Comparative effect of cryoprotectant combinations on the conservation of somatic cells derived from jaguar, Panthera onca, towards the formation of biologic banks.

Due to the reduction of the jaguar population, the formation of somatic cell cryobanks represents an interesting tool for its conservation. Nevertheless, the success of these cryobanks depends on the cryoprotectants used in cryopreservation. We evaluated the effects of the intracellular cryoprotectants (10% dimethyl sulfoxide, DMSO; 10% ethylene glycol, EG) in the absence or presence of an extracellular cryoprotectant (0.2 M sucrose, SUC) on the morphology, confluence, viability, and metabolism of somatic cells derived from five jaguars belonging to Brazilian zoos. The morphology was presented in a descriptive manner, while the confluence, viability and metabolic activity were presented as means and compared using statistical tests. Non-cryopreserved cells were used as control and compared to frozen/thawed cells using cryoprotectants. No difference was observed for the morphology and confluence among non-cryopreserved and cryopreserved cells, regardless of the cryoprotectants. Only cryopreserved cells in EG (45.8%±12.9) had a reduction in their viability when compared to non-cryopreserved cells (97.8%±1.1). Only cryopreserved cells in DMSO with SUC (76.0%±2.7) or absence of SUC (77.0%±3.7) maintained their metabolic activity after thawing, when compared to non-cryopreserved cells (100.0%±6.7). Therefore, combinations of DMSO in the absence and presence of SUC were efficient in the cryopreservation of somatic cells of jaguars.

Increased susceptibility to pentylenetetrazol following survival of cerebral malaria in mice.

Malaria is considered a neglected disease and public health problem, affecting >200 million people worldwide. In the present study we used the Plasmodium berghei ANKA (PbA) model of experimental cerebral malaria (CM) in C57BL/6 mice. After rescue from CM and parasite clearance, animals were submitted to a seizure susceptibility test (45 days after infection) using a low dose of pentylenetetrazol (PTZ, 30 mg/kg) and monitored with use of behavioral and electroencephalography (EEG) methods. Mice rescued from CM presented a reduced latency to myoclonic and tonic-clonic seizures and an increased duration of tonic-clonic seizures. In addition, quantitative analysis of EEG revealed a decrease in relative power at beta frequency band in PbA-infected animals after PTZ injection. Our results suggest that CM may lead to increased susceptibility to seizures in mice.

Anticonvulsant activity of Tetrahydrolinalool: behavioral, electrophysiological, and molecular docking approaches.

Tetrahydrolinalool (THL) is an acyclic monoterpene alcohol, produced during linalol metabolism and also a constituent of essential oils. As described in the literature, many monoterpenes present anticonvulsant properties, and thus we became interested in evaluating the anticonvulsant activity of Tetrahydrolinalool using in mice model as well as in silico approaches. Our results demonstrated that THL increased latency to seizure onset and also reduced the mortality, in picrotoxin induced seizure tests. The results may be related to GABAergic regulation, which was also suggested in seizure testing induced by 3-mercapto-propionic acid. In the strychnine-induced seizure testing, none of the groups pretreated with THL modulated the parameters indicative of anticonvulsant effect. The electrophysiological results revealed that THL treatment reduces seizures induced by pentylenetetrazole. The in silico molecular docking studies showed that the interaction between THL and a GABAA receptor model formed a stable complex, in comparison to the crystaligraphic structure of diazepam, a structurally related ligand. In conclusion, all the evidences showed that THL presents effective anticonvulsant activity related to the GABAergic pathway, being a candidate for treatment of epileptic syndromes.

Morphological, Ultrastructural, and Immunocytochemical Characterization and Assessment of Puma (Puma concolor Linnaeus, 1771) Cell Lines After Extended Culture and Cryopreservation.

Cell lines are valuable tools to safeguard genetic material from species threatened with extinction that is mainly due to human action. In this scenario, the puma constitutes a species whose population is being rapidly reduced in the ecosystems it inhabits. For the first time, we characterized puma skin-derived cell lines and assessed these cells after extended culture (experiment 1) and cryopreservation (experiment 2). Initially, we identified and characterized four dermal fibroblast lines using morphology, ultrastructure, and immunofluorescence assays. Moreover, we evaluated the effects of culture time (1st, 3rd, and 10th passages) and cryopreservation on their morphology, ultrastructure, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis. The cells showed a typical spindle-shaped morphology with centrally located oval nuclei. The cells were identified as fibroblasts by staining for vimentin. In vitro culture after the 1st, 3rd, and 10th passages did not alter most of the evaluated parameters. Cells in the 3rd and 10th passages showed a reduction in ROS levels (p < 0.05). The ultrastructure revealed morphological damage in the prolongments, and nuclei of cells derived from the 3rd and 10th passages. Moreover, cryopreservation resulted in a reduction in ΔΨm compared with that of noncryopreserved cells, suggesting that the optimization of cryopreservation methods for puma fibroblasts is essential. In conclusion, we found that viable fibroblasts could be obtained from puma skin, with slight changes after the 10th passage in in vitro culture and cryopreservation. This is the first report on the development of cell lines derived from pumas.

Quantitative and descriptive histological aspects of jaguar (Panthera onca Linnaeus, 1758) ear skin as a step towards formation of biobanks.

Skin of mammals vulnerable to extinction, such as the jaguar, is used as a source of material in conservation strategies. The composition of skin is not uniform among species, and the ability to distinguish similarities in skin morphology in animal groups is fundamental in the application of skin tissue for use in biobanks. The aim of our study was to evaluate the structure, composition and capacity for culture of ear skin from the yellow and black jaguars. Both qualitative and quantitative methods were used, focusing on skin thickness, cell quantification and distribution, collagen density, proliferative activity and viability. Histomorphometrical study of the skin showed a total thickness of 273.2 and 274.6 µm for the yellow and black jaguars, respectively. Melanocytes and fibroblasts were, respectively, 9.7 and 23.0 for the yellow jaguar and 11.3 and 26.8 for the black jaguar. A collagen density of 67.0% and 49.0% was observed for yellow and black jaguars, respectively. Both animals presented a proliferative activity varying between 1.20 and 1.30. All tissues could promote cellular detachment, reaching subconfluence in 10-15 days. This kind of information from histomorphometrical features and cell cultures can be essential for a more targeted application of ear skin cryopreservation in this species, as such information will enable understanding the action of substances on tissues during the conservation process.

Increased xanthine oxidase-related ROS production and TRPV1 synthesis preceding DOMS post-eccentric exercise in rats.

AIMS: It is well-known that unaccustomed exercise, especially eccentric exercise, is associated to delayed onset muscle soreness (DOMS). Whether DOMS is associated with reactive oxygen species (ROS) and the transient receptor potential vanilloid 1 (TRPV1) is still an open question. Thus, the aim of this study was to investigate the association between TRPV1 and xanthine oxidase-related ROS production in muscle and DOMS after a bout of eccentric exercise. MAIN METHODS: Male Wistar rats performed a downhill running exercise on a treadmill at a -16° tilt and a constant speed for 90min (5min/bout separated by 2min of rest). Mechanical allodynia and grip force tests were performed before and 1, 3, 6, 9, 12, 24, 48 and 72h after the downhill running. Biochemical assays probing oxidative stress, purine degradation, xanthine oxidase activity, Ca(2+) ATPase activity and TRPV1 protein content were performed in gastrocnemius muscle at 12, 24, and 48h after the downhill running. KEY FINDINGS: Our statistical analysis showed an increase in mechanical allodynia and a loss of strength after the downhill running. Similarly, an increase in carbonyl, xanthine oxidase activity, uric acid levels and TRPV1 immunoreactivity were found 12h post-exercise. On the other hand, Ca(2+) ATPase activity decreased in all analyzed times. SIGNIFICANCE: Our results suggest that a possible relationship between xanthine oxidase-related ROS and TRPV1 may exist during the events preceding eccentric exercise-related DOMS.

The Impact of Previous Physical Training on Redox Signaling after Traumatic Brain Injury in Rats: A Behavioral and Neurochemical Approach.

Throughout the world, traumatic brain injury (TBI) is one of the major causes of disability, which can include deficits in motor function and memory, as well as acquired epilepsy. Although some studies have shown the beneficial effects of physical exercise after TBI, the prophylactic effects are poorly understood. In the current study, we demonstrated that TBI induced by fluid percussion injury (FPI) in adult male Wistar rats caused early motor impairment (24 h), learning deficit (15 days), spontaneous epileptiform events (SEE), and hilar cell loss in the hippocampus (35 days) after TBI. The hippocampal alterations in the redox status, which were characterized by dichlorofluorescein diacetate oxidation and superoxide dismutase (SOD) activity inhibition, led to the impairment of protein function (Na(+), K(+)-adenosine triphosphatase [ATPase] activity inhibition) and glutamate uptake inhibition 24 h after neuronal injury. The molecular adaptations elicited by previous swim training protected against the glutamate uptake inhibition, oxidative stress, and inhibition of selected targets for free radicals (e.g., Na(+), K(+)-ATPase) 24 h after neuronal injury. Our data indicate that this protocol of exercise protected against FPI-induced motor impairment, learning deficits, and SEE. In addition, the enhancement of the hippocampal phosphorylated nuclear factor erythroid 2-related factor (P-Nrf2)/Nrf2, heat shock protein 70, and brain-derived neurotrophic factor immune content in the trained injured rats suggests that protein expression modulation associated with an antioxidant defense elicited by previous physical exercise can prevent toxicity induced by TBI, which is characterized by cell loss in the dentate gyrus hilus at 35 days after TBI. Therefore, this report suggests that previous physical exercise can decrease lesion progression in this model of brain damage.