WOUND HEALING IN EMBRYOS* 



DEFINITION : The atility of the Interrupted living surface to close. 



PUBPOSE : To determine the ahility and rate of wound closure of the egg, hlastomere, and 

 embryonic epidermis under various environmental conditions. 



MATEBIALS: 



Biological : Ovulating females and early developmental stages of any Anura or Urodela. 



Technical : Stock solutions (see helow) 



Glass needles, pointed lancet 

 Vital dyes: Nile blue sulphate (I/75OO) 

 Neutral red (1/25OO) 



METHOD : 



Precautions : 



1. Avoid infection. Sterilization of the instruments emd solutions will not he 

 necessary If ordinary care is observed. 



2. Keep eggs and embryos in separate #2 Stenders with covers, at room temperatures 

 or below. Provide 10 to 20 cc. of solution per egg or embryo. This relatively 

 large volume will reduce any effects from accumulating metabolites. 



3. In Ca-free experiments, wash the egg or embryo in several changes of Ca-free 

 solution prior to making the Incision. Adherent calcium or calcium diffused 

 from the embryo may alter the results . 



Controls : Holtfreter (19'+5) says: "In an optimal medium, as represented by our 

 Standard Solution, the healing faculty is extraordinary." We may therefore con- 

 sider the healing process in this solution as the control situation with which the 

 process, in other media, is to be compared. 



Procedure : 



Observations are to be made on wound healing of the following embryonic stages: 



a. Body cavity eggs, having vitelline membranes only. 



b. Recently fertilized eggs - 1 hour after insemination. 



c. The k - cell stage, a single blastomere to be injured. 



d. Animal pole of early gastrula. 



e. Epidermis of neurula. 



The effect on wound healing of the following solutions is to be determined: 



a. Distilled water (glass distilled water preferred), - hypotonic. 



b. Standard Solutions diluted to 10^, - hypotonic. 



c. Standard Solution - isotonic. 



d. Standard Solution x 2: - hypertonic. 



e. Standard Solution plus 0.1^ KOH - alkalinized. (Determine the pH. ) 



(standard Solution for this should be made without the buffer.) 



f. Standard Solution plus 0.1^ HCl - acidified. (Determine the pH. ) 



(standard 'Solution for this should be made without the buffer.) 



Prepare an ovulating female frog, Rfjia pipiens, about i+S hours before the time of the 

 experimental work. The various solutions should be made up and distributed to labelled 

 #2 Stenders ready for the introduction of eggs or embryos. 



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) oiay 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-staln eggs or embryos with one of the vital dyes. Nile blue sulphate 



* This exercise has been organized with the generous aid of Dr. J. Holtfreter. 



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