Wound healing poses significant challenges due to compromised vascularity. This study used Oxygen Saturation – Dynamic Contrast Enhanced Multispectral Optoacoustic Tomography (OS-DCE MSOT) to longitudinally monitor wound healing in a mouse model (1,2). OS-DCE MSOT is employed as a high-resolution imaging modality that can differentiate between oxygenated and deoxygenated hemoglobin and oxygen saturation of total hemoglobin, while also evaluating vascular perfusion.
We established a wound model in ten 4-8 week-old nude mice using a 5 mm biopsy punch (3). Our OS MSOT method measured deoxyhemoglobin (Hb), oxyhemoglobin (HbO2), total hemoglobin (HbT = Hb + HbO2), and oxygen saturation (%sO2 = HbO2 / HbT). We performed these measurements with 21% O2 breathing gas, then switched the breathing gas to 100% O2 (4). Our DCE MSOT method dynamically measured the MSOT signal in the wound bed for 60 seconds at 4.8 second temporal resolution, then we intravenously injected indocyanine green (ICG), and continued to monitor the MSOT signal in the wound bed for an additional 9.6 minutes (5). The results were analyzed for wash-out of ICG in the wound bed. OS-DCE MSOT was performed in a single scan session at Day 0 (when the wound was made), and on Day 3, 6, and 9 to monitor the longitudinal wound healing process. We also photographed the wounds to validate wound healing.
The wound bed exhibited a significantly higher Hb, HbO2, and HbT immediately after creating the wound, relative to subsequent days. We interpret this result as showing blood pooling in the wound bed immediately after creating the wound. The Hb, HbO2 and HbT decreased as early as Day 3. %sO2 was significantly higher on Day 0 than subsequent days when using 21% O2 breathing gas. This result indicated that OS MSOT can detect wound healing as early as Day 3.
Changing the breathing gas from 21% O2 to 100% O2 caused a decrease in Hb and an increase in HbO2 in the wound bed, as expected. ΔsO2 showed a low value on Day 0, indicating that the wound bed did not have additional unused capacity to accumulate O2. ΔsO2 significantly increased on Day 3, showing a large increase in the additional capacity to accumulate O2, indicating that the tissue was healing. This result reinforced that OS MSOT can detect wound healing as early as Day 3.
The DCE MSOT signal was significantly different at Day 0 vs. Day 3, 6, or 9 at 2 minutes after injection (p < 0.001). This result indicated that DCE MSOT can qualitatively detect wound healing as early as Day 3. The standard deviation of the MSOT signal of 10 mice was larger at Day 0 than at Day 3, 6, and 9, and the standard deviation was smallest at Day 9. This result indicated that heterogeneity of DCE MSOT may also be a good biomarker of wound healing. B) The wash-out rate was determined from the MSOT signal at 180-642 seconds. This rate was significantly different at Day 0 vs. Day 3 and 9 (p < 0.025). This result indicated that DCE MSOT can quantitatively detect wound healing as early as Day 3.
Photographs were obtained each day to monitor wound healing. The distances across the wound were not significantly different or only weakly significantly different at Day 3 vs. Day 0; significantly different at Day 6 vs. Day 0; and highly significantly different at Day 9 vs. Day 0. These results showed that wound size indicates wound healing by Day 6.
Overall, our results showed that OS-DCE PAI can detect wound healing sooner that measurements of wound size.
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Figure 1. A) We established a standard wound model in ten 4-8 week-old nude mice using a 5 mm biopsy punch, ensuring uniformity in wound size and depth across subjects. Figure 3. B-D) The wound bed exhibited a significantly higher Hb, HbO2, and HbT immediately after creating the wound, relative to subsequent days. We interpret this result as showing blood pooling in the wound bed immediately after creating the wound. Importantly, the Hb, HbO2 and HbT decreased as early as Day 3. E) %sO2 was significantly higher on Day 0 than subsequent days when using 21% O2 breathing gas, while Days 3, 6 and 9 were not significantly different. Similar results were obtained with 100% O2 breathing gas. Unpaired two-tailed T-test, ** p < 0.01, *** p < 0.001. This result indicated that OS MSOT detects wound healing as early as Day 3. F) The MSOT signal of ICG was monitored for 60 seconds in 4.8 second intervals, followed by i.v. injection of ICG, and then monitored for another 582 seconds. Error bars represent the standard deviation of 10 mice. The MSOT signal was significantly different at Day 0 vs. Day 3, 6, or 9 at 2 minutes after injection (p < 0.001). This result indicated that DCE MSOT can qualitatively detect wound healing as early as Day 3. The standard deviation of the MSOT signal of 10 mice was larger at Day 0 than at Day 3, 6, and 9, and the standard deviation was smallest at Day 9. This result indicated that heterogeneity of DCE MSOT may also be a good biomarker of wound healing. G) The wash-out rate was determined from the MSOT signal at 180-642 seconds. This rate was significantly different at Day 0 vs. Day 3 and 9 (p < 0.025). This result indicated that DCE MSOT can quantitatively detect wound healing as early as Day 3.
Author
UT MD Anderson Cancer Center