Some Observations on the Method of Superfusion

Abstract

In 1941 Kwiatkowski reported a new technique for the assay of histamine, the tissue being suspended in air and the appropriate fluid, with or without dissolved drug, being run over the surface of the preparation. This was a development of the method described by Gaddum, Jang and Kwiatkowski (1939) for the detection of sympathomimetic substances in the venous outflow from the perfused rabbit ear. The principle of running fluid containing an active substance over the surface of a tissue suspended in air had been first reported by Finkleman (1930).The technique described by Kwiatkowski involved the injection of 0.1 ml. of drug solution into a side-arm of the tube carrying the fluid; for histamine assay the concentration of drug required in the injected solution was in the region of 10 ng. /ml. The injection of drugs into a moving stream may lead to errors due to uneven mixing, but this has been avoided in the technique recently described by Gaddum (1953). In this method (superfusion) the flow of fluid over the tissue is interrupted for a given period, the tissue is allowed to drain and the surface is then covered with the drug solution applied from a dropping pipette. The drug is allowed to act for a given period before the flow of superfusing fluid is restarted.It is claimed that very small quantities (5 drops, 0.2 ml.) of drug solution can be used and that this amount is " a reasonable compromise between the conflicting claims of the accuracy which might be achieved with more drops and the economy which would be achieved with fewer." The concentrations of drug used for histamine assay were 1-4 ng./ml. Spencer (personal communication) has suggested that the accuracy of the assay may be improved by the use of 0.5-0.8 ml. of drug solution, this fluid being applied immediately after stopping the flow of the superfusing fluid, without any period of drainage. Test solution volumes of 0.6 ml. have been chosen by Adam, Hardwick and Spencer (1954). The absence of flowing fluid may sometimes result in a response-possibly from release of choline esters (Magnus, 1930) or from a temperature change.However, apart from the literature quoted above, there seems to be little information concerning the relationship of the volume of the test solution to the accuracy of the assay, and no direct comparison has been made of the accuracy of the superfusion method with that of the usual bath techniques.Because we were faced with the problem of assaying very small volumes of test solution, it was of interest to us to obtain information on these points. We have accordingly investigated them, using histamine and the guinea-pig ileum.With the " large-bath " method (volume 2-5 ml.), it is possible to use volumes of test solution of the order of 0.2-0.5 ml. if the concentration of histamine is sufficient to give a final concentration in the bath of 10 ng./ml. If the " small-bath" method is used, the tissue is surrounded by the undiluted test solution; the concentration required is in the region of 1-5 ng./ml., but the smallest convenient b...

Superfusion as a Method for the Study of Drug Antagonism

“…But in rabbit's blood, for example, the proportion might be as high as 3:1 (Code, 1937;Holgate, 1953) and the concentration of mepyramine which would be necessary to abolish the activity of histamine under these conditions would very seriously reduce, if not abolish, the response of the ileum to the less active HT. Indeed, Cambridge and Holgate (1953) have shown that the remaining response in the presence of 10 ng./ml. mepyramine to a mixture of histamine and HT in the proportions of 1.5: 1 was, in fact, due to histamine, the effect of HT having been completely abolished.…”

“…After replacement of the antagonist by mepyramine-free Tyrode the response to histamine increased gradually to its initial level. The amount of drug given (40 drops) was well in excess of the minimum previously proved necessary for maximal accuracy (Cambridge and Holgate, 1954). At the beginning of every experiment a sensitizing concentration of agonist was chosen and repeatedly applied for 60 to 90 min.…”

“…Superfusion Apparatus.-The apparatus used was modified from that described previously (Cambridge and Holgate, 1954), and is shown in diagrammatic form in Fig. 1.…”

“…The technique of superfusion was used, since we had found this method both convenient and advantageous when studying the action of agonists (Cambridge and Holgate, 1954). The results obtained not only illustrate the limitations in the use of antagonists on the ileum for assay of HT in mixtures, but have yielded new information on the antagonism between atropine and HT.…”