Lens Regeneration Research Articles

Histological comparison of in vitro and in vivo development of peripheral posterior capsule opacification in human donor tissue

In order to study the mechanisms involved in the development of posterior capsule opacification (PCO) we compared in vivo developed PCO with PCO formed in tissue culture with focus on the periphery of the lens capsule to evaluate lens regeneration potential. We studied three human tissue groups: Cultured lens capsules after mock cataract surgery (n = 6, 30 days), lens capsules from donors that had previously undergone cataract surgery (IOL capsules) (n = 12) and intact lenses (n = 6). All samples were stained with Vimentin, alpha Smooth Muscle Actin, Picro Sirius Red (for collagen) and Paired box protein (Pax6). We found that cultured capsules and less developed IOL capsules consisted mainly of monolayers of mesenchymal cells, while more developed IOL capsules, contained lens epithelial cells (LECs), globular cells and lens fiber cells. Many IOL capsule samples expressed collagen I and III in areas where cells were in contact with the IOL. Pax6 had a similar dispersed distribution in less developed IOL capsules and cultured capsules, while more developed IOL capsules and intact lenses, concentrated Pax6 in LECs at the equatorial lens bow. The similarities between cultured capsules and less developed IOL capsules indicate that our in vitro developed PCO is comparable to early in vivo developed PCO. The similar morphology of more developed IOL capsules and intact lenses seems to indicate an attempt at lens regeneration.

Advances and clinical application of lens regeneration in mammal

Regeneration of the lens is an ideal strategy for cataract patients to restore their adjustable vision and to acquire excellent visual quality. However, mammalian lens regeneration is slow and incomplete, and functional lens regeneration cannot be achieved. The ability of lower amphibians (such as newts) to regenerate the lens provides the impetus for research on the regeneration of the lens of mammal. At present, the main form of mammalian lens regeneration is the differentiation of lens epithelial cells by capsular bag as a scaffold. In recent years, the continuous development of stem cell technology, tissue engineering and biological materials have made great progress in lens regeneration. This article describes the processes in the normal development of the lens, and reviews the research results of lens regeneration at home and abroad, and discusses the possibility of the new techniques and methods related to regenerative medicine applied to lens regeneration. It is hoped to help in the realization of rapid and complete regeneration of functional lens.(Chin J Ophthalmol, 2019, 55: 549-553).

Using the Xenopus Developmental Eye Regrowth System to Distinguish the Role of Developmental Versus Regenerative Mechanisms.

A longstanding challenge in regeneration biology is to understand the role of developmental mechanisms in restoring lost or damaged tissues and organs. As these body structures were built during embryogenesis, it is not surprising that a number of developmental mechanisms are also active during regeneration. However, it remains unclear whether developmental mechanisms act similarly or differently during regeneration as compared to development. Since regeneration is studied in the context of mature, differentiated tissues, it is difficult to evaluate comparative studies with developmental processes due to the latter’s highly proliferative environment. We have taken a more direct approach to study regeneration in a developmental context (regrowth). Xenopus laevis, the African clawed frog, is a well-established model for both embryology and regeneration studies, especially for the eye. Xenopus eye development is well-defined. Xenopus is also an established model for retinal and lens regeneration studies. Previously, we demonstrated that Xenopus tailbud embryo can successfully regrow a functional eye that is morphologically indistinguishable from an age-matched control eye. In this study, we assessed the temporal regulation of retinal differentiation and patterning restoration during eye regrowth. Our findings showed that during regrowth, cellular patterning and retinal layer formation was delayed by approximately 1 day but was restored by 3 days when compared to eye development. An assessment of the differentiation of ganglion cells, photoreceptor cells, and Müller glia indicated that the retinal birth order generated during regrowth was consistent with that observed for eye development. Thus, retina differentiation and patterning during regrowth is similar to endogenous eye development. We used this eye regrowth model to assess the role of known mechanisms in development versus regrowth. Loss-of-function studies showed that Pax6 was required for both eye development and regrowth whereas apoptosis was only required for regrowth. Together, these results revealed that the mechanisms required for both development and regrowth can be distinguished from regrowth-specific ones. Our study highlights this developmental model of eye regrowth as a robust platform to systematically and efficiently define the molecular mechanisms that are required for regeneration versus development.

The role of sensory innervation in cornea-lens regeneration.

Numerous sensory nerves in the cornea contribute to normal tissue homeostasis. Interestingly, cells within the basal corneal epithelium can regenerate new lenses in the frog, Xenopus. In this study, we investigated whether cornea sensory nerves or their neuropeptides are important for supporting cornea-lens regeneration. Attempts to sever the trigeminal nerve trunk, which provides sensory nerve branches to the cornea, did not inhibit lens regeneration. However, using this approach we found that it was not possible to completely disrupt sensory innervation, as these nerves are able to quickly regenerate back to the cornea. On the other hand, attenuation of neuropeptide levels with capsaicin was found to significantly inhibit lens regeneration, as visualized by a reduction of Substance P. These treatments also led to a reduction of cornea sensory innervation. Interestingly, inhibition of the Substance P-preferred receptor NK-1 with Spantide II did not affect lens-regeneration rates. This study provides evidence that cornea nerves support cornea-lens regeneration, which could occur through the release of various neurotrophic factors. Substance P, however, does not appear to be the critical component of this signaling pathway. Further studies are needed to investigate what role other known neurotrophic factors may play in this process.

Aldose reductase inhibition enhances lens regeneration in mice

After cataract surgery, epithelial cells lining the anterior lens capsule can transition to one of two divergent pathways, including fibrosis which leads to posterior capsular opacification (PCO), or lens fiber cell differentiation which leads to regeneration of lens material. We previously showed that the PCO response can be suppressed with aldose reductase (AR) inhibitors. In this present study we show that AR inhibition, both genetic and pharmacologic with Sorbinil, can augment the process of lens regeneration. Extracapsular lens extraction (ECLE) was carried out in C57BL/6 (WT), AR overexpression (AR-Tg), and AR knockout (ARKO) mice, and in some cases in mice treated with the AR inhibitor sorbinil. Whole eyes were harvested approximately 8 weeks after ECLE and evaluated by histological analysis and immunostaining for the fiber cell marker γ-crystallin. All eyes examined for lens regeneration were paraffin embedded for serial sectioning to produce three-dimensional reconstructed models of lens morphology and size. We observed that AR-null mice respond to ECLE by regenerating a lens-like structure with a circular shape and array of cell nuclei reminiscent of the lens bow region typical of the native mammalian lens. Although WT and AR-Tg eyes also produced some regenerated lens material after ECLE, their structures were consistently smaller than ARKO regenerated lenses. WT mice treated with sorbinil showed higher levels of lens regeneration after ECLE compared to WT mice, as assessed by size and three-dimensional morphology. Altogether, this study adds evidence for a critical role for AR in the response of lens epithelial cells to cataract extraction and lens regeneration.

Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina.

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.

Behavior of Stem-Like Cells, Precursors for Tissue Regeneration in Urodela, Under Conditions of Microgravity.

We summarize data from our experiments on stem-like cell-dependent regeneration in amphibians in microgravity. Considering its deleterious effect on many tissues, we asked whether microgravity is compatible with reparative processes, specifically activation and proliferation of source cells. Experiments were conducted using tailed amphibians, which combine profound regenerative capabilities with high robustness, allowing an in vivo study of lens, retina, limb, and tail regeneration in challenging settings of spaceflight. Microgravity promoted stem-like cell proliferation to a varying extent (up to 2-fold), and it seemed to speed up source cell dedifferentiation, as well as sequential differentiation in retina, lens, and limb, leading to formation of bigger and more developed regenerates than in 1g controls. It also promoted proliferation and hypertrophy of Müller glial cells, eliciting a response similar to reactive gliosis. A significant increase in stem-like cell proliferation was mostly beneficial for regeneration and only in rare cases caused moderate tissue growth abnormalities. It is important that microgravity yielded a lasting effect even if applied before operations. We hypothesize on the potential mechanisms of gravity-dependent changes in stem-like cell behavior, including fibroblast growth factor 2 signaling pathway and heat shock proteins, which were affected in our experimental settings. Taken together, our data indicate that microgravity does not disturb the natural regenerative potential of newt stem-like cells, and, depending on the system, even stimulates their dedifferentiation, proliferation, and differentiation. We discuss these data along with publications on mammalian stem cell behavior in vitro and invertebrate regeneration in vivo in microgravity. In vivo data are very scarce and require further research using contemporary methods of cell behavior analysis to elucidate mechanisms of stem cell response to altered gravity. They are relevant for both practical applications, such as managing human reparative responses in spaceflight, and fundamental understanding of stem cell biology.

Potentiality of Lens Regeneration Treatment Using Cells

Potentiality of Lens Regeneration Treatment Using Cells

Methods for Examining Lens Regeneration in Xenopus.

Some vertebrates are able to regenerate the lens following its removal. This includes species in the genus Xenopus (i.e., X. laevis, X. tropicalis, and X. borealis), the only anurans known to undergo lens regeneration. In Xenopus the regenerated lens is derived de novo from cells located within the basal-most layer of the larval corneal epithelium, and is triggered by factors provided by the neural retina. In larval frogs the corneal epithelium is underlain by an endothelium separated from the corneal epithelium except for a small central attachment (i.e., the "stromal-attracting center"). This connection grows larger as the stroma forms and the frogs approach metamorphosis. Here we provide instructions for performing lentectomies (removal of the original lens) to study lens regeneration.

Ex Vivo Eye Tissue Culture Methods for Xenopus.

Lens regeneration can be studied in whole animals following removal of the original lens (lentectomy). However, culturing a whole animal can be impractical for assays involving small molecule inhibitors or proteins. Ex vivo eye tissue culture is an alternative approach for examining lens regeneration. The ex vivo culture system offers certain advantages when compared to the in vivo regeneration assay, as the percentage of cases showing lens differentiation can exceed that seen in whole animals. This culture system also allows for the treatment of eye tissues in small volumes, which helps ensure reproducibility and reduces the amount (and cost) of small-molecule inhibitors or exogenous proteins, etc., necessary to conduct an experiment. Additionally, different eye tissues can be combined, such as nontransgenic and transgenic tissues (e.g., eyecup and cornea) that carry reporters or inducible transgenes. This approach represents a very useful tool in the analysis of lens regeneration or for simply culturing specific eye tissues, and can be used to culture either Xenopus laevis or Xenopus tropicalis eye tissues.

Lens regeneration in children

Lens regeneration in children

Diverse Evolutionary Origins and Mechanisms of Lens Regeneration.

In this review, we compare and contrast the three different forms of vertebrate lens regeneration: Wolffian lens regeneration, cornea-lens regeneration, and lens regeneration from lens epithelial cells. An examination of the diverse cellular origins of these lenses, their unique phylogenetic distribution, and the underlying molecular mechanisms, suggests that these different forms of lens regeneration evolved independently and utilize neither conserved nor convergent mechanisms to regulate these processes.

Tropomyosin 2 heterozygous knockout in mice using CRISPR-Cas9 system displays the inhibition of injury-induced epithelial-mesenchymal transition, and lens opacity

The process of epithelial–mesenchymal transition (EMT) of lens epithelial cells (LECs) after cataract surgery contributes to tissue fibrosis, wound healing and lens regeneration via a mechanism not yet fully understood. Here, we show that tropomyosin 2 (Tpm2) plays a critical role in wound healing and lens aging. Posterior capsular opacification (PCO) after lens extraction surgery was accompanied by elevated expression of Tpm2. Tpm2 heterozygous knockout mice, generated via the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system showed promoted progression of cataract with age. Further, injury-induced EMT of the mouse lens epithelium, as evaluated histologically and by the expression patterns of Tpm1 and Tpm2, was attenuated in the absence of Tpm2. In conclusion, Tpm2 may be important in maintaining lens physiology and morphology. However, Tpm2 is involved in the progression of EMT during the wound healing process of mouse LECs, suggesting that inhibition of Tpm2 may suppress PCO.

Lens regeneration: a historical perspective.

The idea of regenerating injured body parts has captivated human imagination for centuries, and the topic still remains an area of extensive scientific research. This review focuses on the process of lens regeneration: its history, our current knowledge, and the questions that remain unanswered. By highlighting some of the milestones that have shaped our understanding of this phenomenon and the contributions of scientists who have dedicated their lives to investigating these questions, we explore how regeneration enquiry evolved into the science it is today, and how technological advances accelerated our understanding of these remarkable processes.

PD78 Minimally Invasive Capsulorhexis In Children's Cataracts

Introduction:Minimally invasive capsulorhexis is an incision in the anterior capsule in the peripheral zone for cataract extraction. It allows reduction of the size of the lesion, ensuring a better transparency of the visual axis, preserving the capsule almost intact and a layer of lenticular epithelial cells. The procedure could have a potential regenerative effect of the lens in a natural way. The objective of this study is to assess the efficacy and safety of minimally invasive capsulorhexis to promote lens regeneration in children's cataracts.Methods:This technology was identified by the early Awareness and Alert System, “SINTESIS-new technologies” of Agencia de Evaluación de Tecnologías Sanitarias (AETS) Instituto de Salud Carlos III (ISCIII). An early assessment was conducted. The searched databases were: PubMed, Centre for Reviews and Dissemination (CRD), and Cochrane Library. Clinical studies using the procedure published in any language until 29 September 2017 were reviewed.Results:An open-label, randomized trial in pediatric cataract patients (age: 0–2 years) was retrieved. Twelve patients underwent minimally invasive capsulorhexis, while twenty-five patients received the standard treatment. Regarding efficacy, a transparent regenerated biconvex lens was found in 100 percent of eyes three months after surgery, but wasn't found in the control group. 100 percent of capsular openings healed within one month after surgery in the experimental group, but not in the control group. Both groups increased their visual acuity parameters without significant differences. Regarding safety, children receiving the standard technique had a higher incidence of corneal edema (eight percent in the intervention vs thirty percent in the control group), anterior chamber inflammation (seventeen percent vs seventy-four percent), additional laser capsulotomy (zero percent vs eighty-four percent) and increased visual axis opacification (four percent vs eighty-four percent).Conclusions:Minimally invasive capsulorhexis in children's cataracts seems to be a promising new procedure. Preliminary efficacy results were good and safety profile was better than standard treatment. However, it would be necessary to continue further studies to confirm these results.

The Fate of In Situ Lens Regeneration is Determined by Capsulorhexis Size.

Lens regeneration is an optimal strategy for cataract patients to regain visual acuity with accommodation. We recently designed a novel, minimally invasive capsulorhexis surgical method for cataract removal that achieved functional lens regeneration in human infants. However, small anterior capsulorhexis requires advanced surgical expertise. To examine whether the quality of the regenerated lens can be maintained with enlarged anterior capsulorhexis, we investigated the shape and transparency of the regenerated lenses with different anterior capsulorhexis diameters (ACDs). Thirty-six 4-week-old New Zealand albino rabbits were randomly divided into three groups which underwent lens extraction with different ACDs (Group A: 2.0±0.5 mm, Group B: 4.0±0.5 mm, Group C: 6.0±0.5 mm). The anterior capsule opening area (ACOA) was quantified, and the morphology, weight, and histological characteristics of the regenerated lenses were examined. Lens regeneration was observed in all three groups. In Group A, the regenerated lenses were relatively complete and transparent. In Groups B and C, the regenerated lenses were doughnut-shaped and opaque. The speed of lens regeneration in Group A was significantly faster than that in Groups B and C. The ACOA in Group A healed quickly and completely approximately 2 weeks after surgery. However, in Groups B and C, ACOA did not heal completely until 12 weeks after surgery. Histological examination showed that in Group A, most of the lens epithelial cells differentiated into well-organized lens fibers. However, in Groups B and C, the regenerated lens fibers were disorganized. Capsulorhexis size is a critical determinant of integrity and transparency in lens regeneration.

Calcium signaling in human lens epithelial cells after mechanical stimulation

PurposeThe purpose of this study is to explore and identify intra‐ and inter‐cellular calcium (Ca2+) signaling in human lens epithelial cells (LECs) upon local mechanical stimulation, to understand better the role of Ca2+ in intercellular communication related to cataract formation, lens regeneration and posterior capsular opacification (PCO).MethodsThe anterior lens capsule (aLC: basement membrane and associated LECs) were obtained from cataract surgery. Primary human LEC cultures were established by placing adherently the intact human aLC onto Petri dishes. LECs were stained with Fura‐2 dye, the fluorescence of which was imaged to monitor spatio‐temporal changes in cytosolic free Ca2+ concentrations in response to localized, micropipette induced mechanical stimulation.ResultsAnalysis of the intra‐ and inter‐cellular Ca2+ signaling from postoperative aLCs and cultures showed that the Ca2+ signal spreads radially and its propagation speed increases with the degree of cataract. While in aLC Ca2+ signals travels between the first few neighbor LECs in the order of seconds, in confluent culture the propagation is slower and it covers smaller distances. LECs from aLC with less developed cataracts exhibit faster and bigger changes in intracellular Ca2+ concentration. Moreover, in aLC and in LEC cultures, the duration of Ca2+ transients prolongs with increasing distance from stimulation site, whereas their amplitude decreases.ConclusionsThe modifications of Ca2+ homeostasis in LECs, which are associated with different degrees of cataract, affect Ca2+ signaling upon the local mechanical stimulation. Impairment of Ca2+ signaling might be the basis of cataract formation. LEC's cultures have less developed Ca2+ signaling capabilities, what might be reflected on lens regeneration and PCO.

Endogenous bioelectric currents promote differentiation of the mammalian lens.

The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and β1 subunits of Na+/K+‐ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (Vmem). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized Vmem. Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of β‐crystallin, aquaporin‐0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of Vmem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.

An attempt at natural lens regeneration in congenital cataract surgery

Despite recent advances, successful rehabilitation of children with congenital cataracts is hampered by a range of unresolved issues. The purpose of this study was to aquire personal assessment of the successful experience of Lin on 24 eyes that underwent a modified aspiration procedure followed by natural lens regeneration.

Stem cell approach for cataracts challenged.

Lens regeneration after surgery confounds some ophthalmologists.