No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The glial cells of the retina aid in function and maintenance of the retina. The macroglia, Muller cells and the retinal astrocytes, become reactive following injury or disease in the retina, a response that is characterized by hypertrophy, dedifferentiation, loss of functionality, proliferation, and remodeling of tissue and extracellular matrix (ECM). The microglia which are the resident macrophages, also respond to injury/disease becoming activated, undergoing characteristic molecular and morphological changes, which include regulation of secreted factors, changes in inflammatory response and increased phagocytosis. Reactivity in Muller glia is thought to be the result of secreted signals, such as epidermal growth factor, ciliary neurotrophic factor, and broblast growth factor, which are released at the injury site to interact with quiescent glial cells. Furthermore, microglia and macroglia have been shown by some studies to interact following activation. While BMPs are known to be upregulated following injury in the CNS, little information is available concerning their role in reactive gliosis in the retina. We hypothesize that BMP7 indirectly triggers Muller gliosis by activating microglia. Using RT-qPCR, immunofluorescence and western blot, we assessed changes in gliosis markers in the mouse retinal glia following treatment with BMP. Our results showed that BMP7 was able to trigger Muller cell gliosis in the retina in vitro and in vivo. Furthermore, ablation of microglia lead to a subdued gliosis response in the mouse retina following BMP7 exposure. Thus, BMP7 triggers activation of retinal microglia in addition to the Muller glia. IFN-gamma and IL6 could play a role in microglia mediated activation of Muller glia, following exposure to BMP7. We also assessed the role of the Hippo/YAP pathway in the regulation of gliosis in the retina. We demonstrated that YAP was localized to the nucleus of the Muller cells of the retina and was upregulated in IFN-gamma induced gliosis in the mouse retina.

Protein-energy malnutrition (PEM) has been found in up to 16.3% of acute stroke patients upon admission to hospital. Our laboratory has previously shown that PEM impairs functional outcome in a gerbil model of global ischemia, but the mechanism has not been established. The purpose of the current study was to characterize the marked reactive gliosis apparent in a subset of these animals that could represent an increased inflammatory response. A second objective was to validate a screening protocol for assessing completeness of ischemia in this model. Male Mongolian gerbils, aged 11-12 weeks, were randomized to PEM (2% protein) or control diet (12.5% protein) for 28d. PEM animals lost 12.2% of their initial body weight, and feed intake and serum albumin concentration were 12.3% and 17.8% lower than controls, respectively. At day 28, animals underwent 5 min bilateral common carotid artery occlusion (ischemia) or sham surgery. Activity was monitored using infrared beam interruptions for 20h post-surgery to screen for complete ischemia on the basis of persistent hyperactivity. Brain sections were stained with hematoxylin & eosin, and viable hippocampal CA1 neurons were counted at 10d post-ischemia. Immunohistochemistry for glial-fibrillary acidic protein (GFAP) and ricinus communis agglutinin -120 (RCA-120), markers for astrocytes and microglia, respectively, and the inflammatory cytokine TNF-alpha was performed on brain sections at 6h, 24h, 3d and 10d post-surgery (Ischemic, n=8; Sham, n=3). The activity monitoring procedure for detecting complete ischemia validated against hippocampal CA1 neuronal loss at 10d demonstrated an accuracy of 84.6%. Temporal changes in GFAP and RCA-120 immunoreactivity characteristic of reactive gliosis were demonstrated following ischemia, but this was not exacerbated by PEM. TNF-alpha immunoreactivity following ischemia was also unaltered by PEM. Ischemia significantly reduced surviving CA1 neurons at 10 days post-ischemia (two-way ANOVA; p<0.05), but this was not influenced by PEM. Impaired functional outcome in PEM animals following global ischemia can not be accounted for by increased hippocampal CA1 neuron death or by altered glial response.

Protein-energy malnutrition (PEM) has been found in up to 16.3% of acute stroke patients upon admission to hospital. Our laboratory has previously shown that PEM impairs functional outcome in a gerbil model of global ischemia, but the mechanism has not been established. The purpose of the current study was to characterize the marked reactive gliosis apparent in a subset of these animals that could represent an increased inflammatory response. A second objective was to validate a screening protocol for assessing completeness of ischemia in this model. Male Mongolian gerbils, aged 11-12 weeks, were randomized to PEM (2% protein) or control diet (12.5% protein) for 28d. PEM animals lost 12.2% of their initial body weight, and feed intake and serum albumin concentration were 12.3% and 17.8% lower than controls, respectively. At day 28, animals underwent 5 min bilateral common carotid artery occlusion (ischemia) or sham surgery. Activity was monitored using infrared beam interruptions for 20h post-surgery to screen for complete ischemia on the basis of persistent hyperactivity. Brain sections were stained with hematoxylin & eosin, and viable hippocampal CA1 neurons were counted at 10d post-ischemia. Immunohistochemistry for glial-fibrillary acidic protein (GFAP) and ricinus communis agglutinin -120 (RCA-120), markers for astrocytes and microglia, respectively, and the inflammatory cytokine TNF-alpha was performed on brain sections at 6h, 24h, 3d and 10d post-surgery (Ischemic, n=8; Sham, n=3). The activity monitoring procedure for detecting complete ischemia validated against hippocampal CA1 neuronal loss at 10d demonstrated an accuracy of 84.6%. Temporal changes in GFAP and RCA-120 immunoreactivity characteristic of reactive gliosis were demonstrated following ischemia, but this was not exacerbated by PEM. TNF-alpha immunoreactivity following ischemia was also unaltered by PEM. Ischemia significantly reduced surviving CA1 neurons at 10 days post-ischemia (two-way ANOVA; p<0.05), but this was not influenced by PEM. Impaired functional outcome in PEM animals following global ischemia can not be accounted for by increased hippocampal CA1 neuron death or by altered glial response.

No description available.

OBJETIVO GENERAL: Determinar las alteraciones histomorfométricas de la capa granular de la corteza órbitofrontal de Rattus norvegicus, a nivel de las neuronas granulares y glías de la serie astrocítica sometidas a administración crónica de bebidas alcohólicas adulteradas. MATERIAL Y MÉTODOS: Estudio experimental, prospectivo, comparativo y longitudinal. Se utilizaron cuarenta y cinco ratas de laboratorio Rattus norvegicus, variedad albina, obtenidos de los bioterios del CENAN y trasladados al bioterio de la Facultad de Medicina Humana de la UNMSM. Fueron divididos en 3 grupos de trabajo: el grupo control (Grupo O) y los grupos experimentales (Grupo A y Grupo B), el Grupo A recibió la bebida alcohólica denominada “Tumbaloco” (mezcla de pisco, anisado y cognac adulterado) y el Grupo B la bebida alcohólica denominada “Penal” (mezcla de pisco, anisado y cognac adulterado más cerveza). Se enviaron dichas bebidas alcohólicas al Centro de Información, Control Toxicológico y Apoyo a la Gestión Ambiental (CICOTOX) para el análisis cuantitativo de etanol y metanol por el método de espectrofotometría de absorción atómica. Se les administró de manera crónica, por un tiempo de 12 semanas, bebidas alcohólicas adulteradas al grupo experimental y agua al grupo control mediante el uso de una sonda orogástrica. Luego de las 12 semanas se procedió al sacrificio del animal mediante la decapitación, se procedió a la extracción del encéfalo murino en un tiempo no mayor de 4 minutos luego del cual fue colocado en una solución de formol neutro al 10%. Se hizo un corte coronal del encéfalo a nivel de la corteza prefrontal, una parte del tejido fue incluido en parafina para la realización de láminas histológicas con la coloración Hematoxilina & Eosina e inmunohistoquímica con GFAP (marcador específico para astrocitos) la otra parte del tejido fue llevada a congelación para la elaboración de láminas histológicas con la coloración Argéntica del Río Ortega impregnación simple. Dichas láminas histológicas fueron fotografiadas y posteriormente analizadas mediante el procesador de imágenes JMicrovision 1.2.5. RESULTADOS: No existió diferencia estadísticamente significativa en el peso de los animales al inicio y al final del experimento. El área determinada por las neuronas necróticas de la capa granular fue menor en los grupos que recibieron bebidas alcohólicas adulteradas a predominio del grupo que recibió la bebida “Penal”. El mayor porcentaje de neuronas necróticas se observó en el grupo que recibió bebidas alcohólicas adulteradas más cerveza el cual fue estadísticamente superior que el grupo control, el porcentaje de neuronas necróticas en el grupo que recibió sólo bebidas alcohólicas adulteradas también fue estadísticamente superior que el grupo control pero menor que el grupo que recibió bebidas alcohólicas adulteradas más cerveza. Se observó gliosis de la serie astrocítica en los grupos que recibieron bebidas alcohólicas adulteradas también a predominio del grupo que recibió la bebida “Penal”. CONCLUSIONES: El consumo crónico de bebidas alcohólicas adulteradas produce una disminución de las neuronas granulares y un aumento de las glías de la serie astrocítica a nivel de la capa granular de la corteza órbitofrontal. Las alteraciones fueron mayores en el grupo que recibió la bebida alcohólica “Penal”, mezcla de 3 bebidas alcohólicas adulteradas más cerveza.

Therapeutic strategies of stroke can take two directions: to prevent brain damage from stroke or aid in its repair after a stroke. In this thesis, a rat stroke model, which mimics the human small vessel stroke, was used. Two potential repair strategies were investigated with this model, reduction of inflammatory processes with the aid of minocycline treatment and replacing necrotic neurons with new ones with the aid of neurogenesis of endogenous progenitor cells. <p>The stroke model is induced by disrupting the medium-size pial vessels within a 5mm-circular brain surface of adult Wistar rats. This leads to a cone-shaped cortical lesion. Therefore it mimics the clinical situation of lacunar infarction, the most frequent outcome of small vessel stroke. <p>Minocycline, a second-generation tetracycline, prevented cavitation and facilitated the repopulation of the lesion by reactive astrocytes. However, I could not identify the molecular target as the number of activated microglia, infiltrating leukocytes and CD3+ lymphocytes as well as interleukin-1β expression were not significantly altered. Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and immature neurons. After injury, DCX-positive cells appeared in the neocortex at the base of the lesion. These cells exhibit a morphology resembling differentiated post-migratory neurons with long branched processes. Some of the DCX-positive cells were also immunoreactive for βIII-tubulin, another marker of immature neurons. This might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE containing cells were found in the cortex underneath the lesion. These results suggest that the DCX-positive cells may not originate from neural precursors from the SVZ, but might be generated from local progenitor cells. In summary, using the PVD II model, which mimics the lacunar stroke, I found that neuroblasts appeared spontaneously near the lesion in the cerebral cortex and were attempting to upregulate neuronal properties. Reducing inflammation with post-stroke minocycline treatment prevented cavitation. I think both findings open up exciting new avenues for treatment of lacunar infarctions.

Therapeutic strategies of stroke can take two directions: to prevent brain damage from stroke or aid in its repair after a stroke. In this thesis, a rat stroke model, which mimics the human small vessel stroke, was used. Two potential repair strategies were investigated with this model, reduction of inflammatory processes with the aid of minocycline treatment and replacing necrotic neurons with new ones with the aid of neurogenesis of endogenous progenitor cells. <p>The stroke model is induced by disrupting the medium-size pial vessels within a 5mm-circular brain surface of adult Wistar rats. This leads to a cone-shaped cortical lesion. Therefore it mimics the clinical situation of lacunar infarction, the most frequent outcome of small vessel stroke. <p>Minocycline, a second-generation tetracycline, prevented cavitation and facilitated the repopulation of the lesion by reactive astrocytes. However, I could not identify the molecular target as the number of activated microglia, infiltrating leukocytes and CD3+ lymphocytes as well as interleukin-1β expression were not significantly altered. Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and immature neurons. After injury, DCX-positive cells appeared in the neocortex at the base of the lesion. These cells exhibit a morphology resembling differentiated post-migratory neurons with long branched processes. Some of the DCX-positive cells were also immunoreactive for βIII-tubulin, another marker of immature neurons. This might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE containing cells were found in the cortex underneath the lesion. These results suggest that the DCX-positive cells may not originate from neural precursors from the SVZ, but might be generated from local progenitor cells. In summary, using the PVD II model, which mimics the lacunar stroke, I found that neuroblasts appeared spontaneously near the lesion in the cerebral cortex and were attempting to upregulate neuronal properties. Reducing inflammation with post-stroke minocycline treatment prevented cavitation. I think both findings open up exciting new avenues for treatment of lacunar infarctions.

No description available.

O sistema olfatório desempenha um papel relevante na exploração do ambiente e no reconhecimento social e sexual de mamíferos. Por meio deste sistema os animais podem reconhecer, detectar e discriminar uma grande quantidade de odorantes de estruturas químicas variadas, sinais químicos no ambiente essenciais para a sobrevivência e os ferormônios, que desencadeiam comportamentos sociais e reprodutivos. Algumas doenças e certos tipos de injúrias fisiológicas podem provocar a morte destas células, o que pode levar a perda da sensibilidade olfatória, embora já se saiba que este epitélio apresenta grupos de neurônios capazes de regeneração. A partir deste contexto, a terapia celular acaba sendo uma alternativa para o tratamento de patologias as quais acometem o sistema olfatório, como por exemplo, a anosmia que pode causar problemas graves, desde acidentes com gás ou comida estragada até depressão e distúrbios alimentares, causadas pela perda do paladar. Objetivou-se com este trabalho avaliar a recuperação da função olfatória de ratos anósmicos, bulbectomizados e submetidos a terapia celular com células-tronco provenientes do epitélio olfatório de ratos wistar. Para tanto foram utilizados 21 ratos machos Wistar de sessenta dias de idade, onde três foram utilizados para obtenção das células tronco do epitélio olfatório, dois para o controle cirúrgico, e restante foram divididos em 4 grupos: GI, GII, GIII e GIV os quais foram transplantados após 3, 7, 14 e 21 dias após a bulbectomia, respectivamente. A técnica cirúrgica foi realizada com incisão de pele, tecido subcutâneo e periósteo, seguida de abertura em janela de formato ovalado e remoção dos bulbos olfatórios mediante aspiração. Para a comprovação da anosmia após a cirurgia, os ratos foram submetidos ao teste comportamental do \"odor de gato\", e os do grupo controle após o período experimental foram sacrificados e a área encefálica da lesão causada pela cirurgia foi coletada onde foram realizadas análises histopatológicas. Os animais do GI, GII, GIII e GIV após 3, 7, 14 e 21 dias após bulbectomia foram anestesiados e receberam células tronco (1x106) do EOR no mesmo local da realização da bulbectomia, e posteriormente foram submetidos ao teste comportamental do \"odor de gato\". Transcorrido o período experimental, foram eutanasiados e os fragmentos de encéfalo foram coletados para análise histopatológica e imunohistoquímica. Os resultados evidenciam que realização da intervenção cirúrgica demonstrou remoção parcial do BO, com destruição da conexão nervosa entre os bulbos olfatórios e o epitélio olfatório. Ainda, a partir do teste comportamental do \"odor de gato\", e pela análise histopatológica das lesões causadas pela cirurgia, que evidenciou extensa área de necrose, com presença de hemossiderina e astrogliose reativa, constatou-se que a técnica empregada para promoção da bulbectomia foi eficaz para promoção da anosmia. A partir da análise comportamental, dos animais submetidos a terapia celular, os animais do GII e GIII apresentaram modificações no comportamento olfativo, com comportamento olfativo positivo ao \"odor do gato\", aversão comportamento defensivo, enquanto 100% dos animais do GI e GIV não apresentaram nenhuma modificação no comportamento olfativo. As análises por imunohistoquímica evidenciaram marcação positiva para o GFP, o que indica a presença das células tronco transduzidas com eGFP nos locais das lesões e ainda a expressão positiva do GFAP que evidencia a presença de astrogliose reativa com presença de cicatriz glial nos locais das lesões. / The olfactory system plays an important role in the exploration of the environment and in social and sexual behavior in mammals. Disturbances of the olfactory system such as observed in anosmia has also been related to accidents caused by gas leak and intoxications by food poisoning, in addition to eating disorders due to relation of the olfactory system with the taste. Through the olfactory system, animals recognize, detect and discriminate a large amount of odorants in a variety of chemical products, including pheromones, as well as in the environment which may guarantee their survival. Some diseases and injuries cause death of cells from the olfactory system leading to decrease or loss of the smell sensitivity. It is known that the olfactory epithelium has cells capable of regenerating neurons. Therefore, the utilization of these cells in cell therapy represents an alternative for the treatment of the olfactory system disorders. The objective of this study was to evaluate the regenaration of olfactory function of bulbectomized rats that were subjected to cell therapy with cells isolated from the olfactory epithelium. Twenty one male Wistar rats, sixty days of age were included in this study. Three rats were used for isolation of the olfactory epithelium cells, two for the surgical control and the remaining were divided into 4 groups: GI, GII, GIII and GIV corresponding to groups where cells were transplanted after 3, 7, 14 and 21 days after bulbectomy, respectively. The surgical technique was performed with skin, subcutaneous and periosteum incisions, followed by craniectomy and the removal of olfactory bulbs upon aspiration. For proof of anosmia, the rats were subjected to behavioral testing know as \"cat odor\". The animals of the control group were sacrificed and brain, with the lesion area, collected and processed for histopathological analysis. The animals of the experimental groups (GI, GII, GIII and GIV) were anesthetized and received heterologous cells (1x106) from olfactory epithelium, thorugh the same venue of the bulbectomy. These animals subsequently underwent behavioral \"cat odor\" testing. After 3, 7, 14 and 21 days of the cell injection, the rats were euthanized and the animals brains were collected for histopathological and immunohistochemical analyses. The results show that the surgical procedure promoted partial removal of olfactory bulbs, with destruction of the neural connection between the olfactory bulb and the olfactory epithelium. The behavioral \"cat odor\" test and the histopathological exams of the lesions, which revealed a large area of necrosis with presence of hemosiderin and reactive astrogliosis, demonstrated that the bulbectomy technique used to promote anosmia was effective. The behavioral test showed that the animals from GII and GIII presented changes in olfactory sensitivity, with positive \"cat odor\" aversion and defensive reaction. This test did not show change in GI and GIV groups. Immunohistochemistry analysis was positive for GFP, suggesting the presence of eGFP transduced cells at the sites of injury. In addition, the expression of GFAP positive cells demonstrated the presence of reactive astrogliosis with glial scar at the sites of injury.