Participation of bone marrow-derived cells in hippocampal vascularization after status epilepticus

Purpose: Diseases such as temporal lobe epilepsy, brain trauma and stroke can induce endothelial cell proliferation and angiogenesis in specific brain areas. During status epilepticus (SE), bone marrow-derived cells are able to infiltrate and proliferate, dramatically increasing at the site of injury. However, it is still unclear whether these cells directly participate in vascular changes induced by SE.Method: To investigate the possible role of bone marrow-derived cells in angiogenesis after seizures, we induced SE by pilocarpine injection in previously prepared chimeric mice. Mice were euthanized at 8 h, 7 d or 15 d after SE onset.Results: Our results indicated that SE modified hippocampal vascularization and induced angiogenesis. Further, bone marrow-derived GFP(+) cells penetrated through the parenchyma and participated in the formation of new vessels after SE. We detected bone marrow-derived cells closely associated with vessels in the hippocampus, increasing the density of blood vessels that had decreased immediately after pilocarpine-induced SE.Conclusion: We conclude that epileptic seizures directly affect vascularization in the hippocampus mediated by bone marrow-derived cells in a time-dependent manner. (C) 2014 British Epilepsy Association. Published by Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Physiol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Physiol, BR-04023062 São Paulo, BrazilWeb of Scienc

Long-lasting anxiolytic effect of neural precursor cells freshly prepared but not neurosphere-derived cell transplantation in newborn rats

Background: the GABAergic system plays an important role in modulating levels of anxiety. When transplanted into the brain, precursor cells from the medial ganglionic eminence (MGE) have the ability to differentiate into GABAergic interneurons and modify the inhibitory tone in the host brain. Currently, two methods have been reported for obtaining MGE precursor cells for transplantation: fresh and neurosphere dissociated cells. Here, we investigated the effects generated by transplantation of the two types of cell preparations on anxiety behavior in rats.Results: We transplanted freshly dissociated or neurosphere dissociated cells into the neonate brain of male rats on postnatal (PN) day 2-3. At early adulthood (PN 62-63), transplanted animals were tested in the Elevated Plus Maze (EPM). To verify the differentiation and migration pattern of the transplanted cells in vitro and in vivo, we performed immunohistochemistry for GFP and several interneuron-specific markers: neuropeptide Y (NPY), parvalbumin (PV) and calretinin (CR). Cells from both types of preparations expressed these interneuronal markers. However, an anxiolytic effect on behavior in the EPM was observed in animals that received the MGE-derived freshly dissociated cells but not in those that received the neurosphere dissociated cells.Conclusion: Our results suggest a long-lasting anxiolytic effect of transplanted freshly dissociated cells that reinforces the inhibitory function of the GABAergic neuronal circuitry in the hippocampus related to anxiety-like behavior in rats.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Fisiol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Farmacol, BR-04023062 São Paulo, BrazilUNIFESP, Dept Biociencias, BR-11015020 Santos, SP, BrazilUniv Fed Rio Grande do Sul, Inst Ciencias Basicas Saude, Dept Bioquim, BR-90035003 Porto Alegre, RS, BrazilUniversidade Federal de São Paulo, Dept Fisiol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Farmacol, BR-04023062 São Paulo, BrazilUNIFESP, Dept Biociencias, BR-11015020 Santos, SP, BrazilWeb of Scienc

Caracterização in vitro de neuroesferas e seu potencial de regeneração na doença de alzheimer e lesão por stab wound

Objetctive: verify the role of neural stem cells (NSC) in three different aspects: in vitro behaviour, use in memory repair in transgenic animals for Alzheimer's Disease (DA, APPswe/PS1dE9) following transplantation, and expression of proteins related to injury response. Methods: We used in vitro techniques for cell culture of neuroespheres, genome-wide techniques as well as transplantation and behavioral tests. Results: We verified that the APPswe/PS1dE9 transgene affect neurospheres growth rate as well as promoting cellular death. Furthermore, is important to note that differentiated astrocytes from AD animals neurospheres presented hypertrophic morphology, alike the astrocytes from WT animals. Therefore, in this model is possible to identify alterations in NSC as well as soluble amyloid precursor protein secretion during the embrionic stage that can promote proliferation without interfere in the normal development of the animal. When transplanted in transfenic animals for DA, NSCs were able to contribute to neural repair by increasing neurogenesis and secretion of BDNF neurotrophin. Nevertheless there was no effect on memory and habituation in transplanted AD animals. Therefore we demonstrated that even without improving memory at behavioural level, NSC transpantation increased the neurotrophic support in hippocampus and can be responsible for other modifications at molecular level that should be better investigated. From the results of in vitro experiments and to better understand the behaviour of astrocytes in AD we decide to compare the expression pattern of proteins overexpressed at the lesion site of the SW (stab wound). To perform this analysis, we used the genome-wide data from Magdalena Götz's lab to select candidates that could play a role in the stab wound lesion and in AD. Among the top upregulated genes in reactive macroglia (astrocytes and NG2-glia), most of them were related to inflammation and, among these genes we selected four candidates (Osteopontin, CD68 and Galectin-1 and -3). We verified that although they were overexpressed in reactive macroglia after SW injury, the same cells do not express these proteins in AD animals. Whereby SW is an accute injury that is healed within few weeks, we assumed that the downregulation of these proteins could be related to the disease chronicity. Conclusion: By evaluating the in vitro features and role in neurodegeneration processes, we verified that when transplanted, NSC secrete factors that increase the neurotrophic support and during development can play a role in compensating cell death in AD animals. However, in old AD animals macroglial cells do not express proteins that could assist lesion resolution. This work offers insights about the role of NSC and macroglia in Alzheimer's disease.Objetivo: Verificar o papel das células-tronco neurais (CTN) frente à doença de Alzheimer (DA) em três diferentes aspectos: comportamento in vitro, reparação de memória em animais transgênicos para DA por meio de transplante e expressão de proteinas relacionadas com resposta à lesão. Métodos: Foram utilizadas técnicas in vitro de culturas de neuroesferas, ferramentas de genômica ampla além de transplantes e testes comportamentais. Resultados: Verificamos que o transgene APPswe/PS1dE9 exerce efeito no crescimento de esferas, além de promover morte celular. Além disso, é importante notar que astrócitos diferenciados a partir de neuroesferas de animais DA apresentaram morfologia hipertrófica, diferente dos astrócitos de animais WT. Dessa forma, podemos dizer que nesse modelo é possível identificar alterações nas CTN bem como secreção de proteína precursora amilóide solúvel na fase embrionária, que pode promover a proliferação celuar sem prejudicar o desenvolvimento do animal. Quando transplantadas em animais transgênicos para DA, CTNs são capazes de contribuir para a regeneração do tecido nervoso por meio do aumento da neurogênese e da neurotrofina BDNF. Apesar disso, não houve melhora significante na memória e habituação em animais DA transplantados. Sendo assim, demonstramos que apesar de não ter exercido efeito comportamental, as CTN aumentaram o suporte neurotrófico no hipocampo e podem ser responsáveis por outras modificações a nível molecular que devem ser estudadas mais profundamente. A partir dos resultados dos experimentos in vitro e para melhor entendimento do comportamento de astrócitos na DA, decidimos comparar a expressão de proteínas normalmente expressas em uma lesão aguda (stab wound, SW). Para isso, utilizamos os dados da análise genômica ampla do laboratório da Profa. Magdalena Götz para selecionar candidados que poderiam estar envolvidos na lesão por SW e na DA. Dentre os genes mais superexpressos em macroglia (astrócitos e NG2-glia) reativa após SW, a maioria estava relacionada a inflamação e, dentre esses genes, selecionamos quatro candidatos (Osteopontina, CD68, Galectina-1 e -3). Verificamos que, apesar desses marcadores estarem superexpressos por macroglia reativa após SW, os mesmos tipos celulares praticamente não expressam essas proteínas. Como o SW é uma lesão aguda que é cicatrizada em poucas semanas, supomos que a falta dessas proteínas na DA pode contribuir para a cronicidade da doença. Conclusão: Ao verificar as características in vitro e frente à neurodegeneração das CTN concluímos que as mesmas secretam fatores que promovem efeito neurotrófico quando transplantadas e podem colaborar para o desenvolvimento em animais DA. Porém em animais DA idosos células macrogliais deixam de expressar proteínas que poderiam auxiliar na resolução da lesão. Este estudo oferece insights sobre o papel de CTN e macroglia na doença de Alzheimer.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Transplantation of inhibitory precursor cells from medial ganglionic eminence produces distinct responses in two different models of acute seizure induction

Medial ganglionic eminence (MGE) is one of the sources of inhibitory interneurons during development. Following transplantation in postnatal developing brain, MGE cells can increase local inhibition suggesting a possible protection to GABAergic dysfunction in brain disorders, such as epilepsy. Since it has been shown that MGE-derived cells harvested as neurospheres are able to suppress seizures, it might be important to investigate whether these protective effects would change in different seizure models. Here, we used pentylenetetrazole(PTZ) and maximal electroshock (MES)-induced seizure models to test whether the transplantation of MGE cells would increase the threshold to trigger acute seizures. When transplanted into the neocortex (layers 3-4) of neonatal mice (postnatal days 3-4), MGE cells were able to survive and were mainly found in piriform cortex, fimbria, and ventricular wall regions. Additionally, the number of GFP + cells found in the brains of mice induced with PTZ and MES differed significantly and suggests proliferation and larger survival rate of MGE-transplanted cells after PTZ, but not MES-induced seizures. Following transplantation, there was a reduction in the number of animals presenting mild and severe seizures induced by PTZ. Furthermore, MGE-cell transplantation was able to increase threshold to seizures induced by PTZ, but was not able to prevent seizure spread induced by MES. (C) 2017 Elsevier Inc. All rights reserved.FAPESPCNPqUniv Fed Sao Paulo UNIFESP, Lab Neurofisiol, Sao Paulo, BrazilUniv Fed Rio Grande do Sul, Porto Alegre, RS, BrazilUniv Fed Sao Paulo UNIFESP, Lab Neurofisiol, Sao Paulo, BrazilWeb of Scienc