Introdução O gene NTRK2 codifica um receptor pertencente a família de neurotrofinas Tirosina Kinase, conhecido como TrkB. O TrkB é um receptor de membrana com propriedades relacionadas a sinalização e diferenciação celular que tem sido envolvido em transtornos neuropsiquiátricos. Objetivo Estudar as freqüências de alelos variantes do gene NTRK2 em pacientes com epilepsia do lobo temporal (ELT) comparado a controles sem epilepsia. O impacto desses polimorfismos em variáveis clínicas e psiquiátricas dos pacientes com ELT também foi analisado. Métodos Inicialmente, realizamos estudo de caso-controle comparando as freqüências dos polimorfismos do TrkB em 198 pacientes Brasileiros com origem Européia com ELT e 200 controles sem epilepsia. Na segunda parte, foi avaliado o impacto das variantes alélicas em características clínicas e eletroencefalográficas dos pacientes com epilepsia. Os seguintes polimorfismos foram avaliados: rs1867283A>G, rs10868235C>T, rs1147198G>T, rs11140800A>T, rs1187286G>T, rs2289656A>G, rs1624327A>G, rs1443445A>G, rs3780645C>T, rs2378672C>T. Por fim, 163 pacientes com ELT foram avaliados com uma entrevista psiquiátrica (SCID-I) para detecção de transtornos psiquiátricos ao longo da vida e esses achados foram analisados em relação aos polimorfismos do gene NTRK2. Resultados Pacientes com epilepsia do lobo temporal evidenciaram um aumento significativo de Timina em homozigose no SNP rs10868235 do gene NTRK2 quando comparados ao grupo controle (O.R.=1.90; 95%IC=1.17-3.09; p= 0.01). Não foram encontradas outras diferenças entre pacientes e controles. Pacientes com Adenina em homozigose no SNP rs1443445 do gene NTRK2 tiveram uma média de idade de início de crises mais baixa quando comparados aos demais pacientes (p<0.01). Também observamos que a presença de Timina foi significativamente mais freqüente no SNP rs3780645 do gene NTRK2 em pacientes que necessitam politerapia para o controle de crises se comparados aos que estão em monoterapia. Esse achado pode significar uma maior dificuldade em obter o controle das crises nesse grupo de pacientes (O.R.= 4.13; 95%IC= 1.68-10.29; p= 0.001). Após essa análise, estudamos 163 pacientes com ELT em relação a presença ou não de comorbidades psiquiátricas. A avaliação psiquiátrica foi realizada através da aplicação do SCID-I (Entrevista Clínica Estruturada para Detecção de Transtornos Psiquiátricos de Eixo I do DSM-IV). Setenta e seis pacientes (46.6%) apresentaram transtornos de humor. Sexo feminino, transtorno de ansiedade, genótipo A/A no SNP rs1867283 e genótipo C/C no SNP rs10868235 do gene NTRK2 foram todos fatores independentemente associados com transtornos de humor nesses pacientes Transtornos depressivos foram os que mais contribuíram para esses resultados. Após a regressão logística, fatores de risco independentes para transtornos depressivos em pacientes com ELT foram sexo feminino (OR=2.54; 95%IC=1.18- 5.47; p=0.017), presença de transtorno de ansiedade concomitante (OR=3.30; 95%IC=1.58-6.68; p=0.001), genótipo A/A no SNP rs1867283 do gene NTRK2 (OR=2.84; 95%IC=1.19-6.80; p=0.019), e genótipo C/C no SNP rs10868235 do gene NTRK2 (OR=2.74; IC=1.28-5.88; p=0.010). Conclusões Observamos que pacientes com ELT apresentam uma distribuição alélica distinta do gene NTRK2 quando comparados a controles sem epilepsia e que a variabilidade alélica do NTRK2 influenciou a idade de início de crises e talvez a resposta a terapia farmacológica anticonvulsivante. O sexo feminino, transtornos de ansiedade e variações alélicas no gene NTRK2 foram todos fatores de risco independentes para transtornos de humor ou transtornos depressivos em pacientes com ELT. Até onde temos conhecimento, este é o primeiro estudo evidenciando associações de variantes alélicas do gene NTRK2 em ELT. Acreditamos que outros estudos nessa área ajudarão a elucidar melhor os mecanismos envolvidos na epileptogênese do lobo temporal. Se nossos resultados forem confirmados, as variantes alélicas do gene NTRK2 poderiam ser usadas como um biomarcador para transtornos depressivos em pacientes com ELT. / Introduction The NTRK2 gene encodes a member of the neurotrophic tyrosine kinase family receptor known as TrkB. It is a membrane-associated receptor with signaling and cellular differentiation proprieties that has been involved in neuropsychiatric disorders. Objective Study the frequencies of NTRK2 allele variants in patients with temporal lobe epilepsy (TLE) compared to controls without epilepsy. The impact of these polymorphisms on major clinical and psychiatric variables in TLE was also explored. Methods A case-control study comparing the frequencies of the TrkB gene polymorphism in 198 TLE Brazilian with European origin patients and in 200 matching controls without epilepsy. In a second step, the impact of allelic variation on major clinical and electroencephalographic variables in epilepsy was evaluated in the group of TLE patients. The following polymorphisms were evaluated: rs1867283A>G, rs10868235C>T, rs1147198G>T, rs11140800A>T, rs1187286G>T, rs2289656A>G, rs1624327A>G, rs1443445A>G, rs3780645C>T, rs2378672C>T. At last, 163 TLE patients were evaluated with a psychiatry interview (SCID-I) to detect lifelong psychiatric comorbidities and this findings were analyzed in relation to NTRK2 polymorphisms. Results Patients with temporal lobe epilepsy showed a significant increase of thymine homozygosis in the rs10868235 NTRK2 SNP when compared with the control group (O.R.=1.90; 95%CI=1.17-3.09; p= 0.01) . There were no other differences between patients and controls. Patients with adenine homozygosis in the rs1443445 NTRK2 SNP showed an earlier mean age of seizure onset when compared with other patients (p<0.01). Also, we observed that thymine was significantly more frequent in the rs3780645 NTRK2 SNP in patients that needed polytheraphy for seizure control when compared to those in monotherapy. This finding perhaps reflects an increased difficulty to exert seizure control in this group of patients (O.R.= 4.13; 95%CI= 1.68-10.29; p= 0.001). We also analyzed 163 patients in the TLE group in relation to presence of psychiatric comorbidities. Psychiatric evaluation was performed using the SCID-I (Structured Clinical Interview for DSM-IV, Axis I). Seventy six patients (46.6%) showed mood disorders. Female sex, anxiety disorders, A/A genotype in rs1867283 NTRK2, and C/C genotype in the rs10868235 NTRK2 gene were all independently associated with mood disorders in these patients. Depressive disorders mostly accounted for these results. After logistic regression, independent risk factors for depressive disorder in TLE were female sex (OR=2.54; 95%CI=1.18-5.47; p=0.017), presence of concomitant anxiety disorders (OR=3.30; 95%CI=1.58-6.68; p=0.001), A/A genotype in rs1867283 NTRK2 (OR=2.84; 95%CI=1.19-6.80; p=0.019), and C/C genotype in rs10868235 NTRK2 gene (OR=2.74; 1.28-5.88; p=0.010). Conclusions We observed that patients with epilepsy showed a difference in NTRK2 allelic distribution when compared with controls without epilepsy, and that NTRK2 variability influenced age of seizure onset and perhaps pharmacologic response to seizure control. Female sex, anxiety disorders and allelic variations in NTRK2 gene were all independent risk factors for mood disorder or depressive disorders in TLE. . As far as we know, this is the first study showing an association between NTKR2 allele variants in temporal lobe epilepsy. We believe that other studies in this venue will shade some light on the molecular mechanisms involved in temporal epileptogenesis. If our results were confirmed, NTRK2 gene allele variants could be used as a biomarker for depressive disorders in patients with temporal lobe epilepsy.

Introdução O gene NTRK2 codifica um receptor pertencente a família de neurotrofinas Tirosina Kinase, conhecido como TrkB. O TrkB é um receptor de membrana com propriedades relacionadas a sinalização e diferenciação celular que tem sido envolvido em transtornos neuropsiquiátricos. Objetivo Estudar as freqüências de alelos variantes do gene NTRK2 em pacientes com epilepsia do lobo temporal (ELT) comparado a controles sem epilepsia. O impacto desses polimorfismos em variáveis clínicas e psiquiátricas dos pacientes com ELT também foi analisado. Métodos Inicialmente, realizamos estudo de caso-controle comparando as freqüências dos polimorfismos do TrkB em 198 pacientes Brasileiros com origem Européia com ELT e 200 controles sem epilepsia. Na segunda parte, foi avaliado o impacto das variantes alélicas em características clínicas e eletroencefalográficas dos pacientes com epilepsia. Os seguintes polimorfismos foram avaliados: rs1867283A>G, rs10868235C>T, rs1147198G>T, rs11140800A>T, rs1187286G>T, rs2289656A>G, rs1624327A>G, rs1443445A>G, rs3780645C>T, rs2378672C>T. Por fim, 163 pacientes com ELT foram avaliados com uma entrevista psiquiátrica (SCID-I) para detecção de transtornos psiquiátricos ao longo da vida e esses achados foram analisados em relação aos polimorfismos do gene NTRK2. Resultados Pacientes com epilepsia do lobo temporal evidenciaram um aumento significativo de Timina em homozigose no SNP rs10868235 do gene NTRK2 quando comparados ao grupo controle (O.R.=1.90; 95%IC=1.17-3.09; p= 0.01). Não foram encontradas outras diferenças entre pacientes e controles. Pacientes com Adenina em homozigose no SNP rs1443445 do gene NTRK2 tiveram uma média de idade de início de crises mais baixa quando comparados aos demais pacientes (p<0.01). Também observamos que a presença de Timina foi significativamente mais freqüente no SNP rs3780645 do gene NTRK2 em pacientes que necessitam politerapia para o controle de crises se comparados aos que estão em monoterapia. Esse achado pode significar uma maior dificuldade em obter o controle das crises nesse grupo de pacientes (O.R.= 4.13; 95%IC= 1.68-10.29; p= 0.001). Após essa análise, estudamos 163 pacientes com ELT em relação a presença ou não de comorbidades psiquiátricas. A avaliação psiquiátrica foi realizada através da aplicação do SCID-I (Entrevista Clínica Estruturada para Detecção de Transtornos Psiquiátricos de Eixo I do DSM-IV). Setenta e seis pacientes (46.6%) apresentaram transtornos de humor. Sexo feminino, transtorno de ansiedade, genótipo A/A no SNP rs1867283 e genótipo C/C no SNP rs10868235 do gene NTRK2 foram todos fatores independentemente associados com transtornos de humor nesses pacientes Transtornos depressivos foram os que mais contribuíram para esses resultados. Após a regressão logística, fatores de risco independentes para transtornos depressivos em pacientes com ELT foram sexo feminino (OR=2.54; 95%IC=1.18- 5.47; p=0.017), presença de transtorno de ansiedade concomitante (OR=3.30; 95%IC=1.58-6.68; p=0.001), genótipo A/A no SNP rs1867283 do gene NTRK2 (OR=2.84; 95%IC=1.19-6.80; p=0.019), e genótipo C/C no SNP rs10868235 do gene NTRK2 (OR=2.74; IC=1.28-5.88; p=0.010). Conclusões Observamos que pacientes com ELT apresentam uma distribuição alélica distinta do gene NTRK2 quando comparados a controles sem epilepsia e que a variabilidade alélica do NTRK2 influenciou a idade de início de crises e talvez a resposta a terapia farmacológica anticonvulsivante. O sexo feminino, transtornos de ansiedade e variações alélicas no gene NTRK2 foram todos fatores de risco independentes para transtornos de humor ou transtornos depressivos em pacientes com ELT. Até onde temos conhecimento, este é o primeiro estudo evidenciando associações de variantes alélicas do gene NTRK2 em ELT. Acreditamos que outros estudos nessa área ajudarão a elucidar melhor os mecanismos envolvidos na epileptogênese do lobo temporal. Se nossos resultados forem confirmados, as variantes alélicas do gene NTRK2 poderiam ser usadas como um biomarcador para transtornos depressivos em pacientes com ELT. / Introduction The NTRK2 gene encodes a member of the neurotrophic tyrosine kinase family receptor known as TrkB. It is a membrane-associated receptor with signaling and cellular differentiation proprieties that has been involved in neuropsychiatric disorders. Objective Study the frequencies of NTRK2 allele variants in patients with temporal lobe epilepsy (TLE) compared to controls without epilepsy. The impact of these polymorphisms on major clinical and psychiatric variables in TLE was also explored. Methods A case-control study comparing the frequencies of the TrkB gene polymorphism in 198 TLE Brazilian with European origin patients and in 200 matching controls without epilepsy. In a second step, the impact of allelic variation on major clinical and electroencephalographic variables in epilepsy was evaluated in the group of TLE patients. The following polymorphisms were evaluated: rs1867283A>G, rs10868235C>T, rs1147198G>T, rs11140800A>T, rs1187286G>T, rs2289656A>G, rs1624327A>G, rs1443445A>G, rs3780645C>T, rs2378672C>T. At last, 163 TLE patients were evaluated with a psychiatry interview (SCID-I) to detect lifelong psychiatric comorbidities and this findings were analyzed in relation to NTRK2 polymorphisms. Results Patients with temporal lobe epilepsy showed a significant increase of thymine homozygosis in the rs10868235 NTRK2 SNP when compared with the control group (O.R.=1.90; 95%CI=1.17-3.09; p= 0.01) . There were no other differences between patients and controls. Patients with adenine homozygosis in the rs1443445 NTRK2 SNP showed an earlier mean age of seizure onset when compared with other patients (p<0.01). Also, we observed that thymine was significantly more frequent in the rs3780645 NTRK2 SNP in patients that needed polytheraphy for seizure control when compared to those in monotherapy. This finding perhaps reflects an increased difficulty to exert seizure control in this group of patients (O.R.= 4.13; 95%CI= 1.68-10.29; p= 0.001). We also analyzed 163 patients in the TLE group in relation to presence of psychiatric comorbidities. Psychiatric evaluation was performed using the SCID-I (Structured Clinical Interview for DSM-IV, Axis I). Seventy six patients (46.6%) showed mood disorders. Female sex, anxiety disorders, A/A genotype in rs1867283 NTRK2, and C/C genotype in the rs10868235 NTRK2 gene were all independently associated with mood disorders in these patients. Depressive disorders mostly accounted for these results. After logistic regression, independent risk factors for depressive disorder in TLE were female sex (OR=2.54; 95%CI=1.18-5.47; p=0.017), presence of concomitant anxiety disorders (OR=3.30; 95%CI=1.58-6.68; p=0.001), A/A genotype in rs1867283 NTRK2 (OR=2.84; 95%CI=1.19-6.80; p=0.019), and C/C genotype in rs10868235 NTRK2 gene (OR=2.74; 1.28-5.88; p=0.010). Conclusions We observed that patients with epilepsy showed a difference in NTRK2 allelic distribution when compared with controls without epilepsy, and that NTRK2 variability influenced age of seizure onset and perhaps pharmacologic response to seizure control. Female sex, anxiety disorders and allelic variations in NTRK2 gene were all independent risk factors for mood disorder or depressive disorders in TLE. . As far as we know, this is the first study showing an association between NTKR2 allele variants in temporal lobe epilepsy. We believe that other studies in this venue will shade some light on the molecular mechanisms involved in temporal epileptogenesis. If our results were confirmed, NTRK2 gene allele variants could be used as a biomarker for depressive disorders in patients with temporal lobe epilepsy.

Introdução O gene NTRK2 codifica um receptor pertencente a família de neurotrofinas Tirosina Kinase, conhecido como TrkB. O TrkB é um receptor de membrana com propriedades relacionadas a sinalização e diferenciação celular que tem sido envolvido em transtornos neuropsiquiátricos. Objetivo Estudar as freqüências de alelos variantes do gene NTRK2 em pacientes com epilepsia do lobo temporal (ELT) comparado a controles sem epilepsia. O impacto desses polimorfismos em variáveis clínicas e psiquiátricas dos pacientes com ELT também foi analisado. Métodos Inicialmente, realizamos estudo de caso-controle comparando as freqüências dos polimorfismos do TrkB em 198 pacientes Brasileiros com origem Européia com ELT e 200 controles sem epilepsia. Na segunda parte, foi avaliado o impacto das variantes alélicas em características clínicas e eletroencefalográficas dos pacientes com epilepsia. Os seguintes polimorfismos foram avaliados: rs1867283A>G, rs10868235C>T, rs1147198G>T, rs11140800A>T, rs1187286G>T, rs2289656A>G, rs1624327A>G, rs1443445A>G, rs3780645C>T, rs2378672C>T. Por fim, 163 pacientes com ELT foram avaliados com uma entrevista psiquiátrica (SCID-I) para detecção de transtornos psiquiátricos ao longo da vida e esses achados foram analisados em relação aos polimorfismos do gene NTRK2. Resultados Pacientes com epilepsia do lobo temporal evidenciaram um aumento significativo de Timina em homozigose no SNP rs10868235 do gene NTRK2 quando comparados ao grupo controle (O.R.=1.90; 95%IC=1.17-3.09; p= 0.01). Não foram encontradas outras diferenças entre pacientes e controles. Pacientes com Adenina em homozigose no SNP rs1443445 do gene NTRK2 tiveram uma média de idade de início de crises mais baixa quando comparados aos demais pacientes (p<0.01). Também observamos que a presença de Timina foi significativamente mais freqüente no SNP rs3780645 do gene NTRK2 em pacientes que necessitam politerapia para o controle de crises se comparados aos que estão em monoterapia. Esse achado pode significar uma maior dificuldade em obter o controle das crises nesse grupo de pacientes (O.R.= 4.13; 95%IC= 1.68-10.29; p= 0.001). Após essa análise, estudamos 163 pacientes com ELT em relação a presença ou não de comorbidades psiquiátricas. A avaliação psiquiátrica foi realizada através da aplicação do SCID-I (Entrevista Clínica Estruturada para Detecção de Transtornos Psiquiátricos de Eixo I do DSM-IV). Setenta e seis pacientes (46.6%) apresentaram transtornos de humor. Sexo feminino, transtorno de ansiedade, genótipo A/A no SNP rs1867283 e genótipo C/C no SNP rs10868235 do gene NTRK2 foram todos fatores independentemente associados com transtornos de humor nesses pacientes Transtornos depressivos foram os que mais contribuíram para esses resultados. Após a regressão logística, fatores de risco independentes para transtornos depressivos em pacientes com ELT foram sexo feminino (OR=2.54; 95%IC=1.18- 5.47; p=0.017), presença de transtorno de ansiedade concomitante (OR=3.30; 95%IC=1.58-6.68; p=0.001), genótipo A/A no SNP rs1867283 do gene NTRK2 (OR=2.84; 95%IC=1.19-6.80; p=0.019), e genótipo C/C no SNP rs10868235 do gene NTRK2 (OR=2.74; IC=1.28-5.88; p=0.010). Conclusões Observamos que pacientes com ELT apresentam uma distribuição alélica distinta do gene NTRK2 quando comparados a controles sem epilepsia e que a variabilidade alélica do NTRK2 influenciou a idade de início de crises e talvez a resposta a terapia farmacológica anticonvulsivante. O sexo feminino, transtornos de ansiedade e variações alélicas no gene NTRK2 foram todos fatores de risco independentes para transtornos de humor ou transtornos depressivos em pacientes com ELT. Até onde temos conhecimento, este é o primeiro estudo evidenciando associações de variantes alélicas do gene NTRK2 em ELT. Acreditamos que outros estudos nessa área ajudarão a elucidar melhor os mecanismos envolvidos na epileptogênese do lobo temporal. Se nossos resultados forem confirmados, as variantes alélicas do gene NTRK2 poderiam ser usadas como um biomarcador para transtornos depressivos em pacientes com ELT. / Introduction The NTRK2 gene encodes a member of the neurotrophic tyrosine kinase family receptor known as TrkB. It is a membrane-associated receptor with signaling and cellular differentiation proprieties that has been involved in neuropsychiatric disorders. Objective Study the frequencies of NTRK2 allele variants in patients with temporal lobe epilepsy (TLE) compared to controls without epilepsy. The impact of these polymorphisms on major clinical and psychiatric variables in TLE was also explored. Methods A case-control study comparing the frequencies of the TrkB gene polymorphism in 198 TLE Brazilian with European origin patients and in 200 matching controls without epilepsy. In a second step, the impact of allelic variation on major clinical and electroencephalographic variables in epilepsy was evaluated in the group of TLE patients. The following polymorphisms were evaluated: rs1867283A>G, rs10868235C>T, rs1147198G>T, rs11140800A>T, rs1187286G>T, rs2289656A>G, rs1624327A>G, rs1443445A>G, rs3780645C>T, rs2378672C>T. At last, 163 TLE patients were evaluated with a psychiatry interview (SCID-I) to detect lifelong psychiatric comorbidities and this findings were analyzed in relation to NTRK2 polymorphisms. Results Patients with temporal lobe epilepsy showed a significant increase of thymine homozygosis in the rs10868235 NTRK2 SNP when compared with the control group (O.R.=1.90; 95%CI=1.17-3.09; p= 0.01) . There were no other differences between patients and controls. Patients with adenine homozygosis in the rs1443445 NTRK2 SNP showed an earlier mean age of seizure onset when compared with other patients (p<0.01). Also, we observed that thymine was significantly more frequent in the rs3780645 NTRK2 SNP in patients that needed polytheraphy for seizure control when compared to those in monotherapy. This finding perhaps reflects an increased difficulty to exert seizure control in this group of patients (O.R.= 4.13; 95%CI= 1.68-10.29; p= 0.001). We also analyzed 163 patients in the TLE group in relation to presence of psychiatric comorbidities. Psychiatric evaluation was performed using the SCID-I (Structured Clinical Interview for DSM-IV, Axis I). Seventy six patients (46.6%) showed mood disorders. Female sex, anxiety disorders, A/A genotype in rs1867283 NTRK2, and C/C genotype in the rs10868235 NTRK2 gene were all independently associated with mood disorders in these patients. Depressive disorders mostly accounted for these results. After logistic regression, independent risk factors for depressive disorder in TLE were female sex (OR=2.54; 95%CI=1.18-5.47; p=0.017), presence of concomitant anxiety disorders (OR=3.30; 95%CI=1.58-6.68; p=0.001), A/A genotype in rs1867283 NTRK2 (OR=2.84; 95%CI=1.19-6.80; p=0.019), and C/C genotype in rs10868235 NTRK2 gene (OR=2.74; 1.28-5.88; p=0.010). Conclusions We observed that patients with epilepsy showed a difference in NTRK2 allelic distribution when compared with controls without epilepsy, and that NTRK2 variability influenced age of seizure onset and perhaps pharmacologic response to seizure control. Female sex, anxiety disorders and allelic variations in NTRK2 gene were all independent risk factors for mood disorder or depressive disorders in TLE. . As far as we know, this is the first study showing an association between NTKR2 allele variants in temporal lobe epilepsy. We believe that other studies in this venue will shade some light on the molecular mechanisms involved in temporal epileptogenesis. If our results were confirmed, NTRK2 gene allele variants could be used as a biomarker for depressive disorders in patients with temporal lobe epilepsy.

Recentemente suspeitas de que mecanismos epigenéticos poderiam estar relacionados à fisiopatologia da depressão foram levantadas. Estudos recentes indicam que as alterações na transcrição gênica, induzidas por estresse ou por drogas antidepressivas, parecem envolver mecanismos epigenéticos. Nesse sentido, resultados preliminares de nosso grupo de pesquisa indicaram pioneiramente inibição global da metilação de DNA através da administração sistêmica do agente inibidor da DNA metiltransferase (DNMTs), 5-aza-2-deoxicitidina (5-azaD), induz efeito tipo-antidepressivo, dose-dependente, no modelo animal do nado forçado em ratos(Sales et al., 2011). O córtex pré-frontal medial ventral (CPFMv) é uma estrutura límbica intimamente relacionada com a neurobiologia da depressão. Evidências recentes indicam que o efeito tipo-antidepressivo aparece associado a aumento dos níveis da neurotrofina BDNF (brain derived neurotrophic factor) e de seu receptor TrkB no CPFMv, sendo a sinalização intracelular mediada pela ativação da proteína m-TOR. Contudo, não há evidências de que esses mecanismos moleculares estariam envolvidos nos efeitos induzidos pelos inibidores da metilação do DNA. Sabe-se, no entanto, que tanto o BDNF quanto TrkB têm sua expressão regulada por metilação do DNA. Diante disso, o objetivo presente trabalho será investigar a participação da via BDNF-TrkB-mTOR do CPFMv no efeito antidepressivo induzido por inibidores da metilação de DNA. Para tanto, ratos tratados com inibidores da metilação de DNA (5-azaD ou RG-108), em dois momentos diferentes (imediatamente após o PT e 23horas após o PT) foram submetidos ao teste do nado forçado (FST). Outro grupo de animais recebeu uma injeção intra-CPMv de k252a ou Rapamicina, 40 minutos antes do teste e uma injeção de BDNF intra-CPFMv, 30 minutos antes do teste. Em outro experimento, grupos independentes de animais submetidos ao nado forçado foram tratados sistemicamente com RG-108 e receberam injeção intra-CPFMv de K252a (antagonista de Trk) ou de rapamicina (inibidor da m-Tor), a fim de investigar se o efeito dessas drogas depende da via BDNF-TrkB-mTOR no CPFMv. Um grupo independente foi tratado com RG108 e CPFM desses animais foi dissecado para posterior análise da expressão de BDNF, TrkB e m-TOR, bem como da metilação de DNA. O tratamento com RG108 e 5azaD sistêmico reduziu o tempo de imobilidade dos animais submetidos ao nado forçado nos dois tempo de administração. A administração intra-CPFMv de BDNF promoveu efeito antidepressivo no FST, e esse efeito foi bloqueado pela administração de k252a ou Rapamicina no CPFMv. No mesmo sentido, o efeito antidepressivo do RG108 sistêmico foi bloqueado pela administração intra-CPFMv de k252a ou Rapamicina. Entretanto, a medida dos níveis de metilação global no CPFMv não apresentou alteração como tratamento com RG108, e também não mostrou alteração nos níveis de BDNF presente no CPF. O tratamento com RG108 não alterou a expressão, bem como a ativação de TRkB e mTOR. Concluímos que os inibidores da metilação do DNA apresentam agudamente efeito tipo antidepressivo rápido, que necessita da funcionalidade integral da via BDNF-TRkB-mTOR. Entretanto, esse efeito parece não alterar a síntese e expressão das proteínas envolvidas nessa via no que diz respeito ao CPFmv. / Recent studies indicate that changes in gene transcription induced by stress or antidepressant drugs appear to involve epigenetic mechanisms. Accordingly, results of our research group pioneered indicated global inhibition of DNA methylation through systemic administration of an inhibitor of DNA methyltransferase (DNMTs), 5-aza-2-deoxycytidine (5-AzaD), induces antidepressant-like effect dose-dependent in the animal model of forced swimming in rats (Sales et al., 2011). The ventral medial prefrontal (vmPFC) cortex is a limbic structure closely related to the neurobiology of depression. Recent evidence indicates that the antidepressant-like effect appears associated with increased levels BDNF (Brain derived neurotrophic factor) and its receptor TrkB in vmPFC, and intracellular signaling mediated by activation of protein mTOR. However, there is no evidence that these molecular mechanisms are involved in the effects induced by inhibitors of DNA methylation. It is known, however, both as BDNF and TrkB expression is regulated by DNA methylation. Thus, the goal of this work is to investigate the role of BDNF-TRkB pathway mTOR-vmPFC in the antidepressant effect induced by inhibitors of DNA methylation. To this end, rats treated with inhibitors of DNA methylation (5-Azad or RG-108), at two different times (immediately after 23hours after the PT and PT) were subjected to the forced swim test (FST). Another group received an intra-vmPFC injection of K252a or Rapamycin 40 minutes before the test, and an injection intra-vmPFC of BDNF 30 minutes before the test. In another experiment, separate groups undergoing the forced swim were treated systemically with RG-108 and received intra-vmPFC of K252a (Trk antagonist) or injection of rapamycin (m-Tor inhibitors) in order to investigate the effect these drugs depends on BDNF-TrkB-mTOR pathway in vmPFC. A separate group was treated with RG108 and mPFC these animals were dissected for analysis of the expression of BDNF and TrkB m-TOR, as well as DNA methylation. The systemic treatment whit 5azaD and RG108 reduced the immobility time of rats subjected to FST administration in both time. The intra-vmPFC BDNF administration promoted antidepressant effect in the FST, and this effect was blocked by the administration of K252a or Rapamycin in vmPFC. Similarly, the antidepressant effect of systemic RG108 was blocked by intra-vmPFC of K252a or Rapamycin administration. However, the measurement of the levels of global methylation in CPFMv did not change as treatment with RG108, and also showed no change in the levels of BDNF present in the CPF. Treatment with RG108 did not alter the expression and activation of TrkB and mTOR. We conclude that inhibitors of DNA methylation present acutely antidepressant-like effect, it needs the full functionality of the BDNF-TrkB-mTOR pathway. However, this effect seems not to alter the synthesis and expression of proteins involved in this pathway at vmPFC.

Einleitung: Die Ausbildung eines Netzhautödems ist eine Hauptursache für die Verschlechterung des Sehvermögens bei ischämisch-hypoxischen und inflammatorischen Netzhauterkrankungen. Neben der erhöhten Permeabilität der Blut-Retina-Schranke trägt eine Wasserakkumulation in Netzhautzellen zur Ausbildung eines Netzhautödems bei. Müllerzellen regulieren die retinale Ionen- und Osmohomöostase, indem sie einen transzellulären Ionen- und Wassertransport vermitteln. Zudem kontrollieren Müllerzellen die Größe des Extrazellularraumes, indem sie bei neuronaler Aktivität eine Zellkörperschwellung – ausgelöst durch eine Verkleinerung der extrazellulären Osmolarität – verhindern. Unter pathologischen Bedingungen ist die Volumenregulation gestört, sodass Müllerzellen bei Hypoosmolarität anschwellen. Diese Müllerzellschwellung und eine Glutamat-induzierte Schwellung retinaler Neurone tragen zur Ausbildung eines zytotoxischen Netzhautödems bei. Neuroprotektive Faktoren wie BDNF (brain-derived neurotrophic factor) und bFGF (basic fibroblast growth factor) stimulieren das Überleben retinaler Neurone und verzögern so die retinale Degeneration. Zielstellung: Es war zu zu ermitteln, ob BDNF die zytotoxische Schwellung von Müller- und Bipolarzellen der Rattennetzhaut verhindert. Material und Methoden: Es wurden Netzhautschnitte und isolierte Müller- und Bipolarzellen von 55 adulten Long-Evans-Ratten (durchschnittlich 8-15 Zellen pro Versuchsreihe) verwendet. Eine osmotische Schwellung von Müller- und Bipolarzellen wurde durch eine Superfusion der Schnitte oder der Zellen mit einer 60%igen hypoosmolaren Lösung in Ab- oder Anwesenheit von Bariumchlorid induziert. Die maximale Querschnittsfläche von Müller- und Bipolarzellsomata wurde vor und nach einer vierminütigen Superfusion mit einem konfokalen Laserscanningmikroskop aufgezeichnet. Die nach der Superfusion ermittelte Querschnittsfläche wurde zu den anfänglich gemittelten Kontrollwerten in Beziehung gesetzt und prozentual als Mittelwert mit Standardfehler bestimmt. Mit Hilfe des Prism-Statistikprogramms (Graphpad) wurden die Ergebnisse mittels einem one-way ANOVA Test und einem nachfolgenden Bonferroni\'s multiple comparison Test sowie durch einen Mann-Whitney U Test statistisch analysiert. Ergebnisse: Bei Anwesenheit von BDNF wurde die osmotische Schwellung von Müllerzellen konzentrationsabhängig sowohl in Netzhautschnitten als auch in isolierten Zellen inhibiert. Ebenso inhibierte BDNF konzentrationsabhängig die Schwellung von Bipolarzellen in Netzhautschnitten, jedoch nicht in isolierten Zellen. In Schnitten von postischämischen Netzhäuten bewirkte BDNF eine Schwellungsinhibition von Müllerzellen, nicht aber von Bipolarzellen. Mit pharmakologischen Blockern wurde die durch BDNF induzierte Signalkaskade untersucht. Die BDNF-Schwellungsinhibition von Müllerzellen wurde durch eine Aktivierung von TrkB bewirkt. Die TrkB-Aktivierung führte in Müllerzellen zu einer Transaktivierung von FGF-Rezeptoren sowie zu einer Aktivierung einer glutamatergen-purinergen Signalkaskade, von der bekannt ist, dass sie die osmotische Müllerzellschwellung unterdrückt. Da bFGF die osmotische Müllerzellschwellung inhibiert, wird die Transaktivierung der FGF-Rezeptoren wahrscheinlich durch eine BDNF-induzierte Freisetzung von bFGF aus Müllerzellen vermittelt. Die Ergebnisse lassen vermuten, dass BDNF indirekt auf Bipolarzellen wirkt, indem es eine Freisetzung von Faktoren wie bFGF aus Müllerzellen induziert. Schlussfolgerungen: Die Schwellungsinhibition von Müller- und Bipolarzellen könnte ein neuroprotektiver Mechanismus von BDNF in der Netzhaut darstellen. Während BDNF direkt TrkB auf Müllerzellen aktiviert, ist die Inhibition der Bipolarzellschwellung indirekt und durch die Ausschüttung von glialen Faktoren wie bFGF vermittelt. Der Verlust des Effektes von BDNF auf die Bipolarzellschwellung in ischämischen Netzhäuten könnte darauf zurückzuführen sein, dass gliotische Müllerzellen keine glialen Faktoren mehr in Reaktion auf BDNF freisetzen. Der Verlust des glialen Einflusses auf die Bipolarzellvolumenhomöostase könnte zur Neurodegeneration in der ischämischen Netzhaut beitragen. / Introduction: Tissue edema is a major blinding complication of ischemic-hypoxic and inflammatory retinal diseases. In addition to the hyperpemeability of the blood-retinal barrier, water accumulation in retinal cells resulting in cellular swelling may contribute to the development of retinal edema. Müller glial cells regulate the retinal ion and water homeostasis by allowing transcellular ion and water fluxes. During neuronal activity Müller cells control the extracellular space volume by autocrine inhibition of cellular swelling caused by the reduction of extracellular osmolarity. However, under pathological conditions, Müller cells are not capable to regulate their volume so that they swell rapidly under hypoosmolarity. The osmotic swelling of Müller glial cells and the glutamate induced swelling of retinal neurons contribute to the development of cytotoxic retinal edema. Various neuroprotective factors including brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) stimulate the survival of retinal neurons and thus delay the retinal degeneration. Objective: The objective of the study is to determine whether BDNF inhibits the osmotic swelling of Müller and bipolar cells of the rat retina. Material and Methods: Retinal slices and freshly isolated Müller and bipolar cells of 55 adult Long-Evans rats (in average 8-15 cells per trial) were used. Osmotic swelling of Müller and bipolar cells was induced by superfusion of retinal slices or isolated cells with a 60% hypoosmotic extracellular solution in the absence or presence of barium chloride. The maximal cross-sectional area of Müller and bipolar cell somata was recorded before and after a four minute-long superfusion by using a laser scanning microscope. To determine the extent of cell soma swelling, the cross-sectional area of the cell body extent after superfusion was related to the former averaged cross-sectional area. Results were given as means with standard error as percent values. Statistical analysis was made with Prism (Graphpad) and the significance was determined by the One-way ANOVA test followed by Bonferroni\'s multiple comparison test and the Mann-Whitney U test, respectively. Results: We found that BDNF inhibits dose-depending the osmotic swelling of Müller cells in retinal slices and of isolated cells. BDNF also inhibited dose-depending the osmotic swelling of bipolar cells in retinal slices; however, it did not inhibit the osmotic swelling in isolated bipolar cells. In slices of postischemic retinas, BDNF inhibited the swelling of Müller cells but not the swelling of bipolar cells. The BDNF induced signal transduction cascade was examined by simultaneous administration of blocking agents with the receptor agonists in the hypoosmotic solution. The BDNF-induced inhibition of the osmotic Müller cell swelling was mediated by activation of TrkB. Activation of TrkB in Müller cells results in transactivation of FGF receptors and in an activation of a glutamatergic-purinergic signal transduction cascade which is known to inhibit the osmotic swelling of the cells. Since bFGF also inhibits the osmotic swelling of Müller cells, it can be assumed that the transactivation of FGF receptors is mediated by a BDNF-induced release of bFGF from Müller cells. The results suggest that the effect of BDNF on bipolar cells is indirect by inducing a subsequent release of glial factor from Müller cells such as bFGF. Conclusion: The results show that BDNF inhibits the osmotic swelling of Müller and bipolar cells. The inhibition of cytotoxic cell swelling may contribute to the neuroprotective action of BDNF in the retina. While BDNF acts directly in Müller cells, the BDNF-induced inhibition of the bipolar cell swelling is indirect and mediated by the release of glial factors such as bFGF from Müller cells. The abrogation of the BDNF-induced inhibition of the osmotic bipolar cell swelling in the postischemic retina could be explained with the impairment of the release of glial factors by Müller cells. The abrogation of the Müller cell-mediated regulation of the bipolar cell volume could contribute to the neuronal degeneration in the ischemic retina.

Einleitung: Die Ausbildung eines Netzhautödems ist eine Hauptursache für die Verschlechterung des Sehvermögens bei ischämisch-hypoxischen und inflammatorischen Netzhauterkrankungen. Neben der erhöhten Permeabilität der Blut-Retina-Schranke trägt eine Wasserakkumulation in Netzhautzellen zur Ausbildung eines Netzhautödems bei. Müllerzellen regulieren die retinale Ionen- und Osmohomöostase, indem sie einen transzellulären Ionen- und Wassertransport vermitteln. Zudem kontrollieren Müllerzellen die Größe des Extrazellularraumes, indem sie bei neuronaler Aktivität eine Zellkörperschwellung – ausgelöst durch eine Verkleinerung der extrazellulären Osmolarität – verhindern. Unter pathologischen Bedingungen ist die Volumenregulation gestört, sodass Müllerzellen bei Hypoosmolarität anschwellen. Diese Müllerzellschwellung und eine Glutamat-induzierte Schwellung retinaler Neurone tragen zur Ausbildung eines zytotoxischen Netzhautödems bei. Neuroprotektive Faktoren wie BDNF (brain-derived neurotrophic factor) und bFGF (basic fibroblast growth factor) stimulieren das Überleben retinaler Neurone und verzögern so die retinale Degeneration. Zielstellung: Es war zu zu ermitteln, ob BDNF die zytotoxische Schwellung von Müller- und Bipolarzellen der Rattennetzhaut verhindert. Material und Methoden: Es wurden Netzhautschnitte und isolierte Müller- und Bipolarzellen von 55 adulten Long-Evans-Ratten (durchschnittlich 8-15 Zellen pro Versuchsreihe) verwendet. Eine osmotische Schwellung von Müller- und Bipolarzellen wurde durch eine Superfusion der Schnitte oder der Zellen mit einer 60%igen hypoosmolaren Lösung in Ab- oder Anwesenheit von Bariumchlorid induziert. Die maximale Querschnittsfläche von Müller- und Bipolarzellsomata wurde vor und nach einer vierminütigen Superfusion mit einem konfokalen Laserscanningmikroskop aufgezeichnet. Die nach der Superfusion ermittelte Querschnittsfläche wurde zu den anfänglich gemittelten Kontrollwerten in Beziehung gesetzt und prozentual als Mittelwert mit Standardfehler bestimmt. Mit Hilfe des Prism-Statistikprogramms (Graphpad) wurden die Ergebnisse mittels einem one-way ANOVA Test und einem nachfolgenden Bonferroni\''s multiple comparison Test sowie durch einen Mann-Whitney U Test statistisch analysiert. Ergebnisse: Bei Anwesenheit von BDNF wurde die osmotische Schwellung von Müllerzellen konzentrationsabhängig sowohl in Netzhautschnitten als auch in isolierten Zellen inhibiert. Ebenso inhibierte BDNF konzentrationsabhängig die Schwellung von Bipolarzellen in Netzhautschnitten, jedoch nicht in isolierten Zellen. In Schnitten von postischämischen Netzhäuten bewirkte BDNF eine Schwellungsinhibition von Müllerzellen, nicht aber von Bipolarzellen. Mit pharmakologischen Blockern wurde die durch BDNF induzierte Signalkaskade untersucht. Die BDNF-Schwellungsinhibition von Müllerzellen wurde durch eine Aktivierung von TrkB bewirkt. Die TrkB-Aktivierung führte in Müllerzellen zu einer Transaktivierung von FGF-Rezeptoren sowie zu einer Aktivierung einer glutamatergen-purinergen Signalkaskade, von der bekannt ist, dass sie die osmotische Müllerzellschwellung unterdrückt. Da bFGF die osmotische Müllerzellschwellung inhibiert, wird die Transaktivierung der FGF-Rezeptoren wahrscheinlich durch eine BDNF-induzierte Freisetzung von bFGF aus Müllerzellen vermittelt. Die Ergebnisse lassen vermuten, dass BDNF indirekt auf Bipolarzellen wirkt, indem es eine Freisetzung von Faktoren wie bFGF aus Müllerzellen induziert. Schlussfolgerungen: Die Schwellungsinhibition von Müller- und Bipolarzellen könnte ein neuroprotektiver Mechanismus von BDNF in der Netzhaut darstellen. Während BDNF direkt TrkB auf Müllerzellen aktiviert, ist die Inhibition der Bipolarzellschwellung indirekt und durch die Ausschüttung von glialen Faktoren wie bFGF vermittelt. Der Verlust des Effektes von BDNF auf die Bipolarzellschwellung in ischämischen Netzhäuten könnte darauf zurückzuführen sein, dass gliotische Müllerzellen keine glialen Faktoren mehr in Reaktion auf BDNF freisetzen. Der Verlust des glialen Einflusses auf die Bipolarzellvolumenhomöostase könnte zur Neurodegeneration in der ischämischen Netzhaut beitragen.:Inhaltsverzeichnis ABKÜRZUNGSVERZEICHNIS IX ABBILDUNGSVERZEICHNIS XI TABELLENVERZEICHNIS XIV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Sehorgan - Das Auge 3 2.2 Retina beim Mensch und Tier – Aufbau und Funktionalitäten 4 2.2.1 Bildprozessor der Tierwelt – Die Retina 10 2.2.2 Die Sehbahn – Visuelle Verarbeitung 10 2.2.3 Die Müllerzelle – Vorkommen und Funktion 13 2.2.4 Tierartlicher Vergleich der Müllerzelle 14 2.2.5 Netzhaut als Modellgewebe 16 2.3 Ödembildung: Netzhautödem – Hirnödem 17 2.3.1 Allgemein: Die Pathogenese des Ödems 17 2.3.2 Das Gehirnödem – Ursachen und Folge 17 2.3.3 Das Netzhautödem – Ursachen und Folge 18 2.3.4 Volumen- und Osmohomöostase der Retina 19 2.3.5 Osmotische Schwellung und Osmoregulation der Müllerzelle 19 2.4 Neuroprotektion durch Neurotrophine und Wachstumsfaktoren 21 2.4.1 Brain-derived neurotrophic factor (BDNF) 21 2.4.2 Basic Fibroblastic Growth Factor (bFGF) 22 2.4.3 Neuroprotektive Effekte von BDNF und bFGF in der Retina 23 2.5 Zielstellung 24 2.6 Veterinärmedizinische Relevanz 25 2.6.1 Augenerkrankungen in der Klein- und Großtiermedizin 25 2.6.2 Schlussfolgerung für die Tiermedizin 31 3 TIERE, MATERIAL UND METHODEN 32 3.1 Versuchstiere 32 3.2 Material 32 3.2.1 Chemikalien und Reagenzien 32 3.2.2 Lösungen 33 3.2.3 Testsubstanzen 33 3.2.4 Pharmakologische Blocker 33 3.2.5 Antikörper 35 3.3 Verwendete Materialien und Geräte 36 3.4 Versuchsaufbau und Durchführung von Schwellungsversuchen 37 3.4.1 Gewebspräparation: Retina 37 3.4.2 Zellschwellungsversuche 39 3.4.3 Aufnahmetechnik 40 3.4.4 Superfusion von Retinaschnitten 40 3.4.5 Superfusion von isolierten retinalen Zellen 41 3.4.6 Das Physiologie-Modell: Isoosmolarität – Hypoosmolarität 42 3.4.7 Das Pathologie-Modell mit Barium 42 3.4.8 Tiermodell der retinalen Ischämie – Reperfusion 43 3.5 Auswertungsverfahren 43 3.5.1 Morphometrie der Somata 44 3.5.2 Statistische Analyse 44 3.6 Immunozytochemische Färbungen 45 4 ERGEBNISSE 46 4.1 Zellidentifikation in Retinaschnitten 46 4.1.1 Färbemethodik – MitoTracker Orange 47 4.1.2 Morphologie der Müllerzelle 47 4.1.3 Morphologie der Bipolarzelle 48 4.1.4 Identifikation von Müller- und Bipolarzellen 48 4.1.5 Morphologie isolierter Müller – und Bipolarzellen 49 4.2 Kontrollversuche 50 4.3 Untersuchung des Schwellungsverhaltens mit Testsubstanzen 51 4.4 Untersuchung des Schwellungsverhalten unter BDNF 52 4.4.1 Morphologie der Müllerzellen in Retinaschnitte unter BDNF 52 4.4.2 Wirkung von BDNF auf Müllerzellen in Retinaschnitten 53 4.4.3 Wirkung von BDNF auf die Schwellung von isolierten Müllerzellen 57 4.4.4 Wirkung von BDNF auf die osmotische Schwellung von Bipolarzellen in Retinaschnitten 58 4.4.5 Konzentrationsabhängigkeit der Wirkung von BDNF auf die osmotische Schwellung von Bipolarzellen in Retinaschnitten 59 4.4.6 Wirkung von BDNF auf die Schwellung von isolierten Bipolarzellen 61 4.4.7 Wirkung von BDNF auf die osmotische Müller- und Bipolarzellschwellung in der postischämischen Retina 62 4.5 Untersuchung der osmotischen Schwellung von retinalen Zellen unter bFGF 63 4.5.1 Wirkung von bFGF auf Müllerzellen in Retinaschnitten 63 4.5.2 bFGF-induzierte Transaktivierung metabotroper Glutamatrezeptoren in Müllerzellen 65 4.5.3 Wirkung von bFGF auf die Schwellung von Bipolarzellen in Retinaschnitten 65 4.6 Immunozytochemischer Nachweis von BDNF und TrkB in Müller- und Bipolarzellen 66 4.6.1 Kontrollaufnahmen 66 4.6.2 Immunzytochemischer Nachweis von BDNF und TrkB in isolierten Müllerzellen 67 4.6.3 Immunzytochemischer Nachweis von BDNF und TrkB in isolierten Bipolarzellen 69 5 DISKUSSION 71 5.1 Osmotische Volumenregulation bei Müller- und Bipolarzellen 71 5.2 Immunozytochemische Lokalisation von BDNF und TrkB 73 5.3 Inhibition der osmotischen Schwellung von Müller- und Bipolarzellen durch BDNF 75 5.4 TrkB-Aktivierung in Müllerzellen durch BDNF 77 5.5 Abhängigkeit des BDNF-Effekts von einer Transaktivierung von FGF-Rezeptoren 78 5.6 Indirekte Wirkung von BDNF auf die Bipolarzellschwellung durch gliale Faktoren 79 5.7 Abhängigkeit der BDNF-Wirkung von der Transaktivierung weiterer Rezeptoren 80 5.8 BDNF-induzierte Aktivierung von Ionenkanälen in Müllerzellen 81 5.9 BDNF-induzierte Signalkaskade der Inhibition der retinalen Zellschwellung 82 5.10 Neuroprotektive Wirkung von BDNF 84 6 ZUSAMMENFASSUNG 86 7 SUMMARY 88 8 BILDQUELLENVERZEICHNIS 90 9 LITERATURVERZEICHNIS 91 DANKSAGUNG 109 / Introduction: Tissue edema is a major blinding complication of ischemic-hypoxic and inflammatory retinal diseases. In addition to the hyperpemeability of the blood-retinal barrier, water accumulation in retinal cells resulting in cellular swelling may contribute to the development of retinal edema. Müller glial cells regulate the retinal ion and water homeostasis by allowing transcellular ion and water fluxes. During neuronal activity Müller cells control the extracellular space volume by autocrine inhibition of cellular swelling caused by the reduction of extracellular osmolarity. However, under pathological conditions, Müller cells are not capable to regulate their volume so that they swell rapidly under hypoosmolarity. The osmotic swelling of Müller glial cells and the glutamate induced swelling of retinal neurons contribute to the development of cytotoxic retinal edema. Various neuroprotective factors including brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) stimulate the survival of retinal neurons and thus delay the retinal degeneration. Objective: The objective of the study is to determine whether BDNF inhibits the osmotic swelling of Müller and bipolar cells of the rat retina. Material and Methods: Retinal slices and freshly isolated Müller and bipolar cells of 55 adult Long-Evans rats (in average 8-15 cells per trial) were used. Osmotic swelling of Müller and bipolar cells was induced by superfusion of retinal slices or isolated cells with a 60% hypoosmotic extracellular solution in the absence or presence of barium chloride. The maximal cross-sectional area of Müller and bipolar cell somata was recorded before and after a four minute-long superfusion by using a laser scanning microscope. To determine the extent of cell soma swelling, the cross-sectional area of the cell body extent after superfusion was related to the former averaged cross-sectional area. Results were given as means with standard error as percent values. Statistical analysis was made with Prism (Graphpad) and the significance was determined by the One-way ANOVA test followed by Bonferroni\''s multiple comparison test and the Mann-Whitney U test, respectively. Results: We found that BDNF inhibits dose-depending the osmotic swelling of Müller cells in retinal slices and of isolated cells. BDNF also inhibited dose-depending the osmotic swelling of bipolar cells in retinal slices; however, it did not inhibit the osmotic swelling in isolated bipolar cells. In slices of postischemic retinas, BDNF inhibited the swelling of Müller cells but not the swelling of bipolar cells. The BDNF induced signal transduction cascade was examined by simultaneous administration of blocking agents with the receptor agonists in the hypoosmotic solution. The BDNF-induced inhibition of the osmotic Müller cell swelling was mediated by activation of TrkB. Activation of TrkB in Müller cells results in transactivation of FGF receptors and in an activation of a glutamatergic-purinergic signal transduction cascade which is known to inhibit the osmotic swelling of the cells. Since bFGF also inhibits the osmotic swelling of Müller cells, it can be assumed that the transactivation of FGF receptors is mediated by a BDNF-induced release of bFGF from Müller cells. The results suggest that the effect of BDNF on bipolar cells is indirect by inducing a subsequent release of glial factor from Müller cells such as bFGF. Conclusion: The results show that BDNF inhibits the osmotic swelling of Müller and bipolar cells. The inhibition of cytotoxic cell swelling may contribute to the neuroprotective action of BDNF in the retina. While BDNF acts directly in Müller cells, the BDNF-induced inhibition of the bipolar cell swelling is indirect and mediated by the release of glial factors such as bFGF from Müller cells. The abrogation of the BDNF-induced inhibition of the osmotic bipolar cell swelling in the postischemic retina could be explained with the impairment of the release of glial factors by Müller cells. The abrogation of the Müller cell-mediated regulation of the bipolar cell volume could contribute to the neuronal degeneration in the ischemic retina.:Inhaltsverzeichnis ABKÜRZUNGSVERZEICHNIS IX ABBILDUNGSVERZEICHNIS XI TABELLENVERZEICHNIS XIV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Sehorgan - Das Auge 3 2.2 Retina beim Mensch und Tier – Aufbau und Funktionalitäten 4 2.2.1 Bildprozessor der Tierwelt – Die Retina 10 2.2.2 Die Sehbahn – Visuelle Verarbeitung 10 2.2.3 Die Müllerzelle – Vorkommen und Funktion 13 2.2.4 Tierartlicher Vergleich der Müllerzelle 14 2.2.5 Netzhaut als Modellgewebe 16 2.3 Ödembildung: Netzhautödem – Hirnödem 17 2.3.1 Allgemein: Die Pathogenese des Ödems 17 2.3.2 Das Gehirnödem – Ursachen und Folge 17 2.3.3 Das Netzhautödem – Ursachen und Folge 18 2.3.4 Volumen- und Osmohomöostase der Retina 19 2.3.5 Osmotische Schwellung und Osmoregulation der Müllerzelle 19 2.4 Neuroprotektion durch Neurotrophine und Wachstumsfaktoren 21 2.4.1 Brain-derived neurotrophic factor (BDNF) 21 2.4.2 Basic Fibroblastic Growth Factor (bFGF) 22 2.4.3 Neuroprotektive Effekte von BDNF und bFGF in der Retina 23 2.5 Zielstellung 24 2.6 Veterinärmedizinische Relevanz 25 2.6.1 Augenerkrankungen in der Klein- und Großtiermedizin 25 2.6.2 Schlussfolgerung für die Tiermedizin 31 3 TIERE, MATERIAL UND METHODEN 32 3.1 Versuchstiere 32 3.2 Material 32 3.2.1 Chemikalien und Reagenzien 32 3.2.2 Lösungen 33 3.2.3 Testsubstanzen 33 3.2.4 Pharmakologische Blocker 33 3.2.5 Antikörper 35 3.3 Verwendete Materialien und Geräte 36 3.4 Versuchsaufbau und Durchführung von Schwellungsversuchen 37 3.4.1 Gewebspräparation: Retina 37 3.4.2 Zellschwellungsversuche 39 3.4.3 Aufnahmetechnik 40 3.4.4 Superfusion von Retinaschnitten 40 3.4.5 Superfusion von isolierten retinalen Zellen 41 3.4.6 Das Physiologie-Modell: Isoosmolarität – Hypoosmolarität 42 3.4.7 Das Pathologie-Modell mit Barium 42 3.4.8 Tiermodell der retinalen Ischämie – Reperfusion 43 3.5 Auswertungsverfahren 43 3.5.1 Morphometrie der Somata 44 3.5.2 Statistische Analyse 44 3.6 Immunozytochemische Färbungen 45 4 ERGEBNISSE 46 4.1 Zellidentifikation in Retinaschnitten 46 4.1.1 Färbemethodik – MitoTracker Orange 47 4.1.2 Morphologie der Müllerzelle 47 4.1.3 Morphologie der Bipolarzelle 48 4.1.4 Identifikation von Müller- und Bipolarzellen 48 4.1.5 Morphologie isolierter Müller – und Bipolarzellen 49 4.2 Kontrollversuche 50 4.3 Untersuchung des Schwellungsverhaltens mit Testsubstanzen 51 4.4 Untersuchung des Schwellungsverhalten unter BDNF 52 4.4.1 Morphologie der Müllerzellen in Retinaschnitte unter BDNF 52 4.4.2 Wirkung von BDNF auf Müllerzellen in Retinaschnitten 53 4.4.3 Wirkung von BDNF auf die Schwellung von isolierten Müllerzellen 57 4.4.4 Wirkung von BDNF auf die osmotische Schwellung von Bipolarzellen in Retinaschnitten 58 4.4.5 Konzentrationsabhängigkeit der Wirkung von BDNF auf die osmotische Schwellung von Bipolarzellen in Retinaschnitten 59 4.4.6 Wirkung von BDNF auf die Schwellung von isolierten Bipolarzellen 61 4.4.7 Wirkung von BDNF auf die osmotische Müller- und Bipolarzellschwellung in der postischämischen Retina 62 4.5 Untersuchung der osmotischen Schwellung von retinalen Zellen unter bFGF 63 4.5.1 Wirkung von bFGF auf Müllerzellen in Retinaschnitten 63 4.5.2 bFGF-induzierte Transaktivierung metabotroper Glutamatrezeptoren in Müllerzellen 65 4.5.3 Wirkung von bFGF auf die Schwellung von Bipolarzellen in Retinaschnitten 65 4.6 Immunozytochemischer Nachweis von BDNF und TrkB in Müller- und Bipolarzellen 66 4.6.1 Kontrollaufnahmen 66 4.6.2 Immunzytochemischer Nachweis von BDNF und TrkB in isolierten Müllerzellen 67 4.6.3 Immunzytochemischer Nachweis von BDNF und TrkB in isolierten Bipolarzellen 69 5 DISKUSSION 71 5.1 Osmotische Volumenregulation bei Müller- und Bipolarzellen 71 5.2 Immunozytochemische Lokalisation von BDNF und TrkB 73 5.3 Inhibition der osmotischen Schwellung von Müller- und Bipolarzellen durch BDNF 75 5.4 TrkB-Aktivierung in Müllerzellen durch BDNF 77 5.5 Abhängigkeit des BDNF-Effekts von einer Transaktivierung von FGF-Rezeptoren 78 5.6 Indirekte Wirkung von BDNF auf die Bipolarzellschwellung durch gliale Faktoren 79 5.7 Abhängigkeit der BDNF-Wirkung von der Transaktivierung weiterer Rezeptoren 80 5.8 BDNF-induzierte Aktivierung von Ionenkanälen in Müllerzellen 81 5.9 BDNF-induzierte Signalkaskade der Inhibition der retinalen Zellschwellung 82 5.10 Neuroprotektive Wirkung von BDNF 84 6 ZUSAMMENFASSUNG 86 7 SUMMARY 88 8 BILDQUELLENVERZEICHNIS 90 9 LITERATURVERZEICHNIS 91 DANKSAGUNG 109

O exercício físico pode atenuar os efeitos do envelhecimento sobre o sistema nervoso central, por meio do aumento da expressão de neurotrofinas, tais como fator neurotrófico derivado do cérebro (BDNF), o qual promove a ramificação dendrítica e melhora da maquinaria sináptica, pela interação com seu receptor TrkB. Receptores TrkB são produzidos no corpo da célula e transportados aos terminais axonais, por meio de SLP1, CRMP2, Rab27B e Sortilina onde são ancorados para realizar seu papel fisiológico. Sabendo que a relação entre o tráfego de receptores de neurotrofinas e o treinamento físico ainda é pouco conhecida, o objetivo do presente trabalho é analisar os níveis do receptor TrkB, bem como de seus transportadores anterógrados e retrógrados, no sistema nervoso central de ratos idosos, modelos de neurodegeneração, expostos a diferentes protocolos de treinamento físico moderado. Os ratos do primeiro grupo experimental foram expostos a 1mg/kg/dia de Rotenona ou DMSO durante 4 semanas, depois, juntamente com a exposição à rotenona, realizaram treinamento físico moderado em esteira, 5 vezes por semana, durante 40 minutos; ou permaneceram em repouso. Os ratos do segundo grupo experimental realizaram 6 semanas de treinamento, sendo em seguida expostos à rotenona por 4 semanas, e subdividos em dois grupos, um que continuou o exercício e outro que ficou sedentário. Os resultados encontrados sugerem que o treinamento físico parece reverter ou prevenir de maneira geral os danos presentes na neurodegeneração considerando as proteínas do tráfego de BDNF e seu receptor, e ainda, que a magnitude e direção destas alterações está diretamente relacionada ao protocolo de treinamento físico, bem como, a região do sistema nervoso central analisada / Physical exercise can attenuate the effects of aging on the central nervous system by increasing the expression of neurotrophins such as brain-derived neurotrophic factor (BDNF), which promotes dendritic branching and enhances synaptic machinery, through interaction with its receptor TrkB. TrkB receptors are synthesized in the cell body and are transported to the axonal terminals, through SLP1, CRMP2, Sortilin and Rab27B, to where receptors are anchored to perform its physiological role. However, the aspects of the neurotrophin receptors traffic after physical training is still a matter of investigation. Thus, the present study aims to analyze the expression levels of TrkB receptor and their anterograde carriers in aged Lewis rats, model of neurodegeneration, and its relationship with moderate exercise training. Rats from the first experimental group were exposed to 1mg/kg/day of Rotenone (ROT) or DMSO for 4 weeks, and then subjected or not to moderate exercise running on treadmill, five days a week, 40 minutes a day, combined with the drug. Rats from the second experimental group were trained for 6 weeks, followed by exposure to rotenone during 4 weeks, rats were then subdivided into two groups, one that continued the exercise and the other became sedentary. Results suggest that exercise training appears to reverse or prevent the impairment related to neurodegeneration considering the proteins involved in BDNF signaling, and also that the magnitude and direction of these changes in directly related to the physical training protocol, as well as the area of the central nervous system analyzed

L’objectif de ce travail a été de tester l’hypothèse selon laquelle l’effet bénéfique de l’administration de la forme recombinante de l’activateur tissulaire du plasminogène (rt-PA) chez le patient victime d’un accident vasculaire cérébral (AVC) ischémique ne résulte pas uniquement de son action fibrinolytique mais aussi de sa capacité à augmenter le brain-derived neurotrophic factor (BDNF) dans le cerveau. Dans ce but, nous avons conduit une première étude visant à évaluer chez l’animal sain, l’effet du rt-PA sur les taux cérébraux de BDNF. Dans une seconde approche, nous avons étudié l’effet du rt-PA sur les taux sériques de BDNF chez le patient victime d’un AVC ischémique et chez l’animal soumis à une ischémie cérébrale focale. Le sang a été prélevé chez l’Homme à l’admission (J0), J1, J7 et J90 après l’AVC, et chez le Rat avant et après (1h, 4h et 24h) l’ischémie. Le BDNF a été mesuré dans le cerveau par technique de Western blot et dans le sang par technique ELISA. Dans les deux études le rt-PA (Actilyse®) a été administré sous forme d’un bolus suivi d’une perfusion d’une heure. La première étude montre que 1) le rt-PA augmente les taux de BDNF dans l’hippocampe, 2) le traitement par MK801 (un antagoniste des récepteurs NMDA) mais pas par l’acide tranexamique (un inhibiteur de la plasmine) annule l’effet du rt-PA sur les taux de BDNF. La deuxième étude met en évidence que 1) la récupération neurologique est meilleure chez les patients recevant le rt-PA, 2) le traitement par rt-PA augmente les taux sériques de BDNF à J1 et J7 chez l’Homme, mais ne modifie pas les taux sanguins de BDNF chez l’animal, 3) les taux de BDNF ne sont pas corrélés à la récupération neurologique mais sont inversement corrélés au score cardiovasculaire du patient. En conclusion, nos résultats suggèrent que le rt-PA peut exercer un effet protecteur extra-fibrinolytique en augmentant les taux de BDNFm par une potentialisation de l’activité glutamatergique. Même si le rt-PA induit une meilleure récupération neurologique et augmente les taux circulants de BDNF chez les patients victimes d’un AVC, l’absence de corrélation entre ces deux paramètres n’est pas en faveur de l’utilisation du BDNF circulant comme un marqueur prédictif de récupération neurologique, mais pourrait être un reflet de la capacité de l’endothélium à sécréter le BDNF. / Our objective was to test the hypothesis that the beneficial effect of the administration of the recombinant form of tissue plasminogen activator (rt-PA) in ischemic stroke patient not only results from its fibrinolytic activity but also from its ability to increase brain-derived neurotrophic factor (BDNF) in the brain. To this end, we conducted an initial study to evaluate the effect of rt-PA on brain BDNF levels in healthy animals. In a second study, we investigated the effect of rt-PA on serum BDNF levels in ischemic stroke patients and in animals subjected to permanent focal cerebral ischemia. Blood samples were obtained from patient on admission (D0), D1, D7 and D90 after stroke and in rats before and after (1h, 4h and 24h) ischemia. BDNF was measured in the brain by Western blot and in the blood by ELISA. In both studies, the rt-PA (Actilyse®) was administered as a bolus followed by an infusion of one hour. The first study evidences that 1) rt-PA increases the BDNF levels in the hippocampus, 2) treatment with MK801 (a NMDA receptor antagonist) but not with tranexamic acid (a plasmin inhibitor) canceled the effect of rt-PA on BDNF levels. The second study exhibits that 1) neurological recovery was higher in the patients receiving rt-PA, 2) treatment with rt-PA increases serum BDNF at D1 and D7 in patients, but does not change the blood BDNF levels in animals, 3) BDNF levels are not correlated with neurological recovery but are inversely correlated to the patient cardiovascular score. In conclusion, our results suggest that rt-PA may have a protective extra-fibrinolytic effect by increasing in BDNF levels through a potentiation of glutamatergic pathway. Although rt-PA induces a better neurological recovery and increases circulating BDNF levels in stroke patients, the lack of correlation between these two parameters is not in favor of using circulating BDNF as a predictive marker of neurological recovery, but could be a reflect of the endothelium ability to synthesize BDNF.

O exercício físico pode atenuar os efeitos do envelhecimento sobre o sistema nervoso central, por meio do aumento da expressão de neurotrofinas, tais como fator neurotrófico derivado do cérebro (BDNF), o qual promove a ramificação dendrítica e melhora da maquinaria sináptica, pela interação com seu receptor TrkB. Receptores TrkB são produzidos no corpo da célula e transportados aos terminais axonais, por meio de SLP1, CRMP2, Rab27B e Sortilina onde são ancorados para realizar seu papel fisiológico. Sabendo que a relação entre o tráfego de receptores de neurotrofinas e o treinamento físico ainda é pouco conhecida, o objetivo do presente trabalho é analisar os níveis do receptor TrkB, bem como de seus transportadores anterógrados e retrógrados, no sistema nervoso central de ratos idosos, modelos de neurodegeneração, expostos a diferentes protocolos de treinamento físico moderado. Os ratos do primeiro grupo experimental foram expostos a 1mg/kg/dia de Rotenona ou DMSO durante 4 semanas, depois, juntamente com a exposição à rotenona, realizaram treinamento físico moderado em esteira, 5 vezes por semana, durante 40 minutos; ou permaneceram em repouso. Os ratos do segundo grupo experimental realizaram 6 semanas de treinamento, sendo em seguida expostos à rotenona por 4 semanas, e subdividos em dois grupos, um que continuou o exercício e outro que ficou sedentário. Os resultados encontrados sugerem que o treinamento físico parece reverter ou prevenir de maneira geral os danos presentes na neurodegeneração considerando as proteínas do tráfego de BDNF e seu receptor, e ainda, que a magnitude e direção destas alterações está diretamente relacionada ao protocolo de treinamento físico, bem como, a região do sistema nervoso central analisada / Physical exercise can attenuate the effects of aging on the central nervous system by increasing the expression of neurotrophins such as brain-derived neurotrophic factor (BDNF), which promotes dendritic branching and enhances synaptic machinery, through interaction with its receptor TrkB. TrkB receptors are synthesized in the cell body and are transported to the axonal terminals, through SLP1, CRMP2, Sortilin and Rab27B, to where receptors are anchored to perform its physiological role. However, the aspects of the neurotrophin receptors traffic after physical training is still a matter of investigation. Thus, the present study aims to analyze the expression levels of TrkB receptor and their anterograde carriers in aged Lewis rats, model of neurodegeneration, and its relationship with moderate exercise training. Rats from the first experimental group were exposed to 1mg/kg/day of Rotenone (ROT) or DMSO for 4 weeks, and then subjected or not to moderate exercise running on treadmill, five days a week, 40 minutes a day, combined with the drug. Rats from the second experimental group were trained for 6 weeks, followed by exposure to rotenone during 4 weeks, rats were then subdivided into two groups, one that continued the exercise and the other became sedentary. Results suggest that exercise training appears to reverse or prevent the impairment related to neurodegeneration considering the proteins involved in BDNF signaling, and also that the magnitude and direction of these changes in directly related to the physical training protocol, as well as the area of the central nervous system analyzed

Les glioblastomes (GBM) sont des tumeurs astrocytaires au pronostic défavorable. L’échec des thérapies actuelles (chimio et radiothérapies) est principalement lié à la résistance des cellules souches cancéreuses (CSCs). Ces cellules ont besoin de communiquer en permanence avec leur microenvironnement pour leur survie et pour maintenir une niche favorable à leur développement. Le transfert de matériel entre les CSC, les cellules tumorales et le microenvironnement contribue à l’échappement thérapeutique. Des travaux récents révèlent l’importance des récepteurs aux neurotrophines TrkB et TrkC dans la survie et la croissance des CSC de GBM. Nos travaux préliminaires dans le cancer bronchique démontrent que les récepteurs aux neurotrophines sont transférés aux cellules du microenvironnement via les exosomes afin de les contrôler. Cependant, le mécanisme de diffusion de récepteurs oncogéniques à partir de CSC n’a jamais été étudié. Notre objectif principal était donc de déterminer l’implication des récepteurs des neurotrophines dans le transfert du phénotype agressif des CSC vers les cellules du microenvironnement afin de favoriser la résistance thérapeutique du glioblastome. Nos résultats ont permis d’établir un lien entre le stade de différenciation des cellules tumorales, l’expression des neurotrophines et leur interaction avec le microenvironnement tumoral via les exosomes. Le transfert de TrkB au sein des exosomes joue un rôle clé dans la progression tumorale du GBM et dans l’agressivité cellulaire. Néanmoins, le transfert des récepteurs aux neurotrophines via les exosomes pourrait également être impliqué dans les mécanismes de radiorésistance. Des études menées sur des cellules de GBM humain irradiées et traitées par des exosomes démontrent l’implication de ces derniers dans l’échappement thérapeutique. Parmi les cellules du microenvironnement ciblées par les exosomes, les CSM sont celles qui ont été les moins étudiées bien qu’elles possèdent un tropisme spécifique pour le GBM. Nos travaux démontrent que les exosomes de GBM modifient le phénotype des CSM et augmentent leurs capacités prolifératives et migratoires. La fonction exacte du transfert des récepteurs des neurotrophines devra être analysée dans ces différents modèles afin de préciser son importance dans la physiopathologie du glioblastome et sa progression. L’expression des récepteurs aux neurotrophines dans ces exosomes permet d’envisager leur utilisation en tant que biomarqueurs diagnostiques et/ou pronostiques dans le GBM. Mots clés : Glioblastomes, cellules souches cancéreuses, neurotrophines, TrkB, radiothérapie, cellules souches mésenchymateuses, exosomes. / Glioblastoma are tumors derived from astrocytes with a dark prognosis. Current therapies fail to inhibit relapses due to radioresistant properties of cancer stem cells (CSC). Communication between CSC and their microenvironment is required for maintain “stem cells niche” and cell survival . The transfer of materials between CSC, tumor cells and microenvironment contributes to therapeutic resistance. In glioma, recent studies reveal the major role of TrkB and TrkC in survival of CSC. Our previous work, in lung cancer, have shown that neurotrophin receptors exhibits a control on microenvironment cells and angiogenesis through exosome transfer. However, similar mechanism of oncogenic receptor transfer from CSC has never been studied. Our main goal was to determine the involvement of neurotrophin receptors in the transfer of biological aggressiveness to microenvironment cells in order to promote therapeutic resistance in glioblastoma. Our findings suggest a relationship between cell differentiation status, expression of neurotrophin receptors and their interaction with the microenvironment through exosomes. TrkB-containing exosomes play a key role in the control of glioblastoma progression and cell aggressiveness. Mechanisms of radioresistance might also be dependent of the transfer of neurotrophin receptors through exosomes. Indeed, our results on irradiated human GBM cells and treated by exosomes demonstrate the involvement of exosome in radioresistance mechanisms. Although mesenchymal stem cells (MSCs) are considered as stromal components of glioblastoma, their communication with CSC, particularly through exosomes, remain largely undefined. Our results show that GBM-derived exosomes modify the phenotype of MSCs and increase their proliferative and migratory abilities. The putative function of neurotrophin receptors transfer should be analyzed in these models to determine their prime role in glioblastoma pathogenesis and progression. This finding suggest that the neurotrophin receptor expression in exosomes could be used as diagnostis and prognosis biomarkers of GBM.