Investigation of the molecular mechanisms responsible for inducing preconditioning in different regions of the hippocampus through electrical stimulation of the perforating pathway in rats

Orientador: André Schwambach VieiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A Epilepsia do Lobo Temporal (ELT) constitui a forma mais comum de epilepsia focal em adultos. A ELT pode ser classificada como mesial, o subtipo mais frequente de acordo com à origem e à semiologia das crises. O achado histopatológico mais comum na ELT mesial (ELTM) é a esclerose hipocampal. A estimulação elétrica da via perfurante, por um período de 8 horas em ratos acordados causa uma lesão hipocampal semelhante à lesão observada em humanos com epilepsia do lobo temporal mesial, a atrofia hipocampal, sem levar ao Status Epilepticus (SE). No entanto, para que os animais sobrevivam a um período de estimulação de 8 horas, é necessário um pré-condicionamento por estimulação elétrica da via perfurante, consistindo de uma sessão diária de meia hora por dois dias. Porém, os mecanismos moleculares desencadeados por tal pré-condicionamento ainda são desconhecidos. Explorar tais mecanismos poderia possivelmente evidenciar fenômenos associados à epileptogênese que posteriormente se observa neste modelo após estimulação elétrica por 8 horas. Para a realização deste trabalho foi feito a cirurgia de implante de eletrodos em 10 ratos, onde 5 tiveram o estímulo elétrico do pré-condicionamento, e os outros 5 foram o grupo controle. As regiões do Giro Denteado dorsal (GDD), Giro Denteado ventral (GDV), CA3 dorsal (CA3D) e CA3 ventral (CA3v) do hipocampo foram microdissecadas, sendo extraído o RNA, e realizada a análise de transcriptoma por sequenciamento de alto desempenho (RNAseq). Um total de 1.894 genes foram encontrados diferencialmente expressos no GD dos animais do grupo estimulado quando comparado aos animais do grupo controle. Foram 1436 genes no GDD e 987 no GDV. Na região CA3 foram encontrados 1122 genes diferencialmente expressos, 744 na região dorsal (CA3D) e 662 genes na região ventral (CA3V). Nossos dados mostram diversos possíveis mecanismos responsáveis pela indução resistência a crises e ao Status Epilepticus (SE) após o pré-condicionamento, sendo eles, mecanismos inibitórios, como um aumento coordenado da inibição do giro denteado pela via GABAérgica nos neurônios do GD, diminuição do AMPc e de receptor dopaminérgico D1, baixa expressão de eferinas nas regiões GDD, GDV e CA3D e super expressão das semaforinas nas regiões dorsais (GD e CA3). Também foi observado um indício de aumento de atividade excitatória, semelhante à processos epileptogênicos, tanto no GD quanto em CA3. Desta forma o pré-condicionamento implicaria grande remodelamento do GD e CA3, sendo este diferente entre ambas as estruturas, e diferente também quando se compara a região dorsal com a ventral. Estas observações podem indicar possíveis futuras estratégias para a melhor compreensão do modelo experimental de ELTM por meio da estimulação elétrica da via perfurante, particularmente sobre os componentes da circuitaria hipocampalAbstract: Temporal Lobe epilepsy (TLE) is the most common focal epilepsy in adults, and Mesial Temporal Lobo Epilepsy (MTLE) the most frequent subtype. The histopathological most frequent finding in drug resistant MTLE is Mesial Temporal Sclerosis (MTE), with hippocampal sclerosis being the principal component. The electrical stimulation of the perforant pathway for a period of 8 hours in awake rats reproduces hippocampal lesion with a morphology that resembles the human condition without the induction of an episode of status epilepticus (SE). In order for animals to survive such a long period of stimulation in this model it is necessary two days of 30 minutes preconditioning sessions per day by electrical stimulation of the perforant pathway. However, the molecular mechanisms triggered by electrical stimulation of the perforant pathway for a period of 30 minutes on two consecutive days are still unknown. Exploring such mechanisms could possibly highlight phenomena associated with epileptogenesis that is later observed in this model after electrical stimulation for 8 hours. To carry out the project, electrode implantation surgery was performed on 10 rats, where 5 had the electrical preconditioning stimulation, and the other 5 were the control group. The Dorsal Dentate Gyrus (GDD), Ventral Dentate Gyrus (GDV), Dorsal CA3 (CA3D) and Ventral CA3 (CA3v) regions of the hippocampus were microdissected, and RNA was extracted, and transcriptome analysis using high-throughput sequencing (RNAseq) was performed. A total of 1,895 genes were found differentially expressed in the GD of animals from the stimulated group when compared to animals from the control group. There were 1436 genes in GDD and 987 genes in GDV. In the CA3 region 1122 differentially expressed genes were found, 744 in the dorsal region (CA3D) and 662 genes in the ventral region (CA3v). Our data show several possible mechanisms responsible for the induction of seizure resistance after preconditioning, such as inhibitory mechanisms indicated by a coordinated increase in GABAergic inhibition in DG neurons, decreased cAMP and D1 dopaminergic receptor, low expression of epherins in the GDD, GDV and CA3D regions and overexpression of semaphorins in the GDD and CA3D. There was also a sign of increased excitatory activity, similar to an epileptogenic processes, in both GD and CA3. Thus, preconditioning presents a major remodeling of GD and CA3, which is different between both structures, and also different when comparing the dorsal and ventral regions. These observations may indicate possible future strategies for a better understanding of the effects of electric stimulation of the perforant pathway on different hippocampal circuit componentsMestradoFisiologiaMestre em Biologia Funcional e Molecular88882.329750/2019-01CAPE

Transcriptome analyses of the cortex and white matter of focal cortical dysplasia type II: Insights into pathophysiology and tissue characterization

IntroductionFocal cortical dysplasia (FCD) is a common cause of pharmacoresistant epilepsy. According to the 2022 International League Against Epilepsy classification, FCD type II is characterized by dysmorphic neurons (IIa and IIb) and may be associated with balloon cells (IIb). We present a multicentric study to evaluate the transcriptomes of the gray and white matters of surgical FCD type II specimens. We aimed to contribute to pathophysiology and tissue characterization.MethodsWe investigated FCD II (a and b) and control samples by performing RNA-sequencing followed by immunohistochemical validation employing digital analyses.ResultsWe found 342 and 399 transcripts differentially expressed in the gray matter of IIa and IIb lesions compared to controls, respectively. Cholesterol biosynthesis was among the main enriched cellular pathways in both IIa and IIb gray matter. Particularly, the genes HMGCS1, HMGCR, and SQLE were upregulated in both type II groups. We also found 12 differentially expressed genes when comparing transcriptomes of IIa and IIb lesions. Only 1 transcript (MTRNR2L12) was significantly upregulated in FCD IIa. The white matter in IIa and IIb lesions showed 2 and 24 transcripts differentially expressed, respectively, compared to controls. No enriched cellular pathways were detected. GPNMB, not previously described in FCD samples, was upregulated in IIb compared to IIa and control groups. Upregulations of cholesterol biosynthesis enzymes and GPNMB genes in FCD groups were immunohistochemically validated. Such enzymes were mainly detected in both dysmorphic and normal neurons, whereas GPNMB was observed only in balloon cells.DiscussionOverall, our study contributed to identifying cortical enrichment of cholesterol biosynthesis in FCD type II, which may correspond to a neuroprotective response to seizures. Moreover, specific analyses in either the gray or the white matter revealed upregulations of MTRNR2L12 and GPNMB, which might be potential neuropathological biomarkers of a cortex chronically exposed to seizures and of balloon cells, respectively

Transcriptome analyses of the cortex and white matter of focal cortical dysplasia type II: Insights into pathophysiology and tissue characterization

Introduction Focal cortical dysplasia (FCD) is a common cause of pharmacoresistant epilepsy. According to the 2022 International League Against Epilepsy classification, FCD type II is characterized by dysmorphic neurons (IIa and IIb) and may be associated with balloon cells (IIb). We present a multicentric study to evaluate the transcriptomes of the gray and white matters of surgical FCD type II specimens. We aimed to contribute to pathophysiology and tissue characterization. Methods We investigated FCD II (a and b) and control samples by performing RNA-sequencing followed by immunohistochemical validation employing digital analyses. Results We found 342 and 399 transcripts differentially expressed in the gray matter of IIa and IIb lesions compared to controls, respectively. Cholesterol biosynthesis was among the main enriched cellular pathways in both IIa and IIb gray matter. Particularly, the genes HMGCS1, HMGCR, and SQLE were upregulated in both type II groups. We also found 12 differentially expressed genes when comparing transcriptomes of IIa and IIb lesions. Only 1 transcript (MTRNR2L12) was significantly upregulated in FCD IIa. The white matter in IIa and IIb lesions showed 2 and 24 transcripts differentially expressed, respectively, compared to controls. No enriched cellular pathways were detected. GPNMB, not previously described in FCD samples, was upregulated in IIb compared to IIa and control groups. Upregulations of cholesterol biosynthesis enzymes and GPNMB genes in FCD groups were immunohistochemically validated. Such enzymes were mainly detected in both dysmorphic and normal neurons, whereas GPNMB was observed only in balloon cells. Discussion Overall, our study contributed to identifying cortical enrichment of cholesterol biosynthesis in FCD type II, which may correspond to a neuroprotective response to seizures. Moreover, specific analyses in either the gray or the white matter revealed upregulations of MTRNR2L12 and GPNMB, which might be potential neuropathological biomarkers of a cortex chronically exposed to seizures and of balloon cells, respectively