O eixo baço-cérebro na epilepsia experimental e no tratamento com células mononucleares de medula óssea após o status epilépticus

Abstract

In epilepsy, the most common serious neurological disorder , affects more than 65 million people worldwide. Several investigations in both humans and animals have shown the effectiveness of bone marrow mononuclear cells (BMMC) as a therapy. The role of spleen is to regulate the immune system, as it is the central organ of cellular and humoral immunity and direct or indirectly influences the central nervous system. At the first chapter the objective of this work was to investigate in splenectomized animals, that had undergone the pilocarpine protocol and treated with Bone Marrow Mononuclear Cells (BMMC), the biodistribution of the transplanted cells, to evaluate inflammatory factors expression and spacial memory along the different groups. In order to achieve these objectives, the pilocarpine protocol (320 mg/kg) was used to induce status epilepticus (SE) in male wistar rats that were splenectomized seven days prior to SE. The BMMC were extracted from the Bone Marrow of long bones of C57B1/6 EGFP+ transgenic mice and the separations accomplished through Ficoll gradient. BMMC transplant was performed by injecting a 100 µL suspensions containing 1x107 EGFP+ cells at the tail vein of animals that presented SE one hour after the pilocarpine protocol. 12 and 24 hours after the cell transplant animals were euthanized for brain, lung, liver, kidneys and spleen sampling. Both EGFP+ cells biodistribution and inflammatory factors expression (Interleukin 1β, TNF-α, AIF-1 and Interleukin 10) were assessed through qRT-PCR. Spacial memory was assessed through Morris Water Maze (MWM) ten days after SE. The result of this first study demonstrated that cells injected at the tail vein were found in the spleen. We further found that BMMC treatment was capable of diminishing pro-inflammatory cytokines at brain and spleen tissues of animals submitted to SE. Also splenectomy played an anti-inflammatory role similar to the BMMC transplant at brain cytokines expression throughout the first 12 hours. However splenectomy seemed to have impaired BMMC therapy on animals submitted to SE. Splenectomized animals developed more inflammation and did worse on MWM test. These findings suggest failure on learning and spacial memory consolidation abilities as seen on SE animals without BMMC treatment. From these data we proposed to investigate the spleen’s role on epilepsy development. The objective of the second study was to investigate the importance of the spleen in epileptogenesis evaluating crysis frequency, memory, depressive behavior and brain tissue inflammatory factors expression in two temporal lobe epilepsy models (pilocarpine and kainic acid (KA)). For this project male Wistar rats were splenectomized seven days before pilocarpine (100 mg/kg) and KA (5 mg/kg per hour) SE protocols. During SE protocols the first crysis latency, SE presence and number of deaths were evaluated. At the chronic phase (50 days after SE), rats were filmed 24 h/day during seven days. The object recognition paradigm (OR) was performed one week next to video monitoring to evaluate short term memory. The forced swimming test was carried out one week after OR. Brain tissues were sampled for inflammatory factor expression analysis assays from qRT-PCR after all tests were finished. Therefore, data from our second study revealed that splenectomy reduced the latency to the first seizure in this pilocarpine protocol and did not influence at the percentage of SE animals, number of seizures and mortality during SE. However, in KA protocol splenectomy seemed to increase both seizure latency and the mortality percentage during SE. At chronic phase, seizure frequency was not modified by splenectomy in both pilocarpine and KA groups. Seizure duration remained unchanged by splenectomy in both models. Object recognition memory was impaired on SE animals from both protocols, though splenectomized animals showed better performance at this memory task in both protocols. On forced swimming test, epileptic animals displayed depressive-like behavior and splenectomy did not influence forced swimming results. IL-1β and TNF-α expression levels on brain tissue from SE rats that underwent the pilocarpine protocol was higher when compared to those animals from KA protocol and no difference was observed among groups with and without spleen. AIF-1 expression on brain tissue was negative on rats from all the studied groups. And the brain tissue expression of IL-10 was higher on epileptic splenectomized animals from both SE models. Based on the results of our study it was possible to understand that the spleen plays a role not only on cellular therapy and acute SE phase but also on the epileptogenic process on both epilepsy models.