Lesiones de vía biliar poscolecistectomía: conducta terapéutica actual / Post Cholecystectomy Bile Duct Injuries: Current Therapeutic Option

Resumen La colecistolitiasis es una condición común en Chile. La complicación más frecuente de esta condición es la colecistitis aguda. Aproximadamente 60.000 colecistectomías son realizadas anualmente en nuestro país, convirtiéndola en una de las patologías digestivas más frecuentes. El tratamiento de la colelitiasis ha evolucionado las últimas dos décadas. Con el desarrollo de la laparoscopía a final de 1980, la colecistectomía abierta fue definitivamente sustituida en los 90 por la vía laparoscópica. Desafortunadamente, la generalización de este procedimiento produjo un aumento en la incidencia de lesiones de vía biliar, aumentando desde una 0,1-0,2% hasta un 0,5-0,7%. Si bien, con el progresivo entrenamiento de los cirujanos en la cirugía mínimamente invasiva esta cifra ha disminuido nuevamente, la LVB aún constituye una de las complicaciones más graves en cirugía, generando gran impacto en la calidad de vida del paciente, e incluso costos en salud que pueden llegar a ser devastadores. El manejo de una lesión de vía biliar es, para muchos cirujanos, un desafío quirúrgico, que para su resolución requiere de las habilidades y experiencia de un cirujano hepatobiliar y derivación a centros de salud especializados. Sin duda, la acción más importante es extremar de manera rutinaria las medidas de prevención. Sin embargo, una vez que la lesión se ha producido, el futuro del paciente depende, directamente, del buen criterio del cirujano, tanto para realizar un diagnóstico temprano, como del enfrentamiento terapéutico que realice. El objetivo de este trabajo es dar a conocer diferentes conductas preventivas y terapéuticas cuando esta complicación se presenta.

Calculous biliary disease is a common condition in Chile. Over 60.000 cholecystectomies are performed annually, making gallstone disease one of the most common digestive health problems. The treatment of calculous biliary disease has evolved over the last 2 decades. With the development of laparoscopic technology in the late 1980s, new techniques for cholecystectomy were introduced. By the early 1990s, laparoscopic cholecystectomy had replaced open cholecystectomy in the operative management of gallbladder stone disease. Unfortunately, the widespread application of laparoscopic cholecystectomy has led to a concurrent rise in the incidence of major bile duct injuries. Reports have estimated the incidence has risen from 0.1-0.2 to 0.5-0.7%. Although, exponential training of surgeons in minimally invasive surgery has led to decrease this numbers again, bile duct injuries still constitute one of the most serious complications in surgery, generating a great impact on the patient's quality of life, and even health costs that can be devastating. The management of patients following major bile duct injurie is a surgical challenge, often requiring the skills of experienced hepatobiliary surgeons at tertiary referral centers. In this setting, the most important action is to routinely maximize prevention measures. However, once injury has occurred, patient's future depends directly on the correct judgment of the surgeon, both to make an early diagnosis and to choose the best therapeutic confrontation. The objective of this article is to present the different preventive and therapeutic options available when this complication occurs.

Glaucoma de ángulo cerrado secundario a quistes del cuerpo ciliar tratado con ciclofotocoagulación transescleral subliminal. A propósito de un caso / Angle-closure glaucoma secondary to ciliary body cysts treated with subliminal transscleral cyclophotocoagulation. Report of a case

El glaucoma de ángulo cerrado secundario a quistes del cuerpo ciliar es una entidad infrecuente. Se han descrito diferentes modalidades de tratamiento tanto farmacológicas como quirúrgicas. Entre estas, la ciclofotocoagulación transescleral subliminal es una alternativa comprometedora por menores complicaciones oculares y la misma eficacia reductora de la presión intraocular. Presentamos el caso de una mujer de 33 años con antecedente de hipertensión ocular de 6 años de evolución sin control y glaucoma de un mes de evolución, tratada con múltiples fármacos e intervenciones quirúrgicas, con persistencia de la presión intraocular elevada. Acudió a consulta por presentar dolor ocular y fotofobia de ojo derecho de 3 días de evolución previos a su visita. Se le realizó una ultrabiomicroscopia, encontrando quistes en el cuerpo ciliar por lo que se optó por la realización de ciclofotocoagulación transescleral subliminal, consiguiendo una reducción de la presión intraocular mantenida en el seguimiento a los 3 meses (AU)

Angle-closure glaucoma secondary to ciliary body cysts is a rare condition. Different pharmacological and surgical treatment modalities have been described. Among these, subliminal transscleral cyclophotocoagulation is a promising alternative due to fewer ocular complications and the same intraocular pressure reducing efficacy. The case is presented of a 33-year-old woman with a 6-year history of uncontrolled ocular hypertension, and a one-month history of glaucoma, treated with multiple drugs and surgical interventions, with persistent elevated intraocular pressure. She came to the clinic due to eye pain and photophobia of the right eye of 3 days onset prior to her visit. An ultrasound biomicroscopy was performed, finding cysts in the ciliary body. It was then decided to perform subliminal transscleral cyclophotocoagulation, achieving a reduction in intraocular pressure, which was maintained after three months of follow-up (AU)

Angle-closure glaucoma secondary to ciliary body cysts treated with subliminal transscleral cyclophotocoagulation. Report of a case.

Angle-closure glaucoma secondary to ciliary body cysts is a rare condition. Different pharmacological and surgical treatment modalities have been described. Among these, subliminal transscleral cyclophotocoagulation is a promising alternative due to fewer ocular complications and the same intraocular pressure reducing efficacy. The case is presented of a 33-year-old woman with a 6-year history of uncontrolled ocular hypertension, and a one-month history of glaucoma, treated with multiple drugs and surgical interventions, with persistent elevated intraocular pressure. She came to the clinic due to eye pain and photophobia of the right eye of 3 days onset prior to her visit. An ultrasound biomicroscopy was performed, finding cysts in the ciliary body. It was then decided to perform subliminal transscleral cyclophotocoagulation, achieving a reduction in intraocular pressure, which was maintained after three months of follow-up.

Effect of undersized drilling on the stability of immediate tapered implants in the anterior maxillary sector. A randomized clinical trial.

BACKGROUND: To evaluate the effect of undersized drilling on the primary and secondary stability of immediate implants placed in the anterior maxilla. MATERIAL AND METHODS: A comparative randomized clinical trial was carried out in 30 healthy adults. Thirty tapered implants, 16 involving conventional drilling and 14 undersized drilling, were placed immediately after anterior maxillary tooth removal. Insertion torque and implant stability assessed by resonance frequency analysis (RFA) were evaluated at three different timepoints: at implant placement and 6 and 12 weeks post-implantation. The results were compared using parametric statistical tests. RESULTS: All implants showed adequate stability during follow-up. At implant placement, the undersized drilling group exhibited greater insertion torque values than the conventional drilling group, but stability assessed by RFA showed greater mean values in the conventional group. After 6 and 12 weeks of follow-up, both groups showed improved stability, though the RFA values remained comparatively higher in the conventional group. The differences were not statistically significant. CONCLUSIONS: Based on the results obtained, undersized drilling does not appear to afford significantly improved stability of immediate implants placed in the anterior zone of the maxilla during the osseointegration period.

Implementación de un modelo porcino de trasplante hepático en Chile / Implementation of a porcine liver transplant model in Chile

Resumen Objetivo: Presentar la implementación del primer modelo porcino de trasplante hepático (TH) en Chile y sus resultados. Material y Método: Se implementó un protocolo quirúrgico y anestésico en el contexto de una investigación en perfusión normotérmica hepática financiada por un Fondo Nacional de Desarrollo Científico y Tecnológico. Los cerdos fueron seleccionados por peso (35-40 kilos), en cada experimento se utilizó dos, donante y receptor, sometidos a procura y trasplante respectivamente. El análisis se realizó con estadística descriptiva. Resultados: Se realizaron 26 experimentos (marzo de 2018-octubre de 2019). El protocolo consta de 7 etapas: Preparación, Instrumentalización, Procura o Hepatectomía, Tiempo Anhepático, Etapa de Isquemia-Reperfusión, Monitorización y Eutanasia. Las primeras tres son similares en ambos cerdos, y desde la cuarta en adelante corresponde sólo al receptor. La supervivencia a la cirugía fue de 92,3% (24/26) y al seguimiento de 76,9% (20/26). La mortalidad se produjo por inestabilidad cardiovascular postreperfusión portal. El tiempo quirúrgico promedio fue de 170 min, y el tiempo anhepático de 33 min. La PAM cursó una baja postreperfusión con recuperación al final de la monitorización (67,4 mmHg media) y la tendencia en ph fue a acidosis al final del seguimiento (7,21 media). Se requirió drogas vasoactivas en 12 casos. Discusión: Logramos implementar un modelo experimental simplificado y reproducible de TH sin necesidad de circulación extracorpórea ni puente veno-venoso gracias a la estandarización de la técnica quirúrgica y de los cuidados perioperatorios. Conclusiones: La consolidación de un modelo experimental significa el primer paso en investigación preclínica de nuevas tecnologías asociadas al TH en Chile.

Objective: To Present the implementation of the first porcine model of liver transplant in Chile and its results. Materials and Methods: In the context of an investigation based on normothermic perfusion that was subsidized by the National Fund for Scientific and Technological Development, surgical and anesthetic protocols were implemented. Two pigs were selected by weight (35-40 kg), in which one was a donor and the other a receptor; submitted to procure and as a transplant recipient, respectively. The analysis was executed with descriptive statistics. Results: 26 experiments (June 2018-October 2019) were performed. The protocol includes 7 stages: Preparation, Instrumentalization, Procure or Hepatectomy, Anhepatic Time, Phase of Ischemia-Reperfusion, Monitoring and Euthanasia. The first three stages are similar in both pigs; meanwhile the last four are exclusive to the receptor. 92.3% (24/26) survived the surgery and 76.9% (20/26) survived the follow up, including the pilots. Average surgical time was 170 minutes, and the anhepatic time was 33 minutes. The mean arterial pressure decreased after the reperfusion and it improved towards the end of the monitorization (67,4 mmHg average) In the follow up, pigs presented acidosis (7.21 average). Vasoactive drugs were necessary in 12 experiments. Discussion: We managed to implement a simple and reproducible model of porcine liver transplant without extracorporeal circulation or veno-venous by-pass. This was possible due to the standardization of surgical technique and perioperative cares. Conclusions: The consolidation of porcine liver transplant model is the first step in pre-clinical research for the development of new technologies in liver transplant in Chile.

Factores asociados a reingreso hospitalario precoz posterior a resecciones hepáticas y pancreáticas / Hospital readmission rates after hepatic and pancreatic resections

Resumen Objetivo: Determinar la frecuencia de reingreso hospitalario y sus factores asociados en pacientes sometidos a resecciones hepáticas o pancreáticas en nuestro centro. Metodología: Se revisaron registros de pacientes sometidos a resecciones hepáticas o pancreáticas entre 2012 y 2014. Se registraron variables biodemográficas, quirúrgicas y reingresos hasta 30 días posalta. Se excluyó la cirugía de urgencia, pacientes fallecidos durante la hospitalización, pacientes sometidos a resecciones hepáticas menores a dos segmentos, cirugías no anatómicas o cirugía no resectiva. El análisis estadístico univariable se realizó con prueba χ2 para variables categóricas y T Student/Mann Whitney para variables continuas. El análisis multivariable se realizó con regresión logística. Resultados: Se incluyeron 116 pacientes, 50,9% mujeres. La estadía promedio fue de 14 días. El adenocarcinoma pancreático fue el diagnóstico más frecuente (25,9%) y 40,5% de los procedimientos quirúrgicos fueron pancreatoduodenectomías. La tasa de reingreso global fue 18,1%, mayor en pancreatectomías respecto a hepatectomías (23,7 vs 12,2% respectivamente p < 0,05). Los factores asociados a reingreso fueron: resección de páncreas, leucocitos preoperatorios, complicaciones posoperatorias y tiempo de estadía hospitalaria. Tras el análisis multivariable, sólo el tiempo hospitalario se asocia de forma independiente al reingreso precoz [OR 1,2 IC 95% 1,1-1,5 (p = 0,001)]. Conclusión: La estadía hospitalaria prolongada es un factor de riesgo consistente en la literatura para la rehospitalización posterior a resecciones hepáticas o pancreáticas. La tasa de reingreso posterior a resecciones hepáticas o pancreáticas es elevada, incluso en centros de alto volumen. Recomendamos el uso de este parámetro como un nuevo instrumento de medición de calidad en los resultados quirúrgicos en nuestro país.

Aim: To determine readmission rates and its associated factors in patients undergoing pancreatic and hepatic resections at our center. Matherial and Methods: Perioperative variables of patients undergoing pancreatic and hepatic resections between 2012-2014 were reviewed. Demographic and perioepartive data, as well as up —to postoperative day 30— readmisson rates were analyzed. Emergency cases, postoperative mortality and/or patients undergoing less extensive surgery (less than 2 Couinaud's segments, non-anatomical resections and non resective cases such as bilioenteric anastomoses) were excluded. Readmission associated factors were identified using both univariate (χ2 for categorical and t-student's/Mann-Whitney for continuous variables) and multivariate (logistic regression) analysis. Results: 116 cases were included, 50.9 % female. Mean postoperative stay was 14 days. Pancreatic adenocarcinoma was the most frequent diagnosis (25.9%), and the 40.5% of surgical procedures were pancreaticoduodenectomy. Overall 30-day readmission rate was 18.1%, with a 23.7% for pancreatic resections and 12.2% for hepatic resections. According to univariate analysis; readmission associated factors were: pancreatic resection, preoperative White cell count, the development of postoperative complications and postoperative length of stay. On Multivariate analysis only postoperative stay was the only significant associate factor [OR 1,2 CI 95% 1.1- 1.5 (p = 0.001)]. Conclussion: Readmission rates after pancreatic and hepatic resections are elevated, even in high-volume centers. Postoperative length of stay is a consistent risk factor for readmission after these type of surgeries. We highly recommend including this parameter as a quality marker of our surgical results in our country.

Adaptación cultural del cuestionario LINQ (Lung Information Needs Questionnaire) en pacientes con enfermedad respiratoria crónica en un programa de rehabilitación pulmonar en Cali, Colombia / Cultural Adaptation of the LINQ (Lung Information Needs Questionnaire) Questionnaire in Patients with Chronic Respiratory Disease in a Pulmonary Rehabilitation Program in Cali, Colombia / Adaptação cultural do questionário LINQ (Lung Information Needs Questionnaire) em pacientes com doença respiratória crônica em um programa de reabilitação pulmonar em Cali, Colômbia

Objetivo: Adaptar culturalmente el cuestionario LINQ (Lung Information Needs Questionnaire) en pacientes con enfermedad pulmonar crónica. Métodos: Estudio descriptivo, después de la traducción del cuestionario por traductor certificado y de la adaptación cultural, se aplicó en ocho pacientes para identificar las dificultades y dudas relacionadas con el entendimiento de las preguntas, la terminología y para evaluar no solo la calidad de la traducción, sino también para verificar aspectos prácticos de su aplicación. Posteriormente, se realizó una segunda traducción al idioma inglés la cual fue enviada a los autores para asegurar el contenido original del cuestionario y conseguir el aval para su uso. Resultados: La muestra en la fase de adaptación cultural fue constituida por 6 hombres y 2 mujeres con una edad promedio de 61 años. La versión en español del LINQ y la adaptación cultural fue comprensible y fácil de usar y no presentó discrepancias con la versión original durante el proceso de retratraducción. Conclusiones: La traducción al español y la adaptación del LINQ fue adecuada pues los pacientes no manifestaron dificultades para entender y responder las preguntas. Esto podrá facilitar la realización de futuros estudios que evalúen el componente educativo en los programas de rehabiltación pulmonar.

Objective: To perform a cultural adaptation of the LINQ (Lung Information Needs Questionnaire) for patients with chronic pulmonary disease. Method: Descriptive study. After the translation of the questionnaire by a certified translator, and after its cultural adaptation, it was applied in eight patients in order to identify the difficulties and doubts related to an understanding of the questions and of its terminology, not only to evaluate the quality of the translation, but to verify practical aspects of its application, as well. Afterward, a second translation was done from Spanish to English, and sent back to the authors to ensure the original content of the questionnaire was maintained, and to obtain endorsement for its use. Results: During the cultural adaptation phase, the sample was constituted by 6 men and 2 women, with an average age of 61 years. The Spanish version of the LINQ, as well as the cultural adaptation, was easy to use and did not show any discrepancies with the original version during the re-translation process. Conclusions: The LINQ's translation to Spanish and the cultural adaptation proved to be adequate, because the patients did not express any difficulties in understanding and answering the questions. This will facilitate future studies that evaluate the educational component in the pulmonary rehabilitation program.

Objetivo: adaptar culturalmente o questionário LINQ (Lung Information Needs Questionnaire) em pacientes com doença pulmonar crônica. Métodos: Estudo descritivo, depois da tradução do questionário por tradutor certificado e da adaptação cultural, aplicou-se a oito pacientes para identificar as dificuldades e dúvidas relacionadas com o entendimento das perguntas, a terminologia e para avaliar não só a qualidade da tradução, más também para verificar aspectos práticos de sua aplicação. Posteriormente, realizou-se uma segunda tradução ao idioma inglês a qual foi enviada aos autores para assegurar o conteúdo original do questionário e conseguir o aval para seu uso. Resultados: a amostra na fase de adaptação cultural foi constituída por 6 homens e 2 mulheres com uma idade média de 61 anos. A versão em espanhol de LINQ e a adaptação cultural foram compreensíveis e fáceis de usar e não apresentaram discrepâncias com a versão original durante o processo de retradução. Conclusões: a tradução ao espanhol e a adaptação do LINQ foi adequada pois os pacientes não manifestaram dificuldades para entender e responder as perguntas. Isto poderá facilitar a realização de futuros estudos que avaliem o componente educativo nos programas de reabilitação pulmonar.

Resultados a corto y largo plazo con la técnica de Karydakis para la enfermedad pilonidal sacrocoxígea / Short-and long-term results with Karydaki’s technique for sacrococcygeal pilonidal disease

Introduction: Sacrococcygeal pilonidal cyst or sinus is a common condition in young people. There is still discussion about which of the many existing techniques is best suited to resolve this condition. Objective: To analyze the short-and long-term use of Karydaki's technique in our Hospital. Material and Methods: We analyzed prospective protocols from patients with pilonidal cyst with Karydaki's technique from June 2005 to August 2010. Clinical and telephonic follow-up was done to all patients and a satisfaction survey done to a random sample of the series. Results: We analyzed 62 patients with mean age of 24.5 years (15-45), being 33 men (53 percent). Cysts were previously relapsed in 20.9 percent of cases. The average hospital stay was 2.5 days (1-3). Early complications occurred in 14.3 percent of patients being the most common seroma in 8 percent and dehiscence in 6.4 percent. At a mean follow up of 38 months (12-62), relapse occurred in two patients (3.2 percent), one at 6 months and the other at 3 years, being resolved one by a new Karydaki's technique and the other with marsupialization. Among the group surveyed for satisfaction, 75 percent found the technique satisfactory or very satisfactory in terms of aesthetics and 95 percent would recommend this technique. Conclusions: Karydaki's operation achieves with a simple and aesthetic technic a low recurrence and morbidity and a complete recovery after 15 days in most patients.

Introducción: El quiste o seno pilonidal sacrocoxígeo es una patología frecuente en gente joven. Aún hay discusión sobre cual de las múltiples técnicas existentes es la más adecuada para resolver esta patología. Objetivo: Analizar los resultados a corto y largo plazo con la utilización de la técnica de Karydakis en nuestro medio. Material y Método: Se analizan los protocolos prospectivos de pacientes intervenidos por quiste pilonidal con la técnica de Karydakis desde junio de 2005 hasta agosto de 2010. Se realizó seguimiento clínico y telefónico de los pacientes y una encuesta de satisfacción a una muestra aleatoria de la serie. Resultados: Se analizan 62 pacientes con promedio de edad de 24,5 años (15-45), siendo de ellos 33 hombres (53 por ciento). En un 20,9 por ciento de los casos eran quistes recidivados. La hospitalización promedio fue de 2,5 días (1-3). Un 14,3 por ciento presentó complicaciones precoces siendo las más frecuentes el seroma (8 por ciento) y dehiscencia de sutura (6,4 por ciento). El seguimiento promedio fue de 38 meses (i: 12-62). Dos pacientes tuvieron recidivas (3,2 por ciento), a los 6 meses y a los 3 años, resolviéndose una con nueva técnica de Karydakis y la otra con marsupialización. Del grupo encuestado, el 75 por ciento consideró que la técnica fue satisfactoria o muy satisfactoria (estética) y el 95 por ciento de los pacientes la recomendaría. Conclusiones: La operación de Karydakis logra con una técnica simple y estética una baja recidiva y morbilidad y una recuperación definitiva a los 15 días en la mayoría de los pacientes.

Extracellular ATP induces fast and transient non-selective cationic currents and cytosolic Ca2+ changes in human umbilical artery smooth muscle cells.

Ionotropic purinergic receptors (P2X) are expressed in endothelial and smooth muscle cells of blood vessels. ATP acting on smooth muscle P2X receptors is able to induce vasoconstriction in different kind of vessels. However, to our knowledge, there are no reports that directly show the activity of these purinergic receptors in native human vascular smooth muscle cells. In this work, we describe for the first time an ATP-induced current in freshly isolated human umbilical artery (HUA) smooth muscle cells. The current was measured by patch-clamp technique in whole-cell condition on cells clamped at -50 mV. At 100 µM of ATP the current showed a rapid activation and desensitization, and was carried by both Na(+) and Ca(2+). The current was completely blocked by suramin (300 µM) and partially blocked by 100 µM of Zn(2+) without affecting the kinetic of desensitization. All these properties suggest that the ATP-induced ionic currents are mediated through P2X(1)-like receptors. Moreover, we show that ATP transiently increased cytosolic Ca(2+) in "in situ" smooth muscle cells of intact HUA segments and that this response is dependent of extracellular and intracellular Ca(2+). These data expand the knowledge of purinergic receptors properties in vascular smooth muscle cells and the probable role of ATP as a paracrine modulator of contractile tone in a human artery which is fundamental for feto-placental blood flow.

Response of thawed epididymal red deer spermatozoa to increasing concentrations of hydrogen peroxide, and importance of individual male variability.

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H(2)O(2)) levels on red deer spermatozoa after cryopreservation, and the role of male-to-male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 µm H(2)O(2) for 2 h at 37 °C. Intracellular reactive oxygen species (H(2)DCFDA-CM) increased with H(2)O(2) concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 µm. Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 µm H(2)O(2) and above. In a second experiment, samples from seven males were submitted to 0 and 200 µm H(2)O(2) for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H(2)O(2) presence. We found that the kinematic parameters reflected male-to-male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H(2)O(2) or after incubation with H(2)O(2) . Red deer spermatozoa are relatively resilient to H(2)O(2) after thawing, but it seems to be a great male-to-male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa.

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.

Comparison of the TBARS assay and BODIPY C11 probes for assessing lipid peroxidation in red deer spermatozoa.

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C(11) (B581) and BODIPY 665/676 C(11) (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H(2)O(2) ) 0.1 mM or 1 mM, or tert-butyl hydroperoxide (TBH) 0.1 mM or 1 mM. LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mM of TBH or H(2)O(2). Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.

Genistein effects on Ca2+ handling in human umbilical artery: inhibition of sarcoplasmic reticulum Ca2+ release and of voltage-operated Ca2+ channels.

Isoflavones are a group of natural phytoestrogens including the compound genistein. Health beneficial effects have been attributed to the consumption of this compound, but the fact that it has estrogen-like activity has raised doubts regarding its potential risk in infants, newborns, or in the fetus and placenta during pregnancy. This work is aimed at studying genistein effects on Ca2+ handling by smooth muscle cells of the human umbilical artery (HUA). Using fluorometric techniques, we found that in these cells genistein reduces the intracellular Ca2+ peak produced by serotonin. The same result could be demonstrated in absence of extracellular Ca2+, suggesting that the isoflavone reduces Ca2+ release from the sarcoplasmic reticulum. Force measurement experiments strengthen these results, since genistein reduced the peak force attained by intact HUA rings stimulated by serotonin in a Ca2+-free solution. Moreover, genistein induced the relaxation of HUA rings precontracted either with serotonin or a depolarizing high-extracellular K+ solution, hinting at a reduction of extracellular Ca2+ entry to the cell. This was confirmed by whole-cell patch-clamp experiments where it was shown that the isoflavone inhibits ionic currents through voltage-operated Ca2+ channels. In summary, we show that genistein inhibits two mechanisms that could increase intracellular Ca2+ in human umbilical smooth muscle cells, behaving in this way as a potential vasorelaxing substance of fetal vessels. Taking into account that genistein is able to cross the placental barrier, these data show that isoflavones may have important implications in the regulation of feto-maternal blood flow in pregnant women who consume soy-derived products as part of their meals.

Arginine-rich peptides destabilize the plasma membrane, consistent with a pore formation translocation mechanism of cell-penetrating peptides.

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.

Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa.

The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 x g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1mM of the antioxidant Trolox, 100 microM of the oxidant Fe (with ascorbate), or both. The 3x4 treatments were incubated at 37 degrees C and assessed each hour up to 3h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4h was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30+/-3.52% to 67.94+/-5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.

Efectos de la genisteína en los niveles del Ca2+ citosólico en células musculares de arteria umbilical humana / Genistein effects on Ca2+ handling in human umbilical artery: inhibition of sarcoplasmic reticulum Ca2+ release and of voltage-operated Ca2+ channels

Isoflavones are a group of natural phytoestrogens including the compound genistein.Health beneficial effects have been attributed to the consumption of this compound,but the fact that it has estrogen-like activity has raised doubts regarding itspotential risk in infants, newborns, or in the fetus and placenta during pregnancy.This work is aimed at studying genistein effects on Ca2+ handling by smooth musclecells of the human umbilical artery (HUA). Using fluorometric techniques, we foundthat in these cells genistein reduces the intracellular Ca2+ peak produced by serotonin.The same result could be demonstrated in absence of extracellular Ca2+, suggestingthat the isoflavone reduces Ca2+ release from the sarcoplasmic reticulum.Force measurement experiments strengthen these results, since genistein reduced thepeak force attained by intact HUA rings stimulated by serotonin in a Ca2+-free solution.Moreover, genistein induced the relaxation of HUA rings precontracted eitherwith serotonin or a depolarizing high-extracellular K+ solution, hinting at a reductionof extracellular Ca2+ entry to the cell. This was confirmed by whole-cell patchclampexperiments where it was shown that the isoflavone inhibits ionic currentsthrough voltage-operated Ca2+ channels. In summary, we show that genisteininhibits two mechanisms that could increase intracellular Ca2+ in human umbilical smooth muscle cells, behaving in this way as a potential vasorelaxing substance offetal vessels. Taking into account that genistein is able to cross the placental barrier,these data show that isoflavones may have important implications in the regulationof feto-maternal blood flow in pregnant women who consume soy-derived productsas part of their meals(AU)

Las isoflavonas son un grupo de fitoestrógenosnaturales que incluyen la genisteína. Alconsumo de este compuesto se le han atribuidoefectos beneficiosos para la salud, pero su actividadsimilar a los estrógenos permite pensaren efectos indeseados en niños o en el feto o laplacenta durante el embarazo. En este trabajose estudian los efectos de la genisteína sobre elmanejo de Ca2+ por las células de músculo lisode la arteria umbilical humana (AUH).Mediante la utilización de técnicas fluorométricasse observó que la genisteína reduce elpico de Ca2+ intracelular producido por la serotonina en estas células incluso en ausenciade Ca2+ extracelular, lo que sugiere que la isoflavonareduce la liberación de Ca2+ a partir delretículo sarcoplásmico. Los experimentos demedida de fuerza refuerzan estos resultados, yaque la genisteína redujo la fuerza pico desarrolladapor serotonina en anillos intactos deAUH en una solución libre de Ca2+. Además,la genisteína indujo la relajación de anillos deAUH precontraídos con serotonina o con unasolución despolarizante de alto K+ extracelular,lo que apunta a una reducción de la entradade Ca2+ desde el exterior de la célula. Con latécnica de “patch-clamp” en configuración decélula entera, los resultados confirmaron que laisoflavona inhibe corrientes iónicas a través decanales de Ca2+ operados por el voltaje. Enresumen, mostramos que la genisteína inhibedos mecanismos que incrementan el Ca2+intracelular en células de músculo liso deAUH, comportándose de esta manera comoun potencial vasorrelajante de los vasos fetales.Dado que la genisteína atraviesa la barrera placentaria,estos datos muestran que las isoflavonaspodrían tener consecuencias en la regulacióndel flujo materno-fetal en mujeres embarazadasque incluyan productos derivados de lasoja como parte de sus dietas(AU)

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa.

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity.

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 degrees C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 microm Fe(2+)/1 mm ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 +/- 2.9%) and OXI (11.6 +/- 7.6%) (p < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI (p < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 +/- 0.8% OXI vs. 17.4 +/- 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.

DNA status on thawed semen from fighting bull: a comparison between the SCD and the SCSA tests.

The assessment of sperm chromatin status is compulsory in a complete spermiogram. Here we applied the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test to assess the chromatin status of three fighting bulls. Cryopreserved semen (two straws/bull) were analysed by duplicate after thawing and after 6 h at 37 degrees C with and without oxidative stress (1 mm FE(2+)). Results (SCD: percentage of spermatozoa with halo; SCSA: SD-DFI, %DFI and HDS) were analysed for differences between bulls and treatments, sensitivity and specificity (receiver operating characteristic curves) and repeatability (repeatability coefficients as 2SD of duplicate differences).%DFI for the three bulls was below 2% at 0 h, indicating no risk for fertility according to previous reports. It increased slightly for two of the bulls after FE(2+) treatment (%DFI < 5%) and more pronouncedly for the other bull (C, %DFI approximately 10%), which merits further investigation. SCD rendered higher percentage of halos for bull C, but could not discriminate between samples with and without oxidizing treatment (AUC: 0.52). SCSA (%DFI) showed a high discriminating ability between treatments (AUC: 0.96). The repeatability coefficient was also higher for SCD (5.9) than for %DFI (1.8), indicating lower repeatability for SCD. Overall, %DFI might be the most useful parameter for assessing sperm chromatin on fighting bull. SCD might yield different information than SCSA, hence further research is warranted.

Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation.

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 x 10(6) sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 mm vitamin C. The remaining epididymides and the diluted samples were stored at 5 degrees C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.