Ketamine and medetomidine effects on brain: behavioral, histopathological and electrophysiological studies
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2014-10-16
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A anestesia é essencial para a realização de procedimentos cirúrgicos, promovendo analgesia, hipnose e relaxamento muscular; sendo um requisito na realização de vários procedimentos na investigação neurobiológica.
O antagonista dos receptores N-metil D-Aspartato (NMDA), cetamina, e o agonista dos receptores α2-adrenergicos, medetomidina, são exemplos de fármacos frequentemente utilizados em clínicas veterinárias e em investigação científica para induzir anestesia, analgesia e sedação. Normalmente, estas drogas são utilizadas em combinação, melhorando a estabilidade hemodinâmica e reduzindo os requisitos anestésicos. No entanto, está descrito que a anestesia com antagonistas dos receptores NMDA pode induzir défices de memória em recém-nascidos, sendo ainda incerto o efeito de uma única administração de cetamina na memória dos adultos. Além disso, existe uma lacuna na literatura relativamente ao efeito da administração de cetamina em combinação com medetomidina no cérebro. Assim, o objetivo principal deste trabalho foi explorar o impacto de uma única administração de diferentes doses de cetamina isolada ou em associação com medetomidina no cérebro de murganhos adultos. Para atingir esse objectivo foram realizados quatro estudos diferentes: dois usando testes comportamentais, histopatológicos e imuno-histoquímicos (capítulo 3 e 4) e dois usando testes electrofisiológicos (capítulo 5 e 6).
No primeiro estudo, descrito no capítulo 3, foram avaliados os efeitos de diferentes doses de cetamina sozinha e combinada com medetomidina na memória e na neurodegeneração. Este estudo incluiu doses anestésicas de cetamina combinada com medetomidina (25 mg/kg de cetamina + 1 mg/kg de medetomidina e 75 mg/kg de cetamina + 1 mg/kg de medetomidina), doses subanestésicas de cetamina isolada (25 mg/kg e 75 mg/kg) e uma dose sedativa de medetomidina isolada (1 mg/kg). Alguns murganhos foram testados numa bateria de testes comportamentais (testes “T-maze”, “vertical pole” e “open field”) e outros utilizados para histopatologia (hematoxilina e eosina) e imuno-histoquímica (expressão de procaspase-3, caspase-3 activada e de factor neurotrófico derivado do cérebro).
O segundo estudo, descrito no capítulo 4, complementou o primeiro, visto incluir uma dose anestésica de cetamina isolada (150 mg/kg) e testes comportamentais mais complexos (teste “radial-maze”) que permitiram avaliar diferentes tipos de memória. A avaliação
neurodegenerativa incluiu ainda regiões do cérebro que no primeiro estudo não foram avaliadas.
No terceiro estudo, abordado no capítulo 5, foram estudados os efeitos de diferentes concentrações de cetamina, medetomidina e da combinação cetamina/medetomidina na transmissão sináptica basal e na plasticidade sináptica (facilitação por pulso pareado e potenciação a longo prazo). Os fármacos foram aplicados na solução que contém as fatias de hipocampo. Para a transmissão sináptica basal e para a facilitação de pulso pareado foram testadas concentrações entre 1 e 600 μM de cetamina, entre 1 e 200 μM de medetomidina e desde 30 μM + 1 μM até 600 μM + 24 μM da combinação de cetamina+medetomidina. Para a potenciação de longo prazo foram testadas concentrações entre 3 e 100 μM de cetamina, entre 0,1 e 0,4 μM de medetomidina e a combinação de 3 μM de cetamina com 0,1 μM de medetomidina.
No quarto estudo, descrito no capítulo 6, duas doses de cetamina (25 mg/kg e 75 mg/kg) foram administradas em murganhos adultos para avaliar o impacto deste fármaco na potenciação a longo prazo no hipocampo, 24 horas após a anestesia. Este estudo complementou os estudos anteriores visto a cetamina ter sido administrada diretamente nos murganhos por injeção intraperitoneal.
Os resultados dos capítulos 3 e 4 mostraram que uma única administração de cetamina e medetomidina, isoladamente ou em combinação, não afecta a memória nem a neurodegeneração. Estes resultados foram apoiados pelos resultados obtidos no capítulo 6, que mostraram que a cetamina não afeta a potenciação a longo prazo, um mecanismo essencial para a formação da memória, 24 horas após a anestesia. Além disso, a combinação de cetamina com medetomidina forneceu boa estabilidade hemodinâmica durante a anestesia e preveniu a hiperlocomoção observada aquando da administração isolada de cetamina.
Os resultados do capítulo 5 mostraram que a cetamina e a medetomidina, isoladamente ou em combinação, afectam principalmente a plasticidade de longo prazo em detrimento da plasticidade de curto prazo, aquando da aplicação dos fármacos directamente no hipocampo. Este fato sugere a importância dos mecanismos pós-sinápticos, avaliados por potenciação de longo prazo, em detrimento de mecanismos pré-sinápticos, avaliados por facilitação por pulso pareado, na ação desses fármacos no hipocampo.
Em conclusão, este trabalho mostrou que uma única administração de cetamina e medetomidina, isoladamente ou em combinação, não afecta a memória de murganhos adultos. Assim sendo, a combinação de cetamina/medetomidina mostrou-se uma combinação
anestésica segura para fins clínicos e de pesquisa científica quando são utilizados murganhos adultos.
Anesthesia is essential to perform surgical procedures, providing analgesia, hypnosis and muscular relaxation. Moreover, it is a requirement for several procedures in neurobiological research. The N-metil D-Aspartate (NMDA) receptor antagonist, ketamine, and the α2-adrenoceptor agonist, medetomidine, are examples of drugs frequently used in veterinary clinics and in research to induce anesthesia, analgesia and sedation. Usually, these drugs are used in combination, improving perioperative hemodynamic stability and reducing the anesthetic requirements. However, it was described that anesthesia with NMDA receptor antagonists may induce deficits of memory in neonates, being uncertain the effects of a single administration of ketamine in adult brain. Moreover, there is a lack of knowledge regarding the effect of a single administration of ketamine combined with the medetomidine on brain. Therefore, the main purpose of this thesis is to explore the impact of a single administration of different doses of ketamine alone or combined with medetomidine on brain of adult mice. To achieve this aim were performed four different studies: two using behavioral, histopathological and immunohistochemistry tests (chapter 3 and 4) and two using electrophysiological tests (chapter 5 and 6). In the first study, described in chapter 3, were evaluated the effects of different doses of ketamine alone and it combined with medetomidine on memory and neurodegeneration. This study included anesthetic doses of ketamine combined with medetomidine (25 mg/kg of ketamine + 1mg/kg of medetomidine, 75 mg/kg of ketamine + 1mg/kg of medetomidine), subanesthetic doses of ketamine alone (25mg/kg and 75 mg/kg) and a sedative dose of medetomidine alone (1 mg/kg). Some mice were tested in a battery of behavioral tests (T-maze, vertical pole and open field test), and others were used for histopathological (hematoxylin and eosin staining) and immunohistochemical analyses (expression of procaspase-3, activated caspase-3 and brain-derived neurotrophic factor). The second study, described in chapter 4, complemented the first study, since it included an anesthetic dose of ketamine alone (150 mg/kg) and behavioral tests more complexes (radial-maze test), which allowed to assess different types of memory. Moreover, the neurodegenerative evaluation included some regions of the brain that in the first study were not evaluated. In the third study, described in chapter 5, were evaluated the effects of different concentrations of ketamine, medetomidine and ketamine/medetomidine combination on basal synaptic transmission and synaptic plasticity (paired-pulse facilitation and long-term potentiation), in hippocampal slices of adult mice. These drugs were applied directly in the bath of slices. For basal synaptic excitatory transmission and paired-pulse facilitation were tested concentrations from 1 μM to 600 μM of ketamine, from 1 μM to 200 μM of medetomidine and from 30 μM +1 μM to 600μM +24 μM of ketamine + medetomidine combination. For long-term potentiation were tested concentrations from 3 μM to 100 μM of ketamine, from 0.1 μM to 0.4 μM of medetomidine and the combination of 3 μM of ketamine with 0.1μM of medetomidine. In the fourth study, described in chapter 6, different doses of ketamine (25 mg/kg and 75 mg/kg) were administered in adult mice to assess the impact of this drug on hippocampal long-term potentiation 24 hours after anesthesia. This study complemented the previous study (chapter 5) because the ketamine was administered directly in mice by an i.p. injection, similarly to first and second study. The results of chapter 3 and 4 showed that a single administration of ketamine and medetomidine, alone or in combination, did not affect the memory neither neurodegeneration. These findings were supported by the results obtained in chapter 6, that showed that the ketamine did not affected the long-term potentiation, an essential mechanism for memory formation, 24 hours after anesthesia. Moreover, the combination of ketamine with medetomidine provided a good hemodynamic stability and prevented the hyperlocomotion that arose during the administration of ketamine alone. The results of chapter 5 showed that ketamine and medetomidine alone or in combination mainly affected the long-term plasticity (long-term potentiation) rather than paired-pulse facilitation (short-term plasticity), at the exact moment of drugs applications. This fact suggested the importance of postsynaptic mechanisms, assessed by long-term potentiation, in detriment of presynaptic mechanisms, assessed by paired-pulse facilitation, in the action of these drugs on hippocampus. In conclusion, this thesis showed that a single administration of ketamine and medetomidine, alone or in combination, did not affect memory in adult mice. Thus, the ketamine/medetomidine combination showed to be a safe anesthetic combination for clinical and research purposes when adult mice are used.
Anesthesia is essential to perform surgical procedures, providing analgesia, hypnosis and muscular relaxation. Moreover, it is a requirement for several procedures in neurobiological research. The N-metil D-Aspartate (NMDA) receptor antagonist, ketamine, and the α2-adrenoceptor agonist, medetomidine, are examples of drugs frequently used in veterinary clinics and in research to induce anesthesia, analgesia and sedation. Usually, these drugs are used in combination, improving perioperative hemodynamic stability and reducing the anesthetic requirements. However, it was described that anesthesia with NMDA receptor antagonists may induce deficits of memory in neonates, being uncertain the effects of a single administration of ketamine in adult brain. Moreover, there is a lack of knowledge regarding the effect of a single administration of ketamine combined with the medetomidine on brain. Therefore, the main purpose of this thesis is to explore the impact of a single administration of different doses of ketamine alone or combined with medetomidine on brain of adult mice. To achieve this aim were performed four different studies: two using behavioral, histopathological and immunohistochemistry tests (chapter 3 and 4) and two using electrophysiological tests (chapter 5 and 6). In the first study, described in chapter 3, were evaluated the effects of different doses of ketamine alone and it combined with medetomidine on memory and neurodegeneration. This study included anesthetic doses of ketamine combined with medetomidine (25 mg/kg of ketamine + 1mg/kg of medetomidine, 75 mg/kg of ketamine + 1mg/kg of medetomidine), subanesthetic doses of ketamine alone (25mg/kg and 75 mg/kg) and a sedative dose of medetomidine alone (1 mg/kg). Some mice were tested in a battery of behavioral tests (T-maze, vertical pole and open field test), and others were used for histopathological (hematoxylin and eosin staining) and immunohistochemical analyses (expression of procaspase-3, activated caspase-3 and brain-derived neurotrophic factor). The second study, described in chapter 4, complemented the first study, since it included an anesthetic dose of ketamine alone (150 mg/kg) and behavioral tests more complexes (radial-maze test), which allowed to assess different types of memory. Moreover, the neurodegenerative evaluation included some regions of the brain that in the first study were not evaluated. In the third study, described in chapter 5, were evaluated the effects of different concentrations of ketamine, medetomidine and ketamine/medetomidine combination on basal synaptic transmission and synaptic plasticity (paired-pulse facilitation and long-term potentiation), in hippocampal slices of adult mice. These drugs were applied directly in the bath of slices. For basal synaptic excitatory transmission and paired-pulse facilitation were tested concentrations from 1 μM to 600 μM of ketamine, from 1 μM to 200 μM of medetomidine and from 30 μM +1 μM to 600μM +24 μM of ketamine + medetomidine combination. For long-term potentiation were tested concentrations from 3 μM to 100 μM of ketamine, from 0.1 μM to 0.4 μM of medetomidine and the combination of 3 μM of ketamine with 0.1μM of medetomidine. In the fourth study, described in chapter 6, different doses of ketamine (25 mg/kg and 75 mg/kg) were administered in adult mice to assess the impact of this drug on hippocampal long-term potentiation 24 hours after anesthesia. This study complemented the previous study (chapter 5) because the ketamine was administered directly in mice by an i.p. injection, similarly to first and second study. The results of chapter 3 and 4 showed that a single administration of ketamine and medetomidine, alone or in combination, did not affect the memory neither neurodegeneration. These findings were supported by the results obtained in chapter 6, that showed that the ketamine did not affected the long-term potentiation, an essential mechanism for memory formation, 24 hours after anesthesia. Moreover, the combination of ketamine with medetomidine provided a good hemodynamic stability and prevented the hyperlocomotion that arose during the administration of ketamine alone. The results of chapter 5 showed that ketamine and medetomidine alone or in combination mainly affected the long-term plasticity (long-term potentiation) rather than paired-pulse facilitation (short-term plasticity), at the exact moment of drugs applications. This fact suggested the importance of postsynaptic mechanisms, assessed by long-term potentiation, in detriment of presynaptic mechanisms, assessed by paired-pulse facilitation, in the action of these drugs on hippocampus. In conclusion, this thesis showed that a single administration of ketamine and medetomidine, alone or in combination, did not affect memory in adult mice. Thus, the ketamine/medetomidine combination showed to be a safe anesthetic combination for clinical and research purposes when adult mice are used.
Descrição
Tese de Doutoramento em Ciências Veterinárias
Palavras-chave
Cetamina , Modelo animal (murganho) , Memória , Medetomidina , Neurodegeneração , Plasticidade sináptica