Dissertação (Mestrado em Engenharia Elétrica) - Centro Universitário FEI, São Bernardo do Campo, 2018

O crescimento e as dimensões das estruturas oculares em cães de diversas raças têm sido objeto de estudo. Sabe-se que quanto mais longilíneo o crânio, maior o comprimento axial do bulbo ocular. O objetivo deste trabalho foi acompanhar o desenvolvimento das dimensões dos componentes oculares (comprimento axial, espessura da lente, profundidade da câmara anterior e da câmara vítrea) e relacionar as medidas com o sexo, a idade, tamanho, medidas do crânio e peso de cães da raça Cavalier King Charles Spaniel, uma raça braquicéfala. Foram realizadas 117 medidas biométricas oculares em cães variando entre 15 dias e 36 meses de idade, não sedados, sentados ou deitados em posição esternal, utilizando-se ultrassonografia modo-B com transdutor microconvexo de 8 MHz. No momento de cada medida ocular os cães foram pesados e as medidas de comprimento, altura, distâncias fronto-occipital, fronto-nasal, bizigomática e circunferência do crânio foram registradas. As estruturas oculares mostraram uma curva de rápido crescimento entre 15 dias e 4 meses de idade e uma curva suave de crescimento até os 12 meses, idade em que cessou o crescimento do cão (altura e comprimento). Os machos apresentaram medidas maiores de altura, comprimento e crânio do que fêmeas, porém não houve diferença significativa entre os parâmetros de biometria ocular de machos e fêmeas. O valor médio de comprimento axial do bulbo para cães adultos (acima de 12 meses) foi de 18,10 ± 0,48 mm, para a espessura da lente, de 7,15 ± 0,16 mm, para profundidade da câmara anterior, de 2,05 ± 0,37 mm e para a profundidade da câmara vítrea, de 8,91 ± 0,30 mm. Não houve diferença entre as medidas dos olhos direito e esquerdo. Os resultados sugerem que a curva de crescimento ocular acompanha a curva de crescimento do cão, fato semelhante ao que ocorre em diferentes espécies estudadas por outros autores. Em cães adultos, não foi observada relação entre as medidas dos componentes oculares e as medidas de altura, comprimento, peso e tamanho do crânio. Foi estabelecida uma tabela de crescimento correlacionando comprimento axial do bulbo e idade do cão com a finalidade de padronizar esses dados para a raça. / Ocular biometry and ocular growth has been studied in dogs of different breeds. It\'s already known that dogs with longer skulls have longer axial length of the eye. This study aimed to evaluate the development of ocular dimensions (axial length of the bulbus, lens thickness, anterior and vitreous chamber depth) in Cavalier King Charles Spaniels, a braquicephalic breed, and its relationship to age, gender, weight, height and lenght of the dog and dimensions of the head. Ocular dimensions were obtained from 117 measurements between 15 days and 3 years old, in standing nonsedated animals using B-mode ultrasound with an 8 MHz curvilinear probe. At the same time the dogs were weighted and height, length and head dimensions (head circumference, fronto-occipital, fronto-nasal distance and bizigomatic distances) were recorded. The ocular parameters showed a rapid growth curve from 15 days to 4 months and then a slow curve until 12 months, same age that the height and length ceased its growth. Males showed significant higher measurements of height, length and head parameters than females, but no difference in ocular biometry was found between males and females. The mean value for axial lenght for adults (over 12 months) was 18,10 ± 0,48 mm, for lens thickness was 7,15 ± 0,16 mm, for anterior chamber depth was 2,05 ± 0,37 mm and for vitreous chamber depth was 8,91 ± 0,30 mm. There was no significant difference between left and right eyes. Results suggest that eye growth curves accompanies dogs height, length, head size growth curves, what is similar to the data found in different species studied by other authors. There was no relation between eye parameters and dog\'s height, length, head size or weight in adult individuals. A table was established correlating axial length of the bulbus and age to be used as a reference for the breed.

Therapeutic Ocular Surface Medium (TOSM) is a potential new treatment for patients with ocular surface disorders such as dry eye and persistent epithelial defect (PED). New therapies are needed as many patients with dry eye and PED continue to suffer despite maximal standard therapy, and while efficacious autologous serum therapy is not routinely available. Like serum, TOSM contains tear components and was expected to have some of the physiological effects of tears. Clinical and in vitro studies were used to evaluate two similar formulations of TOSM. To comply with local pharmacy manufacturing policies, components were omitted from TOSM v1 to produce TOSM v2. In pilot studies, conducted over 1 month, TOSM v1 improved dry eye signs and symptoms and healed over a quarter of PED. In a 2 month randomised double-masked trial, TOSM v2 improved the signs and symptoms of dry eye but was not superior to saline (placebo). No serious or irreversible side-effects occurred. The altered composition of TOSM v2 may have reduced its efficacy. However, a significant improvement in blepharitis (eyelid margin disease) and conjunctival impression cytology (an objective measure of ocular surface health) was found with TOSM v2. Improvement in blepharitis is an encouraging finding as it has not been reported in other dry eye trials. It was hypothesised that TOSM would benefit ocular surface disorders by improving ocular surface health. In vitro, primary and cell line human corneal epithelial cells were supported by TOSM v1 and TOSM v2. Outgrowth from limbal explants and corneal reepithelialisation following wounding occurred with TOSM v2. This and the impression cytology findings support our hypothesis. Further, ocular surface damage with dry eye and PED may activate the corneal wound healing response. For wound healing, compared to human serum, TOSM v1 and TOSM v2 had beneficial effects in vitro on epithelial cells and human corneal fibroblasts. This may translate into a reduction in potentially vision-threatening corneal scarring in vivo with TOSM. However, ocular surface disorders are a heterogenous group and wound healing is a complex process such that different preparations of TOSM may be needed for use in different disorders and at different stages of the disease process.

Current understanding of sensitivity and sensation experienced across the ocular surface remains limited. This project explored the regional variation of corneal sensitivity and transducer function, interaction of sensory and autonomic nerves in the lacrimal functional unit, and the ocular surface sensitivity in Dry Eye and with silicone hydrogel (SH) lens wear. Experiments were undertaken, using Belmonte esthesiometer to deliver pneumatic mechanical, chemical and thermal stimuli and Cochet-Bonnet esthesiometer for tactile stimuli, to the cornea and conjunctiva. Psychophysical methods were used to determine the thresholds of stimulus detection, and the magnitude of sensations to suprathreshold stimulation was estimated assuming Steven’s power law. Additionally, tear secretion in response to corneal sensory input was determined by tear meniscus height measured using Optical Coherence Tomography. Sensitivity to pneumatic cool and mechanical stimuli varied slightly across the cornea while chemical sensitivity was not different between regions. The transducer function was also similar between central and peripheral cornea but different between stimulus modalities. In comparison, the reflex tearing response to suprathreshold stimuli was greater with central corneal stimulation. Also, corneal and conjunctival hypersensitivity was found in the dry eye symptomatic group, and it appeared to be associated with symptom severity, tear film stability and corneal epitheliopathy. Refitting with SH lenses after an initial no-lens interval led to increased conjunctival pneumatic mechanical sensitivity, while corneal tactile sensitivity showed a decrease. In addition, corneal staining induced by certain lens-solution combination appeared to be accompanied by increased corneal and conjunctival sensitivity. In conclusion, the position-invariant corneal sensitivity to pneumatic mechanical, chemical and thermal stimuli suggests that the distribution of human corneal sensory fibres may be more homogeneous than previously hypothesised. The mechanisms mediating the sensory aspect of corneal nociception may be similar across the cornea, while, perhaps due to the importance of the visual axis, the tear reflex response to central and peripheral cornea seems to be driven by different neural circuitry, perhaps at the higher levels of the sensory processing pathway. It appears that alteration in sensory processing of the ocular surface occurs in Dry Eye and accompanies SH lens-solution-induced corneal staining. This altered sensitivity seems to be more prominent in the conjunctiva than in the cornea.

Current understanding of sensitivity and sensation experienced across the ocular surface remains limited. This project explored the regional variation of corneal sensitivity and transducer function, interaction of sensory and autonomic nerves in the lacrimal functional unit, and the ocular surface sensitivity in Dry Eye and with silicone hydrogel (SH) lens wear. Experiments were undertaken, using Belmonte esthesiometer to deliver pneumatic mechanical, chemical and thermal stimuli and Cochet-Bonnet esthesiometer for tactile stimuli, to the cornea and conjunctiva. Psychophysical methods were used to determine the thresholds of stimulus detection, and the magnitude of sensations to suprathreshold stimulation was estimated assuming Steven’s power law. Additionally, tear secretion in response to corneal sensory input was determined by tear meniscus height measured using Optical Coherence Tomography. Sensitivity to pneumatic cool and mechanical stimuli varied slightly across the cornea while chemical sensitivity was not different between regions. The transducer function was also similar between central and peripheral cornea but different between stimulus modalities. In comparison, the reflex tearing response to suprathreshold stimuli was greater with central corneal stimulation. Also, corneal and conjunctival hypersensitivity was found in the dry eye symptomatic group, and it appeared to be associated with symptom severity, tear film stability and corneal epitheliopathy. Refitting with SH lenses after an initial no-lens interval led to increased conjunctival pneumatic mechanical sensitivity, while corneal tactile sensitivity showed a decrease. In addition, corneal staining induced by certain lens-solution combination appeared to be accompanied by increased corneal and conjunctival sensitivity. In conclusion, the position-invariant corneal sensitivity to pneumatic mechanical, chemical and thermal stimuli suggests that the distribution of human corneal sensory fibres may be more homogeneous than previously hypothesised. The mechanisms mediating the sensory aspect of corneal nociception may be similar across the cornea, while, perhaps due to the importance of the visual axis, the tear reflex response to central and peripheral cornea seems to be driven by different neural circuitry, perhaps at the higher levels of the sensory processing pathway. It appears that alteration in sensory processing of the ocular surface occurs in Dry Eye and accompanies SH lens-solution-induced corneal staining. This altered sensitivity seems to be more prominent in the conjunctiva than in the cornea.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 116-130). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 116-130). Also available in print.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The BRB consists of both capillary endothelial cells (REC) and retinal pigment epithelial cells (RPE). Both cell types have been suggested as potential activators of circulating T cells. In this study, an <I>in vitro</I> model using cultured rat REC and syngeneic T cells was developed. Furthermore RPE and for the purpose of comparative studies, AEC were also successfully cultured. It was demonstrated that activation of T lymphocyte LFA-1 is a critical event governing the adhesion of T cells to RPE and REC as IFN-γ induced up-regulation of RPE and REC ICAM-1 expression did not increase binding of resting T lymphocytes. The enhanced adhesion of activated lymphocytes (but not resting lymphocytes) to normal and IFN-γ treated RPE and REC was inhibited by LFA-1 mAb and to a lesser extent by ICAM-1 mAb but not OX34 (CD2). Treatment of lymphocytes with the anti-VLA-4 mAb resulted in differential effects on binding to AEC and REC. MAb to VLA-4 significantly blocked enhanced adhesion of activated T cells to AEC but not to REC. The results also demonstrated that VLA-4 mAb significantly inhibited unactivated T cell binding to IFNγ+TNFα+LPS stimulated AEC but not REC, suggesting that VLA-4 may also function in an activation-independent manner. It was shown that activation of T cells can enhance their migratory activity across cultured REC monolayers. Migration was decreased by both adhesion receptor-dependent mechanisms i.e., mAb to LFA-1 (but not ICAM-1) and adhesion receptor-independent mechanisms by means of PGE<SUB>2</SUB>. The results of this thesis have shown that activation of LFA-1 is required for functioning of the LFA-1/ICAM-1-mediated lymphocyte adhesion and migration. In addition to the role of adhesion molecules, inflammatory mediator PGE<SUB>2</SUB>, but not NO<SUP>o</SUP>, was found to be important in regulation of T cell adhesion and migration across REC.

<p>The visual system in humans provides the ability to interpret our surroundings from many distances. This complex system serves as a powerful sense which can drastically impact the quality of life when threatened or eliminated. While the mechanisms involved in visual interpretation are largely understood, many of the mechanisms of ocular diseases remain elusive. The most common ocular disorders are refractive errors, where failure of normal growth processes results in eye components with shape and sizes that are not matched to provide uncorrected sharp visual acuity without correction. Myopia, or nearsightedness, is a refractive error with prevalence rates of epidemic proportions in some urban Asian settings, and rising in other developed countries. Pathological, or high myopia, has an increased risk for potentially blinding ocular morbidities which can be irreversible and further negatively impact quality of life. Myopia, like other common ocular disorders, results from a combination of environmental and genetic factors. Over 20 candidate genomic regions have been identified as involved in myopic development progression. </p><p>One such locus, <italic>MYP3</italic>, on chromosome 12q21-23 spans nearly 44 Mb with more than 200 protein-encoding genes mapped within. Sizable candidate disease genomic regions typically require refinement to identify genes or variants within them which may contribute to disease development. Without an understanding of the underlying mechanistic framework of a disease, as is the case with myopia, biological inferences are difficult to make in prioritizing candidates, which can make finding true disease causing variants seem like finding a needle in a haystack. A better understanding of human ocular growth, as it relates to refractive error, may lead to more knowledgeable approaches to identifying the cause(s) of myopic development and associated ocular diseases. </p><p>To identify genes involved in ocular growth and development, whole genome expression patterns were assessed in human ocular tissues of fetal versus adult eyes, and adult posterior versus peripheral tissues. No database exists of fetal ocular tissue gene expression. In addition to providing insights into expression patterns during ocular development, these tissues were also compared as a surrogate to study rapid eye growth states such as in myopia. Only ocular tissue types with clinical phenotypes associated with myopic development were considered. Human retina/retinal pigment epithelium (RPE), choroid, sclera, cornea* and optic nerve* tissues were isolated from fetal (N=9; *N= 6) and adult (N=6) normal donor eyes. The Illumina® whole genome expression microarray platform was used to assess differential expression of genes. Fetal tissues were compared to their adult counterparts while adult posterior tissues were compared to their peripheral counterparts, and the differences in each were assessed using Ingenuity Pathway Analysis (IPA) for enriched functional groups and canonical pathways. Statistical significance for all tissue comparisons was determined using the Benjamin and Hochberg False Discovery Rate (FDR, 5%). Differentially expressed genes were compared to previously identified candidates for myopic development.</p><p>Additionally, qualitative and quantitative association studies in a large family (N=82) based high myopia cohort by genotyping 768 single nucleotide polymorphisms (SNPs) in the peak linkage area was performed to fine map the <italic>MYP3</italic> linkage peak. Qualitative testing for high myopia (&#8804; -5 diopter (D) affected, > -5 D unaffected) and quantitative testing on the average (avg) dioptric sphere (SPH) was performed. Five candidate SNPs were genotyped in a replicate high myopia cohort for independent validation. Additionally, the most significant SNPs were screened in a previously genotyped twin cohort as a second independent validation cohort.</p><p>Ocular growth expression data were used to help prioritize the resulting association candidates as supporting evidence and was not used on its own to identify or exclude candidates. Candidate genes (within 100 kilobases (kb) of highly associated SNPs) identified through either qualitative or quantitative association testing were screened in the most disease relevant tissues (retina/ retinal pigment epithelium (RPE), choroid and sclera) for differential expression during ocular growth and by physical regions of the tissues within the eye. Genes that were identified by microarray studies as being differentially expressed in one or more tissue were validated using quantitative real time PCR (RT-qPCR). </p><p>Significant gene expression changes with fold changes > 1.5 were found in adult versus fetal retina/RPE (N=1185), choroid (N=6446), sclera (N=1349), and cornea (N=3872), but not the optic nerve nor any of the central versus peripheral tissues. In all adult versus fetal tissues, differentially expressed genes belonging to cancer, development, and cell death/growth functional groups, as well as signaling canonical pathways were enriched. Seventeen genes previously associated with increased susceptibility for non-syndromic high myopia were in the most significant functional assignments for at least one adult versus fetal ocular tissue. In adult central versus peripheral tissues, there was considerably more variation by tissue in enriched functions and canonical pathways of differentially expressed genes. The only functional category shared by all three tissue types was development. </p><p><italic>MYP3</italic> association testing yielded several genetic markers as nominally significant in association with high myopia in qualitative testing including <italic>rs3803036</italic> (p=9.1X10-4), a missense mutation in <italic>PTPRR</italic>; and <italic>rs4764971</italic> (p = 6.1X10-4), an intronic SNP in <italic>UHRF1BP1L</italic>. After correction for multiple testing, quantitative tests found statistically significant SNPs<italic> rs4764971</italic> (p = 3.1x10-6), also found by qualitative testing; <italic>rs7134216</italic> (p = 5.4X10-7), in the 3<super>1</super> UTR of <italic>DEPDC4</italic>; and <italic>rs17306116<iitalic> (p < 9X10-4), intronic within <italic>PPFIA2<italic>. The intronic SNP in <italic>UHRF1BP1L</italic>, <italic>rs4764971</italic>, was validated for association with the quantitative trait of sphere (SPH) using an independently collected non-syndromic, high myopia cohort. SNPs within <italic>PTPRR</italic> (for quantitative association) and <italic>PPFIA2</italic> (for qualitative and quantitative association) both approached significance in the independent high myopia cohort.</p><p>As with screening genes previously implicated in myopic development, qualitative and quantitative association candidates were screened in the independent whole genome expression array analyses, comparing normal rapidly growing fetal to normal grown adult ocular tissues. <italic>PTPRR</italic> and <italic>PPFIA2<italic>, candidates from qualitative and quantitative association respectively, were both validated by RT-qPCR with differential expression in at least one disease relevant ocular tissue. <italic>PTPRR</italic> and <italic>PPFIA2<italic> belong to the same gene family- that of protein tyrosine phosphatase (PTP) genes. This family of genes relays extracellular signals that regulate cell growth, division, maturation and function, and its differential expression is consistent with our myopia surrogate model. </p><p>Many genes implicated in either syndromic or non-syndromic myopia were present in the most significantly enriched adult versus fetal functional and/or canonical pathways together. The adult versus fetal choroid and cornea tissue types had the most overlap with known non-syndromic myopic-associated genes in the most significantly enriched functional groups. Further exploration of the connections amongst these known genes may elucidate possible mechanistic roles for disease progression and/or reveal related novel candidate genes. Differentially expressed genes in central versus peripheral tissues yielded minimal overlap with genes implicated in myopia; however, in addition to broadening our understanding of the spatial variances in these tissues they may contain clues to the development and/or progression of other ocular diseases such as retinopathy of prematurity development.</p><p>The overlap with previously identified myopia-associated genes supports the model of eye growth for studying myopic development in human tissues. This expression data can be used both in prioritizing candidate genes other proposed genomic myopia loci, and also in detailed pathway analyses to identify potential biological mechanisms for candidates within these loci. Our most strongly associated candidate gene both in the discovery and replicate cohort was <italic>UHRF1BP1L</italic>, which was not differentially expressed in our data; however, interacting genes regulate the expression of at least one differentially expressed gene, indicating a possibly pathway connection. It is possible that differential expression may have been missed by the microarray data, or it may not be differentially expressed and affects myopic development through alternative or indirect means. While the expression data is a useful tool in prioritizing and inferring mechanistic roles for candidates, it cannot be used to exclude candidates. Deeper study of the pathways of candidate genes for myopic development may reveal connections to genes involved in ocular growth. Despite these potential limitations, two of the three novel candidates, <italic>PTPRR</italic> and <italic>PPFIA2</italic>, were supported by genomic convergence with the expression data, in addition to our discovery genetic association data. The other novel candidate, <italic>UHRF1BP1L</italic>, was validated in an independent Caucasian high-grade myopia cohort. Further validation and refinement of these three novel <italic>MYP3<italic> candidate genes is necessary to make further claims about their possible involvement in myopic progression.</p> / Dissertation