Intramuscular Injection of Combined Calf Blood Compound (CFC) and Homeopathic Drug Tr14 Accelerates Muscle Regeneration In Vivo

Figure 1. Gene expression analysis of myosin heavy chain 1 (Myh1) (A), neuronal cell adhesion molecule (NCAM) (B), and Pax7 (C) by qPCR at days 1, 3, and 7 following injury. The mRNA values of the treated groups were normalized to the housekeeping gene, 18S, and calculated relative to the untreated control group (marked as the interrupted line). Representative data of four different individuals in triplicate (total number of measurements, n = 12) are shown with SEM. P values < 0.05 indicated statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2. Histological sections at day 1 after surgery. By HE-staining, an increased number of mononucleated cells and more edema (black arrows) were observed in the treated groups compared to the control group. The boxes in the first columns show the area magnified in column 2 and the boxes in column 2 show the area magnified in column 3.

Figure 3. Histological sections at day 3 after surgery. By HE-staining, inflammatory infiltrates were observed in all groups. Isolated regenerated myofibers (black triangles) and newly formed arteriole (black arrow) were observed in muscles treated with the combined therapy. The boxes in the first columns show the area magnified in column 2 and the boxes in column 2 show the area magnified in column 3.

Figure 4. Quantitative histological analyses for the site of laceration. (A) Necrotic area of the injured muscle at days 1, 3, and 7 following surgery in the experimental groups. (B) Numbers of centrally nucleated, regenerative cells per sample at day 7 following muscle injury. (C) Diameter of regenerative cells in the different groups at day 7. (D) Comparison of the diameter of centrally nucleated cells on day 3 and day 7 in rats treated with CFC + Tr14. Data are presented as mean + SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5. Histological sections at day 7 following surgery. By HE-staining, the accelerated regeneration in the group treated with calf blood compound (CFC) only or CFC combined with Tr14 was revealed relative to the control group, sodium chloride group, and the Tr14 group. Regenerated myofibers with centralized nuclei are highlighted (black triangles). The boxes in the first columns show the area magnified in column 2 and the boxes in column 2 show the area magnified in column 3.

Figure 6. Immunohistological sections stained for myogenin positive cells at day 3 and 7 following surgery. Regenerated myofibers with high myogenin expression are highlighted (black triangles). The boxes in the first columns show the area magnified in column 2.

Figure 7. Surgical procedure. (A) Situs after skin incision. (B) Exposed rectus femoris muscle (star) and patella (arrow) after skin incision. (C) Filament-marking of the muscle lesion. (D) Lesion induction in the rectus femoris muscle. (E) Intraoperative intramuscular injection treatment. (F) Situs after cutaneous suture.

Figure 8. Intramuscular injections were performed according to the groups, with intramuscular injections of NaCl, CFC, Tr14, CFC + Tr14, or no injection in the control group (n = 10 for each group and time point). Injection volume was 100 µl. Injection concentrations were: 1) CFC (Actovegin®): 0.965 µg/g bodyweight (BW), Tr14; 2) and Tr14 (Traumeel®): 7.64 pl/g BW. Rats receiving both agents were treated in a 2:1 ratio (CFC:Tr14).