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Identifier 000399702
Title Ανάπτυξη και συγκριτική αξιολόγηση νέων μεθόδων για τον μη επεμβατικό οπτικό χαρακτηρισμό και την ανάλυση φυσιολογικών και παθολογικών νευρικών ιστών
Alternative Title Development and comparative evaluation of new methods for noninvasive optical characterization and analysis of the physiological and pathological nerve tissues
Author Βαζγιουράκη, Ελευθερία Μ.
Thesis advisor Γραβάνης, Α
Φωτάκης, Κ.
Χαραλαμπόπουλος, Ι.
Abstract Multiple sclerosis (MS) is characterized by inflammation and demyelinating lesions in white matter (WM) of the central nervous system (CNS), leading to permanent neurological disabilities (sensory and motor deficiencies). Research on MS has been performed mainly with the aid of the experimental allergic encephalomyelitis (EAE) mouse model. Current diagnostic imaging of myelin alterations is conducted using microscopy of fixed tissue or a variety of modern methods. Fluorescence microscopy is a powerful diagnostic tool, which is usually coupled to immunofluorescence techniques, using the highly specific binding of an antibody to its antigen for labelling specific cellular proteins or other molecules within the cell. This method requires the administration of fluorescent dyes. Unfortunatelly, many of them tend to disrupt the lipid structure of myelin, while considerable shrinkage of myelin sheaths may take place after fixation and dehydration. Moreover, fluorophores are usually expensive and may lose their ability to fluoresce, due to their chemical deterioration under high-power light limiting the examination time of the sample. A variety of other modern optical methods (coherent anti-stokes raman scattering, CARS; third harmonic generation microscopy) have been used to image neuronal status, axonal alterations, or immune cell trafficking in the EAE mice. Although these imaging systems and methods are quite powerful to detect abnormal areas during MS and EAE, they are expensive, not portable and complicated. Therefore, laser energy and power onto the sample have to be optimized in order to achieve maximum penetration depth of light into the tissue under examination, without affecting the sample. Spectral imaging is a method which combines two well-established technologies, spectroscopy and imaging. Spectral imaging results in the collection of a three-dimensional data set, consisting of an image group, through the use of the same specimen field, acquired at different wavelength bands. An important advantage of spectral imaging is that it enables the inspection at narrow spectral bands, along a wide spectral range, permitting the direct assessment of invisible or low-contrast features of diagnostic importance. Additionally, it permits the measurement of the spectrum at each point or area of the image. An optimum set-up for the acquisition of multiple spectral images is the coupling of imaging detectors to monochromators. Imaging spectroscopy is generally performed with custom-built instruments due to the lack of commercial instrumentation. In the present work, a custom-made imaging monochromator coupled to a commercial microscope is used in order to evaluate spinal cord myelin damage. The critical component of the system is an interference filter wheel-based imaging monochromator. The monochromator incorporates a 5 MPixel monochrome CMOS sensor [complementary metal–oxide semiconductor (CMOS)] with 24 interference filters covering the spectral range from visible (400 nm) to near-infrared (1200 nm). The system is sensitive in low light conditions and is easily monitored by the end-user. The developed software is designed in LabView (National Instruments) programming interface and is used for the calibration of the system, for the control of the camera and monochromator and for the spectral image capturing and analysis. ~ 15 ~ The system operates in two modes: spectroscopic and spectrometric. The spectroscopic mode enables the real-time visualization of desired spectral images while the spectrometric mode performs synchronized spectral scanning, image capturing and calculation of a full spectrum per image pixel. The above-mentioned procedures (real-time visualization, spectral scanning, image capturing and calculation of the spectra) are controlled through the software. EAE was induced in female C57BL/6 (B6) mice (6–10 weeks) by immunization with 100 mg MOG35–55 peptide emulsified in complete Freud’s adjuvant (CFA Sigma) s.c. at the base of the tail, and i.p. injections of 200 ng pertussis toxin (Sigma) at the time of immunization and 48 h later. Mice reaching clinical score 3–4, ten (10) days after immunization were introduced in the study. Five female C57/BL6 mice were used as control-naive mice. Sections of 40 or 50 μm sections were cut longitudinally in a vibratome. The sections were incubated with anti-myelin basic protein (anti-MBP) antibody 1:500 (ab980; Chemicon) and anti-cluster of differentiation 3 (anti-CD3) antibody (eBiosciences, purified rat anti-mouse CD3), followed by incubation with secondary antibodies (Alexa Fluor 488, and 633, Invitrogen) 1:1000 in TBST. Finally, samples were coverslipped using Antifade Gold with DAPI (4',6-diamidino-2-phenylindole, Invitrogen). Before of each measurement set, the multispectral imaging microscope system is calibrated. After its successful calibration, spinal cord tissue is scanned for lesion sites and the demyelinated areas are marked and cross-checked with conventional fluorescent imaging methods. Analysis of the collected data show that the differential parameter (DP(λ)) is a significant, wavelength dependent, diagnostic parameter indicating areas with myelin loss. DP(λ) is estimated for each wavelength range of observation. Observation and analysis time, including calibration, lasts for a few minutes. Tissue imaging with multispectral imaging microscope shows that the backscattered signal is increased in the WM because of the abundant numbers of myelinated axons, while anterior horn neuron cell bodies and dendrites show low reflectance. Moreover, the boundaries between white and grey matter of the spinal cord are clearly depicted. Through selective imaging, the tissue becomes gradually transparent at long wavelengths (infrared spectral region), with the limits between WM and GM hardly detectable in images captured at wavelengths over 800nm. DAPI is an intrinsically fluorescent stain, which binds the DNA, thus labelling nucleated cells. The density of the fluorescent molecules is increased in a lesioned area, indicating elevated inflammatory cell numbers from the invasion of immune cells, for example, after induction of the autoimmune reaction and permeabilization of the blood–brain barrier with pertussis toxin to facilitate entry of activated immune cells within the CNS. In addition to the use of DAPI as a fluorescent stain, the use of fluorescent antibodies against MBP is also informative. Indeed, MBP is a major component of myelin, which surrounds the axons of neurons in the CNS. The area of the lesion is depicted by a lower MBP fluorescent signal, indicating decreased levels of myelin, presented as black holes (arrows) in the fluorescent background. Staining of spinal cord sections with antibodies against the CD3 antigen, a marker of T lymphocytes in inflamed areas, shows the infiltration of these immune cells associated with myelin loss. The different reflectance between the normal and the lesioned tissue is clearly depicted in images captured with multispectral imaging system. The accuracy of our system to detect areas with myelin loss was accurate with that of the gold-standard method of immunofluorescence microscopy, through comparative imaging of the same lesions with our microscope system. At the area of the lesion, the backscattered signal is minimized ~ 16 ~ and the area is demonstrated as a black hole in a significantly lighter background. Comparison of the images captured with a fluorescence microscope and multispectral imaging microscope system clearly shows that the latter accurately detects the area of the lesion. At the area of the lesion, accumulations of infiltrated cells displace the neighbouring myelinated fibres, and the sheaths of the myelin are disrupted and dilated, resulting in illusive demyelination. It is thus possible that loss of the backscattered signal is attributed to the loss of reflectivity of myelin, to the existence of the concentrated inflammatory cells of the immune response which absorb the incident light, and to the increased density of inflamed blood vessels. A part of the incident light is transmitted directly to the area of the lesion almost unaffected by the damaged myelin fibres, and another part is backscattered depending upon the absorption coefficient and concentration of the accumulated cells and blood vessels within the lesion. Consequently, the area of the lesion loses the prominent reflectance signal shown in the normal WM. The morphology of the area with myelin loss detected with conventional imaging methods is comparable to that obtained with our system. The estimation of the DP(λ) parameter indicates that the maximum discrimination between damaged and normal areas is shown at 500nm. Spectral differences observed between normal and lesioned WM reflect the different densities of various tissue structures and compositions. Indeed, at the area of the lesion, myelin sheaths are dilated/destroyed, thus incident light is less scattered. Comparison of our method to classical immunofluorescence microscopy methods shows that the sensitivity of our system to detect spinal cord areas with myelin loss is 90.4% and the positive predictive value is 92.2%. In the last experimental part of this study, the multispectral imaging method is applied for the detection of the destruction of the retina layers of the mouse eye induced through AMPA excitotoxicity. The selective imaging of sections of the control mice (PBS) shows that the retina layers have different spectral and imaging characteristics the one from other. The characteristics of the backscattered signal are altered in the case of induced excitotoxicity and the control group. The backscattered signal of the multispectral imaging at slices with AMPA is almost lost and this phenomenon is associated with partial or complete necrosis of the cells.
Language Greek
Issue date 2016-03-24
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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