EYE FIELD OPERATIONS 291 



3. Make the reciprocal transplant indicated in "2", i. e. , from A. punctatum to 

 A. tigrinum, including the entire optic vesicle and overlying lens ectoderm. 



4. Make a large "U" shaped incision through the ectoderm covering the optic 

 vesicle of A. punctatum at stage #27, with the open end of the "U" (uncut 

 margin) toward the anterior, remaining as a hinge. Deflect this flap of ecto- 

 derm forward, and scoop out the optic vesicle, cutting it at the origin from 

 the brain. Quickly remove the overlying ectoderm from a donor embryo op- 

 tic vesicle (same stage but either A. tigrinum or A. mexicanum) and cut it 

 out without damage. Place it in the previously prepared host region, beneath 

 the flap of host ectoderm. So far as possible attempt to orient the donor 

 optic vesicle in the same position as the original host vesicle. 



WOLFFIAN REGENERATION 



This term is derived from the work of Wolff who, in 1895, found that when he removed 

 the lens from the eye of the European Triton that a new lens would regenerate. Such re- 

 generation is presumed to occur from the dorsal rim of the iris by a budding process and 

 has been demonstrated for a large group of Urodeles, with but few exceptions. Its occur- 

 rence among the Anura is questioned. Among the Urodeles it has been described for 

 Triturus taeniatus, T. cristatus, Salamandra and the Axolotl (Wachs, 1914); for Triturus 

 viridescens (Stone and Chace, 1941); for Triturus torosus (Dinnean, 1942); for Ambly- 

 stoma tigrinum up to stage #43 (Ballard, 1936). It has also been seen in Amblystoma 

 jeffer sonianum, A. microstomum, and A. opacum as well as in the Japanese fire sala- 

 mander, Triturus pyrrhogaster . In fact, Triturus (of all species) seems the most reli- 

 able, Amblystoma undependable, and the Anura generally negative. 



1. Select larvae of A. tigrinum (up to stage #43), A. opacum, or any species of 

 Triturus. The operation should be performed on a minimum of 1 specimens, 

 all of the same age and stage. 



2. Anesthetize the larva in 1/3, 000 MS 222 (or 0. 04% chloretone) and place it in a 

 Permoplast depression made to fit so that the larva lies on its side. 



3. Fasten the larval body into position with strips of lens paper held to the Permo- 

 plast with insect pins. 



4. To remove the lens from the right eye use a sharply-tapered glass needle (strong 

 and pointed) and pierce the cornea at one side and run the needle beneath the cor- 

 nea, across the lens, and out through the far side of the cornea. Avoid injury to 

 the underlying lens. Gently scrape a sharp scalpel against the cornea, over the 

 needle, thereby using the two instruments to cut through the cornea. 



5. The lens is generally glass-clear. After separating the lens from the iris, with 

 the glass needle, pick the lens out with #5 watchmaker's forceps. If this proves 

 difficult, it may be possible to reach under the lens with the glass needle and lift 

 it out. There should be no hemorrhage. Specimens in which the retina is injured 

 should be discarded. The slit in the cornea will close and heal by itself. Healing 

 is most rapid in bicarbonate-free Standard or Growing Medium. Examine the re- 

 moved lens under the microscope. Keep the operated larvae in separate, prop- 

 erly marked, finger bowls at controlled temperatures. 



6. At weekly intervals anesthetize the operated larvae and examine the eye for signs 

 of regeneration. Note changes in the pupil immediately after the operation and 

 at regular intervals of 3 to 4 days thereafter. These changes are indicative of 

 changes in respect to the lens within. 



7. After one raonth, during which the larvae are maximally fed, they should be fixed 

 in 10% formaldehyde and the eyes dissected. The unoperated eye may be consid- 

 ered as the control. Estimate the ratio of the diameter of the lens against the 

 diameter of the eye, for both the control and the operated sides, to determine the 

 degree of regeneration. 



8. If there are abundant larvae, the progress of Wolffian regeneration can be best 

 studied by sectioning the eyes at 4 to 5 day intervals after the operation. 



