Biological Cardiac Tissue Effects of High-Energy Heavy Ions – Investigation for Myocardial Ablation

Figure 1. Analysis of bleeding and siderophages in samples of pig hearts six months after targeted C-ion irradiation of the atrioventricular junction (AVJ). Samples from target region (T), entrance channel (EC) and outfield (OUT) were analyzed. Bleeding was quantified in HE stained sections by detection of erythrocytes (e). In irradiated AVJ regions, bleeding is persistent even after 6 months (a–d: 0–55 Gy; black arrows), whereas in samples from EC and OUT regions, only background levels of erythrocytes were detected (white arrows). ***p = 0.0005; *p = 0.0305. Hemosiderin deposits in siderophages were visualized with Prussian Blue staining (f–i; 0–55 Gy; brown arrows). Siderophages were counted per fields of view. Only after the highest dose of 55 Gy, a significant increase was measured (j). In samples from EC and OUT regions, siderophage levels did not differ from sham irradiated control tissues. ****p-value < 0.0001; ***p-value = 0,0001. Tukey Box plot and median (+/−SD). Significance was tested with one-way ANOVA. 0 (N = 1), 25 (N = 2), 40 (N = 1) and 55 Gy (N = 2); n = 3 (0 Gy); n = 4 (t); n = 2 (out); n = 2 (ec); k = 310.

Figure 2. Analysis of microvessels in samples of pig hearts six months after targeted C-ion irradiation of the atrioventricular junction (AVJ). Vessels numbers (capillaries) and diameters per fields of view were measured in cross sections from target region (T) as well as entrance channel (EC) and outfield (OUT) regions. Representative cross sections are shown in (a) (0 Gy) and (b) (55 Gy); capillaries are indicated by arrows. The total number of microvessels is significantly reduced six months after irradiation in target regions (c), but not in ec or out regions. The diameter of microvessels is increased in target regions (d), whereas ec and out regions are not changed compared to sham irradiated control levels. Tukey Box plot and median (+/−SD; − = median, + = mean); One-way ANOVA. ****p < 0.0001.

Figure 3. Inflammation in samples of pig hearts six months after targeted C-ion irradiation of the atrioventricular junction (AVJ). Samples from target region (T) as well as entrance channel (EC) and outfield (OUT) regions were HE-stained and examples are shown in (a–d) (0–55 Gy). Arrows indicate immune cell infiltrations. For a more specific identification, sections were stained with an CD45-antibody to visualize T-cells ((e) 40 Gy and (f) 55 Gy; pink: CD45 positive cells; examples shown by arrows), which matches well with the HE staining.

Figure 4. Fibrosis induction in samples of pig hearts six months after targeted C-ion irradiation of the atrioventricular junction (AVJ). The AVJ was irradiated with 25, 40 or 55 Gy. Examples of Masson- Goldner Trichrome stained sections are shown in (a–h) (0–55 Gy); left panel shows cross sections, right panel shows longitudinal sections. Blue/black- nuclei; red/brown- cytoplasm; red/pink- erythrocytes; green- collagen. In samples from sham irradiated animals, myocardium is intact as indicated by wide areas covered with brown cardiomyocytes and collagen found only in connective tissue in the septa (a,b). In irradiated AVJ samples, interstitial collagen deposition is aggravated (c–h), indicated by an increased area stained in green and less intact cardiomyocytes. To distinguish between healthy/normal connective tissue and pathological collagen deposited by myofibroblasts, sections were immunostained for α-smooth muscle actin (α-SMA). In sham-irradiated tissue, only the perivascular myofibroblasts are positive (i), whereas in irradiated tissue, myofibroblasts between cardiomyocytes are visible ((j), 40 Gy and (k), 55 Gy; α-SMA: pink; examples are marked by arrows).

Figure 5. Structural changes in cardiomyocytes from samples of pig hearts six months after targeted C-ion irradiation of the atrioventricular junction (AVJ). Analysis was performed in HE-stained longitudinal sections of myocardium. Examples are shown in (a–f). In A, sham irradiated myocardium with intact cardiomyocytes is shown. Regular striation is indicated by blue arrows. With increasing dose cardiomyocyte damage is more pronounced ((b) 25 Gy, (c and d) 40 Gy, (e and f) 55 Gy). After 25 Gy, regions with normal cardiomyocytes are present (blue arrows), but in parallel, changes like wavy fibers (white arrow), vacuolization (black arrows) or collagen deposition (green arrowheads) occur. After 40 and 55 Gy, damage to cardiomyocytes is more severe, as shown by larger amounts of vacuolization, loss of striation (*), loss of polarity, bleeding and more collagen deposition. (c,d) as well as (e,f) are from different regions of one animal, respectively.

Figure 6. Cardiac Troponin T distribution in myocardium of pigs six months after targeted C-ion irradiation of the atrioventricular junction (AVJ). Analysis was performed in immunohistochemical stainings (cardiac Troponin T (cTropT, pink/ violet). (a and b) sham irradiated tissue (0 Gy, (a) longitudinal section, (b) cross section). In the longitudinal section, the typical striation of cardiomyocytes is visible (black arrows; insert). In irradiated tissue (55 Gy, (c) longitudinal, (d) cross section), localization of cTropT is partly lost in myofilaments or occurs in the cytoplasm (blue arrows).

Figure 7. Analysis of skin in the entrance channel during C- ion irradiation of the atrioventricular junction (AVJ) in the heart. Sections were stained with HE (a,b), or immunostained with an antibody against Cytokeratin 10 (keratinocytes, (c,d) pink, thick arrows), or Ki67 (proliferating cells, (e,f) thin arrows). No differences between irradiated skin in EC (calculated dose ~17 Gy) and in OUT samples (0 Gy) were detectable, indicating the absence of side effects.