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Intestinal lengthening and reversed segment in a piglet short bowel syndrome model Geert Iede Koffeman, MD,a,b,* Jan B.F. Hulscher, MD, PhD,c Ivo G. Schoots, MD, PhD,d Thomas M. van Gulik, MD, PhD,d Hugo A. Heij, MD, PhD,a and Wim G. van Gemert, MD, PhDa a

Department of Pediatric Surgery, Pediatric Surgical Center Amsterdam AMC/VUmc, Amsterdam, The Netherlands Department of Surgery, St Lucas Andreas Hospital, Amsterdam, The Netherlands c Department of Pediatric Surgery, University Medical Center Groningen, Groningen, The Netherlands d Department of Surgery, Academic Medical Center, Amsterdam, The Netherlands b

article info

abstract

Article history:

Background: Treatment of short bowel syndrome (SBS) remains difficult, entailing severe

Received 15 March 2014

morbidity and mortality. Accepted surgical treatment modalities for SBS are the Bianchi

Received in revised form

intestinal lengthening procedure and reversed-segment procedure. We seek to investigate

7 December 2014

the short-term effects regarding growth, nutrition, and microscopic and functional adap-

Accepted 11 December 2014

tation after the intestinal lengthening and RS procedures in a piglet SBS-model.

Available online 29 December 2014

Material and methods: Twenty-four piglets (Sus scrofa,
30 kg) were divided into four groups (n ¼ 6 each) as follows: sham, SBS, Bianchi lengthening procedure (BIA), and reversed-

Keywords:

segment (RS). At day one either sham laparotomy (sham) or 75% small bowel resection

Short bowel syndrome

(SBS, BIA, and RS) was performed. After 2 wk sham laparotomy (sham and SBS), BIA, or RS

Longitudinal intestinal lengthening

procedure was performed. After 8 wk all animals were terminated. During the experi-

procedure

mental time course, the following parameters were assessed: body weight, intestinal length, diameter, and weight, fat absorption, and biochemical parameters from serum and urine. Citrulline was used as a marker of absorptive enteral mass to demonstrate massive functional bowel loss. Intestinal biopsies were obtained for histologic analysis and electrophysiological measurements to analyze glucose absorptive capacity. Results: Eight weeks after bowel resection, piglet growth was reduced in SBS, BIA, and RS piglets as demonstrated by reduced weight (51
4 kg, 47
2 kg, and 53
1 kg, respectively) compared with sham (69
3 kg; P < 0.01), with no demonstrable difference between SBS and treatment groups. Malabsorption and malnutrition occurred in SBS, BIA, and RS piglets reflected by increased fecal fat loss per 24 h (35
4%, 30
2%, and 32
4%, respectively versus 18
1% in sham; P < 0.01) and reduced serum albumin levels (24
1 g/L, 22
1 g/L, and 24
1 g/L, respectively versus sham 33
1 g/L; P < 0.01), but there was no significant difference between SBS and treatment groups. Serum citrulline levels reflected massive functional bowel loss (SBS 36
7 mmol/L, BIA 23
1 mmol/L, and RS 24
2 mmol/L) compared with sham (64
5 mmol/L; P < 0.01). Electrophysiological measurements demonstrated reduced glucose absorption after intestinal resection, which did not return to base levels within the experimental time course. However, the intestine of BIA and RS piglets adapted more profoundly than SBS piglets, as reflected by a greater crypt depth

* Corresponding author. Department of Pediatric Surgery, Pediatric Surgical Center Amsterdam AMC/VUmc, Amsterdam 1061AE, The Netherlands. E-mail address: [email protected] (G.I. Koffeman). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.12.024

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(490
25 mm and 492
21 mm versus 388
20 mm; P < 0.01); and BIA piglets showed greater villus length (884
58 mm) than RS or SBS piglets (715
30 mm and 737
64 mm, respectively; P < 0.01) after 8 wk. Conclusions: Despite increased histologic intestinal adaptation, neither intestinal lengthening nor RS procedure demonstrated significantly improved absorption, nutrition, or weight gain for the treatment of SBS during the study period. Reduced glucose uptake on electrophysiology measurements and persistent low levels of citrulline may indicate reduced small bowel enterocyte functioning during the initial phase of intestinal adaptation. ª 2015 Elsevier Inc. All rights reserved.

1.

Introduction

Therapy of short bowel syndrome (SBS) is aimed at optimizing enteral and parenteral nutrition (PN) therapy, but also at prevention, early detection, and treatment of complications of PN such as cholestasis and central venous catheter-related infections [1,2]. In the long term, achieving enteral autonomy should be the primary objective. When first line therapy, including surgical interventions such as restoring bowel continuity and adhesiolysis, is inadequate to achieve this goal, further surgical therapy may be indicated. Various surgical therapies have been developed to treat SBS symptoms and accelerate adaptation. These may generally be divided into autologous intestinal reconstruction procedures and bowel transplantation. Bowel transplantation has demonstrated great improvements in survival but is still associated with severe morbidity and mortality. Furthermore, application is constrained by the limited number of donors, especially in pediatric patients [3]. Autologous procedures may offer better long-term results with reduced morbidity and mortality [4]. The longitudinal intestinal lengthening procedure, as originally described by Bianchi [5], is one of the most widely applied autologous surgical procedures for pediatric SBS [6,7]. Another older, less complex procedure is the reversed-segment (RS) procedure [8], sometimes used in combination with the Bianchi procedure. Over the last years, the STEP (serial transverse enteroplasty) procedure [9] has also gained popularity. The Bianchi longitudinal intestinal lengthening procedure makes use of the bifurcated vascularization of the intestine. It uses sequential longitudinal transection and bowel anastomosis, with serial anastomosis to double remaining intestinal length (Fig. 1). Thereby, it increases intestinal length and reduces intestinal diameter with resultant increased transit time and chymal-mucosal contact. The antiperistaltic reversed segment (RS), reduces transit time by surgical isolation of an intestinal segment, 180 rotation, and anastomosis; thus creating an antiperistaltic segment (Fig. 2). RS thereby increases the nutrient load, and thus possibly enhances adaptation. Both procedures have shown clinical value [4e10]. A recent systematic review described that some 70% of children could be weaned from PN after the Bianchi procedure, while an overall intestinal lengthening of 1.48 fold (range 1.25e2.0) could be achieved [9]. For the RS, 45% of adult patients achieved intestinal autonomy, whereas remaining patients showed a reduction in PN dependency in the largest reported clinical series (n ¼ 38) using this technique [10].

Despite such promising clinical results, the working mechanism of both procedures requires further elucidation. We present an experimental study in a validated porcine model for SBS [11] investigating growth and nutritional status, as well as early microscopic and functional changes of the intestine after longitudinal lengthening and antiperistaltic RS.

2.

Material and methods

Twenty-four piglets (Sus scrofa, female,
30 kg, aged 8 wk) were included after 1-wk acclimatization and divided into four groups (n ¼ 6 each) as follows: SBS, sham, Bianchi lengthening procedure (BIA), and RS; (Table 1). The pigs were housed in individual pens, side by side, on a grid floor. Room temperature was maintained at 20 C, with lightedark cycles of 12 h. Animals were fed 1.4 kg of standard grower feed per day (Hope Farms, Woerden, the Netherlands, 9.34 MJ/1000 g, 17.5% protein, 4.5% fat, and 4% fiber) with water ad libitum, except postoperatively. The protocol was approved by the Animal Ethics Committee of Amsterdam (the Netherlands). All animals were handled in accordance with the guidelines prescribed by Dutch legislation and the International Guidelines on the protection, care, and handling of laboratory animals. On day one, surgery was performed, either a sham laparotomy (sham-group), or creation of the SBS by means of 75% small intestinal resection (remaining groups). Surgical intervention occurred again after 2 wk, either sham operation (sham group and SBS group), or Bianchi lengthening procedure (BIA group) or reversed segment (RS group) in the experimental groups; with termination in all groups after 8 wk.

2.1.

Surgical procedures

Creation of the SBS has been described previously [11,12]. Briefly, animals were fasted for 12 h, then analgesia (flunixinum 2 mg/kg and buprenorphine 0.01 mg/kg) was administered. Anesthesia was induced with midazolam (1 mg/kg), ketamine (10 mg/kg), and atropine (0.02 mg/kg). An endotracheal tube was inserted and anesthesia maintained by ventilation with isoflurane (1.5% by volume) and O2/N2O, with sufentanil (15 mg/kg) ketamine (15 mg/kg), and clonidine (0.5 mg/kg). Pancuronium (0.1e0.5 mg/kg) was given for muscle relaxation. Perioperatively, antibiotics (ceftriaxone 30 mg/kg and ampicillin 15 mg/kg) and analgesia (flunixinum 2 mg/kg and buprenorphine 0.01 mg/kg) were administered.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 4 3 3 e4 4 3

The abdomen was shaved, cleaned with betadine, and draped with sterile sheets. Laparotomy was performed by midline incision. The small intestine was measured twice along its antimesenteric border and the mid 75% was removed (resection of all small bowel between 150 cm from the ligament of Treitz to 150 cm before the ileocecal valve) followed by end-toend anastomosis using polydioxane 4-0 suture (Ethicon Inc, Cincinnati, Netherlands). Urine and blood samples were collected before incision, and intestinal biopsies were obtained at the transection site. A catheter was inserted into the cephalic vein for intravenous fluid administration. The Bianchi procedure was performed as described previously [12,13]. Briefly, on day 14, laparotomy was performed via the old scar. At 150 cm from Treitz ligament, the intestine was lengthened from 40 cme80 cm, by means of serial bowel transection and anastomosis using a GIA stapling device (Ethicon Endo-surgery Inc). RS was performed at 150 cm from the ligament of Treitz, a 10 cm segment of intestine was incised, rotated 180 , and anastomosed by end-to-end polydioxane 4.0 (Ethicon) anastomosis with a running suture. Sham laparotomy involved solely laparotomy, intestinal transection, and biopsy removal at 150 cm from the ligament of Treitz, followed by anastomosis and abdominal closure. Anesthesia and analgesia were performed as mentioned previously. Length of bowel used for RS and BIA was determined based on experimental and clinical literature [4e10] and a pilot study testing bowel alteration lengths and study design [12]. Postoperative care was the same in all groups; initially animals were kept on nil by mouth, with intravenous administration of Ringer lactate, NaCl 0.9%, and glucose 5% (30 mL/kg/h). The first and second day after operation, 100 mL of intralipid 20% (8.4 MJ/1000 mL) was administered intravenously. After 72 h postoperatively, animals recommenced feeding on standard grower diet mixed with water to make a soft paste.

435

Intravenous fluids were obtained from Fresenius Kabi, s’Hertogenbosch, the Netherlands.

2.2.

Analysis of growth and nutritional status

At day 1, day 14, and day 56, the following parameters were measured: body weight, fecal fat loss [14] (fecal collection 24 h preoperatively with fecal fat measurement), albumin, and also routine biochemical parameters (serum Naþ, Kþ, creatinine, urea, aspertate aminotransferase, alanine aminotransferase, g-GT, Mg, Fe, vitamin A, vitamin B1, erythrocytes, hemoglobin, leucocytes measured as routine samples in the AMC Academic Hospital Biochemical Laboratory) and essential amino acids (including citrulline) in blood and urine [15,16].

2.3.

Histologic examination

Histologic examination was performed to examine adaptation of the intestinal mucosa and muscle layers. Intestinal ileum biopsies of 5 cm were fixed in 4% formaldehyde within 5 min of excision and processed for routine microscopy examination. After 8 wk (termination), biopsies were also taken at 50-cm intervals through the small intestine. Specimens were stained with hematoxylin and eosin and examined under digital microscopy. LEICA QWin (Leica microsystems, Wetzlar, Germany) was used to capture and analyse microscopic images. From each microscopy, sample villus height and width, crypt depth, longitudinal and circular muscle width were assessed in 10 measurements by one investigator blinded for the experimental group status of the animal (G.I.K.).

2.4.

Electrophysiological measurements

Intestinal adaptation leads to villi and crypt growth/hypertrophy and eventual greater nutrient uptake by increased surface area, to compensate for lost bowel

Fig. 1 e Longitudinal intestinal lengthening procedure. (A) Separation of bifurcated intestinal mesenterium vascularization (B): longitudinal bowel division (C) divided bowel (D): anastomosis.

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Fig. 2 e RS. (A) Selection of small bowel segment (B): transection of bowel (C): reversal of segment (D): anastomosis.

length. It remains less clear what occurs regarding functional changes per centimeter2. Short circuit current (Isc) electrophysiological measurements can be used to reflect aspects of enterocyte function. To test intestinal functioning during the adaptation process, changes in intestinal crypt chloride secretion and villi glucose uptake were measured using electrophysiological measurements as described previously [17]. In vitro electrophysiological experiments of intestinal biopsies were performed on day one, after 2 wk, and 8 wk. During each experiment, six sheets of anti-mesenterial ileum without Peyer’s patches

Table 1 e Study design.

were studied. Tissue was rinsed with ice-cold Ringer’s solution, stripped of the muscular layers, and suspended between Ussing chambers within 15 min, with an exposed surface area of 0.5 cm2. The samples were perfused with Ringer’s solution (composition in mmol/L: NaCl 117.5, KCl 5.7, NaHCO3 25.0, NaH2PO4 1.2, and CaCl2 2.5, MgSO4 1.2) and gassed with humidified 5% CO2/95%O2. Solutions were maintained at 37 C via water jackets and recirculated with a polystatic pump. The pH was 7.3 and osmolality 290 mosmol/kg after carbogenation. After a half-hour acclimatization period (at which time a steady flux state had

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Table 2 e Intestinal morphology. Variable Intestinal length (cm) Median Time ¼ 0 wk Range (cm) % Resection Intestinal bowel (cm) Diameter jejunum 100 cm from Treitz Time ¼ 0 wk Time ¼ 2 wk Time ¼ 8 wk Diameter Ileum (cm) 100 cm from cecum Time ¼ 0 wk Time ¼ 2 wk Time ¼ 8 wk *

Sham

SBS

Bianchi

RS

1630 (1475e1920) d

1560 (1370e1673) 80.7

1565 (1450e1635) 80.8

1580 (1483e1600) 81.0

2.62 (R2.5e3.0) 2.8 (R2.5e3.0) 3 (R3)

2.5 (R2.5) 3.6 (R3.1e4.0) 4.18 (R3.6e4.0)

2.7 (R2.5e3.0) 2.9 (R2.5e3.0) 3.33 (R3.5e4.0)

2.53 (R2.0e3.0) 3.42 (R3.0e4.0) 4.33 (R3.5e5.0)

2.58 (R2.5e3.0) 3.83 (R3.0e4.5) 5.42 (R4.5e7.0) 2.5 (R2.5) 3.83 (R3.0-4.0) 3.0  2* (R 3.0)

2.5 (R2.5) 3.58 (R3.0e4.0) 4.42 (R4.0e5.5) 2.5 (R2.5) 3.5 (R3.0e4.0) 4.5 (R4.0e5.0)

Bianchi lengthened intestine.

been achieved), active (crypt) Cl-secretion was measured after secretagogue administration (carbachol 105 M: inducing Ca2þ/PKC mediated secretion and forskolin 105 M: inducing cyclic adenosine monophosphatemediated crypt secretion) to the serosa. Ten minutes later, active uptake (villi) was measured after glucose (20 mmol/L D-glucose) administration to the mucosa. In all experiments, transepithelial potential difference was measured with Ag/AgCl-electrodes by voltage deflections induced by 10 mA bipolar current pulses through platinum wires. The electrodes and the platinum wires were connected with the chambers via KCl-agar bridges. The Isc and transepithelial resistance were calculated according to Ohm’s law. The potential difference was continuously monitored by a computer program, customized for our laboratory using LabVIEW (National Instruments, Austin, Texas). Chemicals were obtained from SigmaeAldrich Chemie BV (Zwijndrecht, the Netherlands).

2.5.

3.

Results

Massive small bowel resection was performed in three groups with average resection length >80% of total small bowel length (Table 2). One sham piglet demonstrated respiratory failure preoperatively because of complications during intubation and was put down; results were removed from further analysis.

3.1.

Growth and nutritional status

The SBS, BIA, and RS groups displayed comparable growth retardation, steatorrhea, and malnutrition when compared with that of the sham group (Figs. 3e5). Further biochemical results (serum Na, K, creatinine, urea, aspertate aminotransferase, alanine aminotransferase, g-GT, Mg, Fe, vitamin A, vitamin B1, erythrocytes, hemoglobin, and leukocytes) showed no significant differences between groups (data not shown).

Statistics 3.2.

Histologic changes

Statistical analysis (two-way analysis of variance with Bonferroni post-test) was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, www.graphpad.com.

BIA and RS piglets demonstrated greater histologic adaptation compared with that of the SBS and sham piglets after 8 wk (Figs. 6e9). Crypt depth in BIA and RS piglets was 490
25 mm

Fig. 3 e Weight gain (all groups n [ 6 except sham: n [ 5).

Fig. 4 e Fecal fat loss/24 h (all groups n [ 6 except sham: n [ 5).

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Fig. 5 e Serum albumin (all groups n [ 6 except sham: n [ 5). and 492
21 mm, respectively, versus 388
20 mm in SBS (both P < 0.01). Furthermore, BIA piglets showed a greater villus length in Bianchi lengthened ileum (884
58 mm) than RS (in RS ileum) or SBS piglets (715
30 mm and 737
64 mm, respectively; P < 0.01) after 8 wk. Furthermore, the BIA piglets showed an enlargement of longitudinal muscle width, significant in comparison with that of the RS and SBS piglets, though not circular muscle width (Figs. 10e13, Table 3). Diameter of the bowel was also increased in BIA piglets when compared with other groups; both in the Bianchi lengthened ileum and the jejunum and remaining ileum (Table 1).

3.3. Functional status of the bowel during the adaptation process 3.3.1.

Electrophysiology

Changes in Isc (DIsc) induced by D-glucose administration showed decreases in SBS, BIA, and RS groups at 2 wk (85.3
14.0, 67.5
26.3, and 51.2
18.1 mA/cm2) and at 8 wk (85.05
13.3, 105.43
8.5, and 79.0
6.8 mA/cm2; P  0.05), compared with initial values (day 1). There was no significant change in Isc values at these time points for the sham group (14.1
10.18 mA/cm2 and 13.7
15.8 mA/cm2), nor between SBS, BIA, and RS groups. D-glucose-induced percentage DIsc in SBS, BIA, and RS groups demonstrated reductions at 2 wk (43.3
7.2,

55.8
17.1, and 58.5
9.6%) and 8 wk (24.8
5.0, 44.4
9.6, and 35.5
6.3%; P  0.05). Sham results remained unchanged, 91.8
13.1% and 94.3
23.4% at 2 and 8 wk, respectively (Fig. 14). Forskolin-induced DIsc changes (Ca2þ/PKC-mediated crypt secretion) in SBS, BIA, and RS groups at 2 wk were 2.4
3.2, 0.5
1.9, and 0.1
0.5 mA/cm2 and at 8 wk were 3.9
3.1, 3.3
1.5 mA/cm2, and 3.1
3.7 mA/cm2, respectively, compared with initial values (day 1). Sham group was unchanged (0.1
1.6 and 1.6
3.1 mA/cm2) at 2 and 8 wk. Carbachol-induced Isc changes (cyclic adenosine monophosphate-mediated crypt secretion) showed no significant differences in the study period or between groups (data not shown).

3.3.2.

Serum citrulline

Intestinal surface area and uptake were severely reduced after resection as indicated by citrulline levels at 8 wk as follows: SBS: 35.60
7.00 mmol/L, BIA: 23.32
1.13 mmol/L, RS 24.64
2.59 mmol/L versus 64.46
5.43 mmol/L in the sham group; Figure 15. There were no significant differences between groups regarding other essential amino acids (data not shown).

4.

Discussion

This study set out to investigate the effects of the longitudinal lengthening procedure (BIA) and RS procedure on growth and nutritional status, as well as on several histologic and functional characteristics associated with adaptation of the small intestine. By such means, we attempted to gain insight into the initial phases of intestinal adaptation after BIA and RS, and the means by which surgical therapy alters adaptational processes. Intestinal adaptation is characterized by compensatory growth of the intestinal remnant, proportional to the amount of resected intestine. There is mucosal hypertrophy but also hyperplasia of the circular and longitudinal smooth muscle layers. These adaptive changes were also observed in the

Fig. 6 e Histology (hematoxylin and eosin staining): sham/SBS ileum at 8 wk. (Color version of figure is available online.)

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Fig. 7 e Histology (hematoxylin and eosin staining): RS/Bianchi ileum at 8 wk. (Color version of figure is available online.)

present study, in which there were clear histologic signs of adaptation in the BIA and RS groups as follows: greater crypt depth in the RS group and the BIA group and significantly greater villi length in the BIA group compared with SBS and SHAM groups. Furthermore, BIA and RS groups showed increased jejunal muscular width (longitudinal and circular) compared with SBS. This suggests additional morphologic alterations as a result of increased chymal fluid resistance induced by greatly reduced bowel diameter (Bianchi) and reversed peristalsis (RS). Such would concur with postulated theoretical reasoning of the underlying mechanistic causes of these technique’s efficacy. The SBS group intestine has no such induced changes in chymal fluid dynamics and therefore muscular width is comparable with measurements in the sham group. The cause of reduced ileum muscular width (longitudinal and circular) in the SBS group compared with other groups remains unclear, as otherwise SBS results track with Bianchi and RS-group results. Though there was evident morphologic adaptation in the form of crypt hyperplasia and muscle hypertrophy, this did not lead to advantageous growth or improved nutritional uptake in the study time period. In contrast to some previous

studies [18,19], we were not able to demonstrate a clinical advantage in terms of weight gain or albumin levels after surgical interventions (BIA and RS) when compared with the SBS group; or of one surgical intervention over the other. Similar results have been reported by other authors [19e23] demonstrating mixed results for both therapeutic techniques in the piglet model. This lack of clear clinical benefit may be the result of many factors influencing bowel adaptation. Although adaptation leads to higher levels of crypt cell proliferation and mucosal hyperplasia, there may initially be reduced function; with lower uptake of nutrients such as glucose [21]. The increased proliferation results in functionally immature enterocytes populating the villi, especially in the mid ileum. In the early phase of adaptation, this might be an important factor hampering recovery and growth. Lauronen et al. [21] show that specific disaccharidase activities are still reduced at 14 wk after resection in a piglet SBS-model. Initially, expression of GLC5 surface transport molecules may also be reduced [21]. This offers an explanation of the lower glucose uptake measured in our study’s electrophysiology experiments without obvious crypt secretion alteration. Comparable electrophysiological results have

Fig. 8 e Villus size in ileum (all groups n [ 6 except sham: n [ 5).

Fig. 9 e Crypt size in ileum (all groups n [ 6 except sham: n [ 5).

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Fig. 10 e Longitudinal muscle width in jejunum (all groups n [ 6 except sham: n [ 5).

been shown previously by Whang et al. [24] and Wolvekamp [25], who both demonstrated reduced sodium-dependant glucose uptake in rats at 2 and 3 wk after resection, respectively. They concluded this to be a sign of reduced brush border Naþ/glucose co-transporters in enterocytes. However, Wolvekamp shows a return to initial electrophysiological functional values after 10 wk in ileum measurements suggesting resolution of differentiated enterocyte function over time. Improvements in function may therefore lag behind morphology, while the increased mass of functionally immature enterocytes have yet to differentiate. These factors may offer an explanation for the observed adaptational morphology changes without clinical benefit observed in our study. Our study demonstrates that serum citrulline may correlate well with the magnitude of intestinal loss but may be less useful regarding intestinal functional recovery in the initial stages. Lower plasma citrulline levels are associated with a reduced function of enterocytes [15]. In the present study, the persistent low levels of citrulline observed in the BIA and RS groups may therefore also be indicative of a decreased enterocyte function. Serum citrulline levels were reduced in SBS, BIA, and RS groups after intestinal resection. Interestingly, the BIA and RS groups’ citrulline levels remained at resection levels, whereas the SBS group showed some recovery. This might imply that after SBS induction, natural adaptation may initially have been delayed after Bianchi or RS. Other experimental studies also show lengthy periods of

Fig. 11 e Circular muscle width jejunum (all groups n [ 6 except sham: n [ 5).

Fig. 12 e Longitudinal muscle width in ileum (all groups n [ 6 except sham: n [ 5).

reduced serum citrulline after resection [26]. Citrulline recovery may further be linked to weight recovery and other clinical parameters, which did not occur in the time frame of our study. Whether citrulline could function as a marker for enterocyte loss and subsequent recovery in SBS patients remains to be investigated.

4.1.

Limitations of study

4.1.1.

Study length

Our study length was initially based on Sigalet’s [11] experimental design. We subsequently performed a pilot study [12] suggesting that an 8-wk time frame should allow clear group differences regarding clinical outcome to emerge. Other experimental studies use varying study periods. These might depend on study aims, design, and assessed treatment modalities; published study periods vary between 2 wk and 5 mo [8,11,18e27]. This great variance underlines the difficulties in predicting an adequate time frame to demonstrate experimental treatment effect (let alone outcomes in patient application). Though clear SBS physiology was demonstrated during the initial 2 study weeks, the following 6-wk time frame after treatment initiation showed no clinical advantage in the treatment arms despite greater adaptation. This may be due to 1) a too short time period after treatment initiation for clinical advantage to emerge, 2) treatment interference with

Fig. 13 e Circular muscle width in ileum (all groups n [ 6 except sham: n [ 5).

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Table 3 e Microscopy results in jejunum and ileum (H&E staining). Variable Microscopy jejunum (mm) Villus size Time ¼ 0 wk Time ¼ 2 wk Time ¼ 8 wk Crypt depth Time ¼ 0 wk Time ¼ 2 wk Time ¼ 8 wk Longitudinal muscle width Time ¼ 0 weeks Time ¼ 2 weeks Time ¼ 8 weeks Circular muscle width Time ¼ 0 wk Time ¼ 2 wk Time ¼ 8 wk Microscopy ileum (mm) Villus size Time ¼ 0 wk Time ¼ 2 wk Time ¼ 8 wk Crypt depth Time ¼ 0 wk Time ¼ 2 wk Time ¼ 8 wk Longitudinal muscle width Time ¼ 0 weeks Time ¼ 2 weeks Time ¼ 8 weeks Circular muscle width Time ¼ 0 wk Time ¼ 2 wk Time ¼ 8 wk

Sham

SBS

Bianchi

RS

d d 548.4 (
35.6)

526.1 (
88.9) 630.5 (
87.3) 673.3 (
55.5)

563.9 (
33.8) 731.9 (
69.4) 784.4 (
64.5)

561.3 (
16.7) d 623.9 (
50.5)

d d 370.9 (
48.7)

363.9 (
16.8) 422 (
28.9) 418.9 (
27.0)

361.5 (
14.0) 454.3 (
25.7) 479.4 (
16.7)

400.7 (
18.9) 411.6 (
27.6) 472.1 (
11.7)

d d 375 (
33.5)

290.3 (
19.7) 429.2 (
45.5) 460.0 (
55.4)

280.1 (
25.0) 459.4 (
19.5) 722.5 (
80.0)

362.0 (
36.2) 482.3 (
29.3) 614.8 (
67.7)

d d 290.7 (
14.6)

226.4 (
12.6) 410.8 (
48.0) 505.7 (
65.8)

197.1 (
23.2) 394.1 (
36.9) 432.4 (
57.7)

266.4 (
51.7) 383.2 (
18.6) 417.3 (
50.4)

386.2 (
12.5) 468.9 (
18.0) 489.5 (
38.4)

472.1 (
29.1) 626.5 (
40.3) 715.2 (
29.6)

399.1 (
10.1) 743.1 (
45.9) 780.9 (
87.2)

474.3 (
20.9) 662.0 (
48.3) 724.7 (
70.9)

281.5 (
16.8) 295.9 (
17.4) 295.6 (
10.0)

305.2 (
21.0) 364.8 (
18.0) 388.4 (
20.3)

302.0 (
20.2) 439.5 (
9.7) 425.9 (
18.3)

317.9 (
12.4) 432.9 (
14.7) 473.5 (
20.5)

349.7 (
36.5) 512.4 (
51.2) 496.1 (
43.9)

371.6 (
36.9) 354.7 (
46.0) 456.8 (
40.9)

449.4 (
57.1) 645.9 (
54.7) 686.9 (
90.6)

429.0 (
30.4) 576.6 (
39.4) 535.2 (
44.0)

215.7 (
16.8) 358.1 (
31.6) 382.1 (
49.1)

257.7 (
26.2) 288.6 (
27.7) 415.1 (
30.7)

295.0 (
31.1) 410.6 (
29.9) 438.6 (
43.3)

272.6 (
39.8) 394.8 (
39.4) 429.5 (
35.0)

H&E ¼ hematoxylin and eosin.

natural adaptation, and 3) limited clinical advantage for the applied treatment modalities. A longer observation time might have resulted in significant growth as a result of the demonstrated enhanced intestinal histologic adaptation. However, our aim was to obtain a greater understanding of the changes induced by SBS in the initial adaptation phases and the influence hereon after

surgical therapy. Our results concur with those described by Digalakis et al. [8], who investigated RS in a similar pig model, also describing clear histologic adaptation, a favorably modified transit time but no clinical benefits after a similar length of observation. In humans, the adaptation process may continue for months to years, the encompassed time span for animals is uncertain.

Fig. 14 e Electrophysiology results: % Isc change induced by glucose administration (all groups n [ 6 except sham: n [ 5).

Fig. 15 e Serum citrulline (all groups n [ 6 except sham: n [ 5).

442 4.1.2.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 4 3 3 e4 4 3

Bowel alterations

Several further factors may have contributed to the demonstrated lack of clinical benefit regarding growth and nutritional status after either BIA or RS when compared with the SBS piglets. The presented study was preceded by a pilot study in which the optimal length of the Bianchi procedure was determined to be lengthening from 40 cme80 cm [12]. Although patient characteristics usually limit intestinal lengthening and/or RS size and treatment selection in the clinical setting, we found that operative trauma and ileus induced morbidity limit experimental bowel length alteration and expected treatment effect in our pilot study [12]. Other studies using a similar porcine model show [18,19] an increased weight gain in BIA piglets compared with that in SBS piglets though over a longer study period. Also, in these studies, the extent of the lengthened bowel was smaller, that is, 20 cme40 cm instead of 40 cme80 cm. Whether this difference could have affected the results in the present study is uncertain. In humans, at least 20 cme40 cm of remnant small bowel is necessary to perform BIA, with a minimum diameter of the (dilated) bowel of 3 cme5 cm. This dilatation is another possible factor influencing our results; in the present experiment, bowel dilatation was rarely present. The greater surgical trauma of increased lengthening size may also be of influence in explaining the reduced growth curve demonstrated in our study. This is supported by low albumin levels, leucocytosis, and citrulline levels in BIA piglets compared with SBS and RS piglets. Results from the present study suggest that villi and crypt size are enhanced but enterocyte function seems hampered after BIA or RS. Novel therapies should therefore focus on improving the enterocyte absorptive capacity to reduce malabsorption in the initial phases after resection. Based on the findings in the present study, the short-term physiological benefits of BIA and RS can be questioned. This seems in line with recent data from centralized bowel revalidation programs, in which bowel lengthening techniques was only rarely used to obtain intestinal autonomy [28]. Provocatively, one might argue that the present data imply that bowel lengthening techniques should be reserved for those patients with distended bowel loops and recurrent bacterial overgrowth only. As enterocyte function and growth are not initially improved, performing highly complex surgery might best be reserved for such selected cases. Surgical therapeutic advances for SBS remain in development driven by these concerns, [29,30] leading to less complex and traumatic techniques. Since its introduction in 2003, the STEP procedure has shown increasing popularity as an alternative to the Bianchi procedure for SBS children [5e7,9,31]. The underlying principles to enhance uptake and avoid stasis are similar to the Bianchi procedure (autologous lengthened intestine by decreased diameter). Though, just as for the Bianchi procedure, the exact way the STEP aids in intestinal adaptation and intestinal uptake is not clearly understood. An important difference may be a smaller anastomotic wound in the STEP compared with Bianchi; though with alteration of both circular and longitudinal muscle orientation. At the time we performed the present study, published patient studies were yet limited for

the STEP. Therefore, our experimental study focuses on those techniques with the greatest body of clinical proof at that time. The gaining popularity of the STEP merits further investigation, including studies in the animal model. This is not possible in the present study. Future studies under consideration by our team would investigate STEP application, while maintaining the applied study duration to aid data comparability. Repeat procedures and combinations of Bianchi and STEP are now used, with further variations on intestinal lengthening continuing to develop [32,33]. Results gained from this study may also aid in better understanding and enhancing these new applications of intestinal lengthening’s effect and development. Warranted clinical choice in surgical technique remains hampered by uncertainties in most applicable patient and technique selection and timing, whereas complications and operative difficulties are prevalent in all SBS-therapeutic techniques [4e7,9,10,28e31,34]. This may be because of the limited patient series published to date, in combination with the wide variety of patient characteristics. To date, the limited patient evidence remains debatable. We hope this study aids in treatment choice by shedding light on the underlying physiologic mechanisms involved.

5.

Conclusions

In conclusion, implementation of the BIA procedure and the antiperistaltic RS for the treatment of SBS in the piglet model leads to increased morphologic intestinal adaptation, but not to clinical improvement within the first 6 wk after surgery. Reduced glucose uptake on electrophysiology measurements and persistent low levels of citrulline may indicate reduced small bowel enterocyte functioning during the initial phase of intestinal adaptation.

Acknowledgment Authors’ contributions: G.I.K., T.M.V.G., H.A.H., and W.G.V.G. contribute to the conceiving and designing of the study. G.I.K. and I.G.S. collected the data. G.I.K., J.B.F.H., and I.G.S. did the analyzing and interpreting of the data. G.I.K. wrote the article. G.I.K., J.B.F.H., I.G.S., T.M.V.G., H.A.H., and W.G.V.G. approved the final version of the article. J.B.F.H., I.G.S., T.M.V.G., H.A.H., and W.G.V.G. did the critical revisions.

Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.

references

[1] Vanderhoof JA, Langnas AN. Short-bowel syndrome in children and adults. Gastroenterology 1997;113:1767. [2] AGA technical review on short bowel syndrome and intestinal transplantation. Gastro 2003;124:1111.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 4 3 3 e4 4 3

[3] Lao OB, Healey PJ, Perkins JD, Horslen S, Reyes JD, Goldin AB. Outcomes in children after intestinal transplant. Pediatrics 2010;125:e550. [4] Jones BA, Hull MA, Kim HB. Autologous intestinal reconstruction surgery for intestinal failure management. Curr Opin Organ Transpl 2010;15:341. [5] Bianchi A. Longitudinal intestinal lengthening and tailoring: results in 20 children. J R Soc Med 1997;90:429. [6] Reinshagen K, Kabs C, Wirth H, et al. Long-term outcome in patients with short bowel syndrome after longitudinal intestinal lengthening and tailoring. J Pediatr Gastroenterol Nutr 2008;47:573. [7] Sudan D, Thompson J, Botha J, et al. Comparison of intestinal lengthening procedures for short bowel syndrome. Ann Surg 2007;246:593. [8] Digalakis M, Papamichail M, Glava C, et al. Interposition of a reversed jejunal segment enhances intestinal adaptation in short bowel syndrome: an experimental study in pigs. J Surg Res 2011;171:551. [9] Frongia G, Kessler M, Weih S, Nickkholgh A, Mehrabi A, Holland-Cunz S. Comparison of LILT and STEP procedures in children with short bowel syndrome da systematic review of the literature. J Pediatr Surg 2013;48:1794. [10] Beyer-Berjot L, Joly F, Maggiori L, et al. Segmental reversal of the small bowel can end permanent parenteral nutrition dependency: an experience of 38 adults with short bowel syndrome. Ann Surg 2012;256:739. [11] Sigalet DL, Lees GM, Aherne F, et al. The physiology of adaptation to small bowel resection in the pig: an integrated study of morphological and functional changes. J Pediatr Surg 1990;25:650. [12] Koffeman GI, Schoots IG, Aronson DC, et al. The effect of intestinal lengthening on adaptation in a piglet SBS-model: a pilot study. Eur Surg Res 2002;34(Suppl 1):58. [13] Chahine AA, Ricketts RR. A modification of the Bianchi intestinal lengthening procedure with a single anastomosis. J Pediatr Surg 1998;33:1292. [14] Jeejeebhoy KN, Ahmed S, Kozak G. Determination of fecal fat containing both medium and long chain triglycerides and fatty acids. Clin Biochem 1970;3:157. [15] Crenn P, Coudray-Lucas C, Thullier F, Cynober L, Messing B. Postabsorptive plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal failure in humans. Gastroenterology 2000;119:1496. [16] Teerlink T, Van Leeuwen PAM, Houdijk AP. Plasma amino acids determined by liquid chromatography within 17 minutes. Clin Chem 1994;40:245. [17] Schoots IG, Koffeman GI, Bijlsma PB, van Gulik TM. Hypoxia/ reoxygenation impairs glucose absorption and cAMPmediated secretion more profoundly than glutamine absorption and Ca 2þ/PKC-mediated secretion in rat ileum in vitro. Surgery 2006;139:244. [18] Sigalet DL, Lees GM, Aherne FX, et al. Nutritional effects of surgical and medical treatment for short bowel syndrome. J Parenter Eternal Nutr 2001;25:330.

443

[19] Buie WD, Thurston OG, van Aerde JE, Aherne FX, Thomson AB, Fedorak RN. Jejunum is preferable for construction of a Bianchi bowel-lengthening procedure in swine short bowel. J Pediatr Surg 1993;28:102. [20] Sigalet DL, BAwazir O, Martin GR, et al. Glucagon-like peptide-2 induces a specific pattern of adaptation in remnant jejunum. Dig Dis Sci 2006;51:1557. [21] Lauronen J, Pakarinen MP, Kuusanmaki P, et al. Intestinal adaptation after massive proximal small-bowel resection in the pig. Scand J Gastroenterol 1998;33:152. [22] Smith MW. Expression of digestive and absorptive function in differentiating enterocytes [review]. Annu Rev Physiol 1985;47:247. [23] Taqi E, Wallace LE, de Heuvel E, et al. The influence of nutrients, biliary-pancreatic secretions, and systemic trophic hormones on intestinal adaptation in a Roux-en-Y bypass model. J Pediatr Surg 2010;45:987. [24] Whang EE, Dunn JC, Joffe H, et al. Enterocyte functional adaptation following intestinal resection. J Surg Res 1996; 60:370. [25] Wolvekamp MCJ, Durante NMC, Meyssen MAC, et al. The value of in vivo electrophysiological measurements for monitoring functional adaptation after massive small bowel resection in the rat. Gut 1993;34:637. [26] Gutierrez IM, Fisher JG, Offir BI, et al. Citrulline levels following distal versus proximal small bowel resection. J Pediatr Surg 2014;49:741. [27] Weih S, Nickkholgh A, Kessler M, et al. Models of short bowel syndrome in pigs: a technical review. Eur Surg Res 2013; 51:66. [28] Modi BP, Langer M, Ching YA, et al. Improved survival in a multidisciplinary short bowel syndrome program. J Pediatr Surg 2008;43:20. [29] Bianchi A. From the cradle to enteral autonomy: the role of autologous gastrointestinal reconstruction. Gastroenterology 2006;130(2 Suppl 1):S138. [30] Rege AS, Sudan DL. Autologous gastrointestinal reconstruction: review of the optimal nontranplant surgical options for adults and children with short bowel syndrome. Nutr Clin Pract 2013;28:65. [31] Jones BA, Hull MA, Potanos KM, et al. Report of 111 consecutive cases enrolled in the International Serial Transverse Enteroplasty (STEP) Data Registry: a retrospective observational study. J Am Coll Surg 2013; 3:881. [32] Suri M, Dicken B, Nation PN, Wizzard P, Turner JM, Wales PW. The next step? Use of tissue fussion technology to perform the serial transverse enteroplasty-proof of principle. J Pediatr Surg 2012;47:938. [33] Cserni T, Takayasu H, Muzsnay Z, et al. New idea of intestinal lengthening and tailoring. Pediatr Surg Int 2011;27: 1009. [34] Miyasaka EA, Brown PI, Teitelbaum DH. Redilation of bowel after intestinal lengthening procedures -an indicator for poor outcome. J Pediatr Surg 2011;46:145.