Botulinum toxin improved intestinal adaptation to short gut in a twenty-one-day-old weanling rat

Abstract

Necrotizing enterocolitis (NEC) is a severe intestinal disease of multifactorial origin that primarily affects premature infants. Approximately 27% of NEC babies develop short gut (SG) secondary to extensive intestinal resection, and 10% will have chronic dependence on total parenteral nutrition. We evaluated the Botox treatment in SG model rats. Twenty-day-old weanling male rats (weight range 38-70 g, n=72) were divided into four groups (n=18 each): 1) Control (fed a regular liquid diet); 2) Botox (Control submitted to laparotomy and intestinal injection of Botox®); 3) SG (short gut); and 4) SG and Botox (SG+Botox®). After seven post-operative days, samples were collected for biometrics [body weight (BW), intestine weight (IW) and IW/BW ratio (IBR), and intestine length (IL) and height (IH)], histometric analysis [villous height (VH), crypt depth (CD), muscular thickness (MT), and PCNA index)], and intestinal transit time (ITT). BW, IW, and IL decreased in SG (P<0.05). IH, VH, and PCNA index increased in Botox groups [Control = SG < Botox and SG+Botox (P<0.05)], CD increased in Botox, SG, and SG+Botox (P<0.005), and MT was higher in SG and SG+Botox. Botox groups had lower ITT (P<0.05). Botox provided dilatation and histological changes in SG. These findings suggested that Botox improved adaptation and might be applied in SG with promising results.

Figure 1. Steps of the surgical procedure. A, Ileocecal valve (arrow); B, Intestinal arcades (approximately 5-6); C, Amount of intestinal resection between clamps; D, Anastomosis (arrow); E and F, Botox application in the intestinal borders.

Figure 2. Daily total weight from day 0 to day 7 of the procedure of all experimental groups. There was no statistical difference between Control and Botox or between SG and SG+Botox.

Figure 3. Location of injected Botox and measurement of intestinal height at day 7. A. Botox; B, SG+Botox; C, normal intestinal height; and D, intestinal height with Botox. SG: short gut.

Figure 4. Histometric results including grouped analysis amongst the four experimental groups. ***P<0.0005, ****P<0.0001 (ANOVA and Tukey's post hoc test). Photomicrographs of the small intestinal villi for the four experimental groups: A, Control; B, Botox; C, SG; and D, SG+Botox. Black bars: villous height, red bars: crypt depth. Bottom panels: black bars: length of the muscle layer (M) of the gut wall, arrows: PCNA-positively stained nuclei of epithelial cells. Scale bars: middle panels 50 μm and bottom panels 100 μm.

Figure 5. Intestinal transit time from the stomach (S) to the colon (C) amongst the four experimental groups. S: stomach; PJ: proximal jejunum; MJ: middle jejunum, DJ: distal jejunum; JIT: jejunum ileum transition; PI: proximal ileum; DI: distal ileum; PC: proximal colon; and DC: distal colon. *P<0.05 (ANOVA and Tukey's post hoc test).