The Clinical Feature Of And The Diagnostic Value Of In-Vivo Confocal Laser Endomicroscopy On Patients With Non Erosive Reflux Disease

BackgroundNon-erosive reflux disease (NERD) is a common condition defined by the presence of typical symptoms of gastroesophageal reflux and the absence of mucosal breaks seen by standard endoscopy; NERD accounts for more than50%of cases of GERD. In Asia, up to70%of patients with gastroesophageal reflux disease (GERD) suffer from this disease. The fundamental pathogenesis in GERD is the exposure of the esophageal epithelium to acidic gastric contents, resulting in histopathologic injury and/or symptoms. There is a good correlation between esophageal acid exposure and endoscopic changes of the membrane. Duration of esophageal acid exposure correlates with severity of erosive esophagitis, and the number of prolonged acid reflux episodes and esophageal exposure to acid and pepsin is increased in more severe reflux. However, NERD can occur with or without abnormal esophageal acid exposure during24-h ambulatory monitoring. Unfortunately, the sensitivity of24-h pH monitoring for diagnosing NERD is unsatisfactory. The "gold standard" for the diagnosis of NERD is still lacking.Multiple intraluminal impedance (MⅡ) monitoring is a recently developed technique for examining gastroesophageal reflux content.24-hour esophageal MⅡ monitoring combined with pH-metry (MII-pH) allows for detection of all reflux events in terms of acidity (acidic, weakly acidic, weakly alkaline) and composition (liquid, gas or mixed) and bolus movements and thus establish the association of the reflux with symptoms. Study has shown that combined pH-impedance monitoring was more accurate than use of pH alone for the detection of both acidic and weakly acidic refluxes. Succedently, normal values of reflux patterns in healthy subjects have been established in Western and Chinese population. However, the value of MII-pH monitoring in patients with NRED has not been elucidated. We tried to figure out the problem in our study. The aim of the first part of the study is to evaluate the prevalence and the characteristics of reflux episodes in patients with NERD using24h MII-pH monitoring.NERD patients have shown microscopic changes in the distal esophageal epithelium that include elongation of the papillae, proliferation of basal cells and dilated intercellular spaces (DIS) within the squamous epithelium. DIS of the esophageal epithelium is considered a sensitive marker of tissue damage in GERD patients and the most appropriate marker of damage evaluation in NERD. On the other hand, upper gastrointestinal endoscopy is the main clinical tool for visualizing esophageal lesions. However, micro-alterations in NERD may not be visualized by standard endoscopy. Previous studies have reported patients with GERD showed an increased abnormal intrapapillary capillary loops and were more likely to demonstrate micro erosions and increased vascularity at the squamocolumnar junction. However, there is little evidence in vivo of micro-alterations of the esophagus epithelium in patients with NERD.Confocal laser endomicroscopy (CLE) is a newly developed endoscopic technique that allows the observation of living cells, tissue as well as vascular networks of the mucosal layer in the gastrointestinal tract during ongoing endoscopy. The highly magnified images (approximately1000-fold) of the gastrointestinal tract mucosa can permit real-time histological analysis of the site during endoscopy. Therefore, CLE can provide precise assessment of the esophageal squamous epithelial cells and IPCLs without the need of biopsy. However, to date, there have few studies have focused on the use of CLE to assess the micro-alterations of NERD in vivo. The aim of the latter two part of the present study is to investigate the use of electron microscope and CLE for in vivo evaluation the micro-alterations of the esophagus not observed by standard endoscopy in patients with NERD. In addition, we sought to evaluate the influence of different reflux episodes on the micro-alterations of NERD by CLE in vivo.Part1Characteristics of Reflux Episodes in patients with Non-Erosive Reflux Disease using Twenty-four-hour Multichannel Intraluminal Impedance and pH MornitoringBackground&Aims:Non-erosive reflux disease is the most common phenotypic presentations of gastroesophageal reflux disease. Twenty-four-hour multichannel intraluminal impedance and pH (MⅡ-pH) esophageal monitoring considered the only currently available tool to monitor all types of gastroesophageal reflux. Through comparing the characteristics of physical and chemical characteristics of gastroesophageal reflux episodes between patients with NERD and ERD and controls using MⅡ-PH monitoring, the current study sought to study the pattern of different reflux events in patients with NERD, to obtain objective evidence of alterations-to explain the possible pathogenesis of NERD and to provide evidence to treat the diseases.MethodsPatients with NERD and ERD diagnosed by symptoms, Reflux Disease Questionnaire (RDQ) and upper endoscopy from Gastroenterology Clinic, QiLu Hospital of Shandong University. Subjects were enrolled if the score was≧12. Standard white light endoscopy was used to diagnose NERD by no esophageal mucosal injury and ERD by esophageal mucosal breaks. Patients without any reflux symptoms who were willing to participate in the study were recruited as controls. All subjects underwent twenty-four-hour MⅡ-pH esophageal monitoring respectively. The MⅡ-pH catheter contains six impedance segments and one esophageal pH sensors placed3,5,7,9,15, and17cm above the lower esophageal sphincter (LES) and the pH electrode placed at5cm above the LES. Analysis of the pH-impedance parameters included DeMeester Score, number of reflux episodes according to gas-liquid composition and pH (acid, weakly acid and non acid) between the upright and recumbent positions.24-h bolus exposure and proximal extent was also compared between the two groups. ResultsOverall,96patients (46NERD,29ERD and21controls) were eligible for final analysis (mean age,49.4years; males,56.7%). For the DeMeester score, NERD patients had a significantly higher number of DeMeester score (62±65vs.21.62±17.68, P=0.002) than that in the control group, but similar to patients with ERD (P=0.742and P=0.083respectively). The total number of gastro-oesophageal reflux episodes was90of which51%were acid,34%were weakly acidic and16%non acid, NERD patients had a higher incidence of total mixed reflux episodes (69±33vs.28±9, P=0.024) than that in control group. Also the numbers of acid and weakly acid reflux episodes in NERD and ERD patients were higher than that in HV(P=0.043and P=0.047respectively), especially in supine position within ERD patients (P=0.046, P=0.036respectively). However, the incidence of mixed reflux episodes and gas reflux episodes were higher in NERD group than that in ERD group and HV (P=0.032, P=0.043, respectively). NERD patients had a higher incidence of total mixed reflux episodes (69±33vs.28±9, P=0.024) and gas reflux episodes (66±90vs.10±4, P=0.015). With regard to the body position, similar results had seen in the supine position (mixed reflux episodes:14±13vs.6±2, P=0.004, gas reflux episodes20±35vs.3±4, P=0.032) as well as acidic (12±10vs.4±2, P=0.001) than did controls. No significant differences in the numbers of every reflux events in upright position were found between the NERD patients and the control group, as well as weakly acid, non-acid, liquid and gas refluxes in supine position. The time of bolus clearing in NERD group was longer than that in ERD group (P=0.038) and HV (P=0.016). Patients with NERD had more percentage of proximal extent in supine position (P=0.007) than that of with ERD but similar to the HV group (P=0.087). The acid exposure time (%time with esophageal pH<4) in patients with NERD and in control group was7.82±2.91and4.09±2.93, respectively (P=0.001). When compared the reflux events of every patterns between the patients with NERD and EE by gender, female patients with NERD had more non-acid reflux, mixed reflux and gas reflux.Conclusion MⅡ-pH proved to be considerably more useful tool in recording reflux episodes. Patients with NERD have more mixed and non-acid reflux patterns, more24h bolus exposure time and have more proximal extent than ERD patients. Acid reflux in esophagus may play important role in the pathogenesis of NERD.Part2The pathological and histological characteristics of the mucosa at the distal esophagus in patients with non-erosive gastroesophageal reflux diseaseBackground&Aims:Non erosive reflux disease (NERD) often shown micro-alterations in the distal esophagus epithelium, including elongation of the papillae, proliferation of basal cells and dilated intercellular spaces (DIS) within the squamous epithelium, that did not seen by white light endoscopy. Advances in biomedical optics and diagnostic endoscopic tools have led to the improvement of conventional endoscopy for the detection of micro-alterations in the gastrointestinal tract. DIS of esophageal epithelium has been revealed at transmission electron microscopy both in the rabbit acid-perfused esophagus and in esophageal biopsies from GERD patients. However, the relationship between the DIS and the reflux episodes in patients with NERD was not clear. The aim of this study was to observe the pathological histology manifestation of the mucosa at the distal esophagus in patients with NERD and to analyze the relationship between histological features and reflux episodes monitored by multiple intraluminal impedance-pH (MII-pH).MethodsOutpatients were included in and excluded off according to the criteria in part1. All subjects completed24h MII-pH monitoring and endoscopic examination. After routine observation had been finished, biopsy specimens were obtained from every quadrant of the distal2cm esophagus from each individual. At least one sample block in every quadrant was included in the H&E stained specimen analysis. For histopathology, the specimens were fixed, embedded and sectioned vertically and transversely to facilitate the comparison between histology and confocal images. For TEM, the specimens were prefixed, coded and embed, then sectioned transversely to facilitate the comparison between TEM and confocal images. Photographs of at least10fields were magnified at×10000. At least10randomly selected intercellular spaces were measured in each image, for at least10measurements in each case. Every transect was drawn at a distance of not closer than1μm.ResultsOf the106subjects who began the study,55were selected randomly for TEM according to the protocol. Six samples of the23NERD patients,5of the ERD and two of the controls were failed for TEM images and were excluded from the final analysis. Finally,36sample blocks (17NERD patients,14ERD patients and9controls) entered the final TEM analysis. Patients with NERD showed more minimal change lesions in distal esophageal epithelium on endoscopic images. NERD patients and controls showed regular squamous epithelium cells. However, NERD patients showed obscure cellular borders and dilated intracellular spaces in the squamous epithelium, with decreased number of desmosomes on TEM images. Intercellular spaces of esophageal epithelial cell obtained by TEM in volunteers, NERD patients and ERD patients were0.90±0.20μm,1.41±0.46μm,1.53±0.42μm, respectively, with significant differences between the control group and the NERD group (P=0.005). For the routine histological examination of the esophagus, NERD patients were more likely to have histological evidence of esophagitis than controls, but the differences were not significant (P=0.245). In total,43.5%(20/46) of patients with NERD had microscopic esophagitis and56.5%(26/46) were presented with normal histology findings, whereas the proportion of the subjects of controls with microscopic esophagitis and normal were28.6%(6/21) and71.4%(15/21) respectively. Spearman correlation analysis showed that the minimal changes of the esophagus epithelium correlated to acid refluxConclusionsMinimal change lesions are commonly seen in the screening endoscopic examinations for individuals in patients with NERD. TEM is a sensitive method to diagnose the micro-alterations of NERD. Dilated intercellular spaces were seen in both NERD and ERD cases. DIS occurs along the distal esophageal epithelium in NERD patients and could be responsible for the enhancment of acid reflux. Part3The Diagnostic Value of in-vivo Confocal Laser Endomicroscopy in Patients with Non Erosive Reflux DiseaseBackground&Aims:Objectively diagnosing non-erosive reflux disease (NERD) is still a challenge. Confocal laser endomicroscopy is an emerging technology that poses the endoscopist with challenges for identifying micro-features in the human esophagus. CLE allows subsurface histological diagnosis at a cellular and subcellular level in vivo and provides instantaneous histopathology during ongoing endoscopy. We aimed to evaluate the use of confocal laser endomicroscopy (CLE) to examine in vivo the micro-alterations of the esophagus in patients with NERD and its relationship with reflux episodes monitored by multiple intraluminal impedance-pH (MII-pH).Methods:Patients with gastroesophageal reflux symptoms completed Reflux Disease Questionnaires (RDQ). NERD was determined by negative gastroscopy. Patients without reflux symptoms were recruited as controls. Pilot clinical study was conducted before the prospective controlled blinded study. All subjects were examined by white light endoscopy and followed by the standard CLE. The micro-alterations seen on CLE images and the correlation between CLE features, the correlation between CLE and transmission electron microscope (TEM) data and reflux episodes were evaluated was also analyzed.Results:On CLE images, NERD patients had more intrapapillary capillary loops (IPCLs) per image than did controls (8.29±3.52vs.5.69±2.31,P=0.010), as well as the diameter of IPCLs (19.48±3.13vs.15.87±2.21μm, P=0.041) and intercellular spaces of squamous cells (3.40±0.82vs.1.90±0.53μm, P=0.042). The ROC analysis indicated that IPCLs number (optimal cut-off>6per image, AUC0.722,95%CI0.592-0.853, sensitivity67.7%, specificity71.6%), IPCLs diameter (optimal cut-off>17.2μm, AUC0.847,95%CI0.747-0.947, sensitivity81%, specificity76%) and the intercellular spaces of squamous cells (optimal cut-off>2.40μm, AUC0.935,95%CI0.875-0.995, sensitivity 85.7%, specificity90.5%) diagnosed NERD with reasonable accuracy. Combined features of DIS plus increased IPCLs provided100%specificity in the diagnosis of NERD patients. Multivariate progressive regression analysis showed that acidic reflux, especially in the supine position, was related to the increased number and dilation of IPCLs in the squamous epithelium (β=0.063, t=2.895, P=0.038and β=0.156, t=1.023, P=0.04).Bland-Altman plot for the intracellular spaces observed by CLE and with TEM. Pearson's correlation coefficient showed that the intracellular spaces observed by CLE were highly correlated with TEM findings of the biopsy specimens from the same site (r=0.75,P<0.001).Conclusions:CLE represents a useful and potentially significant improvement over standard endoscopy to examine the micro-alterations of the esophagus in vivo. Acidic reflux is responsible for the micro-alterations in the esophagus of patients with NERD.Significance:1. Combined intraluminal impedance monitoring-pH allows detection of all reflux events regarding acidity (acid, weakly acidic, weakly alkaline) and composition (liquid, gas, mixed). It shows a higher sensitivity in the detection of proximal reflux. Through comparing the characteristics of physical and chemical characteristics of gastroesophageal reflux episodes between patients with NERD and ERD using MII-PH monitoring, the current study investigated the pattern of reflux events with NERD patients, obtained objective evidence of alterations in the patients of the two groups and to explain the possible pathogenesis of NERD and provide evidence in treatment of the diseases.2. The value of pathology and histology of the mucosa in diagnosing non-erosive gastroesophageal reflux disease is still a challenge. This study had observed the pathological histology manifestation of the mucosa at the distal esophagus in patients with NERD and analyzed the relationship between histological features and reflux episodes monitored by multiple intraluminal impedance-ph monitoring, more evidence have added in expanding the manifestation NERD.3. As an emerging technology that allows subsurface histological diagnosis at a cellular and subcellular level in vivo, confocal laser endomicroscopy provides instantaneous histopathology during ongoing endoscopy. This study firstly evaluated the role of CLE in diagnosing in vivo the micro-alterations of the esophagus in patients with NERD and its relationship with reflux episodes. The result showed that CLE represents a useful and potentially techniche over standard endoscopy to examine the micro-alterations of the esophagus in vivo.