Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis.

Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO4 exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8-iso-PGF2α and 4-hydroxynonenal (4-HNE). While both treatments induced changes indicative of spermptosis (caspase-3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.

Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes.

The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques (t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation (P < 0.01), PS translocation to the outer membrane (P < 0.001), loss of mitochondrial membrane potential (P < 0.05), and increase in intracellular Na+ (P < 0.01). Average values of markers of capacitative changes were not affected by cryopreservation; however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry.

Flow cytometry in Spermatology: A bright future ahead.

Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high-throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information.The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single-cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry-based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology.

The Impact of Reproductive Technologies on Stallion Mitochondrial Function.

The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant. Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen-thawed stallion sperm. Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies.

Effect of overnight staining on the quality of flow cytometric sorted stallion sperm: comparison with tradtitional protocols.

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre-sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.

Caspase activation, hydrogen peroxide production and Akt dephosphorylation occur during stallion sperm senescence.

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.

Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na.

In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na+ -K+ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (p < 0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p < 0.05) in intracellular Na+ . These changes occurred in relation to activation of caspase 3 (p < 0.001). Cryopreservation reduced the activity of the Na-K+ pump and inhibition of the Na+ -K+ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na+ -K+ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.

The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions.

Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at -196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an 'autophagy-like' mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.

[Idiopathic intracranial hypertension in childhood]. / Hipertensión intracraneal idiopática en la infancia.

INTRODUCTION: We present the cases of idiopathic, intracranial hypertension (HII) in children studied in our hospital over the past 15 years (14 cases), reviewing the clinical features, complementary tests, associated causes, treatment and evolution. PATIENTS AND METHODS: Diagnosis of HII was based on a modification of Ahlskog's criteria (1982) (the child was sedated to avoid increased cranial pressure due to crying): no focal signs, CSF pressure greater than 200 mm of H2O, normal composition of CSF and normal neuro-imaging findings. Two groups were established according to age. The first group included those under 24 months old and the second group those over 24 months. Differences were seen in the presenting, symptoms, neurological examination and treatment. RESULTS: In the first group the cause of consultation was irritability and a bulging fontanelle. They recovered after simply restricting fluids and treating the associated cause. In the second group, consultation was for headache and vomiting, and sometimes diplopia also. Recovery was satisfactory (except in one case) when corticosteroids were added to the above treatment. The complementary tests carried out depended on the clinical history, general and neurological examination and associated cause (MR, vitamin A levels, nutritional index, etc.). A possible aetiological relationship was found in 75% of cases. CONCLUSIONS: We emphasize the favourable course and good prognosis of HII in infants as compared to adults.

¿Qué profesional médico es el más adecuado para impartir cuidados en salud a niños en Atención Primaria en países desarrollados? Revisión sistemática / What medical professional is the most adequate to provide health care to children in primary care in developed countries? Systematic review

Introducción: existe controversia sobre el tipo de profesional más adecuado –pediatras (PED) o médicos de familia/generales (MF/MG)– para prestar atención sanitaria a niños y adolescentes en Atención Primaria (AP). No existen revisiones sistemáticas previas que hayan estudiado este aspecto. El objetivo de este estudio es comparar la atención sanitaria proporcionada por PED y MF/MG en los siguientes aspectos de la práctica clínica: la prescripción de antibióticos (ATB), la indicación de pruebas diagnósticas, el manejo de la otitis media (OMA), del asma, del síndrome febril y de diversas alteraciones psicopatológicas, así como la realización de actividades preventivas. Material y métodos: diseño de estudio: revisión sistemática. Fuente de los datos: hasta diciembre de 2008 se revisaron las bases de datos MEDLINE y CENTRAL, el metabuscador TRIP Database y el buscador Google Académico para recuperar artículos originales y revisiones sistemáticas que compararan la práctica clínica de ambos tipos de profesionales. No se efectuó restricción por idioma. Selección de estudios: se incluyeron estudios de cualquier tipo de diseño (transversal, cohortes, casos y controles, experimentales) que compararan la práctica clínica del PED y el MF/MG. Se excluyeron todas las referencias que no contuvieran investigación original (cartas al director o editoriales). Asimismo, se evaluó la calidad metodológica de cada estudio con el instrumento “OSTEBA; Fichas de lectura crítica”. Dicha calidad era valorada de forma independiente por dos revisores, que llegaban a un consenso en caso de discrepancia. La extracción de datos fue realizada por siete parejas de revisores de forma independiente. Las discrepancias se resolvieron mediante consenso. Resultados: como promedio, los MF/MG prescribieron más ATB que los PED en infecciones del tracto respiratorio superior de probable etiología vírica –odds ratio (OR): 1,4; intervalo de confianza del 95% (IC 95%): 1,1-1,8–. Los PED tuvieron más probabilidades de adherirse a las recomendaciones de guías de práctica clínica sobre el manejo del síndrome febril (OR: 9; IC 95%: 3-25) y del trastorno por déficit de atención con/sin hiperactividad (OR: 5; IC 95%: 3-11), y una mayor capacidad de resolución para otras enfermedades de elevada prevalencia durante la infancia y la adolescencia (como asma y OMA). Los PED presentaban porcentajes de vacunación superiores a los de los MF/MG en todos los estudios que evaluaron este resultado. Conclusión: en vista de los resultados expuestos, parece recomendable mantener la figura del PED en los equipos de AP y reforzar su función específica como primer punto de contacto del niño con el sistema sanitario (AU)

Introduction: There is controversy about which health professional is the most adequate –pediatricians (PED) or family practitioners/general physicians (FP/GP)– to provide health care services to children and adolescents in Primary Care (PC). There are not previous systematic reviews approaching this subject in the previously published literature. The objective of this study is to compare health care provided between PED and FP/GP in the following aspects of the clinical practice: antibiotic (ATB) prescription; diagnostic test indication; acute otitis media (AOM), asthma, febrile syndrome and several psychopathological conditions’ management; and preventive measures accomplishment. Material and methods: study design: systematic review. Data sources: MEDLINE and CENTRAL databases, TRIP Database and Google Scholar, were searched until December 2008 to retrieve original papers and systematic reviews comparing the clinical practice of both kinds of health professionals. No language restriction was made. Studies’ selection: studies of any kind of design were included (cross-sectional, cohorts, case-controls and experimental) comparing the clinical practice of PED and FP/GP. The references without original research were excluded (letters to the editor, editorials). The methodological quality of each study was assessed with the tool “OSTEBA; Critical Appraisal Cards”. Two reviewers assessed the quality of the studies independently, achieving consensus in case of discrepancy. Seven pairs of reviewers made the data extraction independently. Discrepancies were achieved by consensus. Results: On average, FP/GP prescribed more ATB than PED in upper respiratory tract infections of probable viral etiology –odds ratio (OR): 1.4; 95% confidence interval (95% CI): 1.1-1.8–; PED were more likely to adhere to clinical guidelines recommendations on febrile syndrome management (OR: 9; 95% CI: 3-25) and on attention deficit disorder with/without hyperactivity (OR: 5; 95% CI: 3-11), and showed more resolution capacity on other highly prevalent conditions in children and adolescents (such as asthma and AOM). PED showed higher vaccination coverage than FP/GP in all the studies assessing this result. Conclusion: based on the presented results, it seems reasonable to recommend maintaining the PED figure in PC health centers and reinforcing its specific task as the first point of contact of the child with the health care system (AU)

Los aerosoles con suero salino hipertónico al 3% podrían disminuir la duración de la hospitalización en lactantes con bronquiolitis / No disponible

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El riesgo de muerte súbita al acostarse en prono no es el mismo para todos los lactantes: podemos influir aún más en su disminución / No disponible

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Implicar a los padres en la decisión de utilizar antibióticos en la otitis media aguda disminuye su uso / Involving parents in the decision whether to use antibiotics in acute otitis media reduces its use

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Lectura crítica de artículos / Critical appraisal of scientific literature

La medicina basada en la evidencia nos ofrece herramientas de gran utilidad para poder resolver problemas clínicos mediante el análisis eficiente de la literatura científica. Herramientas metodológicas que, si adquirimos y ejercitamos, nos ayudarán a valorar cualquier evidencia científica y a integrarla con nuestros conocimientos y experiencia clínica para poder decidir sobre su aplicabilidad e idoneidad en un paciente concreto. En esta exposición repasaremos los principios generales de la valoración crítica de la literatura científica. También revisaremos los principales criterios que se deben considerar en la valoración de la validez y aplicabilidad de los estudios de evaluación de intervenciones sanitarias y de pruebas diagnósticas (AU)

The evidence based medicine offers us very useful tools to solve clinical problems by means of the efficient analysis of scientific literature. If we acquire and exercise these methodological tools, we will be able to value any scientific evidence and integrate it with our knowledge and clinical experience, and also we will be able to decide on its applicability and suitability in a concrete patient. In this article we will review the general principles of the critical appraisal of scientific literature. Also we will review the main criteria to consider in the analysis of the validity and applicability of the studies of evaluation of health interventions and diagnostic tests (AU)

Neumonía adquirida en la comunidad y control radiológico / Community acquired pneumonia and its radiographic control

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