An optimized method for adult zebrafish brain-tissue dissociation that allows access mitochondrial function under healthy and epileptic conditions.

Mitochondria play key roles in brain metabolism. Not surprisingly, mitochondria dysfunction is a ubiquitous cause of neurodegenerative diseases. In turn, acquired forms of epilepsy etiology is specifically intriguing since mitochondria function and dysfunction remain not completely enlightened. Investigation in the field includes models of epileptic disorder using mainly rodents followed by mitochondrial function evaluation, which in general evidenced controversial data. So, we considered the efforts and limitations in this research field and we took into account that sample preparation and quality are critical for bioenergetics investigation. For these reasons the aim of the present study was to develop a thorough protocol for adult zebrafish brain-tissue dissociation to evaluate oxygen consumption flux and reach the bioenergetics profile in health and models of epileptic disorder in both, in vitro using pentylenetetrazole (PTZ) and N-methyl-D-Aspartic acid (NMDA), and in vivo after kainic acid (KA)-induced status epilepticus. In conclusion, we verify that fire-polished glass Pasteur pipette is eligible to brain-tissue dissociation and to study mitochondrial function and dysfunction in adult zebrafish. The results give evidence for large effect size in increase of coupling efficiency respiration (p/O2) correlated to treatment with PTZ and spare respiratory capacity (SRC) in KA-induced model indicating oxidative phosphorylation (OXPHOS) variable alterations. Further investigation is needed in order to clarify the bioenergetics role as well as other mitochondrial functions in epilepsy.

Forebrain glutamate uptake and behavioral parameters are altered in adult zebrafish after the induction of Status Epilepticus by kainic acid.

The development of new antiepileptic drugs is a high-risk/high-cost research field, which is made even riskier if the behavioral epileptic seizure profile is the unique approach on which the development is based. In order to increase the effectiveness of the screening conducted in the zebrafish model of status epilepticus (SE), the evaluation of neurochemical markers of SE would be of great relevance. Epilepsy is associated with changes in the glutamatergic system, and glutamate uptake is one of the critical parameters of this process. Therefore, we evaluated the levels of glutamate uptake in the zebrafish brain and analyzed its correlation with the progression of behavioral changes in zebrafish at different times after the administration of kainic acid (5 mg/kg). The results showed that the zebrafish suffered with lethargy while swimming for up to 72 h after SE, had reduced levels of GFAP cells 12 h after SE, reduced levels of S100B up to 72 h after SE, and reduced levels of glutamate uptake in the forebrain between 3 h and 12 h after SE. The forebrain region of adult zebrafish after SE present similar changes to the neurochemical limbic alterations that are seen in rodent models of SE. This study demonstrated that there is a time window in which to use the KA zebrafish model of SE to explore some of the known neurochemical alterations that have been observed in rodent models of epilepsy and epileptic human patients.

Comparação das metodologias óptica e de impedância para a determinação de parâmetros hematológicos no analisador Sysmex-XN

Objetivo: Comparar as metodologias de impedância e óptica para contagem de eritrócitos (RBC) e dosagem de hemoglobina (HGB) como forma de corrigir a contagem de eritrócitos em casos de pacientes que apresentam índice de concentração de hemoglobina corpus- cular média (CHCM) acima do ponto de corte. Métodos: Para comparar as metodologias, foram utilizadas amostras com CHCM normal e aumentado. Os parâmetros avaliados foram RBC, HGB e CHCM. Resultados: As amostras com valores de CHCM aumentado demonstraram uma correlação significativa entre RBC e HGB, porém o CHCM apresen- tou uma correlação fraca. Observamos que a metodologia por impedância obteve maior sucesso na correção do CHCM quando as amostras apresentavam aglutinação. Conclusão: A metodologia óptica é uma boa alternativa para correção de valores aumentados de CHCM em situações específicas, mas para casos de aglutinação não se mostrou efetiva.

Objetctive: Compare the impedance and optical methodologies for red blood cells (RBC) count and hemoglobin (HGB) measurement as a way to correct the RBC count in cases of patients who have a MCHC index above the cutoff. Methods: To compare the methodologies, samples with normal and augmented MCHC were used. The parameters evaluated were RBC, HGB and MCHC. Results: Samples with increased MCHC values demonstrated a significant correlation between RBC and HGB, but the MCHC showed a weak correlation. We observed that the impedance methodology was more successful in correcting the MCHC when the samples presented agglutination. Conclusion: Optical methodology is a good alternative for correction of increased CHCM values in specific situations, but for cases of agglutination it has not been shown to be effective.

Anxiolytic effects of diphenyl diselenide on adult zebrafish in a novelty paradigm.

Anxiety-related disorders are frequently observed in the population. Because the available pharmacotherapies for anxiety can cause side effects, new anxiolytic compounds have been screened using behavioral tasks. For example, diphenyl diselenide (PhSe)2, a simple organoselenium compound with neuroprotective effects, has demonstrated anxiolytic effects in rodents. However, this compound has not yet been tested in a novelty-based paradigm in non-mammalian animal models. In this study, we assessed the potential anxiolytic effects of (PhSe)2 on the behavior of adult zebrafish under novelty-induced stress. The animals were pretreated with 0.1, 0.25, 0.5, and 1µM (PhSe)2 in the aquarium water for 30min. The fish were then exposed to a novel tank, and their behavior was quantified during a 6-min trial. (PhSe)2 treatment altered fish behavior in a concentration-dependent manner. At 0.01 and 0.25µM, (PhSe)2 did not elicit effects on fish behavior. At 0.5µM, moderate behavioral side effects (e.g., lethargy and short episodic immobility) were noted. At the highest concentration tested (1µM), dramatic side effects were observed, such as burst behavior and longer periods of immobility. The results were confirmed by spatiotemporal analysis of each group. Occupancy plot data showed dispersed homebase formation in the 0.25µM (PhSe)2-treated group compared with the control group (treated with 0.04% DMSO). Furthermore, animals treated with 0.25µM (PhSe)2 showed a reduction in latency to enter the top and spent more time in the upper area of the tank. These data suggest that (PhSe)2 may induce an anxiolytic-like effect in situations of anxiety evoked by novelty.

Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization.

Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.