To observe the intensity of the inflammatory reaction caused by neonatal urine and meconium on the intestinal wall of rats in order to understand etiology of intestinal damage in gastroschisis.

OBJECTIVES: The aim of this experimental study was to observe the intensity of the inflammatory reaction caused by neonatal urine and meconium on the intestinal wall of rats to better understand etiology of intestinal damage in gastroschisis.
MATERIALS AND METHODS: A total of 24 adult Wistar rats were used as experimental models to simulate the effect of exposed bowel in cases of gastroschisis. The peritoneal cavity of the rats was injected with substances which constitute human amniotic fluid to study the effect on the bowel. Sterile urine and meconium were obtained from newborn humans. The rats were divided into four groups according to the material to be injected. In Group I (Control group) 3 mL of distilled water was injected, in Group II (Urine group) 3 mL of neonatal urine was injected, in Group III (Meconium group) 5% meconium suspension was injected, while in Group IV, a combination of 5% meconium suspension and urine was injected. A total of 3mL solution was injected into the right inferior quadrant twice a day for 5 days. The animals were sacrificed on the 6(th) day by a high dose of thiopentone sodium. A segment of small bowel specimen was excised, fixed in paraffin, and stained with hematoxylin-eosin for microscopic analysis for determination of the degree of inflammatory reaction in the intestinal wall. All pathology specimens were studied by the same pathologist.
RESULTS: The maximum bowel damage was seen in Group II (Urine group) in the form of serositis, severe enteritis, parietal necrosis, and peeling. A lesser degree of damage was observed in Group III (Meconium group) as mild enteritis (mild lymphoid hyperplasia). The least damage was seen in Group IV (Combination of meconium and urine) and Group I (Control group).
CONCLUSION: The intraabdominal injection of neonatal human urine produces significant inflammatory reactions in the intestinal wall of rats.

INTRODUCTION

Gastroschisis is a defect of the anterior abdominal wall with protrusion of bowel loops outside the peritoneal cavity. These bowel loops are in direct contact with the amniotic fluid and exhibit varying degrees of inflammatory reaction. The bowel is, therefore, thickened, edematous, foreshortened, and occasionally atretic. This is responsible for the morbidity associated with gastroschisis. Multiple experimental studies have attempted to determine the causative factor for these bowel abnormalities.

Sherman et al.,[1] have quantified the inflammatory reaction of bowel to substances in amniotic fluid. Indian studies corroborating his findings have not been reported. Urine which is the major component of the amniotic fluid compartment has been blamed for damage to the intestinal wall by many authors.[234] These observations are based on the physiological principle that all of the fetus's urine is excreted into the amniotic cavity. Meconium has been proposed by many as the errant factor. Increasing evidence of physiologic in utero defecation supports this hypothesis.[5] The intensity of the inflammatory reaction is a defining factor in their prognosis from which springs the importance of establishing its etiology and identifying factors which may be associated with worse prognosis.

We performed an experimental study on adult Wistar rats based on the above hypothesis. We observed the intensity of the inflammatory reaction caused by neonatal urine and meconium on rat intestinal wall to identify the possible causative factor for bowel damage.

MATERIALS AND METHODS

Permission of the ethics committees

Permission of the Institutional Animal Ethics Committee (AEC/01/13) and Human Ethics Committee (EC/OA-60/2013) of Seth G. S. Medical College and K.E.M Hospital, Mumbai was taken before commencement of the study. Animals were bred in the Center for Animal Studies of the Institute for the study. The study was conducted in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines.[6]

Experimental animals

Adult male albino Wistar rats (n = 24) weighing between 250 and 350 g were used.

Husbandry conditions

The facility was air-conditioned with 12-15 filtered fresh air changes per day, with ambient temperature of 22 ± 3°C with relative humidity of 30%-70%.

Accommodation

A total of 4animals were housed in one cage with stainless steel top grill, having facilities for food, and drinking water in polypropylene bottles with stainless steel sipper tube. The cages were cleaned at regular intervals.

Diet and water

Rodent feed in the form of pellets was provided ad libitum. Filtered pure drinking water was supplied. The study animals were housed for 6 days.

Collection of urine and meconium

Informed consent for collection of urine and meconium was taken from parents of new born babies. The urine and meconium of six normal newborn babies born at K.E.M Hospital, Mumbai, who were not being fed, were collected according to Clinical and Laboratory Standards Institute Guidelines.[7]

All of the urine and meconium underwent bacteriological analysis at the institute's laboratory. Sterile samples were used. Urine and meconium were stored at a temperature of −20°C for 5 days to prevent bacterial contamination. The urine was defrosted and the meconium diluted with distilled water before injecting in rats for the study.

Experimental procedure

All rats were injected with 3mL of different substances in the right lower quadrant of the abdomen, twice a day for 5 days [Figure 1]. The rats were divided into four groups, depending upon the biological material to be injected into their peritoneal cavities:

Figure 1

Injection of biological material into the peritoneal cavity

Group I — Control group (n = 6): distilled water.

Group II — Urine group (n =6): neonatal urine.

Group III — Meconium group (n=6): 5% neonatal meconium suspension.

Group IV — Combined meconium and urine group (n = 6): combination of meconium and urine.

All animals were sacrificed 24 h after the last injection (6th day of the experiment) by a high dose of thiopentone sodium. A segment of small intestine was removed via a xyphopubic incision. This specimen was studied after staining with standard hematoxylin-eosin stains.

The microscopic analysis was done by a single pathologist who noted the intensity of inflammatory reaction on a standardized data sheet. The inflammatory reaction was grouped into serositis, enteritis, parietal necrosis, and peeling. The pathologist was blinded about the injection.

RESULTS

All rats tolerated the injection well except two rats who developed a hematoma at the injection site. There were no other lesions observed which could have been related to the daily punctures. There was no intraabdominal injury. There were no macroscopic abnormalities of the bowel loops in any of the animals in any group. A total of 24 specimens were obtained belonging to four groups [Figure 2].

Figure 2

Macroscopic aspect of the bowel loops after laparotomy

Results of histopathologic analysis

Group I (Control group)

Mild submucosal congestion in the form of enteritis was observed in three cases (50%). None of the animals exhibited parietal necrosis or peeling [Table 1].

Table 1

Results of histopathological analysis

Group II (Urine group)

Serositis was seen in two animals (33%). Severe enteritis was observed in four (66%) in the form of hypertrophy of lymphoid follicles. Parietal necrosis in one (16%) and peeling in one (16%) were observed [Figure 3].

Figure 3

Histopathological finding of serositis in urine group

Group III (Meconium group)

There was serositis in one animal (16%) and mild enteritis in three animals (50%) in the form of submucosal congestion and mild lymphoid hyperplasia. This was the next most affected group.

Group IV (Combined urine and meconium group)

There was mild enteritis in three animals (50%) in the form of mucosal congestion. The extent of bowel changes was similar to that of Group I (control group).

The maximum bowel changes were seen in the urine group and then the meconium group, suggesting that urine was the factor of amniotic fluid causing maximum bowel damage in this study.

DISCUSSION

In gastroschisis, direct contact of the intestines with amniotic fluid causes intestinal damage. However, the exact component of amniotic fluid which is responsible for the intestinal shortening, thickening, and reduced peristalsis is yet unknown, though highly speculated.[34] There are multiple experimental studies to evaluate the etiologic factor. Urine, the principal component of amniotic fluid, has often been blamed for the intestinal damage. The fetus physiologically urinates into the amniotic cavity.[23] Hence, it has most commonly been implicated. However, this cannot be conclusively proved. The lack of an intense intestinal wall inflammatory process in patients with urinary ascites questions the validity of the theory of urine as the causative factor for bowel damage.[8] Many authors have performed different experiments and tried to correlate urine with damage to the walls of herniated viscera in hen embryos, but they felt that it was not possible to categorically determine whether only urine was responsible for the intestinal damage.[34]

It is also thought that other substances that make up human amniotic fluid, including meconium,[910] may be responsible for the event. Innumerable experiments have demonstrated that intrauterine defecation can be considered physiological.[11] The earlier belief was that meconium is only passed in utero during times of fetal distress. Ciftci et al.,[11] challenged this thought by showing that meconium stained amniotic fluid is not related to meconium passage after fetal distress but reflects impaired clearance of amniotic fluid, which already has meconium caused by in utero physiologic defecation.

Akgur et al.,[9] have shown that intraperitoneal injection of neonatal human diluted meconium causes intestinal damage in adult rat intestines, whereas intraperitoneal neonatal human urine does not have any effect. Olguner et al.,[12] showed that intraamniotic neonatal human urine has no effect on intestines, while intraamniotic neonatal human diluted meconium causes intestinal damage in chick embryos that is identical to the histopathologic features of human gastroschisis specimens described by Amoury et al.,[13] and with the histopathologic features of chick gastroschisis specimens described by other investigators.

In the study by Amoury et al.,[13] and Aktug et al.,[14] primary pathology was observed in serosal surface of intestines obtained from human gastroschisis cases. The main characteristic of the specimens was serosal peel mainly composed of fibrin and collagen found with intraperitoneal meconium-injected rat intestines and in intraamniotic meconium-introduced chick intestines.[9]

Morrison et al.,[15] showed that human fetal gastroschisis is associated with an acute inflammatory exudate, composed predominantly of activated neutrophils with markedly elevated interleukin 8 levels in the amniotic fluid. It has been shown that interleukin 8 is present in human meconium in large concentrations and that it is a powerful inducer of neutrophil chemotaxis.[16]

Api and Olguner[17] concluded that the degree of intestinal damage in gastroschisis correlates with the concentration of intraamniotic meconium. The deleterious effects of intraamniotic meconium could be prevented by lowering the intraamniotic meconium concentration. This could be achieved by two methods: amniotic fluid exchange in cases of normal amniotic fluid volume[1416] and amnioinfusion in cases of oligohydramnios.[18] Amniotic fluid exchange works by partially replacing the meconium bearing amniotic fluid with a solution devoid of meconium, amnioinfusion prevents intestinal damage by diluting the amniotic fluid.

Correia-Pinto et al.,[5] showed that prevention of meconium contamination with anal ligation markedly prevented peel development, and reduced bowel adherence significantly as well as tissue edema. However, bowel shortening could not be prevented. Bowel shortening was the parameter of bowel damage least affected by meconium exposure in his study.

Olguner and Hakguder[19] have suggested that urine has a protective effect on neonatal bowel and showed that neonatal urine contains a high amount of urinary tryps in inhibitor (UTI) compared with adult human urine. UTI has been shown to exert inhibitory effects on interleukin 8. Therefore, far from being destructive, fetal urine in the amniotic fluid might be beneficial because human urine contains UTI.[19]

In our study, the urine group (Group II) showed maximum damage to intestinal wall; some amount of damage was seen in the meconium group (Group III). However the combination of urine and meconium (Group IV) did not cause much harm as the results were similar to those of the control group (Group I). In our experiment, bowel damage was predominantly caused by urine. This strongly supports the hypothesis that urine exposure plays a significant role in the pathogenesis of bowel damage in gastroschisis.

We own up to intrinsic limitations of our preliminary study. We attempted to observe the effect of human urine and human meconium on rat intestine. The response may differ from that found in humans. Use of rat urine and meconium instead of human urine and meconium perhaps would not simulate the actual gastroschisis conditions at all and was dismissed. Results might be inconclusive due to limitations of sample size which may be confirmed by increasing sample size and reproducing similar results on repeat study. Even the control group showed some pathological changes. Possibly, the mechanism used to kill the animals or the repeated punctures of the peritoneal cavity might have led to minor inflammatory abnormalities of the intestinal wall, further confounding the findings.

In conclusion, further studies with larger sample sizes are required to validate our hypothesis that urine is the more damaging component of amniotic fluid in cases of gastroschisis.