1. Heparin-Binding EGF-Like Growth Factor Increases Intestinal Microvascular Blood Flow in Necrotizing Enterocolitis 
Xiaoyi Yu, Andrei Radulescu, Nicholas Zorko, Gail E. Besner 
Gastroenterology 
Volume 137, Issue 1, Pages (July 2009)
DOI: /j.gastro
Copyright © 2009 AGA Institute Terms and Conditions

2. Figure 1
(A) Illustration of intestinal submucosal and villous blood flow. The major sites of resistance to flow (flow regulation) are in the small mesenteric (piercing) arteries and in the submucosal 1A and 2A arterioles arising from the piercing arteries. The 3A arterioles arising from the 2A arterioles proceed to the villi. Modified and reprinted with permission from Nowicki.1 (B) The submucosal vessels (mainly 1A and 2A arterioles) were defined as vessels in the zone between the base of the crypt and the muscularis mucosa (enclosed by white rectangle).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
HB-EGF decreases intestinal histologic injury in rat pups subjected to experimental NEC. Shown are representative H&E-stained sections demonstrating (A) grade 0, normal intestine from pup fed with breast milk for 3 days; (B) grade 1, epithelial cell lifting or separation from pup subjected to stress but treated with HB-EGF for 3 days; (C) grade 2, sloughing of epithelial cells to midvillous level in pup subjected to stress for 3 days; (D) grade 3, necrosis of entire villus in pup subjected to stress for 3 days. Original magnification 100×.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
Incidence of experimental NEC. Shown are the grades of injury for individual pups subjected to stress with or without HB-EGF added to the feeds. In this experiment, pups were subjected to stress for up to 72 hours, at which time all surviving animals were killed. Each point represents an individual animal. The starting number of animals in the stress group was 80 and in the stress + HB-EGF group was 73. Animals were excluded from analysis if they died a significant amount of time before harvesting of intestines, because this led to tissue autolysis. Points above the dashed line indicate histologic injury consistent with NEC (grade 2 or higher injury). *P < .001 compared with stress alone.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
Effect of HB-EGF on villous microcirculatory blood flow in whole-mount specimens visualized by fluorescent microscopy. Shown are representative examples of fluorescent microscopic images of the villous microvasculature as observed in a single image of whole-mount slides obtained from the ileum (original magnification 10×). The villous microvascular blood flow is shown by FITC-labeled high-molecular-weight dextran (green) staining. Shown are images from (A) a pup fed with breast milk for 3 days, showing intact villous microvascular blood flow; (B) a pup subjected to stress for 3 days, showing decreased villous microvascular blood flow; and (C) a rat pup subjected to stress for 3 days but with HB-EGF added to the feeds, showing significant preservation of villous microvascular blood flow.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
Effect of HB-EGF on villous microcirculatory blood as shown by confocal microscopy. Shown are representative images of villous microcirculatory blood flow as observed using FITC-dextran angiography in a 3-dimensional confocal projection of whole-mount slides obtained from the ileum (original magnification 40×). The red staining represents nuclear staining with propidium iodide, and the green staining indicates the microvasculature. The representative panels show intestinal samples from (A) control pups fed with breast milk for 3 days, (B) pups subjected to stress for 3 days, and (C) pups subjected to stress for 3 days but with HB-EGF added to the feeds. D represents quantification of villous microcirculatory blood flow. The number of animals represented by each bar is 13, with the exception of bars 5 and 6, which represent 4 animals each. For each animal studied, 2 measurements were subjected to quantification, with green and red channels separated and signal intensity calculated using Zeiss LSM 5 software. In each scanned file, the ratio of green channel signal intensity (representing villous microcirculatory blood flow) to red channel signal intensity (representing nuclear staining) was obtained. *P < .001 compared with control; **P < .001 compared with stress day 3. Also, refer to Supplementary Movie 1. A corresponds to movie BMF, B corresponds to movie stress, and C corresponds to movie stress + HB-EGF.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

7. Figure 6
Time course for the development of experimental NEC. Shown are the grades of injury for individual pups subjected to stress for 1, 2, 3, or 4 days. Each point represents an individual animal. Points lying above the dashed line indicate histologic injury consistent with NEC (grade 2 or higher injury).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

8. Figure 7
Effect of HB-EGF on intestinal villous microvascular structure as visualized by scanning electron microscopy. Shown are intestinal vascular corrosion casts and scanning electron microscopy images (original magnification: upper panels, 200×; lower panels, 350×) obtained from the ileum of (A and D) a breast milk–fed pup with normal villous central arterioles, capillaries, and venules; (B and E) a pup stressed for 3 days, showing injured villous arterioles, capillaries, and venules; and (C and F) a pup stressed for 3 days but treated with HB-EGF added to the feeds, showing comparable villous microvasculature to that of the breast-fed control, with significant preservation of villous blood flow and microvascular architecture.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

9. Figure 8
Effect of HB-EGF on intestinal submucosal blood flow. Shown are representative images of submucosal microcirculatory blood flow as observed in a 3-dimensional confocal projection of cryosection slides obtained from the ileum (original magnification 40×). The vascular network, including 1A, 2A, and 3A arteries/arterioles, is shown using FITC-dextran angiography. The red staining represents nuclear staining with propidium iodide, and the green staining shows the microvasculature. The representative panels show intestinal samples from (A) control pups fed with breast milk for 3 days, (B) pups subjected to stress for 1 day, (C) pups subjected to stress for 2 days, (D) pups subjected to stress for 3 days, and (E) pups subjected to stress for 3 days but with HB-EGF added to the feeds. F represents quantification of submucosal microcirculatory blood flow. The number of animals represented by each bar is 10, with the exception of bars 5 and 6, which represent 6 and 4 animals, respectively. For each animal studied, 2 measurements were subjected to quantification, with green and red channels separated and signal intensity calculated using Zeiss LSM 5 software. In each scanned file, the ratio of the green channel signal intensity (representing submucosal microcirculatory blood flow) to the red channel signal intensity (representing nuclear staining) was obtained. *P < .001 compared with control; **P < .001 compared with stress day 3. Also, refer to Supplementary Movie 2. A corresponds to movie BMF, D corresponds to movie stress, and E corresponds to movie stress + HB-EGF.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions