Micro-Raman spectroscopy of lipid halo and dense-core amyloid plaques: aging process characterization in the Alzheimer's disease APPswePS1ΔE9 mouse model.

The deposition of amyloid plaques is considered one of the main microscopic features of Alzheimer's disease (AD). Since plaque formation can precede extensive neurodegeneration and it is the main clinical manifestation of AD, it constitutes a relevant target for new treatment and diagnostic approaches. Micro-Raman spectroscopy, a label-free technique, is an accurate method for amyloid plaque identification and characterization. Here, we present a high spatial resolution micro-Raman hyperspectral study in transgenic APPswePS1ΔE9 mouse brains, showing details of AD tissue biochemical and histological changes without staining. First we used stimulated micro-Raman scattering to identify the lipid-rich halo surrounding the amyloid plaque, and then proceeded with spontaneous (conventional) micro-Raman spectral mapping, which shows a cholesterol and sphingomyelin lipid-rich halo structure around dense-core amyloid plaques. The detailed images of this lipid halo relate morphologically well with dystrophic neurites surrounding plaques. Principal Component Analysis (PCA) of the micro-Raman hyperspectral data indicates the feasibility of the optical biomarkers of AD progression with the potential for discriminating transgenic groups of young adult mice (6-month-old) from older ones (12-month-old). Frequency-specific PCA suggests that plaque-related neurodegeneration is the predominant change captured by Raman spectroscopy, and the main differences are highlighted by vibrational modes associated with cholesterol located majorly in the lipid halo.

Nonlinear and vibrational microscopy for label-free characterization of amyloid-ß plaques in Alzheimer's disease model.

Given the long subclinical stage of Alzheimer's disease (AD), the study of biomarkers is relevant both for early diagnosis and the fundamental understanding of the pathophysiology of AD. Biomarkers provided by Amyloid-ß (Aß) plaques have led to an increasing interest in characterizing this hallmark of AD due to its promising potential. In this work, we characterize Aß plaques by label-free multimodal imaging: we combine two-photon excitation autofluorescence (TPEA), second harmonic generation (SHG), spontaneous Raman scattering (SpRS), coherent anti-Stokes Raman scattering (CARS), and stimulated Raman scattering (SRS) to describe and compare high-resolution images of Aß plaques in brain tissues of an AD mouse model. Comparing single-laser techniques images, we discuss the origin of the SHG, which can be used to locate the plaque core reliably. We study both the core and the halo with vibrational microscopy and compare SpRS and SRS microscopies for different frequencies. We also combine SpRS spectroscopy with SRS microscopy and present two core biomarkers unexplored with SRS microscopy: phenylalanine and amide B. We provide high-resolution SRS images with the spatial distribution of these biomarkers in the plaque and compared them with images of the amide I distribution. The obtained spatial correlation corroborates the feasibility of these biomarkers in the study of Aß plaques. Furthermore, since amide B enables rapid imaging, we discuss its potential as a novel fingerprint for diagnostic applications.

A fingerprint of amyloid plaques in a bitransgenic animal model of Alzheimer's disease obtained by statistical unmixing analysis of hyperspectral Raman data.

The global prevalence of Alzheimer's disease (AD) points to endemic levels, especially considering the increase of average life expectancy worldwide. AD diagnosis based on early biomarkers and better knowledge of related pathophysiology are both crucial in the search for medical interventions that are able to modify AD progression. In this study we used unsupervised spectral unmixing statistical techniques to identify the vibrational spectral signature of amyloid ß aggregation in neural tissues, as early biomarkers of AD in an animal model. We analyzed spectral images composed of a total of 55 051 Raman spectra obtained from the frontal cortex and hippocampus of five bitransgenic APPswePS1ΔE9 mice, and colocalized amyloid ß plaques by other fluorescence techniques. The Raman signatures provided a multifrequency fingerprint consistent with the results of synthesized amyloid ß fibrils. The fingerprint obtained from unmixed analysis in neural tissues is shown to provide a detailed image of amyloid plaques in the brain, with the potential to be used as biomarkers for non-invasive early diagnosis and pathophysiology studies in AD on the retina.

Scale/topography of substrates surface resembling extracellular matrix for tissue engineering.

Biomaterial matrices are being developed that mimic the key characteristics of the extracellular matrix, including presenting adhesion sites and displaying growth factors in the context of a viscoelastic hydrogel. This review focuses on two classes of materials: those that are derived from naturally occurring molecules and those that recapitulate key motifs of biomolecules within biologically active synthetic materials. We also discussed some of the most significant biological features of the ECM, and several engineering methods currently being implemented to design and tune synthetic scaffolds to mimic these features. Understanding the cell-protein-material interaction is fundamental for developing more powerful tools in tissue engineering and regenerative medicine strategies. The design of model substrates including the presence of well-defined properties (chemistry, topography, stiffness) and even the gradient of these properties in three dimensional environments must lead in the near future to learn more about the specific roles of protein adsorption and the very dynamic process related to the cell fate of synthetic substrates: cell adhesion, matrix reorganisation, deposition and degradation at the cell-material interface. These materials will open new doors to biosurgical therapeutics in tissue engineering and regenerative medicine.

Human adult stem cells from diverse origins: an overview from multiparametric immunophenotyping to clinical applications.

Stem cells are known for their capacity to self-renew and differentiate into at least one specialized cell type. Mesenchymal stem cells (MSCs) were isolated initially from bone marrow but are now known to exist in all vascularized organ or tissue in adults. MSCs are particularly relevant for therapy due to their simplicity of isolation and cultivation. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSCs for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; specific surface antigen expression in which ≥95% of the cells express the antigens recognized by CD105, CD73, and CD90, with the same cells lacking (≤2% positive) the antigens CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR. In this review we will take an historical overview of how umbilical cord blood, bone marrow, adipose-derived, placental and amniotic fluid, and menstrual blood stem cells, the major sources of human MSC, can be obtained, identified and how they are being used in clinical trials to cure and treat a very broad range of conditions, including heart, hepatic, and neurodegenerative diseases. An overview of protocols for differentiation into hepatocytes, cardiomyocytes, neuronal, adipose, chondrocytes, and osteoblast cells are highlighted. We also discuss a new source of stem cells, induced pluripotent stem cells (iPS cells) and some pathways, which are common to MSCs in maintaining their pluripotent state.

Mitochondrial calcium regulates rat liver regeneration through the modulation of apoptosis.

UNLABELLED: Subcellular Ca(2+) signals control a variety of responses in the liver. For example, mitochondrial Ca(2+) (Ca(mit)(2+)) regulates apoptosis, whereas Ca(2+) in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca(mit)(2+) also affects liver regeneration. The Ca(2+)-buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin-mitochondrial targeting sequence-green fluorescent protein (PV-MITO-GFP). This construct properly localized to and effectively buffered Ca(2+) signals in the mitochondrial matrix. Additionally, the expression of PV-MITO-GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl-2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2-associated X protein (bax), apoptotic peptidase activating factor 1, and caspase-6]. PV-MITO-GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca(mit)(2+) buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl-2 and the decreased expression of bax. CONCLUSION: Together, these results reveal an essential role for Ca(mit)(2+) in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis.