ß-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells.

The beta-nerve growth factor (ß-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama ß-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with ß-NGF, ß-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of ß-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3ß-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. ß-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that ß-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that ß-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

Distribution of GnRH and Kisspeptin Immunoreactivity in the Female Llama Hypothalamus.

Llamas are induced non-reflex ovulators, which ovulate in response to the hormonal stimulus of the male protein beta-nerve growth factor (ß-NGF) that is present in the seminal plasma; this response is dependent on the preovulatory gonadotrophin-releasing hormone (GnRH) release from the hypothalamus. GnRH neurones are vital for reproduction, as these provide the input that controls the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. However, in spontaneous ovulators, the activity of GnRH cells is regulated by kisspeptin neurones that relay the oestrogen signal arising from the periphery. Here, we investigated the organisation of GnRH and kisspeptin systems in the hypothalamus of receptive adult female llamas. We found that GnRH cells exhibiting different shapes were distributed throughout the ventral forebrain and some of these were located in proximity to blood vessels; sections of the mediobasal hypothalamus (MBH) displayed the highest number of cells. GnRH fibres were observed in both the organum vasculosum laminae terminalis (OVLT) and median eminence (ME). We also detected abundant kisspeptin fibres in the MBH and ME; kisspeptin cells were found in the arcuate nucleus (ARC), but not in rostral areas of the hypothalamus. Quantitative analysis of GnRH and kisspeptin fibres in the ME revealed a higher innervation density of kisspeptin than of GnRH fibres. The physiological significance of the anatomical findings reported here for the ovulatory mechanism in llamas is still to be determined.

Injury to the axillary and suprascapular nerves in rotator cuff arthropathy and after reverse shoulder arthroplasty: a prospective electromyographic analysis.

BACKGROUND: Neurologic pre- and postoperative injuries to the axillary and/or suprascapular nerve (SSN) have a higher incidence than expected and may lead to significantly decreased functional outcomes and increased risk of reverse shoulder arthroplasty (RSA) failure. METHODS: Patients who underwent a RSA for rotator cuff tear arthropathy (RCTA) were included from December 2014 to December 2015. This study focused on the clinical (Constant score), radiographic, and pre- and postoperative electromyographic evaluations at 3 and 6 months. RESULTS: Twenty patients met the inclusion criteria. One was lost to follow-up. Preoperatively, 15 patients showed changes on electromyography (9 SSN and 15 axillary nerve lesions); all of them were chronic and disuse injuries. The mean preoperative relative Constant score (rCS) of all included patients was 39 ± 9 (range, 19-64). At 3 months postsurgery, the prevalence of acute injuries for both nerves was 31.5%. At 6 months postsurgery, 2 axillary nerve injuries and 6 SSN injuries remain unchanged, and the rest improved or normalized. The mean postsurgery rCS of the entire cohort at 6-month follow-up was 78 ± 6.5. Mean postoperative rCS for acute postoperative nerve injury was 71 ± 3 for the axillary nerve and 64 ± 5 for SSN. CONCLUSIONS: Axillary and SSN injuries in RCTA have a much higher incidence than expected. Most of these axillary lesions are transient, with an almost complete recovery seen on electromyography at 6 months and with scarce functional impact. However, SSN lesions appear to behave differently, with poor functional results and having a lower potential for a complete recovery.

Midterm outcomes of bone grafting in glenoid defects treated with reverse shoulder arthroplasty.

BACKGROUND: Large glenoid defects are a difficult reconstructive problem for shoulder surgeons. The purpose of this study was to determine the complications, rate of healing, and functional results of glenoid bone grafting in primary or revision surgery with reverse shoulder arthroplasty. METHODS: We retrospectively reviewed 23 patients with glenoid bone loss who underwent primary or revision surgery using a glenoid bone graft with a minimum follow-up of 2 years. Range of motion and the Constant, American Shoulder and Elbow Surgeons, and visual analog scale scores were obtained from preoperative assessment and the latest follow-up visit. Radiographic evaluation included analysis of plain radiographs as well as preoperative and follow-up computed tomography. RESULTS: Three patients were excluded from the study. Allografts were used in 13 cases and autografts in 7 cases. The mean Constant score improved from 30.7 ± 9.4 to 51.3 ± 13.4 (P < .001). At a mean follow-up of 26 months, computed tomography imaging revealed that the glenoid bone graft was fully incorporated in 95% of cases. No statistically significant differences were found on analysis of the clinical and radiographic outcomes related to the graft source. There was a 20% postoperative complication rate: 1 case of aseptic glenoid component loosening, 1 surgical wound hematoma, 1 acromial fracture, and a symptomatic grade 3 scapular notching. CONCLUSIONS: The use of bone grafts in glenoid defects is a useful technique by which, in the majority of cases, single-stage reconstruction surgery may be performed, even in the presence of severe bone loss. Incorporation rates are high, with satisfactory clinical outcome.

Hematoma retrofaríngeo: a propósito de un caso / A case of retropharyngeal hematoma

El hematoma retrofaríngeo de causa traumática es una entidad clínica muy poco frecuente. Por la proximidad con la vía aérea superior y por tratarse de una lesión ocupante de espacio a nivel de los planos profundos del cuello, la misma puede producir la rápida instalación de una insuficiencia respiratoria aguda de tipo obstructivo. Presentamos el caso de un paciente de sexo masculino, de 72 años, que sufre un siniestro de tránsito, moto contra moto, como conductor sin casco. Es asistido en la vía pública por unidad de emergencia móvil prehospitalaria (UEMPH), que constata: traumatismo encéfalo-craneano (TEC) sin pérdida de conocimiento. Se otorga el alta a domicilio desde el lugar del accidente. Reconsulta en domicilio por cuadro de dificultad respiratoria de rápida instalación y edema de cuello. Valorado nuevamente por UEMPH en domicilio, se constata: polipnea, estridor, disfonía. Se realiza traslado en ambulancia a emergencia del Hospital de Clínicas donde se realiza intubación orotraqueal (IOT), asistencia respiratoria mecánica (ARM) y con diagnóstico tomográfico de hematoma retrofaríngeo ingresa a la unidad de cuidados intensivos (UCI). En la evolución se realizó traqueostomía quirúrgica (TQTq) y tratamiento conservador con buena evolución. El hematoma retrofaríngeo traumático es una entidad poco frecuente de gravedad potencial o real que debe conocerse para no demorar su diagnóstico. Una vez que la vía aérea se encuentra asegurada, se presentan dos opciones: drenaje del hematoma u observación clínica. La opción quirúrgica no ha mostrado ventajas respecto al tratamiento conservador.

Retropharyngeal edema caused by trauma is a rather unusual clinical entity. Given its close proximity to the upper airway and being a lesion that occupies space in the deep planes of the neck it may result in a fast installation of acute respiratory distress.The study presents the case of a male patient of 72 years old who suffered a car accident, where two motorbikes took part, and he was not wearing a helmet. He was seen in the site by the mobile emergency services who found: traumatic brain injury (TBI) with no unconsciousness. He was released to go home. The patient consulted again from his domicile for difficulties in breathing of rapid installation and neck swelling. He was assessed by emergency services in his domicile which found: polypnea, stridor and dysphonia. He was sent to the Emergency Unit at the Clinicas University Hospital in an ambulance where orotracheal intubation was performed, he received mechanical ventilation. The scan diagnosis was retropharyngeal edema and he was admitted in the ICU. Upon evolution a surgical tracheostomy was performed and a conservative treatment resulted in a favorable evolution. Traumatic retropharyngeal edema is rather an unusual condition with a potential or real severity that needs to be known to avoid delaying diagnosis. Once the airway is ensured two options are possible: draining the area or clinical observation. The surgical option has evidenced no advantages when compared to a conservative treatment.

O hematoma retrofaríngeo de origem traumática é uma entidade clínica muito pouco frequente. Por sua proximidade com a via aérea superior e por ser uma lesão que ocupa espaço na área dos planos profundos do pescoço, pode produzir rapidamente uma insuficiência respiratória aguda de tipo obstrutivo.Apresentamos o caso de um paciente de sexo masculino, de 72 anos, que teve um acidente de trânsito, motocicleta contra motocicleta, no qual era condutor e estava sem capacete. Foi atendido na via pública por uma unidade de emergência móvel pré-hospitalar (UEMPH), que constatou: traumatismo cranioencefálico (TCE) sem perda de consciência. No lugar do acidente recebeu alta. Consultou novamente no domicilio por quadro de dificuldade respiratória de instalação rápida e edema de pescoço. Avaliado novamente por uma UEMPH no domicilio, foi constatada: polipneia, estridor, disfonia. Foi transferido em uma ambulância ao pronto-socorro do Hospital das Clínicas aonde foi submetido a intubação orotraqueal (IOT), assistência respiratória mecânica (ARM); com diagnóstico tomográfico de hematoma retrofaríngeo foi internado na unidade de cuidados intensivos (UCI). Foram realizados uma traqueostomia cirúrgica (TQTc) e tratamento conservador com boa evolução. O hematoma retrofaríngeo traumático é uma entidade pouco frequente, com gravidade potencial ou real e que deve ser conhecido para evitar demora no seu diagnóstico. Quando a via aérea estiver assegurada, existem duas opções: drenagem do hematoma ou observaçao clínica. A alternativa cirúrgica não tem apresentado vantagens com relação ao tratamento conservador.

Activación química de ovocitos de alpaca vitrificados después de la maduración in vitro / Chemical activation of alpaca oocytes vitrified after in vitro maturation

Se evaluó la viabilidad posdescongelamiento de ovocitos de alpaca vitrificados luego de la maduración in vitro. Los ovocitos fueron recuperados de ovarios obtenidos en el camal Municipal de Huancavelica, Perú, madurados in vitro por 24-25 h en una cámara modular con 5% de O2, 5% de CO2 y 90% de N2 en medio TCM-199 suplementado con Piruvato de Na, HEPES, sulfato de gentamicina, FSH, estradiol 17-beta y suero fetal bovino. Los ovocitos fueron separados de las células de la granulosa con hialuronidasa al 0.1% y distribuidos en tres tratamientos: T1 (n=107), ovocitos expuestos a las soluciones crioprotectoras y vitrificados; T2 (n=121), ovocitos expuestos a las soluciones crioprotectoras no vitrificados (control de toxicidad de los crioprotectantes); T3 (n=232), grupo control de ovocitos no expuestos a las soluciones vitrificantes. Los ovocitos fueron vitrificados en microgotas sobre un papel de aluminio flotando en nitrógeno líquido, utilizando una solución de equilibrio con 4% de etilenglicol y una solución de vitrificación con 35% de etilenglicol, 5% de polivinilpirrolidona y 0.4 M de trehalosa. Las microgotas vitrificadas se almacenaron en nitrógeno líquido y fueron descongeladas 1-4 después. Todos los ovocitos fueron cultivados en SOF-HEPES con 5 uM de ionomicina de Ca por 4 min a temperatura ambiente y luego cultivados por 3 h en 6-DMAP a 38.5 °C en una atmósfera húmeda con 5% de O2, 5% de CO2 y 90% de N2. Luego se cultivaron por 8 d en medio SOF-IVC. Se encontró 58.4, 68.7 y 97.3% de ovocitos morfológicamente normales para T1, T2 y T3, respectivamente. Las tasas de segmentación fueron de 39.9, 49.5 y 62.3%, y las tasas de blastocistos fueron de 0, 0 y 9.2% para T1, T2 y T3, respectivamente. Los resultados demuestran que los ovocitos de alpaca pueden ser vitrificados con el método de superficie sólida y que son viables morfológicamente y fisiológicamente posdescongelamiento.

The aim of this study was to evaluate the viability of vitrified/thawed alpaca oocytes after in vitro maturation. Alpaca oocytes were retrieved from ovaries obtained in theslaughterhouse of Huancavelica, Peru and matured in vitro for 24-25 h in a modular chamber with 5% O2, 5% CO2 and 90% N2 in TCM-199 medium supplemented with sodium pyruvate, HEPES, gentamycin sulphate, FSH, estradiol 17-beta and fetal calf serum. Then, oocytes were fully denuded of cumulus cells with 0.1% hyaluronidase and assigned to three treatments: T1 (n=107), oocytes exposed to cryoprotectans and vitrified; T2 (n=121), oocytes exposed to cryoprotectans without vitrification; and T3 (n=232), control group of oocytes not exposed to vitrification solutions. Alpaca oocytes were vitrified in microdrops on a precooled aluminum foil floating in liquid nitrogen, using an equilibrium solution with 4% ethylene glycol and a vitrification solution with 35% ethylene glycol, 5% polyvinyl-pyrrolidone and 0.4 M trehalose. The vitrified microdrops were stored in liquid nitrogen and were thawed 1-4 d later. All oocytes were cultured on SOF-HEPES with 5 umM Ca ionomycin by 4 min at room temperature followed by 3 h incubation in 6-DMAP at 38.5 ºC in a 5% O2, 5% CO2 and 90% N2 in humidified atmosphere. Subsequently, were cultivated on mSOF medium during 8 days. The rates of oocytes survival were 58.4, 68.7 and 97.3% in T1, T2 and T2 respectively. The rates of cleavage were 39.9, 49.5 and 62.3% and rates of development to blastocysts were 0, 0 and 9.2% in T1, T2 and T3 respectively. The results showed that alpaca oocytes were morphologically and physiologically viable after vitrification by solid surface method.

Efecto del sitio de deposición del plasma seminal sobre la tasa de ovulación y formación del cuerpo lúteo en alpacas / Effect of the deposition site of seminal plasma on the ovulation and corpus luteum development in alpacas

El estudio tuvo por objetivo evaluar el sitio de deposición del plasma seminal sobre la tasa de ovulación y formación del cuerpo lúteo en alpacas. Se seleccionaron 91 hembras no lactantes con folículos .7 mm detectados por ecografía transrectal. Se sincronizola onda folicular con la aplicación de 5 mg de LH, y 12 días después, se determinó la presencia de un folículo dominante (.7 mm). Las hembras se distribuyeron al azar en seis tratamientos: G1 =16 (Plasma seminal vía intramuscular), G2=15 (PBS vía intramuscular), G3=16 (Plasma seminal vía intrauterina), G4=15 (PBS vía intrauterina), G5 =15 (Plasma seminal vía intrauterina con curetaje), G6 =14 (PBS vía intrauterina con curetaje). La tasa de ovulación y presencia de cuerpo lúteo se determinó mediante ecografía el día D2 y D8 (D0 = inicio del tratamiento). Se tomó muestras de sangre los días D0, D3 y D9 para determinar perfiles séricos de progesterona mediante RIA. La tasa de ovulación fue de 93.8, 37.5 y 66.5% para G1, G3 y G5, respectivamente, y no se registraron ovulaciones en los otros grupos. Se encontró que las alpacas G1 producirían 25 veces más ovulaciones con respecto al G3. Los resultados sugieren que la absorción del factor inductor de ovulación (FIO) del plasma seminal es vía sistemica y que el curetaje facilitaría la absorción del FIO incrementando el efecto ovulatorio del plasma seminal.

The present study was carried out to evaluate the effect of deposition site of seminal plasma on the ovulation rate and size of corpus luteum in alpacas. Non-lactating females bearing a .7 mm follicle detected by ultrasound were selected (n = 91). Follicular wave was synchronized by injecting 5 mg of LH and then, the presence of a dominant follicle .7 mm was determined 12 days later through ultrasound evaluation. Alpacas were assigned to one of six experimental groups: G1 (n=16) seminal plasma (SP) by intramuscular injection . i.m.; G2 (n=15) phosphate-buffered saline (PBS) i.m.; G3 (n=16) SP by intrauterine administration . i.u.; G4 (n=15) PBS i.u.; G5 (n=15) SP i.u. with curettage; G6 (14) PBS by i.u. with curettage. Ovulation rate and corpus luteum were determined by ultrasound evaluation on D2 and D8 (D0 = day of treatment). Serum samples were taken from the jugular vein on D0, D3 and D9 to determine progesterone profiles by radioimmunoassay. Ovulation rate was 93.7, 37.5, and 66.5% for G1, G3 and G5 respectively, while none ovulations were observed in the other three groups. Alpacas in G1 would produce 25 more ovulations than G3. Results of the present study suggested that the ovulation-inducing factor (OIF) present in seminal plasma is absorbed systemically and that the mechanical action of curettage would contribute to the absorption of the OIF present in seminal plasma.

Efecto de fracciones del plasma seminal, según su peso molecular, sobre la inducción de la ovulación en llamas (Lama glama) / Effect of plasma seminal fractions, based on molecular weight, on the induction of ovulation in llamas (Lama glama)

El presente estudio fue realizado con el propósito de evaluar el efecto de varias fracciones de plasma seminal de llamas, según su peso molecular (PM), sobre la inducción de la ovulación. Se utilizó 54 llamas hembras sin cría y en óptimo estado reproductivo, con folículo dominante ≥7 mm de diámetro determinado por ecografía transrectal por tres días consecutivos. Los animales se distribuyeron al azar a uno de los seis tratamientos: A) fracción de plasma seminal con PM ≥30 kDa, B) fracción de plasma seminal con PM entre 10 y 30 kDa, C) fracción de plasma seminal con PM entre 5 y 10 kDa, D) fracción de plasma seminal con PM <5 kDa, E) plasma seminal completo, y F) PBS. Los animales fueron tratados con una solución de 1.5 ml de la fracción de plasma seminal o placebo correspondiente por vía intramuscular. Las llamas fueron evaluadas por ecografía en el día 2 y 9 post-tratamiento para determinar la tasa de ovulación y el tamaño del cuerpo lúteo. Los resultados indicaron una tasa de ovulación del 100 por ciento en los grupos A y E, 11.1 por ciento en B y 0 por ciento en los demás tratamientos. No se encontró diferencias estadísticas entre grupos respecto al diámetro luteal. Los resultados obtenidos permiten señalar que la fracción de plasma seminal con peso molecular mayor a 30 kDa induce la ovulación en llamas.

The present study was conducted to evaluate the effect of different fractions of llama seminal plasma, based on the molecular weight (MW), on the ovulation. Fifty four female adult llamas without calf at foot and in good reproductive conditions were used. The animals were bearing a ≥7 mm dominant follicle determined by ultrasound diagnosis during a three consecutive days. Females were randomly distributed in six groups: A, fraction of seminal plasma with MW ≥30 kDa; B, fraction of seminal plasma with MW ≥10 and <30 kDa; C, fraction of seminal plasma with MW ≥5 and <10 kDa ; D, fraction of seminal plasma with MW <5 kDa; E, complete plasma seminal; and F, PBS. Animals were treated with a 1.5 ml solution of the corresponding fraction or placebo via i.m. Ovarian ultrasound was performed at day 2 and 9 post-treatment to determine the ovulation rate and the size of the corpora lutea. Ovulation rate was 100 por ciento in groups A and E, 11.1 por ciento in group B and 0 por ciento in the other groups. No statistical differences were observed between groups on the size of the corpus luteum. The results indicated that the plasma seminal fraction with molecular weight ≥30 kDa induce ovulation in llamas.