The peripheral sensory nerves that innervate the cornea can be easily damaged by trauma, surgery, infection or diabetes. Several growth factors and axon guidance molecules, such as Semaphorin3A (Sema3A) are upregulated upon cornea injury. Nerves can regenerate after injury but do not recover their original density and patterning. Sema3A is a well known axon guidance and growth cone repellent protein during development, however its role in adult cornea nerve regeneration remains undetermined. Here we investigated the neuro-regenerative potential of Sema3A on adult peripheral nervous system neurons such as those that innervate the cornea. First, we examined the gene expression profile of the Semaphorin class 3 family members and found that all are expressed in the cornea. However, upon cornea injury there is a fast increase in Sema3A expression. We then corroborated that Sema3A totally abolished the growth promoting effect of nerve growth factor (NGF) on embryonic neurons and observed signs of growth cone collapse and axonal retraction after 30 min of Sema3A addition. However, in adult isolated trigeminal ganglia or dorsal root ganglia neurons, Sema3A did not inhibited the NGF-induced neuronal growth. Furthermore, adult neurons treated with Sema3A alone produced similar neuronal growth to cells treated with NGF and the length of the neurites and branching was comparable between both treatments. These effects were replicated in vivo, where thy1-YFP neurofluorescent mice subjected to cornea epithelium debridement and receiving intrastromal pellet implantation containing Sema3A showed increased corneal nerve regeneration than those receiving pellets with vehicle. In adult PNS neurons, Sema3A is a potent inducer of neuronal growth in vitro and cornea nerve regeneration in vivo. Our data indicates a functional switch for the role of Sema3A in PNS neurons where the well-described repulsive role during development changes to a growth promoting effect during adulthood. The high expression of Sema3A in the normal and injured adult corneas could be related to its role as a growth factor.

Extracellular vesicles (EVs), a diverse group of cell-derived exocytosed particles, are pivotal in mediating intercellular communication due to their ability to selectively transfer biomolecules to specific cell types. EVs, composed of proteins, nucleic acids, and lipids, are taken up by cells to affect a variety of signaling cascades. Research in the field has primarily focused on stem cell-derived EVs, with a particular focus on mesenchymal stem cells, for their potential therapeutic benefits. Recently, tissue-specific EVs or cell type-specific extracellular vesicles (CTS-EVs), have garnered attention for their unique biogenesis and molecular composition because they enable highly targeted cell-specific communication. Various studies have outlined the roles that CTS-EVs play in the signaling for physiological function and the maintenance of homeostasis, including immune modulation, tissue regeneration, and organ development. These properties are also exploited for disease propagation, such as in cancer, neurological disorders, infectious diseases, autoimmune conditions, and more. The insights gained from analyzing CTS-EVs in different biological roles not only enhance our understanding of intercellular signaling and disease pathogenesis but also open new avenues for innovative diagnostic biomarkers and therapeutic targets for a wide spectrum of medical conditions. This review comprehensively outlines the current understanding of CTS-EV origins, function within normal physiology, and implications in diseased states.

Extracellular vesicles (EVs) have been recognized as promising candidates for developing novel therapeutics for a wide range of pathologies, including ocular disorders, due to their ability to deliver a diverse array of bioactive molecules, including proteins, lipids, and nucleic acids, to recipient cells. Recent studies have shown that EVs derived from various cell types, including mesenchymal stromal cells (MSCs), retinal pigment epithelium cells, and endothelial cells, have therapeutic potential in ocular disorders, such as corneal injury and diabetic retinopathy. EVs exert their effects through various mechanisms, including promoting cell survival, reducing inflammation, and inducing tissue regeneration. Furthermore, EVs have shown promise in promoting nerve regeneration in ocular diseases. In particular, EVs derived from MSCs have been demonstrated to promote axonal regeneration and functional recovery in various animal models of optic nerve injury and glaucoma. EVs contain various neurotrophic factors and cytokines that can enhance neuronal survival and regeneration, promote angiogenesis, and modulate inflammation in the retina and optic nerve. Additionally, in experimental models, the application of EVs as a delivery platform for therapeutic molecules has revealed great promise in the treatment of ocular disorders. However, the clinical translation of EV-based therapies faces several challenges, and further preclinical and clinical studies are needed to fully explore the therapeutic potential of EVs in ocular disorders and to address the challenges for their successful clinical translation. In this review, we will provide an overview of different types of EVs and their cargo, as well as the techniques used for their isolation and characterization. We will then review the preclinical and clinical studies that have explored the role of EVs in the treatment of ocular disorders, highlighting their therapeutic potential and the challenges that need to be addressed for their clinical translation. Finally, we will discuss the future directions of EV-based therapeutics in ocular disorders. Overall, this review aims to provide a comprehensive overview of the current state of the art of EV-based therapeutics in ophthalmic disorders, with a focus on their potential for nerve regeneration in ocular diseases.

The protective function and transparency provided by the corneal epithelium are dependent on and maintained by the regenerative capacity of limbal epithelial stem cells (LESCs). These LESCs are supported by the limbal niche, a specialized microenvironment consisting of cellular and non-cellular components. Disruption of the limbal niche, primarily from injuries or inflammatory processes, can negatively impact the regenerative ability of LESCs. Limbal stem cell deficiency (LSCD) directly hampers the regenerative ability of the corneal epithelium and allows the conjunctival epithelium to invade the cornea, which results in severe visual impairment. Treatment involves restoring the LESC population and functionality; however, few clinically practiced therapies currently exist. This review outlines the current understanding of the limbal niche, its pathology and the emerging approaches targeted at restoring the limbal niche. Most emerging approaches are in developmental phases but show promise for treating LSCD and accelerating corneal regeneration. Specifically, we examine cell-based therapies, bio-active extracellular matrices and soluble factor therapies in considerable depth.

The cornea is a highly specialized organ that relies on its mechanical stiffness to maintain its aspheric geometry and refractive power, and corneal diseases such as keratoconus have been linked to abnormal tissue stiffness and biomechanics. Dynamic optical coherence elastography (OCE) is a clinically promising non-contact and non-destructive imaging technique that can provide measurements of corneal tissue stiffness directly in vivo. The method relies on the concepts of elastography where shear waves are generated and imaged within a tissue to obtain mechanical properties such as tissue stiffness. The accuracy of OCE-based measurements is ultimately dependent on the mathematical theories used to model wave behavior in the tissue of interest. In the cornea, elastic waves propagate as guided wave modes which are highly dispersive and can be mathematically complex to model. While recent groups have developed detailed theories for estimating corneal tissue properties from guided wave behavior, the effects of intraocular pressure (IOP)-induced prestress have not yet been considered. It is known that prestress alone can strongly influence wave behavior, in addition to the associated non-linear changes in tissue properties. This present study shows that failure to account for the effects of prestress may result in overestimations of the corneal shear moduli, particularly at high IOPs. We first examined the potential effects of IOP and IOP-induced prestress using a combination of approximate mathematical theories describing wave behavior in thin plates with observations made from data published in the OCE literature. Through wave dispersion analysis, we deduce that IOP introduces a tensile hoop stress and may also influence an elastic foundational effect that were observable in the low-frequency components of the dispersion curves. These effects were incorporated into recently developed models of wave behavior in nearly incompressible, transversely isotropic (NITI) materials. Fitting of the modified NITI model with ex vivo porcine corneal data demonstrated that incorporation of the effects of IOP resulted in reduced estimates of corneal shear moduli. We believe this demonstrates that overestimation of corneal stiffness occurs if IOP is not taken into consideration. Our work may be helpful in separating inherent corneal stiffness properties that are independent of IOP; changes in these properties and in IOP are distinct clinically relevant issues that affect the cornea health.

Semaphorin3A is considered a classical repellent molecule for developing neurons and a potent inhibitor of regeneration after nervous system trauma. Vinaxanthone and other Sema3A inhibitors are currently being tested as possible therapeutics to promote nervous system regeneration from injury. Our previous study on Sema3A demonstrated a switch in Sema3A’s function toward induction of nerve regeneration in adult murine corneas and in culture of adult peripheral neurons. The aim of the current study is to determine the direct effects of Vinaxanthone on the Sema3A induced adult neuronal growth. We first demonstrate that Vinaxanthone maintains its anti-Sema3A activity in embryonic dorsal root ganglia neurons by inhibiting Sema3A-induced growth cone collapse. However, at concentrations approximating its IC50 Vinaxanthone treatment does not significantly inhibit neurite formation of adult peripheral neurons induced by Sema3A treatment. Furthermore, Vinaxanthone has off target effects when used at concentrations above its IC50, and inhibits neurite growth of adult neurons treated with either Sema3A or NGF. Our results suggest that Vinaxanthone’s pro-regenerative effects seen in multiple in vivo models of neuronal injury in adult animals need further investigation due to the pleiotropic effect of Sema3A on various non-neuronal cell types and the possible effect of Vinaxanthone on other neuroregenerative signals.