Study of Osteocyte Behavior by High-Resolution Intravital Imaging Following Photo-Induced Ischemia

Abstract

Ischemic injuries and local hypoxia can result in osteocytes dysfunction and play a key role in the pathogenesis of avascular osteonecrosis. Conventional imaging techniques including magnetic resonance imaging (MRI) and computed tomography (CT) can reveal structural and functional changes within bony anatomy; however, characterization of osteocyte behavioral dynamics in the setting of osteonecrosis at the single cell resolution is limited. Here, we demonstrate an optical approach to study real-time osteocyte functions in vivo. Using nicotinamide adenine dinucleotide (NADH) as a biomarker for metabolic dynamics in osteocytes, we showed that NADH level within osteocytes transiently increase significantly after local ischemia through non-invasive photo-induced thrombosis of afferent arterioles followed by a steady decline. Our study presents a non-invasive optical approach to study osteocyte behavior through the modulation of local environmental conditions. Thus it provides a powerful toolkit to study cellular processes involved in bone pathologies in vivo.

Keywords: NADH; osteocyte; osteonecrosis; two-photon.

Figure 1

Schematic illustration of two photon imaging system. Abbrv: M, mirror; DC, dichroic mirror; MO, microscope objective; PMT, photomultiplier tube; 2D Galvo, two dimension galvanometer

Figure 2

Intravital two-photon imaging of osteocytes: (A) Osteocytes were connected to one another through a network of cytoplasmic projections; (B) Extracellular matrix; (C) Magnification of the inset in Figure 2A; (D) Overlap of Figure 2A,B.

Figure 3

Photo-thrombosis of the arterioles: (A) Before photo-thrombosis, white circle demonstrates the target site of the laser beam; (B) after photo-thrombosis, many black dots (un-labeled RBC) were noted within the vessel; (C) velocity measurement of the blood flow.

Figure 4

The changes of osteocytes after photo-thrombosis: (A) Osteocytes before they were stained with Calcein and PI at 0 h 3 h and 6 h after photo-thrombosis; (B) Percentage of dead osteocytes; (C) The intensity ratio of PI/Calcein. ** p < 0.01, by a two-tailed unpaired t-test; ns, not significant.

Figure 5

Intensity quantification of NADH auto-fluorescence after photo-thrombosis: (A–D) NADH auto-fluorescence (blue) changes inside the bone at the different time point (E) quantitative analysis of NADH auto-fluorescence change.

Figure 6

Histological examination of classic avascular osteonecrosis: (A) Immunohistochemical manifestation of HIF-1α in different postoperative stages; (B) tunnel staining of femoral heads, the positive osteocytes are shown with black arrows; (C) HIF-1α mRNA levels analyzed by real-time PCR; (D) the percentage of Tunnel positive osteocytes. ** p < 0.01, by a two tailed unpaired t-test.