Steven Thomas

In Vivo Detection of Morphological and Microvascular Changes of the Colon in Association with Colitis Using Fiberoptic Confocal Imaging (FOCI)

Using a well-established rodent model of inflammatory bowel disease (IBD), the present study examined changes in the microvasculature of the colonic mucosa in association with ulcerative colitis (UC). The results were compared to microscopic alterations in tissue morphology to establish a temporal relationship between microcirculatory dysfunction and IBD pathology. Mild colitis was induced in rats by the oral consumption of 5% dextran sulfate sodium (DSS) in drinking water. Control animals were provided with water ad libitum. After 3, 5, and 7 days of oral ingestion of DSS, anesthetized rats were laparotomized. The mucosal surface of the distal colon was then examined using fiber optic confocal imaging (FOCI; excitation 488 nm argon ion laser, detection above 515 nm). Changes in the mucosal architecture were examined following the topical application of the fluorescent dye, tetracycline hydrochloride. Tetracycline hydrochloride, an antibiotic used widely in clinical medicine, enabled imaging of the crypts at the surface of the mucosa. Spatial changes in the microvascular structure were assessed following the intravenous administration of fluorescein isothiocyanate dextran (FITC-dextran). Confocal images were correlated with clinical parameters, including weight loss, occult blood, and stool consistency. Attenuation of the colonic epithelium was detected on day 3 colitis. Morphological changes including crypt loss, crypt distortion, and inflammatory cell infiltrate were detected on day 5 and day 7 colitis. Dual channel imaging showed the mucosal capillary network outlining the stromal confines of the mucus-secreting glands in control tissue. Experimental colitis resulted in diffuse hypervascularity and tortuosity of the capillary vessels. Evidence of increased vessel leakiness (leakage of FITC-dextran from the lumen) was first detected on day 5 colitis. Complete disruption of the normal honeycomb pattern of the vessels and capillary dilation was evident after 7 days of DSS ingestion. These findings suggest that the pathogenesis of ulcerative colitis is associated with changes in the vascular architecture as demonstrated in vivo using confocal microscopy.

Role of confocal endomicroscopy in the diagnosis of celiac disease.

Background and Aims: Confocal laser endomicroscopy (CLE) is a recent development that enables surface and subsurface imaging of living cells in vivo at 1000× magnification. The aims of the present study were to define confocal features of celiac disease (CD) and to evaluate the usefulness of the CLE in the diagnosis of CD in children in comparison to histology. Patients and Methods: Nine patients (8 girls) with a median age of 8.35 years (range 2–12.66 years) and a median weight of 28.3 kg (range 11–71 kg) were suspected with CD and 10 matched controls underwent oesophagogastroduodenoscopy using the confocal laser endomicroscope (EC3870CILK; Pentax, Tokyo, Japan). Histologic sections were compared with the confocal images of the same site by 2 experienced paediatric histopathologists and endoscopists, all of whom were blinded to the diagnosis. Results: The median procedure time was 17 minutes (range 8–25 minutes). Confocal features of CD were defined and a score was developed. A total of 1384 confocal images were collected from 9 patients and 10 controls. Five images from each patient and control were selected and compared with the biopsy specimen of the same site. The sensitivity, specificity, and positive predictive value for the confocal images in comparison to the histology were 100%, 80%, and 81%. The kappa inter-observer agreement between the 2 endoscopists was 0.769 (P = 0.018) and between the 2 histopathologists was 0.571 (P = 0.05). Conclusions: Confocal endomicroscopy offers the prospect of diagnosis of CD during ongoing endoscopy. It also enables targeting biopsies to abnormal mucosa and thereby increasing the diagnostic yield, especially when villous atrophy is patchy in the duodenum.

A new method in the diagnosis of reflux esophagitis: confocal laser endomicroscopy

BACKGROUND The diagnosis of GERD is made by using a combination of clinical symptoms, pH study, endoscopy, and histology. Histologic changes include basal cell hyperplasia and papillary elongation. Confocal laser endomicroscopy (CLE) enables surface and subsurface imaging of living cells in vivo at ×1000 magnification and up to 250 μm below the tissue surface. In the esophagus, the distance between the surface to papillary (S-P) tip can be measured by using CLE. OBJECTIVE To measure the S-P distance in the esophagus in patients with reflux esophagitis and controls by using CLE and comparing with histologic measurements. DESIGN Retrospective analysis of a prospective database. SETTING Endoscopy unit of a tertiary-care childrens hospital. PATIENTS This study involved 7 patients (5 female) with a median age of 7.6 years (range 1.8-15.5 years) and median weight of 23 kg (range 13.2-71 kg) and 16 controls with a median age of 12.0 years (range 2.2-15.3 years) and median weight of 38.2 kg (range 10.7-83 kg). INTERVENTION S-P distance was measured both by CLE and histology and was corrected for height for both patients and controls and the results compared. MAIN OUTCOME MEASUREMENTS To determine if there were significant differences in the S-P distance in patients with esophagitis and controls. RESULTS The median confocal and histologic measurements for S-P distance, corrected for patient height, were 0.19 μm/cm (range 0.10-0.49 μm/cm) and 0.58 μm/cm (range 0.29-0.76 μm/cm) and for controls were 0.44 μm/cm (range 0.20-0.93 μm/cm) and 1.07 μm/cm (range 0.76-0.1.57 μm/cm), respectively. LIMITATIONS Small numbers involved in the study, reliance on only papillary elongation in arriving at a diagnosis. CONCLUSION Measurement of the S-P distance by CLE will enable real-time diagnosis of GERD-related esophagitis during ongoing endoscopy.

Microarchitecture of the Normal Gut Seen with Conventional Histology and Endomicroscopy

For ex vivo histological examination of the gastrointestinal tract, fractions of an organ or small pieces of tissue are needed. Several steps are used in the fixation, staining, and mounting process to ensure production of good-quality histology on glass slides. The most frequently used stain in routine histology is the haematoxylin and eosin (H&E) stain. The most frequently used tissue-staining methods are shown in ⊡ Table 6.1. The final histopathological diagnosis is always based on examination of the whole sample and the structure and architecture of that sample. In cytology, single cells and nuclei are used for making a diagnosis, so staining procedures in cytology are much faster and easier to perform.

Development and Current Technological Status of Confocal Laser Endomicroscopy

This chapter aims to explain the principles of confocal microscopy, the reasons for the key features in the present form of endomicroscopy, and how they account for what is seen during practical endomicroscopy of the GI tract.

Fluorescence confocal endomicroscopy in biological imaging

In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices. There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo microscopy systems. A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system). Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204 sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of a high-density optical fibre bundle (~30,000 fibres) of <1mm diameter to transfer the confocal imaging plane to tissue in intact small animal organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and sub-cellular details could be readily visualised in vivo at high resolution. In rodent disease models, in vivo endomicroscopy with appropriate fluorescent agents allowed examination of thrombosis formation, tumour microvasculature and liver metastases, diagnosis and staging of ulcerative colitis, liver necrosis and glomerulonephritis. Miniaturised confocal endomicroscopy allows rapid in vivo molecular and subsurface microscopy of normal and pathologic tissue at high resolution in small and large whole animal models. Fluorescein endomicroscopy has recently been introduced into the medical device market as a clinical imaging tool in GI endoscopy and is undergoing clinical evaluation in laparoscopic surgery. This medical usage is encouraging in-situ endomicroscopy as an important pre-clinical research tool to observe microscopic and molecular system biologic events in vivo in animal models for various human diseases.