308 



REGENERATION 



LIMB REGENERATION 



The Urodeles show regenerative powers throughout life but the Anura exhibit them until 

 metamorphosis and then only to a limited degree thereafter. It is instructive to study the 

 regeneration of limbs and digits of Urodele larvae. It must be remembered, however, 

 that the post-operative larvae must be treated normally with respect to food, oxygen, 

 space, etc. If x-ray facilities are available, the effect of such irradiations upon regen- 

 erating limbs can be studied (see Butler, 1934 and Butler & Puckett, 1940, and illustra- 

 tions on the following page). 



Procedure : 



1. Select Amblystoma or Triturus larvae beyond stage #38, when the limbs are 

 developing or are already developed. It is essential that larvae of the same 

 age and stage be used in order to determine the effect of cuts at various 

 levels and angles. 

 2. Anesthetize the larvae in 1/10,000 

 MS 222 and make the following 

 cuts with iridectomy scissors. 



a. Cut off the right forelimb at 

 the level of the wrist. 



b. Cut off the right forelimb be- 

 tween the wrist and the pec- 

 toral girdle. One amputation 

 above the elbow. 



c. Repeat the above two cuts but 

 make them at the greatest 

 possible angles. 



d. After cutting the larger 

 skeletal elements (humerus, 

 radius, and ulna) the skele- 

 ton within a few hours may 

 begin to protrude distal to 

 the original level of the am- 

 putation. In such an event, 

 the larva should again be 

 anesthetized and the protrud- 

 ing skeletal elements trimmed 

 off, even with the soft tissues; 

 otherwise healing may be 

 faulty and the results of the 

 experiment difficult to evaluate. 



Left forelimb of advanced Amblystoma, indicating 

 possible levels of amputation. 



(from Schotte' & Butler '41; Jour. Exp. Zool. 87:297) 



Modified Wijhe method for regenerating cartilage (from V. R. Gregg and E. G. Butler, 

 Princeton University). 



1. Fix 24 hours or more in 5 cc of formalin and 95 cc of 70% alcohol. 



2. Without washing, stain 10-60 minutes in 0. 25% methylene blue to which 

 0. 5% HCl has been added. The time depends upon the size of the limb. 



3. Without washing, destain in 95% alcohol, changing as it becomes discolored", 

 and until no more color comes from the tissue. Handle gently. 



4. Dehydrate in absolute, 3 changes of 1 5 minutes each. 



5. Clear in oil of wintergreen until clear. Can leave in this indefinitely. 



6. Mount whole as follows: 



a. Wash off all excess salicylate with 60 min. in xylol. 



b. Place a drop of (Damar) balsam on slide, warm to almost drying in oven 

 at 50°C. for 30+ minutes. 



c. Transfer stained specimen onto semi-dried balsam drop, add thick bal- 

 sam drop, add thick balsam to cover at 2-4 hour intervals, and leave 

 overnight. 



