A functional study on small intestinal smooth muscles in jejunal atresia.

AIM: The present study was aimed to assess the contractile status of neonatal small intestinal smooth muscle of dilated pre-atretic part of intestinal atresia to resolve debatable issues related to mechanisms of persistent dysmotility after surgical repair.
MATERIALS AND METHODS: A total of 34 longitudinally sectioned strips were prepared from pre-atretic dilated part of freshly excised 8 jejunal atresia type III a cases. Spontaneous as well as acetylcholine- and histamine-induced contractions were recorded in vitro by using organ bath preparations. Chemically evoked contractions were further evaluated after application of atropine (muscarinic blocker), pheniramine (H1 blocker), and lignocaine (neuronal blocker) to ascertain receptors and neuronal involvement. Histological examinations of strips were made by using Masson trichrome stain to assess the fibrotic changes.
RESULTS: All 34 strips, except four showed spontaneous contractions with mean frequency and amplitude of 5.49 ± 0.26/min and 24.41 ± 5.26 g/g wet tissue respectively. The response to ACh was nearly twice as compared to histamine for equimolar concentrations (100 μM). ACh (100 μM) induced contractions were attenuated (by 60%) by atropine. Histamine (100 μM)-induced contractions was blocked by pheniramine (0.32 μM) and lignocaine (4 μM) by 74% and 78%, respectively. Histopathological examination showed varying degree of fibrotic changes in muscle layers.
CONCLUSIONS: Pre-atretic dilated part of jejunal atresia retains functional activity but with definitive histopathologic abnormalities. It is suggested that excision of a length of pre-atretic part and early stimulation of peristalsis by locally acting cholinomimetic or H1 agonist may help in reducing postoperative motility problems in atresia patients.

INTRODUCTION

Atresia refers to a congenital obstruction with complete occlusion of the intestinal lumen and accounts for 95% of obstructions in neonates.[1] Intestinal atresia is rare gut congenital disease with prevalence of 2.8 for 10,000 births.[2] It is detected by prenatal ultrasonography in second or third trimester and confirmed at birth by features of intestinal obstruction. Although, new operative techniques and perioperative managements including nutritional therapy have led to an improvement, but prolonged intestinal motility disorders remain common problems in postoperative period.[34] Previous studies indicated that abnormalities of the myenteric ganglia, nerve growth factor, and interstitial cells of Cajal (ICCs) in preatretic, as well as postatretic segments of intestinal atresia may be related to the causes of intestinal motility problems.[56] These studies also hypothesized that interruption of antenatal peristalsis may disturb the normal development of the enteric nervous system which provide an anatomical substrate for the motility disorders observed after surgical repair. Other study on chick intestinal atresia model also showed abnormalities in intramural nerves of the proximal dilated segment.[7] However, no studies, so far, evaluated the contractile status of intestinal smooth muscle in small bowel atresia cases. Thus, it is uncertain whether the normal contraction is retained in preatretic dilated segment. Therefore, the objective of the present study was to assess contractile and histological status of preatretic part of jejunal atresia which may help in understanding of underlying pathophysiology of postoperative motility problems and surgical management.

MATERIALS AND METHODS

This in vitro contraction study was carried out on total 34 intestinal strips prepared from freshly excised specimens from eight jejunal atresia patients operated by jejunoileal-anastomosis. All the cases were of jejunal atresia type IIIa. The freshly excised specimens were immediately collected in a wide mouth bottle containing ice-cold (4-6°C) Krebs-Ringer solution bubbled with 100% oxygen and quickly transferred to the laboratory in the department of physiology for contractile studies. The composition of Krebs-Ringer solution was (mmol/L): NaCl-119; KCl-4.7; CaCl2.2H2O-2.5; KH2PO4-1.2; MgSO4.7H2O-1.2; NaHCO3-5; and glucose-11. All the experiments were conducted as per the guidelines laid down by the ethical committee of the Institute for handling human tissues.

The removed segments were thoroughly cleaned, and the adventitious layer was removed. Thereafter, 2-3 mm wide and 15-20 mm long, longitudinally sectioned strips were prepared from just adjacent preatretic dilated part. These strips were considered as a sample.

The prepared strips were mounted in Krebs-Ringer filled organ bath (10 mL) maintained at 30 ± 1°C and continuously bubbled with 100% O2. The strip was placed under an initial tension of 0.5 g and then left to equilibrate for 60 min, with replacement of Krebs-Ringer solution every 15 min. Isometric contractions were amplified by bridge amplifier and recorded onto a personal computer with the help of force transducer and computerized data acquisition system (Power Lab 4/ST system, AD instruments, Australia).

After stabilization, the initial recording was made for 30 min to assess the presence of any spontaneous contraction. Thereafter, different chemically induced contractions were recorded as detailed below:

Acetylcholine (ACh)-induced contractions were obtained for four different cumulative concentrations (0.1, 1, 10, and 100 μM), (n = 10).

ACh (100 μmol/L)-induced contractions were recorded before and after treatment of muscarinic antagonist atropine (10 μM), (n = 10).

Histamine-induced contractions were obtained for four different cumulative concentrations (0.1, 1, 10, and 100 μM), (n = 12).

Histamine (100 μM)-induced contractions were recorded before and after treatment with H1 receptor blocker pheniramine (0.32 mM), (n = 10) and also after neuronal blocker lignocaine (4 mM), (n = 10).

After the recording, the strips were removed and placed on blotting paper for lightly soaking of the extra water and weighed in a fine balance to express the contractile tension per unit weight of tissue (g/g wet tissue). Thereafter, the strips were preserved in 10% formalin solution for histopathological studies. In histological preparations, Masson trichrome stain was used for detection of fibrosis which appeared as green color by this stain. These methods have been already standardized earlier in our laboratory.[8]

Statistical analysis

The recorded tensions (g weight) were expressed as unit mass of jejunal tissue (g/g of wet tissue). The values were pooled to calculate the mean ± standard error of the mean and statistical significance for differences were evaluated by using Student's t-test and one-way ANOVA as applicable. P < 0.05 was considered as significant.

RESULTS

Spontaneous contractions

All 34 strips showed spontaneous activity except four strips (all four from the single case). The spontaneous contractions were mostly of phasic type [Figure 1] and varied from strip to strip in their amplitude and frequency with mean frequency and amplitude of 5.49 ± 0.26/min and 24.41 ± 5.26 g/g wet tissue respectively [Table 1].

Figure 1

Representative recordings from spontaneous contractions without any chemical interventions (top panel), acetylcholine (middle panel) and histamine (bottom) induced contractions for four cumulative doses (0.1, 1, 10, and 100 μM) are shown. Vertical and horizontal calibrations represent tension (g) and time (min) respectively

Table 1

Total number of samples tested, responded and mean ± SD values of amplitude of contractions (g/g wet tissue) for spontaneous, ACh (100 μM) and histamine (100 μM) induced contractions

Acetylcholine and histamine-induced contractions

Most of the tested strips responded to both ACh, as well as histamine. The responses started within 15-25 seconds after the application of the drug. After application of each higher dose, the tone of the intestinal strips was sharply increased [Figure 1]. It has been observed that ACh-induced contractions were higher as compared to the histamine-induced contractions for the equimolar concentrations (approximately twice for highest dose) [Table 1].

Effect of atropine on acetylcholine-induced contractions

After treatment with atropine (10 μM), ACh (100 μM) produced only 39.97 ± 19.97 % (n = 10) of its initial contraction. Thus, there was nearly 60% blockade of ACh response by atropine (*P < 0.05, Student's t-test for paired observations) [Table 2].

Table 2

Mean ± SD values for ACh (100 μM) and histamine (100 μM) induced contractions before and after treatment with their respective blockers that is, Atropine (10 μM) for ACh and pheniramine (0.32 mM) and lignocaine (4 mM) for histamine, obtained from 10 different samples (n = 10)

Effect of pheniramine and lignocaine on histamine-induced contractions

Histamine-induced contractions were greatly attenuated by pheniramine (approximately 74%), as well as by lignocaine (78%). After pretreatment with pheniramine (0.32 mM) and lignocaine (4 mM), histamine (100 μM/L) produced only 26.10 ± 12.85 % (n = 10) and 22.12 ± 5.63 % (n = 10) of initial contractions, respectively (*P < 0.05, Student's t test for paired observation) [Table 2].

Microscopic examination

The muscle layers showed fibrosis of varying degree in most of the specimens on Masson trichrome stain [Figure 2]. The histological abnormalities were more in the samples which did not show spontaneous contractions.

Figure 2

Photo-microphotograph (×100) showing fibrosis (Masson trichrome stain, green color) of outer longitudinal muscle layer in a sample of jejunal atresia

DISCUSSION

The present study was carried out to understand the functional status of preatretic dilated part of jejunal atresia by recording contractility of intestinal smooth muscle strips (total 34 strips prepared from eight specimens) in vitro. In the present study, strips from almost all cases except one showed spontaneous contractions. Spontaneous contraction is one of the important parameters to understand the functional status of the intestine. Earlier in vitro studies with normal intestinal muscle strips also showed spontaneous contractions.[910] The origin of spontaneous contraction is supposed to be related to the activity of ICC, which are considered as pacemaker cells of the gastrointestinal tract (GIT).[11] These cells provide promising explanations for GIT motor physiology and pathophysiology of many gastrointestinal diseases with abnormal motility. In our study, the rate of the spontaneous contractions was nearly 5.5 ± 0.3 contractions/min which is similar to the average rate of spontaneous contractions in small intestine in normal adult (4.9 ± 0.7 contractions/min) as reported elsewhere.[10] However, in the absence of availability of age and sex matched control specimens, it is difficult to comment whether the rate observed in these atresia cases is absolutely normal or not. Nevertheless, the presence of spontaneous contraction in preatretic part of intestinal atresia indicates the preservation of natural pacemaker activity to a great extent.

The present functional study demonstrated that the muscle strips from preatretic part of jejunal atresia respond to both ACh and histamine in a dose depended manner and the response to ACh was higher than with equimolar concentration of histamine. This type of cholinergic predominance in the normal adult intestine has been reported earlier.[1213] These results indicate the retention of contractile function of jejunal smooth muscle. However, a definitive conclusion about the contractile status needs to be avoided on account of nonavailability of contractile study on normal (control) small intestine in the neonate.

ACh-induced contractions were blocked (approximately 60%) by the atropine, indicating that the cholinergic contractions in the preatretic part were largely mediated through muscarinic receptors. The similar action of cholinergic agonists has been documented on healthy colonic smooth muscle. Further, it may be noted that ACh is known to mediate its effect through M2/M3 muscarinic receptors.[1214] Thus, the observation of our study provides the evidence for preserved physiologic cholinergic contractile mechanisms in preatretic part of intestinal atresia.

In this study, histamine-induced contractions were greatly reduced (approximately 74%) by preapplication of pheniramine (H1 blocker) and thus indicated the involvement of predominantly H1 receptors. Furthermore, the attenuation (approximately 78%) of histamine-induced contractions by lignocaine also signifies the involvement of neural plexuses. Predominant participation of H1 receptors in mediating normal intestinal smooth muscle contraction has also been documented elsewhere.[15] The present study too indicated the existence of similar mechanism.

Histologically, fibrotic changes in musculature were observed in most of the samples of preatretic part of jejunal atresia in the present study. The extent of fibrosis was more in the strips which did not show any spontaneous contractions. The fibrotic changes in the intestinal atresia specimens are likely to impair contractile functions. However, the major limitation of the present study was the unavailability of control specimens that is age and sex matched normal neonate intestinal tissue, due of obvious ethical reasons. Because of lack of control, it was very difficult to ascertain the contractile status in an absolute term. However, on the basis of abnormal histopathological findings, it is clear that preatretic part is abnormally developed tissue. Thus, excision of a length of the preatretic part is likely to improve motility problem in the postoperative period.

Further, it was hypothesized, in previous studies, that interruption of antenatal peristalsis may disturb the normal development of the enteric nervous system which provides an anatomical substrate for the motility disorders.[6] Therefore, the present study may be promising to induce cholinergic or histaminergic contractions of a preatretic segment of intestine bypassing the neural elements.

CONCLUSIONS

The result of the present study showed evidence of preserved pacemaker activity, cholinergic and histaminergic contractile mechanisms to a large extent in addition to fibrotic changes. Therefore, it is suggested that excision of a length of the preatretic part and use of locally acting cholinomimetic drugs or H1 agonists may improve motility in the postoperative period.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.