8 DEVELOPMENT OF THE VEINS IN THE EMBRYO PIG. 



used, and Popoff noted that the injection mass might run with or against the blood-stream 

 from the heart, according to the point of injection; he found also that part of the blood- 

 vessels of the yolk-sac might be injected by the action of the heart driving the fluid through 

 the aorta. In chicks, after the fourth day of incubation, Popoff injected directly into the 

 aorta by means of a hypodermic syringe. 



The history of the different methods of injecting embryos as they have developed in 

 this laboratory may be of interest. The work was begun by Dr. Mall and has been con- 

 tinued by his pupils. At first the work was a direct development from the methods of 

 making injections of the vascular system in adult animals, and this has been summarized 

 by Dr. Flint (1900) in his article on the adrenal. After injecting the blood-vessels of the 

 adrenal in the adult, Flint applied a similar technique to the vessels in fetal stages, namely, 

 he tied a canula into the aorta and injected Prussian blue by means of a gravity flask. For 

 younger stages, he tied a canula into the apex of the heart, and finally in still younger 

 stages into the umbilical artery or vein. By this last procedure he succeeded in injecting 

 embryos as small as 2.5 to 3 cm. in length. 



The next step in the injection of the embryos came in my study of the lymphatic vessels 

 of the skin, which were filled with Prussian blue by means of a fine hypodermic needle. In- 

 asmuch as lymphatic vessels can seldom be seen, good results are only obtained by knowing 

 the level of the lymphatic plexus in order to puncture the vessels. Extensive injections of 

 the lymphatics in stages before the valves develop can best be made by considering the 

 lines of growth, that is to say, by finding the position of larger trunks, which are the primary 

 vessels of an area and which drain an extensive zone. In these studies it became necessary 

 to get the relation of the lymphatics to the blood-vessels, and I found that by puncturing the 

 liver directly with a syringe and filling it with fluid, an injection of the main arteries could 

 be made by means of the heart -beat. Thus was emphasized the necessity of having fresh 

 embryos with the heart still beating. During the same work i 1*K>2) I repeated Budge's 

 injection of the extra-embryonal ccelom and the tissue space connected with it. To make 

 these injections I used a glass canula connected by means of a rubber tube to a pressure fla.sk 

 of a very low column of mercury. This is easily arranged by putting a little mercury into 

 a U-tube, one end of which is attached through a rubber tube to the canula and the other to 

 a rubber bulb which can be compressed by a screw-clamp. These injections were made 

 with India ink, which flows more easily than Prussian blue, since it has a finer granule. 

 This manipulation was watched under a compound microscope, a procedure which was first 

 emphasized by W. G. MacC'allum. Since then all injections of the young embryos have 

 been made with the aid of the binocular microscope. 



In 1905 Dr. Mall published a study of the development of the blood-vessels of the 

 brain made from a series of embryos in which the injections were made either through the 

 aorta or by a direct puncture of the liver. In this paper Dr. Mall analyzed completely the 

 method of injecting the blood-vessels of the embryo through the liver, and showed that by 

 this method either the main arteries or the main veins could be injected at will. To fill 

 the arteries he injected a little ink into the liver and got their injection by means of the 

 heart-beat. On the other hand, he obtained the main veins in a dead embryo by an over- 

 distention of the liver. In these injections India ink was used. 



Finally complete capillary injections were obtained by H. M. Evans. He found that 

 by injecting India ink into the umbilical arteries with a hypodermic syringe, the needle 

 being pointed toward the embryo, the entire capillary bed of the embryo could be filled. 

 By the aid of this method Evans was able to demonstrate the validity of Thoma's theory 



