Journal of Pediatric Surgery 49 (2014) 1791–1794

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Intestinal lengthening in an innovative rodent surgical model Veronica F. Sullins a, Andrew Scott a, Justin P. Wagner a, Doug Steinberger b, Steven L. Lee a, Benjamin M. Wu b, James C.Y. Dunn a,b,⁎ a b

Division of Pediatric Surgery, Department of Surgery, University of California, Los Angeles, CA, USA Department of Bioengineering, University of California, Los Angeles, CA, USA

a r t i c l e

i n f o

Article history: Received 27 August 2014 Accepted 5 September 2014 Key words: Mechanical enterogenesis Bowel lengthening Distraction enterogenesis Short bowel syndrome Biodegradable lengthening device Spring lengthening device

a b s t r a c t Purpose: Current animal models of mechanical lengthening separate intestinal segments from enteric continuity. Such models are difficult to use for repeated lengthening procedures and result in intestinal tissue loss during restoration into continuity. We sought to create a novel surgical model to allow multiple lengthening procedures for the purpose of maximizing the net increase in tissue after intestinal lengthening. Methods: A Roux-en-y jejunojejunostomy with a 6-cm blind-ended Roux limb was created in the proximal jejunum of rats. Encapsulated 1-cm polycaprolactone springs were placed into the closed end of the roux limb and secured with a vessel loop. After 4 weeks, lengthened segments and normal jejunum were retrieved for histologic analysis. Results: Jejunal segments were lengthened from 1.0 cm to 3.0 cm. Lengthened segments had increased smooth muscle thickness, fewer submucosal ganglia, and similar numbers of myenteric ganglia compared to normal intestine. When compared to normal jejunal mucosa, lengthened segments demonstrated unchanged villus height and increased crypt depth. Conclusions: We created an innovative surgical model for intestinal lengthening and successfully lengthened jejunal segments with a degradable spring. The Roux-en-y model may allow the use of a degradable spring for the treatment of short bowel syndrome. © 2014 Elsevier Inc. All rights reserved.

Short bowel syndrome (SBS) is a disorder of malabsorption due to inadequate intestinal length. Neonatal diagnoses such as necrotizing enterocolitis, intestinal atresias, midgut volvulus, abdominal wall defects, complicated meconium ileus, and aganglionosis lead to SBS and mortality has remained 20%–40% [1–3]. Despite medical and surgical advancements, patients with SBS suffer significant long-term morbidity [4,5]. Surgical treatment options are limited to bowel lengthening and transit slowing procedures and many patients are not optimal candidates [4,6,7]. Limitations in treatment options for SBS have led researchers to focus on lengthening existing intestine using mechanical force, or distraction enterogenesis. Strategies of intestinal lengthening include the use of hydraulic pistons, saline distension, tissue expanders, implanted screws, and spring devices [8–13]. All current models isolate intestinal segments from continuity during lengthening, which is associated with loss of tissue upon restoration and does not allow repeated lengthening of the isolated segment. We therefore sought to create a surgical model for intestinal lengthening that may serve as a platform for repeat lengthening procedures to maximize the net increase in tissue length.

⁎ Corresponding author at: Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, Box 709818, Los Angeles, CA 90095-7098, USA. Tel.: +1 310 206 2429; fax: +1 310 206 1120. E-mail address: [email protected] (J.C.Y. Dunn). http://dx.doi.org/10.1016/j.jpedsurg.2014.09.022 0022-3468/© 2014 Elsevier Inc. All rights reserved.

1. Materials and methods The use of animals was approved by the Animal Research Committee (Institutional Review Board Number 2002-037-22). All materials were FDA approved for use in humans. Intestinal lengthening was achieved using springs made from polycaprolactone (PCL), a biodegradable polymer used in absorbable sutures and other medical devices [14]. PCL springs were fabricated as previously described [13], placed into size 5 gelatin capsules (Torpac Inc., Fairfield, NJ) and coated with cellulose acetate pthalate (Eastman Chemicals, Kingsport, TN) for delayed expansion. 1.1. Surgical procedure Adult female Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA) weighing N 250 g were anesthetized with inhaled oxygen and isoflurane (n = 8). The abdomen was entered through a midline laparotomy incision and the jejunum was located and transected approximately 10 cm from the ligament of Treitz. To create a defunctionalized Roux limb, a longitudinal, antimesenteric enterotomy was made 6 cm from the open proximal end of the distal segment. The proximal jejunal segment was then anastomosed in an end-to-side configuration to the distal enterotomy with 6-0 Prolene (Ethicon, Johnson & Johnson; Somerville, NJ) in a simple interrupted fashion to restore enteric continuity. A small opening was created in the mesentery 1 cm from the end of the Roux limb and a vessel loop

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was placed around the jejunum. Coated and encapsulated PCL springs with spring constants between 1 and 2 N/m were placed into the Roux limb, and the end was closed with 6-0 Prolene suture (Ethicon, Johnson & Johnson; Somerville, NJ) in a simple interrupted fashion. The vessel loop was tied loosely and secured with surgical clips, positioning the spring between the end of the defunctionalized limb and the vessel loop (Fig. 1). The bowel was carefully placed back into the abdomen to ensure that there was no twisting of the mesentery, and the abdominal wall was closed in layers. Encapsulated 1-cm PCL tubes were placed into defunctionalized Roux limbs to serve as controls (n = 3). Lengthened segments were retrieved after 4–6 weeks and measured for length. Animal weights were recorded weekly.

1.2. Histologic analysis Mechanically lengthened and normal jejunal tissues were fixed in 10% buffered formalin overnight followed by embedding in paraffin. Care was taken to align tissue cross sections perpendicularly in order to obtain accurate histologic measurements. Tissue blocks were cut into 5-μm sections then stained with hematoxylin and eosin. Sections were examined and recorded at 40 × and 100 × magnification using light microscopy (Leica Microsystems). Muscularis propria thickness, villus height, crypt depth and circumference were measured for each specimen. Adjacent unstained tissue sections were prepared and stained for S100-positive glial cells as previously described [15]. Using fluorescent light microscopy, the number of ganglia was assessed at 100 × magnification in submucosal and myenteric plexuses and expressed as total number of ganglia per cross section. The density of ganglia was calculated and expressed as number of ganglia per mm circumference.

1.3. Statistical analysis Data were expressed as mean values ± standard deviations. Twotailed, paired and unpaired Student’s t-tests were used for statistical analyses where appropriate.

2. Results Experimental segments were lengthened from 1.0 to 3.0 ± 0.5 cm (p b 0.0001). In the control segments there was a slight increase in length from 1.0 to 1.4 ± 0.1 cm (p b 0.05). The increase in length between the experimental and control segments was statistically significant (195 ± 47% versus 40 ± 10%, p b 0.0001). PCL spring devices had either partially degraded or were not present in the lengthened segment at the time of exploration. Rats demonstrated a mean weight gain of 39 ± 21 g at the time of sacrifice. 2.1. Histologic analysis Lengthened jejunum had a greater circumference (1.51 ± 0.32 cm versus 1.02 ± 0.14 cm, p = 0.01) and thicker muscularis propria (297 ± 58 versus 104 ± 28 μm, p b 0.0001) when compared to normal jejunum (Fig. 2). Examination of the mucosa revealed increased crypt depth (270 ± 77 versus 162 ± 27 μm, p = 0.02) in the lengthened jejunum. Differences in villus height were not statistically significant (404 ± 96 versus 475 ± 123 μm, p = 0.34). The total number of ganglia in lengthened jejunum was decreased in the submucosa (14.5 ± 5.7 versus 29.6 ± 2.3 ganglia, p b 0.001) and was unchanged in the myenteric plexus (45.5 ± 7.5 versus 42.8 ± 6.7 ganglia, p = 0.55) (Fig. 3). When comparing ganglion density, lengthened jejunum had decreased density of both submucosal ganglia (1.2 ± 0.7 versus 3.1 ± 0.3 ganglia per mm, p b 0.001) and myenteric ganglia (3.3 ± 0.6 versus 4.2 ± 0.4 ganglia per mm, p b 0.05). 3. Discussion Multiple devices designed for distraction enterogenesis have shown promising results. However, all models require that a segment of intestine be separated from enteric continuity. We previously showed that an isolated jejunal segment could be lengthened nearly 3-fold with a degradable PCL spring [13]. We then restored the lengthened segment into enteric continuity and demonstrated regained motor and absorptive function [16]. While the success of restoring lengthened jejunum is encouraging, its clinical impact is diminished by the loss of tissue that occurs when intestinal anastomoses are created. Size constraints

Fig. 1. Photographs and diagram of the Roux-en-Y procedure. The top photographs show the spring device, the bowel configuration at device placement, and lengthened bowel. The bottom diagram is a schematic representation of the lengthening model. Px = proximal, Ds = distal, S = spring, A = anastomosis.

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Fig. 2. Photographs of hematoxylin and eosin-stained A) normal and B) lengthened jejunum under light microscopy at 40× magnification. Lengthened jejunum demonstrated increased muscularis propria thickness and increased crypt depth. Villus height was similar. Scale bars represent 200 μm.

preclude insertion of a longer spring device, and restoring multiple isolated segments into continuity would result in several serial anastomoses and significant tissue loss. These limitations could be overcome by repeated lengthening of a single, non-isolated intestinal segment. Here we described lengthening of a jejunal segment that remained in contact with luminal contents within the intestinal tract. In this model, after initial lengthening, another device could be placed into the blind end of the lengthened Roux limb for repeat lengthening. When restoring back into continuity, only one or two anastomoses would be necessary, thus maximizing the gain in intestinal length. Another advantage of the Roux limb is that the implanted spring achieves maximal lengthening without inducing obstruction. Our choice of PCL to make the spring device is based on its material properties as a biocompatible, biodegradable polymer often used in medical devices and sutures [14,17]. Degradation times vary but it has been shown that PCL-based implants are still present up to 2 years after subcutaneous implantation [17]. Therefore, it was not surprising to find that in the isolated segment model of intestinal lengthening, the PCL springs were retrieved intact. The degradation rate of PCL in the presence of bile and digestive enzymes is not known. Our defunctionalized Roux limb model permits some reflux of chyme into the segment that is undergoing lengthening, allowing us to test both function and degradation of the device in the presence of digestive enzymes. In addition to demonstrating successful 3-fold lengthening, we

found that the PCL springs had either degraded into pieces or completely disappeared after 4 weeks. This finding suggests that intraluminal placement of the PCL spring device results in lengthening followed by degradation without requiring additional procedures. While measurements of villus height in mechanically lengthened tissue have been varied when compared to normal tissue, the other histologic results are consistent with our previously reported findings and the findings of other investigators [8,12]. Increased crypt depth and thickened muscularis propria are typical features of tissue subjected to mechanical force. These histologic characteristics, together with findings of increased markers of cellular proliferation, suggest that intestinal growth is induced when using distraction techniques [8]. The mechanism of nerve degeneration after mechanical lengthening has yet to be explained. Research shows that mucosal and submucosal layers are more sensitive to ischemia while the muscularis is most sensitive to hypoxia [18,19]. Analysis of rat ileum found a decreased density of submucosal and, to a lesser extent, myenteric ganglion cells in response to ischemia and reperfusion injury [20]. This would suggest that mechanical lengthening induces a relative ischemia across the bowel wall that more substantially affects the submucosa. In chick embryos, researchers found that in the absence of ischemia, dilation causes injury to enteric nerves, with more severe injury to the submucosal ganglia than the myenteric plexus [21]. It was interesting to find that while the number of ganglia in the submucosa decreased in comparison

Fig. 3. Photographs of immunohistochemical staining for S-100 in A) normal and B) lengthened jejunum under fluorescent light microscopy at 100 × magnification. S marks the serosal surface and L marks the luminal surface. Lengthened jejunum had fewer submucosal ganglia (arrowheads) and similar numbers of myenteric ganglia (arrows). Scale bars represent 200 μm.

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to normal jejunum, the number of ganglia in the myenteric plexus did not change. Whether this decrease is due to ischemia, bowel dilation, or an alternate mechanism may be the focus of future research. Though the etiology of nerve degeneration remains unclear, our observations support the notion that submucosal nerves are more sensitive to insult than myenteric nerves. We previously saw decreased numbers of both submucosal and myenteric ganglia in an isolated segment of jejunum that was lengthened by a spring device [13,15]. Compared to the isolated segment model, it is likely that by permitting mucus to escape, our new lengthening model exerts less transmural force on the segment being stretched, allowing the more robust myenteric neurons to persist. Preservation of the myenteric plexus in our model may also be caused in part by conservation of nerves on one side of the segment undergoing lengthening, as opposed to complete isolation. As we have previously seen, in this new model we expect some degree of nerve regeneration when the lengthened segment is restored into continuity [16]. There was an expected decrease in density of ganglia in both the submucosal and myenteric plexuses. While in the submucosal plexus this decrease is due to both the degeneration of nerves and the increase in circumference, in the myenteric plexus it is solely caused by an increase in bowel circumference. The numbers and density of ganglia in this model may not predict small bowel motility after restoration into continuity. In summary, we successfully created a defunctionalized Roux-en-y model of distraction enterogenesis to maximize the length of tissue gained and improve the applicability of an encapsulated PCL spring device. When exposed to enteric enzymes, the PCL spring device lengthened the tissue 3-fold, and then degraded within 4 weeks. This innovative model may serve as a platform for testing the capacity of lengthened intestine to undergo repeat lengthening. Our findings suggest that use of a biodegradable lengthening device for mechanical enterogenesis may be a promising treatment modality in the management of SBS. Acknowledgments We extend many thanks to Elvin Chiang for his assistance with immunostaining and Nan Ye Lei for his assistance with photography. This work was supported by Sun West and the Department of Pediatrics at Harbor-UCLA Medical Center.

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Intestinal lengthening in an innovative rodent surgical model.

Current animal models of mechanical lengthening separate intestinal segments from enteric continuity. Such models are difficult to use for repeated le...
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