Micro Vascular Morphometry

Abstract

Julius Cohnheim (1889) was one of the first to describe the microvasculature of tissues. However, it was not until sixty years later that it could be quantified (Chalkley, et al 1949). Popular methods to visualise microvasculature had been colloidal carbon injection (Hilmas and Gillette 1974) and perfusion with a mixture of India Ink and gelatin (Jee and Arnold 1960). Perfused specimens were either cleared in glycerol and made transparent or embedded in wax and sectioned to be examined under the microscope. Recently colloidal carbon and gelatin has been replaced by a mixture of fixative, copper pigment and latex (Reinhold et al 1983) or photographic emulsion (Reempts et al 1983). Once the microvasculature is “labelled”, it is photographed and the images so obtained are analysed either with a point counting grid according to the Chalkley method (Hilmas and Gillette 1974) or the area or perimeter of the stained vessel cross sections measured using an image analysing computer system (Wilkinson et al 1981). Most of the studies have emphasised the importance of perfusing the microvasculature at near physiological pressures. The introduction of polymerising casting media which withstand electron bombardment in the scanning electron microscope has opened up an elegant avenue to study microvascular patterns (Murakami 1971). It soon became evident that complete filling of microvasculature does not occur at physiological pressures (Lametschwandtner et al 1990). It would thus be difficult to measure a modest reduction in microvascular volume using perfusion at a physiological pressure. If the purpose of a study is to detect an anatomical reduction in the microvasculature then complete filling of the microcirculation would be essential. Microcorrosion casts can demonstrate the extent of filling of the microcirculation in a given tissue. It is, however, difficult to quantitate three dimensional vascular casts. The histology of cast specimens on the other hand can be quantified. The use of vascular casts in microvascular morphometry has been described (Narayan et al 1991). However, in this technique it is not possible to distinguish between vascular spaces, air bubbles or spaces due to loss of tissue as all these appear clear in the histology of a mercox (CL-2B-S Blue, Vilene Hospital, Japan) cast specimen. The present paper describes a technique to measure only mercox filled vascular spaces in a histological section. An attempt has also been made to correlate the perfusion pressure and its effects on capillary filling, with the resulting variation in microvascular morphometry. Murine brains, bone marrow, liver and kidney were used in this study.