Clonación de genotipos adultos y juveniles de Quercus suber y Q. ilex tolerantes a Phytophthora cinnamomi

El síndrome de “la seca” está generando desde hace décadas la pérdida de masas de Quercus mediterráneos provocando grandes pérdidas económicas y ecológicas. Por ello, en 2019 el subgrupo de “Mejora Genética y Fisiología” del Grupo de Trabajo sobre Seca del Ministerio para la Transición Ecológica y el Reto Demográfico impulsó el Programa Nacional para la Conservación y Mejora de Recursos Genéticos de Encina y Alcornoque. Este programa contempla la clonación y la conservación de material tolerante a Phytophthora cinnamomi y/o sequía, seleccionado en anteriores proyectos de investigación. Aplicando técnicas de cultivo in vitro, embriogénesis somática y proliferación de yemas axilares, se pretende clonar una selección de individuos dentro de las progenies más tolerantes, y los mejores genotipos adultos evaluados a través de esas progenies. Las hojas son el explanto elegido para inducir embriones somáticos en material juvenil y adulto de alcornoque; en material de encina adulto el explanto inicial es el tegumento del embrión inmaduro. Las líneas embriogénicas generadas han sido conservadas a largo plazo mediante su crioconservación (almacenamiento en nitrógeno líquido). La proliferación de yemas axilares se emplea para clonar genotipos juveniles de ambas especies. La clonación del material permitirá disponer de copias suficientes que permitan la evaluación de su tolerancia en ensayos de campo

Mechanisms involved in the increased sensitivity of the rabbit basilar artery to atrial natriuretic peptide in diabetes

Atrial natriuretic peptide (ANP) is a vasodilator with significant regional differences and controversial effects in the cerebral circulation, a vascular bed particularly prone to diabetes-induced complications. The present study has investigated how alloxan-induced diabetes modifies the mechanisms involved in the response of the rabbit basilar artery to ANP. ANP (10-12-10−7 M) relaxed precontracted basilar arteries, with higher potency in diabetic than in control rabbits. In arteries from both groups of animals, endothelium removal reduced ANP-induced relaxations. Inhibition of NO-synthesis attenuated ANP-induced relaxation but this attenuation was lower in diabetic than in control rabbits. In control rabbits, indomethacin displaced to the left the concentrationresponse curve to ANP, without significantly modifying the Emax value. In diabetic rabbits, indomethacin significantly enhanced arterial relaxations to ANP. In KCl-depolarised arteries, relaxation to ANP was almost abolished both in control and in diabetic rabbits. Iberiotoxin inhibited relaxations to ANP in both groups of rabbits. Glibenclamide and 4-aminopyridine inhibited the ANP-induced relaxations more in diabetic than in control rabbits. Basilar arteries from diabetic rabbits showed decreased natriuretic peptide receptor C expression and no changes in natriuretic peptide receptor A, large conductance calcium-activated K+ channels (BKCa), ATPsensitive K+ channels (KATP) and voltage-sensitive K+ channels (KV) expression. These results suggest that diabetes enhances the sensitivity of the rabbit basilar artery to ANP by mechanisms that at least include reduced expression of natriuretic peptide receptor C, and enhanced activity of KATP and KV channels. Furthermore, diabetes reduces endothelial NO and prostacyclin which mediate arterial relaxation to ANP

Molecular mechanisms underlying the neuroprotective role of atrial natriuretic peptide in experimental acute ischemic stroke.

Along with its role in regulating blood pressure and fluid homeostasis, the natriuretic peptide system could be also part of an endogenous protective mechanism against brain damage. We aimed to assess the possibility that exogenous atrial natriuretic peptide (ANP) could protect against acute ischemic stroke, as well as the molecular mechanisms involved. Three groups of rats subjected to transient middle cerebral artery occlusion (tMCAO, intraluminal filament technique, 60 min) received intracerebroventricular vehicle, low-dose ANP (0.5 nmol) or high-dose ANP (2.5 nmol), at 30 min reperfusion. Neurofunctional condition, and brain infarct and edema volumes were measured at 24 h after tMCAO. Apoptotic cell death and expression of natriuretic peptide receptors (NPR-A and NPR-C), K+ channels (KATP, KV and BKCa), and PI3K/Akt and MAPK/ERK1/2 signaling pathways were analyzed. Significant improvement in neurofunctional status, associated to reduction in infarct and edema volumes, was shown in the high-dose ANP group. As to the molecular mechanisms analyzed, high-dose ANP: 1) reduced caspase-3-mediated apoptosis; 2) did not modify the expression of NPR-A and NPR-C, which had been downregulated by the ischemic insult; 3) induced a significant reversion of ischemia-downregulated KATP channel expression; and 4) induced a significant reversion of ischemia-upregulated pERK2/ERK2 expression ratio. In conclusion, ANP exerts a significant protective role in terms of both improvement of neurofunctional status and reduction in infarct volume. Modulation of ANP on some molecular mechanisms involved in ischemiainduced apoptotic cell death (KATP channels and MAPK/ERK1/2 signaling pathway) could account, at least in part, for its beneficial effect. Therefore, ANP should be considered as a potential adjunctive neuroprotective agent improving stroke outcome after successful reperfusion interventions

Cerebroprotective Effect of 17β-Estradiol Replacement Therapy in Ovariectomy-Induced Post-Menopausal Rats Subjected to Ischemic Stroke: Role of MAPK/ERK1/2 Pathway and PI3K-Independent Akt Activation

Despite the overwhelming advances in the understanding of the pathogenesis of stroke, a devastating disease affecting millions of people worldwide, currently there are only a limited number of effective treatments available. Preclinical and clinical studies show that stroke is a sexually dimorphic disorder, affecting males and females differently. Strong experimental evidence indicates that estrogen may play a role in this difference and that exogenous 17β-estradiol (E2) is neuroprotective against stroke in both male and female rodents. However, the molecular mechanisms by which E2 intervenes in ischemia-induced cell death, revealing these sex differences, remain unclear. The present study was aimed to determine, in female rats, the molecular mechanisms of two well-known pro-survival signaling pathways, MAPK/ERK1/2 and PI3K/Akt, that mediate E2 neuroprotection in response to acute ischemic stroke. E2 pretreatment reduced brain damage and attenuated apoptotic cell death in ovariectomized female rats after an ischemic insult. Moreover, E2 decreased phosphorylation of ERK1/2 and prevented ischemia/reperfusion-induced dephosphorylation of both Akt and the pro-apoptotic protein, BAD. However, MAPK/ERK1/2 inhibitor PD98059, but not the PI3K inhibitor LY294002, attenuated E2 neuroprotection. Thus, these results suggested that E2 pretreatment in ovariectomized female rats modulates MAPK/ERK1/2 and activates Akt independently of PI3K to promote cerebroprotection in ischemic stroke. A better understanding of the mechanisms and the influence of E2 in the female sex paves the way for the design of future successful hormone replacement therapies

Recommendations guide for experimental animal models in stroke research

Introduction: The progress of effective therapies for stroke has become a challenging task for both researchers and clinicians. Some pitfalls in clinical trials might have their origins in the pre-clinical experimental ischaemic models for the evaluation of potential neuro-protective agents. Methods: We aim to standardise the methods for the development of stroke animal models throughout Spain, to produce document with appropriate recommendations and best practice in order to improve experimental methods in the field of stroke research. Results: Members of several experienced stroke research groups prepared a guide with recommendations in the application of focal cerebral ischaemic models. The main features of this guide are based on the selection of the most appropriate animal model, taking in account the objective of the study, the species, strain, age, sex of animals, as well as risk factors. The experimental design must include a sham control group and the sample size calculation. Animal randomisation and blind analysis, masked assessment of outcomes, monitoring of body temperature and cerebral blood flow, and the reporting of reasons for excluding animals from the study, as well as the mortality rate, are other main points to fulfil in the application of stroke models. Conclusions: Standardised methods are essential to increase the success of the preclinical findings in the stroke neuroprotection field to be able to translate to the clinical practice. Resumen: Introducción: La búsqueda de una terapia neuroprotectora efectiva para el ictus sigue siendo un reto para investigadores y clínicos. Una de las causas principales por la que a nivel clínico han fracasado terapias eficaces en ensayos experimentales reside probablemente en el desarrollo y modo de evaluación en estudios preclínicos de los agentes neuroprotectores en los modelos animales de isquemia cerebral. Métodos: Para unificar la metodología en la aplicación de los modelos experimentales a nivel nacional y mejorar la investigación en este campo, se ha elaborado un documento entre varios grupos españoles expertos en investigación neurovascular que constituye una guía de recomendaciones para el uso de los mismos. Resultados: Sus aspectos fundamentales se basan en la selección del modelo más adecuado en función del objetivo del estudio, teniendo en cuenta el tipo de especie y la cepa animal, la edad, el sexo y los factores de riesgo. La realización del diseño experimental incluye un grupo sham control y el cálculo previo del tamaño muestral. Otros aspectos muy importantes a seguir son la aleatorización en la asignación de los animales en cada grupo, el análisis ciego de los parámetros estudiados, el registro de la temperatura y flujo sanguíneo cerebral, así como la notificación y causas de animales excluidos en el estudio y la tasa de mortalidad. Conclusiones: Es esencial adquirir compromisos metodológicos para la optimización del empleo de los modelos animales de isquemia cerebral que incremente el rendimiento de hallazgos positivos en la fase preclínica y puedan trasladarse a la práctica clínica. Keywords: Experimental design, Translational research, Cerebral ischaemia, Animal models, Palabras clave: Diseño experimental, Investigación traslacional, Isquemia cerebral, Modelos animale

Diabetes modifies the role of prostanoids and potassium channels which regulate the hyperactivity of the rabbit renal artery to BNP

Diabetic nephropathy is associated with increased risk of cardiovascular disease. BNP plays an important role in cardiovascular pathophysiology and therapeutics. The aim of the present study was to investigate the influence of experimental diabetes on the mechanisms that regulate the relaxant response of the rabbit renal artery to BNP. Arterial relaxations to BNP were enhanced in diabetic rabbits. Indomethacin enhanced BNP-induced relaxation in control rabbits, but showed no effect in diabetic rabbits. BNP-induced release of thromboxane A2 or prostacyclin were not different in both groups of animals. Iberiotoxin had no effect on relaxations to BNP in both groups of animals. Charybdotoxin displaced to the right the concentration-response curve to BNP in both group of animals, and inhibited BNP-induced relaxation only in diabetic rabbits. Glibenclamide did not modify the BNP-induced relaxations in control rabbits, but inhibited it in diabetic rabbits. These results suggest that diabetes induces hypereactivity of the rabbit renal artery to BNP by mechanisms that at least include 1) a reduced vasoconstrictor influence of arachidonic acid metabolites via cyclooxygenase 2, which is not related with changes in thromboxane A2 and prostacyclin release from the arterial wall; and 2) a selectively increased modulatory activity of KATP and endothelial IKCa channels