WOUND HEALING IN EMBRYOS 243 



Wounds inflicted on the surface of a single cell will generally heal in a matter of minutes. 

 The closure of a wound in a coated epithelium (ectoderm or endoderm) may take several 

 hours, depending in part on the number of cells injured or removed. 



To facilitate the observations of surface movements in relation to wound healing, it is 

 advisable to pre-stain eggs or embryos with one of the vital dyes. Nile blue sulphate 

 1/7500 is toxic if the embryos are left in it very long but it can be used for rapid staining 

 as in this experiment. A concentration of 1/7 50, 000 of this vital dye is nontoxic and em- 

 bryos may be left in it indefinitely (Zorzoli, 1946). 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 Staining"). 



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

 cleaved 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 observation should be 

 repeated 4 or 5 times for each stage and each solution. 



There are several possible variations in the wound: 



1. 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). 



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

 and note the consequences on healing. 



3. Inflict a wound on a single blastomere of a four-cell stage and allow its contents 

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



4. Peel off from 1/4 to 3/4 of the epidermis of neurulae and later stages and deter- 

 mine the ability to recover. 



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

 which may well alter the wound healing process. Reference is made to: 



1. Crowding, with the consequent accumulation of metabolites. 



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



3. Temperature. 



4. Presence of monovalent metallic ions. 



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

 and "f" above. 



OBSERVATIONS 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 solution 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 sub- 

 sequent 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 1943 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. " 



