Validation of a zebrafish developmental defects assay as a qualified alternative test for its regulatory use following the ICH S5(R3) guideline.

Zebrafish is a popular toxicology model and provides an ethically acceptable small-scale analysis system with the complexity of a complete organism. Our goal is to further validate this model for its regulatory use for reproductive and developmental defects by testing the compounds indicated in the "Guideline on detection of reproductive and developmental toxicity for human pharmaceuticals" (ICH S5(R3) guideline.) To determine the embryotoxic and developmental risk of the 32 reference compounds listed in the ICH S5(R3) guideline, the presence of morphological alterations in zebrafish embryos was analyzed at two different stages to calculateLC50 and EC50 values for each stage. Teratogenic Indexes were established as the ratio between LC50 and EC50 critical for the proper compound classification as teratogenic when it is ≥ 2. A total of three biological replicates have been conducted to study the reproducibility of the assay. The chemicals' concentration in the medium and internally in the zebrafish embryos was evaluated. In this study, the 3 negative compounds were properly categorized while 23 compounds out of the 29 reference ones (sensitivity of 79.31%) were classified as teratogenic in zebrafish. The 6 that had false-negative results were classified 4 as inconclusive, 1 as not toxic, and 1 compound resulted toxic for zebrafish embryos under testing conditions. After the bioavailability experiments, some of the obtained inconclusive results were refined. The developmental defects assay in zebrafish gives an accuracy of 89.66%, sensitivity of 88.46%, specificity and repeatability of 100% compared to mammals; therefore, this is a well-integrated strategy using New Alternative Methods, to minimize the use of animals in developmental toxicity studies.

MiR-219a-5p Enriched Extracellular Vesicles Induce OPC Differentiation and EAE Improvement More Efficiently Than Liposomes and Polymeric Nanoparticles.

Remyelination is a key aspect in multiple sclerosis pathology and a special effort is being made to promote it. However, there is still no available treatment to regenerate myelin and several strategies are being scrutinized. Myelination is naturally performed by oligodendrocytes and microRNAs have been postulated as a promising tool to induce oligodendrocyte precursor cell differentiation and therefore remyelination. Herein, DSPC liposomes and PLGA nanoparticles were studied for miR-219a-5p encapsulation, release and remyelination promotion. In parallel, they were compared with biologically engineered extracellular vesicles overexpressing miR-219a-5p. Interestingly, extracellular vesicles showed the highest oligodendrocyte precursor cell differentiation levels and were more effective than liposomes and polymeric nanoparticles crossing the blood-brain barrier. Finally, extracellular vesicles were able to improve EAE animal model clinical evolution. Our results indicate that the use of extracellular vesicles as miR-219a-5p delivery system can be a feasible and promising strategy to induce remyelination in multiple sclerosis patients.

[Neonatal hypoxia-ischemia: cellular and molecular brain damage and therapeutic modulation of neurogenesis]. / Hipoxia-isquemia neonatal: bases celulares y moleculares del daño cerebral y modulacion terapeutica de la neurogenesis.

INTRODUCTION: Perinatal asphyxia remains a major cause of both mortality and neurological morbidity. Neonatal encephalopathy affects to 1-3/1,000 newborns, leading to significant brain damage and childhood disability. The only standard therapy is moderate hypothermia, whose efficacy, despite proved, is limited, being partially effective. DEVELOPMENT: The capacity of hypothermia in promoting cell proliferation in the neurogenic niches of the central nervous system remains subject of investigation. The use of therapeutic agents such as erythropoietin and cannabinoids and mesenchymal stem cells have shown promising results in experimental models of perinatal asphyxia, being able of modulate neurogenesis, neuronal plasticity and neuroreparation processes after hypoxic-ischemic brain injury. CONCLUSIONS: The effects of these therapies in clinics are still unknown, so as if the newborn cells will be able to effectively integrate in the existing neuronal networks or if they will develop their proper functions in a brain-damaged microenvironment, thus being necessary new works focused on the evaluation of the real potential of these therapies in the modulation of neurogenesis after neonatal hypoxia-ischemia.

TITLE: Hipoxia-isquemia neonatal: bases celulares y moleculares del daño cerebral y modulacion terapeutica de la neurogenesis.Introduccion. La asfixia perinatal continua siendo una de las mayores causas de morbimortalidad neurologica. La encefalopatia neonatal derivada constituye una causa importante de daño cerebral, que afecta de manera moderada-grave a 1-3 de cada 1.000 recien nacidos y comporta un alto riesgo de deficits neurologicos permanentes. La unica aproximacion terapeutica actual consiste en la hipotermia moderada, cuya eficacia, aunque constatada, no siempre consigue una recuperacion funcional total. Desarrollo. Se desconoce con certeza si la hipotermia tiene la capacidad de promover la proliferacion celular en los nichos neurogenicos cerebrales, donde permanecen celulas madre neuronales con capacidad de proliferacion y diferenciacion. El empleo de agentes terapeuticos, como la eritropoyetina o los cannabinoides, y de celulas madre mesenquimales ha mostrado resultados prometedores en diversos modelos experimentales de asfixia perinatal y es capaz de modular los procesos de neurogenesis, de plasticidad neuronal y de neurorreparacion tras un daño cerebral hipoxico-isquemico. Conclusiones. Aun se desconocen los efectos de estas terapias en modelos clinicos y si las celulas recien formadas seran capaces de integrarse de forma efectiva en las redes neuronales existentes o si podran desarrollar sus funciones adecuadamente en un microambiente de lesion cerebral, por lo que se hace necesario el desarrollo de nuevos trabajos enfocados a evaluar el potencial real de estos agentes en la modulacion terapeutica de la neurogenesis tras una hipoxia-isquemia neonatal.

Using the endocannabinoid system as a neuroprotective strategy in perinatal hypoxic-ischemic brain injury.

One of the most important causes of brain injury in the neonatal period is a perinatal hypoxic-ischemic event. This devastating condition can lead to long-term neurological deficits or even death. After hypoxic-ischemic brain injury, a variety of specific cellular mechanisms are set in motion, triggering cell damage and finally producing cell death. Effective therapeutic treatments against this phenomenon are still unavailable because of complex molecular mechanisms underlying hypoxic-ischemic brain injury. After a thorough understanding of the mechanism underlying neural plasticity following hypoxic-ischemic brain injury, various neuroprotective therapies have been developed for alleviating brain injury and improving long-term outcomes. Among them, the endocannabinoid system emerges as a natural system of neuroprotection. The endocannabinoid system modulates a wide range of physiological processes in mammals and has demonstrated neuroprotective effects in different paradigms of acute brain injury, acting as a natural neuroprotectant. The aim of this review is to study the use of different therapies to induce long-term therapeutic effects after hypoxic-ischemic brain injury, and analyze the important role of the endocannabinoid system as a new neuroprotective strategy against perinatal hypoxic-ischemic brain injury.