Differences in cerebral response to esophageal acid stimuli and psychological anticipation in GERD subtypes - An fMRI study

This study was approved by the ethical committee of Peking University Health Science Center (reference number 0565), and all subjects gave informed consent in writing before commencement of the study.

We randomly enrolled 44 right-handed GERD patients who exhibited typical GER symptoms of heartburn and acid regurgitation at least twice a week together with 12 healthy controls. Among these, 5 patients and 1 control did not complete the study due to failure in cooperating with the testing sequence. The remaining 39 patients and 11 controls completed the protocol. After gastroscopy, ambulatory 24-hr esophageal pH monitoring and PPI trials, GERD patients were divided into 9 cases of RE (7 males/2 females, 56.7 ± 5.9 yrs), 11 cases of NERD+ (6 males/5 females, 44.5 ± 3.9 yrs), 8 cases of NERD-SI+ (4 males/4 females, 58.1 ± 3.8 yrs), and 11 cases of NERD-SI- (5 males/6 females, 47.9 ± 2.2 yrs). Criteria for exclusion from the study included such diseases as peptic ulcer, digestive cancer, previous abdominal surgery, Barrett's esophagus, IBS, diabetes mellitus, and the use of sedatives, selective serotonin reuptake inhibitors or other medication that might affect symptom perception. The patients who had taken PPIs during the previous 4 weeks were also excluded. The 11 healthy volunteers (5 males/6 females, 38.0 ± 3.7 yrs) were enrolled as controls after it was determined they had no gastrointestinal disorders through assessment of health history, reflux diagnostic questionnaires (RDQ), endoscopy and 24-hr pH monitoring.

All the patients and controls completed a RDQ and Symptom Check List-90 (SCL-90) psychological questionnaire, followed by gastroscopy and ambulatory 24-hr pH monitoring. The concept of GERD and general pathogenesis of acid reflux causing heartburn was explained to all subjects. Then they underwent an fMRI study.

GERD symptoms were evaluated with the RDQ, which includes two sections to assess the frequency and extent of symptoms including heartburn, acid regurgitation, food reflux and chest pain. These two sections have a total of 24 points. When the subject's score is ≥12, he is considered to have GERD. Patients were required to complete the questionnaire based on their symptoms over the preceding four weeks.

All subjects underwent gastroscopy (Olympus GIF) after fasting overnight. The esophagus was carefully evaluated for presence of mucosal injury. The extent of the esophageal mucosal damage was assessed using the Los Angeles grading system. The stomach and duodenum were also inspected to exclude possible lesions. Routine biopsies were taken in the gastric antrum and duodenal bulb to exclude eosinophilic gastroenteritis.

The extent of esophageal acid exposure was determined using the ambulatory Digitrapper MK III pH monitoring system (Synectic Medical, LTD, Sweden). After fasting overnight, a catheter with two pH probes was inserted via the nose into the esophagus; the proximal pH sensor was placed 5 cm above the upper limit of the lower esophageal sphincter (LES). Patients were asked to record their daily activities. Excess esophageal acid exposure was defined as pH < 4 over more than 5% of the total recording time [31], and analysis of recorded data was performed using standard commercially available software. Patients with pathological acid reflux but without esophagitis were classified as NERD+. Individuals without pathologic acid reflux and without esophagitis were classified as NERD-. Subsequently, the latter group was divided into the NERD-SI+ group (with positive symptom index) and NERD-SI- group (with negative symptom index). The symptom index (SI) was defined as the number of times a symptom occurs in association with acid reflux (pH <4) within a 5-minute time period divided by the total number of times the symptom occurs [32], and SI ≥50% is considered positive. In the NERD-SI- group, the patients were asked to undergo a PPI trial (omeprazole 20 mg twice daily for 7 days), and those whose RDQ scores showed over 50% improvement were placed in the NERD-SI-group. If the score did not show 50% improvement, the subject was excluded from this study [7]. The NERD-SI- patients were evaluated with the fMRI study sequence at least two weeks after the PPI trial.

The Symptom Check List-90 was used for psychological assessment. It is a self-reported symptom inventory reflecting the psychological distress of an individual, which includes 90 items that cover nine psychological areas such as somatization, obsessive-compulsive disorder, interpersonal relationship sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation and psychoticism. It is interpreted using 9 primary symptom types and 3 global indices of distress. Higher scores indicate more distress.

Statistic comparisons were performed using SPSS 11.0. Data are shown as mean ± SE and skew data is shown as quartile values. One-way ANOVA was used to analyze the differences among groups based on age, DeMeester scores, psychological measures and cerebral response data. Because of the small sample size in each group, we used Bonferroni adjustments to reduce the risk of type-I error. The MannWhitney U test was used to test the skew data. The mean difference is significant at the 0.05 level.

GERD patients were divided into four subgroups as RE, NERD+, NERD-SI+ and NERD-SI-. The average age of RE and NERD-SI+ patients was higher than that of the controls (P < 0.05). GERD subgroups showed no statistic difference in GER symptoms. RE and NERD+ patients had more esophageal acid exposure than NERD-SI+, NERD-SI- and control groups, but there was no statistical difference in acid exposure in RE and NERD+ patients (Table 1).

Table 2 shows the scores for the SCL-90 questionnaire. All GERD subgroups had higher scores for most items than controls, but after Bonferroni adjustment only the "positive items" and somatization scores showed significant differences (P = 0.10~0.000). NERD-SI- subjects showed the highest scores among the GERD subgroups, but this was not statistically significant.

Four GERD patients (2 RE, 1 NERD+ and 1 NERD-SI-) reported heartburn during acid infusion and the symptom was eased by saline infusion, but no other subject (all controls and other GERD patients) reported any discomfort during acid infusion. Regarding BOLD signals, there was no significant cerebral activation in the controls during intraesophageal acid perfusion. In contrast, the PFC region was significantly activated in all subtypes of GERD. In addition, the ACC regions were activated in RE and NERD-SI-groups but not in NERD+ and NERD-SI+ patients. The IC regions were activated in RE, NERD+ and NERD-SI-groups but not in NERD-SI+ patients (Figure 2A, Table 3). Table 4 and Figure 3A show the temporal and signal intensity characteristics and Figure 4A shows the activity volume of BOLD responses in the PFC, IC and ACC regions to intraesophageal acid stimulation in the GERD patients. The RE subgroup had the shortest peak-time and most extended volume in the PFC region after acid was infused, and the maximal fMRI signal intensity (MAX %) of this group was higher than that of the other three GERD subgroups. The activity volume of PFC in NERD-SI+ was more extended than that of NERD+ group and NERD-SI- group. In the ACC regions, the RE group also showed the highest MAX% and volume value, and these value were higher than that of the NERD-SI- group. The temporal characteristics showed no differences in the IC region among the RE, NERD+ and NERD-SI- groups.

During induced psychological anticipation stimulation, the PFC was significantly activated both in the control and GERD groups. Activation of the IC region was found in the RE, NERD-SI+ and NERD-SI- subgroups but not in the NERD+ group. Only NERD-SI+ and NERD-SI- patients showed ACC activation (Figure 5A). In the PFC region, the NERD-SI- subgroup had the shortest onset time and peak-time. The activity volumes of NERD-SI+ and NERD-SI- were more extended than those of controls and other GERD groups. Moreover, as compared with the RE subgroup and the controls, all NERD subjects had longer 50% offset-time, although the differences were not statistically significant after Bonferroni adjustment. In the IC region, the NERD-SI+ group showed the shortest peak-time. The temporal characteristics of the ACC response showed no difference in the NERD-SI+ and NERD-SI- groups (Figure 3B, 4B; Table 3, 4).

fMRI signal activation differences in actual esophageal acid perfusion and induced psychological anticipation stimuli were assessed in each GERD subtype. All five groups showed cerebral activation during the anticipation step, and multiple differences in activation area and temporal characteristics emerged. During anticipation in the RE group, the ACC was deactivated while the PFC was activated, but the signal intensity of the PFC was significantly lower in under induced anticipation than in actual acid perfusion stimulation (p = 0.010). In the NERD+ group, the IC was deactivated during anticipation. But there was no significant difference in the PFC activation in NERD+. In contrast with the RE and NERD+ groups, more cerebral areas including the IC (BA 13) and the ACC (BA24) were activated in the NERD-SI+ group during psychological anticipation. In the NERD-SI- group, although the activated area did not show much change, the onset-time and peak-time in the PFC and IC areas were obviously shorter in anticipation stimulation than actual acid infusion (p < 0.001, p < 0.001, p = 0.038, p = 0.038, respectively). The onset-time in the ACC was also shorter with induced anticipation (p = 0.030) (Figure 5B).

The RE group showed the most extensive activated areas (PFC, ACC and IC) and took a shorter time to reach signal activation peak in PFC during esophageal acid perfusion. Moreover, the reactive intensity of magnetic signals (MAX% values in both PFC and ACC regions) and cerebral activity volume were highest in the RE group as compared with the other GERD subtypes, indicating that this group has the most pronounced hypersensitivity to intraesophageal acid stimulus among the four subtypes of GERD. Some studies have reported on visceral sensation in RE mainly by evaluating symptoms resulting from intraesophageal acid perfusion or balloon distention, but results have been conflicting [22, 23]. In the current study, we analyzed cerebral activation and confirmed the RE group has much higher visceral sensitivity than NERD+, NERD-SI+ and NERD-SI-. The chronic tissue injury in the esophageal mucosa of RE patients may expose neural fibers to excessive acid, which may in turn enhance chemosensitivity [10]. Moreover, esophageal pain hypersensitivity to experimental acid infusion can be reversed by acid suppression via the proton pump inhibitors [13]. Long-term acid exposure may therefore be assumed to be the main mechanism for RE hypersensitivity.

The NERD+ group showed moderate ROI activation (PFC and IC) during esophageal perfusion. The average activation was more prominent in this group than in healthy controls, but no significant differences in activation time or signal changes were found in NERD+ versus other GERD subtypes. These findings are partly in agreement with previous observations by Marrero et al. who showed enhanced sensitivity to esophageal acid perfusion in patients with symptoms of GERD, and abnormal 24-hr esophageal pH monitoring studies, but no histological evidence of esophageal inflammation (NERD+ group) as compared with healthy controls [37].

NERD-SI+ showed the PFC activation with esophageal stimulation. When compared with NERD+, the extent of activation in the PFC was greater (p < 0.05) and the onset-time was shorter, although without significant difference. Fundamentally, positive symptom index means "acid-sensitive esophagus" [7], in the current study, we found the NERD-SI+ group showed more esophageal sensitively than the controls and NERD+ group.

In the NERD-SI- group, the three ROI areas were all activated and were more extensive as compared with activation in NERD+. In a recent study of NERD and functional heartburn patients, there was a correlation between pain threshold and acid exposure, and increased esophageal sensitivity was associated with a lower DeMeester score. Thus reflux negative (including NERD-SI- and functional heartburn) patients had lower pain thresholds as compared both with reflux positive patients and controls [38]. Our findings were consistent with this result.

In summary, during esophageal acid perfusion the four subgroups of GERD showed distinctly different activation patterns and all were hypersensitive as compared with controls. Among these groups, the RE group showed the most obvious acid sensitivity.

Under induced psychological anticipation, all of the GERD subgroups and controls developed cerebral activation. This result is partly in accordance with the study by Yaguez et al. which showed that anticipated visceral pain elicited cortical responses in healthy subjects [29]. These findings imply that the cerebral perception and sensation of visceral stimuli can be modulated by psychological factors. In fact, there have been increasing numbers of reports which support the view that emotional states, in particular anxiety and anger, are closely associated with greater perceptual and physiologic responses to visceral stimuli. Wright et al revealed that the perception of symptoms is increased when the NERD- patients are stressed [39]. Fass et al also demonstrated that acute auditory stress can exacerbate heartburn symptoms in GERD patients by enhancing perceptual response to intraesophageal acid exposure, which is associated with greater emotional responses to the stressor [21]. Sabate et al suggested anxiety and coping were significantly related with IBS patients' pain thresholds [40]. Some studies have shown that limbic areas in healthy volunteers and rheumatic arthritis patients, such as the ACC and medial PFC are associated with pain-related anxiety or depression during pain processing [41‐43]. A few studies have addressed anticipation of visceral stimulation. Berman et al reported reduced brainstem inhibition during anticipated pelvic visceral pain correlates with enhanced brain response to the visceral stimulus in women with IBS [30]. Yaguez et al reported actual and anticipated esophageal balloon distention could elicit similar cortical responses [29]. In our study, analysis showed there were different patterns among four groups of patients. RE showed deactivation of the ACC and NERD+ showed the IC area deactivated, while NERD-SI+ showed a greater activation in the ACC and the IC and NERD-SI- showed shorter onset-time and peak-time for activation in the PFC and the IC during anticipation. These results suggest psychological factors play a more important role in NERD-SI+ and NERD-SI- groups than in RE and NERD+.

NERD and RE are generally considered to represent a spectrum of GERD which ranges from mild (NERD) to severe (RE) [44]. To the best of our knowledge, this is the first study to demonstrate that subtypes of NERD, including NERD+, NERD-SI+ and NERD-SI-, display heterogeneous perception and visceral sensation using fMRI. During esophageal acid stimulation, the four subgroups of GERD presented different activation patterns and all were hypersensitive as compared with controls. Among these groups, RE showed the most obvious acid sensitivity. The four subgroups also showed different patterns under induced psychological anticipation, and psychological factors play a more important role in NERD-SI+ and NERD-SI- groups. This likely corresponds to the fact that patients in these two subgroups respond to PPI therapy poorly, and have important implications for improved understanding of the pathogenesis of the four subgroups of GERD, and particularly of the NERD subgroups.

There were some limitations in the current study. First, the mean ages of subjects in our study were not matched precisely among the subgroups and controls. Studies regarding the relationship between age and cerebral activation suggest that older control subjects show significantly less activation in PFC than younger controls [45]. In GERD, older patients showed reduced esophageal chemosensitivity to acid as compared to younger patients [46]. In our study, when age mismatch was taken into consideration, the GERD patients were even more sensitive to intraesophageal acid stimulation and psychological anticipation than controls. The second limitation of the current study is that the PPI responses in the NERD groups (except for the NERD-SI- group) were not checked. However, we enrolled these NERD patients according to the results of RDQ symptom questionnaire, gastroscopy and 24-h pH monitoring and none of these patients had PPI therapy prior to testing. The effectiveness of PPI on visceral sensation and perception might be an aspect to evaluate further.