Optical imaging of mouse articular cartilage using the glycosaminoglycans binding property of fluorescent-labeled octaarginine.
OBJECTIVE
The aim of the current study was to examine the cartilage-specific binding property of polyarginine peptides (R4, 8, 12, and 16) and specifically to test octaarginine peptides for the optical imaging of articular cartilage in experimentally induced arthritis in mice.


METHODS
Four rhodamine-labeled polyarginine peptides each with a different-length arginine chain (R4, 8, 12, or 16) were injected into the knee joints of C57BL/6J mice (n=20). The joints were excised 1h later and the fluorescent signal intensity in cartilage cryosections was compared for the four peptides. To examine the substrate of R8 in cartilage, femoral condyles obtained from another set of mice were treated with chondroitinase ABC (Ch'ase ABC), keratanase or heparitinase then immersed in R8-rhodamine. Fluorescent signals were examined by fluorescent microscopy. Next, R8-rhodamine was injected into the right knee joints of three control and three collagen antibody-induced arthritis (CAIA) mice, and fluorescent intensity in normal and degenerative cartilage was semi-quantitatively analysed on the histological sections using image software. Finally, femoral condyles from normal mice (n=2) and CAIA mice (n=2) were immersed in R8-rhodamine and calcein, then imaged using optical projection tomography (OPT).


RESULTS
Fluorescent signals were specifically detected in the cartilage pericellular matrix from the surface to the tide mark but were completely absent in the calcified layer or bone marrow. The number of arginine residues significantly influenced peptide accumulation in articular cartilage, with R8 accumulating the most. The fluorescent signal in the femoral condylar cartilage diminished when it was treated with Ch'ase ABC. R8 accumulation was significantly decreased in the degenerative cartilage of CAIA mice, and this was demonstrated both histologically and in three-dimensional (3D)-reconstruction image by OPT.


CONCLUSIONS
R8 may be a useful new experimental probe for optical imaging of normal and arthritic articular cartilage.
Inagawa
K
K
Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
Oohashi
T
T
Nishida
K
K
Minaguchi
J
J
Tsubakishita
T
T
Yaykasli
K O
KO
Ohtsuka
A
A
Ozaki
T
T
Moriguchi
T
T
Ninomiya
Y
Y