In vivo detection of experimental ulcerative colitis in rats using fiberoptic confocal imaging (FOCI).

Fiberoptic confocal imaging (FOCI) is a noninvasive microscopic technique that enables subsurface imaging of living tissue in vivo. The aim of the present study was to assess the suitability of FOCI for the in vivo detection of early subsurface changes in the mucosal architecture of the colon in a rat model of ulcerative colitis. Mild colitis was induced in Sprague-Dawley rats (180-250 g) by the oral ingestion of 5% (w/v) dextran sulfate sodium (DSS; Mr 40,000 Da) in drinking water. Control animals were provided with water ad libitum. After three, five or seven days of oral consumption of DSS, the mucosal surface of the colon of anesthetised rats was surgically exposed. Morphological changes in the mucosa were examined (Optiscan F900e personal confocal system with rigid endomicroscope attachment; excitation 488 nm argon ion laser, detection above 515 nm) following the topical application of a fluorescent dye (fluorescein, eosin, or acridine orange). Confocal images were correlated with conventional histology and clinical parameters including occult blood and stool consistency. Histological evaluation of colon sections demonstrated that DSS-induced colitis was characterized by focal loss of mucous crypts, loss of epithelial cells, and neutrophilic infiltration into the mucosa. The extent of mucosal damage was positively correlated with the time of ingestion of DSS. Morphological changes associated with disease activity could be detected microscopically in vivo using FOCI but were not evident by visual inspection of the colon surface. Acridine orange enabled imaging of the colonic crypts at the surface of the mucosa. Morphological changes associated with colitis, including inflammatory cell infiltrate, crypt loss, and crypt distortion, could be detected using this fluorophore. Application of fluorescein and eosin enabled subsurface imaging of the lamina propria surrounding the crypts; however, no change in structure was detected in association with colitic disease activity. This study has shown that the topical application of acridine orange enables in vivo imaging of early colitis in a rat model. FOCI may be suitable for the diagnosis and monitoring of human inflammatory bowel disease.

Autofluorescence of skin burns detected by fiber-optic confocal imaging: evidence that cool water treatment limits progressive thermal damage in anesthetized hairless mice.

BACKGROUND: Although full-thickness burns present no difficulty to clinical judgment, accurate assessment of burn depth immediately after injury in partial thickness burns has always been difficult. METHODS: Thermal burns (applied by a 3-mm-diameter brass rod heated to 50 degrees--80 degrees C for 20 seconds) were induced on the skin of anesthetized hairless mice. Anesthesia was maintained throughout all experiments. Both burns and normal skin were investigated noninvasively in vivo using fiber-optic confocal imaging (FOCI) microscopy (excitation, 488 nm; detection, 505 nm). RESULTS: Autofluorescence was detected in burned skin, and the depth of the autofluorescent region was found to correlate with the intensity of heat applied. Cool water treatment (for 20 minutes immediately after burn induction) significantly reduced the progressive increase in autofluorescence in deeper layers of the skin over the 4-hour postburn observation period. Histology showed burn-associated changes at a lower temperature than that at which autofluorescence was first detected in vivo by FOCI. However, there was a good correlation (r = 0.78) between depth of damage revealed by FOCI compared with that by histology. CONCLUSION: These results suggest that FOCI may be used to provide an index of burn depth.

In vivo detection of small subsurface melanomas in athymic mice using noninvasive fiber optic confocal imaging.

Fiber optic confocal imaging, following intravenous administration of fluorescently labeled antibodies and Texas Red-dextran, enabled in vivo detection of melanoma and surrounding blood vessels in athymic mice. Human melanoma cells (three cell lines) and cultured normal human skin cells were implanted intradermally into the haunch skin of anesthetized athymic BALB/C mice and allowed to grow to a maximum size of 2 mm diameter. Using three different fluorescein-isothiocyanate-labeled antimelanoma antibodies, single channel confocal images of melanoma cells were obtained in vivo. Using noninvasive techniques, the overall in vivo melanoma detection rate for tumors within 0.2 mm of the skin surface was 84% (27 of 32 tumors). Normal cultured human skin cells were found to have little or no fluorescence after administration of the fluorescein-isothiocyanate-labeled antibodies and tumors were not labeled by an isotype control antibody. Dual channel imaging of the implanted melanoma tumor and surrounding dermal vasculature in vivo showed increased blood vessel density at the melanoma site. Conventional immunoperoxidase histology confirmed that fiber optic confocal imaging was able to detect melanoma tumors up to 0.2 mm below the skin surface, in vivo.

Localization of p450scc and 5alpha-reductase type-2 in the cerebellum of fetal and newborn sheep.

Prenatally, neuroactive steroids that modulate GABAergic activity may be synthesized de novo within the fetal brain. We have examined changes in immunoreactivity staining for the steroidogenic enzymes cholesterol P450 side-chain cleavage (P450scc), and 5alpha-reductase type-2 in the cerebellum of late gestation (130-145 days gestation) fetal sheep and newborn lambs (1-4 weeks of age). Both enzymes were predominantly localized in the Purkinje cell body and dendrites of the fetal and newborn cerebellum, with weaker immunoreactivity in a few cells of the inner granular layer. P450scc immunoreactivity was present in Purkinje neurons expressing either of the neuronal microtubule associated proteins MAP1b/5 or MAP2a/b, but was absent from GFAP and HNK-1 positive cells. Soma of Purkinje neurons were also immunopositive for 5alpha-reductase type-2 in the fetuses, but expression decreased to just detectable levels in the 1-2 and 2-4 week old lambs. Both MAP1b/5- and MAP2a/b-positive Purkinje neurons showed 5alpha-reductase type-2 expression in the fetus, whereas the residual 5alpha-reductase staining in the newborn lamb was present only in MAP2a/b-positive Purkinje neurons. Allopregnanolone in the cerebellum decreased from 21.8+/-1.9 ng/g wet weight in fetuses at 140-145 days gestation to 6.7+/-0.5 ng/g in 2-4 week old lambs (P<0. 05). Thus, synthesis of neuroactive steroids from cholesterol is possible in cerebellar neurons in late gestation and persists into neonatal life, 5alpha-reductase type-2 expression is greater in the fetus compared to the neonate, and allopregnanolone concentrations in the cerebellum decrease significantly after birth.

Expression of menkes copper-transporting ATPase, MNK, in the lactating human breast: possible role in copper transport into milk.

The Menkes copper ATPase (MNK) is a copper efflux ATPase that is involved in copper homeostasis. Little is known about the intracellular localization and cell-specific function of the MNK in human tissues. To investigate a possible role for this protein in lactation, we measured its expression in sections of tissue from nonlactating and lactating human breast. Western blot analysis showed that MNK expression was greater in lactating tissue than in nonlactating tissue. By confocal immunofluorescence, the MNK was detected in luminal epithelial cells of the alveoli and ducts but not in myoepithelial cells. In the nonlactating breast epithelial cells, the MNK had a predominantly perinuclear distribution. In lactating breast tissue, the distribution of the MNK was markedly altered. Lactating epithelial cells showed a granular, diffuse pattern, which extended beyond the perinuclear region of the cell. This pattern was similar to that observed in a previous study in which cultured CHO cells were exposed to high copper concentrations. Our results suggest that relocalization of the MNK is a physiological process, which may be mediated by copper levels in the breast or by hormones and other events taking place during lactation. A vesicular pathway for copper from the Golgi into milk, similar to that of calcium, is proposed.(J Histochem Cytochem 47:1553-1561, 1999)