*2.2. Irradiation Procedure*

An orthovoltage X-ray device performed the irradiation with a current of 20 mA and a voltage of 150 kV. For the irradiation, the rats received intramuscular anesthesia using ketamine (100 mg per kg) and medetomidine (0.2 mg per kg). Rats were positioned on the belly and transferred to the irradiation unit in a closed isolation cage to protect the rats from pathogens. An area of 7 <sup>×</sup> 2 cm<sup>2</sup> including the right flap or the area of the prospective right flap in case of preoperative irradiation was irradiated (Figure 2). The rest of the body of the rat was covered with lead shields as protection from irradiation. The left flap served as nonirradiated internal control. Postoperative irradiation was performed on the first day after the operation. Postoperative fractional irradiation was performed on the first, second and third day after the operation. Single stage preoperative irradiation was performed

*J. Pers. Med.* **2022**, *12*, x FOR PEER REVIEW 3 of 14

4 weeks before the operation and fractional preoperative irradiation on the three following days starting 4 weeks preoperatively (Figure 3). *J. Pers. Med.* **2022**, *12*, x FOR PEER REVIEW 3 of 14

**Figure 1.** Harvest of two modified McFarlane flaps: (**A**) preoperative markings parallel and 1 cm lateral to the spine; (**B**) two flaps harvested; (**C**) reinsertion of the flaps. **Figure 1.** Harvest of two modified McFarlane flaps: (**A**) preoperative markings parallel and 1 cm lateral to the spine; (**B**) two flaps harvested; (**C**) reinsertion of the flaps. and third day after the operation. Single stage preoperative irradiation was performed 4 weeks before the operation and fractional preoperative irradiation on the three following days starting 4 weeks preoperatively (Figure 3).

days starting 4 weeks preoperatively (Figure 3). **Figure 2.** Irradiation procedure: (**A**) rat positioned for irradiation; (**B**) irradiated area. **Figure 2.** Irradiation procedure: (**A**) rat positioned for irradiation; (**B**) irradiated area.

**Figure 2.** Irradiation procedure: (**A**) rat positioned for irradiation; (**B**) irradiated area.

Animals were divided in 5 groups (*n* = 5). Group 1 included the control group with-

ative irradiation with 20 Gy and rats of group 5 fractional preoperative irradiation with

Group 2 Single stage postoperative irradiation 20 Gy 1 day after flap harvest

Group 3 Fractional postoperative irradiation 12 Gy each day 1, 2 and 3 after flap

Group 4 Single stage preoperative irradiation 20 Gy 28 days before flap harvest

Group 5 Fractional preoperative irradiation 12 Gy each day 28, 27 and 26 before

Indocyanine green angiography using an IC-Flow™ Imaging System (Diagnostic Green, Farmington Hills, MI, USA) was performed directly after flap harvest and on day 1, 7 and 14 after the operation in groups 1, 2 and 3 (Figure 4). Therefore, indocyanine green was injected via the tail vein (1 mg per kg). An intensity of less than 20% of the maximum

**Figure 3.** Study protocol. **Figure 3.** Study protocol.

*2.3. Groups*

3x12 Gy (Table 1).

**Table 1.** Groups.

*2.4. Imaging*

**Group.** Irradiation regimen

harvest

flap harvest

intensity was defined as malperfused area.

Group 1 None=control

## *2.3. Groups*

Animals were divided in 5 groups (*n* = 5). Group 1 included the control group without irradiation. The rats of group 2 received postoperative irradiation with 20 Gy. The rats of group 3 had postoperative irradiation with 3 × 12 Gy. Rats of group 4 received preoperative irradiation with 20 Gy and rats of group 5 fractional preoperative irradiation with 3 × 12 Gy (Table 1).

#### **Table 1.** Groups.


#### *2.4. Imaging*

perature [16].

*2.5. Blood Samples*

*2.6. Statistical Analysis*

icance was set at *p* < 0.05.

**3. Results**

Indocyanine green angiography using an IC-Flow™ Imaging System (Diagnostic Green, Farmington Hills, MI, USA) was performed directly after flap harvest and on day 1, 7 and 14 after the operation in groups 1, 2 and 3 (Figure 4). Therefore, indocyanine green was injected via the tail vein (1 mg per kg). An intensity of less than 20% of the maximum intensity was defined as malperfused area. *J. Pers. Med.* **2022**, *12*, x FOR PEER REVIEW 5 of 14

**Figure 4.** Indocyanine green angiography after flap harvest in group 2**. Figure 4.** Indocyanine green angiography after flap harvest in group 2.

Standard clinical imaging was performed directly after the operation and on day 1, 3, 7, 10 and 14 after the operation. The animals were sacrificed 14 days after the operation. As previously described [16], near-infrared reflectance-based imaging was performed on the same days using Snapshot NIR® (Kent Imaging Inc.; Calgary, AB, Canada) to measure tissue oxygenation in superficial tissue. Near-infrared light is transmitted onto the skin surface and reflected off the blood within the tissue [16]. There is a difference of oxygenated and deoxygenated light absorption of hemoglobin that depends on the wavelength. In conclusion, the ratio from oxygenated to deoxygenated blood and therefore the viability can be determined by this method. Poorly perfused skin has a lower percentage of oxygenated hemoglobin than well-perfused skin [16,18]. Infrared thermography images were obtained on the same days by a smartphone-compatible thermographic camera (FLIR ONE Pro, FLIR Systems, Inc.; Wilsonville, OR, USA). The camera uses a long-wave infrared sensor. It has an effective temperature range from −20 to 400 °C with a resolution of 0.1 °C with a sensitivity that detects temperature differences as low as 70 mK. Image processing is used to merge the photo with a thermal image and thus measure the tem-Standard clinical imaging was performed directly after the operation and on day 1, 3, 7, 10 and 14 after the operation. The animals were sacrificed 14 days after the operation. As previously described [16], near-infrared reflectance-based imaging was performed on the same days using Snapshot NIR® (Kent Imaging Inc.; Calgary, AB, Canada) to measure tissue oxygenation in superficial tissue. Near-infrared light is transmitted onto the skin surface and reflected off the blood within the tissue [16]. There is a difference of oxygenated and deoxygenated light absorption of hemoglobin that depends on the wavelength. In conclusion, the ratio from oxygenated to deoxygenated blood and therefore the viability can be determined by this method. Poorly perfused skin has a lower percentage of oxygenated hemoglobin than well-perfused skin [16,18]. Infrared thermography images were obtained on the same days by a smartphone-compatible thermographic camera (FLIR ONE Pro, FLIR Systems, Inc.; Wilsonville, OR, USA). The camera uses a long-wave infrared sensor. It has an effective temperature range from −20 to 400 ◦C with a resolution of 0.1 ◦C with a sensitivity that detects temperature differences as low as 70 mK. Image processing is used to merge the photo with a thermal image and thus measure the temperature [16].

groups for staining of DNA double-strand breaks in lymphocytes as previously described [19]. Briefly, lymphocytes were isolated using Pancoll Separating Solution (Pan Biotech, Aidenbach, Germany). After separation, the lymphocytes were incubated overnight with the antibody detecting the phosphorylated variant of the histone H2AX (γ-H2AX) (Puri-

Flap size and malperfused/necrotic areas were calculated using Image J (U.S. National Institutes of Health, Bethesda, MD, USA). The statistical analysis was performed using Microsoft Excel (Microsoft, Redmond, WA, USA) and Prism 8 (GraphPad Software, San Diego, CA, USA). The normal distribution was identified graphically using QQ plots. For the comparison of the medians of the same group at different timepoints and the comparison of different groups at the same timepoint, mixed effect models with the Geisser– Greenhouse correction following the Tukey test were used. The level for statistical signif-

All animals tolerated the operative procedure without complications. Postoperative irradiation resulted in weight loss of approximately 10% (group 2, weight day at 0: 322 ±

fied anti-H2A.X Phospho (Ser139); BioLegend, San Diego, CA, USA).

#### *2.5. Blood Samples*

Blood samples were taken 30 min after irradiation in the postoperative irradiation groups for staining of DNA double-strand breaks in lymphocytes as previously described [19]. Briefly, lymphocytes were isolated using Pancoll Separating Solution (Pan Biotech, Aidenbach, Germany). After separation, the lymphocytes were incubated overnight with the antibody detecting the phosphorylated variant of the histone H2AX (γ-H2AX) (Purified anti-H2A.X Phospho (Ser139); BioLegend, San Diego, CA, USA).

#### *2.6. Statistical Analysis*

Flap size and malperfused/necrotic areas were calculated using Image J (U.S. National Institutes of Health, Bethesda, MD, USA). The statistical analysis was performed using Microsoft Excel (Microsoft, Redmond, WA, USA) and Prism 8 (GraphPad Software, San Diego, CA, USA). The normal distribution was identified graphically using QQ plots. For the comparison of the medians of the same group at different timepoints and the comparison of different groups at the same timepoint, mixed effect models with the Geisser–Greenhouse correction following the Tukey test were used. The level for statistical significance was set at *p* < 0.05.
