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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References

Background

Gastrointestinal (GI) symptoms are common in soldiers in combat or high-pressure operational situations and often lead to compromised performance. Underlying mechanisms are unclear, but neuroendocrine dysregulation, immune activation and increased intestinal permeability may be involved in stress-related GI dysfunction.

Aim

To study the effects of prolonged, intense, mixed psychological and physical stress on intestinal permeability, systemic inflammatory and stress markers in soldiers during high-intensity combat-training.

Methods

In 37 male army medical rapid response troops, GI symptoms, stress markers, segmental intestinal permeability using the 4-sugar test (sucrose, lactulose, mannitol and sucralose) and immune activation were assessed during the 4th week of an intense combat-training and a rest period.

Results

Combat-training elicited higher stress, anxiety and depression scores (all < 0.01) as well as greater incidence and severity of GI symptoms [irritable bowel syndrome symptom severity score (IBS-SSS), P < 0.05] compared with rest. The IBS-SSS correlated with depression (r = 0.41, < 0.01) and stress (r = 0.40, < 0.01) ratings. Serum levels of cortisol, interleukin-6, and tumour necrosis factor-α, and segmental GI permeability increased during combat-training compared with rest (all P < 0.05). The lactulose:mannitol ratio was higher in soldiers with GI symptoms (IBS-SSS ≥75) during combat-training than those without (IBS-SSS <75) (< 0.05).

Conclusions

Prolonged combat-training not only induces the expected increases in stress, anxiety and depression, but also GI symptoms, pro-inflammatory immune activation and increased intestinal permeability. Identification of subgroups of individuals at high-risk of GI compromise and of long-term deleterious effects of operational stress as well as the development of protective measures will be the focus of future studies.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References

The most frequently reported symptoms unrelated to trauma in soldiers in high-pressure operational situations are gastrointestinal (GI), including abdominal pain, diarrhoea, constipation and nausea besides a range of cognitive changes.[1-4] Many cases of diarrhoea in the battlefield may be infectious, but often an infectious cause is not documented and stress-related functional GI disorders (FGID), either postinfectious or not, are a legitimate differential diagnosis.[1, 5] Recent data emerging from the Gulf, Iraq and Afghanistan conflicts have highlighted the considerable significance of GI symptoms on military efficiency and manpower loss in front lines.[3, 4, 6] Although considerable evidence exists on the late consequences of combat stress and anxiety in veterans, much less is known about the effects of ongoing and protracted stress in the battlefield. In other acute stress settings, a rapid onset of GI dysfunction has been observed, as well as an increased susceptibility to inflammatory and infectious intestinal disease.[7]

For ethical reasons, mechanistic studies of stress in humans are largely restricted to experimental and acute paradigms, with obvious limitations on the clinical applicability of the results given the well-known differences between acute and chronic stress.[7, 8] Both physical and psychological stress increase intestinal permeability (IP) and induce enteric neuronal dysfunction in animal models, as well as in experimental human settings, such as athletic exercise with or without high ambient temperatures.[7, 9] The limbic-hypothalamic-pituitary-adrenal (HPA) and noradrenergic systems are the core endocrine stress mechanisms in humans and provide bidirectional neuroendocrine links between the brain and gut. Activation of the HPA axis during acute stress results in increased secretion of corticotrophin-releasing hormone (CRH), which can modulate gut neuroimmune responses directly as well as via mucosal immune cells and inflammatory mediators, such as tumour necrosis factor (TNF)-α and interleukin-1 and -6 (IL-1, IL-6). This can lead to disruption of intestinal barrier integrity and further release of inflammatory mediators.[9-12]

Potential clinical consequences of increased IP include altered immune responses, increased susceptibility to enteric microbial infections, elevated pain sensitivity, GI motility changes, altered cognition and emotion via gut-brain signalling, which are seen in the functional and inflammatory bowel disorders known to be susceptible to stress exacerbation.[7, 13-16] Visceral hyperalgesia and sensitization have been described in a majority of patients with irritable bowel syndrome (IBS) and have also recently been shown in veterans of the Persian Gulf War with IBS-like GI symptoms.[16, 17] Although the underlying cause for non-infectious GI symptoms in soldiers remains unclear, considering the changes in immune activation and intestinal permeability (IP) evident in stress-related gastrointestinal disorders in animal models and clinical settings,[7-9, 12, 13] increased IP secondary to stress and immune activation may well be implicated. We hypothesized that combat-training in soldiers would lead to increased stress and consequently elevated pro-inflammatory mediators and greater intestinal permeability and GI symptoms compared with the resting period. The aim of this study was to investigate the effects of the prolonged, mixed physical and psychological stress of combat-training in soldiers on GI symptoms, segmental intestinal permeability and inflammatory mediators.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References

Subjects

Thirty-nine Asian male soldiers undergoing a 6-week combat-training were recruited from the Medical Response Force (MRF) of the Singapore Armed Forces (SAF).

Baseline tests

All soldiers were screened in the first days of their combat-training. For study inclusion, soldiers were required to be physically and psychologically healthy and without a significant past or current medical history including GI disease and chronic GI symptoms before the beginning of combat-training. Assessment was by the detailed and standardised questionnaires listed below and haematology and biochemistry tests. Ongoing medication of any kind or previous abdominal surgery were exclusion criteria. Participation in this study was voluntary, written informed consent was obtained and parental consent was also obtained for those younger than 21 years. The study protocol was approved by the Ethics Committees of the Singapore National Healthcare Group and the DSO National Laboratories Institutional Review boards. This study was registered at ClinicalTrails.gov under #NCT01417416.

Combat-training course and study design

The 6-week MRF combat-training course is a period of high-intensity stress and serves as a transition from the relatively stress-free classroom-based basic medical course to an environment of high mixed physical and psychological demand. The combat-training course involves combat-simulation and immediate action medical evacuation exercises in protective gear for chemical, biological, radiological and nuclear emergencies at average ambient temperatures around 30 °C and a relative humidity above 80%. Blood sampling for stress and immune mediators, and urine sampling for IP were performed after 4 continuous weeks of combat-training and in the rest period 12 days after completion of the combat-training as a within-subject control. All subjects refrained from alcohol, aspirin and NSAIDs for at least 5 days prior to tests and from smoking in the morning before testing, and fasted from 22:00 h to 06:00 h before the tests.

Questionnaires: anxiety, depression, stress and bowel function

The Perceived Stress Scale-10 item (PSS-10) questionnaire was used for quantification of stress,[18] the Hospital Anxiety and Depression (HAD) scale for anxiety and depression[19] and the IBS-Symptoms Severity Score (IBS-SSS) for evaluation of bowel function and symptoms.[20] Ratings were performed independently by all subjects before blood sampling during the fourth week of combat training and in the subsequent rest period.

Stress markers and inflammatory mediators

Fasting blood samples for CRH and cortisol concentrations were collected between 06:00 and 06:15 h. On the day of sampling, the soldiers rose at 05:30 h as usual and avoided any exercise before blood was drawn from a forearm vein by experienced phlebotomists. The blood was centrifuged at 4 °C at 1200 g for serum and 1600 g for plasma, aliquots were immediately frozen and stored at −80 °C. Serum cortisol was quantified by Elecsys Cortisol Assay (Roche Diagnostics, Indianapolis, IN, USA). Plasma CRH was measured by radioimmunoassay using a methanol extraction method (Endolab; Canterbury Health Laboratories, Christchurch, New Zealand).[21] IL-6, TNF-α, and IL-10 were measured using commercial enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN, USA).

Intestinal permeability

Segmental intestinal permeability was assessed by quantifying the urinary excretion of the orally ingested sugar probes including sucrose, lactulose, mannitol, and sucralose. Sucrose is used to determine the gastroduodenal permeability, lactulose and mannitol are used for estimating small intestinal permeability, and sucralose is an indicator of whole gut permeability mainly reflecting colonic permeability. The practical procedure is well established.[22] Briefly, after the blood sampling, subjects voided their bladders and then ingested a premixed solution of sucrose 40 g, lactulose 7.5 g, mannitol 2 g and sucralose 1 g in 500 mL of tap water. Urine samples were collected as described previously in two portions, 5 h (0–5 h) and 24 h (5–24 h) after ingestion, based on recommendations current at the time of study design.[23-25] Urine from both periods was stored in separate containers kept at 4 °C with sodium fluoride as a preservative. Upon completion, the urine samples were stored at −80 °C until shipping on dry ice to the Division of Digestive Disease of Rush University Medical Center, Chicago, USA. Concentrations of sucrose, lactulose, mannitol and sucralose were quantified by capillary column gas chromatography.[22] The fractional excretion of each sugar was estimated as the ratio of its total urinary excretion to the total oral dose. The ratio of lactulose:mannitol (L/M) was calculated as the fractional excretion of lactulose divided by that of mannitol.

Statistics

spss Statistics 17.0 software (SPSS Inc, Chicago, IL, USA) was used for statistical analysis. Data are presented as median and inter-quartile ranges (IQR). Comparisons between the combat-training and rest periods were performed using the paired sample t-test if sample data were normally distributed or the Wilcoxon test (paired-samples) if sample data were not normally distributed. The differences between soldiers with IBS-SSS ≥75 and IBS-SSS <75 were assessed using the non-parametric two-independent sample test. The differences in frequency of clinical GI symptoms between the two periods were analysed using the McNemar test. Simple correlations between parameters were evaluated by Pearson's rank correlation coefficient or the Spearman's rank correlation coefficient if sample data were not normally distributed. Multiple linear regression analysis was used to investigate correlations between GI symptoms and covariants, including stress, immune markers, and increased IP. Predictors for the change in IP during combat-training were also analysed using multiple linear regression. The choice of variables in the multiple regression analysis was based on theoretical considerations and the findings of previous studies.[16, 26] To assess the effect of the relevant factors on GI symptom severity in soldiers, the stress and IP markers were chosen as independent variables. Co-linearity diagnostic statistics were used to rule out the existence of multiple co-linearity in linear regression models. A tolerance value >0.4 is considered as noncolinearity. A two-tailed < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References

Thirty-nine soldiers of mean age 20.7 years (range: 19–23) from the same MRF troop were included. Two soldiers were excluded because of fever on sampling days. No soldier had clinical evidence of infectious gastroenteritis during the study and there were no further study dropouts. Three soldiers ingested sugar-containing food on the day of urine collection and hence their permeability data were excluded from analysis.

Stress, anxiety and depression levels

The soldiers showed higher anxiety, depression and stress scores during the combat-training period than at rest (Figure 1a). The morning serum cortisol was significantly increased in soldiers during combat-training compared with rest (Figure 1b). Plasma CRH concentrations were similar during combat-training compared with the resting period [2.25 (1.68–3.10) vs. 2.10 (1.65–3.30) pmol/L, P = 0.48].

image

Figure 1. Stress markers in soldiers (n = 37) during combat-training and rest periods. (a) Anxiety, depression and stress. (b) Serum cortisol. Data are shown as medians and IQR.

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Gastrointestinal symptoms

None of the participating soldiers had significant GI symptoms including chronic or recurrent abdominal pain or discomfort, abnormal bowel function (diarrhoea, constipation) before combat-training. Twenty-six (70%) soldiers reported significant GI symptoms after 4 weeks of combat-training, including abdominal pain and/or discomfort, abnormal bowel habits (constipation, alternating constipation and diarrhoea), as well as abdominal pain and/or discomfort together with abnormal bowel habits (Figure 2a). During the subsequent resting period, 11 (30%) soldiers reported the above GI symptoms (Figure 2a). The number of soldiers with GI symptoms was higher during combat-training than at rest (< 0.05).

image

Figure 2. GI symptoms in soldiers as well as anxiety, depression, and stress intensity in soldiers with different GI symptom severity (n = 37). (a) The frequency of GI symptoms in soldiers during combat-training and rest periods. No soldiers had GI symptoms before the start of training (exclusion criterion). (b) HAD anxiety, depression and PSS-10 stress scores in soldiers with IBS-SSS <75 (n = 23) and IBS-SSS ≥75 (n = 14) during combat-training. Data are shown as medians and IQR.

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None of the soldiers had an IBS-SSS ≥75 before the combat-training. During combat-training, 14 (37%) soldiers had an IBS-SSS >75, 12 (32%) had an IBS-SSS between 75 and 174 (mild IBS), and 2 (5%) had an IBS-SSS of 175–300 (moderate IBS). In the resting period, 6 (16%) soldiers had an IBS-SSS >75, and none had an IBS-SSS ≥175. Correspondingly, the average IBS-SSS was significantly greater, at the upper limit of normal, in soldiers during stress than at rest [62.5 (40.0–121.3) vs. 45.0 (30.0–60.0), < 0.05]. The subgroup of soldiers with greater GI symptoms during combat-training (i.e. IBS-SSS ≥75) also had higher anxiety, depression and stress scores than those with IBS-SSS <75 (Figure 2b).

Intestinal permeability changes

5-h urinary sucrose excretion was significantly increased in soldiers during combat-training compared with rest (Table 1a). The urinary mannitol excretion was not significantly different between combat-training and rest periods during both the 5-h and 24-h urine collections; the 5-h and 24-h urinary lactulose excretion was also similar between combat-training and rest periods (Table 1a). Consequently, there were no differences in the L/M ratios between the combat-training and rest periods in both the 5-h and 24-h urines (Table 1a). In the subgroup of soldiers with an IBS-SSS ≥75 during combat-training compared with those with a score <75, mannitol was lower and lactulose was greater (both not significant). Thus, the L/M ratios in soldiers with IBS-SSS ≥75 were greater than in those with IBS-SSS <75 during both the 5-h and 24-h periods (Table 1b). At rest, there were no differences in L/M ratios between the subgroups of soldiers with an IBS-SSS ≥75 (n = 6) and <75 (n = 28) during both the 5-h and 24-h periods (Table 1b). The 5-h and 24-h urinary sucralose excretion was significantly higher during combat-training than at rest (Table 1a).

Table 1. Intestinal permeability in all the soldiers (n = 34) and grouped according to symptom severity during combat-training and rest periods
(a) Urinary sucrose, mannitol and lactulose, the ratio of lactulose:mannitol (L/M), and sucralose excretion in soldiers during the rest and combat-training periods
VariableCombat-training median (IQR)Resting median (IQR)P-value
Urinary sucrose (5-h)0.52% (0.40–1.32%)0.31% (0.25–0.53%)<0.01
Urinary mannitol (5-h)15.55% (12.96–18.59%)14.44% (11.11–18.58%)0.53
Urinary mannitol (24-h)25.32% (22.64–27.82%)20.58% (17.66–28.83%)0.14
Urinary lactulose (5-h)0.94% (0.75–1.03%)0.85% (0.67–1.08%)0.38
Urinary lactulose (24-h)2.62% (2.28–3.13%)2.49% (1.95–3.10%)0.63
L/M ratio (5-h)0.06 (0.05–0.07)0.06 (0.05–0.07)0.98
L/M ratio (24-h)0.107 (0.093–0.118)0.12 (0.09–0.15)0.19
Urinary sucralose (5-h)0.80% (0.61–1.00%)0.27% (0.21–0.32%)<0.01
Urinary sucralose (24-h)2.00% (1.46–2.44%)0.58% (0.43–0.89%)<0.001
(b) Urinary excretion of mannitol and lactulose and the ratio of lactulose:mannitol (L/M) in soldiers with IBS-SSS≥ or <75 during combat-training
VariableCombat-training [median (IQR)]Resting [median (IQR)]
IBS-SSS ≥75 (n = 14)IBS-SSS <75 (n = 20)P-valueIBS-SSS≥75 (n = 6)IBS-SSS<75 (n = 28)P-value
Urinary mannitol (5-h)14.05% (12.65–15.70%)16.95 (15.10–19.68%)0.111.02% (5.02–17.94%)15.68% (12.32–18.50%)0.61
Urinary mannitol (24-h)29.96% (24.77–29.35%)25.04% (22.64–27.70%)0.0717.63% (14.14–20.18%)20.66% (17.84–28.77%)0.45
Urinary lactulose (5-h)0.97% (0.76–1.05%)0.94% (0.84–1.02%)0.390.86% (0.66–1.16%)0.83% (0.68–1.06%)0.89
Urinary lactulose (24-h)2.95% (2.41–3.27%)2.61% (2.33–2.84%)0.252.39% (1.68–3.38%)2.36% (1.97–2.97%)0.20
L/M ratio (5-h)0.06 (0.06–0.09)0.06 (0.05–0.06)0.090.06 (0.05–0.08)0.07 (0.06–0.07)0.42
L/M ratio (24-h)0.13 (0.11–0.14)0.10 (0.08–0.11)<0.050.11 (0.09–0.15)0.12 (0.12–0.13)0.27

Inflammatory markers

Pro-inflammatory cytokine serum levels of IL-6 and TNF-α were significantly higher during combat-training than at rest (Figure 3). Serum IL-10 concentrations were similar in soldiers between the two periods [4.50 (1.33–6.02) vs. 2.54 (1.11–5.49) pg/mL, P = 0.56].

image

Figure 3. Serum inflammatory cytokines in soldiers during the combat-training and rest periods (n = 37). (a) IL-6 concentrations (pg/mL). (b) TNF-α (pg/mL) concentrations. Data are shown as medians and IQR.

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Correlations between GI symptoms, intestinal permeability and stress levels

In a simple regression model, IBS-SSS showed significant correlations with PSS-10 stress (r = 0.41, < 0.05) and HAD depression (r = 0.41, < 0.05) ratings, as well as a weaker but still significant correlation with serum cortisol (r = 0.39, < 0.05) during combat-training, but not in the rest period. In a multiple regression model with IBS-SSS as the dependent variable and stress ratings, IL-6, 5-h sucrose, and 24-h sucralose excretion as independent co-variants, only stress ratings were significantly and independently correlated with IBS-SSS (Table 2a). Due to the co-linearity between the stress and depression ratings and cortisol, the correlation between IBS-SSS and depression or cortisol was analysed separately in a similar multiple regression model with IL-6, 5-h sucrose or 24-h sucralose as independent co-variants. Both depression ratings and serum cortisol levels were significantly and independently correlated with IBS-SSS.

Table 2. Multiple regression assay (MRA) for the co-variants of IBS-SSS and 5-h urinary sucrose respectively. (a) MRA using IBS-SSS score as the dependent variable in soldiers during combat-training (n = 34); stress ratings, serum IL-6, 5-h urinary sucrose, and 24-h urinary sucralose excretion are independent variables. (b) MRA using changes in 5-h urinary sucrose excretion in soldiers between combat-training and rest periods as the dependent variable (n = 34) and serum cortisol, IL-6, TNF-α, and plasma CRH levels as independent variables
VariablesSC (β)S.E.P-value
(a)
Stress0.4351.158<0.05
IL-60.1842.440.30
Sucrose (5-h)0.0418.7050.81
Sucralose (24-h)0.11717.5840.50
(b)
Cortisol−0.5230.002<0.01
TNF-α0.3020.060.07
CRH0.2650.0850.09
IL-6−0.1810.10.26

The increase in 5-h urinary sucrose excretion during combat-training showed a negative correlation with the baseline serum cortisol level at rest. In the multiple linear regression analysis using the increase in urinary 5-h sucrose excretion during the combat-training compared with the resting period as the dependent variable and CRH, cortisol and IL-6 during rest as independent variables, resting cortisol also showed a significant negative correlation, TNF-α and CRH showed marginal positive correlations (Table 2b).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References

Gastrointestinal symptoms in soldiers in modern operational and combat settings are among the most common causes of front-line clinic visits, are often incapacitating and have a major impact on individual performance as well as on the overall military mission.[4, 6] However, underlying mechanisms apart from infectious diarrhoea have not been extensively investigated. Stress responses relating to the GI-tract have been mainly characterized using animal models or in acute human experimental or athletic conditions, but few human studies on on-going, protracted intense stress have been undertaken due to ethical considerations.[7, 27] Stress is widely accepted as a trigger and modifier of a variety of GI diseases such as IBS, inflammatory bowel disease (IBD), peptic ulcers and endurance sport-related injury, which all feature increased gut permeability as a possible underlying pathophysiological mechanism.[15, 16, 28] It is well known that the nervous, endocrine and immune systems are intricately intertwined in stress responses and some parallels can be drawn to inflammatory disorders, with related chronic sequelae.[11, 29] Increased intestinal permeability due to stress-driven neuroimmune activation could explain several aspects of combat stress-related GI dysfunction, including an increased susceptibility to GI infections.[30]

To investigate the role of ongoing chronic, mixed psychological and physical stress, we performed a prospective, longitudinal study in healthy soldiers during protracted combat-training and a rest period, documenting basic cognitive changes, GI symptoms, as well as immune activation and elevated intestinal barrier permeability. In the current study, considerably increased gastrointestinal symptoms were reported by the soldiers during combat-training compared with the rest period. IBS-like symptoms, such as abdominal pain and discomfort, diarrhoea and/or constipation developed de novo in 70% of previously asymptomatic soldiers during combat-training and persisted in 30% into the posttraining rest period 12 days later. Overall, IBS-like symptoms relating to combat-training stress scored by the IBS-SSS were on the threshold to ‘mild’. Those soldiers with more severe GI symptoms had greater anxiety, depression, and stress levels, and intestinal permeability was also increased. Although no specific precedent data exist, a high incidence of GI symptoms is consistent with our expectations and is likely to apply to any high-pressure operational setting and in any country. Reports from front-line combat settings indicate that the intensity of combat stress-related symptoms is often much higher, frequently necessitating hospitalization or medical evacuation.[1, 6]

Experimental studies have shown a multidirectional link between stress, immune activation, increased IP, visceral hypersensitivity and GI symptoms.[9, 11, 31] In this study, increased permeability was shown in all segments of the GI tract using commonly used, non-invasive sugar markers.[22, 32] Combat-training increased sucrose excretion approximately 160% above resting levels, indicating substantially elevated proximal GI permeability. Sucrose excretion mainly reflects absorption in the gastroduodenal region as it is rapidly hydrolysed by sucrose-isomaltase upon entering the duodenum,[33] a minor amount may represent more distal absorption of sucrose having escaped duodenal hydrolysis. A previous study has confirmed that over 95% of sucrose is excreted within 6 h post-ingestion, indicating that the 5-h urine collection was sufficient to reflect changes in excretion quite completely.[32] Sucrose permeability testing has not been reported during stress or in IBS, but is increased in dyspeptic patients with erosive or ulcerative upper GI disease.[34, 35] In a previous study of paediatric patients with IBS or recurrent abdominal pain, the ratio of sucrose/lactulose excretion was increased compared with controls.[26] It should be noted that elevated sucrose excretion is indicative of any form of disruption of the gastroduodenal mucosal barrier due to increased permeability or of decreased sucrase activity, which may or may not be accompanied by structural injury.

The ratio of lactulose:mannitol is routinely used to estimate small intestinal permeability, as both sugars are largely degraded by colonic bacteria. A greater ratio of lactulose:mannitol has repeatedly been shown in IBS and multiple mechanisms may be causally implicated, including neuroimmune activation and CRH-dependent pathways.[11, 16, 24] However, a clear delineation of small and large intestinal permeability changes using these markers and the commonly used urine collection periods has recently been questioned, as on the one hand, labelling studies have shown almost 50% of the labelled liquid sugars reach the colon by 2 h postingestion, and on the other hand, cumulative 90% excretion of both sugars is attained only after circa 18 h.[11, 32, 36] This suggests that the urine collected after 5 h may also represent some colonic absorption, even if most of the sugars are removed by fermentation in the colon. Both large and small intestinal permeability are increased in animal studies of stress and increased colonic permeability has been demonstrated by methods other than sugar excretion in IBS.[9, 37-39] The elevated lactulose:mannitol ratio only in the soldiers with greatest GI symptoms during combat-training indicates a probable association between increased more distal intestinal permeability and clinically perceived changes in GI function. 24-h urinary sucralose is used as a marker of whole-gut permeability including both small bowel and colonic permeability, because over 90% of sucralose reaches the colon within 21 h after ingestion and sucralose is resistant to colonic fermentation.[32, 40] Despite the relatively shorter presence in the proximal intestine than in the colon, we found significant absorption of sucralose in the 0–5 h of urine collection, suggesting relevant small intestinal absorption of sucralose. This finding is compatible with a previous study that showed that sucralose underwent substantial absorption in ileostomy patients.[41] We therefore assume that the increase in whole-gut permeability of approximately 190% during combat-training is due to greater small and large intestinal permeability. Increased colonic permeability has previously been reported in animal stress models and in stress-related GI diseases in humans.[9, 39] The increase in gut permeability during combat-training was similar in magnitude to that reported in IBS (~150%), as assessed by 51Cr-EDTA, compared with healthy controls.[42]

The main mediators of the effects of stress and immune activation on intestinal permeability widely overlap and are increasingly also shown to modulate the intestinal microbiota.[11, 14, 29, 30, 43] This study confirmed the co-incidence of increased stress-related cognitive and systemic factors and elevated pro-inflammatory cytokines, and demonstrated the novel link to abnormal intestinal permeability in ongoing mixed physical and psychological stress in humans. The well-studied stress-activated neuroimmune cascade involves central and peripheral CRH, extrinsic autonomic nerves, the enteric nervous system and pro-inflammatory mediators released from immune cells.[11, 27, 43, 44] Mast cells and other immune cells release the mediators, which include cytokines, biogenic amines, nerve growth factor and proteases. Several of the mediators, especially IL-6 and TFN-α, regulate epithelial permeability by affecting tight junction proteins, intestinal motility, bacterial translocation, sensory sensitization and central nervous system modulation.[45-47] Many of these pathophysiological changes have been shown in patients with IBS.[48] In our soldiers during combat-training, an increased IBS-SSS correlated with greater cortisol, stress, anxiety and depression. As expected, the rise in background, morning cortisol was considerably lower than that shown during acutely stressful interventions, such as sigmoidoscopy in IBS patients.[49] Baseline factors predictive of adverse reactions to stress were assessed within the limitations of the protocol. In previous studies during intense military training increased pretraining serum and salivary cortisol predicted a subsequent impairment in cognitive function during military training.[50, 51] It appears likely that the responsiveness of an individual's stress–neuroimmune axis at rest and during stress influences downstream GI permeability changes and related GI symptoms and vice versa. Possible reasons for the absence of parallel responses in cortisol and CRH could be the resetting of the gain of the feedback relationship under chronic stress, as is known in other chronically stressful conditions such as depression, or the obscuring of varying individual feedback relationships in the group data. Due to the limitations of this study regarding the timing and frequency of sampling, further conclusive studies are required.

Limitations of this study are largely due to the necessary priority of minimizing any disruption of the combat-training schedule of the soldiers. This determined the timing and frequency of blood and urine sampling, the absence of mucosal tissue and faecal samples for quantification of mediators and also the inability to perform the baseline measurements before the start of combat-training. Clearly, multiple saliva samplings would have provided a more comprehensive and accurate reflection of systemic cortisol. More frequent urine collection periods are recommended in future studies, to allow better estimation of permeability of different regions of intestine. Only male soldiers were part of the troops studied and hence no data on females are presented. Lastly, documentation of a wider range of GI symptoms than IBS-SSS would be useful for better discrimination of selective GI effects and a more prolonged observation period posttraining for full resolution of symptoms would be recommended based on the results of this study.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References

In this prospective, longitudinal study in soldiers, protracted combat-training stress induced GI symptoms associated with increased upper and lower intestinal barrier permeability and elevated systemic pro-inflammatory cytokine concentrations. This human study provides a confirmation of data from earlier animal models and validates the hypothesis that protracted and intense physical and psychological stress in humans modulates GI permeability and elicits clinical GI symptoms. An important underlying mechanism for the frequent GI symptoms seen in soldiers under operational stress probably involves increased GI permeability due to neuroimmune activation, but remains to be confirmed in larger studies. Identification of a subgroup of individuals with greater propensity for GI adverse events may allow selection of those at-risk of long-term deleterious effects of chronic stress, such as functional GI disorders, as well as the development of protective measures. These results are likely to apply to other highly stressful operational situations.

Authorship

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References

Guarantor of the article: Clive H. Wilder-Smith.

Author contributions: Xinhua Li: acquisition of data; analysis and interpretation of data; drafting of the manuscript; statistical analysis; administrative, technical, or material support. Enci Mary Kan: acquisition of data; administrative, technical, or material support. Jia Lu: study design; obtained funding, study supervision. Yang Cao: acquisition of data; technical, or material support. Reuben K Wong: study supervision; critical revision of the manuscript for important intellectual content. Ali Keshavarzian: technical support; study design; critical revision of the manuscript for important intellectual content. Clive H. Wilder-Smith: study concept and design; obtained funding; study supervision; critical revision of the manuscript for important intellectual content; analysis and interpretation of data. All authors approved the final version of the manuscript.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References

Declaration of personal interests: None.

Declaration of funding interests: This work was supported by a study grant from the Defence Research and Technology Office, Ministry of Defence, Singapore. The authors express their gratitude to LTC (DR) Michael Ong, CPT (DR) Ryan Choo and CPT (DR) Muhd Taufiq of the Singapore Armed Forces for their invaluable time and effort in the co-ordination of the combat-training protocol for this study.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Authorship
  9. Acknowledgements
  10. References