Effect of Emblica officinalis (Gaertn) on lens regeneration in the frog, Rana cyanophlyctis (Schneider)

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1 Indian Journal of Experimental Biology Vol. 47, March 2009, pp Effect of Emblica officinalis (Gaertn) on lens regeneration in the frog, Rana cyanophlyctis (Schneider) Jayshree Banot, Garima Lata, 0 P Jangir*, Manshi Sharma, Vijay Singh Rathore, S K Saini & Amit Nagai Developmental Biology Laboratory, Department of Zoology, Dungar College, Bikaner , India Received 13 May 2008, revised 27 November 2008 Emblica officinalis (Amla) accelerated cell proliferation and dedifferentiation of pigmented epithelial cells of dorsal iris and consequently induced lens regeneration in R.cyanophlyctis. Further it enhanced the percentage of lens regeneration not only in young tadpoles but also is adult frogs. Lens regeneration ability declined with the age of animals in both control as well as treated groups. Keywords: Emblica officinalis, Frog, Lens regeneration, Tadpole Lens regeneration provides a clear example of transdifferentiation of differentiated cellular type having a distinct pattern of metabolic activities to another cellular type, which is morphologically and biochemically distinct from original. Emblica officinalis (Hindi:Amla) is full of medicinal properties l 2 Ascorbic acid, a constituent of E. officinalis has positive effect on inflammation and wound healing. Ascorbic acid enhances proliferation and dedifferentiation of iris pigmented epithelial cells in culture medium, and also increases the formation of lentoids 3 It is used to treat diabetic cataract, watering of eyes, cough, asthma, bronchitis, peptic ulcer and cardiac disorders 4 Further research of contemporary and traditional medical literature indicates that Emblica either in combination with other herbs or alone has been useful in the amelioration of colds, warts, skin afflictions, influenza, elevated cholestrol and as an immune restorative in cancer conditions. These multifarious medicinal properties of Emblica prompted the study of its effect on lens regeneration in the frog Rana cyanophlyctis. Materials and Methods The experiments were carried out on 180 animals of different developmental stages of Rana cyanophlyctis. Tadpoles of different developmental stages were raised from the spawn of R. cyanophlyctis collected from local ponds during mansoon season. *Correspondent author Telephone: opjangir2003@yahoo.com The lentectomy of anaesthesized animals was done under the stereoscopic binocular microscope. A longitudinal slit was made in the cornea of the right eye extending across the middle of the pupillary space. The lens was extracted through incision (Fig la, b). The left eye was kept intact. Out of these operated animals, 90 were treated with Emblica and remaining 90 were kept untreated and served as control. The mode of treatment was given in two ways depending upon the stage of the animal: (1) Young and mature tadpoles were reared in Emblica solution (5 m1 Emblica extract/500 m1 water). (2) 0.12 m1 Emblica was injected intra-peritoneally into adult frogs. The doses were given on alternate days upto the day of termination of experiment (40 th day after operation). The animals of different stages viz. young and mature tadpoles and adult frogs were categorized into 3 groups: Groups A, Band C The operated animals of both treated and untreated groups were preserved at different time intervals in Bouin's solution for histological evaluation. After 24 to 30 hr of preservation in Bouin's solution, animals were transferred to 70% alcohol for histological slide preparation. Eyes were removed from the preserved animals and dehydrated in alcohol series, cleaned in xylene and embedded in paraffin wax. These were sectioned and stained in haematoxyline and counter stained with eosin.

2 158 INDIAN J EXP BIOL, MARCH 2009 Preparation of Emblica extract - Arn1a powder (50g) was mixed with tapwater and kept for 12 hr. The solution was boiled to make it 250 ml of the whole solution. This extract was treated as standard solution. To make the working solution 5 ml of this standard solution was mixed with 500 ml of tap water. Half of the operated tadpoles (considered as treated group) were reared in the Emblica solution; treated adults were injected 0.12 ml solution of Emblica on alternate days. Results The E. officinalis induced lens regeneration from dorsal iris (Table 1). The results show that lens regenerative power is found in the tadpoles of R. cyanophlyctis however. it declined with the age of the animal. It occurred in 23.3% cases of young three toe stage tadpoles (GroupA, Al) in 16.7% in cases of mature five toe stage tadpoles (Group B, B I) and in 6.7% cases of adult R. cyanophlyctis (Group C, CI). In contrast, Emblica was found to induce and accelerate lens regeneration in different developmental stages of the frog. It occurred in 93.3% cases of young three toe stage tadpoles(group A, A2), in 86.7% cases of mature five toe stage tadpoles (Group B, B2) and 80% in adult frogs(group C, C2) (Table 1). Thus the declining trend of regeneration with age was seen in Emblica treated animals also, The morphological features of regenerated lenses like shape, size and transparency were found similar to that of normal intact lenses. Externally, it was difficult to distinguish the right operated eye (with regenerated lens) from the nonna! intact eye on the day of tennination of experiment, i.e. 40" day. The regenerated lens was found nonna! in its function. which was tested by its nonna! response obtained when intact eye was closed by putting black tape on it. Table 1-Percentage of cases of lens regeneration in untreated (control) and Emblica treared groups young, mature and adult Rana cyanophlyctiss. Stage of animal Sub Groups Day of No. of operated Nanna! No. of Non Regeneration preservation animals preserved Regenerates Lentoids Regenerates (%) Young Tadpoles (3 toe stage) Al 5 5 I 4 OrA (Untreated) 7 5 I I A (Treated) Mature Tadpole (5 toe stage) BI 5 5 I 4 OrB (Untreated) 7 5 I I B (Treated) Adult frog 3 5 Orc CI 5 5 (Untreated) C (Treated) I I I I

3 BANOT er al.: EMBLICA OFFICINA LIS & LENS REGENERATION IN RA NA CYANOPHLYCTls 159 LFC Fig.! (a) - Photograph showing insertion of needle into the right eye of adult frog Raila cyallophlyclis. (b): Newly extracted (Ientectomy) lens (EL) with lens capsule. Fig.2 - Microphotograph of a section passin g through dorsal iris (0 I) of Emblica treated tadpole of R. cyanophlyclis showing depigemcntation of PEes. dorsal iris becomes swollen and knob like. (x 200). Fi g. 3 - Microphotograph of a section passing through lentectomized eye of 3 days Embfica treated tadpole of R. cyallophlycris showing cleft formation in between two laminae of dorsal iris. (x 400). Figs 4,5 - Microphotograph of a section passing through lentectomizcd eye of 5 days Emblica treated tadpole of R. cyallophlyclis showi ng formation of lens vesicle at the tip of dorsal iris. (x 200)

4 160 INDIAN J EX? BIOL, MARCH 2009 The histological changes occurred during lens regeneration in all the three age groups animals were found almost similar. In both, control and Emblica treated animals lens regeneration occurred from pigmented epithelium cells Of dorsal iris. Histological observations revealed that during lens regeneration after lentectomy the two. layers of pigmented epithelium cells of dorsal iris began to thicken and the nuclei of lrls cells changed their shape (Figs 2, 3). The pupillary margin of the iris became knob like. The fonnation of this knob-like structure continued until the free margin became a swollen loop like structure. Scattered mitotic figures were also observed. These changes continued up to day 7 after operation in Emblica treated animals. Then the cells started to dedifferentiate as they threw out their melanosomes. These melanosomes were injested by macrophages that entered the wounded site. Dorsal iris cells continued to divide forming a vesicle-like structure in the region of the removed lens (Figs 4-6). Now the vesicle differentiated into a new regenerated lens (Figs 7, 8). Once the new lens fonned, the cells of dorsal iris ceased mitosis. The newly fonned lens was surrounded by a lens epithelium cells of which cuboidal and slightly taller than before. In addition, lens fiber formation begain in the inner surface of the vesicular lens. Cells began to elongate and entered the lumen of the vesicle. The lumen was filled by primary lens fiber nuclei before the secondary lens fibers began to fonn (Fig. 8). Later, the secondary lens fibers began to differentiate and grow around the central nucleus and the regenerated lens becames a better-defined structure. In the next stage the regenerated lens got detached from the dorsal iris and returned to its nonnal position (Fig. 9). At last, the nuclei of the secondary lens fibers progressively disappeared. Figures 9, 10 show operated eyes of Emblica treated animals with regenerated lens. Figure 10 shows the placement of regenerated lens into nonnal position by day 40 after operation. In untreated operated adult frogs, lens regeneration occurred in only two cases in which a nodulated lentoid structure was found to develop. Histological study revealed that the structure so developed was almost similar in its origin to nonnal lens, originated from dorsal iris in lentectomized iris but was of smaller size. Whereas, in treated adult frogs regenerated l.enses were similar to normal intact lenses. Only in some cases regenerated lenses were oflentoid type. Discussiou The results of the present study showed that E. officinalis induced lens regeneration in not only in young and mature tadpoles but also in adult R.. cyanophlycti, and that lens regeneration capacity declined with the agc of animals; similar trend was also found both in untreated and Emblica treated groups. In both of the groups results clearly show that lens regeneration declined with the age of the animals. Similar trend was found both in untreated and in Emblica treated groups. The mechanism of Emblica effect on lens regeneration is not clear. The literature on this l 'veda drug is very scanty. However, it is reported L'JI ascorbic acid (a constituent of Emblica) has accelerating effect on wound healing ' 2 and enhances proliferation and dedifferentiation of iris pigmented epithelial cells in culture medium'. Thus accelerating effect on lens reb ration in all the developmental stages of the animal R. cyanophlyctis is a peculiar feature observed in the present study. Lens regeneration from non-ocular tissue (dorsal iris) has been well-documented in urodele amphibians"'. However, it has been observed that the ability of iris and retinal pigmented epithelial cell (PECs) to transdifferentiate into lens is not restricted to urodele amphibians but is widely conserved in vertebrates 9. lo Good lens regeneration from PEes in vivo has been seen in a few species of fishes and some other amphibians. Vitamin A is known to induce and accelerate lens regeneration in amphibian tadpoles, froglets, chick embryos and swiss albino mice"'''. In cell culture study, PECs of almost all vertebrates can switch differentiation to acquire the characteristics of Fig. 6 - Microphotograph of a section passing through lentectomized eye of 5 days Emblica treated tadpole showing well defined lens vesicle. (x200). Fig. 7 - Photograph of a hand section passing through the operated eye showing regenerated lens attached with dorsal iris of 7 day Embiica treated tadpole of R. cyanophlyclis. (xlo) Fig. 8 - Microphotograph of section through the eye of 15 days Emblica treated froglet showing newly fonned regenerated lens. Lens epithelium and differentiating lens fibers ean be seen (x200). Fig. 9- Photograph of a 40 days old Ernblica treated tadpole of R. cyanophlyctis, showing well developed regenerated lens in the enucleated eye ball whose lens was extracted on day O. (x5). Fig. 10- Photograph of an operated tadpole ( lententomized) showing nonnallooking eye with regenerated lens. [EL: Extracted lens 01: Dorsal iris, DDC: Dedifferentiated cells, KLS: Knob like structure, LV : Lens vesicle, c: Cornea, RL : Regenerated lens, DLF : Differentiating lens fibers, R : Retina, EWRL : Eye with regenerated lens, CL : Cleft, LFC : Lens forming cells

5 BANOT er al.: EMBLICA OFFICINA LIS & LENS REGENERATION IN RANA CYANOPHLYCTIS 161 R D

6 162 INDIAN J EXP BIOL, MARCH 2009 lens I3 - l6 Pigmented epithelial cells dissociated from fully grown human eyes readily trans-differentiated into lens phenotypes in the manner observed in chick embryo PECs I3 Lentectomy stimulates the iris epithelial cells of the newt's eye to undergo DNA synthesis and proliferate l7 - l9 Concomitantly with these processes, melanosomes disappear from the pigmented epithelial cells of iris and thus undergo dedifferentiation. Similar events have also been reported in present experiment. After completion of the phase of dedifferentiation, some cells retreat from the cell cycle, elongate and proceed to synthesize lens specific proteins and transform into lens fibers l8 l9 It is found that Emblica can enhances dedifferentiation of iris pigmented epithelial cells of tadpoles and adults of R. cyanophlyctis resulting in lens regeneration. This suggests that Emblica officinalis may be usefully employed for investigations of the molecular mechanisms responsible for homeotic transformation. Lens regeneration appears to be a suitable system for such investigation. The discovery opens tqe possibility that researchers may one day enhance the endogenous regenerative capacity of mammals by inducing cellular dedifferentiation in vivo. References Hellman L & Burns J J, Metabolism of L - ascorbic acid-l C14 in man, J Bioi Chem, 230 (1958) Jagetia G C, Rajankant G K & Rao S K, Evaluation of the effect of ascorbic acid treatment on wound healing in mice exposed to different doses of fractionated gamma radiation, Radiat Res, 159 (2003) Kosaka M, Kodama R & Eguchi G, In vitro culture system for iris-pigmented epithelial cells for molecular analysis of transdifferentiation, Exp Cell Res, 245 (1998) 'Suryanarayana P, Kumar P A, Saraswat M. Petrash J M & Reddy G B, Inhibition of aldose reductase by tannoid principles of Emblica officinalis: implications for the prevention of sugar cataract, Mol Vis, 10 (2004) Reyer R W, Regeneration of the lens in the amphibian eye, Q Rev Bioi, 29 (1954) I. 6 Reyer R W, The amphibian eye development and regeneration, in Handbook of sensory physiology~ V. VIlIS P. (1977) Eguchi G & Itoh Y, Regeneration of the lens as a phenomenon of cellular transdifferentiation ; regulability of the differentiated state of the vertebrate pigment epithelial cell, Trans Opthamel Soc, 102 (3) (1982) Eguchi G, Instability in cell commintment of vertebrate pigmented epithelial cells and their transdifferentiation into lens cells, CUTT Top Dev Bioi, 20 (1988) Itoh Y & Eguchi G, In vitro analysis of cellular metaplasia from pigmented epithelial cells to lens phenotypes: A unique model system for studying cellular and molecular mechanisms of "transdifferentiation", Dev Bioi, lis (2) (1986 b) Okada T S, Lens studies continue to provide landmarks of embryology (Developmental Biology) Perspectives, J Bio Sci, 25 (2) (2000) Jangir O.P, Modi & Manshi S. Effect of vitamin A on lens regeneration in pigs, Indian J Exp Bioi, 43 (2005) Shekhawat D S, Jangir 0 P, Acharya P, Suthar, Lens regeneration in mice under the influence of vitamin A, J Biosci, 26 (200 1) Eguchi G & Okada T S, Differentiation of lens tissue from the progeny of chick retinal pigment cells cultured in vitro: A demonstration of a switch of cell types in c10nal cell culture, Proc Natl Acad Sci, 70 (5) (1973) Eguchi G, Transdifferentiation, Transdifferentiation in vertebrate cells in cell culture, (1998) Eisenberg S & Yamada T, A study of DNA synthesis during the transfonnation of the iris into lens in the lentectomized newt, J Exp Zool, 162 (1971) Yamada T & Roesel M E, Activity of DNA replication in the iris epithelium by lens removal, J Exp Zool, 171 (1969) Reyer R W, DNA synthesis and incorporation of labeled iris cells into the lens during lens regeneration in,adult newt, Dev Boil, 24(1971) Yamada T, Cellular synthetic activities in induction of tissue transformation cell differentiation, Ciba Found Symp, (1967) Yamada T Cellular and subcellular events in Wolffian lens regeneration, Curr Top Devlop Bioi, (1967) 247.