Quantitative photoacoustic imaging for early detection of muscle ischemia injury

Abstract

Acute lower extremity ischemia is a limb-and life-threatening problem. The timing of clinical intervention is critical to achieving optimal outcomes. However, there has been a lack of effective techniques capable of evaluating muscle and limb damage. Microcirculatory injury is the initial pathological change during ischemic muscle injury. Here, we performed photoacoustic imaging (PAI) in real time to quantitatively detect the degree of microcirculatory injury of ischemic muscles in a rat model in which Evans blue (EB), which strongly binds to albumin in blood, was used as a nontoxic molecular PA probe. The right lower hind limbs of Sprague-Dawley (SD) rats were subjected to 2 or 3 hours of tourniquet-induced ischemia. Then, PA imaging of the tibialis anterior (TA) muscles in the anterior compartment was performed for 0-24 h after the release of compression. Twenty-four hours after reperfusion, rats were euthanized and examined for pathology, edema and muscle viability. Imaging at 680 nm on rats revealed that there was significant signal enhancement in the TA muscles of the two injury groups compared to the control group, and the 3-h injury group had significantly higher PA signal intensity than the 2-h injury group at each time point. Histopathology results obtained from both the normal and the damaged muscles correlated well with the PAI findings. In conclusion, PA imaging is a promising modality for quantitatively detecting limb and muscle ischemic injury and may pave the road for further clinical application.

Keywords: Muscle; extremity; ischemic injury; microcirculatory injury; photoacoustic imaging.

Figure 1

Photograph of the Endra Nexus 128 PA scanner (Ann Arbor, MI) system used for in vivo photoacoustic imaging of rat muscle.

Figure 2

PA image at 680 nm of a rat tibialis anterior (TA) muscles (24 h after ischemia injury, with no contrast agent injection). A. Optical photograph of TA muscles. B. 3D PA image of normal TA muscles (L: left hind limb), the white dotted line indicated the vascularity and muscle texture. C. 3D PA image of injured muscles (R: right hind limb); the PA signal significantly decreased and the vascularity in the muscle cannot be identified.

Figure 3

Accumulation of EB-albumin in the tibialis anterior (TA) muscles. A. Representative 3D PA images of the TA muscle injury region obtained at 0.5 h, 6 h and 24 h after intravenous injection of EB. The PA images showed the accumulation and distribution of the EB probe within the damaged muscle region (red dotted line) in two injury groups (R: right hind limb). B. Quantification of PA intensities analysis at 680 nm in the region of TA muscles at different time points after intravenous injection of EB. C. Quantitative analysis of PA signal increases after intravenous injection with EB at 6 h compared with 0 h. All results represent mean ± S.D (**P<0.01; ***P<0.001, n=5).

Figure 4

Wet: dry weight ratios. Overall compression resulted in a dramatic increase in the muscle wet:dry weight ratios. Additionally, the 3-h ischemia injury group demonstrated a significant increase in the wet:dry weight ratio compared with the control and 2-h groups, All results represent mean ± S.D (*P<0.05; **P<0.01, n=5).

Figure 5

Histologic findings in muscle tissues in the rat model. Compared to the 2 h group, 3 h ischemia caused greater muscle damage, interstitial edema and hemorrhage. Red arrows show the red cells that infiltrated. Triangles show the collapsed muscle fibers. Blue arrows show the infiltration of neutrophils (HE staining) and myeloperoxidase (anti-MPO staining). Magnification × 400.

Figure 6

Apoptosis measured by TUNEL staining in the TA muscles from each experimental group. TUNEL: TdT-mediated dUTP Nick-End Labeling, a marker for apoptosis and DAPI: 4’,6-diamidino-2-phenylindole. All results are given as the mean ± S.D, n=5 rats (5 slices in each rat) in each group (**P<0.01; ***P<0.001).