EYE FIELD OPERATIONS 299 



WOLFFIAN REGENERATION 



This term Is derived from the work of- Wolff who, in l895, 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 hudding 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. crlstatus. Salaimandra and the Axolotl (Wachs, 191*+); for Triturus 

 viridescens (Stone and Chace, 19^1); for Triturus torosus (Dinnean, 19*<-2); for Amblystoma 

 tigrlnum up to stage #i*-5 (Ballard, I956). It has also been seen in Amblystoma Jefferson- 

 lanum, A. mlcrostomum, and A. opacum as well as in the Japanese fire salamander, Triturus 

 pyrrhogaster. In fact, Triturus (of all species) seems the most reliable, Amblystoma un- 

 dependable, and the Anura generally negative. 



1. Select larvae of A. tigrlnum (up to stage #'+5), A. opacum, or any species of 

 Triturus. The operation should be performed on a minimum of 10 specimens, all 

 of the same age and stage. 



2. Anesthetize the larva in l/5,000 MS 222 (or 0.04^ chloretone) and place it in 

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



5. Fasten the larval body into position with strips of lens paper held to the 

 Permoplast with insect pins. 



h. 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 cornea, 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 lena 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 removed lens under the microscope. Keep the 

 operated larvae in separate, properly marked, finger bowls at controlled tem- 

 peratures. 



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

 signs of regeneration. Note changes in the pupil immediately after the opera- 

 tion and at regular Intervals of 3 to U days thereafter. These changes are 

 indicative of changes in respect to the lens within. 



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

 fixed in 10^ formaldehyde and the eyes dissected. The unoperated eye may be 

 considered 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 1+ to 5 day intervals after the operation. 



There has been considerable discussion, in recent years, about the possibility and 

 the method of lens regeneration in later larval stages (Schotte and Hummel, 1959 and Stone 

 and Sapir, 1914-0). The latter authors investigated Urodeles, Anura, and Fish, all of which 

 were at least 25 and many as much as 80 mm. In length, and they came to the conclusion 

 that at these advanced stages the rim of the iris does not possess the power to regenerate 

 a new lens. In fact, there is evidence of species variation at the earlier larval stages 

 for Stone and Dinnean (1914-0) found no Wolffian regeneration in A. punctatum at any time. 

 Stone and Sapir (191+0) state: "Among the Triturus this unique phenomenon has been proven 

 beyond all doubt. It opens up an interesting field of study to determine more clearly 

 what factors inhibit and release the regeneration of a lens from the rim of the Iris in 

 this group of salamanders." 



