共聚焦激光显微内镜对结肠粘膜损伤的形态和功能诊断
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摘要
背景和目的
肠粘膜屏障是由包括肠上皮细胞、细胞间连接及分泌性屏障所组成的结构,在维护机体内稳态、调节免疫方面发挥重要作用。肠粘膜屏障损伤是包括炎症性肠病、乳糜泻在内的多种胃肠道疾病的重要发病机制。近些年来,一度被认为缺少器质性改变依据的功能性胃肠病,如肠易激综合征的发病机制也被认为和肠粘膜屏障受损有关。
对肠粘膜屏障损伤的研究离不开内窥镜检查和肠粘膜通透性的检测。而当前常规的白光内镜对隐匿性的活动性炎症诊断欠敏感,有报道显示普通白光内镜下表现为正常的患者,约30%经病理活检提示相当部分仍残留有急性活动性炎症,而这种隐匿性的急性炎症往往是炎症性肠病患者复发的危险因素；肠粘膜通透性检测方面：目前的检测方法大体分为侵入性和非侵入性两种。前者如Ussing灌流室,需要对肠粘膜进行多块的活检,并且需要新鲜的标本,还无法广泛展开；后者一般采用口服示踪分子,检测经肠道重吸收后进入循环的含量。所采用的示踪分子需要满足三个条件：1、无毒或低毒；2、正常情况下不被吸收；3、可以在尿液中定量检测。目前多采用检测尿液中小分子糖类的方法,根据不同糖类分子的吸收特性,可以反映不同肠道节段的粘膜通透性,常规采用乳果糖／甘露醇比值代表小肠通透性,采用24小时三氯蔗糖排泄量代表结肠通透性。另外常用的示踪分子是放射性51Cr-EDTA,可以检测全肠道通透性,但因为具有一定放射性和需要临时配置的特点,相比糖分子检测使用范围较小
共聚焦激光显微内镜是一种新型的消化内窥镜,可以在常规内窥镜检查的同时,提供放大500-1000倍的粘膜图像,达到与横切面病理切片近似的灰度图像。除此之外,由于其光学特性,共聚焦激光显微内镜的显影需要应用荧光素作为对比剂。在溃疡性结肠炎病人中可以观察到荧光素渗漏入结肠隐窝腔,而荧光素渗漏与炎症的病理分级密切相关。因此本研究目的在于通过共聚焦激光显微内镜结肠粘膜形态和功能两方面的特征,获取更准确的结肠粘膜损伤的诊断。
方法
第一部分：收集2008.06.01-2009.04.01在山东大学齐鲁医院进行随访的73名溃疡性结肠炎病人作为研究对象。所有病人自愿选择共聚焦激光显微内镜代替常规内镜检查,并且签署知情同意书。首先应用白光内镜模式下炎症分级(Baron分级)评分将所观察到的粘膜表现分为四级：0级正常肠粘膜,1级介于0级和2级之间,2级碰触肠粘膜易出血,3级肠粘膜自发性出血。其中0级和1级代表正常和慢性非活动性炎症；2级和3级代表急性活动性炎症。之后用共聚焦激光显微内镜的显微镜模式将炎症分为四级：A级隐窝大小正常,排列规则；B级,隐窝大小正常,排列紊乱；C级,隐窝排列更趋紊乱,隐窝开口扩张,可有荧光素渗出；D级,正常隐窝结构大量破坏,可有隐窝脓肿。其中A级和B级代表正常和慢性非活动性炎症；C级和D级代表急性活动性炎症。除隐窝结构之外,观察结肠隐窝腔内荧光素渗漏情况,按照有无荧光素渗漏分为两组。对共聚焦激光显微内镜下间质微血管改变分为三级：无改变,轻到中度改变和重度改变。除主观分级外,将获取的共聚焦激光显微内镜静态图像做客观定量分析,以单位图像面积内隐窝数量作为隐窝结构的客观指标,以灰度值作为荧光素渗漏的客观指标。观察部位进行靶向活检,常规送病理,由三位病理医师独立作出炎症分级诊断。所用炎症分级按照Geboes分级法,共分为6级,≤3级作为非活动性炎症,>3代表急性活动性炎症。将白光内镜下和显微内镜下各炎症活动度指标与病理诊断分别作相关性分析。
第二部分：收集2009.04.01-06.30的溃疡性结肠炎患者10人,符合罗马Ⅲ诊断标准的肠易激综合征患者12人,以及健康查体或结肠息肉电切后随访的对照8人。所有病人空腹口服三联糖分子(乳果糖10克,甘露醇5克,三氯蔗糖5克)溶液,后留取24小时尿液,行气象色谱分析法检测肠粘膜通透性。以前5小时尿液中乳果糖／甘露醇比例作为小肠通透性指标,24小时三氯蔗糖排泄量作为结肠通透性指标。之后所有病人接受共聚焦激光显微内镜检查,对病人结肠每10cm观察荧光素渗漏表现,记录荧光素渗漏涉及的范围,荧光素渗漏的程度,出现荧光素渗漏与注射荧光素之间的时间。将荧光素渗漏情况与肠粘膜通透性检测结果行相关性分析。对部分患者,于荧光素注射之前在回盲部局部喷洒吖啶黄,记录单位层面上上皮细胞空隙数量,注射荧光素之后重新计数上皮细胞空隙数量,观察荧光素注射前后肠粘膜上皮空隙的数量变化。分别于荧光素渗漏和无渗漏部位行靶向活检,获取的组织予透射电镜观察,重点观察基底膜完整性,以及炎症细胞穿过基底膜的情况,分析荧光素渗漏与基底膜完整性破坏间的相关性。
结果
第一部分：共聚焦激光显微内镜相比白光内镜对炎症程度的判断更加精确,尤其对于白光内镜下表现正常或仅有轻度炎症的病变,共聚焦激光显微内镜对急性活动性炎症的判断更加敏感。在50个病理诊断有急性活动性炎症的患者中,白光内镜判断有19人为正常或慢性炎症；而应用共聚焦激光显微内镜观察,所有50例急性活动性炎症均被诊断为C级和D级。共聚焦激光显微内镜相比白光内镜对炎症活动度的判断更为准确(P=0.005)。在共聚焦激光显微内镜下隐窝形态改变与病理分级间存在显著相关性(Spearman rho, P<0.001)。荧光素渗漏更多见于急性活动性炎症中(P<0.001)。血管改变与炎症活动度间亦存在显著正相关,共聚焦激光显微内镜下表现为重度血管病变的患者,病理诊断均显示为急性活动性炎症。客观量化分析显示：单位共聚焦激光显微内镜图像内隐窝数量与隐窝形态分级存在显著负相关(P<0.001)；单位面积内隐窝数量与病理评分间存在明显负相关(P=0.001),而灰度分析未能支持荧光素渗漏(P=0.194)。
第二部分：荧光素渗漏表现与结肠粘膜通透性检测存在显著相关性,有荧光素渗漏患者的24小时三氯蔗糖排泄率显著高于无荧光素渗漏者(total sucralose secretion:FLIL,60.64±18.07mg vs.non-FLIL,33.26±8.54mg,P=0.013)。荧光素渗漏时间依赖性不明显,有荧光素渗漏者出现的时间可短在注射荧光素钠后2分钟内出现,而无荧光素渗漏者,即使注射荧光素后20分钟亦无表现。通透性增高的程度与荧光素渗漏的范围和程度呈正相关。结肠上皮细胞空隙在荧光素注射前后无显著变化(Wilcoxon test, P=0.9)。荧光素渗漏部位粘膜透射电镜显示多见基地膜疏松、断裂和淋巴细胞穿过等表现(Contingency Coefficient, value=0.441, P=0.007),同时,基底膜异常与三氯蔗糖排泄率间存在显著相关性,有基底膜异常者三氯蔗糖排泄率高于基底膜正常者(57.66±21.24 vs.42.71±16.77, P=0.04)。
结论
一、共聚焦激光显微内镜可以准确判断结肠粘膜炎症活动程度；
二、对于白光内镜下表现正常或非活动期的溃疡性结肠炎患者,部分仍残留有隐匿的急性活动性炎症,共聚焦激光显微内镜可以提供更准确的判断;
三、隐窝结构改变、荧光素渗漏和血管改变是共聚焦激光显微内镜判断炎症活动度的良好指标；
四、共聚焦激光显微内镜下荧光素渗漏可以作为粘膜通透性增高的标志；
五、荧光素渗漏与粘膜通透性相关；
六、荧光素渗漏的途径可能为细胞间途径,上皮细胞空隙对荧光素保持不可通透状态；
七、基底膜结构异常是荧光素渗漏的重要途径。
研究意义
本研究提出了共聚焦激光显微内镜下结肠粘膜炎症活动度的分级标准,经检验可以准确区分正常粘膜、慢性非活动性炎症和急性活动性炎症,对临床上溃疡性结肠炎患者炎症活动度的判断提供了更敏感的指标。首次观察到共聚焦激光显微内镜下结肠隐窝腔荧光素渗漏现象,并发现与结肠通透性存在相关性,为肠道粘膜病变患者在体检测肠粘膜通透性提供了可能。将消化内镜检查从单纯形态学诊断向形态与功能结合方向推动。首次在人体上证实上皮细胞更新脱落所形成的空隙无通透性。
Backgrounds and aims:
The intestinal barrier consists of intestinal epithelium, paracellular junction and secretive barrier. The intestinal barrier plays a crucial role in the maintenance of homeostasis and regulation of immune activity. Barrier dysfunction in the pathogenesis of several gastrointestinal diseases have been drawing much attentions, such as inflammatory bowel disease (IBD), Celiac disease. And recently the functional GI disorders including irritable bowel syndrome (IBS), which was conventionally believed to be lack of organic alterations, are also found to be associated with barrier dysfunction.
Studies on barrier dysfunction inevitably involve assessment by endoscopy and gastrointestinal permeability test. Currently the hiding active inflammation could not be easily detected by using conventional white lighe endoscopy. It was reported that 30% of patients appearing normal under conventional endoscopy were revealed as having acute active inflammation by histopathology. And the hiding acute active inflammation was proved to be an independent risk factor to predict relapse. In the area of intestinal permeability tests, both the invasive and non-invasive methods are not easy to carry out. The gold standard invasive method, Ussing chamber, is in need of dozens of fresh biopsy samples, which is difficult to carry out on human. Consequently the non-invasive assessment of urine markers after oral intaking. The markers used in the intestinal permeability test should meet 3 conditions:1, non or low toxic; 2, not absortable in normal conditions; 3, quantitatively detectable in urine. The most commonly used markers are saccharides. The non-invasive tests rely on oral administration and subsequent urinary collection of probes that can selectively characterize permeability from different regions fo the gut. In many studies, a triple sugar test involving administration of 3 saccharides was used to assess small intestinal permeability and colonic permeability. Traditionally, the lactulose to mannitol ratio is used to express small intestinal permeability while 24 hours of sucralose excretion is used to express colonic permeability. The other common probe is (51)Cr-EDTA. This probe is capable of whole gut permeability assessment. However, its radioactivity and requirement of temporary storage limit the applications.
Confocal laser endomicroscopy (CLE) is an endoscopic device providing in vivo microscopic morphology during ongoing endoscopic procedures. The mostly applied staining agent in CLE is fluorescein sodium, which is intravenously applied and distributed immediately throughout mucosal tissues. In our previous study on classification of inflammation activity in ulcerative (UC) by CLE, the fluorecein leakage into lumen (FLIL) was associated with active inflammation in UC. One possible explanation of FLIL is increased colonic permeability, since fluorecein sodium and sucralose have the similar molecular mass (376.27 and 397.64, respectively). The primary goal of this study is aimed to validate the association between FLIL in colonic mucosa and colonic permeability.
Methods
PartⅠ
From June 1,2008, to April 30,2009, we recruited consecutive patients previously diagnosed as having UC who visited the outpatient department of Qilu Hospital for colonoscopy surveillance. After being informed about the purpose of the study, those who were willing to choose CLE instead of conventional colonoscopy were included in the study. The CLE procedures did not differ from those of conventional colonoscopy, except for the additional evaluation of mucosal inflammation in the distal colon, including the sigmoid colon and rectum, by the Baron Score. After Baron endoscopy scores for inflammation were recorded, the distal tip of the endoscope was placed gently onto the observed mucosa with the endomicroscopy mode turned on. At least 2 to 3 Z-stacks of images (scanning from the superficial to the deep layer of targeted mucosa) were obtained, and the endoscopists evaluated the crypt architecture simultaneously. The crypt architecture was classified into 4 types. Types A and B are considered normal and chronic inflammation respectively, and types C and D indicate acute inflammation. Microvascular alterations were evaluated by real-time CLE assessment of inflammation activity as well. In addition to crypt architecture and microvascular alterations, we introduced a new marker, fluorescein leakage into lumen (FLIL), to define acute inflammation seen on CLE. Images of observed mucosa were stored digitally on laser discs for further evaluation, In the end, a targeted biopsy was performed for histological analysis. Objective post-CLE analysis involved evaluation of all Z-stacks of images of the observed mucosa for number of crypts per CLE image (CPCI) and fluorescein density of CLE images (FDCI). CPCI was considered the objective variable to show the reliability of the objective real-time crypt architecture analysis, as was FDCI for FLIL. Three pathologists (CJZ, WQH, HC) from 2 independent hospitals evaluated the slices for histology assessment of inflammation in UC according to the Geboes Index. The scale includes 6 grades:structural (architectural changes), chronic inflammatory infiltrates, lamina propria neutrophils and eosinophils, neutrophils in epithelium, crypt destruction, and erosion or ulceration. Each grade is divided into 4 to 5 subgroups.
PartⅡ
Consecutive patients who were scheduled for colonoscopy from April 1st to June 30th,2009 were informed about the purpose of this study. Those prefering CLE over conventional colonoscopy were enrolled and gave their written informed consents. After successful intubation into cecum,5 ml of 10%fluorescein sodium was intravenously given. The distal tip of CLE was gently placed onto mucosa while laser turned on. Laser volume were fixed on 500 nm and brightness on median of all the procedures. Each procedure included CLE observation per 10 cm of colon during the withdrawl of the colonoscope as well as conventional white light colonoscopy examination. Images of each z-stack were stored in a specific folder. Two biosies were taken from each patient, one was for routine histopathology, and another for transmission electron microscopy. For patients without FLIL, biopsies were taken from the distal colon, and biopsies were taken from the targeted mucosa of those with FLIL. In addition to evaluation of FLIL, analysis of epithelial gaps in colon was also conducted in some patients. Number of gaps were counted by per z-stack of images as reported. We assumed that decreased numbers of gaps under CLE after fluorescein injection indicates epithelial gaps are permeable, and constant gaps despite of fluorescein injection indicates impermeability of epithelial gaps. Two days before CLE procedures, patients fasted overnight and emptied their bladder before drinking a test solution containing 5 g mannitol,10 g lactulose, and 5g sucralose diluted in 100 ml of tap water. Then the patients were asked to collect 24 hours of urine into the sealed and disinfected containers. Urine was then collected for the following 24 hours. The ratio of lactulose and mannitol (L/M) recovery during the first 5 hours was used as an index of small intestinal permeability and the total mass of sucralose excretion (mg) during the 24 h was used as an index of colonic permeability. Samples from mucosa with or without FLIL were sent for transmission electronic microscopy (TEM) analysis.
Results
PartⅠ
Assessment of crypt architecture and fluorescein leakage with CLE both showed good correlation with histology results (Spearman rho, both P< 0.001). CLE seemed to be more accurate than conventional white-light endoscopy for evaluating macroscopical normal mucosa. More than half of the patients with normal mucosa seen on conventional white-light endoscopy showed acute inflammation on histology, whereas no patients with normal mucosa or with chronic inflammation seen on CLE showed acute inflammation on histology. Assessment of microvascular alterations by CLE showed good correlation with histology findings (P<0.001). On post-CLE objective assessment, subjective architectural classifications were supported by number of crypts per image (P< 0.001) but not fluorescein leakage results by grey scale(P= 0.194).
PartⅡ
FLILs were found in 21 z-stacks of CLE images during 18 procedures. Among the 21 z-stacks, 17 were found in the cecum mucosa, and the rest were in the distal segments including sigmoid colon (3) and rectum(l). Time durations between fluorescein injection and FLIL were from 2 to 11 minutes. Distribution of procedures with FLILs among patients'groups are:7 in UC,8 in IBS and 3 in HC. Chi-square test showed no significant difference of FLIL prevalence among 3 groups (X2=2.326, P=0.312). Though average colonic permeability (parameter:sucralose excretion) is higher in patients with UC than IBS and HC, the differencs were not statistically significant P=0.083). However, patients with FLIL found during CLE have significantly higher average colonic permeability than those without (total sucralose secretion:FLIL,60.64±18.07mg vs. non-FLIL,33.26±8.54mg, P=0.013). There was no significant difference between numbers of epithelial gaps before and after fluorescein injection (Wilcoxon test, P=0.9). In the 12 patients without FLIL under CLE examinations, TEM showed normal basement membrane in 10 patients. In the 18 patients with FLIL, TEM showed normal basement membrane in 6 patients (Contingency Coefficient, value=0.441, P=0.007). The abnormalities of basement membrane includes decreased thickness, breaked and infiltrated by lymphocyte. Patients with abnormal TEM findings had higher colonic permeability (57.66±21.24 vs.42.71±16.77, P=0.04).
Conclusions
●Confocal laser endomicroscopy is reliable in assessment of colonic mucosal inflammation.
●For those with hiding acute active inflammation but appearing normal under conventional white light endoscopy, confocal laser endomicroscopy could provide more accurate diagnosis.
●Crypt architecture, fluorescein leakage and microvascular alterations are promising markers for acute inflammation under confocal laser endomicroscopy.
●Fluorescein leakage under confocal laser endomicroscopy is associated with increased colonic permeability.
●Paracellular pathway, and abnormal basement membrane are more likely to the pathways of fluorescein leakage than epithelial gaps.
Significance
A practical criteria upon classification of inflammatory activity in colonic mucosa by confocal laser endomicrsocopy combining crypt architecture, microvascular alterations and fluorescein leakage was established in this study. Fluorescein leakage into crypt lumen is the first time to be raised in the assessment of colonic permeability. And epithelial gaps were firstly approved to be impermeable in an in vivo manner by confocal laser endomicroscopy.
肠粘膜屏障是由包括肠上皮细胞、细胞间连接及分泌性屏障所组成的结构,在维护机体内稳态、调节免疫方面发挥重要作用。肠粘膜屏障损伤是包括炎症性肠病、乳糜泻在内的多种胃肠道疾病的重要发病机制。近些年来,一度被认为缺少器质性改变依据的功能性胃肠病,如肠易激综合征的发病机制也被认为和肠粘膜屏障受损有关。
对肠粘膜屏障损伤的研究离不开内窥镜检查和肠粘膜通透性的检测。而当前常规的白光内镜对隐匿性的活动性炎症诊断欠敏感,有报道显示普通白光内镜下表现为正常的患者,约30%经病理活检提示相当部分仍残留有急性活动性炎症,而这种隐匿性的急性炎症往往是炎症性肠病患者复发的危险因素；肠粘膜通透性检测方面：目前的检测方法大体分为侵入性和非侵入性两种。前者如Ussing灌流室,需要对肠粘膜进行多块的活检,并且需要新鲜的标本,还无法广泛展开；后者一般采用口服示踪分子,检测经肠道重吸收后进入循环的含量。所采用的示踪分子需要满足三个条件：1、无毒或低毒；2、正常情况下不被吸收；3、可以在尿液中定量检测。目前多采用检测尿液中小分子糖类的方法,根据不同糖类分子的吸收特性,可以反映不同肠道节段的粘膜通透性,常规采用乳果糖／甘露醇比值代表小肠通透性,采用24小时三氯蔗糖排泄量代表结肠通透性。另外常用的示踪分子是放射性51Cr-EDTA,可以检测全肠道通透性,但因为具有一定放射性和需要临时配置的特点,相比糖分子检测使用范围较小
共聚焦激光显微内镜是一种新型的消化内窥镜,可以在常规内窥镜检查的同时,提供放大500-1000倍的粘膜图像,达到与横切面病理切片近似的灰度图像。除此之外,由于其光学特性,共聚焦激光显微内镜的显影需要应用荧光素作为对比剂。在溃疡性结肠炎病人中可以观察到荧光素渗漏入结肠隐窝腔,而荧光素渗漏与炎症的病理分级密切相关。因此本研究目的在于通过共聚焦激光显微内镜结肠粘膜形态和功能两方面的特征,获取更准确的结肠粘膜损伤的诊断。
方法
第一部分：收集2008.06.01-2009.04.01在山东大学齐鲁医院进行随访的73名溃疡性结肠炎病人作为研究对象。所有病人自愿选择共聚焦激光显微内镜代替常规内镜检查,并且签署知情同意书。首先应用白光内镜模式下炎症分级(Baron分级)评分将所观察到的粘膜表现分为四级：0级正常肠粘膜,1级介于0级和2级之间,2级碰触肠粘膜易出血,3级肠粘膜自发性出血。其中0级和1级代表正常和慢性非活动性炎症；2级和3级代表急性活动性炎症。之后用共聚焦激光显微内镜的显微镜模式将炎症分为四级：A级隐窝大小正常,排列规则；B级,隐窝大小正常,排列紊乱；C级,隐窝排列更趋紊乱,隐窝开口扩张,可有荧光素渗出；D级,正常隐窝结构大量破坏,可有隐窝脓肿。其中A级和B级代表正常和慢性非活动性炎症；C级和D级代表急性活动性炎症。除隐窝结构之外,观察结肠隐窝腔内荧光素渗漏情况,按照有无荧光素渗漏分为两组。对共聚焦激光显微内镜下间质微血管改变分为三级：无改变,轻到中度改变和重度改变。除主观分级外,将获取的共聚焦激光显微内镜静态图像做客观定量分析,以单位图像面积内隐窝数量作为隐窝结构的客观指标,以灰度值作为荧光素渗漏的客观指标。观察部位进行靶向活检,常规送病理,由三位病理医师独立作出炎症分级诊断。所用炎症分级按照Geboes分级法,共分为6级,≤3级作为非活动性炎症,>3代表急性活动性炎症。将白光内镜下和显微内镜下各炎症活动度指标与病理诊断分别作相关性分析。
第二部分：收集2009.04.01-06.30的溃疡性结肠炎患者10人,符合罗马Ⅲ诊断标准的肠易激综合征患者12人,以及健康查体或结肠息肉电切后随访的对照8人。所有病人空腹口服三联糖分子(乳果糖10克,甘露醇5克,三氯蔗糖5克)溶液,后留取24小时尿液,行气象色谱分析法检测肠粘膜通透性。以前5小时尿液中乳果糖／甘露醇比例作为小肠通透性指标,24小时三氯蔗糖排泄量作为结肠通透性指标。之后所有病人接受共聚焦激光显微内镜检查,对病人结肠每10cm观察荧光素渗漏表现,记录荧光素渗漏涉及的范围,荧光素渗漏的程度,出现荧光素渗漏与注射荧光素之间的时间。将荧光素渗漏情况与肠粘膜通透性检测结果行相关性分析。对部分患者,于荧光素注射之前在回盲部局部喷洒吖啶黄,记录单位层面上上皮细胞空隙数量,注射荧光素之后重新计数上皮细胞空隙数量,观察荧光素注射前后肠粘膜上皮空隙的数量变化。分别于荧光素渗漏和无渗漏部位行靶向活检,获取的组织予透射电镜观察,重点观察基底膜完整性,以及炎症细胞穿过基底膜的情况,分析荧光素渗漏与基底膜完整性破坏间的相关性。
结果
第一部分：共聚焦激光显微内镜相比白光内镜对炎症程度的判断更加精确,尤其对于白光内镜下表现正常或仅有轻度炎症的病变,共聚焦激光显微内镜对急性活动性炎症的判断更加敏感。在50个病理诊断有急性活动性炎症的患者中,白光内镜判断有19人为正常或慢性炎症；而应用共聚焦激光显微内镜观察,所有50例急性活动性炎症均被诊断为C级和D级。共聚焦激光显微内镜相比白光内镜对炎症活动度的判断更为准确(P=0.005)。在共聚焦激光显微内镜下隐窝形态改变与病理分级间存在显著相关性(Spearman rho, P<0.001)。荧光素渗漏更多见于急性活动性炎症中(P<0.001)。血管改变与炎症活动度间亦存在显著正相关,共聚焦激光显微内镜下表现为重度血管病变的患者,病理诊断均显示为急性活动性炎症。客观量化分析显示：单位共聚焦激光显微内镜图像内隐窝数量与隐窝形态分级存在显著负相关(P<0.001)；单位面积内隐窝数量与病理评分间存在明显负相关(P=0.001),而灰度分析未能支持荧光素渗漏(P=0.194)。
第二部分：荧光素渗漏表现与结肠粘膜通透性检测存在显著相关性,有荧光素渗漏患者的24小时三氯蔗糖排泄率显著高于无荧光素渗漏者(total sucralose secretion:FLIL,60.64±18.07mg vs.non-FLIL,33.26±8.54mg,P=0.013)。荧光素渗漏时间依赖性不明显,有荧光素渗漏者出现的时间可短在注射荧光素钠后2分钟内出现,而无荧光素渗漏者,即使注射荧光素后20分钟亦无表现。通透性增高的程度与荧光素渗漏的范围和程度呈正相关。结肠上皮细胞空隙在荧光素注射前后无显著变化(Wilcoxon test, P=0.9)。荧光素渗漏部位粘膜透射电镜显示多见基地膜疏松、断裂和淋巴细胞穿过等表现(Contingency Coefficient, value=0.441, P=0.007),同时,基底膜异常与三氯蔗糖排泄率间存在显著相关性,有基底膜异常者三氯蔗糖排泄率高于基底膜正常者(57.66±21.24 vs.42.71±16.77, P=0.04)。
结论
一、共聚焦激光显微内镜可以准确判断结肠粘膜炎症活动程度；
二、对于白光内镜下表现正常或非活动期的溃疡性结肠炎患者,部分仍残留有隐匿的急性活动性炎症,共聚焦激光显微内镜可以提供更准确的判断;
三、隐窝结构改变、荧光素渗漏和血管改变是共聚焦激光显微内镜判断炎症活动度的良好指标；
四、共聚焦激光显微内镜下荧光素渗漏可以作为粘膜通透性增高的标志；
五、荧光素渗漏与粘膜通透性相关；
六、荧光素渗漏的途径可能为细胞间途径,上皮细胞空隙对荧光素保持不可通透状态；
七、基底膜结构异常是荧光素渗漏的重要途径。
研究意义
本研究提出了共聚焦激光显微内镜下结肠粘膜炎症活动度的分级标准,经检验可以准确区分正常粘膜、慢性非活动性炎症和急性活动性炎症,对临床上溃疡性结肠炎患者炎症活动度的判断提供了更敏感的指标。首次观察到共聚焦激光显微内镜下结肠隐窝腔荧光素渗漏现象,并发现与结肠通透性存在相关性,为肠道粘膜病变患者在体检测肠粘膜通透性提供了可能。将消化内镜检查从单纯形态学诊断向形态与功能结合方向推动。首次在人体上证实上皮细胞更新脱落所形成的空隙无通透性。
Backgrounds and aims:
The intestinal barrier consists of intestinal epithelium, paracellular junction and secretive barrier. The intestinal barrier plays a crucial role in the maintenance of homeostasis and regulation of immune activity. Barrier dysfunction in the pathogenesis of several gastrointestinal diseases have been drawing much attentions, such as inflammatory bowel disease (IBD), Celiac disease. And recently the functional GI disorders including irritable bowel syndrome (IBS), which was conventionally believed to be lack of organic alterations, are also found to be associated with barrier dysfunction.
Studies on barrier dysfunction inevitably involve assessment by endoscopy and gastrointestinal permeability test. Currently the hiding active inflammation could not be easily detected by using conventional white lighe endoscopy. It was reported that 30% of patients appearing normal under conventional endoscopy were revealed as having acute active inflammation by histopathology. And the hiding acute active inflammation was proved to be an independent risk factor to predict relapse. In the area of intestinal permeability tests, both the invasive and non-invasive methods are not easy to carry out. The gold standard invasive method, Ussing chamber, is in need of dozens of fresh biopsy samples, which is difficult to carry out on human. Consequently the non-invasive assessment of urine markers after oral intaking. The markers used in the intestinal permeability test should meet 3 conditions:1, non or low toxic; 2, not absortable in normal conditions; 3, quantitatively detectable in urine. The most commonly used markers are saccharides. The non-invasive tests rely on oral administration and subsequent urinary collection of probes that can selectively characterize permeability from different regions fo the gut. In many studies, a triple sugar test involving administration of 3 saccharides was used to assess small intestinal permeability and colonic permeability. Traditionally, the lactulose to mannitol ratio is used to express small intestinal permeability while 24 hours of sucralose excretion is used to express colonic permeability. The other common probe is (51)Cr-EDTA. This probe is capable of whole gut permeability assessment. However, its radioactivity and requirement of temporary storage limit the applications.
Confocal laser endomicroscopy (CLE) is an endoscopic device providing in vivo microscopic morphology during ongoing endoscopic procedures. The mostly applied staining agent in CLE is fluorescein sodium, which is intravenously applied and distributed immediately throughout mucosal tissues. In our previous study on classification of inflammation activity in ulcerative (UC) by CLE, the fluorecein leakage into lumen (FLIL) was associated with active inflammation in UC. One possible explanation of FLIL is increased colonic permeability, since fluorecein sodium and sucralose have the similar molecular mass (376.27 and 397.64, respectively). The primary goal of this study is aimed to validate the association between FLIL in colonic mucosa and colonic permeability.
Methods
PartⅠ
From June 1,2008, to April 30,2009, we recruited consecutive patients previously diagnosed as having UC who visited the outpatient department of Qilu Hospital for colonoscopy surveillance. After being informed about the purpose of the study, those who were willing to choose CLE instead of conventional colonoscopy were included in the study. The CLE procedures did not differ from those of conventional colonoscopy, except for the additional evaluation of mucosal inflammation in the distal colon, including the sigmoid colon and rectum, by the Baron Score. After Baron endoscopy scores for inflammation were recorded, the distal tip of the endoscope was placed gently onto the observed mucosa with the endomicroscopy mode turned on. At least 2 to 3 Z-stacks of images (scanning from the superficial to the deep layer of targeted mucosa) were obtained, and the endoscopists evaluated the crypt architecture simultaneously. The crypt architecture was classified into 4 types. Types A and B are considered normal and chronic inflammation respectively, and types C and D indicate acute inflammation. Microvascular alterations were evaluated by real-time CLE assessment of inflammation activity as well. In addition to crypt architecture and microvascular alterations, we introduced a new marker, fluorescein leakage into lumen (FLIL), to define acute inflammation seen on CLE. Images of observed mucosa were stored digitally on laser discs for further evaluation, In the end, a targeted biopsy was performed for histological analysis. Objective post-CLE analysis involved evaluation of all Z-stacks of images of the observed mucosa for number of crypts per CLE image (CPCI) and fluorescein density of CLE images (FDCI). CPCI was considered the objective variable to show the reliability of the objective real-time crypt architecture analysis, as was FDCI for FLIL. Three pathologists (CJZ, WQH, HC) from 2 independent hospitals evaluated the slices for histology assessment of inflammation in UC according to the Geboes Index. The scale includes 6 grades:structural (architectural changes), chronic inflammatory infiltrates, lamina propria neutrophils and eosinophils, neutrophils in epithelium, crypt destruction, and erosion or ulceration. Each grade is divided into 4 to 5 subgroups.
PartⅡ
Consecutive patients who were scheduled for colonoscopy from April 1st to June 30th,2009 were informed about the purpose of this study. Those prefering CLE over conventional colonoscopy were enrolled and gave their written informed consents. After successful intubation into cecum,5 ml of 10%fluorescein sodium was intravenously given. The distal tip of CLE was gently placed onto mucosa while laser turned on. Laser volume were fixed on 500 nm and brightness on median of all the procedures. Each procedure included CLE observation per 10 cm of colon during the withdrawl of the colonoscope as well as conventional white light colonoscopy examination. Images of each z-stack were stored in a specific folder. Two biosies were taken from each patient, one was for routine histopathology, and another for transmission electron microscopy. For patients without FLIL, biopsies were taken from the distal colon, and biopsies were taken from the targeted mucosa of those with FLIL. In addition to evaluation of FLIL, analysis of epithelial gaps in colon was also conducted in some patients. Number of gaps were counted by per z-stack of images as reported. We assumed that decreased numbers of gaps under CLE after fluorescein injection indicates epithelial gaps are permeable, and constant gaps despite of fluorescein injection indicates impermeability of epithelial gaps. Two days before CLE procedures, patients fasted overnight and emptied their bladder before drinking a test solution containing 5 g mannitol,10 g lactulose, and 5g sucralose diluted in 100 ml of tap water. Then the patients were asked to collect 24 hours of urine into the sealed and disinfected containers. Urine was then collected for the following 24 hours. The ratio of lactulose and mannitol (L/M) recovery during the first 5 hours was used as an index of small intestinal permeability and the total mass of sucralose excretion (mg) during the 24 h was used as an index of colonic permeability. Samples from mucosa with or without FLIL were sent for transmission electronic microscopy (TEM) analysis.
Results
PartⅠ
Assessment of crypt architecture and fluorescein leakage with CLE both showed good correlation with histology results (Spearman rho, both P< 0.001). CLE seemed to be more accurate than conventional white-light endoscopy for evaluating macroscopical normal mucosa. More than half of the patients with normal mucosa seen on conventional white-light endoscopy showed acute inflammation on histology, whereas no patients with normal mucosa or with chronic inflammation seen on CLE showed acute inflammation on histology. Assessment of microvascular alterations by CLE showed good correlation with histology findings (P<0.001). On post-CLE objective assessment, subjective architectural classifications were supported by number of crypts per image (P< 0.001) but not fluorescein leakage results by grey scale(P= 0.194).
PartⅡ
FLILs were found in 21 z-stacks of CLE images during 18 procedures. Among the 21 z-stacks, 17 were found in the cecum mucosa, and the rest were in the distal segments including sigmoid colon (3) and rectum(l). Time durations between fluorescein injection and FLIL were from 2 to 11 minutes. Distribution of procedures with FLILs among patients'groups are:7 in UC,8 in IBS and 3 in HC. Chi-square test showed no significant difference of FLIL prevalence among 3 groups (X2=2.326, P=0.312). Though average colonic permeability (parameter:sucralose excretion) is higher in patients with UC than IBS and HC, the differencs were not statistically significant P=0.083). However, patients with FLIL found during CLE have significantly higher average colonic permeability than those without (total sucralose secretion:FLIL,60.64±18.07mg vs. non-FLIL,33.26±8.54mg, P=0.013). There was no significant difference between numbers of epithelial gaps before and after fluorescein injection (Wilcoxon test, P=0.9). In the 12 patients without FLIL under CLE examinations, TEM showed normal basement membrane in 10 patients. In the 18 patients with FLIL, TEM showed normal basement membrane in 6 patients (Contingency Coefficient, value=0.441, P=0.007). The abnormalities of basement membrane includes decreased thickness, breaked and infiltrated by lymphocyte. Patients with abnormal TEM findings had higher colonic permeability (57.66±21.24 vs.42.71±16.77, P=0.04).
Conclusions
●Confocal laser endomicroscopy is reliable in assessment of colonic mucosal inflammation.
●For those with hiding acute active inflammation but appearing normal under conventional white light endoscopy, confocal laser endomicroscopy could provide more accurate diagnosis.
●Crypt architecture, fluorescein leakage and microvascular alterations are promising markers for acute inflammation under confocal laser endomicroscopy.
●Fluorescein leakage under confocal laser endomicroscopy is associated with increased colonic permeability.
●Paracellular pathway, and abnormal basement membrane are more likely to the pathways of fluorescein leakage than epithelial gaps.
Significance
A practical criteria upon classification of inflammatory activity in colonic mucosa by confocal laser endomicrsocopy combining crypt architecture, microvascular alterations and fluorescein leakage was established in this study. Fluorescein leakage into crypt lumen is the first time to be raised in the assessment of colonic permeability. And epithelial gaps were firstly approved to be impermeable in an in vivo manner by confocal laser endomicroscopy.
引文
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11. Kerckhoffs AP, Akkermans LM, de Smet MB, Besselink MG, Hietbrink F, Bartelink IH, Busschers WB, Samsom M, Renooij W. Intestinal Permeability in Irritable Bowel Syndrome Patients:Effects of NSAIDs. Dig Dis Sci 2009.
12. Piche T, Barbara G, Aubert P, Bruley des Varannes S, Dainese R, Nano JL, Cremon C, Stanghellini V, De Giorgio R, Galmiche JP, Neunlist M. Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome: involvement of soluble mediators. Gut 2009;58:196-201.
13. Zhou Q, Zhang B, Nicholas Verne G. Intestinal membrane permeability and hypersensitivity in the irritable bowel syndrome. Pain 2009; 146:41-6.
14. Teshima CW, Meddings JB. The measurement and clinical significance of intestinal permeability. Curr Gastroenterol Rep 2008; 10:443-9.
15. Kiesslich R, Burg J, Vieth M, Gnaendiger J, Enders M, Delaney P, Polglase A, McLaren W, Janell D, Thomas S, Nafe B, Galle PR, Neurath MF. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004; 127:706-13.
16. Polglase AL, McLaren WJ, Skinner SA, Kiesslich R, Neurath MF, Delaney PM. A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-GI tract. Gastrointest Endosc 2005;62:686-95.
17. Kitabatake S, Niwa Y, Miyahara R, Ohashi A, Matsuura T, Iguchi Y, Shimoyama Y, Nagasaka T, Maeda O, Ando T, Ohmiya N, Itoh A, Hirooka Y, Goto H. Confocal endomicroscopy for the diagnosis of gastric cancer in vivo. Endoscopy 2006;38:1110-4.
18. Becker V, Vercauteren T, von Weyhern CH, Prinz C, Schmid RM, Meining A. High-resolution miniprobe-based confocal microscopy in combination with video mosaicing (with video). Gastrointest Endosc 2007;66:1001-7.
19. Hurlstone DP, Thomson M, Brown S, Tiffin N, Cross SS, Hunter MD. Confocal endomicroscopy in ulcerative colitis: differentiating dysplasia-associated lesional mass and adenoma-like mass. Clin Gastroenterol Hepatol 2007;5:1235-41.
20. Meining A, Saur D, Bajbouj M, Becker V, Peltier E, Hofler H, von Weyhern CH, Schmid RM, Prinz C. In vivo histopathology for detection of gastrointestinal neoplasia with a portable, confocal miniprobe: an examiner blinded analysis. Clin Gastroenterol Hepatol 2007;5:1261-7.
21. Goetz M, Kiesslich R. Confocal endomicroscopy: in vivo diagnosis of neoplastic lesions of the gastrointestinal tract. Anticancer Res 2008;28:353-60.
22. Guo YT, Li YQ, Yu T, Zhang TG, Zhang JN, Liu H, Liu FG, Xie XJ, Zhu Q, Zhao YA. Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo: a prospective study. Endoscopy 2008;40:547-53.
23. Hurlstone DP, Baraza W, Brown S, Thomson M, Tiffin N, Cross SS. In vivo real-time confocal laser scanning endomicroscopic colonoscopy for the detection and characterization of colorectal neoplasia. Br J Surg 2008;95:636-45.
24. Hurlstone DP, Tiffin N, Brown SR, Baraza W, Thomson M, Cross SS. In vivo confocal laser scanning chromo-endomicroscopy of colorectal neoplasia:changing the technological paradigm. Histopathology 2008;52:417-26.
25. Leong RW, Nguyen NQ, Meredith CG, Al-Sohaily S, Kukic D, Delaney PM, Murr ER, Yong J, Merrett ND, Biankin AV. In vivo confocal endomicroscopy in the diagnosis and evaluation of celiac disease. Gastroenterology 2008; 135:1870-6.
26. Pech O, Rabenstein T, Manner H, Petrone MC, Pohl J, Vieth M, Stolte M, Ell C. Confocal laser endomicroscopy for in vivo diagnosis of early squamous cell carcinoma in the esophagus. Clin Gastroenterol Hepatol 2008;6:89-94.
27. Zhang JN, Li YQ, Zhao YA, Yu T, Zhang JP, Guo YT, Liu H. Classification of gastric pit patterns by confocal endomicroscopy. Gastrointest Endosc 2008;67:843-53.
28. Chang-Qing Li X-JX, Tao Yu, Xiao-Meng Gu, Xiu-Li Zuo, Cheng-Jun Zhou, Wei-Qing Huang, Hua Chen, Yan-Qing Li. Classification of Inflammation Activity in Ulcerative Colitis by Confocal Laser Endomicroscopy. Am J Gastroenterol advance online publication 2009.
29. Kiesslich R, Goetz M, Angus EM, Hu Q, Guan Y, Potten C, Allen T, Neurath MF, Shroyer NF, Montrose MH, Watson AJ. Identification of epithelial gaps in human small and large intestine by confocal endomicroscopy. Gastroenterology 2007; 133:1769-78.
30. Goke M, Podolsky DK. Regulation of the mucosal epithelial barrier. Baillieres Clin Gastroenterol 1996; 10:393-405.
31. Mahida YR, Galvin AM, Gray T, Makh S, McAlindon ME, Sewell HF, Podolsky DK. Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells. Clin Exp Immunol 1997;109:377-86.
32. Muller CA, Autenrieth IB, Peschel A. Innate defenses of the intestinal epithelial barrier. Cell Mol Life Sci 2005;62:1297-307.
33. Vilela EG, de Abreu Ferrari Mde L, de Gama Torres HO, Martins FP, Goulart EM, Lima AS, da Cunha AS. Intestinal permeability and antigliadin antibody test for monitoring adult patients with celiac disease. Dig Dis Sci 2007;52:1304-9.
34. Duerksen DR, Wilhelm-Boyles C, Parry DM. Intestinal permeability in long-term follow-up of patients with celiac disease on a gluten-free diet. Dig Dis Sci 2005;50:785-90.
35. Marshall JK, Thabane M, Garg AX, Clark W, Meddings J, Collins SM. Intestinal permeability in patients with irritable bowel syndrome after a waterborne outbreak of acute gastroenteritis in Walkerton, Ontario. Aliment Pharmacol Ther 2004;20:1317-22.
36. Quigley E. Gut permeability in irritable bowel syndrome: more leaks add to slightly inflamed bowel syndrome conspiracy theory. Gastroenterology 2009; 137:728-30.
37. Smecuol E, Bai JC, Sugai E, Vazquez H, Niveloni S, Pedreira S, Maurino E, Meddings J. Acute gastrointestinal permeability responses to different non-steroidal anti-inflammatory drugs. Gut 2001;49:650-5.
38. Kiesslich R, Gossner L, Goetz M, Dahlmann A, Vieth M, Stolte M, Hoffman A, Jung M, Nafe B, Galle PR, Neurath MF. In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2006;4:979-87.
39. Becker V, von Delius S, Bajbouj M, Karagianni A, Schmid RM, Meining A. Intravenous application of fluorescein for confocal laser scanning microscopy: evaluation of contrast dynamics and image quality with increasing injection-to-imaging time. Gastrointest Endosc 2008; 68:319-23.
40. Li CQ, Xie XJ, Yu T, Gu XM, Zuo XL, Zhou CJ, Huang WQ, Chen H, Li YQ. Classification of Inflammation Activity in Ulcerative Colitis by Confocal Laser Endomicroscopy. Am J Gastroenterol 2009.
41. Brown GR, Lindberg G, Meddings J, Silva M, Beutler B, Thiele D. Tumor necrosis factor inhibitor ameliorates murine intestinal graft-versus-host disease. Gastroenterology 1999;116:593-601.
42. Masungi C, Mensch J, Van Dijck A, Borremans C, Willems B, Mackie C, Noppe M, Brewster ME. Parallel artificial membrane permeability assay (PAMPA) combined with a 10-day multiscreen Caco-2 cell culture as a tool for assessing new drug candidates. Pharmazie 2008;63:194-9.
43. Watson AJ, Chu S, Sieck L, Gerasimenko O, Bullen T, Campbell F, McKenna M, Rose T, Montrose MH. Epithelial barrier function in vivo is sustained despite gaps in epithelial layers. Gastroenterology 2005; 129:902-12.
44. Laukoetter MG, Bruewer M, Nusrat A. Regulation of the intestinal epithelial barrier by the apical junctional complex. Curr Opin Gastroenterol 2006;22:85-9.
45. McAlindon ME, Gray T, Galvin A, Sewell HF, Podolsky DK, Mahida YR. Differential lamina propria cell migration via basement membrane pores of inflammatory bowel disease mucosa. Gastroenterology 1998; 115:841-8.
2. Riley SA, Mani V, Goodman MJ, Dutt S, Herd ME. Microscopic activity in ulcerative colitis: what does it mean? Gut 1991;32:174-8.
3. Ibarra-Palomino J, Barreto-Zuniga R, Elizondo-Rivera J, Bobadilla-Diaz J, Villegas-Jimenez A. [Application of chromoendoscopy to evaluate the severity and interobserver variation in chronic non-specific ulcerative colitis]. Rev Gastroenterol Mex 2002;67:236-40.
4. Kunihiro M, Tanaka S, Sumii M, Ueno Y, Ito M, Kitadai Y, Yoshihara M, Shimamoto F, Haruma K, Chayama K. Magnifying colonoscopic features of ulcerative colitis reflect histologic inflammation. Inflamm Bowel Dis 2004; 10:737-44.
5. Teshima CW, Meddings JB. The measurement and clinical significance of intestinal permeability. Curr Gastroenterol Rep 2008; 10:443-9.
6. Kiesslich R, Goetz M, Lammersdorf K, Schneider C, Burg J, Stolte M, Vieth M, Nafe B, Galle PR, Neurath MF. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007;132:874-82.
7. Li CQ, Xie XJ, Yu T, Gu XM, Zuo XL, Zhou CJ, Huang WQ, Chen H, Li YQ. Classification of Inflammation Activity in Ulcerative Colitis by Confocal Laser Endomicroscopy. Am J Gastroenterol 2009.
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4. Kunihiro M, Tanaka S, Sumii M, Ueno Y, Ito M, Kitadai Y, Yoshihara M, Shimamoto F, Haruma K, Chayama K. Magnifying colonoscopic features of ulcerative colitis reflect histologic inflammation. Inflamm Bowel Dis 2004; 10:737-44.
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6. Kiesslich R, Goetz M, Lammersdorf K, Schneider C, Burg J, Stolte M, Vieth M, Nafe B, Galle PR, Neurath MF. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007; 132:874-82.
7. Watanabe O, Ando T, Maeda O, Hasegawa M, Ishikawa D, Ishiguro K, Ohmiya N, Niwa Y, Goto H. Confocal endomicroscopy in patients with ulcerative colitis. J Gastroenterol Hepatol 2008;23 Suppl 2:S286-90.
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16. Smecuol E, Bai JC, Sugai E, Vazquez H, Niveloni S, Pedreira S, Maurino E, Meddings J. Acute gastrointestinal permeability responses to different non-steroidal anti-inflammatory drugs. Gut 2001;49:650-5.
17. Zeng J, Li YQ, Zuo XL, Zhen YB, Yang J, Liu CH. Clinical trial:effect of active lactic acid bacteria on mucosal barrier function in patients with diarrhoea-predominant irritable bowel syndrome. Aliment Pharmacol Ther 2008;28:994-1002.
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1. Munkholm P, Langholz E, Hollander D, Thornberg K, Orholm M, Katz KD, Binder V. Intestinal permeability in patients with Crohn's disease and ulcerative colitis and their first degree relatives. Gut 1994;35:68-72.
2. Oriishi T, Sata M, Toyonaga A, Sasaki E, Tanikawa K. Evaluation of intestinal permeability in patients with inflammatory bowel disease using lactulose and measuring antibodies to lipid A. Gut 1995;36:891-6.
3. Laukoetter MG, Nava P, Nusrat A. Role of the intestinal barrier in inflammatory bowel disease. World J Gastroenterol 2008; 14:401-7.
4. McGuckin MA, Eri R, Simms LA, Florin TH, Radford-Smith G Intestinal barrier dysfunction in inflammatory bowel diseases. Inflamm Bowel Dis 2009; 15:100-13.
5. Silva MA. Intestinal dendritic cells and epithelial barrier dysfunction in Crohn's disease. Inflamm Bowel Dis 2009; 15:436-53.
6. Spiller RC, Jenkins D, Thornley JP, Hebden JM, Wright T, Skinner M, Neal KR. Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome. Gut 2000;47:804-11.
7. Dunlop SP, Hebden J, Campbell E, Naesdal J, Olbe L, Perkins AC, Spiller RC. Abnormal intestinal permeability in subgroups of diarrhea-predominant irritable bowel syndromes. Am J Gastroenterol 2006; 101:1288-94.
8. Camilleri M, Gorman H. Intestinal permeability and irritable bowel syndrome. Neurogastroenterol Motil 2007;19:545-52.
9. Shulman RJ, Eakin MN, Czyzewski DI, Jarrett M, Ou CN. Increased gastrointestinal permeability and gut inflammation in children with functional abdominal pain and irritable bowel syndrome. J Pediatr 2008;153:646-50.
10. Zeng J, Li YQ, Zuo XL, Zhen YB, Yang J, Liu CH. Clinical trial:effect of active lactic acid bacteria on mucosal barrier function in patients with diarrhoea-predominant irritable bowel syndrome. Aliment Pharmacol Ther 2008;28:994-1002.
11. Kerckhoffs AP, Akkermans LM, de Smet MB, Besselink MG, Hietbrink F, Bartelink IH, Busschers WB, Samsom M, Renooij W. Intestinal Permeability in Irritable Bowel Syndrome Patients:Effects of NSAIDs. Dig Dis Sci 2009.
12. Piche T, Barbara G, Aubert P, Bruley des Varannes S, Dainese R, Nano JL, Cremon C, Stanghellini V, De Giorgio R, Galmiche JP, Neunlist M. Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome: involvement of soluble mediators. Gut 2009;58:196-201.
13. Zhou Q, Zhang B, Nicholas Verne G. Intestinal membrane permeability and hypersensitivity in the irritable bowel syndrome. Pain 2009; 146:41-6.
14. Teshima CW, Meddings JB. The measurement and clinical significance of intestinal permeability. Curr Gastroenterol Rep 2008; 10:443-9.
15. Kiesslich R, Burg J, Vieth M, Gnaendiger J, Enders M, Delaney P, Polglase A, McLaren W, Janell D, Thomas S, Nafe B, Galle PR, Neurath MF. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004; 127:706-13.
16. Polglase AL, McLaren WJ, Skinner SA, Kiesslich R, Neurath MF, Delaney PM. A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-GI tract. Gastrointest Endosc 2005;62:686-95.
17. Kitabatake S, Niwa Y, Miyahara R, Ohashi A, Matsuura T, Iguchi Y, Shimoyama Y, Nagasaka T, Maeda O, Ando T, Ohmiya N, Itoh A, Hirooka Y, Goto H. Confocal endomicroscopy for the diagnosis of gastric cancer in vivo. Endoscopy 2006;38:1110-4.
18. Becker V, Vercauteren T, von Weyhern CH, Prinz C, Schmid RM, Meining A. High-resolution miniprobe-based confocal microscopy in combination with video mosaicing (with video). Gastrointest Endosc 2007;66:1001-7.
19. Hurlstone DP, Thomson M, Brown S, Tiffin N, Cross SS, Hunter MD. Confocal endomicroscopy in ulcerative colitis: differentiating dysplasia-associated lesional mass and adenoma-like mass. Clin Gastroenterol Hepatol 2007;5:1235-41.
20. Meining A, Saur D, Bajbouj M, Becker V, Peltier E, Hofler H, von Weyhern CH, Schmid RM, Prinz C. In vivo histopathology for detection of gastrointestinal neoplasia with a portable, confocal miniprobe: an examiner blinded analysis. Clin Gastroenterol Hepatol 2007;5:1261-7.
21. Goetz M, Kiesslich R. Confocal endomicroscopy: in vivo diagnosis of neoplastic lesions of the gastrointestinal tract. Anticancer Res 2008;28:353-60.
22. Guo YT, Li YQ, Yu T, Zhang TG, Zhang JN, Liu H, Liu FG, Xie XJ, Zhu Q, Zhao YA. Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo: a prospective study. Endoscopy 2008;40:547-53.
23. Hurlstone DP, Baraza W, Brown S, Thomson M, Tiffin N, Cross SS. In vivo real-time confocal laser scanning endomicroscopic colonoscopy for the detection and characterization of colorectal neoplasia. Br J Surg 2008;95:636-45.
24. Hurlstone DP, Tiffin N, Brown SR, Baraza W, Thomson M, Cross SS. In vivo confocal laser scanning chromo-endomicroscopy of colorectal neoplasia:changing the technological paradigm. Histopathology 2008;52:417-26.
25. Leong RW, Nguyen NQ, Meredith CG, Al-Sohaily S, Kukic D, Delaney PM, Murr ER, Yong J, Merrett ND, Biankin AV. In vivo confocal endomicroscopy in the diagnosis and evaluation of celiac disease. Gastroenterology 2008; 135:1870-6.
26. Pech O, Rabenstein T, Manner H, Petrone MC, Pohl J, Vieth M, Stolte M, Ell C. Confocal laser endomicroscopy for in vivo diagnosis of early squamous cell carcinoma in the esophagus. Clin Gastroenterol Hepatol 2008;6:89-94.
27. Zhang JN, Li YQ, Zhao YA, Yu T, Zhang JP, Guo YT, Liu H. Classification of gastric pit patterns by confocal endomicroscopy. Gastrointest Endosc 2008;67:843-53.
28. Chang-Qing Li X-JX, Tao Yu, Xiao-Meng Gu, Xiu-Li Zuo, Cheng-Jun Zhou, Wei-Qing Huang, Hua Chen, Yan-Qing Li. Classification of Inflammation Activity in Ulcerative Colitis by Confocal Laser Endomicroscopy. Am J Gastroenterol advance online publication 2009.
29. Kiesslich R, Goetz M, Angus EM, Hu Q, Guan Y, Potten C, Allen T, Neurath MF, Shroyer NF, Montrose MH, Watson AJ. Identification of epithelial gaps in human small and large intestine by confocal endomicroscopy. Gastroenterology 2007; 133:1769-78.
30. Goke M, Podolsky DK. Regulation of the mucosal epithelial barrier. Baillieres Clin Gastroenterol 1996; 10:393-405.
31. Mahida YR, Galvin AM, Gray T, Makh S, McAlindon ME, Sewell HF, Podolsky DK. Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells. Clin Exp Immunol 1997;109:377-86.
32. Muller CA, Autenrieth IB, Peschel A. Innate defenses of the intestinal epithelial barrier. Cell Mol Life Sci 2005;62:1297-307.
33. Vilela EG, de Abreu Ferrari Mde L, de Gama Torres HO, Martins FP, Goulart EM, Lima AS, da Cunha AS. Intestinal permeability and antigliadin antibody test for monitoring adult patients with celiac disease. Dig Dis Sci 2007;52:1304-9.
34. Duerksen DR, Wilhelm-Boyles C, Parry DM. Intestinal permeability in long-term follow-up of patients with celiac disease on a gluten-free diet. Dig Dis Sci 2005;50:785-90.
35. Marshall JK, Thabane M, Garg AX, Clark W, Meddings J, Collins SM. Intestinal permeability in patients with irritable bowel syndrome after a waterborne outbreak of acute gastroenteritis in Walkerton, Ontario. Aliment Pharmacol Ther 2004;20:1317-22.
36. Quigley E. Gut permeability in irritable bowel syndrome: more leaks add to slightly inflamed bowel syndrome conspiracy theory. Gastroenterology 2009; 137:728-30.
37. Smecuol E, Bai JC, Sugai E, Vazquez H, Niveloni S, Pedreira S, Maurino E, Meddings J. Acute gastrointestinal permeability responses to different non-steroidal anti-inflammatory drugs. Gut 2001;49:650-5.
38. Kiesslich R, Gossner L, Goetz M, Dahlmann A, Vieth M, Stolte M, Hoffman A, Jung M, Nafe B, Galle PR, Neurath MF. In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2006;4:979-87.
39. Becker V, von Delius S, Bajbouj M, Karagianni A, Schmid RM, Meining A. Intravenous application of fluorescein for confocal laser scanning microscopy: evaluation of contrast dynamics and image quality with increasing injection-to-imaging time. Gastrointest Endosc 2008; 68:319-23.
40. Li CQ, Xie XJ, Yu T, Gu XM, Zuo XL, Zhou CJ, Huang WQ, Chen H, Li YQ. Classification of Inflammation Activity in Ulcerative Colitis by Confocal Laser Endomicroscopy. Am J Gastroenterol 2009.
41. Brown GR, Lindberg G, Meddings J, Silva M, Beutler B, Thiele D. Tumor necrosis factor inhibitor ameliorates murine intestinal graft-versus-host disease. Gastroenterology 1999;116:593-601.
42. Masungi C, Mensch J, Van Dijck A, Borremans C, Willems B, Mackie C, Noppe M, Brewster ME. Parallel artificial membrane permeability assay (PAMPA) combined with a 10-day multiscreen Caco-2 cell culture as a tool for assessing new drug candidates. Pharmazie 2008;63:194-9.
43. Watson AJ, Chu S, Sieck L, Gerasimenko O, Bullen T, Campbell F, McKenna M, Rose T, Montrose MH. Epithelial barrier function in vivo is sustained despite gaps in epithelial layers. Gastroenterology 2005; 129:902-12.
44. Laukoetter MG, Bruewer M, Nusrat A. Regulation of the intestinal epithelial barrier by the apical junctional complex. Curr Opin Gastroenterol 2006;22:85-9.
45. McAlindon ME, Gray T, Galvin A, Sewell HF, Podolsky DK, Mahida YR. Differential lamina propria cell migration via basement membrane pores of inflammatory bowel disease mucosa. Gastroenterology 1998; 115:841-8.