18 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. 



In the present study the above procedure was the routine method of injection 

 employed. Pig embryos, brought from the abattoir, contained in the uterus, were 

 found to be wholly satisfactory material. If not permitted to cool excessively in 

 transit the embryos lived for at least two hours in a 38 incubator. On being 

 received at the laboratory a section of the uterine wall containing the placenta was 

 excised, with the embryo left connected by the umbilical structures. As soon as 

 the technical preparations for injection were completed the amnion was opened and 

 the embryo placed upon a padded block at the proper level. The first needle was 

 then inserted into the easily discernible central canal of the spinal cord and the 

 second into the left cerebral ventricle or into the mesencephalic ventricle. By 

 elevation of the reservoir connected with the first needle the cerebro-spinal fluid 

 was replaced by the injection solution. As soon as the replacement was complete 

 the needles were withdrawn and the embryo and its uterine portion replaced in 

 the incubator. The heart of the embryo could be easily observed in the smaller 

 forms and served as the index of a continued circulation. 



The incubation of the embryos was continued for varying periods of time, but 

 it was soon ascertained that a period of over 30 minutes generally resulted in a 

 complete spread of the injection solution. For comparison the period of incuba- 

 tion was lengthened and shortened, but the best results were usually obtained with 

 a 45-minute incubation after the replacement. 



Injections of the necessary true solutions were made, in the routine experiment, 

 with a 1 per cent concentration of potassium ferrocyanide and iron-ammonium 

 citrate in distilled water. By a 1 per cent solution is meant a salt concentration of 

 this amount (potassium ferrocyanide, 0.5 gm.; iron-ammonium citrate, 0.5 gm.; 

 water, 100 c.c.). The resultant true solution should be practically isotonic with the 

 body-fluids. In this way any injurious consequences due to hypertonic or hypo- 

 tonic solutions were apparently overcome. The factors of osmosis and diffusion 

 also had to be considered in this connection. 



Other concentrations of the so-called "ferrocyanide mixture" were used, but 

 only for the sake of comparison or for the purpose of investigating some particular 

 phase of the problem. The results obtained by the use of these concentrations were 

 not relied upon as affording standards for the normal pathway of the cerebro-spinal 

 fluid. 



In addition to the replacement type of injection, many observations were 

 carried out on pig embryos, with a simple syringe-injection of the ferrocyanide solu- 

 tion into the central canal of the spinal cord or into the cerebral ventricles. It 

 proved to be a very simple matter to regulate the pressures by this method, and 

 three arbitrary standards (mild, moderate, and strong) were found to be of value 

 in a comparison of the extent of the spread obtained by replacement and by injection. 



The prussian-blue reaction (formation of ferric ferrocyanide) was obtained in 

 these experiments by fixing the whole embryo in an agent containing hydrochloric 

 acid. For histological study the best results were obtained by immersing the 

 specimen from 1 to 10 minutes in a 10 per cent formaldehyde solution containing 



