Treatment with Sildenafil and Donepezil Improves Angiogenesis in Experimentally Induced Critical Limb Ischemia

Abstract

Objectives. In this study, we aimed to demonstrate the role of sildenafil (an antagonist of phosphodiesterase type 5 (PDE-5)) and donepezil (a specific and reversible inhibitor of acetylcholinesterase (Ach)) in increasing ischemia-induced angiogenesis. Method. Critical limb ischemia was induced by ligation of the common femoral artery followed by ligation of the common iliac artery. The operated animals were divided into 3 groups: receiving sildenafil, receiving donepezil, and surgery alone; the contralateral lower limb was used as a negative control. The results were controlled based on clinical score and Doppler ultrasound. Gastrocnemius muscle samples were taken from all animals, both from the ischemic and nonischemic limb and were used for histopathological and immunohistochemical examination for the evaluation of the number of nuclei/field, endothelial cells (CD31), dividing cells (Ki-67), and vascular endothelial growth factor (VEGFR-3). Results. An increasing tendency of the number of nuclei/field with time was observed both in the case of sildenafil and donepezil treatment. The formation of new capillaries (the angiogenesis process) was more strongly influenced by donepezil treatment compared to sildenafil or no treatment. This treatment significantly influenced the capillary/fiber ratio, which was increased compared to untreated ligated animals. Sildenafil treatment led to a gradual increase in the number of dividing cells, which was significantly compared to the negative control group and compared to the ligation control group. The same effect (increase in the number of Ki-67 positive cells) was more obvious in the case of donepezil treatment. Conclusion. Donepezil treatment has a better effect in ligation-induced ischemia compared to sildenafil, promoting angiogenesis in the first place, and also arteriogenesis.

Figure 1

Techniques used for the histopathological study. Gastrocnemius muscle cross-sections: H&E staining for the evaluation of the number of nuclei/field; immunohistochemical staining for endothelial cells (CD31), dividing cells (Ki-67) and vascular endothelial growth factor (VEGFR-3).

Figure 2

Evaluation of the number of nuclei/field in gastrocnemius muscle sections from Wistar rats, at the 4 experimental times (H&E staining, scale bar is 50 μm). Quantification of the number of nuclei/field: the graph shows the mean and standard deviation for each experimental variant. Statistical differences are marked: ^∗P < 0.0125 compared to the negative control group, ^#P < 0.0125 compared to the ligation control group.

Figure 3

Histochemical evaluation of capillary density in gastrocnemius muscle sections from Wistar rats, at the 4 experimental times (scale bar is 50 μm). Quantification of the number of CD31 positive cells/mm²: the graph illustrates the mean and standard deviation for each experimental variant. Statistical differences are marked: ^∗P < 0.0125 compared to the negative control group, ^#P < 0.0125 compared to the ligation control group, and ^@P < 0.0125 compared to the sildenafil treated group.

Figure 4

Evaluation of the capillary/muscle fiber ratio: the graph illustrates the mean and standard deviation for each experimental variant. Statistical differences are marked: ^∗P < 0.0125 compared to the negative control group, ^#P < 0.0125 compared to the ligation control group.

Figure 5

Histochemical evaluation of dividing cells in gastrocnemius muscle sections from Wistar rats, at the 4 experimental times (scale bar is 50 μm). Quantification of the number of Ki-67 positive cells/field: the graph illustrates the mean and standard deviation for each experimental variant. Statistical differences are marked: ^∗P < 0.0125 compared to the negative control group, ^#P < 0.0125 compared to the ligation control group, and ^@P < 0.0125 compared to the sildenafil treated group.

Figure 6

Histochemical evaluation of vascular endothelial growth factor in gastrocnemius muscle sections from Wistar rats, at the 4 experimental times (scale bar is 50 μm). Quantification of the number of CD31 positive cells/field: the graph shows the mean and standard deviation for each experimental variant. Statistical differences are marked: ^∗P < 0.0125 compared to the negative control group and ^#P < 0.0125 compared to the ligation control group.