Limitations and advances in new treatments and future perspectives of corneal blindness

Limitações e avanços em novos tratamentos e perspectivas futuras na cegueira corneal

Rosalia Antunes-Foschini Leidiane Adriano Adriana de Andrade Batista Murashima Amanda Pires Barbosa Luis Fernando Nominato Lara Cristina Dias Marina Zilio Fantucci Denny Marcos Garcia Monica Alves Eduardo Melani Rocha About the authors

ABSTRACT

This review is intended to describe the therapeutic approaches for corneal blindness, detailing the steps and elements involved in corneal wound healing. It also presents the limitations of the actual surgical and pharmacological strategies used to restore and maintain corneal transparency in terms of long-term survival and geographic coverage. In addition, we critically review the perspectives of anabolic agents, including vitamin A, hormones, growth factors, and novel promitotic and anti-inflammatory modulators, to assist corneal wound healing. We discuss the studies involving nanotechnology, gene therapy, and tissue reengineering as potential future strategies to work solely or in combination with corneal surgery to prevent or revert corneal blindness.

Keywords:
Blindness; Corneal diseases; Corneal transplantation; Genetic therapy; Cell- and tissue-based therapy; Stem cells

RESUMO

O presente trabalho traz uma revisão das abordagens terapêuticas para a cegueira da córnea. O estudo detalha as etapas e os elementos envolvidos na cicatrização da córnea. Ele mostra as limitações das estratégias cirúrgicas e farmacológicas usadas para restaurar e manter a transparência da córnea em termos de sobrevida a longo prazo e alcance geográfico. As perspectivas dos agentes anabólicos, incluindo vitamina A, hormônios, fatores de crescimento e novos moduladores pró-mitóticos e anti-inflamatórios para auxiliar a cicatrização da ferida na córnea, são revisadas criticamente. Aqui, apresentamos estudos envolvendo nanotecnologia, terapia gênica e reengenharia de tecidos como possíveis estratégias futuras para atuar de maneira isolada ou combinada com a cirurgia da córnea para prevenir ou reverter a cegueira corneana.

Descritores:
Cegueira; Doenças da córnea; Transplante de córnea; Terapia genética; Terapia baseada em transplante de células e tecidos; Células-tronco

INTRODUCTION

In the first part of this review, we challenge the common sense of three assumptions concerning corneal blindness and reinforce that a) corneal blindness is not a minor epidemiologic problem; b) although the major causes are predictable, the current prevention measures against corneal blindness are not followed or not effective; and c) corneal transplantation, which is the major therapeutic strategy, is limited in terms of access and long-term effectiveness, which is because approximately 180,000 corneal transplants are performed per year across the world; however,16 million people are blind due to corneal diseases and the average half-life of a corneal transplant is lower than 15 years(11 Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, et al. Global survey of corneal transplantation and eye banking. JAMA Ophthalmol. 2016;134(2):167-73.

2 Garg P, Krishna PV, Stratis AK, Gopinathan U. The value of corneal transplantation in reducing blindness. Eye (Lond). 2005;19(10): 1106-14.

3 Borderie VM, Boelle PY, Touzeau O, Allouch C, Boutboul S, Laroche L. Predicted long-term outcome of corneal transplantation. Ophthalmology. 2009;116(12):2354-60.
-44 Flaxman SR, Bourne RRA, Resnikoff S, Ackland P, Braithwaite T, Cicinelli MV, Das A, Jonas JB, Keeffe J, Kempen JH, Leasher J, Limburg H, Naidoo K, Pesudovs K, Silvester A, Stevens GA, Tahhan N, Wong TY, Taylor HR; Vision Loss Expert Group of the Global Burden of Disease Study. Global causes of blindness and distance vision impairment 1990-2020: a systematic review and meta-analysis. Lancet Glob Health. 2017;5(12):e1221-e1234.5. Comment in: Lancet Glob Health. 2017;5(12):e1164-5.). In the second part, we review alternative therapeutic approaches to corneal transplantation to treat corneal blindness, including the modalities of lamellar keratoplasty, ocular surface reconstruction, and potential novel medications designed to modulate corneal wound healing. For this purpose, we conducted a literature review of recent medical articles. The mechanisms underlying corneal wound healing and therapeutic approaches to prevent or treat corneal blindness were addressed with variable completeness, depending on the uniqueness and relevance, based on an extensive search of the literature. Therefore, in this paper, we intend to offer a review of the state-of-the-art corneal blindness treatment approaches, adding a critical evaluation of the clinical relevance, whenever possible(55 Grant MJ, Booth A. A typology of reviews: an analysis of 14 review types and associated methodologies. Health Info Libr J. 2009;26(2):91-108.). This is justified by the fact that reverting corneal blindness by corneal transplantation is a limited strategy, as mentioned earlier. In summary, this review demonstrates the alternative corneal surgical modalities and their limitations and investigates the perspectives of novel therapeutic strategies for corneal blindness based on the current understanding of corneal wound healing.

Lessons learned from the past

In the XIX century, a Brazilian ophthalmologist, Gama Lobo, reported about a disease in four children, slave descendants, with infections involving the lungs, mucosal tissues, and eyes. The children were very thin and weak and cried without producing tears. He named the new disease as Ophthalmia Braziliana and hypothesized that it was caused by eating few meals or being deprived of essential nutrients in the food(66 Gama Lobo M da. Da ophthalmia braziliana (About the Brasilian ophthalmia). Gaz Méd Lisboa. 1865;28(17):466-9.). In 1934, after the discovery of vitamin A, Mellanby demonstrated that rats deprived of this vitamin for 10 days showed an absence of tears, corneal melting, and, just as importantly, degeneration of trigeminal ganglions (TGs)(77 Mellanby E. Xerophthalmia, trigeminal nerve degeneration and vitamin A deficiency. J Pathol Bacteriol. 1934;38(3):391-407.,88 Wolbach SB, Howe PR. Tissue changes following deprivation of fat-soluble a vitamin. J Exp Med. 1925;42(6):753-77.). Several items must have coincided for an eventual lethal outcome in the patients described by Gama Lobo, but a key nutritional element required for vision, corneal integrity, and body health was found to be vitamin A or retinoic acid, and the condition associated with its deprivation is known as keratomalacia(66 Gama Lobo M da. Da ophthalmia braziliana (About the Brasilian ophthalmia). Gaz Méd Lisboa. 1865;28(17):466-9.,88 Wolbach SB, Howe PR. Tissue changes following deprivation of fat-soluble a vitamin. J Exp Med. 1925;42(6):753-77.

9 Sommer A. Xerophthalmia and vitamin A status. Prog Retin Eye Res. 1998;17(1):9-31.
-1010 Sommer A, Green WR, Kenyon KR. Clinicohistopathologic correlations in xerophthalmic ulceration and necrosis. Arch Ophthalmol. 1982;100(6):953-63.).

Vitamin A supports not only the corneal tissue but also the lacrimal functional unit (LFU) that protects the cornea(1111 Stern ME, Gao J, Siemasko KF, Beuerman RW, Pflugfelder SC. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res. 2004;78(3):409-16.). Vitamin A deprivation may be a health problem in the XXI century, whereas the application of this nutrient could be an adjuvant topical anabolic therapy for corneal wound healing, indicating two hypotheses that require further investigation(1212 Faustino JF, Ribeiro-Silva A, Dalto RF, Souza MM, Furtado JM, Rocha GM, et al. Vitamin A and the eye: an old tale for modern times. Arq Bras Oftalmol. 2016;79(1):56-61.,1313 Kim EC, Choi JS, Joo CK. A comparison of vitamin a and cyclosporine a 0.05% eye drops for treatment of dry eye syndrome. Am J Ophthalmol. 2009;147(2):206-13 e3.).

In Sweden, the ophthalmologist Henrik Sjögren described a series of 19 female patients with inflammation of the ocular surface as having tear deficiency, dry mouth, and, in some cases, polyarthralgia. He termed this condition as keratoconjuctivitis sicca, and decades later, it was redefined as a systemic disease named after him as Sjögren’s syndrome (SS)(1414 Sjogren H. Keratoconjunctivitis sicca. In: Trans Ophthalmol Section Swedish Med Assoc, 1929-1931. Acta Ophthalmol. 1932;10:405-6.,1515 Murube J. The first definition of Sjogren's syndrome. Ocul Surf. 2010;8(3):101-10.). SS is one of the most common autoimmune diseases worldwide(1616 Qin B, Wang J, Yang Z, Yang M, Ma N, Huang F, et al. Epidemiology of primary Sjögren's syndrome: A systematic review and meta-analysis. Ann Rheum Dis. 2015;74(1):1983-9.,1717 Sanchez-Guerrero J, Perez-Dosal MR, Cardenas-Velazquez F, Pérez-Reguera A, Celis-Aguilar E, Soto-Rojas AE, et al. Prevalence of Sjogren's syndrome in ambulatory patients according to the American-European Consensus Group criteria. Rheumatology (Oxford). 2005;44(2):235-40.). The etiology of this disease remains unknown, and no possible cures have yet been developed(1818 Garcia-Carrasco M, Fuentes-Alexandro S, Escarcega RO, Salgado G, Riebeling C, Cervera R. Pathophysiology of Sjogren's syndrome. Arch Med Res. 2006;37(8):921-32.,1919 Nikolov NP, Illei GG. Pathogenesis of Sjogren's syndrome. Curr Opin Rheumatol. 2009;21(5):465-70.). However, several studies have clarified that inflammatory events occurring in the ocular surface and in the lacrimal gland (LG) and tear deficiency are associated with hormonal status and the state of the neural network, confirming the model of LFU(1111 Stern ME, Gao J, Siemasko KF, Beuerman RW, Pflugfelder SC. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res. 2004;78(3):409-16.,2020 Tracey KJ. Physiology and immunology of the cholinergic antiinflammatory pathway. J Clin Invest. 2007;117(2):289-96.,2121 Ferrari G, Bignami F, Giacomini C, Capitolo E, Comi G, Chabane L, et al. Ocular surface injury induces inflammation in the brain: in vivo and ex vivo evidence of a corneal-trigeminal axis. Invest Ophthalmol Vis Sci. 2014;55(10):6289-300.). Furthermore, in severe cases, SS can induce corneal melting or opacity per se (2222 Tuli SS, Schultz GS, Downer DM. Science and strategy for preventing and managing corneal ulceration. Ocul Surf. 2007;5(1):23-39.

23 Kenyon KR. Decision-making in the therapy of external eye disease: noninfected corneal ulcers. Ophthalmology. 1982;89(1):44-51.
-2424 Gudas PP Jr., Altman B, Nicholson DH, Green WR. Corneal perforations in Sjogren syndrome. Arch Ophthalmol. 1973;90(6):470-2.). Since its first description, a clear aspect about SS is its predominance in women and the role of sex hormones in its physiopathology, emphasizing the prospect of the therapeutic use of androgens and other anabolic hormones for ocular surface diseases(2525 Sullivan DA, Rocha EM, Aragona P, Clayton JÁ, Ding J, Golebiowski B, et al. TFOS DEWS II Sex, gender, and hormones report. Ocul Surf. 2017;15(3):284-333.).

The above-described lessons teach us two points; first, the neural network integrates the cornea and the LG by the sensorial and autonomic nerves in the LFU. It maintains the constitutive and regulated exocrine secretion, including anabolic agents such as hormones, vitamin A, and growth factors, which are crucial for corneal integrity and homeostasis. Second, the anabolic agents and growth factors present in the LFU are useful in the therapeutic approaches to prevent or treat corneal blindness.

Corneal wound healing mechanisms

To understand the role of growth factors and anabolic agents in preventive and therapeutic approaches for corneal diseases, it would be helpful to review the steps and the players involved in the process of corneal wound healing. The cornea is a transparent organ in front of the eye, with a spherical toroidal or aspheric format and an average central thickness of 520 µm and an average peripheral thickness of 650 µm. Although it possesses such a fragile profile, being almost 90% transparent and typically composed of water, it works as a shield for the eye globe(2626 Pepose JL. The cornea. In: adler FH, Hart WM, eds. Adler's Physiology of the Eye: clinical application. 9th ed. St. Louis: Mosby Year Book; 1992.). The protective role provided by the tear film is broadly recognized and described as deficient in keratomalacia, SS, and children’s dry eye, where tear deficiency is an early manifestation and the outcome is corneal opacity or perforation(2222 Tuli SS, Schultz GS, Downer DM. Science and strategy for preventing and managing corneal ulceration. Ocul Surf. 2007;5(1):23-39.,2727 Lemp MA, Wolfley DE. The lacrimal apparatus. In: In: adler FH, Hart WM, eds. Adler's Physiology of the Eye, 9th ed. St. Louis: MosbyYear Book; 1992.

28 Velasco Cruz A, Attié-Castro F, Fernandes S, Cortes JF, Pierre-Filho PT, Rocha EM, et al. Adult blindness secondary to vitamin A deficiency associated with an eating disorder. Nutrition. 2005;21(5):630-3.
-2929 Mac Cord Medina F, Silvestre de Castro R, Leite SC, Rocha EM, Rocha GM. Management of dry eye related to systemic diseases in childhood and longterm follow-up. Acta Ophthalmol Scand. 2007;85(7):739-44.).

Corneal restoration during wound healing exhibits the following five properties: a) avascularity, b) high sensitivity, c) epithelial renewal supported by limbal stem cells, d) a distinct corneal layer wound response, and e) cross-talk between the cornea and the LFU(3030 Tseng SC, Tsubota K. Important concepts for treating ocular surface and tear disorders. Am J Ophthalmol. 1997;124(6):825-35.

31 Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suther T, et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature. 2006;443(7114):993-7.
-3232 Mergler S, Valtink M, Takayoshi S, Okada Y, Miyajima M, Saika S, et al. Temperature-sensitive transient receptor potential channels in corneal tissue layers and cells. Ophthalmic Res. 2014;52(3):151-9.).

Corneal wound healing can be divided into three phases(3333 Agrawal VB, Tsai RJ. Corneal epithelial wound healing. Indian J Ophthalmol. 2003;51(1):5-15.,3434 Zieske JD, Gibbons IK. Agents that affect corneal wound healing: modulation of structure and function. In: Albert DM, Jakobiec FA, eds. Principles and Practice of Ophthalmlogy : basic sciences. Philadelphia: WB Saunders; 1994.) (Figure 1). In the first step, the hyperacute phase, the cornea loses mass and integrity. The proinflammatory storm is characterized by the secretion of chemotactic factors. Corneal necrosis and clearance occur by collagenolytic destruction and leukocyte permeability and attraction. The symptoms in this phrase include pain and blurred vision. The process is initiated in the first 12 h and may last approximately 7 days, with ocular surface inflammation (redness, tearing, and discomfort) and opacity and the wound being covered by fibrinoid material, building a matrix for the second phase(3333 Agrawal VB, Tsai RJ. Corneal epithelial wound healing. Indian J Ophthalmol. 2003;51(1):5-15.,3535 Azar DT, Gipson IK. Repair of the corneal epithelial adhesion structures following keratectomy wounds in diabetic rabbits. Acta Ophthalmol. Suppl 1989;192:72-9.

36 Lu L, Reinach PS, Kao WW. Corneal epithelial wound healing. Exp Biol Med (Maywood). 2001;226(7):653-64.
-3737 Ormerod LD, Garsd A, Reddy CV, Abelson MB, Kenyon KR. Dynamics of corneal epithelial healing after an alkali burn. A statistical analysis. Invest Ophthalmol Vis Sci. 1989;30(8):1784-93.).

Figure 1
Schematic and clinical illustration of the three phases of the corneal wound healing process.

The second, subacute phase occurs between an average of 7 to 21 days after the trauma. This phase can be identified using typical biomarkers, viz., keratocyte and epithelial cell proliferation. The inflammatory signs are milder, and anabolic and growth factors and anti-inflammatory cytokines comprise the predominant early mediators of inflammation(3838 Wilson SE, Liu JJ, Mohan RR. Stromal-epithelial interactions in the cornea. Prog Retin Eye Res. 1999;18(3):293-309.

39 Hardarson T, Hanson C, Claesson M, Stenevi U. Time-lapse recordings of human corneal epithelial healing. Acta Ophthalmol Scand. 2004;82(2):184-8.
-4040 Zieske JD. Extracellular matrix and wound healing. Curr Opin Ophthalmol. 2001;12(4):237-41.).

In this phase, the adjacent healthy epithelial cells lose the structures that make them a compact and interconnected layer (tight junctions and hemidesmosomes) and migrate to cover the wound. These corneal epithelial cells provide paracrine secretion, produced by the epithelial cells or filtered from the tear film that are now regulated to carry anabolic agents and growth factors to induce the extracelluar matrix reconstitution(3838 Wilson SE, Liu JJ, Mohan RR. Stromal-epithelial interactions in the cornea. Prog Retin Eye Res. 1999;18(3):293-309.,4141 Zhou L, Beuerman RW, Huang L, Barathi A, Foo YH, Li SF, et al. Proteomic analysis of rabbit tear fluid: defensin levels after an experimental corneal wound are correlated to wound closure. Proteomics. 2007;7(17):3194-206.). This process induces keratocyte mitosis and dedifferentiation in myofibroblasts or fibroblasts, depending on the interactions between cytokines and growth factors(4242 Hassell JR, Birk DE. The molecular basis of corneal transparency. Exp Eye Res. 2010;91(3):326-35.). Fibroblast growth factor-2 (FGF-2) is associated with cell proliferation in the wounded cornea, and transforming growth Factor-β (TGF-β) is associated with the synthesis of the fibrotic extracellular matrix and keratocyte dedifferentiation, which induces faster and stronger, but also more opaque, corneal scars(4343 Etheredge L, Kane BP, Hassell JR. The effect of growth factor signaling on keratocytes in vitro and its relationship to the phases of stromal wound repair. Invest Ophthalmol Vis Sci. 2009;50(7):3128-36.). Insulin-like growth factors I and II (IGF-I/II) and also insulin in pharmacological levels are capable of synthesizing collagen and proteoglycan, combining the elements into a more organized extracellular matrix, resulting in a more transparent stroma(4242 Hassell JR, Birk DE. The molecular basis of corneal transparency. Exp Eye Res. 2010;91(3):326-35.,4444 Musselmann K, Alexandrou B, Kane B, Hassell JR. Maintenance of the keratocyte phenotype during cell proliferation stimulated by insulin. J Biol Chem. 2005;280(38):32634-9.). During this phase, the inflammatory signs and symptoms reduce gradually and the visual symptoms of visual haze and glare persist.

The third phase is initiated by the 3rd week and lasts for several months and is characterized by extracellular matrix tissue remodeling and homeostasis recovery, including transparency, surface regularity, and the shielding function of the cornea, thus consolidating the healing process. This phase includes edema reduction, collagen secretion, and restoration of nerve fibers, basal membrane, intercellular channels, and epithelial cells. It is marked by symptom attenuation and visual acuity improvement(3333 Agrawal VB, Tsai RJ. Corneal epithelial wound healing. Indian J Ophthalmol. 2003;51(1):5-15.,4545 Ljubimov AV, Saghizadeh M. Progress in corneal wound healing. Prog Retin Eye Res. 2015;49:17-45.).

The outcome is dependent on the severity and persistency of the aggression and a combination of external and systemic factors(3333 Agrawal VB, Tsai RJ. Corneal epithelial wound healing. Indian J Ophthalmol. 2003;51(1):5-15.,3434 Zieske JD, Gibbons IK. Agents that affect corneal wound healing: modulation of structure and function. In: Albert DM, Jakobiec FA, eds. Principles and Practice of Ophthalmlogy : basic sciences. Philadelphia: WB Saunders; 1994.,4545 Ljubimov AV, Saghizadeh M. Progress in corneal wound healing. Prog Retin Eye Res. 2015;49:17-45.). In the first phase, poor outcomes include progressive stromal erosion, perforation, and corneal melting. In the second and third phases, the process may result in intense and deep opacity, neovascularization, and altered neural network replacement (Figure 2). In these cases, loss of the optic function of the cornea and persistent pain and inflammation are observed in the clinical setting(3333 Agrawal VB, Tsai RJ. Corneal epithelial wound healing. Indian J Ophthalmol. 2003;51(1):5-15.,4646 Philipp W, Speicher L, Humpel C. Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas. Invest Ophthalmol Vis Sci. 2000;41(9):2514-22.).

Figure 2
Quadrant representation of the progression of corneal wound healing with one favorable (homeostatic) and two unfavorable outcomes (opaque scar or melting and perforation).

These unfavorable outcomes are present in several diseases and also account for the prevalence of corneal opacity and blindness (Figure 3).

Figure 3
Illustration of corneal opacity: A) direct observation, B) slit lamp image of a corneal scar with neovascularization, and c) the presence of an epithelial defect limited by fluorescein staining. Although none are favorable for vision improvement, both distinct outcomes can occur (i.e., neovascularization and scarring versus chronic epithelium defects and corneal ulceration), although the reasons and mechanisms for their differences are unknown.

Alternatives to corneal transplantation and novel treatments for corneal opacity and their limitations

In this section, we review the surgical alternatives to fix corneal diseases that cause changes in its shape and transparency. The alternatives range from the less invasive and preventive techniques to the most invasive and applied in severe cases. In the second part, we review the currently available options in topical drug therapy.

Surgical alternatives to corneal transplantation

Changes in corneal shape, also known as ectasia, can cause blindness, which does not necessarily result in corneal opacity but induces blindness due to severe refractive problems. Keratoconus is the major type of ectasia whose frequency in the population varies from 0.4 to 86 cases per 100,000 inhabitants(4747 Gokhale NS. Epidemiology of keratoconus. Indian J Ophthalmol. 2013;61(8):382-3.). The cause of keratoconus is unknown, but it is probably multifactorial. Although keratoconus does not frequently induce corneal opacity or neovascularization, it disturbs the curvature, and biomechanical properties of the cornea, potentially leading to bilateral visual impairment and blindness, making it one of the most frequent reasons for corneal transplantation(4848 Saad A, Gatinel D. Topographic and tomographic properties of forme fruste keratoconus corneas. Invest Ophthalmol Vis Sci. 2010;51(11):5546-55.,4949 Sibley D, Hopkinson CL, Tuft SJ, Kaye SB, Larkin DFP; National Health Service Blood and transplant ocular tissue advisory group and contributing ophthalmologists (OTAG Study 26). Differential effects of primary disease and corneal vascularisation on corneal transplant rejection and survival. Br J Ophthalmol. 2020;104(5):729-734.). Briefly, conservative treatments include glasses, hard contact lenses, and intrastromal corneal rings, before its severity reaches the need for corneal transplantation. All these treatments are capable of reverting the blindness caused by keratoconus, and more recently, the corneal crosslinking induced by ultraviolet light and riboflavin (vitamin B 2) topical application is being advocated as a strategy to prevent the progression of keratoconus. Despite the high prevalence and the impact of keratoconus on the patient’s life, access to these treatments is hindered by the economic and technological barriers. Furthermore, their long-term efficacy and stability are modest, considering that the disease manifests at a young age and the need for lifetime support(5050 Rebenitsch RL, Kymes SM, Walline JJ, Gordon MO. The lifetime economic burden of keratoconus: a decision analysis using a markov model. Am J Ophthalmol. 2011;151(5):768-73 e2.,5151 McGhee CN, Kim BZ, Wilson PJ. Contemporary treatment paradigms in keratoconus. Cornea. 2015;34(10):S16-23.).

Alternative techniques to penetrating transplantation

Lamellar corneal transplantations, replacing only the altered layers, constitute a group of growing alternatives to penetrant keratoplasty (PK). These types of transplantations were conceived by Barraquer in Colombia in the 1960s(5252 Barraquer JI. Lamellar keratoplasty. (Special techniques). Ann Ophthalmol. 1972;4(6):437-69.). Currently, both the anterior (deep anterior lamellar keratoplasty, DALK) and the posterior modalities, Descemet’s membrane endothelial keratoplasty (DMEK) and its variations, of these lamellar techniques are in use and replacing PK in the majority of referral centers throughout the world(5353 Reddy JC, Hammersmith KM, Nagra PK, Rapuano CJ. The role of penetrating keratoplasty in the era of selective lamellar keratoplasty. Int Ophthalmol Clin. 2013;53(2):91-101.

54 Shimazaki J, Ishii N, Shinzawa M, Yamaguchi T, Shimazaki-Den S, Satake Y. How much progress has been made in corneal transplantation? Cornea. 2015;34 Suppl 11:S105-11.

55 Godefrooij DA, Gans R, Imhof SM, Wisse RP. Trends in penetrating and anterior lamellar corneal grafting techniques for keratoconus: A national registry study. Acta Ophthalmol. 2016;94(5):489-93.
-5656 Coster DJ, Lowe MT, Keane MC, Williams KA; Australian Corneal Graft Registry Contributors. A comparison of lamellar and penetrating keratoplasty outcomes: a registry study. Ophthalmology. 2014;121(5):979-87.). Clinical trials and meta-analysis conducted till date have demonstrated similar outcomes and prognoses of PK compared to those of lamellar corneal transplantations for treating common corneal diseases, such as pseudophakic bullous keratopathy (PBK), and the same challenges of PK: inflammation and vascularization(4949 Sibley D, Hopkinson CL, Tuft SJ, Kaye SB, Larkin DFP; National Health Service Blood and transplant ocular tissue advisory group and contributing ophthalmologists (OTAG Study 26). Differential effects of primary disease and corneal vascularisation on corneal transplant rejection and survival. Br J Ophthalmol. 2020;104(5):729-734.,5555 Godefrooij DA, Gans R, Imhof SM, Wisse RP. Trends in penetrating and anterior lamellar corneal grafting techniques for keratoconus: A national registry study. Acta Ophthalmol. 2016;94(5):489-93.,5757 Nanavaty MA, Wang X, Shortt AJ. Endothelial keratoplasty versus penetrating keratoplasty for Fuchs endothelial dystrophy. Cochrane Database Syst Rev. 2014(2):CD008420.,5858 Keane M, Coster D, Ziaei M, Williams K. Deep anterior lamellar keratoplasty versus penetrating keratoplasty for treating keratoconus. Cochrane Database Syst Rev. 2014(7):CD009700.). Studies have also reported promising results for endothelial lamellar corneal transplantations compared with PK in terms of visual acuity, final refractive error, less invasiveness, graft survival, and recovery period(5959 Greenrod EB, Jones MN, Kaye S, Larkin DF; National Health Service Blood and transplant ocular tissue advisory group and contributing ophthalmologists (ocular tissue advisory group audit study 16). Center and surgeon effect on outcomes of endothelial keratoplasty versus penetrating keratoplasty in the United Kingdom. Am J Ophthalmol. 2014;158(5):957-66.,6060 Ang M, Soh Y, Htoon HM, Mehta JS, Tan D. Five-year graft survival comparing descemet stripping automated endothelial keratoplasty and penetrating keratoplasty. Ophthalmology. 2016;123(8):1646-52.). These modalities replace the corneal endothelium that does not regenerate spontaneously in humans. However, endothelial lamellar corneal transplantations cannot overcome the two major limitations in reverting corneal opacity and blindness at the population level, i.e., the scarcity of corneas for grafting and the dependence on highly specialized centers to provide the treatment(5959 Greenrod EB, Jones MN, Kaye S, Larkin DF; National Health Service Blood and transplant ocular tissue advisory group and contributing ophthalmologists (ocular tissue advisory group audit study 16). Center and surgeon effect on outcomes of endothelial keratoplasty versus penetrating keratoplasty in the United Kingdom. Am J Ophthalmol. 2014;158(5):957-66.,6060 Ang M, Soh Y, Htoon HM, Mehta JS, Tan D. Five-year graft survival comparing descemet stripping automated endothelial keratoplasty and penetrating keratoplasty. Ophthalmology. 2016;123(8):1646-52.).

Ocular surface reconstruction and keratoprosthesis

The pioneering studies of Thoft and Friend conducted during the 1970s opened the possibility of promoting the epithelial regeneration of the cornea(6161 Thoft RA, Friend J. The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci. 1983;24(10):1442-3.). The concepts developed from their studies were translated and applied to ocular surface reconstruction for critical cases involving neovascularization, fibrosis, and limbal deficiency(6262 Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology. 1989;96(5):709-22.

63 Kinoshita S, Kiorpes TC, Friend J, Thoft RA. Limbal epithelium in ocular surface wound healing. Invest Ophthalmol Vis Sci. 1982; 23(1):73-80.

64 Kwitko S, Marinho D, Barcaro S, Bocaccio F, Rymer S, Fernandes S, et al. Allograft conjunctival transplantation for bilateral ocular surface disorders. Ophthalmology. 1995;102(7):1020-5.

65 Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349(9057):990-3.

66 Azuara-Blanco A, Pillai CT, Dua HS. Amniotic membrane transplantation for ocular surface reconstruction. Br J Ophthalmol. 1999;83(4):399-402.
-6767 Tsubota K, Toda I, Saito H, Shinozaki N, Shimazaki J. Reconstruction of the corneal epithelium by limbal allograft transplantation for severe ocular surface disorders. Ophthalmology. 1995;102(10):1486-96.). In addition, the usefulness of the amniotic membrane and ex vivo corneal limbal epithelial stem cell expansion was demonstrated in further studies(6868 Tsubota K, Satake Y, Kaido M, Shinozaki N, Shimmura S, Bissen-Miyajima H. Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation. N Engl J Med. 1999; 340(22):1697-703.,6969 Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv Ophthalmol. 2000;44(5):415-25.).

At the same institution where Thoft and Friend started the project for ocular surface reconstruction, but a little early, Doane et al. initiated studies to produce a keratoprosthesis capable of replacing the central cornea, which can be applied in very severe cases of ocular surface scarring with anatomical and functional damage(7070 Doane MG, Dohlman CH, Bearse G. Fabrication of a keratoprosthesis. Cornea. 1996;15(2):179-84.).

Both ocular surface reconstruction and its alternative for treating corneal blindness in the most severe cases of surface scarring, the keratoprosthesis, gained technological adjustments and resources in the past few decades. Besides the necessary training and sophisticated equipment, these strategies had limited survival curves in terms of maintaining transparency, visual acuity, or eye globe integrity(7171 Santos MS, Gomes JA, Hofling-Lima AL, Rizzo LV, Romano AC, Belfort JR. R. Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency. Am J Ophthalmol. 2005;140(2):223-30.

72 Scocco C, Kwitko S, Rymer S, Marinho D, Bocaccio F, Lindenmeyer R. HLA-matched living-related conjunctival limbal allograft for bilateral ocular surface disorders: long-term results. Arq Bras Oftalmol. 2008;71(6):781-7.

73 Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363(2):147-55.

74 Basu S, Ali H, Sangwan VS. Clinical outcomes of repeat autologous cultivated limbal epithelial transplantation for ocular surface burns. Am J Ophthalmol. 2012;153(4):643-50.
-7575 Cauchi PA, Ang GS, Azuara-Blanco A, Burr JM. A systematic literature review of surgical interventions for limbal stem cell deficiency in humans. Am J Ophthalmol. 2008;146(2):251-9.). The survival curve of the Boston keratoprosthesis indicated a half-life of 3 years, and that of a limbal transplant from an allogeneic donor was less than 1 year(7171 Santos MS, Gomes JA, Hofling-Lima AL, Rizzo LV, Romano AC, Belfort JR. R. Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency. Am J Ophthalmol. 2005;140(2):223-30.

72 Scocco C, Kwitko S, Rymer S, Marinho D, Bocaccio F, Lindenmeyer R. HLA-matched living-related conjunctival limbal allograft for bilateral ocular surface disorders: long-term results. Arq Bras Oftalmol. 2008;71(6):781-7.
-7373 Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363(2):147-55.,7676 Stolz AP, Kwitko S, Dal Pizzol MM, Marinho D, Rymer S. Experience with Dohlman-Doane keratoprosthesis: case reports. Arq Bras Oftalmol. 2008;71(2):257-61.,7777 Sayegh RR, Ang LP, Foster CS, Dohlman CH. The Boston keratoprosthesis in Stevens-Johnson syndrome. Am J Ophthalmol. 2008;145(3):438-44.). Therefore, ocular surface reconstruction with allogeneic limbal stem cell transplantation and keratoprosthesis for reverting corneal blindness are hampered by limited survival. However, when autologous limbal transplant was possible from grafts obtained from the healthy contralateral eye, the survival was longer than 5 years in more than 80% of the cases(7373 Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363(2):147-55.). Nevertheless, autologous limbal transplant is much less common in clinical practice because of its much less requirement. Transplantation of cultivated limbal epithelial cells and small limbal autologous transplants have been used to avoid the limitations of the scarcity of autologous epithelial stem cells in the damaged cornea or the immune reactions of allogeneic cells; however, again, these techniques are restricted to higher technology centers and selected cases of ocular surface diseases and still require long-term analysis(7878 Sangwan VS, Basu S, MacNeil S, Balasubramanian D. Simple limbal epithelial transplantation (SLET): a novel surgical technique for the treatment of unilateral limbal stem cell deficiency. Br J Ophthalmol. 2012;96(7):931-4.

79 Pellegrini G, Rama P, Di Rocco A, Panaras A, De Luca M. Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine. Stem Cells. 2014;32(1):26-34.

80 Basu S, Sureka SP, Shanbhag SS, Kethiri AR, Singh V, Sangwan VS. Simple limbal epithelial transplantation: long-term clinical outcomes in 125 cases of unilateral chronic ocular surface burns. Ophthalmology. 2016;123(5):1000-10.
-8181 Fernandez-Buenaga R, Aiello F, Zaher SS, Grixti A, Ahmad S. Twenty years of limbal epithelial therapy: an update on managing limbal stem cell deficiency. BMJ Open Ophthalmol. 2018; 3(1):e000164.).

Corneal neurotization and other grafting strategies

As indicated previously (Part I), corneal trophism and avascularity are typically sustained by robust corneal innervation(8282 Bonini S, Rama P, Olzi D, Lambiase A. Neurotrophic keratitis. Eye (Lond). 2003;17(8):989-95.

83 Ferrari G, Hajrasouliha AR, Sadrai Z, Ueno H, Chauhan SK, Dana R. Nerves and neovessels inhibit each other in the cornea. Invest Ophthalmol Vis Sci. 2013;54(1):813-20.
-8484 Labetoulle M, Baudouin C, Calonge M, Merayo-Llves J, Boborides KG, Akova YA, et al. Role of corneal nerves in ocular surface homeostasis and disease. Acta Ophthalmol. 2019;97(2):137-45.). Loss of innervation leads to fragility in corneal transparency(8383 Ferrari G, Hajrasouliha AR, Sadrai Z, Ueno H, Chauhan SK, Dana R. Nerves and neovessels inhibit each other in the cornea. Invest Ophthalmol Vis Sci. 2013;54(1):813-20.). In this context, a surgical technique (neurotization/neurotisation, as it appears with both spellings in the medical literature) has been proposed to restore corneal innervation and revert neurotrophic keratitis(8585 Wolkow N, Habib LA, Yoon MK, Freitag SK. Corneal neurotization: review of a new surgical approach and its developments. Semin Ophthalmol. 2019:34(7-8):473-87.). Neurotization is a surgical procedure in which autologous nerve tissue grafting between the neurotrophic cornea and the peripheral nervous system is intended to restore corneal sensation(8686 Catapano J, Fung SS, Halliday W, Jobst C, Cheyne D, Ho ES, et al. Treatment of neurotrophic keratopathy with minimally invasive corneal neurotisation: long-term clinical outcomes and evidence of corneal reinnervation. Br J Ophthalmol. 2019;103(12):1724-31.

87 Malhotra R, Elalfy MS, Kannan R, Nduka C, Hamada S. Update on corneal neurotisation. Br J Ophthalmol. 2019;103(1):26-35.

88 Jowett N, Pineda Ii R. Corneal neurotisation by great auricular nerve transfer and scleral-corneal tunnel incisions for neurotrophic keratopathy. Br J Ophthalmol. 2019;103(9):1235-8.
-8989 Ting DS, Figueiredo GS, Henein C, Barnes E, Ahmed O, Mudhar HS, et al. Corneal neurotization for neurotrophic keratopathy: clinical outcomes and in vivo confocal microscopic and histopathological findings. Cornea. 2018;37(5):641-6.).

Other grafting strategies involve the salivary gland duct transposing to the OS or minor salivary gland grafts to the orbital cavity to provide basal biological fluid to regenerate and sustain the epithelial surface(9090 Geerling G, Borrelli M. Adnexal surgery for severe ocular surface disease. Semin Ophthalmol. 2005;20(2):101-12.

91 Geerling G, Sieg P. Transplantation of the major salivary glands. Dev Ophthalmol. 2008;41:255-68.

92 MacLeod AM, Robbins SP. Submandibular gland transfer in the correction of dry eye. Aust N Z J Ophthalmol. 1992;20(2):99-103.

93 Sant' Anna AE, Hazarbassanov RM, de Freitas D, Gomes JA. Minor salivary glands and labial mucous membrane graft in the treatment of severe symblepharon and dry eye in patients with Stevens-Johnson syndrome. Br J Ophthalmol. 2012;96(2):234-9.
-9494 Wakamatsu TH, Sant'Anna AE, Cristovam PC, Alves VA, Wakamatsu A, Gomes JA. Minor salivary gland transplantation for severe dry eyes. Cornea. 2017;36(1):S26-S33.). However, the confidence in this surgical strategy to revert corneal blindness is limited by the lack of controlled trials and long-term results.

Topical drug therapy for corneal blindness

The major pharmacological strategies used to prevent and treat corneal blindness as a single or adjuvant treatment include anti-inflammatory, anabolic and growth factors, and neurotrophic and neurotransmitter analogs.

Topical corticosteroids are hazardous options in cases of corneal infection, severe inflammation, and delayed wound healing(9595 Bian F, Pelegrino FS, Henriksson JT, Pflugfelder S, Volpe EA, Li DQ, et al. Differential effects of dexamethasone and doxycycline on inflammation and MMP production in murine alkali-burned corneas associated with dry eye. Ocul Surf. 2016;14(2):242-54.

96 Mirabelli P, Peebo BB, Xeroudaki M, Koulikovska M, Lagali N. Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model. Exp Eye Res. 2014;125:118-27.

97 Donshik PC, Berman MB, Dohlman CH, Gage J, Rose J. Effect of topical corticosteroids on ulceration in alkali-burned corneas. Arch Ophthalmol. 1978;96(11):2117-20.
-9898 Renfro L, Snow JS. Ocular effects of topical and systemic steroids. Dermatol Clin. 1992;10(3):505-12.). However, topical corticosteroids are still the best choice to prevent corneal transplant rejection and subsequent failure(9999 Jabbehdari S, Rafii AB, Yazdanpanah G, Hamrah P, Holland EJ, Djalilian AR. Update on the management of high-risk penetrating keratoplasty. Curr Ophthalmol Rep. 2017;5(1):38-48.). Corticosteroids modulate inflammatory cytokines, thereby reducing neovascularization and opacity(9595 Bian F, Pelegrino FS, Henriksson JT, Pflugfelder S, Volpe EA, Li DQ, et al. Differential effects of dexamethasone and doxycycline on inflammation and MMP production in murine alkali-burned corneas associated with dry eye. Ocul Surf. 2016;14(2):242-54.). Therefore, excluding the contraindications, corticosteroids remain the gold standard adjuvant therapy for modulating corneal wound healing.

Among the natural biological fluids with anabolic, lubricant, and nutritional properties for treating corneal diseases and promoting wound healing in the most severe cases are the autologous serum (AS) and platelet-rich plasma (PRP)(100100 Fox RI, Chan R, Michelson JB, Belmont JB, Michelson PE. Beneficial effect of artificial tears made with autologous serum in patients with keratoconjunctivitis sicca. Arthritis Rheum. 1984; 27(4):459-61.

101 Schulze SD, Sekundo W, Kroll P. Autologous serum for the treatment of corneal epithelial abrasions in diabetic patients undergoing vitrectomy. Am J Ophthalmol 2006;142(2):207-11.

102 Yoon KC, Heo H, Im SK, You IC, Kim YH, Park YG. Comparison of autologous serum and umbilical cord serum eye drops for dry eye syndrome. Am J Ophthalmol 2007;144(1):86-92.

103 Young AL, Cheng AC, Ng HK, Cheg LL, Leung GY, Clam DS. The use of autologous serum tears in persistent corneal epithelial defects. Eye (Lond). 2004;18(6):609-14.
-104104 Bennett JE. The management of total xerophthalmia. Trans Am Ophthalmol Soc. 1968;66:503-29.). However, due to the lack of similar comparative parameters for analyzing the outcomes of several studies together and the short duration of most of the clinical trials, it is not possible to conclude that any of the abovementioned fluids are superior therapeutic strategies(105105 Pan Q, Angelina A, Marrone M, Stark WJ, Akpek EK. Autologous serum eye drops for dry eye. Cochrane Database Syst Rev. 2017; 28(2):CD009327.,106106 Sharma N, Goel M, Velpandian T, Titiyal JS, Tandon R, Vajpayee RB. Evaluation of umbilical cord serum therapy in acute ocular chemical burns. Invest Ophthalmol Vis Sci. 2011;52(2):1087-92.).

The topical use of recombinant nerve growth factor eye drops to restore the neural network in neurotrophic keratitis has been investigated for several years and was recently approved for commercial use as Oxervate® (Cenegermin)(107107 Lambiase A, Rama P, Bonini S, Caprioglio G, Aloe L. Topical treatment with nerve growth factor for corneal neurotrophic ulcers. N Engl J Med. 1998;338(17):1174-80. Comment in: N Engl J Med. 1998;338(17):1223-3.

108 Bremond-Gignac D, Daruich A, Robert MP, Chiambaretta F. Recent innovations with drugs in clinical trials for neurotrophic keratitis and refractory corneal ulcers. Expert Opin Investig Drugs. 2019;28(11):1013-20.
-109109 Pflugfelder SC, Massaro-Giordano M, Perez VL, Hamrah P, Deng SX, Espandar L, et al. Topical recombinant human nerve growth factor (cenegermin) for neurotrophic keratopathy: A multicenter randomized vehicle-controlled Pivotal Trial. Ophthalmology. 2020;127(1):14-26.). The other topical medication is ReGeneraTing Agent (RGTA)®, a tissue protector that mimics the extracellular matrix and speeds up the corneal wound healing process in refractory conditions by binding with healing agents and protecting against lytic enzymes(108108 Bremond-Gignac D, Daruich A, Robert MP, Chiambaretta F. Recent innovations with drugs in clinical trials for neurotrophic keratitis and refractory corneal ulcers. Expert Opin Investig Drugs. 2019;28(11):1013-20.). Based on the limited and short-term controlled observations, the variability of the surgical techniques and the short 8 weeks of observations of the topical therapies (Cenegermin and RGTA), these approaches are being received with caution, and the reports indicate that further studies are required in terms of neurotrophic keratitis, which, as previously mentioned, is one of the most challenging causes of corneal neovascularization and opacity and where corneal transplantation has a very limited prognosis(4949 Sibley D, Hopkinson CL, Tuft SJ, Kaye SB, Larkin DFP; National Health Service Blood and transplant ocular tissue advisory group and contributing ophthalmologists (OTAG Study 26). Differential effects of primary disease and corneal vascularisation on corneal transplant rejection and survival. Br J Ophthalmol. 2020;104(5):729-734.,109109 Pflugfelder SC, Massaro-Giordano M, Perez VL, Hamrah P, Deng SX, Espandar L, et al. Topical recombinant human nerve growth factor (cenegermin) for neurotrophic keratopathy: A multicenter randomized vehicle-controlled Pivotal Trial. Ophthalmology. 2020;127(1):14-26.

110 Fleeman N, Mahon J, Nevitt S, et al. Cenegermin for treating neurotrophic keratitis: an evidence review group perspective of a NICE single technology appraisal. Pharmacoecon Open. 2019; 3(4):453-61.
-111111 Koaik M, Baig K. Corneal neurotization. Curr Opin Ophthalmol. 2019;30(4):292-8.).

The abovementioned descriptions indicate that corneal blindness, and its various causes, cannot be largely reverted by PK or its surgical alternatives in combination with or replaced by adjuvant drug therapy in terms of large-scale or long-lasting strategies(11 Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, et al. Global survey of corneal transplantation and eye banking. JAMA Ophthalmol. 2016;134(2):167-73.,22 Garg P, Krishna PV, Stratis AK, Gopinathan U. The value of corneal transplantation in reducing blindness. Eye (Lond). 2005;19(10): 1106-14.,5757 Nanavaty MA, Wang X, Shortt AJ. Endothelial keratoplasty versus penetrating keratoplasty for Fuchs endothelial dystrophy. Cochrane Database Syst Rev. 2014(2):CD008420.,112112 Balarabe AH, Mahmoud AO, Ayanniyi AA. The Sokoto blind beggars: causes of blindness and barriers to rehabilitation services. Middle East Afr J Ophthalmol. 2014;21(2):147-52.

113 Hong J, Shi W, Liu Z, Pineda R, Cui X, Sun X, et al. Limitations of keratoplasty in China: A survey analysis. PLOS ONE. 2015; 10(7):e0132268.
-114114 Lamm V, Hara H, Mammen A, Dhaliwal D, Cooper DK. Corneal blindness and xenotransplantation. Xenotransplantation. 2014; 21(2):99-114.) (Table 1). The lessons learned from the past as mentioned above (Session 2) indicate that vitamin A deficiency is probably not just a cause of dry eye and corneal melting but also disrupts the neural network, which is a crucial support for corneal integrity and still extremely difficult to restore with the current therapeutic strategies as discussed above. Furthermore, in conditions where the tear film is missing (dry eye), not just dryness but also suppression of the protective mediators present in tears, including growth factors and hormones, results in delay or induces a scarring corneal wound healing.

Table 1
Human primer sequences used for real-time PCR

Future perspectives of corneal blindness: drugs, cell genetic reprogramming, tissue reengineering, and combined strategies

After identifying that treatment is not simple or widely accessible and that the cure is not possible in several cases due to the time restrictions of the treatments, it is necessary to identify the pathophysiological events associated with corneal opacity. Destruction of the cornea occurs in one of the following two ways: a) melting and perforation caused due to inflammation and necrosis and/or b) scarring and neovascularization caused due to denervation. Depending on the intensity of each process, it may cause corneal damage to one of the poles (ulceration or neovascularization) or restrict it somewhere between the two (Figure 3). Therefore, inflammation and denervation are the events that need to be reverted to prevent corneal blindness.

The present knowledge about the therapeutic options to assist corneal wound healing to prevent or revert corneal blindness is detailed below in the following topics: a) regenerative drugs (growth factors and hormonal agents); b) novel analgesic and anti-inflammatory drugs delivered as eye drops or using c) nanotechnology; d) cell genetic reprogramming (e.g., viral vector gene transfer or other strategies of gene therapy) of the cornea or its natural delivery system, the LG; e) tissue reengineering (e.g., combined allogeneic transplantation, including embryonic tissues); and combined approaches.

Regenerative drugs

Sex and other hormones are involved in the maintenance of the cornea and the ocular surface and in the response to diseases(2525 Sullivan DA, Rocha EM, Aragona P, Clayton JÁ, Ding J, Golebiowski B, et al. TFOS DEWS II Sex, gender, and hormones report. Ocul Surf. 2017;15(3):284-333.). Estrogens elevate the inflammatory response in the LGs of female individuals compared to that in male individuals of several species(136136 Sullivan DA, Sato EH. Potential therapeutic approach for the hormonal treatment of lacrimal gland dysfunction in Sjogren's syndrome. Clin Immunol Immunopathol. 1992;64(1):9-16.). In contrast, androgens, insulin, and other hormones exert anti-inflammatory and anabolic effects on the cornea and LGs(2525 Sullivan DA, Rocha EM, Aragona P, Clayton JÁ, Ding J, Golebiowski B, et al. TFOS DEWS II Sex, gender, and hormones report. Ocul Surf. 2017;15(3):284-333.). Diseases involving the absence or impaired action of hormones that risk compromising the transparency and integrity of the cornea include diabetes mellitus (DM) and thyroid autoimmune disease, among others(2525 Sullivan DA, Rocha EM, Aragona P, Clayton JÁ, Ding J, Golebiowski B, et al. TFOS DEWS II Sex, gender, and hormones report. Ocul Surf. 2017;15(3):284-333.,137137 Rocha EM, Mantelli F, Nominato LF, Bonini S. Hormones and dry eye syndrome: an update on what we do and don't know. Curr Opin Ophthalmol. 2013;24(4):348-55.). Therefore, the therapeutic use of hormones may assist the process of corneal wound healing and restore the ocular surface homeostasis.

The anabolic effects of growth factors, such as NGF and IGF-I, and hormones, such as insulin and androgen topical therapy, include improvement of tear secretion and reduction of the duration of ulcers(109109 Pflugfelder SC, Massaro-Giordano M, Perez VL, Hamrah P, Deng SX, Espandar L, et al. Topical recombinant human nerve growth factor (cenegermin) for neurotrophic keratopathy: A multicenter randomized vehicle-controlled Pivotal Trial. Ophthalmology. 2020;127(1):14-26.,138138 Yanai R, Nishida T, Chikama T, Morishige N, Yamada N, Sonoda KH. Potential new modes of treatment of neurotrophic keratopathy. Cornea. 2015;34 Suppl 11:S121-7.

139 Rocha EM, Cotrim AP, Zheng C, Riveros PP, Baum BJ, Chiorini JÁ. Recovery of radiation-induced dry eye and corneal damage by pre treatment with adenoviral vector-mediated transfer of erythropoietin to the salivary glands in mice. Hum Gene Ther. 2013;24(4):417-23.

140 Abdelkader H, Patel DV, McGhee C, Alany RG. New therapeutic approaches in the treatment of diabetic keratopathy: A review. Clin Exp Ophthalmol. 2011;39(3):259-70.

141 Saragas S, Arffa R, Rabin B, Kronish J, Miller D, Mayman C. Reversal of wound strength retardation by addition of insulin to corticosteroid therapy. Ann Ophthalmol. 1985;17(7):428-30.
-142142 Sullivan DA, Sullivan BD, Ullman MD, Rocha EM, Krenzer KL, Cermak JM, et al. Androgen influence on the meibomian gland. Invest Ophthalmol Vis Sci. 2000;41(12):3732-42.). Of interest, the healthy LG is not only a target but also has the capacity to produce and secrete growth factors and hormones such as insulin and convert testosterone into a more powerful hormone, dihydrotestosterone, by type 1 and 2 5-alfa-reductase(143143 Rocha EM, Wickham LA, da Silveira LA, Krenzer KL, Yu FS, et al. Identification of androgen receptor protein and 5 alpha-reductase mRNA in human ocular tissues. Br J Ophthalmol. 2000;84(1):76-84.,144144 Cunha DA, de Alves MC, Stoppiglia LF, Jorge AG, Módulo CM, Carneiro EM, et al. Extra-pancreatic insulin production in RAt lachrymal gland after streptozotocin-induced islet beta-cells destruction. Biochim Biophys Acta. 2007;1770(8):1128-35.).

The conceptual support for using insulin as a topical corneal therapy is based on the observation that DM induces neurotrophic keratopathy and causes slower wound healing, lower tear secretion, and changes in the cornea and LG structures(145145 Azar DT, Spurr-Michaud SJ, Tisdale AS, Gipson IK. Altered epithelial-basement membrane interactions in diabetic corneas. Arch Ophthalmol. 1992;110(4):537-40.

146 Dogru M. Tear secretion and tear film function in insulin dependent diabetics. Br J Ophthalmol. 2000;84(10):1210.
-147147 Modulo CM, Jorge AG, Dias AC, Braz AM, Bertazolli-Filho R, Jordão AA, et al. Influence of insulin treatment on the lacrimal gland and ocular surface of diabetic rats. Endocrine. 2009;36(1):161-8.). Insulin deprivation leads to LG malfunction and corneal damage, and it has been observed that topical or systemic insulin replacement can restore tear flow and the corneal structure in diabetic human and animal models(148148 Rocha EM, Lima MH, Carvalho CR, Saada MJ, Velloso LA. Characterization of the insulin-signaling pathway in lacrimal and salivary glands of rats. Curr Eye Res. 2000;21(5):833-42.

149 Rocha E, Cunha D, Carneiro E, Boschero AC, Saad MJ, Velloso LA. Identification of insulin in the tear film and insulin receptor and IGF-I receptor on the human ocular surface. Invest Ophthalmol Vis Sci. 2002;43(4):963-7.

150 Cruz-Cazarim ELC, Cazarim MS, Ogunjimi AT, Petrilli R, Rocha EM, Lopez RV. Prospective insulin-based ophthalmic delivery systems for the treatment of dry eye syndrome and corneal injuries. Eur J Pharm Biopharm. 2019;140:1-10.

151 Dias AC, Modulo CM, Jorge AG, Braz AM, Jordão Jr. AA, Bertazolli Filho R, et al. Influence of thyroid hormone on thyroid hormone receptor beta-1 expression and lacrimal gland and ocular surface morphology. Invest Ophthalmol Vis Sci. 2007;48(7):3038-42.
-152152 Zagon IS, Klocek MS, Sassani JW, McLaughlin PJ. Use of topical insulin to normalize corneal epithelial healing in diabetes mellitus. Arch Ophthalmol. 2007;125(8):1082-8.).

As mentioned in section 3, insulin has a corneal wound healing property compared with keratocytes that is not as rapid as that exhibited by growth factors, including IGF1, but is less scarring(141141 Saragas S, Arffa R, Rabin B, Kronish J, Miller D, Mayman C. Reversal of wound strength retardation by addition of insulin to corticosteroid therapy. Ann Ophthalmol. 1985;17(7):428-30.,153153 Yanai R, Yamada N, Inui M, Nishida T. Correlation of proliferative and anti-apoptotic effects of HGF, insulin, IGF-1, IGF-2, and EGF in SV40-transformed human corneal epithelial cells. Exp Eye Res. 2006;83(1):76-83.). Studies have suggested that insulin could be used as a supportive treatment to prevent corneal diseases in diabetic subjects and as a potential promoter of corneal wound healing in patients with dry eye disease(152152 Zagon IS, Klocek MS, Sassani JW, McLaughlin PJ. Use of topical insulin to normalize corneal epithelial healing in diabetes mellitus. Arch Ophthalmol. 2007;125(8):1082-8.,153153 Yanai R, Yamada N, Inui M, Nishida T. Correlation of proliferative and anti-apoptotic effects of HGF, insulin, IGF-1, IGF-2, and EGF in SV40-transformed human corneal epithelial cells. Exp Eye Res. 2006;83(1):76-83.).

Studies conducted using diabetic animal models have demonstrated that insulin topical therapy could improve neurotrophic corneal ulcers and dry eye disease; however, a recent clinical trial in humans revealed that insulin topical therapy showed similar outcomes as those of artificial tears after 4 weeks of treatment(150150 Cruz-Cazarim ELC, Cazarim MS, Ogunjimi AT, Petrilli R, Rocha EM, Lopez RV. Prospective insulin-based ophthalmic delivery systems for the treatment of dry eye syndrome and corneal injuries. Eur J Pharm Biopharm. 2019;140:1-10.,154154 Wang AL, Weinlander E, Metcalf BM, Barney NP, Gamm DM, Nehls SM, et al. Use of topical insulin to treat refractory neurotrophic corneal ulcers. Cornea. 2017;36(11):1426-8.,155155 Aniah Azmi N, Bastion MC. Short-term results of trial of topical insulin for treatment of dry eyes in diabetics. Eye Contact Lens 2020;46(1):S25-32.). The limiting aspects pertaining to the storage and delivery of the small and unstable insulin peptide to the ocular surface have been addressed using nanotechnology, where the number of microparticles, stably enveloping the therapeutic molecule, and the time to modulate the wound healing process can lead to a promising strategy for treating corneal diseases and dry eye disease(150150 Cruz-Cazarim ELC, Cazarim MS, Ogunjimi AT, Petrilli R, Rocha EM, Lopez RV. Prospective insulin-based ophthalmic delivery systems for the treatment of dry eye syndrome and corneal injuries. Eur J Pharm Biopharm. 2019;140:1-10.,156156 Lopez RF, Rocha EM, Cruz EL, Cazarim MS. Microparticles, methods of production, ophthalmic composition, and use. Brazil, 2015. BR 1020150058560. In: Paulo UoS, ed. Revista de Propriedade Industrial. Brazil: Souza, M.A., 2015;v.).

In the inner face of the cornea, the topical use of Rho kinase inhibitors restored endothelial pump function and reduced edema in PBK when used as a single or adjuvant treatment in combination with various modalities of deep lamellar corneal transplantation(157157 Macsai MS, Shiloach M. Use of topical rho kinase inhibitors in the treatment of fuchs dystrophy after descemet stripping only. Cornea. 2019;38(5):529-34.,158158 Kinoshita S, Koizumi N, Ueno M, Okumura N, Imai K, Tanaka H, Yamamoto L, et al. Injection of cultured cells with a ROCK inhibitor for bullous keratopathy. N Engl J Med. 2018;378(11):995-1003.). This topical corneal treatment increased the endothelial cell density and was able to minimize the waiting period for a corneal transplant and replaced it with lower risk procedures(157157 Macsai MS, Shiloach M. Use of topical rho kinase inhibitors in the treatment of fuchs dystrophy after descemet stripping only. Cornea. 2019;38(5):529-34.).

Novel analgesic and anti-inflammatory drugs

Recent studies have demonstrated that cannabinoid analogs can reduce pain sensations and leukocyte migration to corneas burned with silver nitrate(159159 Yang Y, Yang H, Wang Z, Varadaraj K, Kumari SS, Mergler S, et al. Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury. Cell Signal. 2013;25(2):501-11.,160160 Thapa D, Cairns EA, Szczesniak AM, Togueri JT, Caldwell MD, Kelly ME. The Cannabinoids Delta(8)THC, CBD, and HU-308 Act via distinct receptors to reduce corneal pain and inflammation. Cannabis Cannabinoid Res. 2018;3(1):11-20.). As these outcomes were shown to be comparable or superior to those of topical corticosteroids in reducing corneal pain, inflammation, and opacity without causing the side effects of ocular hypertension and corneal toxicity associated with topical steroids and nonsteroidal anti-inflammatory drugs (NSAIDs), cannabinoid analogs could be considered as a useful adjuvant corneal topical therapy that require further studies(2222 Tuli SS, Schultz GS, Downer DM. Science and strategy for preventing and managing corneal ulceration. Ocul Surf. 2007;5(1):23-39.). Of interest, in 2020, the Brazilian Health Surveillance Agency approved the therapeutic use of cannabidiol for treating refractory diseases, including neuropathic pain. Other alternatives that can be used to inhibit corneal inflammation and pain include transient receptor of potential vanilloid-1 (TRPV-1) antagonists, such as resiniferatoxin, whose analgesic effects have been confirmed, and it also did not slow down the process of corneal wound healing in animal studies by blocking the sodium/calcium channels(161161 Bates BD, Mitchell K, Keller JM, Chan CC, Swaim WD, Yaskovich E, et al. Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist. Pain. 2010;149(3):522-8.).

Nanotechnology

There are several examples where delivery systems and microenvironment packing therapeutic molecules can render them more stable and available in the ocular tissue. Earlier, we mentioned the example of insulin topical therapy, although several other molecules are being designed and tested(150150 Cruz-Cazarim ELC, Cazarim MS, Ogunjimi AT, Petrilli R, Rocha EM, Lopez RV. Prospective insulin-based ophthalmic delivery systems for the treatment of dry eye syndrome and corneal injuries. Eur J Pharm Biopharm. 2019;140:1-10.,162162 Souza JG, Dias K, Pereira TA, Bernardi DS, Lopez RF. Topical delivery of ocular therapeutics: carrier systems and physical methods. J Pharm Pharmacol. 2014;66(4):507-30.). Another example is fungal keratitis (FK), a neglected disease (Orpha: 519930), which is strongly related to corneal trauma and has limited treatment options and poor prognosis(163163 Ibrahim MM, Vanini R, Ibrahim FM, Fioriti LS, Furlan EM, ProvinzanoM, Castro RS, et al. Epidemiologic aspects and clinical outcome of fungal keratitis in southeastern Brazil. Eur J Ophthalmol. 2009;19(3):355-61.,164164 Ibrahim MM, Vanini RV, Ibrahim FM, Martins WP, Carvalho RT, Castro RS, et al. Epidemiology and medical prediction of microbial keratitis in southeast Brazil. Arq Bras Oftalmol. 2011;74(1):7-12.). FK therapy can also benefit from nanotechnology, where chitosan solutions or chitosan/poloxamer gel preparations for formulating the antifungal fluconazole, available for systemic use, can be an option for topical use, with corneal permeability and a sustained presence at the target sites(165165 Gratieri T, Gelfuso GM, de Freitas O, Rocha EM, Lopez RF. Enhancing and sustaining the topical ocular delivery of fluconazole using chitosan solution and poloxamer/chitosan in situ forming gel. Eur J Pharm Biopharm. 2011;79(2):320-7.).

Cell reprogramming by gene therapy

Therapeutic strategies using cell reprogramming by gene therapy can promote the overexpression and local delivery of growth factors, anabolic hormones, or other intended adjuvant molecules to revert corneal inflammation or opacity, as detailed below. These therapeutic genes can reprogram the corneal cells or the LG(166166 Oliveira LA, Kim C, Sousa LB, Schwab IV, Rosenblatt MI. Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector. Arq Bras Oftalmol. 2010;73(5):447-53.). Previous studies have shown that the salivary gland can be reprogrammed by viral vector gene therapy to work as a bioreactor and delivery system of hormones and other therapeutic molecules to treat severe oral dryness caused due to SS or radiotherapy at the experimental and clinical levels(167167 Goldfine ID, German MS, Tseng HC, Wang J, Bolaffi JL, Chen JW, et al. The endocrine secretion of human insulin and growth hormone by exocrine glands of the gastrointestinal tract. Nat Biotechnol. 1997;15(13):1378-82.

168 Baum BJ, Alevizos I, Zheng C, Cotrim AP, Liu S, McCullagh L, et al. Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction. Proc Natl Acad Sci U S A. 2012;109(47):19403-7.

169 Alevizos I, Zheng C, Cotrim AP, Liu S, McCullagh L, Billings ME, et al. Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction. Gene Ther. 2017;24(3):176-86.
-170170 Voutetakis A, Bossis I, Kok MR, Zhang W, Wang J, Cotrim AP, et al. Salivary glands as a potential gene transfer target for gene therapeutics of some monogenetic endocrine disorders. J Endocrinol. 2005;185(3):363-72.). Furthermore, hormone gene therapy can be used to transfer the hormone erythropoietin (Epo), which preserved LG secretions and corneal epithelial integrity after the application of an adenovirus containing the Epo gene to the salivary gland(139139 Rocha EM, Cotrim AP, Zheng C, Riveros PP, Baum BJ, Chiorini JÁ. Recovery of radiation-induced dry eye and corneal damage by pre treatment with adenoviral vector-mediated transfer of erythropoietin to the salivary glands in mice. Hum Gene Ther. 2013;24(4):417-23.).

Corneal neovascularization reduces corneal transparency and the prognosis of corneal transplantation, and the actual treatment approaches for neovascularization are little effective and not long-lasting(171171 Maddula S, Davis DK, Maddula S, Burrow MK, Ambati BK. Horizons in therapy for corneal angiogenesis. Ophthalmology. 2011; 118(3):591-9.,172172 Koenig Y, Bock F, Horn F, Kruse F, Straub K, Cursiefen C. Short- and long-term safety profile and efficacy of topical bevacizumab (Avastin) eye drops against corneal neovascularization. Graefes Arch Clin Exp Ophthalmol. 2009;247(10):1375-82.). The key elements needed to prevent corneal neovascularization are a) constant vigilance for soluble vascular endothelial growth factor (VEGF) receptors on the ocular surface that can inhibit corneal neovascularization(3131 Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suther T, et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature. 2006;443(7114):993-7.) and b) the presence of corneal nerves working as anti-neovessel elements in the cornea(8383 Ferrari G, Hajrasouliha AR, Sadrai Z, Ueno H, Chauhan SK, Dana R. Nerves and neovessels inhibit each other in the cornea. Invest Ophthalmol Vis Sci. 2013;54(1):813-20.).

The neovascularization and opacity caused due to alkali burns in rat corneas were prevented in rats injected with an adenovirus containing the genes of soluble VEGF receptors (VEGFRs) in the LG. After 7 days, the corneas protected by the VEGFRs expressed in the LG were more transparent than those treated with an adenovirus with null genes or saline(173173 Nominato LF, Dias AC, Dias LC, Fantucci MZ, Silva LE, Murashima AA, et al. Prevention of corneal neovascularization by adenovirus encoding human vascular endothelial growth factor soluble receptor (s-VEGFR1) in lacrimal gland. Invest Ophthalmol Vis Sci. 2018;59(15):6036-44.). Therefore, LG may function as a target of gene therapy, functioning as a bioreactor for therapeutic molecules to prevent corneal scarring and blindness caused due to opacity or neovascularization(173173 Nominato LF, Dias AC, Dias LC, Fantucci MZ, Silva LE, Murashima AA, et al. Prevention of corneal neovascularization by adenovirus encoding human vascular endothelial growth factor soluble receptor (s-VEGFR1) in lacrimal gland. Invest Ophthalmol Vis Sci. 2018;59(15):6036-44.).

Tissue reengineering

Taking in account the limitations associated with OS reconstruction using the limbus transplant as mentioned above, the possibility of reengineering of corneal cells in vitro is being attempted. In the corneal limbus, the niches of stem cells exhibit mitotic activity mediated by at least three crucial transcription factors as follows: ATP-binding cassette, subfamily B, member 5 (ABCB5), paired box protein PAX6, and WNT7A(174174 Ouyang H, Xue Y, Lin Y, Zhang X, Xi L, Patel S, et al. WNT7A and PAX6 define corneal epithelium homeostasis and pathogenesis. Nature. 2014;511(7509):358-61.,175175 Ksander BR, Kolovou PE, Wilson BJ, Saab KR, Guo Q, Ma J, et al. ABCB5 is a limbal stem cell gene required for corneal development and repair. Nature. 2014;511(7509):353-7.). Therefore, the strategies for preserving or restoring these niches could include cell reprogramming to overexpress these transcription factors to achieve a stable corneal epithelial layer to revert ulcers or keratinization and support the corneal epithelial layer. The approach of gene therapy using these transcription factors combined with tissue reengineering to grow distinct corneal layers in vitro opens the possibility of using the combined approaches to repair or replace corneal layers in therapies used for corneal wound healing(176176 Hayashi R, Ishikawa Y, Sasamoto Y, et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function. Nature 2016;531(7594):376-80.,177177 McCabe KL, Kunzevitzky NJ, Chiswell BP, Xia X, Goldberg F, Lanza R. Efficient generation of human embryonic stem cell-derived corneal endothelial cells by directed differentiation. PLoS One. 2015;10(12):e0145266.).

Biosynthesis and xenotransplantation of corneas have also been explored as possible alternatives to corneal transplantation using tissue reengineering(114114 Lamm V, Hara H, Mammen A, Dhaliwal D, Cooper DK. Corneal blindness and xenotransplantation. Xenotransplantation. 2014; 21(2):99-114.,178178 Shi Y, Bikkuzin T, Song Z, Jin X, Jin H, Li X, et al. Comprehensive evaluation of decellularized porcine corneal after clinical transplantation. Xenotransplantation. 2017;24(6).,179179 Kim MK, Hara H. Current status of corneal xenotransplantation. Int J Surg. 2015;23:255-60.).

In cases where the LG is also damaged by the disease, the potential LG regeneration is limited(180180 Zoukhri D. Mechanisms involved in injury and repair of the murine lacrimal gland: role of programmed cell death and mesenchymal stem cells. Ocul Surf. 2010;8(2):60-9.

181 Malki LT, Dias AC, Jorge AG, Módulo CM, Rocha EM. Lacrimal gland primary acinar cell culture: the role of insulin. Arq Bras Oftalmol. 2016;79(2):105-10.
-182182 Shatos MA, Haugaard-Kedstrom L, Hodges RR, Dartt DA. Isolation and characterization of progenitor cells in uninjured, adult rat lacrimal gland. Invest Ophthalmol Vis Sci. 2012;53(6):2749-59.), and it is known that without the support of the LG, the corneal integrity is severely damaged(183183 Scherz W, Dohlman CH. Is the lacrimal gland dispensable? Keratoconjunctivitis sicca after lacrimal gland removal. Arch Ophthalmol. 1975;93(4):281-3.). Till date, only one study has been capable of demonstrating the restoration of a functional LG from transplanted embryonic tissue using tissue reengineering techniques(184184 Hirayama M, Ogawa M, Oshima M, Sekine Y, Ishida K, Yamashita K, et al. Functional lacrimal gland regeneration by transplantation of a bioengineered organ germ. Nat Commun. 2013;4:2497.). Nevertheless, the strategies used for restoring and reintegrating extensively damaged LFU structures are unknown, which is probably the major challenge in reverting corneal blindness in the long-term in diseases involving the extraocular organs.

Table 2 summarizes the potential molecules and surgical interventions capable of working in a combined preventive and therapeutic manner or as an adjuvant therapy for corneal opacity to minimize the incidence of corneal blindness in the future (Table 2).

Table 2
Potential clinical and surgical novel strategies for corneal opacity treatment

Corneal blindness is a health problem and a therapeutic challenge. If few conditions have found efficient strategies as trachoma, which is being treated with the combined strategy that includes Surgery, Antibiotics, Facial cleaning, and Environmental improvement (SAFE), and vitamin A supplementation can prevent keratomalacia secondary to nutritional problems even in remote regions, there are several conditions causing corneal blindness that are not being efficiently reverted by the currently available therapeutic strategies(201201 Travers A, Strasser S, Palmer SL, Stauber C. The added value of water, sanitation, and hygiene interventions to mass drug administration for reducing the prevalence of trachoma: a systematic review examining. J Environ Public. Health 2013;2013:682093.,202202 Faustino JF, Ribeiro-Silva A, Dalto RF, Souza MM, Furtado JM, Rocha GM, et al. Vitamin A and the eye: an old tale for modern times. Arq Bras Oftalmol 2016;79(1):56-61.). Novel therapeutic strategies using growth factors, anabolic agents, new promitotic, and anti-inflammatory drugs, combined with delivery systems, or corneal or LG genetic reprogramming of cells in association or not with corneal tissue reengineering can reduce the need for corneal transplantation and may function as adjuvants, providing customized therapies supporting more stable and long-lasting therapies for corneal transparency.

ACKNOWLEDGMENT

The authors would like to thank the support of the grants from the following Brazilian governmental institutions: Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) (nº 2015/20580-7 and 2014/22451-7) (São Paulo, SP, Brazil); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (nº: 474450/2012-0) (Brasilia, DF, Brazil); CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) (Finance Code 001) (Brasilia, DF, Brazil); Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clinicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA) (669/2018) (Ribeirão Preto, SP. Brazil); and Research Core of Ocular Physiopathology and Therapeutics from University of São Paulo (NAP-FTO) (nº 12.1.25431.01.7) (Ribeirão Preto, SP. Brazil).

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Publication Dates

  • Publication in this collection
    03 Feb 2021
  • Date of issue
    May-Jun 2021

History

  • Received
    23 Jan 2020
  • Accepted
    02 July 2020
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