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Keywords:

  • laser scanning cytometry;
  • Alzheimer's disease;
  • tissue sections;
  • brain sections;
  • cyclin B1;
  • cell cycle analysis;
  • two- and three-dimensional distribution

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Background

The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections.

Methods

We adapted this technique to immunohistochemical labelled human brain slices.

Results

We were able to identify neurons according to their labelling and to display morphological structures such as the lamination of the entorhinal cortex. Further, we were able to distinguish between neurons with and without cyclin B1 expression and we could assign the expression of cyclin B1 to the cell islands of layer II and the pyramidal neurons of layer V of the entorhinal cortex in Alzheimer's disease effected brain. In addition, we developed a method depicting the three-dimensional distribution of the cells in intact tissue sections.

Conclusions

In this pilot experiments we could demonstrate the power of the LSC for the analysis of human brain sections. © 2006 International Society for Analytical Cytology

The Laser Scanning Cytometer (LSC) was commercially introduced in the early 1990s (1). This microscope-based instrument might fill the gap between high throughput multiparametric cytometry and morphological analysis. The specimen, arranged on a microscope slide, is scanned stepwise and the emitted fluorescence light is guided to an optical bench equipped with four photomultipliers. For each fluorescence event up to six fluorochromes can be detected and quantified simultaneously (2). An additional feature unique to the LSC is the recording of the exact xy position of every object on the slide together with the fluorescence data. This allows the identification of objects as single cells, doublets, debris or artifacts, the relocalisation of selected cells and permits an analysis of the cell and tissue morphology (for instance after cytomorphological counterstaining (3, 4)). The LSC enables us to focus on different issues: quantitative analysis of multiple fluorescence colors, the determination of polyploidy, cell cycle, and apoptosis. The first assays were established to investigate cultured cells and peripheral blood leukocytes, whereas today the spectrum of applications expanded to formalin-fixed, paraffin-embedded material, freshly resected tissue, and fresh frozen tissue (5–9). In general, the preparation of single cells or nuclei from solid tissue blocks was performed with different methods, such as mechanical dissociation, grating cells from a tissue block, touch preparation, touch smears, or fine-needle aspirate biopsies (10–14). Although these methods are well established for tumor material, they are not useful for brain tissue. The preparation of nuclei is unsuitable, as the display of cytoplasmic proteins (e.g. celltype-specific markers or neurotransmitters) is necessary for the discrimination of different celltypes. By preparing single cells, the morphology of the tissue is getting lost, which makes further anatomical and pathological analysis impossible.

The aim of our study was to analyze different celltypes and subpopulations, to display morphological details of the brain tissue and to study the two- and three-dimensional distribution of the cells in the tissue sections, using the LSC technique.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Patients and Specimens

In the present study, the brain of a patient with Alzheimer's disease (AD) was used. The case meets the National Institute of Neurologic and Communicative Disorders and Stroke (NINCDS) and Alzheimer's Disease and Related Disorders Association (ADRDA) criteria for definite diagnosis of AD. According to Braak and Braak (15), the AD case was classified as stage I–II. The whole procedure of case recruitment and performing the autopsy was elaborated in accordance with the convention of the council of Europe on Human Rights and Biomedicine and had been approved by the responsible Ethical Committee of the University of Leipzig. Tissue blocks from the entorhinal cortex (Brodman area 28) were fixed in 4% phosphate-buffered paraformaldehyde (4% PFA in PBS, pH 7.4) for 10 days and cryoprotected in 30% sucrose. For immunohistochemistry, sections of 30 μm and 120 μm thickness were cut on a freezing microtome.

Immunohistochemistry

Free-floating sections of the human brain were pretreated in 10 mM citrate buffer (pH 6.0) heated by microwave. After blocking of unspecific binding sites with 0.3% milk powder, 0.1% gelatine, 1% bovine serum albumin, and 0.05% Tween®20 in 10 mM Tris-buffered saline (pH 7.4), a cocktail of the primary antibodies monoclonal mouse-anti-pan-neuronal neurofilament marker (SMI-311, Sternberger Monoclonals, Baltimore, MD, dilution 1:750) and polyclonal rabbit-anti-cyclin B1 (clone H-433, Santa Cruz, CA, 1:500) were incubated overnight at 4°C in blocking solution. The SMI-311 antibody was labeled with a secondary cyanine 5 (Cy5™)-conjugated goat-anti-mouse antibody (Dianova, Hamburg, Germany, 1:300), and the cyclin B1 signal was amplified using a secondary system consisting of a biotinylated donkey-anti-rabbit antibody (Amersham, Buckinghamshire, UK, 1:1000), the TSA™ Biotin System (Perkin Elmer Life Sciences, Boston, MA), and a cyanine 2 (Cy2™)-conjugated Streptavidin (Dianova, 1:500). The labeled human sections were treated with 50 μg/ml propidiumiodide (PI) in Tris buffered saline containing 100 μg/ml Ribonuclease A (Sigma, St. Louis, MO), for 30 min at 37°C. Sections were mounted onto slides using DAKO® fluorescent mounting medium and stored at 4°C in the darkness until analysis.

Sample Analysis

The measurements were performed with a LSC (CompuCyte Corporation, Cambridge, MA). The instrument settings and the optimal adjustment of the threshold for the analysis of the threefold labeled slices (Cy5™, Cy2™ and PI) have been described earlier (16). While scanning the whole parahippocampal gyrus, PI was taken as trigger signal and for each flourescent event the Cy5™ and the Cy2™ signal was recorded and displayed in the xy-position window. As a control, a specimen of the patient was measured with only the secondary antibodies by omitting the primary antibodies.

The 120-μm thick tissue sections were analyzed in several foci. Subsequently, measurements were merged into one data file to obtain data of the three-dimensional distribution of the cells in the brain section.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

In the present study, we analyzed the two- and three-dimensional distribution of neurons in human brain sections by the LSC. Using the task of the LSC software to acquire the xy-coordinates of each fluorescence event, morphological structures of the object can be displayed. Figure 1 shows the LSC image of the parahippocampal gyrus of an AD patient, each dot representing one fluorescence event. White dots show glial cells, while the blue colored dots in Figure 1A depict neurons as SMI-311 positive cells and reveal morphological details such as the pyramidal cell-layer in the subiculum and layer V and the cell islands of layer II, which are characteristic for the entorhinal cortex. Figure 1B displays cyclin B1 positive and –negative neurons as green, blue dots, respectively, revealing that some pyramidal neurons in the subiculum and layer V and the neurons in the cell islands of layer II express this protein.

thumbnail image

Figure 1. Illustration of the human parahippocampal gyrus within the x-y-position window of the LSC software. A: color coding: blue: SMI-311-positive cells (neurons) showing morphological details such as the pyramidal cell-layer in the subiculum (S), layer V (V), and the cell islands of layer II (arrowhead). B: color coding: blue: cyclin B1-negative neurons; green: cyclin B1-positive neurons. Only a few pyramidal cells in the subiculum and layer V, but all neurons in the entorhinal islands of layer II show a cyclin B1 expression.

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The analysis of the 120 μm thick section renders the three-dimensional distribution of the cells (Fig. 2). The section was scanned in five depth of focus (every 30 μm) with trigger set on the DNA (PI) fluorescence. Cy5™ positive cells (neurons) and Cy5™-Cy2™ double labeled cells (cyclin B1 positive neurons) were differentiated and color gated (center dotplots). This was done for every depth of focus. The xy position plots in the center show the same focus level as seen in the left panel with the discrimination of the different fluorescent colors. By the merge feature of the LSC, which allows the combination of different measurements, the spaces between the layers could be filled resulting in a three-dimensional distribution of cells in brain tissue.

thumbnail image

Figure 2. Analysis of a 120 μm thick section in five depth of focus (every 30 μm, trigger: PI fluorescence). For each focus level cell populations were differentiated and color gated (color coding: green: Cy5™-Cy2™ double labeled cells display cyclin B1 positive neurons; red: Cy5™ positive cells display cyclin B1 negative neurons; black: unlabeled cells). Right panel: combination of different measurements showing the three-dimensional distribution of the cells in the brain section.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Although the LSC is a well established instrument for the analysis of cells and tissues, only a few publications are available on the analysis of tissue sections using this technique. In our study, we were able to identify neurons and also subpopulations of them: neurons that show disregulated cell cycle by cyclin B1 expression and neurons that do not. The xy-position window clearly shows the structure of the parahippocampal gyrus and even single layers of the cortex. The analysis of the cyclin B1 expression indicates that structures particularly affected in AD such as the pyramidal cells in the layer V and the cell islands of layer II, show this mitotic cyclin. This is in agreement with earlier observations (17), drawing upon the hypotheses, that neurons in AD die due to re-entering a lethal cell cycle (reviewed in18).

To our knowledge, we are the first group investigating frozen tissue sections with 30 μm and 120 μm thickness by LSC. The use of thick slices prevents errors occurring with thin sections (5 μm or 10 μm): truncated nuclei, feigning a reduced DNA content and hence disturb the cell cycle analysis. The huge amount of data incurring in three-dimensional analysis are still difficult to handle but this will soon fall into oblivion with the further development of the computer technology. Compared to other methods for the analysis of tissue sections such as histology and microscopy, the LSC affords a standardized and fast measurement of the specimens and a simultaneous quantification of multiple fluorescence signals (19). Thereby not only cell suspensions as in flow cytometry can be investigated, but also cells in their structural context. These features turn the LSC into a powerful tool for research and diagnostical applications.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED