Dry eye disease is a complex ophthalmic disorder that consists of two main subtypes, aqueous deficient dry eye (ADDE) and evaporative dry eye disease (EDED). Due to the complex underlying physiology, human dry eye disease can be difficult to model in laboratory animal species. Thus, the identification and characterization of a spontaneous large animal model of dry eye disease is desirable. Dogs have been described as an ideal spontaneous model of ADDE due to the similar pathophysiology between dogs and humans. Recently, EDED and meibomian gland dysfunction (MGD) have been increasingly recognized and reported in dogs. These reports on EDED and MGD in dogs have identified similarities in pathophysiology, clinical presentations, and diagnostic parameters to humans with the comparable disorders. Additionally, the tests that are used to diagnose EDED and MGD in humans are more easily applicable to dogs than to laboratory species due to the comparable globe sizes between dogs and humans. The reported response of dogs to EDED and MGD therapies are similar to humans, suggesting that they would be a valuable preclinical model for the development of additional therapeutics. Further research and clinical awareness of EDED and MGD in dogs would increase their ability to be utilized as a preclinical model, improving the positive predictive value of therapeutics for EDED and MGD in both humans and dogs.

OCT4 is a core transcription factor involved in pluripotency maintenance in the early mammalian embryo. The POU5F1 gene that encodes the OCT4 protein is highly conserved across species, suggesting conserved function. However, studies in several species including mice, cattle, and pigs, suggest that there are differences in where and when OCT4 is expressed. Specifically, in the horse, several studies have shown that exposure to the uterine environment may be necessary to induce OCT4 expression restriction to the inner cell mass (ICM) of the developing embryo, suggesting that there may be equine-specific extrinsic regulators of OCT4 expression that have not yet been investigated. However, an alternative hypothesis is that this restriction may not be evident in equine embryos because of our inability to culture them to the epiblast stage, preventing the observation of this restriction. In vitro studies have identified that OCT4 is expressed in the immature equine oocyte and in the early equine embryo, but OCT4 expression has not been studied after the formation of the ICM in the equine embryo. Despite the gaps in knowledge about equine-specific functions of OCT4, this factor has been used in studies assessing equine embryonic stem cells and to induce pluripotency in equine somatic cells. This review describes the role of OCT4 in the equine embryo and its applications in equine stem cell research.

As standard in vitro fertilization is not a viable technique in horses yet, many different techniques have been used to create equine embryos for research purposes. One such method is parthenogenesis in which an oocyte is induced to mature into an embryo-like state without the introduction of a spermatozoon, and thus they are not considered true embryos. Another method is somatic cell nuclear transfer (SCNT), in which a somatic cell nucleus from an extant horse is inserted into an enucleated oocyte, creating a genetic clone of the donor horse. Due to limited availability of equine oocytes in the United States, researchers have investigated the potential for combining equine somatic cell nuclei with oocytes from other species to make embryos for research purposes, which has not been successful to date. There has also been a rising interest in producing transgenic animals using sperm exposed to exogenous DNA. The successful creation of transgenic equine blastocysts shows the promise of sperm mediated gene transfer (SMGT), but this method is not ideal for other applications, like gene therapy, because it cannot be used to induce targeted mutations. That is why technologies like CRISPR/Cas9 are vital. In this review, we argue that parthenogenesis, SCNT, and interspecies SCNT can be considered genetic manipulation strategies as they create embryos that are genetically identical to their parent cell. Here, we describe how these methods are performed and their applications and we also describe the few methods that have been used to directly modify equine embryos: SMGT and CRISPR/Cas9.

Microbial keratitis is a common cause of ocular pain and visual impairment worldwide. The ocular surface has a relatively paucicellular microbial community, mostly found in the conjunctiva, while the cornea would be considered relatively sterile. However, in patients with microbial keratitis, the cornea can be infected with multiple pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium sp. Treatment with topical antimicrobials serves as the standard of care for microbial keratitis, however, due to high rates of pathogen resistance to current antimicrobial medications, alternative therapeutic strategies must be developed. Multiple studies have characterized the expression and activity of antimicrobial peptides (AMPs), endogenous peptides with key antimicrobial and wound healing properties, on the ocular surface. Recent studies and clinical trials provide promise for the use of AMPs as therapeutic agents. This article reviews the repertoire of AMPs expressed at the ocular surface, how expression of these AMPs can be modulated, and the potential for harnessing the AMPs as potential therapeutics for patients with microbial keratitis.

Objectives

This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.

Procedures

A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.

Results

Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.

Conclusions

This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.