A demência é caracterizada como um declínio progressivo nas funções cognitivas e perda grave de memória. A doença de Alzheimer (DA) é a forma mais comum de demência. Dentre as características neuropatológicas, a doença caracteriza-se por perdas seletivas neuronais e sinápticas, presença de placas neuríticas extracelulares contendo o peptídeo β- amilóide (Aβ) e emaranhados neurofibrilares (NFTs) compostos de formas hiperfosforiladas da proteína tau. Diversos modelos animais têm sido desenvolvidos para investigar a etiologia da DA, embora ainda não estabelecida, muitos fatores de riscos e alterações patológicas tem sido utilizados para a elaboração de modelos in vivo e in vitro da DA. O ácido ocadáico (AO), um potente inibidor de fosfatase 1 e 2A, ocasiona a deposição de Aβ, perda neuronal e sináptica, e subsequente déficit de memória, características semelhantes às ocorridas na DA. O objetivo deste trabalho foi avaliar uma possível disfunção cognitiva e alterações nos parâmetros bioquímicos astrocitários de ratos submetidos ao ácido ocadáico, intrahipocampal, como modelo de demência. Ratos Wistar machos (90 dias) foram submetidos à infusão intrahipocampal e bilateral de AO (100 ng), e 12 dias após a cirurgia, avaliada a memória pelo teste de memória espacial no labirinto aquático de Morris, e realizados testes bioquímicos. Usando este modelo animal de infusão do AO, nós observamos um déficit cognitivo espacial e alterações neurogliais, particularmente, em marcadores de astrogliose, como a proteína glial fibrilar ácida (GFAP) e S100B. Houve alterações no metabolismo do glutamato, pela diminuição significativa do transportador de aminoácido excitatório 2 (EAAT2/GLT-1) e atividade da glutamina sintetase; bem como, é característico na DA, um aumento no estresse oxidativo, observado pelo aumento das proteínas carboniladas e diminuição no conteúdo de glutationa (glutationa reduzida) no modelo de infusão do AO. Foram observadas também, alterações na via das MAPKs, onde a infusão do AO aumentou a fosforilação da p38MAPK, não alterando outras MAPKs, como a JNK 1/2 e ERK 1/2. Em conclusão, a injeção bilateral de AO foi capaz de induzir um déficit cognitivo espacial e estresse oxidativo no modelo de demência proposto, e pela primeira vez ao nosso conhecimento, alterações astrogliais em modelo de demência induzido pelo AO. Por fim, as alterações astrogliais observadas neste modelo de demência contribuem para a compreensão da fisiopatologia da DA e de outras doenças acompanhadas de déficit cognitivo, bem como contribuem para a identificação de alvos terapêuticos para a DA. / Dementia is characterized as a progressive decline in cognitive functions and severe memory loss. Alzheimer’s disease (AD) is the most common form of dementia. The defining neuropathological characteristics of AD are the presence of extracellular accumulation of aggregated β-amyloid in senile plaques in the brain, and also by intraneuronal aggregates of neurofibrillary (NFT) consisting of hyperphosphorylated tau proteins, wich leads to progressive brain dysfunction. Although the cause of AD remains elusive, many possible risk factors and pathological alterations have been used in the elaboration of in vivo and in vitro models of this disease, including intrahippocampal infusion of okadaic acid (OA). Okadaic acid (OA), a potent inhibitor phosphatase 1 and 2A, leads to the deposition of β-amyloid, subsequent neuronal degeneration, synaptic loss and memory impairments, all of which resemble AD-like pathology. In this study, our aim was evaluate spatial cognitive deficit and neuroglial alterations in rats submitted to the OA-induced dementia model. Male Wistar rats (90 days old) were submitted to bilateral intrahippocampal OA-injection (100 ng), and 12 days after the surgery the rats were submitted to training and test in the Morris water maze, and biochemistry tests were performed. Using this model, we evaluated spatial cognitive deficit and neuroglial alterations, particulary astroglial protein markers such as glial fribillar acidic protein (GFAP) and S100B. There have been changes in the metabolism of glutamate, the significant decrease in the excitatory amino acid transporter 2 (EAAT2/GLT-1) and glutamine synthetase activity, as is characteristic of AD, increased oxidative stress, observed by increased protein carbonyl and decreased glutathione in rats submitted to injection of OA. Were also observed, alterations in the MAPKs pathway, where the infusion of OA increased the phosphorylation of p38MAPK, without altering other MAPKs, such as JNK 1/2 and ERK 1/2. In conclusion, bilateral injection of OA induces spacial cognitive deficit, and causes oxidative stress in this model and demonstrate, for the first time to our knowledge, neuroglial alteriations. Findings contribute to understanding diseases accompanied by cognitive deficit and the OA model of dementia.

No description available.

The mammalian brain is a complex organ, the result of a very specific and regulated differentiation process. Although there are many different cell types in the mammalian brain, neurons make up the bulk of the tissue. Neurons come from the divisions of radial glial progenitors (RGPs), which are columnar stem cells in the developing brain. These cells undergo two types of division: symmetric or asymmetric. Symmetric divisions expand the stem cell population, resulting in two new RGPs. Symmetric divisions are critical for ensuring the stem cell population is not depleted too quickly in development. Asymmetric divisions are neurogenic, producing one RGP and one cell that will either differentiate into one neuron, or an intermediate progenitor (IP) that will divide again and produce two to four neurons (Shitamukai, Konno, and Matsuzaki 2011). Several factors have been linked to this determination, including mitotic spindle orientation, centrosomal inheritance, and exposure to proliferative factors, like sonic hedgehog and Notch (Chenn and McConnell 1995; Gaiano and Fishell 2002; Han 2016). This work will focus on spindle orientation, which has been linked to cell fate in many contexts (Lancaster and Knoblich 2012; Williams and Fuchs 2013; Chenn and McConnell 1995). Spindle orientationmust be tightly controlled in order to expand the RGP cell population in early development, then, with more randomized spindles, to shift to producing neural precursors during cortical expansion (Götz and Huttner 2005). While the exact mechanism is still unknown, the orientation of the mitotic spindle relative to the ventricular surface at the time of division affects what type of division occurs (Lancaster and Knoblich 2012). A related process in RGP neural production is interkinetic nuclear migration (INM), in which the RGP nucleus travels apically and basally in a cell-cycle dependent manner (Noctor et al. 2001; Sauer 1935; Hu et al. 2013). The RGP only divides when the nucleus reaches the apical surface; why this occurs is still not known. INM ensures that only a small population of RGPs is dividing in a controlled manner, allowing for cells to interpret polarity cues and orientthe spindle while dividing. One protein that is important to multiple processes in neuronal development is Kif1A. Kif1A is a kinesin motor that has been shown to be critical for INM, in particular for transporting the nucleus basally after division. When Kif1A expression is reduced using shRNA, RGPs fail to migrate away from the ventricular surface, but continue to go through the cell cycle at a normal rate (Carabalona, Hu, and Vallee 2016). Additionally, RGPs that lack Kif1A also exhibit more horizontal and symmetric divisions. This indicates that Kif1a is involved in asymmetric, oblique divisions that produce neurons. Thus, without Kif1a, RGPs produce fewer neurons, instead expanding the RGP cell population. Another kinesin that may be involved in spindle orientation is Kif13B. Kif13B is in the same kinesin-3 subfamily as Kif1A. While structurally very similar to Kif1A, it does have distinct features. It contains a CAP-gly domain, used for binding to the plus end of microtubules. This domain is absent from other kinesin-3 family members, including the most closely related,Kif13A. Kif13B has been shown to be critical for spindle orientation in polarized Drosophila S2 cells, as well as in neuroblasts (Carabalona, Hu, and Vallee 2016; Siegrist and Doe 2005). Kif13B functions to anchor the mitotic spindle to other factors at the cell cortex during mitosis. This occurs through direct interaction with Discs large (Dlg1), which then connects to other factors at the cell membrane, including G?i, LGN, and NuMA. This is a critical process to ensure daughter cells are properly specified. Many of these factors, including LGN and NuMA have been identified as important spindle regulators in RGP divisions as well. Kif13B binds to Dlg1 and to 14-3-3 ?, which is bound to 14-3-3 ?, bound to NudE and Dynein, connecting the Kif13B to Dynein (Lu and Prehoda 2013). Kif13B, as a kinesin, moves along microtubules towards the plus end. Dynein moves in the opposite direction, towards the minus end. The connection of two opposing motors moving in opposite directions may serve to put tension on the spindle and prevent it from freely moving within the cell. When Kif13B is knocked down or removed in cells, the spindle orients randomly in the cell, not in line with LGN or NuMA at the cell cortex (Siegrist and Doe 2005; Lu and Prehoda 2013). This indicates that in mammalian systems, it likely is important for maintaining orientation, and its loss in RGPs would result in random orientation as well. This would result in more neurogenic divisions in RGPs, which is the opposite of the effect seen with Kif1a shRNA. By using in utero electroporation of embryonic rat brains as well as a mouse model ofKif13b knockout in RGPs, I have shown that Kif13B and Kif1A have opposing roles in neurogenesis. This difference can be traced to an alteration of IP production, which Kif1A shRNA decreases, and Kif13b shRNA increases. This can be further traced to the opposing effects on spindle orientation of dividing RGPs. Kif1a shRNA results in more horizontal spindle angles while Kif13b shRNA or deletion results in more random spindle angles. While the kinesin-3 family members are very similar in structure, there are key differences between them. Kif1A has a cargo binding domain at its C terminus, the pleckstrin homology (PH) domain. Kif13B contains a CAP-gly domain. This difference in tail domains would presumably allow Kif13B to bind to microtubule plus ends, while Kif1A would dissociate from the spindle. This difference, therefore, could explain why these two very similar kinesins appear to be performing the opposite roles in spindle orientation. This work provides evidence for a novel mechanism of regulation of neuron production in the mammalian cortex.

Understanding the biological basis of behavior is crucial for gaining insights into human nature, treating behavioral disorders and improving overall well-being. Efforts to understand the biological basis of behavior have largely emphasized the role of neurons. However, examples across life show that behavior can occur in lieu of or in cell types outside of neurons. This thesis presents work exploring the mechanisms underlying multiple behaviors using the model system Drosophila melanogaster. Chapter 2 provides evidence for the influence of glia on courtship, aggression, and sleep. Chapter 3 characterizes a Drosophila model of autism spectrum disorder, finding that genetic neuronal manipulation of a single gene can cause pathologies beyond the nervous system. Chapter 4 investigates the connection between sleep behavior and oxidative stress response, demonstrating metabolism as a probable mediator of this relationship. Altogether, this work supports a wider definition for the biological basis of behavior.

<p> Mammalian sialidases are hydrolytic enzymes that initiate the removal of terminal a2-3, a2-6 and a2-8 sialic acid residues from various sialylated glycoconjugates. Sialidases are reportedly involved in numerous cellular functions involving proliferation, differentiation, antigenic expression, inflammation and the tumorigenicity of malignant cells. Recently, sialidase has been implicated in various immune signaling pathways, involving immune effector cells, such as activated lymphocytes and macrophages. The human lysosomal sialidase gene encodes a 46 kD glycoprotein which exists in a multienzyme complex with β-galactosidase and PPCA. Neurodegenerative diseases such as Tay-Sachs and Sandhoff are characterized by the progressive storage of glycoproteins and sialylated oligosaccharides in the nervous system. The induction of inflammatory mediators is a critical step in the pathogenesis of neurodegeneration that remains largely undefined. As such, an in vitro model of Tay-Sachs disease was used to identify potential mediators involved in disease progression. In addition, we have used the THP-1 monocytic cell line as a model of human macrophages which play a key role in potentiating a variety of immune responses. </p> <p> Translocation of neul from lysosomes to the cell surface and the resulting interaction with signaling molecules suggests neul is involved in the regulation of immune activities. We have investigated the role of sialidase on CD44 expression, an inflammation-associated glycoprotein found on the cell surface. Our data indicate that sialidase interacts with CD44 on the cell surface which may contribute to disease progression in Tay-Sachs disease. We illustrate that overexpression of sialidase stimulates interleukin-6 (IL-6) secretion in both human Tay-Sachs neuroglia and THP-1 derived macrophages. Moreover, the sialidase inhibitor 2-deoxy-2, 3-dehydro-N-acetylneuraminic acid (DANA) was found to attenuate IL-6 secretion and sialidase expression in THP-1 derived macrophages. </p> / Thesis / Master of Science (MSc)

O estímulo pela atividade física pode mediar a ação do IGF-1 sobre a indução à neuroplasticidade, modular o neurometabolismo e influenciar a memória e a ansiedade. Entretanto, a atividade física também pode induzir à hipoglicemia, condição que pode ser revertida com a administração de rápida fonte de glicose provinda da alimentação. Assim como a atividade física, a dieta exerce forte influência nos processos moleculares, celulares e comportamentais e estudos analisando o efeito exclusivo de dietas ricas em açúcar revelam prejuízos sobre a memória e favorecimento à ansiedade. Portanto, analisar os efeitos moleculares e comportamentais num protocolo que contemple atividade física e dieta não exclusiva de xarope de milho podem corroborar ou discordar das pesquisas que analisam os mecanismos de uma dieta exclusiva. O objetivo desse trabalho foi verificar os efeitos da administração de xarope de glicose do milho a 15% após atividade física aguda forçada em esteira sobre a memória, a ansiedade, a atividade locomotora, a neuroplasticidade e a morte celular de camundongos machos. Foram formados 4 grupos, sendo sedentário controle (SED), treinado controle (TRE), sedentário administrado (SED15%) e treinado administrado (TRE15%). O regime de treinamento consistiu em atividade física forçada em esteira por 40 min a 0,7km/h por 5 dias. A administração de 15% do xarope de milho se deu sempre após atividade física por período de 1½h. Para avaliação de memória aversiva foi utilizado a esquiva passiva no 4º e 6º dias e para avaliação do comportamento relacionado à ansiedade foi utilizado o labirinto em cruz elevado. Para análise de morte celular foi utilizado kit TUNEL que identifica fragmentação do DNA. Os dados comportamentais foram avaliados estatisticamente através do software Prisma e os demais qualitativamente. Houve influência da administração sobre o menor consumo para o grupo TRE15%, prejuízos à memória aversiva para ambos os grupos, indução ao comportamento relacionado à ansiedade nos animais do grupo SED15%, maior exposição às situações de risco para o grupo TRE15%, incidência considerável e difusa de morte celular na amígdala e importante expressão de IGF-1 nos grupos. A administração de 15% de xarope de glicose do milho posterior à exposição na esteira desencadearam importante expressão de IGF-1, mas não suficiente para proporcionar neuroproteção causando consideráveis e difusos pontos de morte celular na amígdala, prejuízos à memória aversiva, medo induzindo comportamento relacionado à ansiedade para SED15% e significativa maior exposição às situações de risco para TRE15% após protocolo agudo de 5 dias / The stimulation by physical activity can mediate the action of IGF-1 on the induction of neuroplasticity, modulating neural energetic metabolism and influencing memory and anxiety behavior. However, physical activity can also to lead to hypoglycemia, a condition that can be reversed by the administration of glucose. As physical activity, diet has a strong influence on molecular, cellular and behavioral processes and studies analyzing exclusively the effects of high-sugar diets reveal prejudices on memory and favors anxiety. Therefore, analyze the molecular and behavioral effects in a protocol that includes physical activity and non-exclusive diet of corn syrup can corroborate or disagree with the studies that analyze the mechanisms of an exclusive diet. The aim of this work was to investigate the effects of 15% of glucose corn-syrup administration after treadmill training on memory, anxiety, neuronal plasticity and cell death in male mice. Sedentary control (SED), Trained control (TRE), Sedentary administrated (SED15%) and Trained administered (TRE15%) were the 4 groups formed randomly. The training regimen consisted on a treadmill training for 40 min at 0.7km/h during 5 days. The administration of the 15% corn syrup were always given after a 1½h period after physical activity. For evaluation of aversive memory the passive avoidance was used in day 4 and day 6 and for assessment of anxiety-related behavior was used the elevated plus maze. For cell death analysis TUNEL kit that identifies DNA fragmentation was used and immunohistochemistry was applied to analyze IGF-1 expression on amygdala. Behavioral data were evaluated statistically using Prisma software and cell death and IFG-1 expression were analyzed qualitatively. Results: There was influence of administration on the lowest feed consumption to TRE15%, losses to aversive memory for both groups, inducing behavior related to anxiety in SED15%, greater exposure to risk situations for TRE15%, considerable incidence and diffuse cell death the amygdala and important expression of IGF-1 in the groups. The administration of 15% glucose corn syrup after treadmill training triggered an important expression of IGF-1, but not enough to provide neuronal protection causing considerable and diffuse points of cell death in the amygdala, losses to aversive memory, fear inducing behavior related to anxiety to SED15% and significantly greater exposure to risk situations for TRE15% after acute protocol 5 days

Dorsal root ganglia (DRG) cell preparations are commonly used to study the properties of sensory neurons in relation to nociception. A typical DRG cell preparation contains both neurons and glial cells, and in addition to a conventional supportive role of glial cells, an increasing volume of literature has reported interactions between neurons and accompanying glial cells. A typical mixed DRG cell preparation can be separated into a neuron-enriched cell fraction and a preparation of purified glial cells. Using these purified cell fractions, we can study the relative contributions and interactions between neurons and glial cells in regulating neurite outgrowth and adenylyl cyclase-dependent cell signalling activity in vitro. / From our previous studies, pretreating DRG cell cultures with pertussis toxin (PTx) caused neurite retraction over a period of 2 h following the initial stimulus of removal from incubator. The purpose of the current study was to investigate whether this PIx-dependent response was specific to anyone of the three subpopulations of DRG neurons. Interestingly, no neurite retraction response was observed in enriched DRG cultures, including cultures enriched with isolectin B4 (IB4)-positive neurons or IB4-negative neurons. Addition of glial cells or conditioned medium from glial cells to IB4-negative cultures was necessary to restore the PTx-dependent neurite retraction response, which was then only observed in large diameter proprioceptive neurons. To conclude, glial cells constitutively release factor/s that stimulate neurite retraction in larger diameter neurons, and is counterbalanced by neuroprotective Gilo protein signalling pathway. / From our studies, we have provided evidence of bidirectional interactions between neurons and glial cells, with glial cells regulating neurite outgrowth and neurons regulating adenylyl cyclase activity in glial cells. These findings reveal the properties of glial cells in regulating neurite outgrowth and in producing prostanoid-stimulated responses. Moreover, our fmdings provide foundation to understand complex neuron-glia interactions in vivo which will eventually help to overcome obstacles in promoting neurite regeneration and in controlling pain. / In a parallel study, we proved that hyperalgesic agents such as prostaglandin E2 (PGE2) and the prostacyclin (PGI2) mimetic (cicaprost) stimulate cAMP production in DRG cell culture via EP4 and IP receptors, respectively. These prostanoids were presumed to act only on the neurons in typical mixed cell cultures, but since we had acquired purified glial cell preparation, we tested for involvement of glial cells in measurement of agonist-stimulated cAMP production. Interestingly, a purified glial cell cultures also produced EP4 and IP-dependent responses. The expression of EP4 and IP receptors by DRG glia was further confirmed by the detection of EP4 and IP-like immunoreactivity and mRNA. Moreover, these agonist-stimulated responses were greatest in the glial cell preparation, and surprisingly weakest in the neuron-enriched cell cultures. Furthermore, the presence of neurons significantly inhibited both EP4 and IP receptor-dependent signalling in glial cells, but was without effect on forskolin (agonist-independent) stimulation of adenylyl cyclase. In order to characterize this neuron-glia interaction, we tested the adenylyl cyclase activities in glial cell cultures which were treated with conditioned medium derived from neurons or were separated from physical contact with neurons plated on transwell membrane. These studies further suggest that the neuron-glia interactions were dependent on both soluble factors and cell-cell contact. / Ng, Kai Yu. / Adviser: Helen Wise. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 152-172). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O estímulo pela atividade física pode mediar a ação do IGF-1 sobre a indução à neuroplasticidade, modular o neurometabolismo e influenciar a memória e a ansiedade. Entretanto, a atividade física também pode induzir à hipoglicemia, condição que pode ser revertida com a administração de rápida fonte de glicose provinda da alimentação. Assim como a atividade física, a dieta exerce forte influência nos processos moleculares, celulares e comportamentais e estudos analisando o efeito exclusivo de dietas ricas em açúcar revelam prejuízos sobre a memória e favorecimento à ansiedade. Portanto, analisar os efeitos moleculares e comportamentais num protocolo que contemple atividade física e dieta não exclusiva de xarope de milho podem corroborar ou discordar das pesquisas que analisam os mecanismos de uma dieta exclusiva. O objetivo desse trabalho foi verificar os efeitos da administração de xarope de glicose do milho a 15% após atividade física aguda forçada em esteira sobre a memória, a ansiedade, a atividade locomotora, a neuroplasticidade e a morte celular de camundongos machos. Foram formados 4 grupos, sendo sedentário controle (SED), treinado controle (TRE), sedentário administrado (SED15%) e treinado administrado (TRE15%). O regime de treinamento consistiu em atividade física forçada em esteira por 40 min a 0,7km/h por 5 dias. A administração de 15% do xarope de milho se deu sempre após atividade física por período de 1½h. Para avaliação de memória aversiva foi utilizado a esquiva passiva no 4º e 6º dias e para avaliação do comportamento relacionado à ansiedade foi utilizado o labirinto em cruz elevado. Para análise de morte celular foi utilizado kit TUNEL que identifica fragmentação do DNA. Os dados comportamentais foram avaliados estatisticamente através do software Prisma e os demais qualitativamente. Houve influência da administração sobre o menor consumo para o grupo TRE15%, prejuízos à memória aversiva para ambos os grupos, indução ao comportamento relacionado à ansiedade nos animais do grupo SED15%, maior exposição às situações de risco para o grupo TRE15%, incidência considerável e difusa de morte celular na amígdala e importante expressão de IGF-1 nos grupos. A administração de 15% de xarope de glicose do milho posterior à exposição na esteira desencadearam importante expressão de IGF-1, mas não suficiente para proporcionar neuroproteção causando consideráveis e difusos pontos de morte celular na amígdala, prejuízos à memória aversiva, medo induzindo comportamento relacionado à ansiedade para SED15% e significativa maior exposição às situações de risco para TRE15% após protocolo agudo de 5 dias / The stimulation by physical activity can mediate the action of IGF-1 on the induction of neuroplasticity, modulating neural energetic metabolism and influencing memory and anxiety behavior. However, physical activity can also to lead to hypoglycemia, a condition that can be reversed by the administration of glucose. As physical activity, diet has a strong influence on molecular, cellular and behavioral processes and studies analyzing exclusively the effects of high-sugar diets reveal prejudices on memory and favors anxiety. Therefore, analyze the molecular and behavioral effects in a protocol that includes physical activity and non-exclusive diet of corn syrup can corroborate or disagree with the studies that analyze the mechanisms of an exclusive diet. The aim of this work was to investigate the effects of 15% of glucose corn-syrup administration after treadmill training on memory, anxiety, neuronal plasticity and cell death in male mice. Sedentary control (SED), Trained control (TRE), Sedentary administrated (SED15%) and Trained administered (TRE15%) were the 4 groups formed randomly. The training regimen consisted on a treadmill training for 40 min at 0.7km/h during 5 days. The administration of the 15% corn syrup were always given after a 1½h period after physical activity. For evaluation of aversive memory the passive avoidance was used in day 4 and day 6 and for assessment of anxiety-related behavior was used the elevated plus maze. For cell death analysis TUNEL kit that identifies DNA fragmentation was used and immunohistochemistry was applied to analyze IGF-1 expression on amygdala. Behavioral data were evaluated statistically using Prisma software and cell death and IFG-1 expression were analyzed qualitatively. Results: There was influence of administration on the lowest feed consumption to TRE15%, losses to aversive memory for both groups, inducing behavior related to anxiety in SED15%, greater exposure to risk situations for TRE15%, considerable incidence and diffuse cell death the amygdala and important expression of IGF-1 in the groups. The administration of 15% glucose corn syrup after treadmill training triggered an important expression of IGF-1, but not enough to provide neuronal protection causing considerable and diffuse points of cell death in the amygdala, losses to aversive memory, fear inducing behavior related to anxiety to SED15% and significantly greater exposure to risk situations for TRE15% after acute protocol 5 days

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (MMed (Biomedical Sciences. Anatomy and Histology))--University of Stellenbosch, 1993. / Glial fibrillary acidic protein (GFAP) is found in varying amounts in the cytoplasm of most normal and neoplastic cells of astroglial origin. Though not glial specific, immunoelectron microscopy has shown that vimentin and GFAP are coexpressed as monomers of glial intermediate filaments. These structures display irreversible assembly and a slow metabolic turnover. Although currently applied as astroglial markers, these intermediate filament proteins may reflect the functional and developmental differentiation status of the cells in which they are expressed. Some authors have tried to apply these aspects as diagnostic parameters for grades of malignancy and anaplasia whilst other workers have indicated variable concentrations of GFAP in different astroglial cell types and entities. Different processing protocols, including the use of epoxy and acrylic resins, omission of osmium tetroxide and variations in concentration and incubation time of primary fixatives, were evaluated to find a compromise between antigen availability and acceptable ultrastructure. Thin sections were labelled on grid for GFAP (Dako A561) and vimentin (Dako M725) by means of the indirect immunogold method. For semi- quantification of relative antigen concentrations, a novel method was devised to calculate the labelling density, percentage heterogeneity of the particle distribution and the surface area investigated. This allowed expression of labelling results as a three figure unit. Standardized post-embedding immunoelectron microscopy was performed on 11 normal and neoplastic human tissue specimens. The tissue was exposed to conventional immersion fixation in glutaraldehyde and osmium tetroxide prior to modified embedding in LR White resin. The validity of these results was verified by correlation with conventional histopathological, immunohistochemical and clinical data obtained for each specimen. The presence of epoxy resin in thin sections was shown to reduce antigen availability to such an extent that very low to negative labelling was encountered. Acrylic LR White resin allowed more acceptable immunodetection, but at the cost of inferior ultrastructure and greater instability of thin sections in the electron beam. This masked the effects of glutaraldehyde fixation on the density of the tissuefixative matrix which included destruction of the vimentin and some GFAP associated epitopes. Although osmium tetroxide was required for acceptable ultrastructure, it reduced the labelling sensitivity by 20% and was responsible for premature curing of acrylic resin during impregnation of tissue. Despite superior resolution gained by electron microscopy and the advantage of semi-quantification of labeling results, the labelling sensitivity of this technique is lesser than that of light microscopical immunohistochemistry. Immunoelectron microscopy confirmed the association between GFAP and glial intermediate filaments in almost all the glial tumours studied, correlating well with GFAP expression in matching specimens demonstrated at light microscopical level. In the absence of intermediate filaments, no positivity for GFAP or vimentin was found in oligodendroglial components of mixed tumours. GFAP positivity in astrocytomas was demonstrated by between 17 and 126 particles / µm2, whilst lower figures were obtained for the glioblastoma (PD = 8) and some of the mixed gliomas (Pd = 6). Rosenthal fibres showed both peripheral and central positive labelling for GFAP, thus providing more evidence for their hypothetical degenerative, astroglial nature. The meningioma studied, was GFAP negative, but produced low density positivity for vimentin. Coexpression of GFAP and vimentin was demonstrated in an astroblastoma and degenerative infant brain tissue, thus supporting the presence of both these proteins development of glial structures. Although sites of likely glial intermediate filament synthesis were found, the antigen availability for vimentin was too low to allow a reliable assessment of specific vimentin localization and determination of the GFAP : vimentin ratio in individual intermediate filaments and/or astroglial fibres. Variations in particle densities (PD) which demonstrated GFAP in the various astroglial entities studied, were considered to be a result of variable technical and tissue processing factors rather than truly significant differences in expression of GFAP in individual intermediate filaments. This lead to the conclusion that the GFAP concentration / glial intermediate filament area is likely to be constant for mature glial intermediate filaments and therefore cannot be used to distinguish between different astroglial cells or entities. Whether each cell has a different number of glial intermediate filaments, has not been established satisfactorily. Following complementary conventional immunohistochemistry and careful orientation of biopsy material, the procedure can be applied to suitable specimens for the electron microscopical localization of high concentrations of aldehyde resistant, cytoplasmic antigens.