Abnormalities of digestive tract innervation in rat fetus treated with ethylenethiourea

PURPOSE: The pathophysiology of abnormalities associated with myenteric plexus lesions remains imperfectly understood. Such abnormalities have been correlated with subocclusive intestinal conditions in children with Hirschsprung’s disease, cases of chronic constipation and, postoperatively, in cases of anorectal anomalies. This study evaluated abnormalities of the myenteric plexus in fetus from female rats that received ethylenethiourea. METHODS: Female rats were exposed to ethylenethiourea on the 11th day of pregnancy (experimental group) or to 0.9% physiological solution (control group). Abnormalities were only found in the experimental group. The digestive tract muscle layer was analyzed morphometrically and changes to the frequencies of nerve plexus cells and interstitial cells of Cajal were evaluated, using hematoxylineosin, S-100 protein, neuron-specific enolase and C-Kit, respectively. RESULTS: Muscle and skeletal abnormalities were observed in 100%, anorectal anomalies in 86%, absent tail in 71%, short tail in 29%, duodenal atresia in 5%, esophageal atresia in 5% and persistent omphalomesenteric duct in 5%. Histopathological analysis showed a thinner muscle layer associated with lower frequencies of ganglion cells and interstitial cells of Cajal, in all gastrointestinal tract. CONCLUSION: Severe nerve plexus abnormalities associated with muscle layer atrophy were observed throughout the gastrointestinal tract in newborn rats exposed to ethylenethiourea.


Anomalies of intestinal nervous plexus, including
Hirschsprung's disease and dysganglionosis are related to defects associated with abnormal migration of the neuroblasts 1 , but for such anomalies the explanation for the pathophysiology and embryological remains imperfectly understood 2 .
This diffuse reduction in the number of ganglion cells along the colon is often seen in chronic constipation, so this type of disease can be considered as a possible reason for chronic idiopathic constipation 3 .
This group of diseases affects social and productive life by intervening in the planning and economy of the health system, as well as increased costs and productive force in the country 4 .
These abnormalities have been correlated with obstructive or subocclusive intestinal conditions that are unrelated to tumors, in cases of children with Hirschsprung's disease, cases of idiopathic chronic constipation 5 and, postoperatively, cases of anorectal anomalies. In particular they have been correlated with cases of low anorectal anomalies that present a good prognosis regarding fecal incontinence but frequently evolve with constipation 6 .
Most of the researches using ethylenethiourea for evaluations of anorectal anomaly are directed to changes in the pelvic region 7 not including the changes of the small intestine.
With the need to expand the knowledge of dysganglionosis, experimental models have been created over the years to study these diseases, but the relationship between changes in the small intestine and anomalies of myenteric plexus remain unclear.
The improving of the knowledge about the chronic diseases such as constipations and dysplasia of the myenteric plexus, is a great importance for clinical practice, needing a more comprehensive assessment.
With the need of better understanding about the changes of the myenteric plexus in the small intestine in fetuses with anorectal anomalies, this study evaluated changes in the myenteric plexus throughout the gastrointestinal tract induced by ingestion of ETU and the alteration that occurred concurrently in the muscle wall bowel. On the 20 th day of pregnancy, cesarean section was performed under dissociative anesthesia consisting of xylazine hydrochloride (3 mg/kg) in association with ketamine hydrochloride (50 mg/kg). Analgesia consisting of tramadol hydrochloride at a dose of 2 mg/kg was provided.

This
After the fetuses had been removed, the rats were sacrificed by deepening the anesthesia. The cross-sections were measured using ten randomly chosen regions in the selected segments of the esophagus and stomach, small intestine and rectum of each animal, and come up with the average of these measurements.
Immunohistochemical analysis was carried out with the aims of evaluating changes to the frequencies of ganglion cells, using S-100 protein and neuron-specific enolase, and evaluating the ICCs, using C-kit antibodies (CD-117).
To determine the changes to the frequencies of ICCs and ganglion cells, an adaptation of the methodology described by Wedel et al. 10 was used. In this, cell counts were obtained within the areas that had been measured using the Samsung CCD color video camera coupled to the optical microscope, and the number

Results
Thirty-two newborn rats were evaluated: 21 in the experimental group and 11 in the control group. None of the 11 newborn rats in the control group presented any abnormalities.
On the other hand, the 21 newborn rats in the experimental group presented a variety of abnormalities in the gastrointestinal tract, musculoskeletal system and spine.
Musculoskeletal abnormalities were observed in 100% of the fetus rats in the experimental group. These included rib deformities, short thoracic limbs, absence of toes and incomplete formation of the pelvic limb, with severe hip lesions.
Spinal abnormalities were seen in 100% of the cases, and these were expressed as abnormalities of the tail. Fifteen of the 21 newborn rats (71%) were tailless, while the tail was short or vestigial in the other six rats (29%). No cases of rachischisis were observed.

Histological evaluation using hematoxylin-eosin
In the experimental group animals, the esophagus, stomach, small intestine, large intestine and rectum all presented nerve plexus abnormalities characterized by absence or necrosis of the ganglion cells.
The muscle layers of the wall on the entire gastrointestinal tract were presented thinner, and the more severe the degree of lesion in the myenteric plexus was, the bigger was the lesion in the muscle (Figure 1).  Table 1).
The data, presented as average ± standard deviations, showed that there was a statistically significant difference between the control and experimental groups (P < 0.01).

Immunohistochemical analysis
An immunohistochemical analysis was performed with the aim of evaluating changes to the frequencies of ganglion cells and ICCs.

Evaluation by immunohistochemistry for neuro-specific enolase and protein S-100 (ganglion cells)
In the experimental group, it was observed that the frequency of ICCs was lower than in the control group. In the esophagus and stomach subgroup, the observed mean was 9.7 (± 1.1) cells/mm 2 in the experimental group and 17.5 (± 2.9) cells/ mm 2 in the control group.

Evaluation by immunohistochemical c-kit or CD-117
The ICCs were evaluated by means of immunohistochemistry using the C-Kit or CD-117 marker ( Figure   4).
In the experimental group, it was observed that the frequency of ganglion cells was reduced in relation to the control group. In the esophagus and stomach subgroup, the observed measurements were 14.6 ± 2.2 cells/mm 2 for the rats exposed to ETU and 23.9 ± 2.9 cells/mm 2 for the controls. This pattern was also observed in the small intestine subgroups, with 12.9 ± 1.4 cells/mm 2 and 21.3 ± 1.2 cells/mm 2 , and in the rectum subgroups, with 13.4 ± 4.1 cells/mm 2 and 21.9 ± 2.3 cells/mm 2 , for the experimental and control groups respectively.  There is evidence that many disorders of gastrointestinal motility caused by neuron malformations are associated with concomitant changes to the ICCs. This observation may suggest that there is mutual influence between changes to the enteric nerve system and ICCs, during the development 10 .
The model studied here was shown to be easily reproducible, given that we found that 18 out of the 21 newborn rats (86%) presented anorectal anomalies. The reproducibility rate was thus similar to the other authors, which ranged from 63.2% 15 to 98% 16 .
Abnormalities of the musculoskeletal system were presented in 100% of the experimental group. They were shown to be more severe in the pelvic limbs when in association with the absence of the tail and anorectal anomalies, and this was also described by Qi et al. 16 .
The spinal abnormalities found in the present study were similar to those found by other authors 17  The changes in the myenteric plexus remained present also in the small intestine and stomach and esophagus of fetuses

Conclusions
The fetuses exposed to ethylenethiourea changes in the myenteric plexus were associated with changes in the thickness of the muscle throughout the gastrointestinal tract.
There was a correlation between the intensity of the injury in intestinal smooth muscle and damage to the myenteric plexus throughout the gastrointestinal tract, showing that the more severe were the myenteric plexus injuries more severe were the injury intestinal muscles layer throughout the gastrointestinal tract.