14 ORIGIN OF THE PULMONARY VESSELS IN THE CHICK. 



knowledge of the origin of these vessels. The period of origin, from the beginning 

 of the proliferation of the angioblasts until the establishment of a lumen in the 

 pre-pulmonic capillaries, represents a relatively short period of incubation and 

 exact stages are not easily defined. The delicate collapsing capillaries are difficult 

 to make out in serial sections unless they are injected. Wax reconstruction of 

 such minute vessels is more or less impracticable because there are no blood- 

 corpuscles in the small capillaries. Angioblast and mesenchyme cells are not 

 easily differentiated and hence interpretation is often difficult. The location and 

 continuity of angioblastic cells with endothelium or other angioblasts are of great 

 value in their identification. The angioblast is larger than the mesenchyme cell, 

 the cytoplasm contains more basophilic substance, and the nucleus is more oval, 

 larger, and more vesicular. In injected specimens mounted in toto the small 

 capillaries are concealed by large systemic vessels packed with granules of ink. 

 This drawback has been overcome by a simple method of paraffin dissection for 

 the younger embryos and direct dissection of the older ones. 



METHODS. 



Three methods were used in this study: (1) Injecting living embryos and clear- 

 ing, by the Spalteholz technique, for dissection in oil of wintergreen ; (2) embedding 

 injected chicks in paraffin for dissection; (3) cutting serial sagittal and cross-sections 

 (10 to 15 microns) for staining. A summary of the development of the technique 

 of injections is found in the work of Sabin (1915). The injection method used in 

 this work is a modification of that devised by Popoff . The injections were made by 

 blowing ink into the vitelline vein of the living embryo by means of a fine glass 

 canula. Popoff (1894) first described this method of injecting small vessels. In 

 his work on the yolk-sac he found that injections of prussian blue greatly facil- 

 itated the study of the capillaries. He did not apply the method to vessels within 

 the embryo proper, but used it for the vessels of the yolk-sac by injections made 

 into the marginal sinus. At that time he noted the influence of the heart and the 

 direction of the blood-flow upon the completeness of his injections. 



My injections were made into the right vitelline vein, which lies over the artery 

 and lends itself readily to injection. The tributaries of this vein join at an angle 

 just before entering the body of the embryo. The point of the canula was intro- 

 duced into the vein at the vertex of this angle, which acts as a guide and offers 

 sufficient resistance to allow the entry of the needle into the vein. The tip of the 

 canula is visible and the extent of the injection under perfect control. MacCallum 

 simplified the injection of small vessels by following its course under a compound 

 microscope. A binocular microscope is of great help in making very dilute injec- 

 tions where danger lies in blowing too much ink into the blood-stream. A small 

 amount of ink diluted with physiological saline does not embarrass the circulation. 

 The heart action mixes the ink thoroughly with blood plasma and gives a complete 

 injection. The ink granules adhere to the endothelium of the vessels, due either 

 to the sticky surface of the endothelium or to direct phagocytosis. Care should be 

 observed that no vessels are torn in preparation for the injection. 



