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We elucidated the distribution of interstitial cells of Cajal (ICC) in human stomach, using cryosection and c-Kit immunohistochemistry to identify c-Kit positive ICC. Before c-Kit staining, we routinely used hematoxylin and eosin (HE) staining to identify every structure of human stomach, from mucosa to longitudinal muscle. HE staining revealed that the fundus greater curvature (GC) had prominent oblique muscle layer, and c-Kit immunostaining c-Kit positive ICC cells were found to have typical morphology of dense fusiform cell body with multiple processes protruding from the central cell body. In particular, we could observe dense processes and ramifications of ICC in myenteric area and longitudinal muscle layer of corpus GC. Interestingly, c-Kit positive ICC-like cells which had morphology very similar to ICC were found in gastric mucosa. We could not find any significant difference in the distribution of ICC between fundus and corpus, except for submucosa where the density of ICC was much higher in gastric fundus than corpus. Furthermore, there was no significant difference in the density of ICC between each area of fundus and corpus, except for muscularis mucosa. Finally, we also found similar distribution of ICC in normal and cancerous tissue obtained from a patient who underwent pancreotomy and gastrectomy. In conclusion, ICC was found ubiquitously in human stomach and the density of ICC was significantly lower in the muscularis mucosa of both fundus/corpus and higher in the submucosa of gastric fundus than corpus.
Interstitial cell of Cajal (ICC) has long been found in gastrointestinal (GI) tract of animal . It is the end of the last century where researchers began to assume ICC as a regulator of GI motility in animal, including humans, throughout from esophagus to anus [2-6]. Many studies indicated that ICC is the genuine pacemaker cells which generate spontaneous electrical activities, known as slow waves in GI tract [7,8]. Slow waves from ICC spread passively via gap junctions to neighboring GI smooth muscle cells and then produce spontaneous contractions like peristalsis [7-9].
ICC is a myoid cell of mesenchymal origin with pacemaking ability . ICC normally expresses the protooncogene c-Kit, therefore, antibodies raised against the gene product (c-Kit) are useful for the identification of ICC [7,8]. c-Kit is a receptor tyrosine kinase, and its activation by ligand [stem cell factor (SCF) or "Steel" factor] is essential for the development of ICC. Therefore, a blockade of c-Kit with neutralizing antibodies  and/or mutations of c-Kit or Steel  impair normal development of ICC in the myenteric plexus region (ICC-MY) of small intestine. It was reported that all subtypes of ICC in human stomach are been developed as early as 4th month developmental stage with their specific functions . To date, at least two types of major ICC have been identified in the GI tract: One is ICC-MY of myenteric region, and the other is a second population of ICC which are distributed outside the myenteric region and is abundant in deep muscular plexus through the muscle layers (intramuscular ICC, ICC-IM). In general, ICC-MY is believed to form two dimensional networks in the neural plexuses and play a role as a pacemaker , whereas ICC-IM which is known to occupy only 5% of the tunica muscularis  is known to form synaptic connection with nerve terminals of enteric motor neurons of the stomach and play a central role, receptor inputs from motor neurons, although there are still controversies [13-15]. In accordance with this notion, significant reduction of density and constructional destruction of ICC with abnormal functions have been suggested to be responsible for many GI motility disorders and diseases such as constipation, infantile hypertrophic pyloric stenosis and acquired megacolon [16,17]. Indeed, ICC is widely accepted today as an essential target to be considered seriously in many of abnormal GI motility diseases.
In addition to the above mentioned aspect on the motility, ICC has also been shown to be related to inflammatory diseases such as inflammation-induced dysmotility, which is ascribed to damage of ICC [18,19]. In fact, pathophysiological changes of ICC have been reported in GI mucosa and submucosa of diverse gastric diseases such as gastritis and adenocarcinoma . To date, however, only a few studies have attempted to identify and elucidate the role of ICC outside the musculature of the GI tract such as mucosa, microvilli, submucosal plexus and muscularis mucosa [1,21,22]. Furthermore, a mutation of proto-oncogene c-Kit has been also reported in a sub-group of GI cancer such as colorectal carcinomas and gastrointestinal stromal tumors (GIST) . c-Kit is essential for the maintenance of normal hematopoiesis, melanogenesis and differentiation of mast cell and ICC . Since c-Kit is known to maintain their proliferation, migration, and survival , the malfunction of c-Kit is thought to be associated also with a certain neoplastic disorders and gastric dysmotility in mucosa, microvilli, submucosal plexus and muscularis mucosa [1,21,22]. There is one more type of muscle in muscular layer of human gastric wall, also called oblique muscle, which is more prominent in fundus than corpus. However, physiological role of this muscle and the presence of ICC within the muscle are not yet well understood. Therefore, we first attempted to clarify the distribution of ICC in whole gastric wall, spanning from mucosa through muscularis mucosa, submucosa, oblique muscle, circular muscle, and myenteric plexus to longitudinal muscle. In fact, pathologic changes such as inflammation and carcinogenesis of superficial mucosa and muscularis mucosa might well impair harmonious movement of mucosa and villi in GI tract. Second, we attempted to identify gastric regional differences by classifying stomach to fundus greater curvature, corpus greater curvature and corpus lesser curvature. Since distribution, density and role of ICC in human stomach are not well understood at present, we designed this study to elucidate the distribution of c-Kit positive ICC with its density in human stomach.
Human gastric fundus and corpus from both greater and lesser curvature were obtained from 14 gastric cancer patients who underwent subtotal gastrectomy. Whole thickness samples were taken from the gastric wall of each patient, in particular from the fundus (n=4) and corpus (n=9) of the greater curvature, and the corpus of the lesser curvature (n=1). Specimens were removed from macroscopically normal tissue far from the neoplastic area immediately after surgical operation. All patients gave written informed consent and this experimental protocol using human stomach was also approved by the Institutional Review Board for Clinical Research of Chungbuk National University, School of Medicine, Korea. Specimens in Krebs-Ringer bicarbonate (KRB) solution were pinned on sylgard plate in order to maintain original shape and length. Then, connective tissues were removed, cut and pinned to rectangular shape (2×3 cm, width and length). Finally, these gastric tissues were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer for 24 hours at room temperature (RT). Two longitudinal sections, 3 cm in length and 3 mm in thickness, were taken from each fixed tissue. The sections were processed on a computerized tissue processor (Tissue-Tek VIP; Sakura Finetek, Torrance, CA, USA) and embedded in paraffin (Paraplast Medium; Leica Biosystems, Maarn, The Netherlands) on a tissue embedding console system (Tissue-Tek TEC; Sakura Finetek, Torrance, CA, USA). Five formalin-fixed paraffin tissue blocks were also retrieved from the archive of the Pathology Department at Chungbuk National University Hospital. They consisted of one tissue block from normal corpus of the stomach, resected at Whipple operation for pancreas adenocarcnoma, and four tissue blocks from gastric adenocarcinoma resected at subtotal or total gastrectomy.
All paraffin embedded tissue blocks were sectioned at 4 µm thickness with a microtome, and slides were then prepared. Paraffin was removed from the slides with xylene treatment. The sections were rehydrated with an ethanol series and stained with hematoxylin and eosin. Final stages of processing were performed with an automated slide stainer (Tissue-Tek Prisma; Sakura Finetek, Torrance, CA, USA) and automated coverslipper (Tissue-Tek Glas; Sakura Finetek, Torrance, CA, USA).
Sections from paraffin embedded gastric tissues were cut at 4 µm thickness and mounted on positively charged slides (Superfrost Plus; VWR Internatinal, West Chester, PA, USA). Deparaffinization and antigen retrieval prior to immunostaining were simultaneously accomplished on an automated PT module (Lab Vision, Fremont, CA, USA). Immunohistochemistry was done on an automated immunostainer (Autostainer 360; Lab Vision, Fremont, CA, USA) according to the manufacturer's protocol. Peroxidase staining was done using UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific, Fremont, CA, USA). Briefly the sections were incubated in hydrogen peroxide block for 10 minutes to reduce nonspecific background staining due to endogenous peroxidase. After washing in phosphate buffered saline plus Tween 20 (20×) (PBS; ScyTek laboratories, Logan, Utah, USA), they were incubated with ultra V block for 5 minutes at RT to block nonspecific binding. The sections were then incubated with polyclonal rabbit anti-human CD117 (Dako, Carpinteria, CA, USA) at a dilution of 1:400 and monoclonal mouse anti-human mast cell tryptase (clone AA1; Dako, Glostrup, Denmark) at a dilution of 1:200 for 40 minutes at RT. After washing in PBS, they were incubated at RT with primary antibody enhancer, followed by washes in PBS and incubation with the HRP polymer for 15 minutes at RT. Sections were then washed in PBS, followed by staining with DAB Plus Chromogen and Substrate. Counterstaining was performed with hematoxylin. Negative controls were prepared by omitting primary antibodies or by substituting them with a nonimmune serum in order to check the specificity of the immunostaining. All sections for histologic and immunohsitochemical analysis were examined using a microscope (BX50; Olympus Corporation), and photographs were taken with attached camera (ProgRes C14; JENOPTIK, Jena, Germany) operated with CapturePro software (JENOPTIK, Jena, Germany).
KRB solution (CO2/bicarbonate-buffered Tyrode) contained (in mM): NaCl 122, KCl 4.7, MgCl2 1, CaCl2 2, NaHCO3 15, KH2PO4 0.93, and glucose 11 (pH 7.3~7.4, bubbled with 5% CO2/95% O2). PBS was purchased from ScyTek laboratories (USA). All drugs used in this study were purchased from Dako (anti-human c-Kit, anti-human mast cell tryptase) and Sigma.
The data are expressed as means±SEM. Statistical significance was estimated by Student's t-test. p<0.05 was considered to be statistically significant.
Fig. 1A shows histology of three regions (fundus greater curvature, corpus greater curvature and corpus lesser curvature) of human stomach. In Fig. 1Ba, we could identify muscle layers of oblique, circular and longitudinal muscle in greater curvature of fundus. As shown in Fig. 1Ba and 1Bb, we routinely identified whole structure of stomach first by HE staining before immunohistochemical analysis and then compared it to the structure revealed by c-Kit immunoreactivity. From these stainings, we verified that oblique muscle layer was more prominent in gastric fundus than gastric corpus in human stomach (Fig. 1B, ,2,2, ,3,3, ,4,4, 5B and 5C). c-Kit positive immunoreactivity was observed in every layer of all three regions in human stomach.
HE-stained structure of human gastric fundus was compared with that of c-Kit immunoreactivity. As shown in Fig. 2 and and4A,4A, c-Kit positive ICC was observed, ranging from muscularis mucosa all the way to longitudinal muscle layer in fundus of greater curvature. In each layer, long spindle-like (fusiform) cell body (arrow head) with multiple processes (arrow) protruded from the central cell body was observed (Fig. 2, ,4A4A and and5A).5A). ICC in septa (ICC-SEP) is indicated by double arrow head. In oblique muscle (Fig. 4Ad) and myenteric border (Fig. 4Af) of human gastric fundus, we could observe typical ICC maintaining fusiform cell body and attached processes together. However, we could not generally identify whole ICC morphology, because we sliced the tissue using cryosection for c-Kit immunostaining. Therefore, dotted dark brown cells with nucleus or individual processes such as dark brown rod are c-Kit positive immunoreactivity usually found, although ICC forms network in fact. In this study, nevertheless, we clearly demonstrated c-Kit positive ICC of muscularis mucosa (Fig. 2Cb, 3Ab, 4Ab, 4Bb and 5Ab). Since ICC in the muscularis mucosa has been reported only in one or two animal studies, the present finding is valuable, showing the possibility that ICC might play a role for harmonious movement of mucosa and microvilli.
As shown in Fig. 3, ,4B,4B, 5A and 5C, c-Kit positive ICC was observed also in every layer of muscularis mucosa, all the way to longitudinal muscle layer in gastric corpus of both greater and lesser curvature. Fusiform cell bodies and multiple processes from them were observed, as shown in Fig. 3Ac, 3Bc, 3Cb, 4Bd, 4Be and 4Bf. In circular muscle, myenteric border and longitudinal muscle (Bf inset) of gastric corpus, long spindle-like cell bodies, processes and even numerous ramifications arising from cell processes were observed, as shown in Fig. 4Bb, 4Bd, 4Be, 3Cb, and 3Ac. Fig. 5A shows ICC of gastric corpus from lesser curvature. In corpus lesser curvature, c-Kit positive ICC was found also in all the layers examined.
Since we could not get enough number of gastric tissues from lesser curvature, the distribution of ICC was compared between fundus (Fig. 5B) and corpus (Fig. 5C) from greater curvature of human stomach. We counted dense cell body with nucleus as one individual ICC under 400× magnification field of microscopy, and the number of ICC twice under double or three side-viewed microscopy together and then averaged it for quantitative analysis. In fundus greater curvature, the averaged number of ICC of muscularis mucosa, submucosa, oblique muscle, circular muscle, myenteric border and longitudinal muscle were 1.7±0.9, 8±0.7, 6±3, 4.8±1.9, 6.3±0.5, 4.9±1.2, rescpectively (n=3, 4, 2, 4, 4 and 4, respectively; Fig. 5A). In corpus greater curvature, the averaged number of ICC of muscularis mucosa, submucosa, circular muscle, myenteric border and longitudinal muscle were 2.3±0.5, 4.1±0.7, 5.1±1.0, 4.3±0.6, 3.8±0.4, respectively (n=8, 10, 10, 10 and 10, respectively; Fig. 5B). In fundus and corpus of stomach, the density of ICC in muscularis mucosa was significantly less than that of other layers of fundus (submucosa and myenteric area) and corpus (circular muscle, myenteric area and longitudinal muscle) (p<0.05; Fig. 5B and 5C). Meanwhile, the density of ICC in muscularis mucosa was less than other layers (fundus oblique, circular and longitudinal muscle, and corporal sub mucosa), but with no significant difference (p>0.05; Fig. 5B and 5C).
Furthermore, no significant difference was observed between two regions except for ICC from submucosa. The averaged number of ICC in submucosa from fundus greater curvature was significantly higher than that from corpus greater curvature (p<0.05; Fig. 5B and 5C).
Distribution of ICC was compared between the corpus (Fig. 6A) of normal person and cancerous tissue (Fig. 6B) obtained from a patient who underwent pancreotomy and gastrectomy. c-Kit positive ICC was observed in every layer. c-Kit positive immunoreactive ICC was found also in gastric mucosa of cancer area as well as normal area.
As shown in Fig. 2Cb, 3Ab, 4Ab, 6Aa and 6Ba, c-Kit positive immunoreactivity was found in all the mucosae from three regions. In addition, ICC was found in cancer area. It was of an interest to find c-Kit positive ICC-like cells [c-Kit (+) ICC-like cells] and they have been suggested to be involved in inflammation, dysplasia and tumorigenesis in human stomach (see discussion).
In the present study, we investigated the distribution of c-Kit positive ICC in fundus greater curvature, corpus greater curvature and corpus lesser curvature of human stomach to determine whether they show regional variations of ICC density along the circumference and along the length in the organ. The results showed that ICC was distributed in every layer of gastric wall in decreasing order of mucosa, muscularis mucosa, submucosa, oblique muscle, circular muscle, myenteric region, longitudinal muscle. There was no significant difference in the density of ICC between fundus and corpus except for ICC in submucosa. The density of ICC in submucosa of fundus was higher than that of corpus, and the density of ICC in muscularis mucosa of each fundus and corpus was significantly lower than other layers.
As well known, ICC expresses a proto-oncogene c-Kit . However, except for marker for ICC, c-Kit and/or SCF have been shown to be expressed also in variety of normal tissues such as brain, placenta and lung  as well as malignant tumors and gastric carcinoma cell line [20,26]. In addition, activation of c-Kit by mutations has been shown also in a number of human malignancies such as human gastric carcinomas [20,27], gastrointestinal GIST , small cell lung cancer , and colorectal cancer . Therefore, ICC seems to have many abilities, and it might be associated also with tumor growth and/or the process of carcinogenesis in gastric mucosa. Although data are not shown here, we also found c-Kit positive ICC-like cells [c-Kit (+) ICC-like cells] which are associated with inflammation and/or regeneration of epithelium in human stomach (manuscript in submission). We confirmed the existence of c-Kit (+) ICC-like cells in human gastric mucosa: They are present mainly in the stroma around repair zone of the glands in chronic gastritis as well as in normal mucosa, whereas they seem to redistribute over the entire mucosa in gastritis with intestinal metaplasia. c-Kit (+) ICC-like cells around repair zone are found tightly attached to epithelial cells, but not to metaplastic epithelial cells. Thus, c-Kit (+) ICC-like cells seem to have a role in epithelial recovery process, and may even be associated with carcinogenesis of human gastric mucosa (manuscript in submission). Further study is in progress to get more evidences.
Near the end of the last century, ICC was suggested to be a regulator of GI motility in animals, including humans, throughout from esophagus to anus [2,3,6], and the distribution was found not even in stomach, small intestine and colon, showing regional differences. For example, the density of ICC-MY was higher at antrum and corpus than at fundus in murine stomach, whereas ICC-IM was denser at fundus and corpus than at antrum . Furthermore, the density of ICC-IM was lower at lesser curvature than greater curvature within the same corpus region [28,29], and the density of ICC-IM was much higher at circular muscle layer than at longitudinal muscle layer within muscle coat. Therefore, the regional unique distribution of ICC seemed to be related to unique physiological function of each GI tract. In fact, the density of ICC in colon was much higher at proximal colon, where strong haustral contractions toward anus begin . Recently, ICC was reported also in human stomach, small intestine and colon [15,31,32], and all subtypes of ICC were shown to develope as early as 4th month development stage . However, still little is known on the regional variation of ICC in human stomach. In the present study, therefore, we attempted to elucidate the distribution of ICC in fundus greater curvature, corpus great curvature and corpus lesser curvature. In fundus and corpus of great curvature, ICC was observed in every layers in decreasing order of mucosa, muscularis mucosa, submucosa, circular muscle, myenteric area, and longitudinal muscle. Only in submucosa layer, ICC density of fundus greater curvature was higher than that of corpus greater curvature (Fig. 5B and 5C), and the density of ICC in muscularis mucosa of each fundus (compared to that of submucosa and myenteric region) and corpus (compared to circular muscle, myenteric region and longitudinal muscle) was also significantly lower than other layers (Fig. 5B and 5C). In the case of oblique muscle, as already known, fundus showed prominently thick oblique muscle layer (Fig. 1A, ,2,2, ,3A3A and and5B),5B), where ICC was overexpressed (Fig. 4Ad). This finding seems to coincide with the report that ICC is overexpressed in muscular hypertrophy. We plan to address on this finding in the future.
There are species differences and regional variations in the morphology and distribution of ICC of GI tract. ICC of bipolar type has been identified in muscle coat of mouse and guinea-pig [13,33] and in fundus of human fetus , whereas multipolar type has been reported in myenteric border of corpus and antrum of mouse and guinea-pig [13,33]. ICC in rat and dog antrum is all bipolar type . In the present study, we failed to find out exactly how many types of ICC were present in human stomach. Nonetheless, we found some regional differences in density of processes and ramifications of ICC. The processes and ramifications of ICC of corpus greater curvature were more prominent in myenteric area (Fig. 3Cb vs Fig. 2Bb, Fig. 4Be vs Fig. 4Af) and longitudinal muscle layer (Fig. 3Ac vs Fig. 2Ab, Fig. 4Bf vs Fig. 4Ag; see inset of Fig. 4Bf) compared with those of fundus greater curvature. The oblique muscle layer was significantly prominent only in fundus, however, did not show consistent result (Fig. 2Ad vs Fig. 4Ad). The corpus lesser curvature did not show much process and ramifications (Fig. 5). The study using cryosection seems to have a limitation in elucidating whole morphology of ICC cells.
In addition to the morphology of ICC, the distribution of ICC has been known to show regional and species differences in animals such as human  and dog . In human stomach, ICC was reported to be present at the border of submucosa in antrum , but absent in the inner part of the corporal circular muscle layer . In the present study, however, we observed ICC practically everywhere, particularly dense in submucosa (Fig. 2Ac, Fig. 3Ab, Fig. 4Ab, Fig. 4Ac, Fig. 4Bc and Fig. 5Ac). We showed the existence of intramuscular ICC in muscle layers of oblique (Fig. 4d, corpus), circular (Fig. 3Bc, corpus) and longitudinal muscles without significant difference in their distribution (Fig. 2Ab, fundus; Fig. 3Ac and Fig. 4Bf, corpus). ICC in septa (ICC-SEP) was reported only in corpus and antrum in human and animal stomach [34,35], however, we found ICC-SEP in fundus (Fig. 4Ae, circular muscle; Fig. 4Ag, longitudinal muscle), corpus (Fig. 4Bd, circular muscle), and corpus lesser curvature (Fig. 5A).
In the present study, we found ICC in every layer of fundus including myenteric region (Fig. 2, ,4A,4A, ,5B).5B). In GI tract, spontaneous contraction is ascribed to pacemaking potential generated by ICC-MY. However, fundus does not show spontaneous motility and has ICC-MY in myenteric border of mouse and guinea-pig [13,33]. Recently, Kim et al. found spontaneous contraction in fundus smooth muscle of human stomach, and circular and longitudinal muscles showed rhythmical spontaneous contraction, respectively (manuscript in submission). However, spontaneous contractility almost disappeared by stepwise increment of isotonic load to 1 g (data not shown). Gastric fundus plays a storage function called gastric receptive relaxation via vagally mediated mechanism . The gastric fundus is innervated by both excitatory cholinergic neurons and NANC (non adrenergic- and non cholinergic) inhibitory neurons, which are associated with gastric receptive relaxation . Nitric oxide (NO) has been recognized as an inhibitory neurotransmitter to mediate smooth muscle relaxation in the mammalian gastrointestinal tract. All these results imply that the basic role of fundus is passive relaxation. Therefore, spontaneous contractility of fundus observed in this study was somewhat unexpected. In fact, ICC has already been shown to exist in human fundus including myenteric area [31,35]: it was pointed out that the difference between human and mouse and guinea-pig was due to the presence of ICC-MY in myenteric border. The above finding is consistent with our present data, since we also found ICC-MY in human gastric fundus (Fig. 2, ,4A4A and and5B).5B). Therefore, the ICC might be responsible for the spontaneous rhythmicity of circular and longitudinal smooth muscle of human gastric fundus. Since it produced spontaneous motility in resting state and disappeared in stretched state, it is highly likely that spontaneous rhythmicity would disappear after food intake to serve as a reservoir, and that spontaneous rhythmicity in time would return to help propel food, taken by receptive relaxation, or remain in fundus to gradually distal stomach.
In this study, we found also c-Kit positive cells in submucosa and muscularis mucosa in human gastric fundus and corpus. Recently, ICC associated with submucosal plexus (ICC-SP) was found in guinea-pig stomach, seemingly contributing to the regulation of the mucosal function such as secretion, absorption and transportation of fluid . In accordance with pacemaker activity of muscularis mucosa, the spontaneous pacemaking motility was reported earlier in rabbit stomach (4.55~4.70 cycles/min) . In fact, the spontaneous motility of muscularis mucosa might also be ascribed to glandular pressure and red blood cell velocity, even in humans , and it could regulate the amount of blood in gastric mucosal venous system . These reciprocal changes might alter the intramucosal pressure, thus affecting the movement of tissue fluid and the function of the gastric cells indirectly. To date, ICC-like cells in muscularis mucosa was reported also in rat stomach including pacemaking response [19,37]. In addition, ICC-SP has a close structural relationship with the muscle fibers of the muscularis mucosa, therefore, they may also be functionally involved in villous movement. In fact, although the presence of ICC-SP in the small intestine has not yet been demonstrated, muscle bundles within the villi are known to be continuous with those of the muscularis mucosa, and nerve fibers from the submucous plexus innervate the muscularis mucosa and smooth muscle fibers in the cores of the intestinal villi . Since ICC-SP and ICC were found in muscularis mucosa of human stomach in this study, they might possibly be associated with spontaneous villous movement and physiological function of gastric mucosa. In this study, we found that the density of submucosal ICC in fundus greater curvature was significantly higher than that of corpus greater curvature (p<0.05; Fig. 5B and C). Since gastric acid is secreted from fundus gastric glands, the ICC of submucosa and muscularis mucosa of fundus might well be related to increased activity of secretion through coordination of mucosal and/or villous movement.
This work was supported by the research grant of the Chungbuk National University in 2008.