Os receptores glutamatérgicos metabotrópicos (mGluRs) estão envolvidos nos processos de plasticidade, neurodegeneração e neuroproteção. Avaliamos a expressão de mGluRs no sistema visual de ratos e pintos em diferentes tempos após enucleação ocular. Os animais foram avaliados pelo método de imuno-histoquímica, immunoblotting e RT-PCR, para detecção dos receptores mGluR1,2/3,5 e 7. Foi observado aumento da imunorreatividade (IR) de mGluR1, 5 e 7 no colículo superior, porém não foi observada diferença no núcleo geniculado lateral. Houve aumento na expressão protéica para mGluR1, 5 e 7 e aumento da expressão gênica para mGluR1,5 e 7; por outro lado, ocorreu uma diminuição de mGluR3. No TeO, foi observado aumento da IR para mGluR1 e 5 e diminuição para mGluR2/3. As análises de immunoblotting confirmaram o aumento observado de mGluR1 e diminuição de mGluR2/3. Os resultados indicam uma modulação diferencial na expressão gênica e protéica dos mGluRs, sugerindo a participação desses receptores em processos plásticos decorrentes de lesões no sistema visual adulto. / The metabotropic glutamate receptors (mGluRs) are involved in neuronal plasticity and neuroprotection. We analyzed the expression of mGluRs in the visual system of rats and chicks in several periods after ocular enucleation. The localization and expression of mGluR1, 5, 2/3 and 7 receptors were evaluated by standard immunoperoxidase, immunoblotting and real-time PCR protocols. The immunorreativity, protein and gene expression of mGluR1, 5 and 7 receptors in the superior colliculus showed an increase, whereas no changes were seen in the lateral geniculate nucleus. For mGluR3, gene expression was decreased. In the TeO, mGluR1 and 5 increased for all survival periods analyzed. Immunoblotting analyses confirmed the increases for mGluR1 and 5, decreases for mGluR2/3. These results indicate that the expression of mGluRs is regulated by the glutamatergic retinal input, and add data on a possible role of these receptors in neuroplasticity in adult animals.

O modelo de enucleação ocular em roedores é frequentemente empregado para o estudo dos efeitos da desaferentação de estruturas retinorrecipientes. Avaliamos a geração de espécies reativas de oxigênio (EROs), no colículo superior (CS) e núcleo geniculado lateral dorsal do tálamo (GLD) após enucleação ocular. A oxidação da dihidroetidina revelou o aumento da geração de EROs no CS e no GLD após a lesão. Os resultados de RT-PCR revelaram aumento da expressão gênica de Nox 2 em ambas as estruturas avaliadas. Em contrapartida, observou-se aumento da expressão gênica de Nox 1 e 4 apenas no CS. Com a finalidade de avaliarmos o envolvimento de EROs no remodelamento estrutural após a lesão, animais foram tratados com apocinina e ensaios de imuno-histoquímica foram realizados utilizando-se anticorpos contra neurofilamentos (NFs) e proteínas associadas a microtúbulos-2 (MAP-2). Os resultados revelaram que a enucleação ocular produz um aumento na imunorreatividade para NFs e MAP-2 no CS e GLD, o que foi atenuado pelo tratamento com apocinina. / Unilateral ocular enucleation represents a useful model to study visual system plasticity. We evaluated the reactive oxygen species (ROS) generation in the main visual relays of the mammalian brain, namely the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG), after ocular enucleation. Dihydroethidium oxidation revealed increased ROS generation in SC and DLG. ROS generation was decreased by the Nox inhibitors DPI and apocynin. Real-time PCR results revealed that Nox 2 was upregulated in both retinorecipient structures after deafferentation, whereas Nox 1 and Nox 4 were upregulated only in the SC. To evaluate the role of ROS in structural remodeling after the lesions, apocynin was given to enucleated rats and immunohistochemistry was conducted for markers of neuronal remodeling into SC and DLG. Immunohistochemical data showed that ocular enucleation produces an increase of neurofilament and microtubule-associated protein-2 immunostaining in both SC and DLG, which was markedly attenuated by apocynin treatment.

Os receptores glutamatérgicos metabotrópicos (mGluRs) estão envolvidos nos processos de plasticidade, neurodegeneração e neuroproteção. Avaliamos a expressão de mGluRs no sistema visual de ratos e pintos em diferentes tempos após enucleação ocular. Os animais foram avaliados pelo método de imuno-histoquímica, immunoblotting e RT-PCR, para detecção dos receptores mGluR1,2/3,5 e 7. Foi observado aumento da imunorreatividade (IR) de mGluR1, 5 e 7 no colículo superior, porém não foi observada diferença no núcleo geniculado lateral. Houve aumento na expressão protéica para mGluR1, 5 e 7 e aumento da expressão gênica para mGluR1,5 e 7; por outro lado, ocorreu uma diminuição de mGluR3. No TeO, foi observado aumento da IR para mGluR1 e 5 e diminuição para mGluR2/3. As análises de immunoblotting confirmaram o aumento observado de mGluR1 e diminuição de mGluR2/3. Os resultados indicam uma modulação diferencial na expressão gênica e protéica dos mGluRs, sugerindo a participação desses receptores em processos plásticos decorrentes de lesões no sistema visual adulto. / The metabotropic glutamate receptors (mGluRs) are involved in neuronal plasticity and neuroprotection. We analyzed the expression of mGluRs in the visual system of rats and chicks in several periods after ocular enucleation. The localization and expression of mGluR1, 5, 2/3 and 7 receptors were evaluated by standard immunoperoxidase, immunoblotting and real-time PCR protocols. The immunorreativity, protein and gene expression of mGluR1, 5 and 7 receptors in the superior colliculus showed an increase, whereas no changes were seen in the lateral geniculate nucleus. For mGluR3, gene expression was decreased. In the TeO, mGluR1 and 5 increased for all survival periods analyzed. Immunoblotting analyses confirmed the increases for mGluR1 and 5, decreases for mGluR2/3. These results indicate that the expression of mGluRs is regulated by the glutamatergic retinal input, and add data on a possible role of these receptors in neuroplasticity in adult animals.

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No. of bitstreams: 2 Dissertação - Célia Regina Malveste Ito - 2017.pdf: 2187840 bytes, checksum: ce24431d5cefc9c1e1356d4d3763413d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-09-26 / Decontamination of the surface of the donor eyeballs is part of the operational norms that eye banks advocate before preservation, and antisepsis procedures are effective, ensuring greater transplantation safety. The objective of the present study was to evaluate the antiseptic effect in reducing the microbiota of the ocular globe of donors of corneas prior to enucleation, with 5% povidone-iodine (PVP-I) and 0.05% chlorhexidine gluconate (GC), In the action times of 5, 10 and 15 minutes, as well as the susceptibility profile of the microbiota isolated from gentamicin. Thirty pairs of corneas received antiseptics, with PVP-I in the right eye and GC in the left, and for each time of action 10 pairs of eyeballs were used. Swabs were collected from the ocular surface before application of the solutions, after and at the time of preservation of the corneal tissue, to evaluate the reduction of the microbiota. After identification of the microbiota, an antibiogram test was performed with gentamicin. The data were computed and evaluated by Chi-square or Fisher's exact test, T-test and McNemar test paired, and the statistical significance level was 5% (p <0.05). In the second collection, after antisepsis, there was a reduction of 39,5% in the total of gram positive bacteria (G +), and of 76,5% in the gram negative (G-) bacteria, with no statistically significant difference (p = 0.183), which demonstrated that the bacterial elimination capacity of the antiseptics was similar. It was observed that, in the second collection, both were more effective for G-, with a statistically significant difference (p <0.001), than for G +, with no statistically significant difference (p = 0.494). In the third collection, after the residual effect of the antiseptics, there was a reduction of 99.1% of all the microorganisms. In the antibiogram test, 88% of the isolated microorganisms were sensitive to gentamicin. It was concluded that the use of antiseptics is essential for the effective decontamination of donated corneas prior to preservation. The residual time of the antiseptics increased the decontamination power of PVP-I and GC, being similar in reducing the microbiota of the ocular globe of the donor of corneas. Gentamycin contained in the cornea preservation medium complements the antisepsis of the donated tissues. / A descontaminação da superfície dos globos oculares doados são normas operacionais que os bancos de olhos preconizam antes da preservação e os procedimentos de antissepsia são eficazes, garantindo uma maior segurança ao transplante. O objetivo do presente estudo foi avaliar o efeito antisséptico na redução da microbiota do globo ocular de doadores de córneas antes da enucleação, com o povidona-iodo (PVP-I) a 5% e gluconato de clorexidina (GC) a 0,05%, nos tempos de ação de 5, 10 e 15 minutos, bem como o perfil de susceptibilidade da microbiota isolada à gentamicina. Trinta pares de córneas receberam antissépticos, sendo o PVP-I no olho direito e o GC no esquerdo, e para cada tempo de ação foram utilizados 10 pares de globos oculares. Foram colhidos swabs da superfície ocular antes da aplicação das soluções, após e no momento da preservação do tecido corneano, para avaliar a redução da microbiota. Após identificação da microbiota, foi realizado teste de antibiograma com gentamicina. Os dados foram computados e avaliados pelos testes Qui-Quadrado ou Exato de Fisher, teste T e Teste McNemar pareado, e o nível de significância estatística foi (p<0,05). Com relação aos dados obtidos na segunda coleta, após o uso de antissépticos, houve uma redução de 39,5% no total de bactérias gram positivas (G+) e de 76,5% nas gram negativas (G-), não havendo diferença estatística significativa (p=0,183), sendo semelhante a capacidade de eliminação bacteriana dos antissépticos. Observa-se que, na segunda coleta, ambos foram mais eficazes para as G-, com diferença estatisticamente significativa (p<0,001), do que para as G+, sem diferença estatisticamente significativa (p=0,494). Na terceira coleta, após o efeito residual dos antissépticos, houve redução de 99,1% de todos os micro-organismos. No teste de antibiograma, 88% dos micro-organismos isolados foram sensíveis à gentamicina. Concluiu-se que o uso de antissépticos é essencial para a efetiva descontaminação das córneas doadas antes da preservação. O tempo residual dos antissépticos aumentou o poder de descontaminação do PVP-I e GC, sendo semelhantes na redução da microbiota do globo ocular do doador de córneas. A gentamicina contida no meio de preservação de córnea complementa a antissepsia dos tecidos doados.

O modelo de enucleação ocular em roedores é frequentemente empregado para o estudo dos efeitos da desaferentação de estruturas retinorrecipientes. Avaliamos a geração de espécies reativas de oxigênio (EROs), no colículo superior (CS) e núcleo geniculado lateral dorsal do tálamo (GLD) após enucleação ocular. A oxidação da dihidroetidina revelou o aumento da geração de EROs no CS e no GLD após a lesão. Os resultados de RT-PCR revelaram aumento da expressão gênica de Nox 2 em ambas as estruturas avaliadas. Em contrapartida, observou-se aumento da expressão gênica de Nox 1 e 4 apenas no CS. Com a finalidade de avaliarmos o envolvimento de EROs no remodelamento estrutural após a lesão, animais foram tratados com apocinina e ensaios de imuno-histoquímica foram realizados utilizando-se anticorpos contra neurofilamentos (NFs) e proteínas associadas a microtúbulos-2 (MAP-2). Os resultados revelaram que a enucleação ocular produz um aumento na imunorreatividade para NFs e MAP-2 no CS e GLD, o que foi atenuado pelo tratamento com apocinina. / Unilateral ocular enucleation represents a useful model to study visual system plasticity. We evaluated the reactive oxygen species (ROS) generation in the main visual relays of the mammalian brain, namely the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG), after ocular enucleation. Dihydroethidium oxidation revealed increased ROS generation in SC and DLG. ROS generation was decreased by the Nox inhibitors DPI and apocynin. Real-time PCR results revealed that Nox 2 was upregulated in both retinorecipient structures after deafferentation, whereas Nox 1 and Nox 4 were upregulated only in the SC. To evaluate the role of ROS in structural remodeling after the lesions, apocynin was given to enucleated rats and immunohistochemistry was conducted for markers of neuronal remodeling into SC and DLG. Immunohistochemical data showed that ocular enucleation produces an increase of neurofilament and microtubule-associated protein-2 immunostaining in both SC and DLG, which was markedly attenuated by apocynin treatment.

En ögonamputation innebär en livslång begränsning av synfunktionen vilket kan förväntas påverka livskvaliteten. Syftet var att identifiera faktorer som påverkade livskvalitet på lång sikt efter en ögonamputation i vuxen ålder samt att beskriva hur livskvaliteten påverkades. Metod: Litteraturstudien följde niostegsmodellen av Polit och Beck (2017). Artiklarnas relevans bedömdes i tre urvalssteg: 1. Huruvida titel/ sammanfattningen matchade syftet. 2. Huruvida artiklarna i sin helhet matchade syftet samt in- och exklusionskriterierna. 3. Kvalitets­bedömning. Databearbetning: Faktorer som påverkade livskvalitet hos ögonamputerade protesbärare sorterades efter likheter och olikheter. Kategorier formulerades och tre karakteristiska teman identifierades bland dessa. Resultatet baserades på sju artiklar. Bland faktorerna som påverkade livskvalitet efter en ögonamputation identifierades tre teman ’Slutgiltig fysisk förändring’, ’Psykiska och känslomässiga påfrestningar’ samt ’Socialt undvikande beteende’. Temat ’Slutgiltig fysisk förändring’ tilldelades kategorierna ’Två blir ett: Enögdheten &amp; monokulärt seende’ och ’Ett camouflerat operationsärr: Ögonhåla &amp; protes’. ’Bearbetning av ögonförlusten’, ’Psykologiska problem’ samt ’Utmattning’ var de tre kategorierna tillhörande temat ’Psykiska och känslomässiga påfrestningar’. Temat ’Socialt undvikande beteende’ tillhörde kategorierna: ’Upplevd begränsad funktionsduglighet’ och ’Osäkerhet i mellanmänskliga relationer’. Slutsatsen: Studien sammanfattar kunskapsläget kring faktorer som påverkar livskvalitet efter en ögonamputation på lång sikt. Kunskap som behövs för att kunna optimera rehabiliteringsprocessen, ge god omvårdnad samt förhindra utanförskap. / Amputation of an eye results in life-long limitations of eye sight function which can be expected to affect quality of life (QoL). The aim of this study was to identify factors having long-term effects on QoL after amputation of an eye in adult patients and to describe in which way QoL was affected. Method: A litterature review following nine steps according to Polit and Beck (2017) was performed. Selection of relevant scientific articles was done in three steps: Evalutating relevance according to 1. title and abstract, 2. Article as a whole concidering aim of the review and criteria for in- and ex-clusion. 3. Study quality. Data analysis: Factors affecting QoL due to eye-amputation and wearing of an artificial eye were sorted according to similarities and differences. That way different categories were formed and among those main themes were identified. The result was based on seven articles. The three main themes that could be identified among factors influencing QoL after amputation of an eye were: ’Definitive physical changes’, ’Mental and emotional strains’ and ’Social avoidance’. Characteristic categories for the theme ’Definitive physical changes’ were ’Two become one: Monocularism &amp; monocular sight’ and ’A camouflaged operation scar: Eye socket &amp; prosthesis’. ’Processing the loss of an eye’, ’Psychological problems’ and ’Fatigue’ were three categories belonging to the theme ’Mental and emotional strains’.  The theme ’Social avoidance’ included categories like ’Experienced limited functioning’ and ’Uncertainty in interpersonal relationsships’. Conclusions: The review tries to sum up available ’recent’ research on factors with long-term effect on QoL after amputation of an eye. Knowledge which is necessary to improve rehabilitation- and caring-interventions as well as to prevent alienation in such patients

La période postnatale et l’expérience sensorielle sont critiques pour le développement du système visuel. Les interneurones inhibiteurs exprimant l’acide γ-aminobutyrique (GABA) jouent un rôle important dans le contrôle de l’activité neuronale, le raffinement et le traitement de l’information sensorielle qui parvient au cortex cérébral. Durant le développement, lorsque le cortex cérébral est très susceptible aux influences extrinsèques, le GABA agit dans la formation des périodes critiques de sensibilité ainsi que dans la plasticité dépendante de l’expérience. Ainsi, ce système inhibiteur servirait à ajuster le fonctionnement des aires sensorielles primaires selon les conditions spécifiques d’activité en provenance du milieu, des afférences corticales (thalamiques et autres) et de l’expérience sensorielle. Certaines études montrent que des différences dans la densité et la distribution de ces neurones inhibiteurs corticaux reflètent les caractéristiques fonctionnelles distinctes entre les différentes aires corticales. La Parvalbumine (PV), la Calretinine (CR) et la Calbindine (CB) sont des protéines chélatrices du calcium (calcium binding proteins ou CaBPs) localisées dans différentes sous-populations d’interneurones GABAergiques corticaux. Ces protéines tamponnent le calcium intracellulaire de sorte qu’elles peuvent moduler différemment plusieurs fonctions neuronales, notamment l’aspect temporel des potentiels d’action, la transmission synaptique et la potentialisation à long terme. Plusieurs études récentes montrent que les interneurones immunoréactifs (ir) aux CaBPs sont également très sensibles à l’expérience et à l’activité sensorielle durant le développement et chez l’adulte. Ainsi, ces neurones pourraient avoir un rôle crucial à jouer dans le phénomène de compensation ou de plasticité intermodale entre les cortex sensoriels primaires. Chez le hamster (Mesocricetus auratus), l’énucléation à la naissance fait en sorte que le cortex visuel primaire peut être recruté par les autres modalités sensorielles, telles que le toucher et l’audition. Suite à cette privation oculaire, il y a établissement de projections ectopiques permanentes entre les collicules inférieurs (CI) et le corps genouillé latéral (CGL). Ceci a pour effet d’acheminer l’information auditive vers le cortex visuel primaire (V1) durant le développement postnatal. À l’aide de ce modèle, l’objectif général de ce projet de thèse est d’étudier l’influence et le rôle de l’activité sensorielle sur la distribution et l’organisation des interneurones corticaux immunoréactifs aux CaBPs dans les aires sensorielles visuelle et auditive primaires du hamster adulte. Les changements dans l’expression des CaBPs ont été déterminés d’une manière quantitative en évaluant les profils de distribution laminaire de ces neurones révélés par immunohistochimie. Dans une première expérience, nous avons étudié la distribution laminaire des CaBPs dans les aires visuelle (V1) et auditive (A1) primaires chez le hamster normal adulte. Les neurones immunoréactifs à la PV et la CB, mais non à la CR, sont distribués différemment dans ces deux cortex primaires dédiés à une modalité sensorielle différente. Dans une deuxième étude, une comparaison a été effectuée entre des animaux contrôles et des hamsters énucléés à la naissance. Cette étude montre que le cortex visuel primaire de ces animaux adopte une chimioarchitecture en PV similaire à celle du cortex auditif. Nos recherches montrent donc qu’une suppression de l’activité visuelle à la naissance peut influencer l’expression des CaBPs dans l’aire V1 du hamster adulte. Ceci suggère également que le type d’activité des afférences en provenance d’autres modalités sensorielles peut moduler, en partie, une circuiterie corticale en CaBPs qui lui est propre dans le cortex hôte ou recruté. Ainsi, nos travaux appuient l’hypothèse selon laquelle il serait possible que certaines de ces sous-populations d’interneurones GABAergiques jouent un rôle crucial dans le phénomène de la plasticité intermodale. / The postnatal period and sensory experience are critical for the development of the visual system. The inhibitory interneurons expressing the γ-aminobutyric acid (GABA) play an important role in the control of neural activity, refinement and treatment of sensory information which reaches the cerebral cortex. During development, when the cerebral cortex is very likely to be influenced by extrinsic factors, GABA acts in the formation of critical period of receptivity as well as in experience dependent plasticity. Thus, this inhibitory system adjusts the functioning of the primary sensory areas according to the specific conditions of activity from the environment, cortical afferents (e.g. of thalamic origin), and sensory experience. Several studies show that differences in the distribution and density of these inhibitory interneurons tend to reflect functional discrepancies between the different neocortical areas. Parvalbumin (PV), Calretinin (CR) and Calbindin (CB) are calcium-binding proteins (CaBPs) found in different sub-populations of GABAergic cortical interneurons. These proteins buffer intracellular calcium levels, which can in turn modulate several neural functions, notably the temporal aspect of action potentials, synaptic transmission and long-term potentiation. Several recent studies are showing that CaBPs immunoreactive (ir) interneurons are also very sensitive to experience and sensory activity during development and adulthood. Therefore, these neurons may have a critical role in intermodal plasticity or compensatory processes between primary sensory cortices. In the hamster (Mesocricetus auratus), after enucleation at birth, the primary visual cortex can be recruited by other sensory modalities such as touch and audition. After this type of visual deprivation, there is establishment of permanent ectopic projections between the inferior colliculus (IC) and the lateral geniculate nucleus (LGN). This phenomenon leads to the rerouting of auditory information to the primary visual cortex (V1) during postnatal development. By using this animal model, the general objective of this thesis is to study the influence and the role of sensory activity on the distribution and organization of cortical interneurons that display immunoreactivity for CaBPs in the primary visual and auditory sensory areas in adult hamsters. Changes in the expression of CaBPs were quantitatively determined by assessing the laminar distribution profiles of cell bodies revealed by immunohistochemistry. In the first experiment, we studied laminar distribution of CaBPs in the primary visual (V1) and auditory (A1) cortices of normal hamsters. PVir and CBir, but not CRir neurons, are distributed in a dissimilar fashion between the two primary cortices devoted to each sensory modality. In the second study, a comparison was performed between control animals and hamsters which were enucleated at birth. The results of this study show that the primary visual cortex of these animals adopts a PVir chemoarchitecture similar to that of the auditory cortex. Our research shows that the abolition of visual activity at birth can influence the expression of CaBPs in V1 of the adult hamster. The present results also suggest that the type of activity in afferents from other sensory modalities can at least in part modulate the cortical circuitry of CaBPs in the host or recruited cortex. Thus, our work supports the hypothesis that sub-populations of GABAergic interneurons may play a critical role in the intermodal cortical plasticity.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

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