Effects of Topical or Intravitreal Application of Anti-Vascular Endothelial Growth Factor on Density of Intestinal Blood Vessels of Mice

Abstract

Background and Objectives: Anti-vascular endothelial growth factor (anti-VEGF) therapy has become the first-line treatment for diabetic macular edema. However, it is still not clear whether anti-VEGF agents act on systemic blood vessels. The aim of this study is to determine whether a direct topical application or intravitreal injection of anti-VEGF will change the intestinal blood vessels of mice. Materials and Methods: C57BL/6 mice were laparotomied under deep anesthesia, and the blood vessels on the surface of the intestines were exposed, examined, and photographed through a dissecting microscope. Vascular changes were evaluated before and at 1, 5, and 15 min after the topical application of 50 µL of the different anti-VEGF agents onto the surface of the intestine (group S) or after the intravitreal injection (group V). The vascular density (VD) was determined for five mice in each group before and after 40 μg/μL of aflibercept (Af), or 25 μg/μL of bevacizumab (Be), or 10 μg/μL of ranibizumab (Ra) were applied. Endothelin-1 (ET1), a potent vasoconstrictor, was used as a positive control, and phosphate-buffered saline (PBS) was used as a control. Results: For group S, no significant changes were observed after PBS (baseline, 1, 5, and 15 min: 46.3, 44.5, 44.8, and 43.2%), Be (46.1, 46.7, 46.7, and 46.3%), Ra (44.7, 45.0, 44.7, and 45.6%), and Af (46.5, 46.2, 45.9, and 46.1%, repeated ANOVA) were applied topically. Significant decreases in the VD were observed after ET1 (46.7, 28.1, 32.1, and 34.0%, p < 0.05) was topically applied. For group V, no significant differences were observed for all anti-VEGF agents. Conclusions: The topical application or intravitreal injections of anti-VEGF agents do not cause a change in the VD of the intestinal vessels, which may be related to its safety.

Keywords: anti-vascular endothelial growth factor agents; direct titration; intravitreal injection; vasoconstriction.

Scheme A1

Vascular density evaluations using two-photon laser-scanning microscopy. Intestine of transgenic mouse expressing green protein was evaluated. Before treatment, normal vascular was seen (a). Fifty-μL of endothelin-1 titration was applied into the space between the surface of intestine and the coverslip. Vascular change was observed at 3.5 min (b) and at 5 min after application (c). Due to the gastrointestinal peristalsis, the same vessels could not be observed. White arrow: vascular wall.

Figure 1

Preparation of mouse and microscope set up. A vertical skin incision is made on a deeply anesthetized C57BL/6 mouse. The intestines are exposed, and the mouse was placed on the stage of a dissecting microscope. A microscope cover glass was attached to the end of an arm of the mouse-holding device and adjusted to avoid excess pressure on the intestines (a,b). Fifty microliters of each agent were applied into the space between the surface of the intestine and the coverslip for the S group (c).

Figure 2

Evaluations of vascular density. The recorded images (a) were converted to black–white images using ImageJ (b). The blood vessels were displayed in black, and the surrounding tissue in white. Each black–white image was analyzed using AngioTool and the vascular density was calculated automatically (c).

Figure 3

Vascular density evaluations for control agents. No obvious vascular changes were observed for PBS-applied blood vessels of the S and V groups. For the ET1-treated group, obvious vasoconstrictions were observed for the S group (second panel from the bottom) from 1 to 15 min. No obvious changes were observed for the V group (bottom panel). ET-1, endothelin-1; PBS, phosphate-buffered saline.

Figure 4

Vascular density evaluations for different anti-VEGF agents. No obvious vascular change was observed for each anti-VEGF agent in the treated group (both S and V groups). VEGF: vascular endothelial growth factor.