, and transcardially perfused with PBS and then PFA. The spine with the ribs was taken out and further subjected to PFA fixation for one day. Then, L1–L6 were excised carefully and immersed in delipidation/decoloration reagent (hexanediol/methylimidazole) for four days (days 1–5). After delipidation and decoloration, L1–L6 were stained with anti-SMA or anti-CD31 for seven days (days 5–12) and subjected to refractive index adjustment in CUBIC-R (days 12–14). The Au signal was visualized by light sheet microscopy and analyzed by Imaris software.

Video 1.. Procedures for the L1-L6 preparation.

After transcardial perfusion with PBS followed by PFA, the spine with the ribs was immediately taken out and further fixed with PFA. Then, L1–L6 were excised by carefully cutting the intervertebral discs between T13-L1 and L6-S1 with a razor, with the T13 rib as a position marker under stereomicroscopy installed with an LED illuminator.

Figure 2.. Cleared lumbar cords.

Brightfield image of the lumbar cords before and after tissue clearing.

Figure 3.. Detection of microinflammation at L5 by Au₁₃ nanoclusters and staining of the artery with anti-SMA antibody.

(A) aEAE mice with clinical score 2.5 and naïve mice were injected i.v. with Au₁₃ nanoclusters or solvent. Figures show cleared L3–L6 from each mouse stained with anti-SMA antibody, which recognizes smooth muscle actin on the artery. The signal of Au₁₃ nanoclusters was visualized as a punctate signal with a diameter of 50–200 nm in the dorsal blood vessels centered on L5 and in the parenchyma of the spinal cords in aEAE mice. Intravascular Au signals were also observed in aEAE and naïve mice injected with Au₁₃ nanoclusters. Scale bars, 1,500 μm. Magenta: Au₁₃ nanocluster; green: SMA (artery). (B) Magnified images of L5 from the aEAE and naïve mice in (A). Scale bars, 1,000 μm. Magenta: Au₁₃ nanocluster; green: SMA (artery).

Figure 4.. Detection of microinflammation at L5 by Au₁₃ nanoclusters and staining of whole blood vessels with anti-CD31 antibody.

aEAE mice with clinical score 3 were injected i.v. with Au₁₃ nanoclusters. Cleared L5 stained with anti-CD31 antibody, which recognizes platelet endothelial cell adhesion molecule-1 on whole blood vessels, is shown. Scale bars, 400 μm. Green: Au₁₃ nanocluster; red: CD31 (blood vessel).

Video 2.. The detection of microinflammation at L5 by Au₁₃ nanoclusters and staining of the artery with anti-SMA antibody.

A 3D movie of the lumbar spinal cords of an aEAE mouse with clinical score 2.5 after injecting i.v. with Au₁₃ nanoclusters. The cleared lumbar spinal cords were stained with anti-SMA antibody, which recognizes smooth muscle actin on the artery. Magenta: Au₁₃ nanocluster; green: SMA (artery).

Video 3.. The detection of microinflammation at L5 by Au₁₃ nanoclusters and staining of whole blood vessels with anti-CD31 antibody.

A 3D movie of the lumbar spinal cords of an aEAE mouse with clinical score 3 after injecting i.v. with Au₁₃ nanoclusters. The cleared lumbar spinal cords were stained with anti-CD31 antibody, which recognizes platelet endothelial cell adhesion molecule-1 on whole blood vessels. Green: Au₁₃ nanocluster; red: CD31 (blood vessel).