Cryodehydration protocol to obtain high-quality permanent anatomical material.

The study of anatomy is largely dependent on cadaveric specimens to fulfill the tridimensional comprehension of each structure as well as the relationship between organs. Given the difficult access to fresh anatomical specimens, the constant renovation of samples for research and educational purposes is unsustainable, beyond the ethical issues involved. The standard technique for preserving specimens involves fixation and later immersion in formaldehyde, which enables a good result, but also presents elevated carcinogenic potential. Therefore, safe and efficient preservation methods are mandatory for anatomical practices and investigations. An accessible and inexpensive alternative for specimen preservation is cryodehydration. Cryodehydrated specimens can be kept dry, with no final odor, reducing the use and exposure to formaldehyde. The objective of this study was to propose a simplified step-by-step cryodehydration protocol to obtain high-quality anatomical specimens. Through consecutive freezing and thawing cycles, cryodehydration caused a weight reduction of 60%-70% and allowed anatomical preservation, while maintaining the main morphological aspects of cavitary and parenchymatous organs, muscles, or even full-body sections. The final specimens presented high durability and can be maintained for decades, preserving all relevant anatomical features.

Behavioral Pharmacology of Five Uncommon Passiflora Species Indicates Sedative and Anxiolytic-like Potential.

BACKGROUND: There are over 500 species in the Passiflora genus, and while some of them are very well known in folk medicine for their anxiolytic effects, very little is known for the other genus representants, which could also present medicinal effects. OBJECTIVE: In this study, we performed an interspecific pharmacological comparison of five investigated Passiflora species, all native to Brazil, namely P. bahiensis, P. coccinea, P. quadrangularis, P. sidaefolia, and P. vitifolia. METHODS: Extracts were administered to mice before behavioral testing, including a general pharmacological screening and anxiolytic-like effect investigation. RESULTS: Three of the species (P. coccinea, P. quadrangularis, and P. sidaefolia) induced a decrease in locomotor activity of mice; P. coccinea also reduced the latency to sleep. Importantly, none of the species interfered with motor coordination. Oral administration evoked no severe signs of toxicity, even at higher doses. Regarding the anxiolytic-like profile, P. sidaefolia reduced the anxious-like behavior in the Holeboard test in a similar way to the positive control, Passiflora incarnata, while not affecting total motricity. CONCLUSION: These results indicated that P. coccinea, P. quadrangularis, and P. sidaefolia reduced the general activity of mice and conferred a calmative/sedative potential to these three species, which must be further elucidated by future investigations.

Ayahuasca blocks ethanol preference in an animal model of dependence and shows no acute toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayahuasca, a psychoactive beverage prepared from Banisteriopsis caapi and Psychotria viridis, is originally used by Amazon-based indigenous and mestizo groups for medicinal and ritualistic purposes. Nowadays, ayahuasca is used in religious and shamanic contexts worldwide, and preliminary evidence from preclinical and observational studies suggests therapeutic effects of ayahuasca for the treatment of substance (including alcohol) use disorders. AIM OF THE STUDY: To investigate the initial pharmacological profile of ayahuasca and its effects on ethanol rewarding effect using the conditioned place preference (CPP) paradigm in mice. MATERIALS AND METHODS: Ayahuasca beverage was prepared using extracts of B. caapi and P. viridis, and the concentration of active compounds was assessed through high performance liquid chromatography (HPLC). The following behavioral tests were performed after ayahuasca administration: general pharmacological screening (13, 130, or 1300 mg/kg - intraperitoneally - i.p., and 65, 130, 1300, or 2600 mg/kg - via oral - v.o.); acute toxicity test with elevated doses (2600 mg/kg - i.p., and 5000 mg/kg - v.o.); motor activity, motor coordination, and hexobarbital-induced sleeping time potentiation (250, 500, or 750 mg/kg ayahuasca or vehicle - v.o.). For the CPP test, the animals received ayahuasca (500 mg/kg - v.o.) prior to ethanol (1.8 g/kg - i.p.) or vehicle (control group - i.p.) during conditioning sessions. RESULTS: Ayahuasca treatment presented no significant effect on motor activity, motor coordination, hexobarbital-induced sleeping latency or total sleeping time, and did not evoke signs of severe acute toxicity at elevated oral doses. Ayahuasca pre-treatment successfully inhibited the ethanol-induced CPP and induced CPP when administered alone. CONCLUSIONS: Our results indicate that ayahuasca presents a low-risk acute toxicological profile when administered orally, and presents potential pharmacological properties that could contribute to the treatment of alcohol use disorders.

Historical evolution of spheroids and organoids, and possibilities of use in life sciences and medicine.

BACKGROUND: An impressive percentage of biomedical advances were achieved through animal research and cell culture investigations. For drug testing and disease researches, both animal models and preclinical trials with cell cultures are extremely important, but present some limitations, such as ethical concern and inability of representing complex tissues and organs. 3D cell cultures arise providing a more realistic in vitro representation of tissues and organs. Environment and cell type in 3D cultures can represent in vivo conditions and thus provide accurate data on cell-to-cell interactions, and cultivation techniques are based on a scaffold, usually hydrogel or another polymeric material, or without scaffold, such as suspended microplates, magnetic levitation, and microplates for spheroids with ultra-low fixation coating. PURPOSE AND SCOPE: This review aims at presenting an updated summary of the most common 3D cell culture models available, as well as a historical background of their establishment and possible applications. SUMMARY: Even though 3D culturing is incapable of replacing other current research types, they will continue to substitute some unnecessary animal experimentation, as well as complement monolayer cultures. CONCLUSION: In this aspect, 3D culture emerges as a valuable alternative to the investigation of functional, biochemical, and molecular aspects of human pathologies.

Irisin modulates genes associated with severe coronavirus disease (COVID-19) outcome in human subcutaneous adipocytes cell culture.

Obesity patients are more susceptible to develop COVID-19 severe outcome due to the role of angiotensin-converting enzyme 2 (ACE2) in the viral infection. ACE2 is regulated in the human cells by different genes associated with increased (TLR3, HAT1, HDAC2, KDM5B, SIRT1, RAB1A, FURIN and ADAM10) or decreased (TRIB3) virus replication. RNA-seq data revealed 14857 genes expressed in human subcutaneous adipocytes, including genes mentioned above. Irisin treatment increased by 3-fold the levels of TRIB3 transcript and decreased the levels of other genes. The decrease in FURIN and ADAM10 expression enriched diverse biological processes, including extracellular structure organization. Our results, in human subcutaneous adipocytes cell culture, indicate a positive effect of irisin on the expression of multiple genes related to viral infection by SARS-CoV-2; furthermore, translatable for other tissues and organs targeted by the novel coronavirus and present, thus, promising approaches for the treatment of COVID-19 infection as therapeutic strategy to decrease ACE2 regulatory genes.

Efficient isolation and proliferation of human adipose-derived mesenchymal stromal cells in xeno-free conditions.

Classical methods used for culture of adipose-derived mesenchymal stromal cells (ADSCs) use xenobiotic components, which may present a potential risk for biological contamination and/or elicit immunological reactions. Therefore, the aim of this study was to establish a xeno-free methodology for the isolation and proliferation of human ADSCs (hADSCs). hADSCs were isolated by enzymatic digestion or mechanical dissociation and cultured in the presence of fetal bovine serum or human platelet lysate. Proliferation curves were performed as a function of time from the cell culture and used to calculate the population doubling time. Immunophenotyping and differentiation tests were used to identify and characterize the hADSCs. Human ADSCs isolated and cultured in conventional or xenobiotic-free conditions peaked at different days but achieved similar maximum proliferation. The hADSCs differentiation ability was similar in all groups. The characterization of hADSCs by flow cytometry showed low contamination of the cultures by other cell types. The xenobiotic-free methodology described in this study is a feasible and reproducible alternative for isolation and proliferation of hADSCs. This methodology is in accordance with the recommendations of the National Health Surveillance Agency, which proposes avoidance of xenobiotic products.

New methodologies for old problems: tridimensional gastrointestinal organoids and guts-on-a-chip / Novas metodologias para velhos problemas: organoides gastrointestinais e guts-on-a-chip tridimensionais

ABSTRACT Objectives: The present review intended to present a critical overview of the methodological and experimental advances concerning tridimensional cell culture models within the scope of gastrointestinal research. Methods: A literature review was performed and some of the main published articles in the area were mentioned. Main results: Classic studies and high impact results were presented, starting from the pioneer works with gastrointestinal organoids, with a small gut organoid, to the achievement of guts-on-a-chip and multi-organ-chips. It was also discussed which implications the construction of such co-cultures bring, as well as future applications arising from these new methodologies. Conclusions: Despite the still discrete number of publications, in quantitative terms, there are qualitative promising and consistent results addressing physiopathological aspects and new therapeutic perspectives of tridimensional in vitro cultures in the gastroenterology field. It is expected, thus, that such new methodological approaches, including organoids and guts-on-a-chip, may contribute decisively to the advance in knowledge on basic aspects, as well as on the translation to new therapeutic approaches in gastrointestinal diseases.

RESUMO Objetivos: A presente revisão visou apresentar uma abordagem crítica dos avanços metodológicos e experimentais referentes a modelos de cultura celular tridimensionais no âmbito do sistema gastrintestinal. Métodos: Foi realizada revisão da literatura com ênfase nos principais artigos publicados na área. Resultados principais: São apresentados trabalhos clássicos e resultados de maior impacto, desde os trabalhos pioneiros com organoides do sistema gastrintestinal, com intestino delgado, até a obtenção de guts-on-a-chip e multi-organ-chips. Discutiu-se, ainda, as implicações decorrentes da elaboração de tais co-culturas, bem como as futuras aplicações decorrentes dessas novas metodologias. Conclusões: Apesar do número ainda discreto de publicações, em termos quantitativos, há, qualitativamente, resultados promissores e consistentes abordando aspectos fisiopatológicos e de novas perspectivas terapêuticas em gastrenterologia decorrentes das culturas tridimensionais in vitro. É esperado, portanto, que essas novas abordagens metodológicas incluindo organoides e guts-on-a-chip possam contribuir decisivamente para o avanço no conhecimento sobre de aspectos básicos, bem como para a translação do conhecimento para novas abordagens terapêuticas em doenças gastrintestinais.

Environmental enrichment and physical exercise revert behavioral and electrophysiological impairments caused by reduced adult neurogenesis.

It is well known that adult neurogenesis occurs in two distinct regions, the subgranular zone of the dentate gyrus and the subventricular zone along the walls of the lateral ventricles. Until now, the contribution of these newly born neurons to behavior and cognition is still uncertain. The current study tested the functional impacts of diminished hippocampal neurogenesis on emotional and cognitive functions in transgenic Gfap-tk mice. Our results showed that anxiety-related behavior evaluated both in the elevated plus maze as well as in the open field, social interaction in the sociability test, and spatial working memory in the spontaneous alternation test were not affected. On the other hand, recognition and emotional memory in the object recognition test and contextual fear conditioning, and hippocampal long-term potentiation were impaired in transgenic mice. Furthermore, we evaluated whether environmental enrichment together with physical exercise could improve or even restore the level of adult neurogenesis, as well as the behavioral functions. Our results clearly demonstrated that environmental enrichment together with physical exercise successfully elevated the overall number of progenitor cells and young neurons in the dentate gyrus of transgenic mice. Furthermore, it led to a significant improvement in object recognition memory and contextual fear conditioning, and reverted impairments in hippocampal long-term potentiation. Thus, our results confirm the importance of adult neurogenesis for learning and memory processes and for hippocampal circuitry in general. Environmental enrichment and physical exercise beneficially influenced adult neurogenesis after it had been disrupted and most importantly recovered cognitive functions and long-term potentiation. © 2016 Wiley Periodicals, Inc.