Original Article
Relationship between headache and mucosal mast cells in pediatric Helicobacter pylori-negative functional dyspepsia
Cephalalgia 33(5) 323–329 ! International Headache Society 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0333102412472070 cep.sagepub.com
Jung Sook Yeom1, Myung Bum Choi1, Ji-Hyun Seo1, Ji Sook Park1, Jae-Young Lim1, Chan-Hoo Park1, Hyang-Ok Woo1, Hee-Shang Youn1, Gyung-Hyuck Ko2, Seung-Chul Baik3, Woo-Kon Lee3, Myung-Je Cho3 and Kwang-Ho Rhee3 Abstract Background: Although many patients with functional dyspepsia experience headache concurrently with dyspeptic symptoms, studies suggesting mechanisms underlying this phenomenon are limited. Herein, we explore the relationship between gastrointestinal inflammatory cells and presence of headache associated with dyspeptic symptoms in children with Helicobacter pylori-negative functional dyspepsia. Methods: Fifty-six patients with H. pylori-negative functional dyspepsia underwent upper endoscopy with biopsy to investigate recurrent epigastric pain or discomfort. Patients were divided into two groups according to self-reported presence of headache associated with dyspeptic symptoms. Inflammatory cells including mast cells, and enteroendocrine cells in the gastroduodenal mucosa were evaluated. Associations between headache presence and cellular changes in the gastroduodenal mucosa were examined. Results: Headache was not associated with the grade of lymphocytes, neutrophil infiltration, or enteroendocrine cell density in the gastroduedenal mucosa. However, headache was significantly associated with high mast cell density in the body (27.81 8.71 vs. 20.30 8.16, p < 0.01) and duodenum (23.16 10.40 vs. 14.84 5.88, p < 0.01). Conclusions: Presence of headache associated with dyspeptic symptoms is strongly related to mucosal mast cell density in pediatric patients with H. pylori-negative functional dyspepsia. Thus, our results may help clinicians understand and treat headache during dyspeptic symptoms in such pediatric patients. Keywords Functional dyspepsia, headache, mast cells, enteroendocrine cells, Helicobacter pylori Date received: 19 July 2012; revised: 30 October 2012; 15 November 2012; accepted: 21 November 2012
Introduction Headache is frequently associated with functional gastrointestinal disorders (1,2). The association between chronic headache, migraine, and irritable bowel syndrome has been confirmed by several clinical observations and epidemiological studies (3,4). In a Danish study, the coincidence rate of irritable bowel syndrome and headache was found to be 60%, though this rate depended on the course of the syndrome (5). Although functional dyspepsia is a common gastrointestinal disorder in children (6),
1 Department of Pediatrics, Gyeongsang National University School of Medicine, Gyeongsang Institute of Health Science, Republic of Korea 2 Department of Pathology, Gyeongsang National University School of Medicine, Gyeongsang Institute of Health Science, Republic of Korea 3 Department of Microbiology, Gyeongsang National University School of Medicine, Gyeongsang Institute of Health Science, Republic of Korea
Corresponding author: Hee-Shang Youn, Department of Pediatrics, Gyeongsang National University School of Medicine, 92 Chilam-dong, Jinju 660-751, Gyeongnam, Republic of Korea. Email:
[email protected]
324 there has yet to be a similar study evaluating the relationship between functional dyspepsia and headache. However, we have encountered many patients with concurrent functional dyspepsia and headache in clinical practice, and a few studies have suggested possible common mechanisms underlying this phenomenon. Schurman et al. (7) examined the relationship between gastroduodenal mucosal inflammation and psychological functioning in pediatric patients with functional dyspepsia. They found a correlation between mast cell density and somatization and hypothesized that physical symptoms, like headache, in functional dyspeptic patients, result from mediators released by mast cells (7). Their hypothesis is attractive because increasing evidence suggests that mast cells play a role in generating dyspeptic symptoms as well as headache via mast cell-nerve interactions (8–10). In addition, mast cells are of particular interest because they have been implicated as an important link between stress, gastrointestinal dysfunctions, and headache. Mast cells may thus explain the interaction between psychological and biological factors in the development and persistence of abdominal pain and headache (11,12). However, it may be difficult to determine whether headache is directly correlated with mucosal mast cell density in pediatric patients with functional dyspepsia because headache was one of several symptoms of somatization in the study by Schurman et al. (7). According to their results, headache should occur simultaneously with dyspeptic symptoms, yet they did not mention such a concurrence in their study. Therefore, the present study aimed to explore the relationship between gastroduodenal inflammatory cells and presence of headache associated with dyspeptic symptoms in functional dyspepsia in children. Although our primary interest was mast cells, we also examined additional characteristics of general inflammation (i.e. neutrophils for acute inflammation and lymphocytes for chronic inflammation) and enteroendocrine cells (i.e. enterochromaffin cells, enterochromaffin-like cells, and G cells) to determine whether such a relationship is unique to mast cells. General inflammation in the gut is associated not only with gastrointestinal diseases including functional dyspepsia (13) but also with headache (14). Additionally, mast cells can recruit these circulating inflammatory cells to the gastric mucosa (15). Most histamine in the body is synthesized by mast cells, and histamine has been suggested to play a key role in generating dyspeptic symptoms and headache (8–10). However, enteroendocrine cells also synthesize important signaling molecules that link the brain and gut, including most of the body’s serotonin and some histamine (16). Therefore, it was impossible to determine which cells were directly related to functional dyspepsia or headache if nonspecific general inflammation or
Cephalalgia 33(5) enteroendocrine cells was combined with mast cell infiltration. We excluded functional dyspeptic patients with Helicobacter pylori infection in order to avoid the extragastric effects of these bacteria.
Methods Patients Seventy-four consecutive school-age children (range six to 12 years) underwent endoscopy to investigate recurrent epigastric pain or discomfort and were diagnosed with functional dyspepsia at the Department of Pediatrics of Gyeongsang National University Hospital from August 1996 to December 2002. Diagnosis was made according to the Rome III pediatric criteria (17). Eighteen patients were excluded because of H. pylori infection. The remaining patients were divided into two groups, a headache-positive (þ) group and a headache-negative () group, according to the self-reported presence or absence of headacheassociated dyspeptic symptoms. This study was approved by the Gyeongsang National University Hospital Institutional Review Board (GNUHIRB2011-107).
Gastroduodenal inflammation Inflammatory cells, neuroendocrine cells, and H. pylori were measured in mucosal biopsy specimens obtained by endoscopy. Two biopsy specimens each were taken from the gastric antrum, body, and duodenal mucosa. The biopsy specimens obtained from the antrum and body were inoculated in 1% urea broth. The urease test was considered positive when the urea broth at 37 C showed a color change from yellow to pink within 48 hours. Specimens were stained with hematoxylin and eosin in the standard fashion to detect H. pylori and for general inflammation grading. We excluded patients if H. pylori was detected by urease test or histopathologic analysis of any specimen. To grade the acute and chronic inflammation of gastroduodenum, neutrophil and lymphocyte infiltration was conducted according to the Sydney system: grade 0, absent; grade 1, mild; grade 2, moderate; and grade 3, severe. Mast cells were enumerated using immunohistochemical techniques. Paraffin-embedded blocks were sectioned at 5 mm thickness, and the endogenous peroxidase activity was inhibited by treating the sections with 0.5% periodic acid for 30 min. The specimens were incubated for 20 hours at 4 C with 1:500 diluted mouse anti-human mast cell tryptase Ab (Dako, Carpinteria, CA, USA). They were then washed three times with 0.05 M phosphate-buffered saline (PBS) for 10 min and incubated with biotinylated goat
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Yeom et al. anti-mouse IgG (Dako) for 90 min. Next, the specimens were incubated with streptavidin (Dako) for one hour. They were then washed and stained with 0.025% 3,30 diaminobenzidine tetrahydrochloride. To evaluate enteroendocrine cells, Grimelius silver staining was performed. Afterward the specimens were reacted with silver solution (30 mg silver nitrate, 90 ml distilled water (DW), and 10 ml 0.1 M pH 5.6 acetate buffer) at 60 C for four hours. We reduced the specimen for one min with Bodian reducing solution (1 g hydroquinone, 5 g sodium sulfite, and 100 ml DW) that had been previously warmed to 60 C and then counterstained with light green solution for 1 min. After scanning tissue sections at low magnification (100) to identify the most involved areas, tryptasepositive mast cells and enterochromaffin-like cells were counted in the lamina propria and epithelium in five consecutive non-overlapping fields at high power (400) using Image J. Average cell counts were determined and expressed as the number of cells per high power field.
Statistical analysis All statistical analyses were performed using SPSS version 12 (SPSS Inc., Chicago, IL). Fisher’s exact test, Student’s t test, and Mann-Whitney U test were used to examine the association between headache and both inflammatory cell density and grading. All p values were two tailed; an a level of
