Post-Injury Regeneration of Optical Retina in Anuran Amphibians

Though many consider the human eye to be among the most advanced when compared to analogous structures in other organisms, certain properties and capabilities of the amphibian visual system surpass that of higher order animals, despite the fact that by complexity standards, the amphibian eye is relatively crude. Most notably, amphibians possess the ability to regenerate structures of the eye—including the retina and the optic nerve—after suffering significant injury to these areas. This idea of regeneration is not exclusive to amphibians, but they are perhaps the best-studied vertebrates regarding this phenomenon, and amphibian anatomy has long been used as a critical reference when examining our own bodies. Could lowly amphibians pave the way for new breakthroughs in human eye treatments?

The basic anatomy of the amphibian eye. Though cruder than that of humans, they exhibit many of the same structures.The structure of the human eye. Note the similarities to the amphibian eye and the implications this could have on human medicine.

The structure of the human eye. Note the similarities to the amphibian eye and the implications this could have on human medicine.

Retinal Development

As we have known for many decades, like many vertebrates, the retina of amphibians is, at first, part of a larger group of structures composing the optic vesicle, which invaginates to form the optic cup (Gaze 1960). Due to inductive signaling from surrounding tissues, a crucial separation then occurs: the optic cup forms two distinct regions: the retinal pigmented epithelium (RPE) and the neural retina (NR) (Araki 2007). It has been shown that many transcription factors are responsible for the dorsal-ventral polarity of the optic cup, and thus, the patterning of the RPE and NR (Hitchcock et al 2004). Many signaling molecules are responsible for retinal development, and subsequently, its regeneration.

The development of the vertebrate eye. The optic vesicle makes contact with different types of tissues at the dorsal (a), distal (b) and ventral (c) regions. These are the dorsal mesenchyme derived from the cephalic neural crest, the surface epidermis and ventral mesenchyme derived from the procaudal mesoderm, respectively. (B) Schematic drawings of transversal images of the developing eye. The optic vesicle neuroepithelium is subdivided into the dorsal and ventral regions, which later develop into the retinal pigmented epithelium (RPE) and neural retina, respectively. From Araki (2007).

Retinal Transdifferentiation

Transdifferentiation (the event where a cell changes to another kind of cell) is the process that many biologists believe governs the regeneration process (Mitashov 1997). Until recently, it had long been common belief that urodele amphibians (newts) were the only species capable of full retinal regeneration post-metamorphosis—in other words, as adults—while anuran amphibians, such as frogs, were only capable of regeneration in tadpole stages of development. However, recent studies have shown that anuran amphibians are also capable of post-metamorphosis retinal regeneration.

The regeneration of the newt retina. Retinal regeneration at different stages after the surgical removal of the retina is shown in the light micrographs from (A) to (H). The retinal pigment epithelial (RPE) cells appear to be irregularly arranged at day 5 (arrow in E), which then become more epithelial at day 10 (arrow in F). At the same time, RPE cells become depigmented. Then, a multistratified epithelium is formed (large arrow in G) and one pigmented single epithelium is also observed (small arrow in G). About 1 month later, a well stratified retinal tissue and a RPE have developed (arrow in H). Hematoxylin and eosin staining. BrdU labeling study indicates that at day 4 only a few cells in the RPE layer are labeled, while at day 5 most of the RPE cells have become labeled (arrow in J). (I and J are from Ikegami et al. 2002). From Araki (2007).

Retinal Regeneration

Yoshii et al (2007) sought to examine the extent of the regenerative ability of anuran retinas. They surgically removed the lens and neural retinas of 120 young adult frogs of the species Xenopus laevis and observed the surgical sites for extended periods of time following the procedure. Full retinal regeneration was observed in 70% of the subjects, and the full regrowth of the lens was also found to have occurred in many of the frogs. There was no significant difference seen in regeneration between frogs of varying ages. They also noted that in most of the unsuccessful cases were due to the inhibition of growth by the incision closure, which was occluding the vitreous cavity.

Early stages of retinal regeneration of X. laevis subjects (3 months post-metamorphosis). (A, B) Day 7 and (C, D, E) Day 10 after retinectomy. The area indicated by asterisk in (A) is shown in (B) at a higher magnification. Arrows indicate RVM (retinal vascular membrane). (B) Black arrow indicates RVM where pigmented cells form an epithelial monolayer sheet. Blue arrow indicates single pigmented cell located between the retinal pigmented epithelium (RPE) (indicated by yellow arrowhead) and the RVM. (C) The areas indicated by a single and by double asterisks in (C) are shown in (D) and (E) at a higher magnification, respectively. (D) shows regenerating retina (arrow) at the marginal zone, continuous to the iris epithelium. Yellow-colored arrowhead indicates the RPE layer. (E) shows regenerating retina on the RVM located more posteriorly. Black arrows indicate capillaries on RVM and blue arrows indicate pigmented cells located in a narrow space between the newly formed epithelium and RPE layer (yellow arrowhead). A pigmented cell (indicated by the lower left blue arrow) extends from RPE layer to the regenerating epithelium. From Yoshii et al (2007).

Yoshii et al also took a closer look at prepared histological samples of X. laevis eyes that received the same treatment and could be more easily examined. By Day 7 after the surgery, a new layer of RPE had formed facing the original layer in the vitreous chamber (the new layer formed the inner layer). Then, transdifferentiation events seemed to occur, and the new RPE changed into a new neural retina.

Later stages of X. laevis retinal regeneration. (A) Day 15, (B, C) Day 20, (D) Day 40 and (E) Day 30 after retinectomy. (A) By Day 15, lens structures are well recognized with intense eosin staining. Regenerating retinas (arrows) are still thin and are separated from RPE layer. (B) By Day 20, the laminar structure of the regenerating retina is partially developed at the periphery (arrow). The area indicated by asterisk is shown in (C) at a higher magnification. (C) A few RPE cells are found in the space between RPE layer and the retinal epithelium as shown by blue arrows. Some cells extend from RPE layer to the retinal layer (indicated by lower blue arrow). Blue-colored arrowheads indicate pigmented cells attached to the retinal layer. (D) By Day 40 a well-stratified retinal structure develops as shown by an arrow. (E) In some cases neither the retina nor lens regenerates, the eye cavity being occluded by connective tissue derived from the choroid tissue. (F, G) Laminar structure of the regenerating retina at Day 30 is well identified with acetylated tubulin staining. Arrows indicate immunoreactive ganglion cell bodies. G is a Nomarsky differential image of the same area. From Yoshii et al (2007).

Finally, Yoshii et al examined important genetic markers in order to better support the notion that transdifferentiation is the critical mechanism for retinal regeneration. Specifically, they looked at RPE65, a marker found in retinal pigmented cells that is also found in humans. The newly formed inner RPE layer stained positively for RPE65, indicating that these new epithelial cells were actually derived from the original RPE cell layer.

In addition, they examined the gene Pax6, an important gene for retinal regeneration. In a normal, unaltered retina, Pax6 is found only in the ganglion and amacrine cells, but not in other retinal or RPE cells. However, the samples with removed retinas stained positive for Pax6 in both the original and newly formed RPE layers by Day 10.

Furthermore, when doubly stained for both RPE65 and Pax6, both RPE layers stained positive, indicating the likely event of transdifferentiation. Retinal stem cells that originate in the ciliary marginal zone (CMZ); namely, the retinal vascular membrane, which was left unaltered in their subjects, were also found to stain positive for RPE65. Quite notably, the main difference between this study other similar studies conducted on anuran species is that they left the retinal vascular membrane in place, whereas it was removed in all previous studies. This could have led to the propagation of the idea that anuran species could not undergo retinal regeneration post-metamorphosis.

RPE65 and Pax6 staining of regenerating retina on Day 10 after removal of retina. (A, B) Pax6 staining and Nomarsky differential image of normal Xenopus retina. Arrows indicate two layers of positively stained ganglion and amacrine cells. (C, D, E, F) A newly formed pigmented layer (asterisk) and the original RPE layer (arrowhead) at Day 10 after retinectomy. Pigmented cells in both layers are mostly doubly stained for RPE65 and Pax6, as shown by arrows. From Yoshii et al (2007).

Conclusions and Implications

Overall, Yoshii et al found that the original RPE layer and stem cells from the retinal vascular membrane in the CMZ both assist in the formation of a new neural retina in anuran amphibians. The RPE layer undergoes transdifferentiation from RPE cells to NR cells. The exact function of the retinal vascular membrane is undetermined, as is the exact signaling mechanism that causes the migration of its stem cells (though FGF-1 is predicted to play a part).

Schematic diagram for X. laevis retinal regeneration. The upper part in (A) shows retinectomized eye cavity, and the lower shows intact one. Cells from two origins regenerate the retina; ciliary marginal cells and the retinal pigmented epithelial cells. The CMZ (ciliary marginal zone) partially remains after retinectomy with the present surgical procedure, and CMZ stem cells initiate migration on the RVM (retinal vascular membrane) to the posterior direction. (B) At the same time, some of RPE cells leave RPE layer, migrate and attach to the RVM, where they form a new RPE layer as indicated in (C). Numerous capillaries (indicated as C) are seen in RVM. RPE cells on the RVM proliferate and transdifferentiate to neural retinal precursor cells (D, E). RPE cells that were positively stained for RPE65 are shown by brown-colored nuclei or pigmented granules in the cytoplasm. From Yoshii et al (2007).

Research into the regenerative properties of amphibians holds exciting potential for possible human applications. Many researchers believe that humans do not lack regenerative capacity—in fact, humans have been found to have retained many genes involved in regenerative processes—but that they are inactive in their default state. Can these genes and transcription factors be successfully activated? This has yet to be determined, and safety concerns are likely to stall human trials for a long time. Many genes have widespread effects, and it is unlikely that we know the breadth of the impact that the activation of one of these genes could have.

References:

Araki, Masasuke (2007). “Regeneration of the amphibian retina: Role of tissue interaction and related signaling molecules on RPE transdifferentiation.” Development, Growth, and Differentiation 49: 109-120.

Gaze, R.M. (1960). “Regeneration of the Optic Nerve in Amphibia.” International Review of Neurobiology 2: 1-38.

Hitchcock et al (2004). “Persistent and injury-induced neurogenesis in the vertebrate retina.” Progress in Retinal and Eye Research 23: 183-194.

Mitashov, Victor I. (1997). “Retinal regeneration in amphibians.” International Journal of Developmental Biology 41: 893-905.

Yoshii et al (2007). “Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: Transdifferentiation of retinal pigmented epithelium regenerates the neural retina.” Developmental Biology 303(1): 45-56.

Leave a Reply

Your email address will not be published. Required fields are marked *