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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: J Gastrointest Surg. 2019 Oct 21;24(1):188–197. doi: 10.1007/s11605-019-04400-z

Burn-Induced Impairment of Ileal Muscle Contractility is Associated with Increased Extracellular Matrix Components

Claire B Cummins 1, Yanping Gu 2, Xiaofu Wang 1, You-Min Lin 3, Xuan-Zheng Shi 3, Ravi S Radhakrishnan 1,*
PMCID: PMC8634548  NIHMSID: NIHMS1751637  PMID: 31637625

Abstract

Introduction:

Severe burns lead to marked impairment of gastrointestinal motility, such as delayed gastric emptying and small and large intestinal ileus. However, the cellular mechanism of these pathologic changes remains largely unknown.

Methods:

Male Sprague Dawley rats approximately 3 months old and weighing 300–350g were randomized to either a 60% total body surface area full-thickness scald burn or sham procedure and were sacrificed 24 hours after the procedure. Gastric emptying, gastric antrum contractility ileal smooth muscle contractility, and colonic contractility were measured. Muscularis externae was isolated from the ileal segment to prepare smooth muscle protein extracts for Western Blot analysis.

Results:

Compared to sham controls, the baseline rhythmic contractile activities of the antral, ileal, and colonic smooth muscle strips were impaired in the burned rats. Simultaneously, our data showed that ileal muscularis ECM proteins fibronectin, and laminin were significantly up-regulated in burned rats compared to sham rats. TGF-β signaling is an important stimulating factor for ECM protein expression. Our results revealed that TGF-β signaling was activated in the ileal muscle of burned rats evidenced by the activation of Smad2/3 expression and phosphorylation. In addition, the total and phosphorylated AKT, which is an important downstream factor of ECM signaling in smooth muscle cells, was also up-regulated in burned rats’ ileal muscle. Notably, these changes were not seen in the colonic or gastric tissues.

Conclusion:

Deposition of fibrosis-related proteins after severe burn are contributors to decreased small intestinal motility.

Keywords: Gastrointestinal Motility, Burns, Ileus, Myoctes, Smooth Muscle

Introduction

Severe burns result in global injury to the gastrointestinal tract, with changes noted from the esophagus to the colon. Clinically, the changes seen include gastric ulcers, feeding intolerance, abdominal distension, delayed gastric emptying and prolonged ileus[1–5]. While these effects have long been documented, no clear pathophysiology has been yet been elucidated. Hormonal changes, aggressive fluid resuscitation, and sympathetic overload have all been proposed as potential mechanisms, but inhibition of these pathways does not yet result in complete resolution of gastrointestinal deficits after burn.[6–8]

Fibrosis is a frequent sequela to the inflammation present after severe burns in a wide variety of tissues. Fibrotic changes are clearly visible in the skin[9–12], but are also present in remote tissues. Several population-based longitudinal studies have been conducted in adults who sustained severe burns as children and found significant increases in mortality related to cardiovascular disease[13, 14], and other studies have linked this to the presence of cardiac fibrosis more than 5 years after the initial injury[15]. Liver function is also closely correlated to mortality risk after burn, and hepatic fibrosis is the likely mechanism through which this occurs[16, 17].

Proliferation of smooth muscle cells is correlated with poor tissue compliance[18, 19]. Proliferation of smooth muscle cells leads to secretion of extracellular matrix (ECM) proteins[20]. In turn, ECM proteins influence the migration and proliferation of smooth muscle cells, creating a positive feedback loop[20, 21]. In inflammatory bowel disease, fibrotic remodeling with deposition of excess ECM proteins to functional consequences such as impairment of colonic motility and absorptive function[22, 23].

Given the inflammatory state after severe burn, the fibrosis seen in other remote tissues, and the global decrease in intestinal mobility, we hypothesized that the decreased intestinal transit seen after severe burn would be partially mediated by deposition of fibrosis-related proteins in smooth muscle.

Methods

Animals

All animal research procedures adhered to the National Institutes of Health guidelines for experimental animal use and were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch at Galveston, TX (Protocol#1509059). Animals were allowed to acclimate for 1 week before experimentation and received food and water ad libitum throughout the study. Animals were kept on a 12:12 light-dark cycle. Temperature was maintained at ~22°C in all animal housing and procedure rooms.

A well-established model for the induction of a 60% TBSA full-thickness burn was used[24, 25]. Male Sprague-Dawley rats (300–350 g) were given analgesia (buprenorphine, 0.05mg/kg, s.c.) and anesthetized with general anesthesia (isoflurane, 3–5%). Rats were placed within a protective mold that exposed ~30% of the TBA and submerged in 95°C to 100°C water to induce a scald burn. The dorsum was immersed to 10 seconds and the abdomen for 2 seconds, resulting in a 30% TBSA injury on both the dorsum and the abdomen (60% TBSA burn in total). Room temperature LR solution (40 mL/kg, i.p.) was administered immediately after the burn for resuscitation. Sham rats were given analgesia (buprenorphine, 0.05 mg/kg, s.c.) and then anesthetized (Isoflurane, 3–5%), placed in the burn mold, and submerged in tap temperature water. However, no post-burn resuscitation bolus was given due to concerns of fluid overload without the presence of increased insensible losses after burn injury. Rats received oxygen during the recovery from anesthesia. All animals were housed individually after the procedure and received food and water ad libitum. No significant changes in weight were seen between the pre-procedure and post-procedure states in either sham or burn rats. Rats were humanely euthanized at 24 hours post burn (hpb) and serum and tissue were collected.

Muscle activity

The muscle activity was measured according to previously reported protocols[26–28]. Sections of the colon, ileum, and gastric antrum were collected. The muscle strips were mounted in 10mL organ baths at 37°C containing carbonated Krebs solution. Muscle strips were mounted along muscle orientation. Muscle contractility was measured using Grass isometric force transducers and amplifiers with a Biopac data-acquisition system (Biopac Systems, Goleta, CA, USA). Strips were adjusted in length to an initial tension of 1g, then allowed to stabilize for 60 minutes. Contractile response to acetylcholine (ACh) was recorded in 15–20 minute intervals. Contractility was measured in terms of increase in area under curve (AUC) in the first 4 minutes of ACh addition against the AUC during 4 minutes before the addition of ACh. AUCs of each muscle strip were normalized with its cross section area which was calculated using the following formula: wet tissue weight (mg)/[tissue length (mm) × 1.05 (muscle density in mg/mm3). Strips isolated from sham and burn rats were measured at the same time.

Gastric Emptying

Gastric emptying of solid food was measured as previously described[29]. Rats were sacrificed after fasting for 12 hours with access to water. Pre-weighed regular chow pellets (1.5–2.0g) were given to the animals. All animals consumed the food within 5–10 minutes. At ninety minutes after the meal, the animals were humanely euthanized. The stomach was collected and the contents were removed, dried for 48 hours, and then weighed. The following formula was used to calculate gastric emptying: Gastric emptying (%) = (1-dried weight of food recovered from the stomach/weight of food intake) ×100.

Western Immunoblotting

Colonic, ileal, and gastric tissues were collected in fresh carbogenated Krebs buffer. The muscularis externae was separated from the mucosa/submucosa layer by microdissection. Tissues were snap frozen and stored in −80°C until use. Samples were homogenized on ice in lysis buffer supplemented with protease inhibitor cocktails (Sigma-Aldrich). After spinning at 12,000 g at 4°C for 15 minutes, the supernatant proteins were collected and resolved by a standard immunoblotting method [30–32]. Antibodies used included phosphorylated STAT3 Tyr705 (Cat#9145), STAT3 (Cat#4904), AKT (Cat#4691), phosphorylated AKT (Cat#4060), MLC2 (#8505), phosphorylated MLC2 (#3671), SMAD2/3 (#8685), and phosphorylated SMAD (#9510) from Cell Signaling (Danvers, MA). SM22 (Cat#ab10135) and calponin (Cat#ab46794) antibodies were purchased from Abcam (Cambridge, UK). Fibronectin (Cat#SC-6952), phosphorylated PKCα (Cat#SC-12356-R), and PKCα (Cat#SC-208) were purchased from Santa Cruz Biotechnology (Dallas, TX). Laminin (Cat#NB300–144SS) antibodies were purchased from Novus Biologicals (Centennial, CO). α-SMA (Cat#A5228) antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Collagen I antibodies (Cat#600-401-103) were purchased from Rockland (Pottstown, PA). All blots were repeated at least 3 times.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 7.0 from GraphPad Software Inc. (La Jolla, CA). Data are presented as mean ± standard error of the mean, with significance denoted as follows: *: P<0.05. Error bars represent the standard error of the mean.

Results

Functional decrease in gastrointestinal motility after burn

Gastric emptying rate was significantly decreased at 24 (p<0.01) hours post burn (hpb) (Figure 1a). Contractility of the gastric antrum was significantly decreased 24 hpb at ACh concentrations of 10−3(p<0.01) and 10−2(p<0.01) (Figure 1b). Similarly, antral contractility to KCl was significantly depressed 24 hpb (p<0.01). Ileal longitudinal muscle contractility was significant decreased at 24 hpb for all ACh concentrations (10−6, p<0.01; 10−5, p<0.01; 10−4, p<0.001, 10−3, p<0.001; 10−2, p<0.05) (Figure 1c). Colonic circular muscle contractility was significantly decreased at 24 hpb at ACh concentrations greater than 10−5 (10−5, p<0.05; 10−4, p<0.05, 10−3, p<0.05; 10−2, p<0.05) (Figure 1d).

Figure 1 –

Figure 1 –

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Functional decrease in gastrointestinal motility after burn. (a) decreased gastric emptying, (b) decreased antral contractility to acetylcholine (ACh) and potassium chloride (KCl), (c) decreased ileal longitudinal muscle contractility to ACh, (d) decreased colonic circular muscle contractility to ACh. All error bars are representative of standard error of the mean. * denotes p<0.05. p-values shown compared to sham group. N=6 for all groups.

Markers of innate smooth muscle contraction

Calponin, an inhibitor of the ATPase activity of myosin in smooth muscle, was significantly elevated in ileal muscle at 24 hpb (p<0.05) (Figure 2a). No significant changes were seen in colonic calponin expression. SM22, a calponin-related protein that is specifically expressed in adult smooth muscle, demonstrated no significant changes in gastric or colonic tissues (Figure 2b). Phosphorylation of myosin light chain (MLC) allows for smooth muscle contraction to begin. There were no significant changes in phosphorylated MLC, total MLC, or the ratio of phosphorylated to total MLC in ileal tissue 24 hpb (Figure 2c). Protein kinase C (PKC) is a central regulator of smooth muscle contraction. A significant decrease in phosphorylated PKC is seen in colonic tissues at 24 hpb (p<0.01) (Figure 2D). No significant change was seen in total PKC levels or the ratio of phosphorylated to total PKC in colonic tissues. The gastric antrum demonstrated no significant changes in phosphorylated PKC, total PKC, or the ratio of phosphorylated to total PKC.

Figure 2 –

Figure 2 –

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Markers of innate smooth muscle contraction. (a) calponin expression in ileal muscle (left) and colonic tissue (right), (b) SM22 expression in colonic tissue (left) and gastric antrum (right), (c) phosphorylated and total myosin light chain (MLC) levels in ileal muscle, (d) phosphorylated and total protein kinase C (PKC) levels in colonic tissue (left) and gastric antrum (right). All error bars are representative of standard error of the mean. * denotes p<0.05. p-values shown compared to sham group. N=6 for all groups. Results are representative of at least 3 independent experiments.

Inflammation in gastrointestinal tissues after burn

Signal transducer and activator of transcription 3 (STAT3) is an inflammatory mediator. Significant elevation of phosphorylated STAT3 was noted at 24hpb in ileal tissue (p<0.05) (Figure 3a). No significant changes were noted in total STAT3 levels or the ratio of phosphorylated to total STAT3. In the gastric antrum and colon, no significant changes in phosphorylated STAT3 were noted (Figure 3b). SMAD 2 and 3 are several of the main signal transducers for the receptors of transforming growth factor beta (TGFß). Phosphorylated SMAD2 was markedly elevated in ileal tissues 24 hpb and the ratio of phosphorylated to total SMAD2 was significantly elevated at 24 hpb (p<0.05) (Figure 3c). Conversely, phosphorylated SMAD 3 was significantly elevated in ileal tissue 24 hpb (p<0.05) with marked elevation of the ratio of phosphorylated to total SMAD3.

Figure 3 –

Figure 3 –

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Inflammation in gastrointestinal tissues after burn. (a) phosphorylated and total levels of signal transducer and activator of transcription 3 (STAT3) in ileal tissue, (b) phosphorylated levels of STAT3 in colonic (left) and gastric antrum (right), (c) phosphorylated and total levels of SMAD 2 (left) and SMAD 3(right) in ileal tissue. All error bars are representative of standard error of the mean. * denotes p<0.05. p-values shown compared to sham group. N=6 for all groups. Results are representative of at least 3 independent experiments.

Fibrosis-related protein deposition in gastrointestinal tissues after burn

Excess deposition of ECM proteins such as collagen I, fibronectin, and laminin are hallmarks of fibrosis development. Collagen I expression was markedly elevated in ileal muscle 24 hpb and fibronectin expression was significantly elevated (p<0.05) (Figure 4a). Laminin was also significantly elevated in ileal muscle 24 hpb (p<0.05), while colonic tissues demonstrated no significant change (Figure 4b).

Figure 4 –

Figure 4 –

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Fibrosis-related protein deposition in gastrointestinal tissues after burn. (a) Collagen 1 (left) and fibronectin (right) expression in ileal muscle, (b) laminin expression in ileal muscle (left) and colonic tissue (right), (c) phosphorylated and total levels of protein kinase b (AKT) in ileal muscle (left) and colonic tissue (right). All error bars are representative of standard error of the mean. * denotes p<0.05. p-values shown compared to sham group. N=6 for all groups. Results are representative of at least 3 independent experiments.

Protein kinase B, also known as AKT, plays a central role in the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Activation of this pathway leads to deposition of fibrosis-related proteins in smooth muscle. Phosphorylated AKT levels were significantly elevated in ileal muscle 24 (p<0.05) (Figure 4c). No significant changes in phosphorylated AKT, total AKT, or the ratio of phosphorylated to total AKT were noted in colonic tissues.

Discussion

Our study demonstrated that the global decrease in gastrointestinal motility is present as early as 24 hours after burn. Additionally, our data shows that this is not likely due to innate smooth muscle inhibition in the gastric antrum, the ileum or the colon. Our study is the first to demonstrate that there is a presence of a large inflammatory burden in the ileum, leading to subsequent deposition of fibrosis-related proteins in the ileal muscle. These changes are not present in the stomach or the colon, indicating that the pathogenesis of decreased intestinal motility after burn is distinct in these separate tissues.

Severe burn resulted in a significant decrease in gastric emptying, gastric antral contractility, ileal contractility, and colonic contractility. This is consistent with other studies which also demonstrate a global decrease in intestinal transit after burn [6]. Gastric emptying rates have been seen to be decreased by 37–42% after burn as soon as 6 hours after injury[7, 33, 34]. Similarly, small intestinal motility is seen to decrease by 24–42% at 6 hours, and colonic motility decreased by approximately 34% [35, 36, 34].

Our results demonstrated a significant increase in calponin in ileal muscle and a significant decrease in phosphorylated PKC in the colon. However, all other factors measured, including MLC and SM22 were not significantly changed by burn. These factors are heavily inter-related and we suspect that innate inhibition of the smooth muscle activation pathways does not play a major role in the pathogenesis of decreased intestinal transit after burn. Calponin and SM22 belong to a group of differentiation determinants in smooth muscle. Calponin and SM22 appear synchronously during the differentiation of all smooth muscle cell populations[37]. Calponin activation also is thought to take place via a PKC-dependent pathway[38]. Phosphorylation of PKC and MLC play a central role in the activation or inhibition of smooth muscle contraction[39–41]. It is therefore likely that true inhibition of these pathways would produce a much more robust response than the one we observed.

Competing theories for the explanation of delayed intestinal transit after burn include sympathetic overload and intestinal edema [6–8]. However, our results are not completely congruent with these previous hypotheses. Aggressive resuscitation following burn injury causes intestinal edema which decreases intestinal contractile activity via decreased MLC phosphorylation[42]. Phosphorylation and activation of PKC has been implicated in sympathetic stimulation of smooth muscle contraction [43]. Without definitive changes in either the levels of phosphorylated PKC and MLC, it seems less likely that sympathetic stimulation or intestinal edema would be solely responsible for the clinical changes in intestinal motor function after burn.

Massive inflammation is classically present after severe burn. Our results demonstrated a significant increase in phosphorylated STAT3 and activation of SMAD 2/3 in ileal tissues. However, no significant changes in STAT3 were noted in colonic or gastric tissues. STAT3 plays a dual role in intestinal inflammation. STAT3 can be protective and anti-inflammatory in the intestinal mucosa through its actions in epithelial and myeloid cells, whereas it promotes inflammation when it acts in T cells [44]. Similar to STAT3, SMAD 2/3 and TGFß can provide a protective role by promoting the restoration of intestinal morphology and barrier function following stress [45, 46]. Dendritic cells mediate intestinal immune tolerance. A lower proportion of colonic dendritic cells produce pro-inflammatory cytokines when compared with their ileal counterparts, likely an adaptation to the greater bacterial load in the colon [47]. The role of the ileum in innate immunity [48] also likely contributes to the increased susceptibility of the ileum to inflammation following burn.

Inflammation is the frequent precursor of fibrosis [49]. Inflammation in the intestine leads to activation of myofibroblasts and increased production of ECM proteins [50]. Our results demonstrated a significant increase in fibronectin and laminin expression in ileal muscle 24 hours after burn injury. Ileal muscle also demonstrated a significant increase in AKT phosphorylation, a key component of the PI3K-AKT pathway [51]. The PI3K-AKT pathway has been implicated in the development of fibrosis in smooth muscle. While histologic specimens were not taken in this study, deposition of fibrosis-related proteins can occur very rapidly following burn injury, as seen in cardiac tissues [52]. While previous studies have focused on the role that burn-induced inflammation plays on the intestinal mucosa [6], our study is the first to demonstrate that these changes are adversely affecting the ileal muscle as well, and are likely contributing factors to the decreased intestinal transit seen after burns.

Conclusion

Fibrotic changes mediated through the inflammatory state after severe burn are contributors to decreased small intestinal motility. However, these changes are not mirrored in gastric or colonic tissues, despite the global nature of adynamic ileus after burn. This indicates that the changes seen in the small intestine may be representative of a unique underlying pathophysiology.

Acknowledgements:

This paper was presented as a plenary presentation through the Surgical Society for the Alimentary Tract at Digestive Disease Week on May 21, 2019

Footnotes

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

The authors declare that they have no conflicts of interest.

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