reviewed paper: Raimondo, S, L. Maltez, J.E. Pereira, G. Koopmans, A. Varegão, S. Geuna (2012) Stereology of Posttraumatic Spinal Cord Injury, NeuroQuantology, March 2012, p. 107 – 116
This review does a great job of pointing out the benefits of, and yet lack of, unbiased stereology research on lesion volume and cell number associated with spinal cord (SC) damage and regeneration. The point is made that both the ‘reliability of quantitative assessments of histological data’ that unbiased stereology provides, and the ‘procedure for counting SC neurons’ are similar to ‘other regions of the nervous system’ and ‘any other brain area’ (Raimondo, 2012, p. 107, second paragraph and p. 112, fourth paragraph). Correspondingly, the definition of and techniques to ascertain cardinal properties in any organ do not change; unbiased stereology affords advantages whether you are trying to estimate: number, length, surface, or volume, to characterize any tissue: connective, epithelial, muscle or nervous.
The authors surveyed PubMed records between 1990 and 2011; only a small percentage of papers reported the use of unbiased stereology to look at normal and pathological SC conditions. That inspired this review (Raimondo) to look at tissue processing and staining as well as stereological methods for the assessment of volume and number in the spinal cord.
Some aspects that affect the implementation of the unbiased stereology probe come up during discussion of tissue processing. If possible, the whole region has to be available for sampling. This is a main tenet of unbiased stereology and in this case, means collecting tissue that encompasses the whole lesion length. The issue of section thickness is also addressed; many spinal cord studies are done with sections ranging from five to 50 microns (Raimondo, p. 108, last paragraph). Pairs of contiguous thin sections are required for the physical fractionator; while thick sections are needed for the optical fractionator, both probes that estimate number.
Rules and considerations for effective unbiased stereology are given, starting with sampling. It is ‘almost always impossible (or at least very inefficient) to analyze the entire structure in all it parts’ … ‘the essence of sampling in morpho-quantitative research is represented by the selection of a small part of a biological structure that allows the investigator to infer conclusions about the entire structure’ (Raimondo, p. 111, first paragraph). Systematic random sampling is the most frequent way to arrive at the volume fraction to be sampled, and involves a random starting section and subsequent selection of sections based on a constant interval (Raimondo, fig. 6). Both the sampling technique and the rules for the stereological probe must ensure that ‘every histological parameter under investigation has the same chance of being selected for the final sample that will lead to the estimate, without making an assumption’ (Raimondo, p. 112, second paragraph). Design-based (unbiased) and model-based stereology are compared. The former takes into account and creates rules to eliminate bias ahead of time while the latter tries to make up for these shortcomings after-the-fact by massaging the data using formulas that require assumptions about the tissue. The consensus that ‘design-based stereology is considered the best approach in quantitative morphology of the nervous system’ is noted (Raimondo, p. 111, last paragraph).
Regarding the actual probes used to estimate number and volume, the optical or physical disector is recommended to estimate SC cell number, and Cavalieri/point-counting is recommended to estimate lesion volume. The optical disector is technically more easy to use than the physical disector; it is easier to use thick sections and focus through their depth than to compare two physical thin sections. Rules of optical disector counting include honoring the inclusion and exclusion planes, using the leading edge of the cell for counting, and using thick sections and an oil objective. The NvVref and fractionator method are compared; the fractionator method is easier since there is no need to measure or estimate the reference volume (Raimondo, p.12-113). Point counting, including Area Fraction Fractionator, can be used to estimate the percentage of one type of tissue, for instance when looking at white matter sparing. The volume of SC cells can be probed during the optical fractionator probe by using the local Nucleator. Of course, any parameter, first or second order, about SC tissue can be estimated using modern unbiased stereological probes.
In conclusion, the authors urge those doing SC injury research who wish to estimate the degree of lesion based on morphological predictors of damage and regeneration to ‘contribute to the progressive disuse of old and bias-keeping morphometrical methods in favour to the adoption of modern and bias-free stereological methods’ (Raimondo, conclusion).