The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.

Diagnosis of vulvar lesions by non-invasive optical analysis: a pilot study.

A procedure that could allow an early in vivo and non-invasive detection of vulvar lesions would be extremely useful. We tested an innovative optical method (Optiprobe), which uses a harmless, visible light source for the in vivo, on-line detection of minimal alterations in the structure of vulvar epithelium. A group of 3 female volunteers without gynecological symptoms were first screened to evaluate optical properties of normal vulvar tissue. Next, a group of 16 patients undergoing gynecological examination for vulvar lesions was evaluated by the Optiprobe at suspected sites before these sites were biopsied for histological analysis. Adjacent, non-involved sites were also measured to provide internal controls. Histological analysis of the biopsies identified one case that did not show obvious alterations, 4 cases of high-grade vulvar intraepithelial neoplasia (VIN), 5 cases of vulvitis, and 6 cases of lichen sclerosis (LS).The optical properties of the VIN cases were significantly different from those of controls, due to a decrease in the absorption spectra and an increase in the scattering spectra. In contrast, a significant increase in the absorption spectra and a decrease in the scattering spectra were observed in the cases of vulvitis. In the LS cases, the absorption spectra were as in controls, whereas the scattering spectra were significantly decreased. We conclude that the Optiprobe provides a useful tool for a rapid and non-invasive detection of vulvar alterations. The method should contribute to reduce the number of biopsies and to facilitate the long-term follow-up of vulvar lesions.

Live imaging of neural structure and function by fibred fluorescence microscopy.

Only a few methods permit researchers to study selected regions of the central and peripheral nervous systems with a spatial and time resolution sufficient to image the function of neural structures. Usually, these methods cannot analyse deep-brain regions and a high-resolution method, which could repeatedly probe dynamic processes in any region of the central and peripheral nervous systems, is much needed. Here, we show that fibred fluorescence microscopy-which uses a small-diameter fibre-optic probe to provide real-time images-has the spatial resolution to image various neural structures in the living animal, the consistency needed for a sequential, quantitative evaluation of axonal degeneration/regeneration of a peripheral nerve, and the sensitivity to detect calcium transients on a sub-second timescale. These unique features should prove useful in many physiological studies requiring the in situ functional imaging of tissues in a living animal.

Diagnosis and grading of gastritis by non-invasive optical analysis.

OBJECTIVES: The precise identification of many diseases of the gastrointestinal tract requires the histological analysis of multiple biopsies of the lining mucosae, thus preventing an immediate diagnosis and the safe screening of the entire organ. To address these limitations, we developed a novel spectroscopic procedure for a real-time, non-invasive optical analysis of mucosae. METHODS: We have used a fibre-optic probe that monitors light propagation through small tissue volumes to evaluate the antral and fundic mucosa of 51 patients that underwent gastroscopy for symptoms of dyspepsia. Several optical coefficients were computed from the recorded light reflectance, and confronted to the diagnosis made by an expert gastroscopist at the time of the clinical examination. Both evaluations were then validated by comparison with the histological diagnosis of a pathologist who screened biopsies taken at the sites of the optical measurements. RESULTS: We report that the optical procedure discriminated normal and pathological gastric mucosae with a higher sensitivity and specificity than endoscopic diagnosis. We also show that the changes in light-scattering coefficient, which permitted the optical diagnosis of gastritis alterations, were indirectly correlated with the extent of inflammatory infiltration of the mucosa and detected mucosal alterations mild enough to escape endoscopic detection. CONCLUSIONS: The results show that, in a normal clinical setting, the optical in vivo analysis provided by our system detects alterations typical of gastritis, and allow for their graded scoring with a specificity and sensitivity that compare well with those of standard histology, while avoiding the invasiveness of the latter procedure. The method is adaptable to the screening of other types of lesions and mucosae and, hence, should prove useful in improving available diagnostic approaches.

[Optical diagnosis of cervical dysplasia]. / Diagnostic optique des lésions dysplasiques du col utérin.

Cervix cancer is a curable disease when diagnosed at an early stage. Screening of cervical lesions by cytology and colposcopy with in situ staining has allowed for substantial progress in early diagnosis and consequently the cure of cervix cancer. Nevertheless, because of its low specificity, this approach generally implies repetitive tissue sampling and, thus a relative long time before the treatment of the lesions. Furthermore, the cost of preparation and analysis of biopsy samples is sufficiently high to represent a burden for industrialized countries and a virtual impossibility for the developing world. To overcome these problems, various biophotonic methods using optical fibers have been developed to allow for detection of cervical epithelial anomalies in a specific, fast and non-invasive way. This process, known as "optical biopsy", is based on the measurement of light-tissue interactions, which are analysed by various mathematical and data processing methods, to provide information on the metabolism and morphology of epithelial tissue. Currently investigated methods can be distinguished according to the type of signal used to probe the tissue (fluorescence, reflectance), the depth of analysed tissue (surface analysis, confocal imaging, tomography), the analysis modalities (spectral measurements or imaging), and the use of additive molecules (contrasting or photosensitizing agents, inorganic fluorophores). While most of the methods remain experimental, constant progress in the understanding of the mechanisms of light behavior in biological environments as well as advances in optical fibers technology, will make a number of these methods soon available for clinical practice to contribute efficiently to the reduction of biopsy number and cost of cervical screening.

In vivo endoscopic tissue diagnostics based on spectroscopic absorption, scattering, and phase function properties.

A fast spectroscopic system for superficial and local determination of the absorption and scattering properties of tissue (480 to 950 nm) is described. The probe can be used in the working channel of an endoscope. The scattering properties include the reduced scattering coefficient and a parameter of the phase function called gamma, which depends on its first two moments. The inverse problem algorithm is based on the fit of absolute reflectance measurements to cubic B-spline functions derived from the interpolation of a set of Monte Carlo simulations. The algorithm's robustness was tested with simulations altered with various amounts of noise. The method was also assessed on tissue phantoms of known optical properties. Finally, clinical measurements performed endoscopically in vivo in the stomach of human subjects are presented. The absorption and scattering properties were found to be significantly different in the antrum and in the fundus and are correlated with histopathologic observations. The method and the instrument show promise for noninvasive tissue diagnostics of various epithelia.

A new optical method for the non-invasive detection of minimal tissue alterations.

Histological analysis, which is used to detect and diagnose most tissue alterations, requires an invasive biopsy procedure and a time-consuming tissue treatment, which limit its efficiency in providing rapid, cost-effective diagnosis and hinder the longitudinal study of tissue alteration. To address these limitations, we have developed a novel procedure, using the features of elastic-scattering spectroscopy, for a real-time, non-invasive analysis of tissues. We have tested whether this approach can detect in vivo changes in mouse skin induced by a single exposure to either complete Freund's adjuvant or 12-O-tetradecanoylphorbol-13-acetate, two drugs known to induce discrete alterations of epidermis and dermis, without obvious changes on the skin surface. Here we report that the evaluation of localized absorption and reduced scattering coefficients permitted the detection of changes in skin regions that showed histological alterations, but not in regions which failed to be modified by the drugs. Results show that the optical in vivo analysis of small regions has sufficient specificity and sensitivity to detect minimal alterations of superficial tissues. In view of the prominent involvement of mucosal alterations in most human diseases, including carcinomas, the method provides a useful complement to standard biopsy, notably for the in vivo screening of early in situ epithelial alterations.