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Implantable Oxygen Biosensor Reveals Post-Occlusion Tissue Reactive Hyperoxia
Jennifer S. Chien, B.S.E, Mahmoud Mohammed, B.E., Hysem Eldik, B.S., Mohamed Ibrahim, M.D., Scott Nichols, PhD, Natalie Wisniewski, PhD, Bruce Klitzman, PhD.
Duke University, Durham, NC, USA.

Introduction:
This study utilized an implantable biosensor to measure tissue oxygenation before, during, and after a transient ischemic insult. The purpose of this study was to use implantable O2 sensors to quantify local O2 content following ischemic challenges and perfusion restoration.
Material & Methods:
All animals were treated according to approved IACUC protocols. First, rats were subjected to unilateral femoral artery and vein ligation and received bilateral biosensor implants in the hind limbs. The biosensors were composed of a porphyrin embedded into a porous poly-hydroxyethylmethacrylate (pHEMA) scaffold and were injected through an 18-gauge needle into the subcutis. The biosensor’s phosphorescence lifetime changed inversely with O2 concentration. Near infrared spectroscopy (NIR) was also used to quantify percent O2 saturation of hemoglobin at the tissue level. Laser Doppler flowmetry was used to quantify blood flow. On post-operative days 28 and 84, the rats underwent a series of systemic hypoxic challenges as well as bilateral hind limb tourniquet application. Response magnitudes, response times and normalized changes were calculated for comparison between techniques. Statistical significance was assessed using ANOVA and two-sample paired t-test at p-value < 0.05.
Results & Discussion:
Laser Doppler flowmetry confirmed a reactive hyperemia following tourniquet release. In addition, both the phosphorescence lifetime biosensor and NIR methods suggested that the tissue O2 content temporarily but significantly exceeded baseline following tourniquet release (p<0.05). We term this phenomenon reactive hyperoxia. (Figure 1). This suggests that the O2 supply transiently exceeds oxygen consumption. Even though tissue O2 consumption was expected to be elevated following ischemia to repay the O2 debt developed during occlusion, the elevated O2 content indicates that the increased consumption is met with an even greater elevated O2 supply. The NIR spectroscopy and phosphorescence lifetime data are consistent. The phosphorescence lifetime biosensor also demonstrates a more prominent response magnitude in general and a faster response over time (71 secs vs. 143 secs in NIR; p = 0.0053), suggesting that the sensing elements may have gained greater proximity to the blood supply via vascular ingrowth through the porous pHEMA scaffold.
Conclusion:
The implantable phosphorescence lifetime biosensor provided real time monitoring of tissue oxygenation, with a rapid and prominent response, excellent biocompatibility and highly localized measurements. Both the phosphorescence lifetime and NIR spectroscopy techniques demonstrated a reactive hyperoxia post ischemia. Direct assessment of tissue oxygenation may provide helpful diagnostic and prognostic information on wound healing, re-vascularization, 3D scaffold design and efficacy of therapeutics promoting tissue oxygenation.


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