Elsevier

Neuroscience

Volume 130, Issue 4, 2005, Pages 813-831
Neuroscience

Review
Design-based stereology in neuroscience

https://doi.org/10.1016/j.neuroscience.2004.08.050Get rights and content

Abstract

Quantitative morphology of the CNS has recently undergone major developments. In particular, several new approaches, known as design-based stereologic methods, have become available and have been successfully applied to neuromorphological research. However, much confusion and uncertainty remains about the meaning, implications, and advantages of these design-based stereologic methods. The objective of this review is to provide some clarification. It does not comprise a full description of all stereologic methods available. Rather, it is written by users for users, provides the reader with a guided tour through the relevant literature. It has been the experience of the authors that most neuroscientists potentially interested in design-based stereology need to analyze volumes of brain regions, numbers of cells (neurons, glial cells) within these brain regions, mean volumes (nuclear, perikaryal) of these cells, length densities of linear biological structures such as vessels and nerve fibers within brain regions, and the cytoarchitecture of brain regions (i.e. the spatial distribution of cells within a region of interest). Therefore, a comprehensive introduction to design-based stereologic methods for estimating these parameters is provided. It is demonstrated that results obtained with design-based stereology are representative for the entire brain region of interest, and are independent of the size, shape, spatial orientation, and spatial distribution of the cells to be investigated. Also, it is shown that bias (i.e. systematic error) in results obtained with design-based stereology can be limited to a minimum, and that it is possible to assess the variability of these results. These characteristics establish the advantages of design-based stereologic methods in quantitative neuromorphology.

Section snippets

Contents

Section 1: Basic information about design-based stereology in the literature

Section 2: Considerations for specimen preparation for design-based stereologic analysis

Section 3: Laboratory equipment for design-based stereologic analyses

Section 4: Potential bias in results of design-based stereologic analyses

Section 5: Parameters that can be assessed by design-based stereology

5.1: Considerations about delineation of brain regions.

5.2: Volume of a brain region

5.3: Number of neurons within a given

Section 1: basic information about design-based stereology in the literature

More than 20 reviews and eight comprehensive books dedicated to design-based stereology and its applications in neurosciences are available. Among the reviews, some address more basic concepts of design-based stereology, whereas others deal with specific details of this methodology. Within the first category, the papers by Gundersen (1986, 1992), Cruz-Orive and Weibel (1990), Coggeshall and Lekan (1996), Mayhew and Gundersen (1996) and Royet (1991) provide introductions to the basic concepts of

Section 2: considerations for specimen preparation for design-based stereologic analysis

Design-based stereologic methods have been developed to make statements about structures such as an identified organ, a definable brain region, a population of cells, or linear biological structures within a tissue. In the case of the brain, if such statements are to be valid for an entire brain region, then the analyzed sample of sections and microscopic fields has to be representative of it. This requires that one has access to the entire brain region, that all cells or linear biological

Section 3: laboratory equipment for design-based stereologic analyses

Recent developments in design-based stereology include techniques and analyses that have become possible only with the introduction of computer-interfaced microscopes and imaging instrumentation, in particular, estimates of local volumes with the Optical Rotator (section 5.5), estimates of the length of linear biological structures with Space Balls (section 5.7), and investigations on the spatial distribution of cells with Nearest-Neighbor analysis (section 5.8). Furthermore, the availability

Section 4: potential bias in results of design-based stereologic analyses

Let us assume a stereologic estimate of the total number of neurons within a certain brain region. If this estimate could be repeated ad infinitum and the mean of the estimates would equal the (unknown) true total number of neurons within this region, the estimates would be unbiased (i.e. without systematic error; for details see West, 2002). Importantly, the use of design-based stereology does not guarantee unbiasedness of the corresponding estimates (Guillery and Herrup, 1997). Rather, the

5.1: considerations about delineation of brain regions

Each stereologic investigation starts with the identification of the boundaries of the brain region of interest (thereafter abbreviated as BROI) on a systematic-random series of sections throughout this BROI (Fig. 1A). In many cases (such as the cerebellar granule cell layer) the boundaries of the BROI can easily be identified on Nissl-stained sections, and one can trace the boundaries on video images displayed on a computer (Glaser and Glaser, 2000). Alternatively, one can perform only a rough

Section 6: variability of estimates obtained with design-based stereology

The results of quantitative histologic investigations performed with design-based stereologic methods are estimates rather than exact measurements. Thus, the results will vary if the same stereologic estimate were to be independently repeated. This implies that information about the variability of stereologic estimates is an important topic.

If the same stereologic estimate would be repeated ad infinitum, one could calculate the coefficient of variation of these estimates (note that the mean of

Section 7: presentation of results obtained with design-based stereology

Investigators and readers, as well as editorial boards and reviewers have all been concerned by the issue of what information and discussion are essential in a stereologic paper. Whereas there is no general answer to this question, the following recommendations might be useful.

The materials and methods of stereologic studies should provide a description of the histologic processing, with consideration of the specific requirements to be met (outlined in section 2) and the potential sources of

Section 8: the future of design-based stereology in neuroscience

Over the recent years design-based stereology has become the state-of-the-art methodology in quantitative histologic analyses, and the application of design-based stereologic methods to the analysis of the CNS has considerably contributed to our understanding of the functional and pathological morphology of the brain. Nevertheless, design-based stereology is still an evolving field and it can be expected that its applications in neuroscience will be subject to many improvements and extensions

Concluding remarks

Many important questions in neuroscience cannot simply be answered with a simple qualitative analysis. Whenever quantitative morphologic approaches are used, the following issues should be considered: are the results representative for the entire BROI? Are the results independent from the size, shape, spatial orientation, and spatial distribution of the cells to be investigated? Is bias (i.e. systematic error) in the results kept to a minimum? Is it possible to assess the variability of the

Acknowledgments

We thank our many colleagues who prompted us to write this review. We would like to thank the Stanley Medical Research Institute and the US-National Alliance of Autism Research (to C.S. and P.R.H.), the Alzheimer Forschung Initiative e.V., the Internationale Stichting Alzheimer Onderzoek and the Hersenstichting (to C.S.), and NIH grants AG02219, AG05138, MH58911, and MH66392 (to P.R.H.). P.R.H. is the Regenstreif Professor of Neuroscience.

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