**2. Results**

#### *2.1. Modulation of Blood Concentration Levels of IL10 after Pharmacological Stimulation with IL10 and Anti-IL10*

To investigate whether blood concentration levels of endogenous IL10 were a ffected by an intravenous administration of IL10 or anti-IL10 via tail vein injection, a Mouse Magnetic Luminex Assay was used to determine the blood concentration levels of IL10 prior to (baseline, BL) and 24 h (24 h) after the respective treatments. A control group received NaCl 0.9%. At BL, blood concentration levels of IL10 were below the detection limit of 1.59 pg/mL in all groups. At 24 h, elevated blood concentration levels of IL10 were found only in the IL10 treatment group with 6.35 ± 2.20 pg/mL (*p* < 0.05), indicating that an external administration of IL10 leads to a sustainable change in endogenous blood concentration levels of IL10 for at least 24 h when applied via tail vein injection. Blood concentration levels of IL10 in mice treated with anti-IL10 or NaCl 0.9% remained below the detection limit (Figure 1).

**Figure 1.** Modulation of blood concentration levels of IL10 after pharmacological stimulation with IL10 and anti-IL10. IL10, anti-IL10, and NaCl were administered via tail vein injection. Blood concentration levels of IL10 were measured before (baseline, BL) and 24 h after pharmacological stimulation. At BL endogenous IL10 levels were below the detection limit of 1.59 pg/mL in all subjects. After an external administration of IL10, blood concentration levels remained significantly increased after 24 h. An effect of anti-IL10 could not be detected, as baseline levels of IL10 remained below the detection limit. \* indicates *p* < 0.05; *n* = 3 in each group.

### *2.2. Alteration of Macrophage Polarization in the Perivascular Bed of Growing Collateral Vessels after Pharmacological Stimulation with IL10 and Anti-IL10*

Adductor muscle samples were harvested 3 days (3 d) and 7 d after FAL to analyze the effect of a treatment with IL10 and anti-IL10 on the polarization of macrophages in the perivascular bed of growing collateral vessels. The samples were sectioned and stained using antibodies targeting known macrophage markers CD68 and CD163 [11,13] (Figure 2a). The two largest collateral vessels of each section were selected and the ratio of macrophages of the alternatively activated phenotype CD163+/CD68+ to the classically activated phenotype CD163−/CD68<sup>+</sup> per visual field was calculated. Mice treated with NaCl had a median ratio of CD163+/CD68+ to CD163−/CD68<sup>+</sup> macrophages of 0.46 (IQR: 0.37–1.20) on day 3 (3 d) and 0.40 (IQR: 0.37–0.55) 7 d after FAL. When treated with IL10 the ratio is skewed towards the alternatively activated phenotype on both 3 d and 7 d after FAL with a ratio of 1.00 (IQR: 0.45–1.44) and 1.19 (IQR: 0.52–1.69). Contrariwise, the ratio is skewed towards the classically activated phenotype after application of anti-IL10 on both 3 d and 7 d after FAL with a ratio of 0.25 (IQR: 0.18–0.35) and 0.27 (IQR: 0.00–0.53), differing significantly from that of the IL10 treatment group (*p* < 0.05) (Figure 2b).

**Figure 2.** Alteration of macrophage polarization in the perivascular bed of growing collateral vessels after pharmacological stimulation with IL10 and anti-IL10. (**a**) Confocal micrographs of macrophage differentiation subtypes 3 d and 7 d after FAL. Sections of adductor muscles segments containing growing collateral vessels (V) were stained using DAPI and macrophage differentiation markers CD68 and CD163. The ratio of the alternatively (CD163+/CD68+) activated phenotype, indicated by white arrows, and classically (CD163−/CD68<sup>+</sup>) activated phenotype varies with indicated application. Scale bar: 25μm. (**b**) Quantification of macrophage polarization in the perivascular bed of growing collateral vessels after pharmacological stimulation with IL10 and anti-IL10 3 d and 7 d after FAL. The distribution of macrophage subtypes was skewed towards the alternatively activated phenotype after IL10 application. When anti-IL10 was injected, the opposite effect was observed, and the distribution was skewed towards the classically activated phenotype. \* indicates *p* < 0.05; 3 d: *n* = 6 in each group; d7: NaCl and IL10 *n* = 4, anti-IL10: *n* = 6.

### *2.3. Evaluation of Hind-Limb Perfusion Recovery after FAL and Pharmacological Stimulation with IL10 and Anti-IL10*

To assess the effect of varying blood concentration levels of IL10 on growing collateral vessels IL10 and anti-IL10 were externally applied in mice after FAL. Hind-limb perfusion was assessed using Laser-Doppler-Imaging before and shortly after FAL, on 3 d, 7 d, and 14 d and compared to a control group receiving NaCl. Immediately after FAL an acute reduction of hind-limb perfusion was observed in all groups (NaCl: 0.13 ± 0.01, IL10: 0.16 ± 0.03, anti-IL10: 0.13 ± 0.01). Hind-limb reperfusion in the NaCl (*n* = 6) group showed an adequate increase to 0.34 ± 0.04, 0.62 ± 0.07, and 0.62 ± 0.07 on 3 d, 7 d, and 14 d respectively. Elevated blood concentration levels of IL10 led to a significantly higher hind-limb perfusion on 3 d and 14 d (*n* = 5, 3 d: 0.54 ± 0.09, *p* < 0.05, 14 d: 0.83 ± 0.7, *p* < 0.05). Although hind-limb perfusion on 7 d was elevated compared to the control group, the difference was not significant (0.77 ± 0.09, *p* = 0.07) (Figure 3a). Contrariwise, application of anti-IL10 showed a significant impairment of hind-limb perfusion on7d(*n* = 5, 0.42 ± 0.4, *p* < 0.05). On 3 d and 14 d, however, no significant difference was observed (3 d: 0.35 ± 0.05, *p* = 0.86, 14d: 0.61 ± 0.07, *p* = 0.89) (Figure 3b).

**Figure 3.** Evaluation of hind-limb perfusion recovery after femoral artery ligation (FAL) and application of IL10 and anti-IL10 (L/R ratio). Hind-limb perfusion was measured prior to and immediately after FAL, on 3 d, 7 d and 14 d. (**a**) IL10 led to a significant acceleration of hind-limb perfusion recovery on 3 d (IL10: 0.54 ± 0.09 vs. NaCl: 0.34 ± 0.04, *p* < 0.05) and 14 d (IL10: 0.83 ± 0.07 vs. NaCl: 0.62 ± 0.07, *p* < 0.05) while (**b**) anti-IL10 led to a brief but significant impairment of hind-limb perfusion recovery on 7 d (anti-IL10: 0.42 ± 0.04 vs. NaCl: 0.62 ± 0.07, *p* < 0.05). (**c**) Representative Laser-Doppler-Images of ligated and non-ligated hind-limbs. \*indicates *p* < 0.05; NaCl: *n* = 6, IL10 and anti-IL10: *n* = 5.

#### *2.4. Macroscopic Observations on Ligated Hind-Limbs after FAL and Application of IL10 and Anti-IL10*

Ligated hind-limbs were inspected prior to FAL, 3 d and 7 d after FAL. Macroscopic observations associated with an acute ischemia of the affected hind-limb, termed critical ischemic events (CIE), were recorded after FAL and treatment with IL10 and anti-IL10. CIE were categorized as follows: inflamed hind-limb, necrotic digits, necrotic hind-limb and amputation. In total (*n* = 115) CIE occurred in 20%. Necrotic digits were observed most frequently in 65%, followed by a necrotic hind-limb in 17%, an amputation in 13% and an inflamed hind-limb in 4% of CIE. The highest incidence of CIE was observed after application of anti-IL10 (anti-IL10: 37.1% vs. NaCl: 11.1% *p* < 0.01). The incidence of CIE when treated with IL10 was similar to the control group (IL10: 13.6% vs. NaCl: 11.1% *p* = 0.73) (Figure 4).

**Figure 4.** After FAL and pharmacological stimulation with IL10 or anti-IL10 the onset of critical ischemic events (CIE) were recorded. The highest incidence of CIE was observed after application of anti-IL10 (anti-IL10: 37.1% vs. NaCl: 11.1% *p* < 0.01, IL10: 13.6%). \*\*indicates *p* < 0.01; NaCl: *n* = 36, IL10: *n* = 44, anti-IL10: *n* = 35
