Meesmann's epithelial corneal dystrophy (MEeD), which clinically presents with microcysts that can cause irritation, blurred vision or photophobia, is a genetic disorder caused by dominant-negative mutations in the KRT3 and KRT12 genes. Eradicating the mutant protein or tipping the balance strongly in favour of the wild type allele are viable options for therapeutic intervention. Here we studied two therapeutic approaches for suppression of the mutant KRTl2 allele and have developed, characterised and initiated in vivo testing using two novel KRTl2 mouse models. For a transient therapeutic approach, short interfering RNAs (siRNAs) were designed and proved capable of mutation-specific inhibition of the alleles responsible for two MEeD causative mutations (p.Leu132Pro and p.Arg135Thr; 70-90%) in vitro. No off-target issues were observed and suppression of endogenously expressed p.Leu132Pro was also shown in an ex vivo model. For a more generic, yet potentially permanent therapeutic approach, total KRTl2 was suppressed (~50%) with an siRNA expressed from a short hairpin by targeting a region homologus to both the WT and mutant mRNAs. KRT 12 was replaced with a co-expressed recoded allele made resistant to the siRNA. To further develop these potential therapeutics, two novel mouse models were generated allowing evaluation of gene modulation in vivo. (1) A humanised dominant negative mutant model that expresses K12 p.Leu132Pro revealed major changes to corneal phenotype in homozygous animals. Microcysts were observed and keratin expression patterns disrupted. Additionally, RNAseq analysis highlighted over 1600 dysregulated genes, which could feature other potential therapeutic targets for the treatment of symptomatic MEeD. Heterozygous mice presented with a subtler phenotype. (2) A Krt12 luciferase reporter mouse model was optimised and will facilitate live animal corneal imaging, thus aiding the development of topical siRNA delivery formulations. These mouse models in conjunction with our gene silencing development programme pave the way for in vivo assessment of RNA i-based therapeutics for the cornea.

Migraine is a complex neurological disorder that affects approximately 38 million Americans. For over 25 years, the neuropeptide calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. In fact, several pharmaceutical companies are tailoring treatments to antagonize CGRP actions. However, due to the complexity of migraine, exactly how and where CGRP acts to contribute to migraine have remained controversial: whereas several studies suggest that CGRP acts in the central nervous system (CNS) in this context, others have indicated a role in the periphery. Central nervous system sites of action include the trigeminal nucleus and several higher brain regions, and peripheral sites include the vasculature and dural mast cells in the meninges. Among the sites of CGRP action, the trigeminal nerve, which is the major somatosensory structure of the face, is of particular interest because it bridges the CNS and the periphery. Migraine is generally thought to involve abnormal signaling in the trigeminovascular system, and about 50% of trigeminal neurons have CGRP immunoreactivity. Although the notion that CGRP has a central site of action in relation to migraine had gained ground over the past decade, the recent discovery that monoclonal antibodies against CGRP can prevent migraine attacks has resurrected the possibility that a peripheral site of action is involved as well. Clarification of the sites of CGRP action in migraine will be crucial to developing an understanding of mechanisms that underlie migraine so that future treatments can be rationally designed. One diagnostic criterion for migraine is photophobia, a painful and often debilitating response to non-noxious levels of light. Our laboratory previously developed a preclinical model of migraine in which the light-aversive behavior of mice is used as a surrogate of photophobia. Specifically, mice were sensitized to CGRP by introducing a nestin/hRAMP1 transgene. In these mice versus control littermates, light aversion in response to central (intracerebroventricular, ICV) injection of CGRP was enhanced in dim light. In wild-type mice, CGRP (ICV) also elicited aversion to very bright light; this did not occur in vehicle-treated mice. Additionally, I have shown that CGRP injected peripherally (intraperitoneal, IP) can induce significant light aversion in wild-type mice. I have begun to identify the sites of action outside of the central nervous system, using four lines of transgenic mice with different patterns of overexpression of CGRP receptors: global hRAMP1 mice (expression in all tissues), nestin/hRAMP1 mice (expression only in nervous tissue), tagln/hRAMP1 (expression only in smooth muscle cells), and tek2/hRAMP1 (expression in endothelial cells). As predicted, in the global hRAMP mice light aversion, in response, to IP-injected CGRP was enhanced. However, in nestin/hRAMP1 mice, only ICV-injected, and not IP-injected, CGRP induced enhanced light aversion. This finding suggests that peripheral CGRP activates neural pathways involved in light aversion, but by an indirect mechanism. To determine where in the periphery CGRP is acting, a pharmacological and genetic approach was taken. Since CGRP is one of the most potent vasodilators in the body, it is well positioned to have vascular effects that induce light aversive behavior. This hypothesis was based on findings that 1) intravenous administration of CGRP in human subjects can cause migraine pain, and 2) perivascular CGRP can sensitize the trigeminal nerve, which could alter synaptic transmission to the central nervous system and 3) CGRP monoclonal antibodies are effective in clinical trial and likely do not cross the blood brain barrier. Thus, there is a mechanism by which CGRP in the periphery can sensitize the trigeminal nerve and alter sensory perception, leading to photophobia. The role of the vasculature in migraine, specifically vasodilation, has been controversial and now the consensus is that it is neither necessary nor sufficient. First, I wanted to test the role of vasodilation in this model. I pharmacologically inhibited CGRP-induced vasodilation using two vasoconstrictors, phenylephrine and endothelin-1. Blocking CGRP-induced vasodilation partially attenuates the light aversive response. Moreover, mice that overexpress the CGRP receptor in smooth muscle, but not endothelial, cells exhibit enhanced light aversion indicating a role for vascular actions of CGRP in this preclinical model of migraine. These results present clear evidence that CGRP has actions on the vasculature to induce light aversion. Additionally, the inability of blocking vasodilation to completely rescue the light aversion suggests that the vasculature may not be the only peripheral target of CGRP in migraine pathophysiology. This work improves the understanding of peripheral CGRP actions in migraine and raises awareness that contribution of the vasculature in migraine should not be ignored. The identification of sites of CGRP action in regions inside and outside of the CNS could lead to improved and more successful therapeutics for migraine.

Calcitonin gene-related peptide (CGRP) has been strongly implicated in the pathophysiology of migraine. CGRP levels are elevated during a migraine attack. Injection of CGRP can trigger a delayed migraine-like headache in migraineurs. Finally, CGRP receptor antagonists are effective antimigraine therapeutics. Consequently, a CGRP-sensitized mouse, nestin/hRAMP1 was genetically engineered to conditionally express a subunit of the CGRP receptor, hRAMP1, in neurons and glia. In response to CGRP, nestin/hRAMP1 mice demonstrated a significant decrease in time in the light zone of a dim light-dark box compared to vehicle-treated nestin/hRAMP1 mice and CGRP-treated control mice. This reflects photophobia-like behavior. Photophobia is a common symptom of migraine, where light exacerbates the headache pain. Furthermore, CGRP decreased motility in the dark zone, which may reflect exacerbation of pain by movement that is often experienced during a migraine. Wildtype mice have also demonstrated this CGRP-induced behavior, but required bright light and habituation to the chamber. While there is a difference in sensitivity in this assay between wildtype and nestin/hRAMP1 mice, it demonstrates that endogenous CGRP receptors are sufficient to convey this behavior. A common antimigraine drug, rizatriptan, attenuated the CGRP-induced behaviors in wildtype mice validating the assay as a migraine model. To explore the relative contributions of CGRP receptors on neurons versus glia, synapsin/hRAMP1 transgenic mice were genetically engineered to express hRAMP1 in neurons only. In contrast to the nestin/hRAMP1 mice, the synapsin/hRAMP1 mice did not show CGRP-induced light aversion upon naïve exposure to a dim chamber. This suggests that neuronal overexpression of hRAMP1 is insufficient to convey a heighted sensitivity to CGRP in the light aversion assay. As a first step to understanding the mechanism underlying CGRP-induced light aversion, a non-behavioral assay was developed to measure photic blink reflexes by measuring orbicularis oculi EMG responses in mice. Bright light increased orbicularis oculi activity, and an air puff induced a blink response. Interestingly. CGRP and bright light increased the duration of squinting following the air puff-induced blink. This pilot suggests that the trigeminal system plays a key role in mediating CGRP-induced light sensitivity. Overall, these studies propose a potential model for the mechanisms involved in migraine and photophobia in which CGRP likely acts through endogenous CGRP receptors on neurons and glia in the trigeminal system to trigger light sensitivity.

Objetivos: Verificar a relação entre a incapacidade relacionada aos sintomas vestibulares e a presença de aura e cronicidade da migrânea. Além disso, investigar a sensibilidade visual em indivíduos com migrânea e sem cefaleia, bem como o controle de equilíbrio postural perante estimulação luminosa. Métodos: mulheres com migrânea e mulheres sem cefaleia foram avaliadas nos estudos apresentados nesta tese. Inicialmente, as mulheres com migrânea foram estratificadas em migrânea com aura, migrânea sem aura e migrânea crônica. Informações sobre sintomas vestibulares foram coletadas e a autopercepção de incapacidade relacionada aos sintomas vestibulares foi avaliada por meio do questionário Dizziness Handicap Inventory. Posteriormente, os grupos migrânea e controle foram avaliados nas posturas bipodal e unipodal sob três condições de luz: (1) ambiente, (2) limiar de desconforto visual e (3) desconforto visual intenso, para analisar variáveis do centro de pressão (CoP). Ambas as condições de desconforto visual foram determinadas com base no relato de desconforto dos participantes do grupo migrânea. Por fim, foram coletados dados sobre a intensidade de desconforto visual durante as atividades diárias. Resultados: Os pacientes com migrânea apresentaram maiores escores do Dizziness Handicap Inventory do que os controles, e a presença de enxaqueca está associada a um maior risco de sintomas vestibulares, bem como a um maior risco de incapacidade moderada a grave. Considerando os subtipos de migrânea, os grupos com aura e migrânea crônica apresentaram pontuações mais elevadas no questionário do que o grupo migrânea sem aura. Ainda, a aura, a intensidade e a frequência da migrânea podem predizer a incapacidade da tontura. Na análise do desconforto visual, o grupo com enxaqueca relatou limiar de desconforto visual de 450 lux e desconforto visual intenso a 2000 lux, enquanto os controles não relataram desconforto visual. O grupo migrânea também apresentou maior intensidade de desconforto para realizar as atividades diárias, principalmente para dirigir e caminhar em dias ensolarados. Na análise estabilométrica, os indivíduos com migrânea apresentaram maior área do centro de pressão (CoP) nas três condições, e maior velocidade e RMS do CoP sob as duas condições de desconforto visual, quando comparados aos controles. Apenas o grupo migrânea apresentou maior área, velocidade e RMS do CoP para ambas as condições de desconforto visual em comparação com a condição ambiente. Conclusões: A prevalência de sintomas vestibulares é maior na migrânea, particularmente nos subtipos com aura e crônica, juntamente com umamaior incapacidade relacionada a esses sintomas. Ademais, também podemos assumir que os pacientes com migrânea apresentam sensibilidade visual durante o período interictal, e o desconforto induzido pela luz pode ser um fator perturbador que piora o equilíbrio, mesmo durante a postura em pé. / Objective: To verify if there was any relationship between the handicap related to vestibular symptoms and the presence of aura and the chronicity of migraine attacks. Subsequently, to investigate the visual sensitivity in migraineurs and non-headache subjects, as well as the response of balance control to light stimulation. Methods: Women with migraine and nonheadache women were assessed in the studies presented in the current thesis. Initially, the migraineurs were stratified as migraine with aura, migraine without aura and chronic migraine. Information regarding vestibular symptoms was collected, and the self-perceived handicap related to vestibular symptoms was assessed through the Dizziness Handicap Inventory questionnaire. On the second moment, both migraine group and control group were evaluated in bipodal and unipodal postures under three light conditions: (1) ambient, (2) visual discomfort threshold and (3) intense visual discomfort, in order to analyze variables of the center of pressure. Both visual discomfort conditions were determined based on the report of discomfort of the migraine individuals. Finally, data about the intensity of visual discomfort during daily activities were collected. Results: Patients with migraine exhibited greater Dizziness Handicap Inventory scores than controls, and the presence of migraine is associated with a greater risk of vestibular symptoms and with a greater risk of moderate-to-severe handicap. Considering the subtypes of migraine, patients with migraine with aura and chronic migraine reached greater scores than those migraineurs without aura. Also, migraine aura, intensity and frequency can predict the dizziness handicap. In the analysis of visual discomfort, the migraine group reported a visual discomfort threshold of 450 lx and intense visual discomfort at 2000 lx, while controls did not report visual discomfort. Migraineurs also presented higher discomfort intensity to perform daily activities, especially to driving and to walking in a sunny day. On the stabilometric analysis, subjects with migraine presented greater center of pressure (CoP) area under the three conditions, and greater CoP velocity and RMS under the visual discomfort light conditions, compared to controls. Only the migraine group showed greater CoP area, velocity and RMS for both visual discomfort light conditions compared to the ambient condition. Conclusions: The prevalence of vestibular symptoms is increased in migraine, particularly in migraine with aura and chronic migraine along with an increased handicap due to those symptoms. Furthermore, we can also assume that patients with migraine present visualsensitivity during the interictal period, and the light-induced discomfort might be a disturbing factor that worsens balance even during quiet standing posture.

Objetivos: Verificar a relação entre a incapacidade relacionada aos sintomas vestibulares e a presença de aura e cronicidade da migrânea. Além disso, investigar a sensibilidade visual em indivíduos com migrânea e sem cefaleia, bem como o controle de equilíbrio postural perante estimulação luminosa. Métodos: mulheres com migrânea e mulheres sem cefaleia foram avaliadas nos estudos apresentados nesta tese. Inicialmente, as mulheres com migrânea foram estratificadas em migrânea com aura, migrânea sem aura e migrânea crônica. Informações sobre sintomas vestibulares foram coletadas e a autopercepção de incapacidade relacionada aos sintomas vestibulares foi avaliada por meio do questionário Dizziness Handicap Inventory. Posteriormente, os grupos migrânea e controle foram avaliados nas posturas bipodal e unipodal sob três condições de luz: (1) ambiente, (2) limiar de desconforto visual e (3) desconforto visual intenso, para analisar variáveis do centro de pressão (CoP). Ambas as condições de desconforto visual foram determinadas com base no relato de desconforto dos participantes do grupo migrânea. Por fim, foram coletados dados sobre a intensidade de desconforto visual durante as atividades diárias. Resultados: Os pacientes com migrânea apresentaram maiores escores do Dizziness Handicap Inventory do que os controles, e a presença de enxaqueca está associada a um maior risco de sintomas vestibulares, bem como a um maior risco de incapacidade moderada a grave. Considerando os subtipos de migrânea, os grupos com aura e migrânea crônica apresentaram pontuações mais elevadas no questionário do que o grupo migrânea sem aura. Ainda, a aura, a intensidade e a frequência da migrânea podem predizer a incapacidade da tontura. Na análise do desconforto visual, o grupo com enxaqueca relatou limiar de desconforto visual de 450 lux e desconforto visual intenso a 2000 lux, enquanto os controles não relataram desconforto visual. O grupo migrânea também apresentou maior intensidade de desconforto para realizar as atividades diárias, principalmente para dirigir e caminhar em dias ensolarados. Na análise estabilométrica, os indivíduos com migrânea apresentaram maior área do centro de pressão (CoP) nas três condições, e maior velocidade e RMS do CoP sob as duas condições de desconforto visual, quando comparados aos controles. Apenas o grupo migrânea apresentou maior área, velocidade e RMS do CoP para ambas as condições de desconforto visual em comparação com a condição ambiente. Conclusões: A prevalência de sintomas vestibulares é maior na migrânea, particularmente nos subtipos com aura e crônica, juntamente com umamaior incapacidade relacionada a esses sintomas. Ademais, também podemos assumir que os pacientes com migrânea apresentam sensibilidade visual durante o período interictal, e o desconforto induzido pela luz pode ser um fator perturbador que piora o equilíbrio, mesmo durante a postura em pé. / Objective: To verify if there was any relationship between the handicap related to vestibular symptoms and the presence of aura and the chronicity of migraine attacks. Subsequently, to investigate the visual sensitivity in migraineurs and non-headache subjects, as well as the response of balance control to light stimulation. Methods: Women with migraine and nonheadache women were assessed in the studies presented in the current thesis. Initially, the migraineurs were stratified as migraine with aura, migraine without aura and chronic migraine. Information regarding vestibular symptoms was collected, and the self-perceived handicap related to vestibular symptoms was assessed through the Dizziness Handicap Inventory questionnaire. On the second moment, both migraine group and control group were evaluated in bipodal and unipodal postures under three light conditions: (1) ambient, (2) visual discomfort threshold and (3) intense visual discomfort, in order to analyze variables of the center of pressure. Both visual discomfort conditions were determined based on the report of discomfort of the migraine individuals. Finally, data about the intensity of visual discomfort during daily activities were collected. Results: Patients with migraine exhibited greater Dizziness Handicap Inventory scores than controls, and the presence of migraine is associated with a greater risk of vestibular symptoms and with a greater risk of moderate-to-severe handicap. Considering the subtypes of migraine, patients with migraine with aura and chronic migraine reached greater scores than those migraineurs without aura. Also, migraine aura, intensity and frequency can predict the dizziness handicap. In the analysis of visual discomfort, the migraine group reported a visual discomfort threshold of 450 lx and intense visual discomfort at 2000 lx, while controls did not report visual discomfort. Migraineurs also presented higher discomfort intensity to perform daily activities, especially to driving and to walking in a sunny day. On the stabilometric analysis, subjects with migraine presented greater center of pressure (CoP) area under the three conditions, and greater CoP velocity and RMS under the visual discomfort light conditions, compared to controls. Only the migraine group showed greater CoP area, velocity and RMS for both visual discomfort light conditions compared to the ambient condition. Conclusions: The prevalence of vestibular symptoms is increased in migraine, particularly in migraine with aura and chronic migraine along with an increased handicap due to those symptoms. Furthermore, we can also assume that patients with migraine present visualsensitivity during the interictal period, and the light-induced discomfort might be a disturbing factor that worsens balance even during quiet standing posture.

Pediatric headache is a global health concern, with up to 60% of youth experiencing persistent headache pain for at least three months (Abu-Arafeh et al., 2010). Often accompanying the headache are symptoms such as sensitivity to light and sound (Hayne & Martin, 2019; Neut et al., 2012). Headache pain and its associated sensitivities often result in a significant reduction in daily function and avoidance of school, leisure, and social activities (Langdon & DiSabella, 2017). Sensitivities to light and sound are important yet understudied symptoms of pediatric headache disorders. The lack of empirical evidence to guide occupational therapists in the treatment of headache-related sensitivities in children and adolescents presents a major gap in the practice area of pediatric chronic pain. Pediatric Auditory and Visual Exposure (PAVE) was developed to address this gap and to provide occupational therapists with an effective, standardized approach to treat headache-related sensitivities in youth. PAVE is a theoretically driven, exposure-based occupational therapy intervention for youth (ages 8–17) presenting with a diagnosed pediatric headache disorder and experiencing light and/or sound sensitivities that interfere with their daily functioning. The intervention consists of six treatment modules employed over the course of several weeks. The primary goal of PAVE is to improve participant daily functioning through reduction of avoidance behavior, sensory sensitivity, and pain intensity, all of which contribute to functional disability.