Variation in the Extraction Efficiency of Estradiol and Progesterone in Moist and Lyophilized Feces of the Black Howler Monkey (Alouatta pigra): Alternative Methods

Several fecal steroid extraction techniques have been developed to measure the ovary function in different species of mammals. However, regardless of the method of extraction and the sample type chosen, it has been observed that they can yield results with different percentages of recuperation. The objective of this study was to determine whether the type of substratum, solvent and extraction method used have any influence on the extraction efficiency in the feces of Alouatta pigra (black howler monkey). For this purpose we used two methods: agitation and ebullition. With each method, we utilized moist and lyophilized feces. The validation of radioimmunoassay method was accurate and precise for quantify estradiol and progesterone in lyophilized feces of A. pigra. To both of which ethanol and methanol, absolute and at 80%, were added, besides the hormones 125I-Estradiol and 125I-Progesterone. The extraction efficiency for 125I-Estradiol was from 87.72 ± 3.97 to 41.24 ± 2.67%, and for 125I-Progesterone from 71.15 ± 4.24 to 42.30 ± 1.19% when we used the agitation method. Whereas with the ebullition method, the extraction efficiency for 125I-Estradiol ranged from 86.89 ± 2.66 to 71.68 ± 3.02% and for 125I-Progesterone from 98.31 ± 1.26 to 85.40 ± 1.98%. Due to the differences found in these assays, which depend on the method used, the type of feces employed and the type of solvent added to them, we recommend the ebullition method and the lyophilized feces of A. pigra for extracting the hormones, since in moist feces there may exist variables which might interfere in the quantification of 125I-Estradiol and 125I-Progesterone

Hematology and serum biochemistry values of free-ranging Iberian wolves (Canis lupus) trapped by leg-hold snares

Hematology and serum biochemistry are important tools in assessing the health and physiological status of wildlife populations. Nevertheless, studies on free-ranging wolves (Canis lupus) are scarce, and no reference values are available neither for Iberian wolves nor for wolves captured with leghold snares. We report 37 hematology and serum biochemistry variables obtained from 26 free-ranging Iberian wolves captured with leg-hold snares between 2007 and 2014, including variables previously not reported in the literature. The values obtained are similar to the published reference intervals for Scandinavian wolves captured by darting from a helicopter, except for higher values for mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), leukocyte count, creatinine kinase (CK), ?-globulins, and total bilirubin (TBIL) and lower values for alkaline phosphatase (ALP). We propose that differences in leukocyte count, CK, and TBIL are related to the method of capture, while differences in RDW, MCHC, ALP, and ?-globulins could reflect physiological adaptations to environmental conditions, sampling, or pre-analytical artifacts. Lymphocyte count was lower and neutrophil/lymphocyte ratio was significantly higher in older, reproductive females, while ALP and phosphorus were higher in juvenile wolves. For the first time, we describe hematology and serum biochemistry values of free-ranging Iberian wolves captured with leg-hold snares. The data reported here is the first published reference for wolves captured with similar methods and for monitoring Iberian wolves populations’ physiological and health status.We thank Nuria Fandos and Carla Ferreira, rangers from Xunta de Galicia and Parque Nacional de los Picos de Europa, and volunteers who helped during the trapping sessions. The wolves were captured under projects financed by Associacao de Conservacao do Habitat do Lobo Iberico (ACHLI) in Portugal and by Picos de Europa National Park, Ministerio de Agricultura, Alimentacion y Medio Ambiente, and Xunta de Galicia in Spain. Sara Roque benefited from grant SFRH/BD/12291/2003 from Fundacao para a Ciencia e a Tecnologia. Jose V. Lopez-Bao was supported by a postdoctoral contract from the Spanish Ministry of Economy and Competitiveness. This is the paper no. 5 from the Iberian Wolf Research Team

The microbiological transformation of some ent-13-epi-manoyl oxide diterpenes by Gibberelle fujikuroi

Incubation of ent-19-hydroxy-13-epi-manoyl oxide with Gibberella fujikuroi afforded ent-12a,19-dihydroxy-13-epi-manoyl oxide and ent-7B,12a19-trihydroxy-13-epi-manoyl oxide, the first of which was transformed by chemical methods into varol (ent-12a-hydroxy-13-epi-manoyl oxide). The incubation of ent-3B-hydroxy-13-epi-manoyl oxide (ribenol) with the same fungus gave the 12B-hydroxy, the 11B-hydroxy and the 11B,12B-dihydroxy derivatives of ribenol, the first of which was identical with varodiol.This work bas been supported by the CICYT, Ministry of Education and Science, Spain. We thank Dr. A. García-Granados (University of Granada) for copies of spectra of varol and varodiol, and Dr. J. R. Hanson (University of Sussex, U.K.) for gifts of Gibberella fujikuroi and for his interest in this work, R. G. thanks the Ministry of Education and Science and the Spanish Research Council (CSIC) for a "Formación de Personal Investigador" fellowship.Peer reviewe

Lesion-associated microglia and macrophages mediate corralling and react with massive phagocytosis for debris clearance and wound healing after LPS-induced dopaminergic depletion

Altres ajuts: Acord transformatiu CRUE-CSICNeuroinflammation contributes to neuronal degeneration in Parkinson's disease (PD). However, how brain inflammatory factors mediate the progression of neurodegeneration is still poorly understood. Experimental models of PD have shed light on the understanding of this phenomenon, but the exploration of inflammation-driven models is necessary to better characterize this aspect of the disorder. The use of lipopolysaccharide (LPS) to induce a neuroinflammation-mediated neuronal loss is useful to induce reliable elimination of dopaminergic neurons. Nevertheless, how this model parallels the PD-like neuroinflammation is uncertain. In the present work, we used the direct LPS injection as a model inductor to eliminate dopaminergic neurons of the substantia nigra pars compacta (SNpc) in rats and reevaluated the inflammatory reaction. High-resolution 3D histological examination revealed that, although LPS induced a reliable elimination of SNpc dopaminergic neurons, it also generated a massive inflammatory response. This inflammation-mediated injury was characterized by corralling, a damaged parenchyma occupied by a vast population of lesion-associated microglia and macrophages (LAMMs) undertaking wound compaction and scar formation, surrounded by highly reactive astrocytes. LAMMs tiled the entire lesion and engaged in long-standing phagocytic activity to resolve the injury. Additionally, modeling LPS inflammation in a cell culture system helped to understand the role of phagocytosis and cytotoxicity in the initial phases of dopaminergic degeneration and indicated that LAMM-mediated toxicity and phagocytosis coexist during LPS-mediated dopaminergic elimination. However, this type of severe inflammatory-mediated injury, and subsequent resolution appear to be different from the ageing-related PD scenario where the architectural structure of the parenchyma is mostly preserved. Thus, the necessity to explore new experimental models to properly mimic the inflammatory compound observed in PD degeneration

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.The study was funded by the Spanish Ministerio de Economía y Competitividad (MINECO; grant SAF2014-56279-R and SAF2017-87459-R to A.M. Planas), Acción Estratégica de Salud (grants AES 1366/13 and AESI 1346/16 to P. Lauzurica), and Fundació la Marató de TV3 (grants 474-082230 and 20153031 to P. Garcia-de-Frutos). F. Miró-Mur had a PERIS award by the Health Department of the Generalitat de Catalunya. A. Otxoa-de-Amezaga has an FPI fellowship (MINECO)