Nesta pesquisa, os aspectos macroestruturais, microestruturas e histoquímicos do complexo ganglionar celíaco foram investigados em 6 cães domésticos saudáveis e 6 cães domésticos acometidos pela diabetes mellitus, com o objetivo de identificar possíveis diferenças estruturais, morfométricas e quantitativas nos neurônios ganglionares celíacos. desta forma, observamos o gânglio celíaco esquerdo originou-se junto à origem da artéria celíaca esquerda e apresentava um formato irregular, seu aspecto era nodular, caracterizando um complexo ganglionar. Em relação a histoquímica, foram encontrados neurônios NADPH-diaphorase positivos, apresentando reação visível. A positividade da reação permitiu-nos concluir que há neurônios potencialmente nitrérgicos no gânglio celíaco de animais sadios e diabéticos. No entanto, não podemos afirmar se estes neurônios são efetivamente nitrérgicos, porque exige a combinação de outras reações imunohistoquímicas. O tamanho do corpo celular do neurônio do gânglio celíaco, representado pela sua área seccional, aumentou significativamente nos animais do grupo diabético quando comparado aos do grupo controle, tendo um fator de aumento de 1,3x. O aumento na área seccional do núcleo no grupo diabético foi de 1,39x maior que no grupo controle, sendo este aumento significativo. Em termos práticos isto significa que nos animais diabéticos, o núcleo e o corpo celular neuronal aumentam em proporções muito próximas. / In this research, the macrostructural, microstructural and histochemical aspects were investigated in 6 clinacally health dogs and 6 diabetic dogs aiming to identify the possible alterations on the strucutre, morphometric and qualitative parameteres of neurons of the celiac ganglionar complex. Our observations revealed that the celiac ganglion was originated close to the origin of the celiac artery and showed a irregular aspect, being caractherized as a complex. Histochemically, neurons NADPH-diaphorase reactives were identified. This positive reaction, on both health and diabetic animals, led us to conclude this neurons were potentially nitrergic, besides, to confirm the nitrergic aspect for this neurons it is necessary to combine immunohistochemical method. Neurons celular body size of the celiac ganglion, represented by its sectional area, increased significantly, at 1.3x rate, on the diabetic animals when compared to the health animals. The increase on the nuclear sectional area of the diabetic animals were significant, at 1.39x rate larger than the other group. These results mean that on the diabetic animals, nuclear and neuronal body celular raise rate increases proportionally.

The general aim of the thesis was to deduce whether selected naturally occurring flavonoids (genistein, epicatechin gallate (EC), epigallocatechin gallate (EGCG), baicalin) attenuate various secondary insults that may cause death of ganglion cells in primary open angle glaucoma (POAG). An ischemic insult to the rat retina significantly causes the inner retina to degenerate indexed by changes of various antigens, proteins and mRNAs located to amacrine and ganglion cells. These changes are blunted in animals treated with genistein as has been shown for ECGC. Studies conducted on cells (RGC-5 cells) in culture showed that hydrogen peroxide, L-buthionine sulfoximine (BSO)/glutamate and serum deprivation (mimicking oxidative stress), rotenone, sodium azide (affecting mitochondria function in specific ways) and light (where the mitochondria are generally affected) all generated reactive oxygen species and caused death of RGC-5 cells. EGCG was able to attenuate cell death caused by hydrogen peroxide, sodium azide and rotenone. Only EC was able to attenuate BSO/glutamate-induced cell death, in addition to cell death caused by hydrogen peroxide and rotenone. Genistein had no positive effect on cell death in experiments carried out on RGC-5 cells. Exposure of RGC-5 cells to flavonoids showed that EC and EGCG increased the mRNA expression of endogenous antioxidants such as HO-1 (heme oxygenase 1) and Nrf-2 (nuclear erythroid factor-2-related factor 2). Light insult, rotenone and sodium azide activate the p38 (protein kinase 38) pathway, while only light and rotenone activate the JNK (c-Jun amino-terminal kinase) pathway. Serum deprivation affects mitochondrial apoptotic proteins causing an increase in the ratio of Bax/Bcl2 (Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2). An insult of light to RGC-5 cells, unlike that induced by sodium azide, is inhibited by necrostatin-1 and causes an activation of AIF (apoptosis-inducing factor) with alpha-fodrin being unaffected. These studies suggest that ganglion cell death caused by insults as may occur in POAG involves various cellular signaling pathways. The selected flavonoids have diverse actions in increasing cellular defense mechanisms, and in negating the effects of ischemia and specific types of oxidative stress. The results argue for the possible use of flavonoids in the treatment of POAG to slow down ganglion cell death.

Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4E(S209A)). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca2+ imaging experiments on dorsal root ganglion neurons, NGF-and IL-6-induced increases in excitability were attenuated in neurons from eIF4ES209A mice. These effects were recapitulated in Mnk1/2(-/-) mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4ES209A and Mnk1/2 (-/-) mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus affects 1 in 1,000 newborns and nearly 1,000,000 Americans, leading to an increase in intercranial pressure due to the build-up of cerebrospinal fluid. There are numerous complications that arise as a result of hydrocephalus, but this study focuses on optic disc edema. The subarachnoid space surrounding the optic nerve contains cerebrospinal fluid. The cerebrospinal fluid increases in hydrocephalus, putting pressure on the optic nerve. The additional intracranial pressure has been proposed to cause axoplasmic stasis within the retinal ganglion cell axons, leading to axonal damage and retinal ischemia. The purpose of this study was to determine the effects of hydrocephalus on the optic disc and retina in several animal models of hydrocephalus. This study uses two genetic and two injury-induced models of hydrocephalus in addition to immunohistochemistry and histological stains to examine the optic disc, thickness of retinal layers, and numbers of retinal cells. This study serves as preliminary work to help build the case that hydrocephalus causes cell loss in the retina, as well as swelling of the retinal ganglion cell axons, leading to axoplasmic stasis and cell death. / Indefinitely

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Indiana University-Purdue University Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) have the ability to self renew indefinitely while maintaining their pluripotency, allowing for the study of virtually any human cell type in a dish. The focus of the current study was the differentiation of hPSCs to retinal ganglion cells (RGCs), the primary cell type affected in optic neuropathies. hPSCs were induced to become retinal cells using a stepwise differentiation protocol that allowed for formation of optic vesicle (OV)-like structures. Enrichment of OV like structures allowed for the definitive identification of RGCs. RGCs displayed the proper temporal, spatial, and phenotypic characteristics of RGCs developing in vivo. To test the ability of hPSC-RGCs to serve as a disease model, lines were generated from a patient with an E50K mutation in the Optineurin gene, causative for normal tension primary open angle glaucoma. E50K RGCs displayed significantly higher levels of apoptosis compared to a control lines. Apoptosis was reduced with exposure to neuroprotective factors. Lastly, hPSC-derived RGCs were studied for their ability to develop functional features possessed by mature in vivo RGCs. hPSC-derived RGCs displayed a few immature functional features and as such, strategies in which to expedite synaptogenesis using hPSC-derived astrocytes were explored. Astrocyte and RGG co-cultures displayed expedited synaptic and functional maturation, more closely resembling mature in vivo RGCs. Taken together, the results of this study have important implications for the study of RGC development and by extension, the advancement of translational therapies for optic neuropathies.

Indiana University-Purdue University Indianapolis (IUPUI) / Retinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Reti- nal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on charac- terizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner express- ing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composi- tion and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to de- termine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs dis- played recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from xvii the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC develop- ment, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.

Glaucoma is a group of ocular disorders characterized by optic nerve damage that leads to vision loss and blindness. Damage to retinal ganglion cells (RGCs), particularly through axonal damage due to an increase in intraocular pressure (IOP), is a proposed mechanism behind glaucomatous injury. Other than increased IOP, vascular changes leading to ischemia are another explanation for glaucoma. A state of ischemia leads to a decrease in nutrients supplied to neurons of the retina and creates a hypoxic environment which is linked to cell death in both IOP- and non-IOP-related injury. Injury during glaucoma not only affects RGCs but also has secondary effects that impact the function of other cells in the retina like amacrine cells (ACs). To better understand how RGCs and ACs respond during glaucomatous injury, this study characterized the changes in viability of these cells under hypoxic conditions over time. With the use of a unique immunopanning technique, RGCs and two subpopulations of ACs (CD15+ and CD57+) were isolated from 12-week-old C57BL/6J mice and cultured for 6 to 9 days. After about a week of culturing, the three cell types were placed under either normoxic (n = 5) or hypoxic (n = 6) conditions, and cell viabilities were measured at 1-hour time intervals over 24 hours. RGC and AC isolations based on the immunopanning technique resulted in high yield and viability, confirming the findings of previous optimization studies. In response to hypoxic conditions, RGCs and the two subpopulations of ACs all experienced a decrease in cell viability over the course of 24 hours. Surprisingly, CD57+ cells showed increased susceptibility to injury and death during isolation. However, the remaining CD57+ cells that stayed alive in culture by the start of the time-course experiment were the most resilient to cell death during hypoxia, showing significantly higher cell viability compared with CD15+ and Thy1.2+ cells. The characterization of CD15+, CD57+, and Thy1.2+ cells in response to hypoxia highlights a difference in resilience across neuronal cell types in the retina. Although CD57+ exhibited greater resilience than its counterparts, the mechanism behind neuroprotection among these cells is still unknown and requires further study. / 2024-01-28T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20244号 / 医博第4203号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 伊佐 正, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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