THE ANATOMICAL RECORD 202521-525 (1982)

Comparison of Lens Regeneration in Adult Newts, Triturus viridescens, Between Light and Dark Preadapted and Non-Preadapted Animals

F R A N K CHAN Health Science l k p u r t m e n t . Cd/ege ( I / I l c d r h liclatrtl l’rt~f~~.ssion.s. U’ichitu S ta te Unicvrsity, U’rchittr. Kansas 6‘7208

A B S T R A C T I t is well known that if the original lens is removed from the eye- ball in certain Urodeles, a new lens will regenerate from the dorsal iris. Experi- ments were designed to study the different effects on lens regeneration in the newt, Triturus viridescens, between the light and dark preadapted animals and light and dark non-preadapted animals. The preadapted animals were preadapted to continuous light or dark for 90 days prior to lentectomy. The newts remained in the same controlled environments postlentectomy for 25 days to test the effect of light preadaptation on lens regeneration. The non-preadapted animals were placed in the light controlled environment immediately after lentectomy for a 25-day regeneration period.

The results indicated that there was no difference in the rate of regeneration be- tween continuous light preadapted and non-preadapted animals. However, the dark non-preadapted animals regenerated new lenses a t a much slower rate than the dark preadapted animals.

The rate of lens regeneration was similar among the controls with 12 hours of light per 24-hour period, the dark preadapted, the continuous light preadapted, and continuous light non-preadapted groups.

Many investigators have demonstrated that the rate of lens regeneration in adult newts varies in different light controlled environ- ments. Borsuk and Popov (1968), using Tri- turus taeniatus, suggested that the best condi- tion for lens regeneration is under natural il- lumination with alternation of day and night. Animals under continuous light did not regen- erate their lenses as fast. Animals in complete darkness regenerated their lenses much more slowly than those in the above two conditions. Chan and McCarthy (1980) preadapted Tri- turirs viridescens in complete darkness for 90 days, after which time the lenses were removed and the animals were returned to the same dark environment in order for lens regenera- tion to occur. They found no significant differ- ence in the rate of regeneration between the dark preadapted group and the control animals which received 12 hours of light per 24 hours. However, they did not place a group of animals in the dark immediately after lentectomy in order to see if preadaptation to the dark envi- ronment makes a difference in the capacity of llie animals to regenerate new lenses.

This paper describes an attempt to answer the following questions: (1) Is there any differ- ence in lens regeneration rate between dark preadapted animals and dark non-preadapted animals? (2) Is there any regeneration rate dif- ference between continuous light preadapted animals and continuous light non-preadapted animals? (3) Can animals regenerate their lens faster under controlled strong continuous light?

M A T E R I A L S A N D ME’TIIODS

The experimental animals, adult male and fe- male Triturus viridescens, were purchased through a local pet shop. They were visually selected for uniform size (approximately 2 gm).

The animals were divided into the following five groups. (1) The control group of 25 animals was kept in an aquarium with 12 hours of light per 24-hour period for 90 days. The eyes of these animals were then lentectomized, and they remained in the same environment for another 25 days for lens regeneration to occur.

0003-276X18212024-0521$02.00 S 1982 ALAN R. LISS. INC.

522 F. CHAN

(2) The dark preadapted group of 25 animals was kept in a darkroom equipped with red safe- lights (GE60A21-R). Unexposed film was placed in the room and periodically developed to determine if there was any exposure to light. The room was kept dark except for 5 minutes every other day when the red safelights were on to allow feeding and cleaning of the animals in their aquarium. The animals were kept in complete darkness for 90 days after which time both eyes were lentectomized; a red safelight was used for dissecting microscope illumina- tion. The animals then remained in this dark environment 25 days for their lenses to regen- erate. (3) The dark non-preadapted group of 25 animals was lentectomized and placed immedi- ately in complete darkness for 25 days in order for their lenses to regenerate. (4) The preadapt- ed continuous light group of 25 animals was placed under continuous light for 90 days. The light consisted of four fluorescent light bulbs totaling 864 foot candles in brightness. The light bulbs were approximately 4 inches above the animals. After 90 days the animals were lentectomized in both eyes and returned to the same environment. (5) The continuous light non-preadapted group of 25 animals was len- tectomized and placed immediately under a continuous light condition, as were those in group 4, for 25 days for lens regeneration to occur.

All groups were kept a t an average tempera- ture of 25-26°C. The temperature was checked regularly as temperature has been shown to af- fect the rate of regeneration (Donaldson and Chan, 1973).

The lenses of all animals were removed through a nasotemporal slit in the cornea. The animals were kept in a moist chamber with operating solution (Rose and Rose, 1965) dilu- ted 1:10 for 2 days and thereafter in aquaria in their respective light controlled environments. The anesthetic used was M S 222 (Ethyl M - Aminobenzoate Methanesulfonate).

Five animals in each group were sacrificed at 10, 15, 20, and 25 days postlentectomy. The heads were fixed in Bouin's solution and decal- cified in Decal. Serial cross sections were cut a t 9 pm and stained with Delafield's hematoxylin and eosin.

The regenerates were staged according to Yamada (1977). The regenerate size was deter- mined by averaging corneal-retinal, dorsal- ventral, and cranial-caudal diameters. The first two diameters were measured with an ocular micrometer in a section passing through the center of the regenerate while the last was de-

termined by multiplying the number of sections of the regenerates by 9 pm.

I{ E s u I .'r s The results showed that the rate of regenera-

tion was similar in the control (A), dark pre- adapted (B), continuous light preadapted (D), and continuous light non-preadapted (E) groups. Among them, the continuous light pre- adapted group appeared to regenerate slightly faster and the dark preadapted group was slightly slower. However, the dark non-pre- adapted group was far behind in the regenera- tion stages a t 10, 15, 20, and 25 days of lens regeneration (Table 1).

On the tenth day of regeneration, most of the regenerates in groups A, D, and E were at stage V. In group B, there were five regener- ates a t stage IV and five at stage V. Only the regenerates in group C, the dark non-preadapt- ed group, remained at stage 11. On the 15th day of regeneration, most of the regenerates were either at stage VII or VIII except the dark non-preadapted group which consisted of six regenerates a t stage I11 and two at stage 11. Most of the regenerates in groups A, B, D, and E reached stage X by the 20th day; but in group C, only six regenerates were at stage IV and four a t stage V. On the 25th day of regener- ation, most of the regenerates were at stage XI except those in group C which had nine at stage VI and one at stage VII (Table 1).

I t appeared that the animals in the dark non- preadapted group consistently regenerated new lenses at a much slower rate than the other groups all through the regeneration process. The rate of regeneration was three stages be- hind the others on the 10th day and about four stages behind on the 15th. 20th and 25th days.

Microscopic examination revealed that each 10-day-old regenerate of the controls, dark pre- adapted, continuous light preadapted, and continuous light non-preadapted showed the thickening of the internal layer of the growing lens vesicle by the proliferation of the iris epithelial cells into two cell layers (Fig. 1: A l , B1, D1, E l ) . However, in each of the 10-day-old dark non-preadapted regenerates, the margin of the dorsal iris was slightly enlarged and there was a small space that separated the two cell layers of the iris (Fig. 1: Cl). By the 15th day, each of the regenerates, except those in the dark non-preadapted group, developed lens fiber complex in the internal layer of the dorsal iris. Twenty-nine of them even developed sec- ondary lens fibers in the external layers (Fig. 1: A2, B2, D2, E2). Most of the regenerates of the

LENS REGENERATION IN ADULT NEWTS 523

dark non-preadapted group remained at stage I11 with the appearance of some depigmented cells a t the middorsal margins of the irises (Fig. 1: C2).

By the 20th day of regeneration, 35 eyes were at stage X with the presence of lens epi- thelium and further accumulation of second- ary lens fibers. The lenses were still attached to the dorsal irises (Fig. 1: A3, B3, D3, E3). Five eyes in the dark preadapted group regenerated slightly slower to stage IX with many large lens fiber nuclei in the centers of the lenses (Table 1). Again, the dark non-preadapted group only reached stages IV and V with the early development of lens vesicles and thicken- ing of the internal layers of the lens vesicles (Fig. 1: C3). By the 25th day, the eyes in the controls, dark preadapted, continuous light preadapted and continuous light non-pre- adapted developed perfect lenses and were de- tached from the dorsal irises (Fig. 1: A4, B4, D4, E4). However, the dark non-preadapted group slowly reached stage VI and started to develop secondary lense fibers in the internal layers of the irises in two eyes (Fig. 1: C4).

Except for the dark non-preadapted group, the size of the regenerates for specific days dif- fered very little in all animals. The average size was 191 pm for the 15-day-old regenerates, 320 pm for the 20-day-old regenerates, and 403 pm for the 25-day-old regenerates. Some of the re- generates of the continuous preadapted ani- mals appeared a little larger than those in all the other groups, The average size of the 25th- day regenerates of the dark non-preadapted group was 159 pm. They were smaller than the 15-day-old regenerates of the other groups.

Vacuoles (cataracts) occurred in the regener- ates in all five groups of animals. I t appeared that more cataracts were found in the dark pre- adapted group, especially in the latter stages of the regeneration (Fig. 1: B3, B4).

The survival rate was 100% in the controls, dark preadapted, continuous light preadapted, and continuous light non-preadapted groups. Of the 20 animals placed in dark non-preadapt- ed environment, two died within 15 days. The rest of them survived through the experiment.

DISCUSSION

The experiment shed some light on the ques- tions asked in the introduction of the paper on the rate of lens regeneration in the adult newts, Triturus viridescens, in different light con- trolled environments.

The dark non-preadapted animals regenera- ted their lenses a t a much slower rate than the

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524 F. CHAN

Fig. 1. Lens regeneration under different conditions of il- lumination. ( A ) Control group: 113) dark preadapted group: IC) dark non-preadapted; (D) continuous light preadapted:

IR) continuous light non-preadapted: ( I 1 10 days postlentect- omy: 12) 15 days: (3) 20 days; ( 4 ) 25 days. X 100.

LENS REGENERATION IN ADUTn' NEWTS 525

other groups, which might suggest that the dark environment could retard the process of lens regeneration. Since the delay was about four stages throughout the 25 days of regener- ation, it also suggests that the darkness could affect all stages of cellular dedifferentiation, transformation, and differentiation. However, i t appeared that the darkness could not stop the process completely; if more time were given, the animals could still regenerate per- fect lenses.

The interesting fact was that if these ani- mals were preadapted in the dark for 90 days before lentectomy they seemed to be able to ad- just to the dark environment and be able to re- generate new lenses at normal rate. I t appear- ed that light was not the necessary factor for lens regeneration because the animals could re- generate perfect lenses in total darkness. These findings concurred with the results shown by Maier and Singer (1977) in their ex- periment in limb regeneration. They discov- ered that the blinded (enucleation of eyeball) newts could regenerate their limbs at the same rate as the nonblinded newts. They speculated that the melatonin produced by the pineal gland might affect the rate of limb regenera- tion. The production of prolactin and serotonin could also promote the mitosis in regeneration processes. Since we could not remove the eye- ball from the newts in our experiment, we could not rule out the effect of retinal cells in lens regeneratin. Moreover, it had been well demonstrated that the retinal factor was im- portant in the stimulation of lens regeneration (Stone and Steinitz, 1953; Reyer, 1954).

In the continuous light experiment, the re- sults indicated that there was no significant difference in the rate of regeneration between the continuous light preadapted animals and continuous light non-preadapted animals. The continuous light preadapted animals regener- ated slightly faster in the early stages, but both groups reached stage X on the 20th day and stage XI on the 25th day.

The size of some of the continuous light pre- adapted regenerates was bigger than the non- preadapted regenerates. However, the average

size between the two groups was not signifi- cantly different.

Chan and McCarthy (1980) reported the high fatality rate of the dark adapted animals in the study of lens regeneration in the adult newts. In the present study, the survival rate was ex- cellent for the dark adapted animals. Of the 40 animals placed in the dark, only two died in the first 15 days. This could be because healthier animals were used in this experiment.

In conclusion, the lens regeneration rate was about the same among the light and dark pre- adapted animals. However, the dark non-pre- adapted animals regenerated their new lenses at a much slower rate than the other groups. The control group with alternation of light and dark was not the fastest group in lens regener- ation as suggested by Borsuk and Popov (1968). Increasing the time of light exposure, as in the light preadapted experiment, did not significantly increase the rate of lens regenera- tion as suggested by Maier and Singer (1977) in the limb regeneration study.

ACKNOWLEDGMENTS

This study was supported by PHS grants ROlEY02663-02 and ROlEY02663-03.

LITERATURE CITED

Rorsuk, R.A., and V.V. Popov (1968) Value of light and dark for regeneration of the crystalline lens in Triturirs taenin- tus . Dokl. Acad. Nauk USSR, 180: 1500-1503.

Chan. F.. and J. McCarthy (19801 The effect of prolonged light and dark adaptation on lens regeneration in the adult newt, Triturus uiridescens. Growth, 44: 12-18.

Donaldson. D.H., and F. Chan (1973) The influence of tem- perature on lens regeneration in the adult newt, Triturics uiridescens. Growth, 37: 69-76.

Maier. C.E.. and M. Singer (1977) The effect of light on fore- limb regeneration in thenewt. J . Exp. Zoal..202: 241-244.

Keyer, R. (1954) Regeneration of the lens in the amphibian eye. Q. Rev. Rial.. 29 1-46.

Kose. F.C., and S.M. Rose (1965) The role of normal epi- dermis in recovery of regenerative ability in x-rayed limbs of Triturus. Growth. 29: 261 -393.

Stone, L.S.. and H. Steinitz (1953i'rhe regeneration of lenses in eyes with intact and regenerating retina in adult Tri- f u r u s iliridescens. J. Exp. 2001.. 124: 435-467.

Yarnada. 1'. (1977) Control Mechanism in Cell-Type Conver- sion in Newt Lens Regeneration, S. Karger. New York. p. 126.