School of Medicine and Health Sciences, University of North Dakota
Morphometry is a precise method of measurement that employs conventional morphological methods to estimate the physical dimensions of an unbiased population of biological structures. The structures may be quite large and visible to the naked eye or they may be small and require substantial magnification. In order to measure capillary basement membranes (CBMs), transmission electron microscopic (TEM) methods are applied to tissue samples. The samples must be selected without bias and carefully prepared, accurately measured, corrected for plane of section, and statistically analyzed to provide a faithful numerical description of the measured CBM population.
Basement membranes (BMs) were identified first in the mid-19th century. They were described as homogeneous and sometimes fibrillar extracellular sheets that supported the digestive and respiratory systems, and as continuous matrix barriers between mucous membranes and their underlying capillaries. These remarkably accurate accounts formed the bases of textbook descriptions of BMs until the 1950s, when the advent of TEM provided substantially increased image resolution of biological samples.
Subsequent TEM studies coupled with the development of new methods to isolate BMs led to biochemical and immunochemical analyses, which showed that BMs were comprised largely of collagen, noncollagenous glycopro-teins, and proteoglycans assembled in a network that resulted in the electron-dense layer (basal lamina) imaged by TEM . Studies of isolated BMs substantially increased our understanding of the production, assembly, and degradation of their molecular components. Importantly, it was shown that although most BMs comprised similar molecular subunits, they were not identical, and a nonunitary concept of BM composition was generally adopted.
BMs also varied widely in their location and morphological presentation, including width (thickness). Some (e.g., the lens capsule and Descemet's membrane in the eye) were extremely thick and could be removed from their underlying tissues with a dissecting microscope. Others, including those of most blood capillaries, were very thin and could not be imaged clearly by light microscopic (LM) techniques. It is now recognized that regardless of location and width, TEM techniques are required to provide adequate images for accurate BM measurements, and this is especially true for the microvasculature where CBMs are particularly narrow.
Although BMs exhibit slow turnover rates, they are nevertheless dynamic structures, and their thickness is altered with age, physiological state, or pathological environment. For example, many microvascular BMs are thickened in diabetes mellitus, leading to renal, retinal, and neurological disorders. Interestingly, BMs of nonvascular tissues (e.g., renal tubules) also show thickness increases in diabetes. Accordingly, it is believed that although BMs from various tissue types are differentially thickened during normal aging or in disease states, the mechanisms leading to such thickening may be similar.
Because CBM thickness is widely regarded as a bio- _
marker for aging and a hallmark for chronic complications 1 ¡.im CBM N^
of diabetes, numerous studies have centered on this parameter. The purpose of this chapter is to provide the reader with background information regarding the morphological heterogeneity of CBMs, and specifically with some methods by which their thickness may be accurately measured.
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