Near Infrared Fluorophore Specific for Annexin A2 Identifies Peripheral Nerve Injury in a Rodent Crush Model
David M Brogan, MD, MSc1; Tony Y Lee, BA2; Samuel Achilefu, Ph.D.2; Christopher J Dy, MD2
1Washington University School of Medicine, St. Louis, MO; 2Washington University in St. Louis, St. Louis, MO
Introduction: Increased permeability in the blood nerve barrier is found after peripheral nerve injury and annexin A-2 has been shown to translocate to Schwann cell surface after injury, influencing vascular permeability as well as leukocyte recruitment. We hypothesize that this change in permeability can facilitate intraneural transit of LS-301, a near-infrared compound that binds to phosphorylated Annexin A-2 to identify nerve injury.
Materials & Methods: Six groups of 8 Lewis rats (48 total) were subjected to a mild or severe unilateral sciatic nerve crush injury. Bilateral sciatic nerves were marked with epineural sutures to define a region of interest(ROI). One of two vascular clamps was applied to the ROI for 60 seconds then imaged with a Licor Pearl Imager. Cypate-3 dye (emission wavelength 700 nm) and LS-301 dye (emission wavelength 800 nm) were intravenously administered and imaging repeated for crush and control sides(Figure 1). Non-survival surgeries and repeat imaging were performed at 5 hrs, 48 hrs and 2 weeks (16 rats per time period- 8 mild crush, 8 severe). In 48 hr and 2 week groups, baseline imaging was performed at non-survival surgery, dye injected and imaging repeated 5 hours later. Multiple paired t-tests were performed to compare control and injured fluorescence with correction for multiple comparisons using Holm-Sidak method.
Results: Fluorescence within the ROI of the mildly injured nerve (M =0.088 ; SD =0.034) and severely injured nerve (M = 0.073; SD = 0.021) was significantly increased compared to their contralateral control (M = 0.045; SD = 0.012 and M=0.026; SD = 0.010 respectively) (p<0.01 and p<0.01) at 5 hrs after injury (Figure 2). Fluorescence was also increased 48 hours after injury and probe injection in mild (M=0.036; SD=0.005) and severe crush (M=0.023; SD = 0.010) compared to the uninjured sides (M=0.016; SD=0.005 and M=0.010; SD=0.005 respectively)(p<0.01 and p<0.01).
- LS-301 demonstrated increased fluorescence in injured nerves at 5 and 48 hrs post injury.
- LS-301 is currently in clinical trials for imaging of solid tumors and its use could be expanded to identify nerve injury intra-operatively.
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