RESUMO
A micromolar concentration of zinc has been shown to significantly change the dynamics of exocytosis as well as the vesicle contents in a model cell line, providing direct evidence that zinc regulates neurotransmitter release. To provide insight into how zinc modulates these exocytotic processes, neurotransmitter release and vesicle content were compared with single cell amperometry and intracellular impact vesicle cytometry with a range of zinc concentrations. Additionally, time-of-flight secondary ion mass spectrometry (ToF-SIMS) images of lipid distributions in the cell membrane after zinc treatment correlate to changes in exocytosis. By combining electrochemical techniques and mass spectrometry imaging, we proposed a mechanism by which zinc changes the fusion pore and the rate of neurotransmitter release by changing lipid distributions and results in the modulation of synaptic strength and plasticity.
Assuntos
Lipídeos/química , Zinco/química , Animais , Catecolaminas/análise , Membrana Celular/metabolismo , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Etilenodiaminas/química , Exocitose/efeitos dos fármacos , Células PC12 , Análise de Componente Principal , Ratos , Espectrometria de Massas por Ionização por Electrospray , Zinco/farmacologiaRESUMO
Laser desorption ionization mass spectrometry (LDI-MS) is used to image brain lipids in the fruit fly, Drosophila, a common invertebrate model organism in biological and neurological studies. Three different sample preparation methods, including sublimation with two common organic matrixes for matrix-assisted laser desorption ionization (MALDI) and surface-assisted laser desorption ionization (SALDI) using gold nanoparticles, are examined for sample profiling and imaging the fly brain. Recrystallization with trifluoroacetic acid following matrix deposition in MALDI is shown to increase the incorporation of biomolecules with one matrix, resulting in more efficient ionization, but not for the other matrix. The key finding here is that the mass fragments observed for the fly brain slices with different surface modifications are significantly different. Thus, these approaches can be combined to provide complementary analysis of chemical composition, particularly for the small metabolites, diacylglycerides, phosphatidylcholines, and triacylglycerides, in the fly brain. Furthermore, imaging appears to be beneficial using modification with gold nanoparticles in place of matrix in this application showing its potential for cellular and subcellular imaging. The imaging protocol developed here with both MALDI and SALDI provides the best and most diverse lipid chemical images of the fly brain to date with LDI.
Assuntos
Química Encefálica , Drosophila melanogaster/química , Ouro/química , Lipídeos/análise , Nanopartículas Metálicas/química , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sublimação Química , Animais , MasculinoRESUMO
We have characterized the use of sublimation to deposit matrix-assisted laser desorption/ionization (MALDI) matrices in secondary ion mass spectrometry (SIMS) analysis, i.e. matrix-enhanced SIMS (ME-SIMS), a common surface modification method to enhance sensitivity for larger molecules and to increase the production of intact molecular ions. We use sublimation to apply a thin layer of a conventional MALDI matrix, 2,5-dihydroxybenzoic acid (DHB), onto rat brain cerebellum tissue to show how this technique can be used to enhance molecular yields in SIMS while still retaining a lateral resolution around 2 µm and also to investigate the mechanism of this enhancement. The results here illustrate that cholesterol, which is a dominant lipid species in the brain, is decreased on the tissue surface after deposition of matrix, particularly in white matter. The decrease of cholesterol is followed by an increased ion yield of several other lipid species. Depth profiling of the sublimed rat brain reveals that the lipid species are de facto extracted by the DHB matrix and concentrated in the top most layers of the sublimed matrix. This extraction/concentration of lipids directly leads to an increase of higher mass lipid ion yield. It is also possible that the decrease of cholesterol decreases the potential suppression of ion yield caused by cholesterol migration to the tissue surface. This result provides us with significant insights into the possible mechanisms involved when using sublimation to deposit this matrix in ME-SIMS.
Assuntos
Encéfalo/patologia , Gentisatos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Masculino , Análise de Componente Principal , Ratos , Ratos Sprague-DawleyRESUMO
Imaging mass spectrometry has shown to be a valuable method in medical research and can be performed using different instrumentation and sample preparation methods, each one with specific advantages and drawbacks. Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) has the advantage of high spatial resolution imaging but is often restricted to low mass molecular signals and can be very sensitive to sample preparation artifacts. In this report we demonstrate the advantages of using gas cluster ion beams (GCIBs) in combination with trifluoracetic acid (TFA) vapor exposure for the imaging of lipids in mouse brain sections. There is an optimum exposure to TFA that is beneficial for increasing high mass signal as well as producing signal from previously unobserved species in the mass spectrum. Cholesterol enrichment and crystallization on the sample surface is removed by TFA exposure uncovering a wider range of lipid species in the white matter regions of the tissue, greatly expanding the chemical coverage and the potential application of TOF-SIMS imaging in neurological studies. Ar4000(+) (40 keV) in combination with TFA treatment facilitates high resolution, high mass imaging closing the gap between TOF-SIMS and matrix-assisted laser desorption ionization (MALDI).
Assuntos
Gases/química , Lipídeos/análise , Imagem Molecular , Ácido Trifluoracético/química , Animais , Encéfalo , Íons/química , Ratos , Espectrometria de Massa de Íon Secundário , Fatores de Tempo , VolatilizaçãoRESUMO
Background: Psoriasis is a systemic inflammatory disease characterized by epidermal proliferation in the skin. Altered lipid metabolism is considered to be a central factor in the psoriatic etiopathogenesis. Thus, it is necessary to visualize chemical specificity of the samples for better medical diagnosis and treatment. Here, we investigate its role in the development of psoriatic lesions, before and after ultraviolet phototherapy, in a case study. Methods: The distribution and morphology of different lipids and fibrous proteins in psoriatic (lesional) tissues were visualized by two complementary label-free imaging techniques: 1) non-linear microscopy (NLM), providing images of lipids/proteins throughout the skin layers at submicrometer resolution; and 2) mass spectrometry imaging (MSI), offering high chemical specificity and hence the detection of different lipid species in the epidermal and dermal regions. A conventional method of histological evaluation was performed on the tissues, with no direct comparison with NLM and MSI. Results: Psoriatic tissues had a higher lipid content, mainly in cholesterol, in both the epidermal and dermal regions, compared to healthy tissues. Moreover, the collagen and elastin fibers in the psoriatic tissues had a tendency to assemble as larger bundles, while healthy tissues showed smaller fibers more homogeneously spread. Although phototherapy significantly reduced the cholesterol content, it also increased the amounts of collagen in both lesional and non-lesional tissues. Conclusion: This study introduces NLM and MSI as two complementary techniques which are chemical specific and can be used to assess and visualize the distribution of lipids, collagen, and elastin in a non-invasive and label-free manner.
RESUMO
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used for lipid profiling of intestine tissue sections from rats fed specially processed cereals and rats fed ordinary feed as a control. This cereal is known to increase the activity of antisecretory factor in plasma and the exact mechanism for the activation process at the cellular level is unclear. ToF-SIMS has been used to track food induced changes in lipid content in intestinal tissue sections to gain insight into the possible mechanisms involved. Data from 20 intestine sections belonging to four different rats from each group of control and specially processed cereals-fed rats were obtained using the stage scan macroraster with a lateral resolution of 5 µm. Data were subsequently subjected to orthogonal partial least squares discriminant analysis. The data clearly show that changes of certain lipids are induced by the specially processed cereal feed. Scores plots show a well-defined separation between the two groups. The corresponding loading plots reveal that the groups separate mainly due to changes of vitamin E, phosphocholine, and phosphosphingolipid fragments, and that for the c18:2 fatty acid. The observed changes in lipids might give insight into the working mechanisms of antisecretory factor in the body, and this has been successfully used to understand the working mechanism of specially processed cereal-induced antisecretory factor activation in intestine.
Assuntos
Ração Animal , Dieta/métodos , Grão Comestível , Intestinos/química , Lipídeos/análise , Animais , Ratos , Espectrometria de Massa de Íon Secundário/métodosRESUMO
Time of flight secondary ion mass spectrometry (ToF-SIMS) was used to image the lipid localization in brain tissue sections from rats fed specially processed cereals (SPC). An IonTof 5 instrument equipped with a Bi cluster ion gun was used to analyze the tissue sections. Data from 15 brain samples from control and cereal-fed rats were recorded and exported to principal components analysis (PCA). The data clearly show changes of certain lipids in the brain following cereal feeding. PCA score plots show a good separation in lipid distribution between the control and the SPC-fed group. The loadings plot reveal that the groups separated mainly due to changes in cholesterol, vitamin E and c18:2, c16:0 fatty acid distribution as well as some short chain monocarboxylic fatty acid compositions. These insights relate to the working mechanism of SPC as a dietary supplement. SPC is thought to activate antisecretory factor (AF), an endogenous protein with regulatory function for inflammation and fluid secretion. These data provide insights into lipid content in brain following SPC feeding and suggest a relation to activating AF.