Frozen-section fluorescence microscopy and stereology in the quantification of neuronal death within dorsal root...

Histochemical and morphological research increasingly relies upon quantification of complex biological systems. For such investigations to be meaningful, quantification techniques must meet the seemingly conflicting requirements of being theoretically robust, yet sufficiently practical to facilitate widespread applicability. Validity ought to be enhanced by theoretical simplicity, use of measured rather than assumed variables, and minimising observer interpretation. Practicality is facilitated by simplifying and reducing measurements, broadening applicability, and reducing costs and analysis time. As a result, quantification systems that rely upon sampling and estimation have been favoured over serial reconstruction techniques. To provide reliable estimates, sampling must be valid at all levels from tissue harvest, to the selection of microscope fields in which quantification is performed by techniques that account for the anisotropic distribution, and variable size of many elements in biological systems. These principles are embodied in the development of a stereological approach to the quantification of neuronal death within dorsal root ganglia after peripheral nerve injury. This frozen section technique is efficient and flexible, since it permits simultaneous morphological examination, TUNEL, or standard fluorescence immunohistochemistry, broadening its applicability. Section shrinkage is minimal, and counting by optical disection has proved to be time-efficient and sufficiently reproducible to reliably detect losses in the order of 5, with minimal inter-observer variation. As is discussed, stereology has not yet met with universal acceptance, but by balancing theoretical validity with practical applicability, it has proved an excellent approach to the investigation of neuronal death within dorsal root ganglia. Frozen-section fluorescence microscopy and stereology in the quantification of neuronal death within dorsal root ganglia.