Confocal laser endomicroscopy in experimental pigs Methods of ex vivo imaging

Summary

Background.
Confocal laser scanning endomicroscopy (CLSE) enables online in vivo cellular surface and subsurface imaging of normal and pathological tissue at high resolution and magnification. The aim of this study was to work out a method of ex vivo in vitro CLSE in experimental pigs and to compare CLSE images with those of “classic” histology.

Material and methods.
Five mature female pigs entered the study. CLSE on an ex vivo in vitro basis was started 10 minutes after pharmacological euthanasia and carried out for 30 minutes. Fluorescein was administrated i.v. as a fluorescence substance.

Results.
CLSE was successful in all tissue samples of all animals (the oesophagus, stomach, small intestine, large bowel). We have succeeded to obtain high quality images within the first 30 minutes that means 40 minutes after the euthanasia of experimental animals. CLSE images corresponded well with those of haematoxylin-eosin staining.

Conclusions. CLSE on an ex vivo in vitro basis in experimental pigs is feasible.

Key words:

confocal laser, endomicroscopy, gastrointestinal, experimental pigs.

After overnight fasting, a sterile 10% water solution of 10 mg fluorescein sodium (Fluorescite, S.A. Alcon-Couvreur N.V., Puurs, Belgium) was administrated rapidly intravenously into the superior caval vein. Ten minutes later, the pigs were sacrificed by means of pharmacological euthanasia (T61, Intervet International BV, Boxmeer, the Netherlands; dose of 2 mL/kg). Immediate autopsy was performed and samples of the oesophagus, stomach, small intestine and large bowel were collected for confocal laser endomicroscopy and parallel light microscopy.

Confocal laser endomicroscopy

Confocal laser endomicroscopy on an ex vivo in vitro basis was started 10 minutes after pharmacological euthanasia and carried out for 30 minutes. The investigation was performed by means of Confocal laser endomicroscopy system Pentax (Tokyo, Japan)/Optiscan (Notting Hill, Australia). It consists of a standard video colonoscope (EC3870K) with miniaturised confocal microscope on its tip, processor (EPK-100) and laser system (ISC OU1000). To prevent a direct contact of the tip of video-endoscope and confocal microscope with a porcine tissue we used a semi-permeable membrane Dialysis tubing visking (Carl Roth, Karlsruhe, Germany). It is made of regenerated cellulose (molecular weight 14 kDa), stable at pH range 5–9. Both tissue and membrane were flushed by a saline solution during investigation. All confocal laser endomicroscopy images were recorded (in average 1000 images per one organ per one animal).

Histology

Specimens of the oesophagus, stomach, small and large bowel were carefully fixed with 10% neutral buffered formalin. Samples were subsequently embedded into paraffin, 1 μm thick horizontal tissue sections were cut and hematoxylin-eosin staining was provided. Optical histology was subsequently compared with confocal laser microscopic images. Stained samples were evaluated using a BX-51 microscope (Olympus Optical Co, Tokyo, Japan)

Ethics

The Project was approved by the Institutional Review Board of the Animal Care Committee at the Institute of Experimental Biopharmaceutics, Academy of Sciences of the Czech Republic, Protocol Number 149/2006. Animals were held and treated in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (17). 

Results

Confocal laser microscopy was successful in all tissue samples of all animals (the oesophagus, stomach, small intestine, large bowel). We have succeeded to obtain high quality images within the first 30 minutes that means 40 minutes after the euthanasia of experimental animals. Confocal laser microscopy images correspond well with those of hematoxylin-eosin staining (see Figures 1–4). Use of semi-permeable cellulose-based membrane (to prevent a direct contact of the tip of video-endoscope and confocal microscope with a porcine tissue) did not affect quality of images. They were fully comparable with those obtained during a direct investigation in humans. 

Discussion

This study has proved that confocal laser endomicroscopy on an ex vivo in vitro basis in experimental animals is feasible. We have worked out this method to overcome some problems and difficulties of confocal laser scanning microscopy in experimental setting. In vivo, it is not possible to investigate different parts of the gastrointestinal tract of a particular animal in the same time. Endoscopy access to the small intestine of a pig is rather difficult because of the transverse pyloric fold (torus pyloricus) in a porcine stomach serving as a “gatekeeper”, thus making the pylorus relatively stenotic (18). Large bowel cleansing for colonoscopy is not feasible in a pig. During confocal laser scanning microscopy in vivo, the endomicroscope has to be placed vertically against the target point on the mucosa during the examination. However, it may be difficult to scan vertically against the mucosa in some parts of the gastrointestinal tract. Further, any movement in the digestive tract during an examination makes confocal laser endomicroscopy difficult (10). Last but not least it is necessary to consider cost of the equipment. Confocal laser endomicroscopy dedicated for animal use only would not be cost-effective for most centres.

Fluorescence confocal laser scanning microscopy can take advantage of dyes that stain specific cellular structures, so that it is closer to traditional histopathological imaging. Suitable agents are fluorescein, acriflavine, tetracycline, or cresyl violet. With regards to fluorescein, no immediate toxicity was observed after topical or systemic application. Fluorescein sodium that has been safely used in ophthalmology for decades gives a good overall impression of the mucosa after partly leaking from circulation into the tissue. It renders many subcellular details such as mucin in colonic goblet cells. A sufficient contrast is also observed in vessels where fluorescein is partly retained because of its plasma protein binding. Due to its pharmacological properties, fluorescein does not stain nuclei (5, 16, 19).

In humans, an endomicroscopic image can be obtained already within 30 seconds after i.v. injection due to very rapid distribution of the substance in the human body. The effect remains for approximately one hour. A side effect is yellowish coloration of the skin and urine of the patient. The skin coloration appears within a few minutes and lasts up to 12 hours. The colour of the urine becomes normal within 36 hours.

In our project, the dose and timing of fluorescein was based on our own study on pharmacokinetics and organ distribution of fluorescein in experimental pigs. The elimination of fluorescein from blood was rapid (half-time 30 minutes). The fast decline in plasma concentration suggests a multi-compartmental model of distribution, with in a rapid drop within the first 10 minutes because of equilibration with extravascular fluid compartments. That is why we decided a 10-minute interval as optimal time for the euthanasia of experimental animals. Fluorescein organ distribution within the gastrointestinal tract was comparable (~ 10–25 μg/g), fluorescein concentration was the highest in the duodenum, the lowest in the oesophagus, and as twice as high in the gastric body compared to gastric antrum (20). Despite these differences, there was no tangible distinction of particular confocal scanning images. Once borderline organ concentration of fluorescein is reached, confocal laser microscopy is possible.

There are only limited data on experimental use of confocal laser scanning microscopy in experimental setting. Götz et al. did great work testing various fluorescent agents and imaging different normal and pathological gastrointestinal organs and tissues using prototype probes in experimental rodents and pigs (7, 21–23). They performed their confocal laser scanning microscopy in vivo.

In conclusion, confocal laser scanning endomicroscopy on an ex vivo in vitro basis in experimental pigs is feasible. It can be used for future imaging studies on injury of various drugs (e.g. non-steroidal anti-inflammatory drugs) to different parts of the gastrointestinal tract.

The endoscopic part of the study was supported by research project MZO 00179906 from the Ministry of Health, Czech Republic. The experimental part of the study was supported by research grant GA ČR 305/08/0535.

The authors are grateful to Mrs. Sylva Cvejnová, Mrs. Helena Medková, Mrs. Ludmila Pavlatová and Mrs. Eva Vrchotová for their excellent technical assistance.

Address for correspondence:

Associate Professor Marcela Kopáčová, MD, PhD.

2nd Department of Medicine, Charles University Teaching Hospital

Sokolská 581, 500 05 Hradec Králové, Czech Republic

e-mail: kopacmar@fnhk.cz