The Site of Action of Histamine and Serotonin along the Vascular Tree : A Topographic Study

While it is an established fact that histamine and serotonin increase the permeabili ty of blood vessels, the exact portion of the vascular tree which is so affected has not been conclusively demonstrated. The present study was undertaken to clarify this point. Our experiments were based on a method to which we refer as "vascular labeling," and which permits one to identify leaking vessels by means of visible accumulations of foreign particles within their walls. The mechanism of the labeling, elucidated by previous electron microscopic studies, is the following. Histamine and serotonin cause the endothelial cells of certain vessels to separate, and thus to create discrete intercellular gaps. Plasma escapes through these gaps, and filters through the basement membrane. If the plasma has been previously loaded (by intravenous injection) with colloidal particles of a black material such as carbon or mercuric sulfide, these part icles--too large to pass through the basement membrane-wi l l be retained and accumulate in visible amounts within the wall of the leaking vessel. This method is used to maximal advantage if the tissue is cleared and examined by transillumination in toto, so that leaking vessels can be accurately identified in their relationship to the vascular tree. As a test tissue we used the rat cremaster, a laminar striated muscle which can be easily excised with its vascular supply virtually intact. The rats were prepared with an intravenous injection of carbon or HgS, and a subcutaneous injection into the scrotum of histamine, serotonin, or NaC1 (as a control). The injected drug diffused into the underlying cremastcr and the vessels became labeled. One hour later, when the carbon had been cleared from the blood stream, the animal was killed. The cremaster was excised, stretched, fixed in formalin, cleared in glycerin, and examined by transillumination under a light microscope. The lesions induced by histamine and serotonin were identical. The leaking vessels, as indicated by the carbon deposits, always belonged to the venous side of the circulation. The heaviest deposits were found in venules 20 to 30 micra in diameter. The deposits decreased towards larger venules up to a maximum diameter of 75 to 80 micra, and towards the finer vessels until the caliber reached approximately 7 micra. Essentially spared by the deposits were the finest vessels, 4 to 7 micra in diameter, and constituting an extensive network oriented along the muscular fibers. By killing animals at varying intervals after the injections, it was found that the carbon particles were slowly removed from the vascular walls by the action of phagocytic cells. After 10 months there was still enough carbon locally to be recognized by the naked eye. 607 on N ovem er 7, 2017 jcb.rress. org D ow nladed fom The process of acute inf lammat ion, according to one of the basic principles of general pathology, is in large measure the result of an increased permeabi l i ty of blood capillaries (1, 2). This concept is so fundamenta l tha t it may appear to lie beyond the range of challenge. To be sure, i t would scarcely be reasonable to question the existence of an increased permeabi l i ty of the blood vessels, an obvious fact which was firmly established by Cohnhe im as early as 1873 (3). O n the o ther hand, the actual site of the leak has never been exactly identified. I t could conceivably appear in the capillaries, in the arterioles, in the venules, or in a combina t ion of these; perhaps it could even vary according to the injurious agent. Surpris ing as it m ay seem, the cur ren t belief tha t the capillaries become m~Jre permeable is founded not on direct evidence, but r a the r on t radi t ion born of cursory observations. This is t rue not only for the process of acute inf lammat ion, bu t also for the mechan ism of act ion of his tamine, serotonin, and the other substances known to affect the permeabi l i ty of blood vessels. The lack of precise informat ion on this point is easily explained by three major technical difficulties which are encountered, at the level of the l ight microscope, when a change as subtle as increased permeabi l i ty is to be recognized and correctly located in the maze of the vascular network. In the first place, the p r imary al terat ion of the endothe l ium lies beyond the resolution of the l ight microscope (4, 5), as Cohnhe im himself implied when he speculated about a "molecu la r change" (6). Fur thermore , histological sections are singularly unsuited for a study of this kind; the terminal ramifications of the arterial and venous trees, and the capil lary network, are difficult or impossible to distinguish from each o ther when seen in cross-section, and even more so, of course, when the vessels have become abnormal in s tructure and caliber. Finally, the t radi t ional experiment , current ly used to demons trate an increased vascular permeabil i ty, does not lend itself to detailed observations. A dye such as t rypan blue is injected into the blood stream; the dye will leak out more readily at the site of vascular damage (1, 2). This procedure is very demonst ra t ive to the naked eye, bu t disappoin t ing unde r the microscope; colored plasma escapes freely out of the damaged vessels, and the result is a diffuse blue haze. Hence there is little hope to p inpoin t the actual site of the lesion along any single type of vessel. The first of these difficulties has been overcome by means of the electron microscope: it is now clear tha t h is tamine and serotonin cause leaks to appear between endothel ial cells, whereas the basement m e m b r a n e remains in tac t and literally filters the plasma which escapes through the opening (4, 5). O n the basis of this observation it becomes a simple mat te r to label leaking vessels in such a m a n n e r tha t they may be identified with the l ight microscope. Ins tead of a dye, a suspension of suitable par t icula te matter , such as ca rbon Mack, should be injected into the blood stream. Then , wherever a leak is present in the endothel ium, p lasma will flow out; bu t the suspended particles, if of appropr ia te size, should be held back by the filtering effect of the basement membrane , and soon accumula te in amounts large enough to be visible with the l ight microscope. This effect should be put to max ima l advantage by examining the tissue as a w h o l e with its vascular tree i n t a c t r a t h e r than in tissue sections. We have applied this procedure to the s tudy of two agents which are typical representatives of the compounds known to increase "cap i l l a ry" permeabi l i ty: h is tamine and serotonin. These are the best known chemical mediators of vascular injury in acute in f lammat ion (7, 8). Topographically the result was quite unexpected: both mediators, when studied in this manner , appea r to exert their specific effect on the venous side of