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Journal of the Anatomical Society of India

Study of Fetal Stomach Under Light Microscope

Author(s): Chimmalgi M and Sant S M

Vol. 54, No. 2 (2005-07 - 2005-12)

Chimmalgi M and Sant S M - B. J. Medical College, Pune

Abstract: In fetuses aged between 15 to 38 weeks, light microscopic structure of the stomach was studied. Tissues from both the body and pyloric parts of the stomach of various age group fetuses were collected, processed and staining was done with H/E and Masson's trichrome stain. Development proceeded in four phases in the body of stomach. At 15- 16 weeks, surface epithelium was 'cuboidal' in body of stomach, rapidly becoming columnar. Glands showed four stages of development. Parietal cells were the first type of cells to appear in the gastric glands, mucous neck cells and chief cells being seen from 23-24 weeks. Muscularis mucosa made its first appearance by the end of 22nd week. Muscularis externa was made up of only circular muscle coat from 15th to 24th week, longitudinal muscle layer being added beyond this period.

Pyloric part showed more extensively folded mucosa with deeper pits. Glands were predominantly mucous with few parietal cells. At any stage of development, both mucosal and muscularis external layers were thicker in the pyloric part than in the body of the stomach. Pyloric part preceded the body in the development at all stages.

Key Words: Body of stomach, pyloric part, gastric glands, gastric pits, parietal cells, chief cells, mucous neck cells.

Development of a unicellular zygote into a newborn has always held the interest of anatomists. Understanding of the processes involved in the formation of various organs and systems has unraveled most cryptic secrets of the nature. Although, at macroscopic level these developmental processes are largely clear to us, details of microscopic development are still elusive. Histogenesis and further microscopic development are studied in almost all systems; however, such studies are fewer in the gastrointestinal tract. Available literature is mainly replete with studies conducted on fetuses of Western origin, Salenius (1962), Lillibridge (1964), Goldstein (1969), Grand et al (1976), William et al (1995) and Rindi et al (2002). Very few documented studies have been done on fetuses of Indian origin which differ largely from their Western counterparts in their rate of growth and maturity. Textbook teaching mainly lays stress upon the germ cell layers contributing to the formation of the gut, macroscopic development, and rotation of the gut, Moore and Persaud (1993).

We have therefore taken up a project to systematically document the normal microscopic structure of entire gastrointestinal tract in fetuses of different age groups belonging to Indian origin. This study concerning the microscopic structure of 'stomach' in fetuses is a part of the main project. Objectives of this study were to:

  1. Study the microscopic structure of the stomach in fetuses of different age groups to know the stages involved in development of adult picture.
  2. To compare and contrast the differences between the different age groups.

Materials and Methods:

Study was conducted in the Department of Anatomy, B. J. Medical College, Pune. Aborted and stillborn fetuses were collected from the Department of Obstetrics and Gynecology, Sassoon General Hospitals, Pune after meticulously following the due formalities. Presence of any congenital anomaly or grossly visible post-mortem decomposition changes resulted in exclusion of such cases from the study.

A total of thirty-eight aborted/stillborn fetuses were selected for the study. Fetuses were grouped according to their ages based on their crown-rump length (CRL) as shown in Table 1, Hamilton and Mossman (1978). Based on their CRL, they were divided into six groups, youngest fetus with CRL of 80mm belonging to 15-18 weeks age group. Oldest fetus with CRL 342mm belonged to 35-38 weeks age group.

Fetuses were dissected immediately. Blocks of tissue 3-5mm in diameter were collected from body and pyloric parts of the stomach along the greater curvature. These tissues were fixed immediately in 10 percent formalin and were processed to prepare 5µ thick sections. These sections were stained with haematoxylin- eosin and Masson's trichrome stain and studied under light microscope. Measurements of different layers and cells were taken using micrometer.

Table 1: Gestation age based on Crown-rump length

? CRL (in mm) Age (in weeks) No. of Fetuses
61-100 15-18 5
101-150 19-22 10
151-200 23-26 11
201-260 27-30 5
261-320 31-34 4
321-390 35-38 3

Observations:

Body of the stomach:

All the four layers of stomach were clearly visible by 15th week. Between 15th to 38th weeks, four successive phases of development were observed. In the first and third phases, submucosa was the thickest layer. In the second and fourth phases, there was predominantly growth in the mucosal and muscularis external layers.

(A) First phase (15-20 weeks or CRL 80-120mm):

All the four layers were arranged parallel to each other with submucosa being the thickest layer (fig. 1). Mucosa showed (a) surface epithelium made of cuboidal to small columnar cells, (b) invagination of the surface epithelium to form gastric pits and (c) few 'acinar' glands at the depth made up of clusters of differentiated cells. Pit to gland ratio was 3:1 to 4:1. Muscularis mucosa was conspicuous by its absence. Lamina propria merged imperceptibly with submucosa, both of these layers showing reticulum of stellate or polygonal cells with rounded central nucleus and clear cytoplasm resembling primitive mesenchymal cells. Connective tissue fibers were not seen. Lamina propria could be differentiated from submucosa by the more cellular nature of the former. Muscularis externa showed circular or oblique smooth muscle fibers. Serosa was made up of simple squamous epithelium. It was seperated from muscularis externa by extensive subserosal connective tissue, resembling submucosal layer, but containing larger blood vessels. Under high power, the surface epithelial cells were cuboidal to small columnar, measuring 7-8µ in height, with vesicular nucleus and clear cytoplasm. Gastric glands showed clusters of eosinophilic cells, cuboidal to pyramidal in shape, measuring 6-8µ with homogenous cytoplasm and condensed or vesicular nucleus. These are the earliest parietal cells that could be identified. No other cell types were found in the glands.

(B) Second phase (21st to 24th weeks or CRL 126-164 mm):

This stage was characterized by mucosa and muscularis externa forming the thickest layers. Initially, in the 21st and early 22nd weeks (CRL 126-140 mm), surface epithelium began to appear columnar at places. Number of the glands increased, which were still clusters of eosinophilic cells. Two or more glands began to open in same fovea. Muscularis mucosa was still not seen at this stage. Muscularis externa gained in thickness and was made up of predominantly circular muscle fibers.

In late 22nd week to 24th week (CRL 141-164 mm) mucosa and muscularis externa were still thicker than the submucosa. Surface epithelium was simple columnar, measuring 25-35µ in length, with a rounded nucleus situated centrally or basally. Cytoplasm showed clear secretory vesicles close to the luminal surface staining positively for mucin with Masson's trichrome stain. Similar mucous cells were found extending into gastric pits and neck part of the gland; but these cells were shorter measuring 12-15µ in length and were having basal nucleus (fig. 2).

The gastric glands began to elongate and now appeared 'tubular'. Gastric pit to gland ratio changed to 2:1. Eosinophilic cells were seen at the bottom of the glands (fig. 2); in the upper parts of the glands mucous secreting pale columnar cells with basal vesicular nucleus were seen. These cells were interspersed with a few cuboidal cells having rounded open faced nucleus and granular cytoplasm, staining purplish with haematoxylin and eosin stain. These mark the first appearance of chief cells. Muscularis mucosa made its first appearance by the end of 22nd week (CRL 148mm). It was seen as condensation under low power. Under high power spindle shaped cells with elongated nuclei were seen. Although these cells resembled smooth muscle fibers, they failed to take up stain for the muscle when they were stained with Masson's trichrome stain (fig. 3). Collagen fibrils began to appear in the submucosa. Other layers were similar to the prior stage.

Arrow pointing at the acinar glands

Fig. 1: 15-20 weeks age [10x, H/E]: Arrow pointing at the acinar glands with first discernible, eosinophilic, early parietal cells.

Arrow shows chief cells with granular cytoplasm

Fig. 2: 24 weeks of age [10x, H/E]: Arrow shows chief cells with granular cytoplasm and asterisk shows basal parietal cells in the elongated glands.

Arrow showing condensation at muscularis mucosa

Fig.3: 22 weeks of age [10x, Masson's trichrome]: Arrow showing condensation at muscularis mucosa that has failed to take up stain for muscle. Surface epithelium and mucous neck cells ae seen taking up green stain for mucin.

(C) Third phase (25th to 27th weeks or CRL 175-215 mm):

Mucosa was similar to as stated above with further addition to the length of the glands. In submucosal layer, there was secondary increase in the thickness and re-arrangement, resulting in folded appearance. Muscularis mucosa was as seen in the earlier stages.Muscularis externa began to show an additional outer longitudinal smooth muscle coat at places.

(D) Fourth phase (28th week onwards or CRL 240mm onwards):

There was tremendous increase in the glandular tissue. This accentuated submucosal folds. Basal parts of the glands became tortuous resulting in transversely cut sections of the glands. There was increase in the number of the basophilic cells in the glands (fig. 4).

Muscularis mucosa was wavy in outline and the muscle fibers began to take up stain with Masson's trichrome stain. Submucosa showed bundles of collagen fibers, interspersed with fibroblasts and well

formed blood vessels. There was increase in thickness of the muscularis externa layer, outer longitudinal layer now being seen consistently. By 32 to 34 weeks (CRL >290 mm), overall picture began to resemble that of an adult. Long tubular glands were seen, showing basophilic cells occupying more basal position, eosinophilic cells in the neck region interspersed with mucous cells. Gastric pit to gland ratio now was 1:1 to 1:2 (fig. 5). Although oblique layer of the muscularis externa was not seen, other layers resembled those of adult stomach.

From 15-21 weeks (CRL 80-140 mm) blood vessels in subserosal and submucosal layers were merely endothelium-lined spaces. Tunica media and adventitia were absent. By the end of 22nd week, they developed connective tissue sheath around them. Smooth muscle fibers appeared in their walls by 28th week, initially in the larger blood vessels of subserosal layer and subsequently in the blood vessels of submucosal layer.

Arrow showing parietal cells

Fig.4: 28 weeks of age [10x, H/E]: Arrow showing parietal cells and asterisk showing chief cells.

Arrow showing condensation at muscularis mucosa

Fig.5: 22 weeks of age [10x, Masson's trichrome]: Arrow showing condensation at muscularis mucosa that has failed to take up stain for muscle. Surface epithelium and mucous neck cells ae seen taking up green stain for mucin.

Pylorus Part of Stomach:

All the four layers were clearly seen by 15th week and at all stages, mucosa and muscularis externa layers were thicker than the similar layers in the fundus. There was no clear demarcation into 'phases' in development as in case of fundus or body. Mucosa was more extensively folded. Surface epithelium was tall columnar from 15 weeks of age unlike that of fundus. It was showing centrally located rounded nucleus and clear cytoplasm staining faintly positive for mucin with Masson's trichrome stain. Glandular cells were also columnar with clear cytoplasm, failing to take up stain for mucin at this stage (fig. 6).

Tubular glands were seen in the pyloric part of the stomach by as early as 20 weeks of age (CRL: 120mm). A few eosinophilic cells were seen in these glands but they were largely absent towards pyloric sphincter.

Condensation at the site of muscularis mucosa and appearance of folding in the submucosal layer were seen after 20 weeks (CRL >128mm). Ratio between gastric pits to gastric gland was 1:1 at this age, approaching 2:1 by 24 weeks.

By 24th week, surface epithelium was filled wih mucin displacing the flattened nucleus basally. Mucin staining cells had reached upto the base of the glands. Muscularis mucosa showed wavy pattern; submucosa and lamina propria showed increased collagen content.

By 27 to 28 weeks (CRL>215mm), there was remarkable increase in the thickness of the mucosa, pit to gland ratio increasing to 3:1 or more (fig. 7). Increase in the thickness of mucosa was much more towards the pyloric sphincter. In the muscularis externa, circular muscle coat was thicker towards the sphincter. Adult pattern had been established by the end of 28 weeks of age (fig. 8).

Arrow showing gland cell failing to take up stain mucin

Fig.6: 15-20 weeks of age [10x, Masson's trichrome]: Arrow showing gland cell failing to take up stain mucin.

Arrow showing long gastric pits

Fig. 7: 28 weeks of age [10x, Masson's trichrome]: Arrow showing long gastric pits.

Image Showing adult pattern

Fig. 8: 136 weeks of age [10x, Masson's trichrome]: Showing adult pattern

Discussion

Microscopic structure of the body and pyloric part of the stomach was studied in the fetuses of the age group 15-38 weeks of intra-uterine life. In the body of the stomach, development proceeded in four phases.

In the first phase between 15-20 weeks, submucosa was the thickest layer. In the second phase from 21st to 24th weeks, there was increase in the thickness of the mucosa and muscularis externa layers. From 25th to 27th weeks, in the third phase, there was again increase in the thickness of submucosa, this time however, the increase in thickness was more due to increase in the intercellular substance as was evident from the appearance of collagen fibrils increasing rapidly to form collagen bundles. In the last phase beginning at 28 weeks and continuing up to term, there was again a tremendous increase in the thickness of the mucosa and muscularis externa layers.

Gastric glands of the body of stomach also showed four stages in their development. These stages did not overlap with the four phases of growth mentioned above. In the first stage, between 15- 20 weeks, the glands were 'acinar'. In the next stage (21st and 22nd weeks) these glands were 'branching', showing 2 or more glands opening into the same fovea. In the third stage, in 23rd to 28th weeks, the glands elongated and became 'tubular'. In the fourth stage, beyond 28th week, increase in the length of these glands made them 'tortuous'.

According to Arey (1974), gastric pits in the fundic and body part of stomach appear by 8th week. It has also been stated that at this time of fetal life, intense reaction with histochemical stains for succinic dehydrogenase in the fundic and body regions heralds the appearance of true parietal cells, Grand et. al (1976). In our study sample, with the youngest fetus being 15-16 weeks old (CRL 80mm), gastric pits were clearly demonstrable. Eosinophilic parietal cells were the first to be seen from the 15 weeks' stage in our study as well. Although, the parietal cells are the earliest to appear, it has been pointed out that acid was rarely extractable from fetal stomach before 32 weeks of gestation, Keene and Hewer (1929).

True enterochromaffin cells were demostrared by 11 weeks in the entire stomach, Singh (1963), Singh (1966). However, by 10th week, enterochromaffin cells are found to be actively secreting, a newly found and interesting gastric peptide, involved in food intake control and growth hormone release, Rindi et al (2002).

By 13th week, cells resembling chief cells appear in the fundus and body. Though they show granules staining black with iron haematoxylin, they cannot be shown histochemically to contain pepsinogen until term, Salenius (1962). It has also been stated that simultaneous with the appearance of chief cells, PAS (Periodic acid Schiff) positive mucous neck cells also appear by 13th week and by 16th week, they actively secrete mucin. We concur with their findings that chief and mucus neck cells appear concomitantly, however, in our study these cells appeared by 22nd week. Keene and Hewer (1929) have demonstrated peptic activity in the human fetal stomach as early in gestation as 16th week. Use of more sensitive histo-chemical methods must have resulted in earlier detection of parietal, chief and mucous neck cells in other studies as compared to our study.

Arey (1974) has demonstrated first appearance of muscularis mucosa at 160mm CRL stage. In our study we could demonstrate its presence by the end of 22nd week (CRL of 148mm). At 148 mm CRL stage the layer was seen only as condensation and failed to take up stain for muscle when stained with Masson's tri-chrome stain. Clearly discernible smooth muscle cells taking up special stain were seen only by 28th week. Absence of differentiated smooth muscle cells at 148 mm CRL stage could be the reason why literature quotes the appearance of this layer at a later stage.

Grand et al (1976) have observed craniocaudal pattern of development in the appearance of pits and differentiation of glands. Pyloric pits appear by 10th week; glands differentiate by 11th week and may be completed by 13th week. In our study, the pyloric picture showed well-formed pits and glands by 15 to 16 weeks. At any stage, pyloric development appeared to precede that of body, this perhaps due to simpler glandular design in pyloric region as compared to fundic/body glands resulting in earlier differentiated appearance. We agree with Goldstein et al (1969) that from 20th week stage, gastric pits were deeper in the pyloric part and showed branching glands. Presence of parietal cells in the pyloric glands have been reported by Salenius (1962) and Lillibridge (1964). In our study although these cells were seen at the junctional zone, closer to the pyloric end the glands were devoid of parietal cells.

At all stages, both in body and pylorus circular muscle layer was seen in the muscularis externa, the longitudinal layer being added only by 28th week.

Thus, to conclude, all the four layers of stomach namely, mucosa, submucosa, muscularis externa and serosa were present by 15th week of gestation. Surface epithelium was cuboidal in the body from 15-20th week; from 21st to 22nd week columnar epithelium began to appear. Four phases of growth were seen in the body: submucosa was the thickest layer between 15-20 weeks, mucosa and muscularis externa being thicker from 21-24 weeks, in the third phase between 25-27 weeks submucosa again was the thickest layer. Mucosa and muscularis externa increased rapidly in the final phase beyond 28 weeks. Gastric glands in the body went through four stages of appearance, namely, acinar (15- 20 weeks), branching (21-22 weeks), tubular (23-28 weeks) and tortuous (>28 weeks). Parietal cells were the first to appear being present since 15th week, chief and mucous neck cells appeared by 22nd week. Muscularis mucosa appeared by the end of 22nd week (CRL 148 mm). Submucosa and lamina propria were reticulum of cells up to 23rd to 24th weeks, connective tissue fibers being seen subsequently. Muscularis externa showed circular muscle layer from 15th week, longitudinal muscle layer being added by 28th week. Pyloric region showed more complex mucosal folds, tall columnar surface epithelium was present since 15th week. Deeper gastric pits were present and glands showed mucous cells and a few parietal cells.

References:

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