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Review
. 2015 Oct 31:12:35.
doi: 10.1186/s12989-015-0110-8.

Assessing particle and fiber toxicology in the respiratory system: the stereology toolbox

Christina Brandenberger  1   2 , Matthias Ochs  3   4   5 , Christian Mühlfeld  6   7   8
Affiliations
Review

Assessing particle and fiber toxicology in the respiratory system: the stereology toolbox

Christina Brandenberger et al. Part Fibre Toxicol. .

Abstract

The inhalation of airborne particles can lead to pathological changes in the respiratory tract. For this reason, toxicology studies on effects of inhalable particles and fibers often include an assessment of histopathological alterations in the upper respiratory tract, the trachea and/or the lungs. Conventional pathological evaluations are usually performed by scoring histological lesions in order to obtain "quantitative" information and an estimation of the severity of the lesion. This approach not only comprises a potential subjective bias, depending on the examiner's judgment, but also conveys the risk that mild alterations escape the investigator's eye. The most accurate way of obtaining unbiased quantitative information about three-dimensional (3D) features of tissues, cells, or organelles from two-dimensional physical or optical sections is by means of stereology, the gold standard of image-based morphometry. Nevertheless, it can be challenging to express histopathological changes by morphometric parameters such as volume, surface, length or number only. In this review we therefore provide an overview on different histopathological lesions in the respiratory tract associated with particle and fiber toxicology and on how to apply stereological methods in order to correctly quantify and interpret histological lesions in the respiratory tract. The article further aims at pointing out common pitfalls in quantitative histopathology and at providing some suggestions on how respiratory toxicology can be improved by stereology. Thus, we hope that this article will stimulate scientists in particle and fiber toxicology research to implement stereological techniques in their studies, thereby promoting an unbiased 3D assessment of pathological lesions associated with particle exposure.

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Figures

Fig. 1
Fig. 1
Lung sampling for stereology. a Lung fixation under defined inflation pressure. b Assessment of the lung volume or reference space by the Archimedes’ principle. c Serial sections of the lung – optionally with isotropic uniform random (IUR) orientation with the orientator. d Systematic uniform random sampling (SURS) of lung sections: in this example, every 3rd section is included in the sampling. The first section is picked at random - either by throwing a dice or with a random number table. e If smaller samples are desired for tissue embedding a further SUR sub-sampling is performed. Again every third tissue piece is selected here and the 1st one chosen randomly. f Selected tissue blocks are embedded for LM or TEM – optionally with IUR orientation using the isector
Fig. 2
Fig. 2
Structures and stereological test probes. The intersection of stereological test probes with the structure of interest provides countable events: test point counts for volumes, test line intersection counts for surfaces, test plane transect counts for length estimation and test volume object counts for number estimation. Note that the sum of the dimension of the test probe and the structure always equals 3. Figure adapted from [96]
Fig. 3
Fig. 3
Sampling strategies for homogeneously and heterogeneously distributed lung lesions. Systematic random sampling such as SURS and fractionator sampling are well recommended for homogeneously distributed lung lesions where small sample sizes are sufficient to reach a high precision of the estimate. However, site-specific lesions might not be adequately represented in systematic uniform random sampled tissue or fields of view. Different sampling strategies are therefore recommended for heterogeneous lesions in dependence on lung lesion distribution: Focal lesions which are randomly distributed over the whole lung are best addressed with an initial random tissue sampling followed by the proportionator approach for image acquisition. This enhances the efficiency greatly. If no proportionator is available, a more rigorous image sampling is required to obtain sufficient information as explained in Example IV for the airways. Site-specific lesions as for example in the bronchioles are best approached with stratified sampling in a two-step procedure within randomly sampled histological sections. First, the volume of the compartment of interest is estimated (for example bronchioles) and second, the lesion in the compartment of choice. An example of such a two-step sampling is presented in the Examples II and III for the parenchyma (protocol paragraphs). Note that this approach is still random, though site-specific. SURS and whole lung estimates could still be applied, but are likely to “dilute” the effect; hence subtle pathological changes might be missed. Region-specific lesions such as centrilobular emphysema might be more challenging to assess. If the region-specific lesion can be defined in both control and treated subjects, stratified sampling is recommended. If not, but the lesion is very prominent, SURS is still a valid alternative in combination with pathological description of the region of the lesion. However, certain limitations of the random sampling approach need to be recognized, particularly if the lesions are only very mild and their region not strictly defined
Fig. 4
Fig. 4
Example of volume and surface area estimation with stereological probes. a Lung volume estimation by Cavalieri method: a point grid with a known area per point is superimposed over mouse lung sections. The number of points multiplied by the area per point and the slice thickness will result in the total lung volume. b Volume estimation of parenchymal (P) and non-parenchymal (NP) lung volume by point counts. Note that a four-fold coarse point grid was included for the counting of parenchymal points. c Volume estimation of alveolar septa (S) and airspace (A) by point counts and estimation of alveolar surface area with line probe intersections (I). d Quantification of mucus per length of basement membrane on AB/PAS mucus positive section (purple). The volume of mucus (M) and epithelium (E + M) is estimated with point counts and the surface area of the basement membrane with the line probe intersections (I). A fifty-fold coarse point grid was included for the counting of lung tissue points (L)
Fig. 5
Fig. 5
Number estimation with disector. a Cell number estimation with the disector. Proliferating cells (BrdU positive) are stained with immunohistochemistry in brown. All cells within the counting frame which are present on the reference section (a) but not on the look-up section (a’) are counted (arrow) and vice versa. b Alveolar number estimation with the disector. Bridges (B) are counted in the reference section (b) and look-up section (b’). Note that any bridges in touch with the red exclusion line are not included (red arrow) in the evaluation and those in touch with the green inclusion line are (green arrow)
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