DEVELOPMENT OF THE VEINS IN THE EMBRYO PlCi. 



of the growth of blood-vessels by showing that tho main arteries of the body could be 

 traced to their antecedent plexuses. 



In general embryos of medium size can be injected best with a hypodermic syringe. 

 For these injections the chorionic sac should be opened immediately before the injection is 

 made; until the arnnion and chorion have fused the amnion should be left intact. To make 

 the injections it is essential to see the vessels clearly and to use a fine, sharp needle. The 

 needles should be the finest obtainable, No. 28. Either the artery or the vein may he 

 punctured, the more complete capillary injections always being made through the artery. 

 For embryos from 7 to 15 mm. long it is not necessary to tie off any of tho vessels of the 

 membranes; above this stage it is practically impossible to obtain complete injections 

 without previously tying off the group of vessels of the membranes which are not used for 

 the injection. The vein which accompanies the artery injected gives a sufficient vent for 

 the fluid. The specimen should be kept moist with amniotic fluid. If extravasations 

 occur in the skin vessels, they are readily seen at the time of injection and in general they 

 are sure to occur if the embryo is not perfectly fresh; that is, perfect injections are never 

 obtained unless the embryo is still warm, the heart beating, and there are no previous 

 extravasations of blood due to rough handling. If any ink escapes on the surface of the 

 skin, it must be washed off in running water before fixation, if the specimen is to be cleared 

 in toto. 



In the study of complete injections made with India ink, all of the vessels can be 

 analyzed in the early stages; but as soon as the vascular system becomes complex, as in 

 embryos of about 15 mm. in length, it becomes practically impossible to distinguish all the 

 arteries from the veins. This can readily be seen, for example, in an injection such as the 

 one shown in figure 10, plate 4. In these stages one can not determine with certainty given 

 vessels without obtaining injections that will bring out just these arteries, or these arteries 

 with their arterial capillaries or again just these veins. This precision of injection can be 

 obtained by the use of silver nitrate as the injecting fluid. For embryos I use a solution of 

 0.25 per cent of silver nitrate in distilled water. The silver solution does not run readily. By 

 varying the amount of fluid injected and the point of injection one can obtain either a 

 partial injection of the entire embryo or a partial injection of any organ. The silver solu- 

 tion kills the endothelium and immediately whitens it, so that it is possible to estimate 

 approximately the extent of the injection and to stop it at any desired point. The flow of 

 the silver solution stops as soon as the pressure is taken off, that is to say, the flow of the 

 solution is due entirely to artificial pressure and is not aided at all by the heart-beat nor by 

 the constriction of the blood-vessels. For general arterial injections it is best to inject with 

 a hypodermic syringe into the umbilical artery. For an embryo 20 mm. in length I use 

 about 1 c.c. of the fluid. To fill the arterial capillaries it is only necessary to use more of 

 the solution. For venous injections, since an injection of silver into the umbilical vein 

 usually fills only the veins of the liver, I use a glass canula and inject directly into the 

 primary head vein. If the embryo is placed in a strong light under the binocular micro- 

 scope, the vein can practically always be made out, especially just behind the ear, by the 

 presence of a little blood in it. An example of such an injection is shown in figure .">, plate _'. 

 As soon as the injection is complete, the embryo should be placed in a bowl of distilled water 

 and exposed to direct sunlight or to the table arc light to reduce the silver nitrate. 



In the study of the vascular system, the Spalteholz method for clearing entire speci- 

 mens, described in his article published in 1914, is invaluable. In general the essentials 

 of the method are, first, fixation in formalin; second, a thorough bleaching of t he tissues with 



