Metodología de desarrollo de software

CSF sampling in man is a standard procedure for investigating neurological disease. It is usually carried out on a one to one basis because of it’s well established technical risks and rarely for research purposes except as an ’extra’ once the cannula is inserted and CSF taken for investigative medical purposes (e.g. Reynolds et al, 1972; Brodie et al, 1985; Loscher et al, 1988). Until the late ’70 and early ’80 most of the studies involving serial sampling were carried out in larger animals such as the cat, dogs or primates (Sarna et al, 1983; Sokomba et a, 1988; Patsalos et al, 1992). This was partly because of difficulties in implanting CSF catheters into small animals such as rats without causing unwanted injury and

partly because the methodology for analyzing small sample volumes and concentrations was not well developed (Sarna et al, 1983; Kornbuher et al, 1986). The rat produces only about 2-3 CSF per minute, the total CSF volume being about 250 ^1 (Sarna et al, 1983; Chaouloff et al, 1986). Therefore, a sample volume of as little as 25 /xl represents 10% of the total volume. Increasing the sample volumes may not only result in e.g. coning but also subarachnoid bleeding but the CSF protein concentration also changes independent of blood contamination. This may be of relevance in the interpretation of data obtained from the CSF (Suckling and Reiber, 1984).

The earliest reported method for serial CSF sampling in fully conscious, freely mobile rats was described in 1979 by Bouman and Van Wimersma Greidamus and was soon followed by others in rats (Swartz and Stenberg, 1980; Danguir et al, 1982; Kiser, 1982; Lai et al, 1983; Sarna et al, 1983; Gunther and Merger, 1984; Kornhuber et al, 1986).. The method of Sarna et al (1983) is now an established technique for serial sampling of CSF in my

institution.

As with serial blood sampling, there are disadvantages with serial CSF sampling. The surgical procedures are delicate and injury to the brain and cerebral blood vessels is an obvious risk. This is harmful to the rat and, as in humans, the resulting contamination of CSF with blood, the increase in blood-brain barrier permeability (through inflammation and infection) and the change in CSF metabolism (e.g. through stress) can give misleading results. It may not always clinically obvious that damage has been inflicted. As the amount of CSF that can be collected at any one time from a rat or guinea pig should not exceed 40

purity and normality of the fluid by microscopical and biochemical means and to analyze the substances under investigation. Another complication is that different sites for cannulation have been proposed, including the cisterna magna (e.g. Bouman and Van Wimersma Greidanus, 1979; Sarna et al, 1983; Lai et al, 1983; Nakamura and Cowley, 1988), the 3rd ventricle (e.g. Danguir et al, 1982; Chaouloff et al, 1986) and the lateral ventricles (e.g. Gwosdow et al, 1985). The composition of CSF varies at these sites, particularly in the water and protein content (McMurray, 1988b). This can affect the concentration of the substance under investigation and give conflicting results if data from one cannulation site is compared with that of others. However, there is much more uniformity in the opinion about CSF cannulation, the majority of workers preferring the cisterna magna because of ease of access (Kornhuber et al, 1986).

As with blood cannulation, in depth validation of the techniques for serial CSF sampling in fully conscious, freely mobile rats is limited. The per/post operative mortality has not been established, nor has the incidence of infection, brain injury or subarachnoid haemorrhage. Sarna et al (1983) noted that 60% of the catheters were patent for use on the third post­ operative day and verified the position of the catheters by radiography. Chaouloff et al (1986) devised a protocol by which they verified that the amount of manipulation and handling of the rat with their technique did not cause stress induced changes in CSF monoamine metabolism. A number of workers observed that sampling was possible for an average of two weeks, but on occasion the catheters were patent for up to four weeks (Schwartz and Steinberg, 1980; Lai et al, 1983; Sarna et al, 1983; Gunther and Merger, 1984; Kornhuber et al, 1986). Of the few reports where some validation aspects were done in some detail. Suckling and Reiber (1984) in guinea pigs, noted that the composition of CSF proteins in

serially obtained samples changed only if the volume of the individual sample exceeded 40

fi\. Kornhuber et al (1986) studied the post-operative weight recovery in detail with and without CSF sampling and the success rate for cannula patency over two weeks. They found that contamination with blood increased with successive sampling. Recently, Westergren and Johansson (1991) examined the extent of infection, meningeal irritation and blood contamination in serially obtained samples using plastic (cf. Sarna et al, 1983) and steel cannulas (cf. Schwartz and Steinberg, 1980; Kornhuber et al, 1986). They noted that infection and meningeal irritation could not be prevented even with aseptic cannulas with the former method while with the latter blood contamination was always present and increased significantly with successive samplings. Meningeal irritation and infection lead to increased permeability of the blood-brain and to increased transfer of substances from blood to CSF.