236 WOUND HEALING IN EMBRYOS 



1/7500 is toxic If the embiyos are left in it very long tut it can be used for rapid 

 staining as in this experiment. A concentration of l/750,000 of this vital dye is non- 

 toxic and embryos may be left in it indefinitely (Zorzoli, 1914-6). The stages are to be 

 immersed in the vital dye for a few minutes, or dye-stained agar or cellophane may be 

 used for locally staining areas on the surface of the egg, (see section of "Vital Stain- 

 ing"). 



The wound is best made with a sharp-pointed glass needle or lancet. In the case of 

 uncleaved eggs the needle may be inserted below the surface coating and the cut made by an 

 upward movement, using the glass needle as a knife blade. The healing capacity of coated 

 epithelia can be best observed in a neurula or somewhat older stage where part of the 

 epidermis is peeled off by means of a pair of glass needles. 



The wound should be inflicted while the specimen is immersed in the solution to be 

 tested. For instance, a body cavity egg is transferred from the body cavity to Standard 

 Solution and then the wound is inflicted. Since the healing process is rapid it will be 

 necessary to observe the wound rather constantly for a period of minutes. This observa- 

 tion should be repeated Ij- or 5 times for each stage and each solution. 



There are several possible variations in the wound: 



a. Compare the healing of wounds in the animal and the vegetal poles; dorsal and 

 ventral epidermis of the neurula (where there is little and much yolk). 



b. Inflict several wounds, some parallel and others at right angles to each other, 

 and note the consequences on healing. 



c. Inflict a wound on a single blastomere of a four-cell stage and allow its con- 

 tents to escape. Note the effect on this and the other blastomeres. 



d. Peel off from l/U to 5/**- of the epidermis of neurulae and later stages and 

 determine the ability to recover. 



There are environmental variables, aside from the osmotic conditions of the medium, 

 which may well alter the wound healing process. Beference la made to: 



a. Crowding, with the consequent accunnilatlon of metabolites. 



b. Increasing concentration of carbon dioxide (or even oxygen). 



c . Temperature . 



d. Presence of monovalent metallic ions. 



e. Extremes of pH. The moderate shift in pH will be determined with solutions 

 "e" and "f" above. 



OBSEBVATIONS AND THE TABULATION OF DATA : 



The healing process as observed in the Standard Solution is to be considered the 

 normal or control situation. It is important, therefore, to establish data in this solu- 

 tion for all stages and operating conditions first. Thereafter, comparisons are to be 

 made with these data. If there is a limit In time and facilities. It is recommended that 

 solutions "b" and "e" in the above list might be omitted. 



Drawings made at the time of wound infliction, a minute thereafter, and at a stated 

 subsequent interval will constitute the record for each of the above observations. 



DISCUSSION : 



The most recent and complete analysis of wound healing is contained in a I9U5 paper 

 by Holtfreter in which he says that "The coated surface layer is of predominant importance 

 for the closing of wounds in single cells and in epithelia." 



Sodium, Potassium, and Magnesium ions, hypertonlcity, and alkalinity all cause lique- 

 faction and dispersion of the surface layer while Calcium, a slightly hypotonic medium, 

 and an intermediary pH, tend to counteract the effect of the former by binding and solidi- 

 fying the egg substances, thus favoring wound healing. Calcium, even in a concentration 

 of 1/100,000,000 is sufficient to aid the healing process and this amount may diffuse out 

 of the embryo from regions other than the wound area. The coated cells, in the wound 

 healing process, tend to spread over the uncoated ones. 



