The use of quantitative immunocytochemistry (QICC) to measure calbindin D28k-like immunoreactivity in the rat brain

We have developed a method of quantitative immunocytochemistry using an iodinated second antibody to visualise the anatomical distribution of primary antibodies in tissue sections, by macroautoradiography. Computer-assisted densitometry was used to analyse the pattern of optical densities within autoradiograms. The amount of antigen present in tissue sections was then quantified by comparison with non-biological standards which were processed in parallel with the tissue sections. Using this technique we have measured calbindin like-immunoreactivity in 4 areas of rat brain and have found that the values obtained are similar to those obtained by radioimmunoassay. A similar approach can be used to quantify autoradiograms by comparison with antigen standards to measure amounts of radiolabelled immunoreactivity and determine concentrations of biologically active molecules in discrete brain areas.     

Quantification of endocannabinoids in rat biological samples by GC/MS: technical and theoretical considerations

In the last several years, interest has increased significantly about the endocannabinoids anandamide and 2-arachidonylglycerol, two lipid messengers that activate cannabinoid receptors. Quantification of these compounds in biological samples presents numerous technical challenges. Because of their low abundance, endocannabinoids are usually quantified by isotope dilution assays using mass spectrometry coupled to either gas chromatography or high-performance liquid chromatography. Although endocannabinoid levels in biological fluids, such as plasma and cerebrospinal fluid, can be directly determined by these techniques, the complex lipid profile of brain tissue samples mandates purification of lipid extracts before GC/MS analysis; this step is not necessary when using HPLC/MS. We have found that when silica gel chromatography is used for endocannabinoid purification, poor recovery and loss of deuterium from the internal standards lead to inaccurate estimation of endocannabinoid levels. By contrast, purification strategies using C(18) solid-phase extraction permits precise and reproducible GC/MS quantification of endocannabinoids in tissue samples.     

Tissue profiling by mass spectrometry: a review of methodology and applications

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has become a valuable tool to address a broad range of questions in many areas of biomedical research. One such application allows spectra to be obtained directly from intact tissues, termed "profiling" (low resolution) and "imaging" (high resolution). In light of the fact that MALDI tissue profiling allows over a thousand peptides and proteins to be rapidly detected from a variety of tissues, its application to disease processes is of special interest. For example, protein profiles from tumors may allow accurate prediction of tumor behavior, diagnosis, and prognosis and uncover etiologies underlying idiopathic diseases. MALDI MS, in conjunction with laser capture microdissection, is able to produce protein expression profiles from a relatively small number of cells from specific regions of heterogeneous tissue architectures. Imaging mass spectrometry enables the investigator to assess the spatial distribution of proteins, drugs, and their metabolites in intact tissues. This article provides an overview of several tissue profiling and imaging applications performed by MALDI MS, including sample preparation, matrix selection and application, histological staining prior to MALDI analysis, tissue profiling, imaging, and data analysis. Several applications represent direct translation of this technology to clinically relevant problems.     

Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Regional trapping of microspheres in the lung compares well with regional blood flow

Microspheres (MS) are often used to measure the distribution of pulmonary blood flow in the assumption that the number of MS trapped in a region is proportional to blood flow. However, regional distribution of trapped MS has not been directly compared with regional blood flow in the lung. Regional trapping of MS was compared with regional flow of erythrocytes (RBC's) in isolated, perfused left lungs of dogs. Radioactivity from labeled MS and RBC's was measured by external detection using a gamma camera. We defined six regions of interest in the image of the left lateral surface of the lung: a dorsocaudal, a caudal, two ventral, an apical, and a central region. In each lung, regional trapping of MS was measured from the image of radioactivity obtained after slow injection of a suspension of MS into the arterial perfusion tubing. A radioactive bolus of labeled RBC's was injected during rapid imaging of the lung to obtain radioactivity vs. time curves from each region. The peaks of the regional radioactivity vs. time curves were used to estimate regional flows, though compensation had to be made for overlap of the washout and washin phases of the bolus of labeled RBC's. The results indicated that there were no differences in the regional distribution of MS compared with the regional distribution of RBC flow in isolated, perfused dog lungs.     

Integrated mass spectrometry imaging and omics workflows on the same tissue section using grid-aided,parafilm-assisted microdissection

BackgroundIn spite of the number of applications describing the use of MALDI MSI, one of its major drawbacks is the limited capability of identifying multiple compound classes directly on the same tissue section.MethodsWe demonstrate the use of grid-aided, parafilm-assisted microdissection to perform MALDI MS imaging and shotgun proteomics and metabolomics in a combined workflow and using only a single tissue section. The grid is generated by microspotting acid dye 25 using a piezoelectric microspotter, and this grid was used as a guide to locate regions of interest and as an aid during manual microdissection. Subjecting the dissected pieces to the modified Folch method allows to separate the metabolites from proteins. The proteins can then be subjected to digestion under controlled conditions to improve protein identification yields.ResultsThe proof of concept experiment on rat brain generated 162 and 140 metabolite assignments from three ROIs (cerebellum, hippocampus and midbrain/hypothalamus) in positive and negative modes, respectively, and 890, 1303 and 1059 unique proteins. Integrated metabolite and protein overrepresentation analysis identified pathways associated with the biological functions of each ROI, most of which were not identified when looking at the protein and metabolite lists individually.ConclusionsThis combined MALDI MS imaging and multi-omics approach further extends the amount of information that can be generated from single tissue sections.General significanceTo the best of our knowledge, this is the first report combining both imaging and multi-omics analyses in the same workflow and on the same tissue section.     

Validation of DTI Tractography-Based Measures of Primary Motor Area Connectivity in the Squirrel Monkey Brain

Diffusion tensor imaging (DTI) tractography provides noninvasive measures of structural cortico-cortical connectivity of the brain. However, the agreement between DTI-tractography-based measures and histological ‘ground truth’ has not been quantified. In this study, we reconstructed the 3D density distribution maps (DDM) of fibers labeled with an anatomical tracer, biotinylated dextran amine (BDA), as well as DTI tractography-derived streamlines connecting the primary motor (M1) cortex to other cortical regions in the squirrel monkey brain. We evaluated the agreement in M1-cortical connectivity between the fibers labeled in the brain tissue and DTI streamlines on a regional and voxel-by-voxel basis. We found that DTI tractography is capable of providing inter-regional connectivity comparable to the neuroanatomical connectivity, but is less reliable measuring voxel-to-voxel variations within regions.     

Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry

Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson’s disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value.Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2 pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1–6; Leu-Enk-Arg), (1–9), (2–13), and (7–13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results.Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1–7) in the cortex and dynorphin B (2–13) in the striatum. Enzyme inhibitors showed region- and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1–7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2–13).This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections.     

Mass spectrometry imaging: Towards a lipid microscope?

Biological imaging techniques are the most efficient way to locally measure the variation of different parameters on tissue sections. These analyses are gaining increasing interest since 20 years and allow observing extremely complex biological phenomena at lower and lower time and resolution scale. Nevertheless, most of them only target very few compounds of interest, which are chosen a priori, due to their low resolution power and sensitivity. New chemical imaging technique has to be introduced in order to overcome these limitations, leading to more informative and sensitive analyses for biologists and physicians.Two major mass spectrometry methods can be efficiently used to generate the distribution of biological compounds over a tissue section. Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) needs the co-crystallization of the sample with a matrix before to be irradiated by a laser, whereas the analyte is directly desorbed by a primary ion bombardment for Secondary Ion Mass Spectrometry (SIMS) experiments. In both cases, energy used for desorption/ionization is locally deposited -some tens of microns for the laser and some hundreds of nanometers for the ion beam- meaning that small areas over the surface sample can be separately analyzed. Step by step analysis allows spectrum acquisitions over the tissue sections and the data are treated by modern informatics software in order to create ion density maps, i.e., the intensity plot of one specific ion versus the (x,y) position.Main advantages of SIMS and MALDI compared to other chemical imaging techniques lie in the simultaneous acquisition of a large number of biological compounds in mixture with an excellent sensitivity obtained by Time-of-Flight (ToF) mass analyzer. Moreover, data treatment is done a posteriori, due to the fact that no compound is selectively marked, and let us access to the localization of different lipid classes in only one complete acquisition.     

Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins.     

In vivo approaches and rationale for quantifying kinetics and imaging brain lipid metabolic pathways

Developing a kinetic strategy to examine rates of lipid metabolic pathways can help to elucidate the roles that lipids play in tissue function and structure in health and disease. This review summarizes such a strategy, and shows how it has been applied to quantify different kinetic aspects of brain lipid metabolism in animals and humans. Methods involve injecting intravenously a radioactive or heavy isotope labeled substrate that will be incorporated into a lipid metabolic pathway, and using chemical analytical and/or imaging procedures (e.g., quantitative autoradiography or positron emission tomography) to determine tracer distribution in brain regions and their lipid compartments as a function of time. From the measurements, fluxes, turnover rates, half-lives and ATP consumption rates can be calculated, and incorporation rates can be imaged. Experimental changes in these kinetic parameters can help to identify changes in the expression of regulatory enzymes, and thus aid in drug targeting. Cases that are discussed are arachidonic acid turnover and imaging of neuroreceptor-initiated phospholipase A2 activation, ether phospholipid biosynthesis, and kinetics of the phosphatidylinositol cycle.     

Radioimmunochemical methods for the quantitative autoradiographic determination of antigens in brain and other tissues

1. We have used horseradish peroxidase-conjugated protein A- and 125I-protein A to develop immunohistochemical and radioimmunohistochemical methods for the localization of antigens in brain and other tissues of the rat. 2. We visualized methionine-enkephalin fibers in the rat brain by incubating tissue sections with a specific polyclonal antibody and peroxidase-conjugated protein A. The method is simple, fast, and less expensive and more sensitive than classical immunohistochemical techniques and the principle could be used to visualize many other tissue antigens. 3. Incubation of tissue samples with specific polyclonal antibodies and 125I-protein A, followed by autoradiography, allows the permanent recording of the radioimmunohistochemical localization of brain methionine-enkephalin, tyrosine hydroxylase, and angiotensin-converting enzyme and of pituitary vasopressin and could be applied to the localization of many other tissue antigens. 4. A new quantitative radioimmunohistochemical technique for methionine-enkephalin allows the determination of the endogenous peptide content in discrete brain nuclei from 16-microns-thick sections. The method is based on the quantitative determination of the amount of 125I-protein A bound to specific tissue areas after incubation with a specific polyclonal antibody, followed by autoradiography and computerized microdensitometry. To quantify the endogenous peptide content, the values obtained are interpolated into a methionine-enkephalin internal standard curve. This standard curve was constructed by measuring endogenous concentrations of methionine-enkephalin by radioimmunoassay in specific brain regions and correlating these values with quantitative autoradiographic determinations in homologous areas of adjacent sections. Similar methods can be developed for other tissue antigens. 5. These new methods allow for the localization and quantification of tissue antigens in very discrete areas of the brain and other tissues and have a wide application in neurobiology and pathology.     

Sensitive liquid chromatography/tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat brain tissue

One prerequisite for therapeutic effects of psychiatric drugs is the ability to pass the blood brain barrier. Hence, it is important to know the concentration of antipsychotic drugs in brain tissue. In general, determinations of lipophilic compounds from lipophilic matricies such as the brain are a challenge. Here we have adapted a plasma assay for antipsychotics for the target organ the brain. Using modified sample preparation and chromatographic strategies, the analytes were extracted from rat brain homogenate and analyzed by LC-MS/MS. The method used a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column with a mobile phase of acetonitrile/5 mM ammonium formate (pH 6.1 adjusted with formic acid) and gradient elution. All analytes were detected in positive ion mode using multiple-reaction monitoring. The method was validated and the linearity, lower limit of quantitation, precision, accuracy, recoveries, specificity and stability were determined. This method was then successfully used to quantify the rat brain tissue concentration of the analytes after chronic treatment with these antipsychotic drugs.     

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.     

Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections

MALDI MS imaging mass spectrometry can be used to map the distribution of targeted compounds in tissue sections with a spatial resolution currently of about 50 microm, providing important molecular information in many areas of biological research. After matrix application, a raster of a section by the laser beam yields ions from compounds in a tissue mass-to-charge range from 1000 to over 100000. Two-dimensional intensity maps can then be reconstructed to provide specific molecular images of a tissue.     

Quantitative mass spectrometry imaging of propranolol and olanzapine using tissue extinction calculation as normalization factor

In order to quantify small molecules at the early stage of drug discovery, we developed a quantitation approach based on mass spectrometry imaging (MSI) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) without the use of a labeled compound. We describe a method intended to respond to the main challenges encountered in quantification through MALDI imaging dedicated to whole-body or single heterogeneous organ samples (brain, eye, liver). These include the high dependence of the detected signal on the matrix deposition, the MALDI ionization yield of specific target molecules, and lastly, the ion suppression effect on the tissue. To address these challenges, we based our approach on the use of a normalization factor called the TEC (Tissue Extinction Coefficient). This factor takes into account the ion suppression effect that is both tissue- and drug-specific. Through this protocol, the amount of drug per gram of tissue was determined, which in turn, was compared with other analytical techniques such as Liquid Chromatography-Mass spectrometry (LC-MS/MS).     

Direct profiling and identification of peptide expression differences in the pancreas of control and ob/ob mice by imaging mass spectrometry

Imaging mass spectrometry (IMS) technology utilizes MALDI MS to map molecules of interest in thin tissue sections. In this study, we have evaluated the potential of MALDI IMS to study peptide expression patterns in the mouse pancreas under normal and pathological conditions, and to in situ identify peptides of interest using MS/MS. Different regions of the pancreas of both control and ob/ob mice were imaged, resulting in peptide-specific profiles. The distribution of ions of m/z 3120 and 3439 displayed a striking resemblance with Langerhans islet's histology and, following MS/MS fragmentation and database searching were identified as C-peptide of insulin and glicentin-related polypeptide, respectively. In addition, a significant increase of the 3120 peak intensity in the obese mice was observed. This study underscores the potential of MALDI IMS to study the contribution of peptides to pancreas pathology.     

In situ metabolomic mass spectrometry imaging: recent advances and difficulties

MS imaging (MSI) is a remarkable new technology that enables us to determine the distribution of biological molecules present in tissue sections by direct ionization and detection. This technique is now widely used for in situ imaging of endogenous or exogenous molecules such as proteins, lipids, drugs and their metabolites, and it is a potential tool for pathological analysis and the investigation of disease mechanisms. MSI is also thought to be a technique that could be used for biomarker discovery with spatial information. The application of MSI to the study of endogenous metabolites has received considerable attention because metabolites are the result of the interactions of a system's genome with its environment and a total set of these metabolites more closely represents the phenotype of an organism under a given set of conditions. Recent studies have suggested the importance of in situ metabolite imaging in biological discovery and biomedical applications, but several issues regarding the technical application limits of MSI still remained to be resolved. In this review, we describe the capabilities of the latest MSI techniques for the imaging of endogenous metabolites in biological samples, and also discuss the technical problems and new challenges that need to be addressed for effective and widespread application of MSI in both preclinical and clinical settings.     

Multiple statistical analysis techniques corroborate intratumor heterogeneity in imaging mass spectrometry datasets of myxofibrosarcoma

MALDI mass spectrometry can generate profiles that contain hundreds of biomolecular ions directly from tissue. Spatially-correlated analysis, MALDI imaging MS, can simultaneously reveal how each of these biomolecular ions varies in clinical tissue samples. The use of statistical data analysis tools to identify regions containing correlated mass spectrometry profiles is referred to as imaging MS-based molecular histology because of its ability to annotate tissues solely on the basis of the imaging MS data. Several reports have indicated that imaging MS-based molecular histology may be able to complement established histological and histochemical techniques by distinguishing between pathologies with overlapping/identical morphologies and revealing biomolecular intratumor heterogeneity. A data analysis pipeline that identifies regions of imaging MS datasets with correlated mass spectrometry profiles could lead to the development of novel methods for improved diagnosis (differentiating subgroups within distinct histological groups) and annotating the spatio-chemical makeup of tumors. Here it is demonstrated that highlighting the regions within imaging MS datasets whose mass spectrometry profiles were found to be correlated by five independent multivariate methods provides a consistently accurate summary of the spatio-chemical heterogeneity. The corroboration provided by using multiple multivariate methods, efficiently applied in an automated routine, provides assurance that the identified regions are indeed characterized by distinct mass spectrometry profiles, a crucial requirement for its development as a complementary histological tool. When simultaneously applied to imaging MS datasets from multiple patient samples of intermediate-grade myxofibrosarcoma, a heterogeneous soft tissue sarcoma, nodules with mass spectrometry profiles found to be distinct by five different multivariate methods were detected within morphologically identical regions of all patient tissue samples. To aid the further development of imaging MS based molecular histology as a complementary histological tool the Matlab code of the agreement analysis, instructions and a reduced dataset are included as supporting information.     

Imaging mass spectrometry of thin tissue sections: a decade of collective efforts

Imaging mass spectrometry (MS) allows to monitor the spatial distribution and abundance of endogenous and administered compounds present within tissue specimens. Several different but complementary imaging MS technologies have been developed allowing the analysis of a wide variety of compounds including inorganic elementals, metabolites, lipids, peptides, proteins and xenobiotics with spatial resolutions from micrometer to nanometer scales. In the past decade, an enormous collective body of work has been done to develop and improve the imaging MS technology. This article gives a historical perspective, an overview of the principle and status of the technology and lists the main fields of applications. It also enumerates some of the critical challenges we need to collectively address for imaging MS to be considered a mainstream analytical method.