Search

Strabismus is an ocular motor disorder characterized by eye misalignment, where clinically the extraocular muscle (EOM) pairs are presumed to be "overacting", and "underacting". There is currently a need for effective treatments that could act to strengthen an underacting muscle to aid in establishing eye alignment. Neurotrophic factors, such as insulin like growth factor-1 (IGF-1) and brain derived neurotropic factor (BDNF) have emerged as potential treatment options that could strengthen extraocular muscle. Neurotrophic factors have the ability to alter properties of EOM myofibers, neuromuscular junctions, and the motor neurons themselves through retrograde transport from the target muscle. A multi-action pharmacological agent that could be applied directly to the readily accessible EOM could present a clinically simple treatment for strabismus. In chapter 2, I build on previous work, which demonstrated that IGF-1 makes myofibers stronger. Rabbit EOM was pre-injected hepatocyte growth factor prior to IGF-1 treatment. With combined treatment, increased muscle force was obtained with lower doses of IGF-1. This work supports the potential therapeutic use of IGF-1 to correct strabismus by increasing muscle force. Despite the strong evidence that IGF-1 treatment strengthens EOM, the efficacy of IGF-1 to alter eye alignment in a binocular animal model is currently unknown. In chapters 3 and 4, I test the hypothesis that persistent bilateral (Chapter 3) or unilateral (Chapter 4) IGF-1 treatment to the medial rectus of a nonhuman primate can alter eye positioning. For these studies, we treated visually normal infant nonhuman primates during the postnatal development of the ocular motor system, which allowed us not only to assess if persistent IGF-1 treatment could disrupt the eye alignment, but also to investigate if aberrant neurotrophic factor signaling during development of the ocular motor system in the EOM is a possible cause of strabismus. In chapter 3, bilateral treatment did not result in strabismus. However, myofiber size increased in both the treated EOM and the untreated antagonist muscles. I hypothesize that over the three months of sustained treatment, the ocular motor system adapted to maintain eye alignment. In chapter 4, unilateral IGF-1 did create strabismus. This novel finding strongly suggests that altered molecular signaling by the EOM could be cause of strabismus. Finally, in Chapter 5, I extend my studies of neurotrophic factor treatment to infant nonhuman primates to test if another promising neurotropic factor, BDNF, could alter eye alignment during development of the ocular motor system. At the end of the three-month treatment period, no strabismus developed. However, there were marked changes to a subpopulation of myofibers that contain slow myosin heavy chain isoform. These results have interesting implications for the role of BDNF in selectively influencing tonic properties of the EOM. This work strongly demonstrates that sustained release of neurotrophic factors can significantly alter extraocular muscle properties, and disrupt eye alignment. Future studies will further investigate both the role of neurotrophic factors in determining normal EOM properties, and test if sustained release with IGF-1 can treat an animal model with strabismus.

Mammalian eye development requires fusion of the upper and lower eyelids in embryogenesis. Many genetic mutations cause defective eyelid closure in mouse embryogenesis, resulting in an eye open at birth (EOB) phenotype. Previous work from our laboratory has shown that loss function mutation of the gene coding for Mitogen-Activated Protein Kinase Kinase Kinase 1 (MAP3K1) leads to the EOB phenotype. This thesis explores the genetic and gene-environmental interaction mechanisms of MAP3K1 in eyelid closure and the etiology and pathogenesis of congenital diseases associated with defective eyelid closure. We have shown that MAP3K1 crosstalks with the EGFR and RhoA-ROCK pathways and regulates the Jun N-terminal kinases (JNKs) for lid closure. The transcription factor c-Jun, a JNK substrate, is also essential for eyelid closure, but whether c-Jun is a component of the MAP3K1 signaling network is not known. In Chapter 2, I show that MAP3K1 is associated with cytoskeleton, activates JNK and induces actin polymerization. MAP3K1 promotes the eyelid inferior epithelial cell elongation that leads to the formation of an epithelium protrusion. The c-Jun, on the other hand, is expressed only in the protruding eyelid epithelium, where it promotes ERK phosphorylation and cell migration. Homozygous deletion of either gene results in defective eyelid closure, but the Map3k1 and c-Jun double heterozygous does not. Hence, MAP3K1 and c-Jun belong to distinct pathways, acting consecutively to regulate eyelid morphogenetic closure.Although genetic codes are the blueprints for organogenesis, environmental insults can modify the developmental programs. Gene-environment interaction is responsible for the majority cases of multifactorial birth defects. In Chapter 3, I show that gene (Map3k1)-environment {2,3,7,8-tetrachlorodibenzodioxin (TCDD)} interaction is an etiology for defective eyelid closure. In utero TCDD exposure causes defective eyelid closure only when the Map3k1 gene is reduced by 50%, and in a manner dependent on the aryl hydrocarbon receptor (AHR). How TCDD-AHR and MAP3K1 axis crosstalk in eyelid morphogenesis is yet to be understood, but likely involves the inhibition of the JNK-c-Jun pathways. Many genetic mutant mice exhibit the EOB phenotype, but the developmental roles of eyelid closure are not known. In Chapter 4, I examine seven EOB strains, which were generated by different strategies of gene ablation that inactivated distinct signaling pathways. Results show that defective eyelid closure leads to corneal erosion/ulceration, Meibomian gland hypoplasia, truncation of the eyelid tarsal muscles, failure of levator palpebrae superiors (LPS) extension into the upper eyelid and the misplacement of the inferior oblique (IO) and inferior rectus (IR) muscles. Some of the abnormalities can be traced to pre-natal fetuses. Hence, in addition to serving as a protective barrier of the cornea, eyelid closure in embryogenesis is required for the development of ocular adnexa. Collectively, my results suggest that (1) eyelid closure is regulated by diverse genes acting through temporal-spatial signaling pathways, (2) gene-environment interaction is likely a real life etiology for causing the eyelid closure defects, and (3) the failure of eyelid closure leads to aberrant development of ocular surface and adnexa, which may account for congenital diseases, such as blepharoptosis and strabismus.

The extraocular muscles (EOM) are both morphologically and functionally spared in the absence of dystrophin, which results in the fatal disease Duchenne muscular dystrophy (DMD). It is currently thought that intrinsic differences between the EOM and other non-cranial skeletal muscles account for this sparing. The work of this thesis examines the sparing of the EOM in different mouse models of DMD and differences between progenitor cells of the EOM and those of non-cranial skeletal muscles. Results of functional and morphological studies indicate that the mdx:utrophin+/- mouse model may serve as a more useful model of disease than the mdx mouse model, and that EOM sparing is not due to autosomal homolog utrophin up-regulation, as the EOM are spared in mouse models that lack both dystrophin and utrophin. Finally, gamma irradiation studies suggest that there is a population of progenitor cells in the EOM that is either better able to survive in the diseased muscle or is more highly proliferative than the progenitor population found in limb. Greater understanding of this population of progenitors in the EOM may provide insight into EOM sparing in DMD, and possibly even therapeutic advancements for treatment of this fatal disease.