Dinámica mitocondrial en las enfermedades neurodegenerativas / Mitochondrial dynamics in neurodegenerative diseases

Resumen Las enfermedades neurodegenerativas son un grupo heterogéneo caracterizado por la disminución gradual, progresiva y selectiva de las funciones del sistema nervioso. La etiología de estas patologías aún se desconoce, sin embargo, se ha propuesto que la función mitocondrial pudiese estar participando en el establecimiento de estas enfermedades, debido al alto requerimiento energético que tienen las neuronas para realizar sus funciones fisiológicas. La mitocondria es un organelo dinámico que puede cambiar su morfología y función en respuesta a diferentes estímulos fisiológicos, por ello se ha empezado a estudiar a la dinámica mitocondrial como uno de los principales reguladores de la supervivencia celular. Este evento comprende diferentes procesos como la generación de nuevas mitocondrias y su eliminación cuando ya no son funcionales, así como los procesos de fusión y fisión mitocondrial y el tráfico de estos organelos en el entorno celular. Todos estos procesos son altamente regulados y tienen como finalidad la óptima funcionalidad de la mitocondria y la homeostasis celular.

Abstract Neurodegenerative diseases are a group of heterogeneous diseases characterized by a gradual, progressive and selective decrease in nervous system functions. The etiology of these pathologies remains unknown; however, mitochondrial function has been proposed as a common factor that could be involved in the establishment of these diseases, owing to the high energy requirement neurons have in order to carry out their physiological functions. Mitochondria are extremely dynamic organelles that can change their morphology and function in response to different physiological stimuli and, for this reason, mitochondrial dynamics have started being studied as one of cell survival main regulators. This event comprises different processes, such as the generation of new mitochondria and their elimination when they are no longer functional, as well as mitochondrial fusion and fission processes and the traffic of these organelles within the cellular environment. All these processes are highly regulated, and their main purpose is optimal functionality of mitochondria and cellular homeostasis.

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

The influence of Aloe vera with mesenchymal stem cells from dental pulp on bone regeneration: characterization and treatment of non-critical defects of the tibia in rats

Abstract Objective This study aimed to evaluate the inflammatory effect and bone formation in sterile surgical failures after implantation of a collagen sponge with mesenchymal stem cells from human dental pulp (hDPSCs) and Aloe vera. Material and Methods Rattus norvegicus (n=75) were divided into five experimental groups according to treatment: G1) control (blood clot); G2) Hemospon®; G3) Hemospon® in a culture medium enriched with 8% Aloe vera; G4) Hemospon® in a culture medium containing hDPSCs and G5) Hemospon® in a culture medium enriched with 8% Aloe vera and hDPSCs. On days 7, 15 and 30, the animals were euthanized, and the tibia was dissected for histological, immunohistochemistry and immunofluorescence analyses. The results were analyzed using nonparametric Kruskal-Wallis test and Dunn's post-test. Results On days 7 and 15, the groups with Aloe vera had less average acute inflammatory infiltrate compared to the control group and the group with Hemospon® (p<0.05). No statistically significant difference was found between the groups regarding bone formation at the three experimental points in time. Osteopontin expression corroborated the intensity of bone formation. Fluorescence microscopy revealed positive labeling with Q-Tracker® in hDPSCs before transplantation and tissue repair. Conclusion The results suggest that the combination of Hemospon®, Aloe vera and hDPSCs is a form of clinical treatment for the repair of non-critical bone defects that reduces the inflammatory cascade's effects.

Endothelial assessment of donated tectonic corneas: a viable option for posterior lamellar transplantation / Avaliação da viabilidade do uso de córneas tectônicas em transplantes lamelares posteriores a partir da análise da vitalidade das células endoteliais

ABSTRACT Purpose: Donated corneas are classified as tectonic if there are defects within any layers of the cornea which would prevent a satisfactory visual outcome after transplantation. This study aimed to evaluate whether some tectonic corneas have sufficient endothelial characteristics to allow their use in posterior lamellar keratoplasty, and explored their reclassification for use in this sight-improving procedure. Methods: A retrospective review of all corneal tissues preserved by the Sorocaba Eye Bank from January to April of 2014 was performed. All donated corneas classified as tectonic were included. Endothelial tissue was defined as healthy and viable for posterior lamellar keratoplasty if endothelial cell density was ≥2000 cells/mm2. Additional parameters analyzed included Descemet folds and stretch marks, loss of endothelial cells, corneal endothelial polymegathism/ pleomorphism, pseudo-guttata, and reflectivity. Results: During the study period, 2,847 corneas were preserved, of which 423 (14.85%) were classified as tectonic. Of these, 87 (20.56%) were reported as having endothelial viability and were included in the posterior lamellar keratoplasty group. Average corneal endothelial cell density of this group was 2,471 SD ± 256 cells/mm2 (range 2012-2967 cells/mm2). Conclusion: A significant number of corneas classified as tectonic showed endothelial viability and were included in the posterior lamellar keratoplasty group (20.56%). Despite stromal and/or epithelial alterations, these corneas could have been potentially distributed for posterior lamellar transplantation to improve vision, thus reducing the corneal transplantation waiting period. This study highlights how corneal tissue reclassification could increase the potential amount of corneal tissue available for optical transplantation.

RESUMO Objetivo: Avaliar a vitalidade endotelial das córneas classificadas como tectônicas e discutir a viabilidade de seu uso na ceratoplastia lamelar posterior. Métodos: Realizou-se uma revisão retrospectiva de todos os tecidos corneanos preservados pelo Banco de Olhos Sorocaba de janeiro a abril de 2014. Todas as córneas doadas classificadas como tectônicas foram incluídas e avaliadas com ênfase na vitalidade endotelial. Os parâmetros de avaliação da lâmpada de fenda de cada córnea e densidade de células endoteliais medidos por microscópio especular foram registrados: córneas que apresentavam vitalidade endotelial apesar de alterações no estroma e/ou no epitélio foram selecionadas e incluídas em um grupo denominado grupo lamelar posterior. O tecido endotelial foi definido como saudável e viável para a ceratoplastia lamelar posterior, se houvesse uma densidade de células endoteliais ≥2.000 células/mm2. Outros parâmetros também foram analisados, incluindo; estrias ou pregas na Descemet, perda de células endoteliais, polimegatismo e pleomorfismo endotelial, pseudo-guttata e reflexividade endotelial. Resultados: Durante o período do estudo, foram preservadas 2.847 córneas, das quais 423 (14,85%) foram classificadas como tectônicas. Dessas, 87 (20,56%) apresentaram vitalidade endotelial e foram incluídos no grupo lamelar posterior. A densidade média das células endoteliais da córnea deste grupo era de 2.471 SD ± 256 células/mm2, variando de 2.012 a 2.967 células/mm2. Conclusão: Um número significativo de córneas classificadas como tectônicas apresentaram vitalidade endotelial e foram incluídas no grupo lamelar posterior (20,56%). Apesar de alterações estromais e/ou epiteliais, estas córneas poderiam ter sido potencialmente distribuídas para transplantes lamelares posteriores com finalidade ótica, otimizando a disponibilidade de tecidos, com impacto positivo na saúde pública.

Effect of varying hypoxia reoxygenation times on autophagy of cardiomyocytes

Abstract Purpose: To investigate the impact of different hypoxia reoxygenation (HR) times on autophagy of rat cardiomyocytes (H9C2). Methods: Rat cardiomyocytes were randomly divided into normal control group (group A), hypoxia group (group B), 2 h HR group (group C), 12 h HR group (group D), and 24 h HR group (group E). LC3 II/LC3 I was determined via western blotting, and cell viabilities of cardiomyocytes were measured using methyl thiazolyl tetrazolium (MTT) assay. Results: Cell viabilities in HR model groups were significantly lower than those of group A (P<0.05). LC3 II/LC3 I levels in groups B to D were significantly higher than those of group A (P<0.05), and group D showed the highest LC3 II/LC3 I levels. Cell viabilities in groups B to D were significantly lower than those of group A (P<0.05), with group D showing the lowest cell viabilities (P<0.05). Conclusions: Hypoxia can induce autophagy in rat cardiomyocytes, which can be further activated by reoxygenation; most notable after 12 h. Hypoxia-induced cell injury can be aggravated by reoxygenation. The lowest cell viability was observed at 12 h after reoxygenation; however, cell viability can be recovered after 24 h.

Cryotherapy: biochemical alterations involved in reduction of damage induced by exhaustive exercise

When exercises are done in intense or exhaustive modes, several acute biochemical mechanisms are triggered. The use of cryotherapy as cold-water immersion is largely used to accelerate the process of muscular recovery based on its anti-inflammatory and analgesic properties. The present study aimed to study the biochemical effects of cold-water immersion treatment in mice submitted to exercise-induced exhaustion. Swiss albino mice were divided into 4 treatment groups: control, cold-water immersion (CWI), swimming exhaustive protocol (SEP), and SEP+CWI. Treatment groups were subdivided into times of analysis: 0, 1, 3, and 5 days. Exhaustion groups were submitted to one SEP session, and the CWI groups submitted to one immersion session (12 min at 12°C) every 24 h. Reactive species production, inflammatory, cell viability, and antioxidant status were assessed. The SEP+CWI group showed a decrease in inflammatory damage biomarkers, and reactive species production, and presented increased cell viability compared to the SEP group. Furthermore, CWI increased acetylcholinesterase activity in the first two sessions. The present study showed that CWI was an effective treatment after exercise-induced muscle damage. It enhanced anti-inflammatory response, decreased reactive species production, increased cell viability, and promoted redox balance, which could decrease the time for the recovery process.

Long non-coding RNA HOTAIR promotes UVB-induced apoptosis and inflammatory injury by up-regulation of PKR in keratinocytes

Excessive exposure to ultraviolet (UV) rays can cause damage of the skin and may induce cancer, immunosuppression, photoaging, and inflammation. The long non-coding RNA (lncRNA) HOX antisense intergenic RNA (HOTAIR) is involved in multiple human biological processes. However, its role in UVB-induced keratinocyte injury is unclear. This study was performed to investigate the effects of HOTAIR in UVB-induced apoptosis and inflammatory injury in human keratinocytes (HaCaT cells). Quantitative real-time polymerase chain reaction was performed to analyze the expression levels of HOTAIR, PKR, TNF-α, and IL-6. Cell viability was measured using trypan blue exclusion method and cell apoptosis using flow cytometry and western blot. ELISA was used to measure the concentrations of TNF-α and IL-6. Western blot was used to measure the expression of PKR, apoptosis-related proteins, and PI3K/AKT and NF-κB pathway proteins. UVB induced HaCaT cell injury by inhibiting cell viability and promoting cell apoptosis and expressions of IL-6 and TNF-α. UVB also promoted the expression of HOTAIR. HOTAIR suppression increased cell viability and decreased apoptosis and expression of inflammatory factors in UVB-treated cells. HOTAIR also promoted the expression of PKR. Overexpression of HOTAIR decreased cell viability and increased cell apoptosis and expression of inflammatory factors in UVB-treated cells by upregulating PKR. Overexpression of PKR decreased cell viability and promoted cell apoptosis in UVB-treated cells. Overexpression of PKR activated PI3K/AKT and NF-κB pathways. Our findings identified an essential role of HOTAIR in promoting UVB-induced apoptosis and inflammatory injury by up-regulating PKR in keratinocytes.

MicroRNA-221 promotes cell proliferation, migration, and differentiation by regulation of ZFPM2 in osteoblasts

Bone fracture is a common medical condition, which may occur due to traumatic injury or disease-related conditions. Evidence suggests that microRNAs (miRNAs) can regulate osteoblast differentiation and function. In this study, we explored the effects and mechanism of miR-221 on the growth and migration of osteoblasts using MC3T3-E1 cells. The expression levels of miR-221 in the different groups were measured by qRT-PCR. Then, miR-221 mimic and inhibitor were transfected into MC3T3-E1 cells, and cell viability and migration were measured using the CCK-8 assay and the Transwell migration assay. Additionally, the expression levels of differentiation-related factors (Runx2 and Ocn) and ZFPM2 were measured by qRT-PCR. Western blot was used to measure the expression of cell cycle-related proteins, epithelial-mesenchymal transition (EMT)-related proteins, ZFPM2, and Wnt/Notch, and Smad signaling pathway proteins. miR-221 was significantly up-regulated in the patients with lumbar compression fracture (LCM) and trochanteric fracture (TF). miR-221 promoted ALP, Runx2, and OPN expressions in MC3T3-E1 cells. miR-221 overexpression significantly increased cell proliferation, migration, differentiation, and matrix mineralization, whereas suppression of miR-221 reversed these effects. Additionally, the results displayed that ZFPM2 was a direct target gene of miR-221, and overexpression of ZFPM2 reversed the promoting effects of miR-221 overexpression on osteoblasts. Mechanistic study revealed that overexpression of miR-221 inactivated the Wnt/Notch and Smad signaling pathways by regulating ZFPM2 expression. We drew the conclusions that miR-221 overexpression promoted osteoblast proliferation, migration, and differentiation by regulation of ZFPM2 expression and deactivating the Wnt/Notch and Smad signaling pathways.

Autofagia, una estrategia de supervivencia celular / Autophagy. A strategy for cell survival

La autofagia es un proceso de reciclado de partes de la célula. Como se describe en esta revisión, ocurre naturalmente preservando a las células de la acumulación de toxinas, moléculas y organelas dañadas y además permite los procesos de desarrollo y diferenciación de los tejidos. En el transcurso de la autofagia, el procesamiento de los sustratos a reciclar genera ATP, lo que constituye una fuente alternativa de energía en situaciones de estrés. En este sentido, bajo condiciones hostiles como hipoxia o falta de nutrientes, el proceso puede dispararse de modo exacerbado llevando a la muerte celular. Algunas alteraciones en su funcionamiento pueden involucrar el desarrollo de diversas patologías, tales como el daño hepático, el cáncer y las enfermedades neurodegenerativas.

Autophagy is a process of recycling parts of the cell. As described in this review, it occurs naturally in order to preserve cells from the accumulation of toxins, damaged molecules and organelles, and to allow processes of tissue development and differentiation. In the course of autophagy, the processing of the substrates to be recycled generates ATP, thus providing an alternative source of energy in stress situations. In this sense, under hostile conditions such as hypoxia or lack of nutrients, the autophagy process can be exacerbated leading to cell death. Some alterations in its functioning may involve the development of various pathologies, including liver damage, cancer and neurodegenerative diseases.

Efeito de campos magnéticos estáticos e compensados na proliferação celular in vitro / Proteomics of the effect of compensated and static magnetic fields on cell proliferation in vitro

Inserido no paradigma da transdisciplinaridade, o presente trabalho foi desenvolvido em etapas, com os seguintes objetivos: a) Construir um dispositivo com base de metal não magnético para ímãs permanentes, visando à geração de um Campo Magnético Estático (CME) ou de um Campo Magnético Compensado (CMC); b) Expor culturas de células mesenquimais a um CME e a um CMC, ou a nenhum campo (controle); c) Analisar a influência destes campos na viabilidade e proliferação celular e nos casos em que houve alteração em pelo menos um destes parâmetros, utilizar a análise proteômica como ferramenta para a compreensão dos mecanismos envolvidos. O dispositivo foi construído utilizando aço inoxidável, capaz de gerar dois tipos de Campos Magnéticos: Compensado (CMC) com intensidade de aproximadamente 0 mT e Estático (CME) com intensidade média de 165 mT. Estes campos foram aplicados a culturas de células mesenquimais de medula óssea de camundongos AJ (MSC/AJ), nos períodos de 0, 24, 48, 72 e 96 h (CMC) e 24 h (CME). Os efeitos sobre a proliferação e a viabilidade foram avaliados por método de contagem manual de células com marcação por azul de tripan. A análise proteômica foi realizada para os experimentos com CMC, com o objetivo de descrever as proteínas envolvidas nas alterações encontradas. A exposição ao CMC tendeu a reduzir a proliferação das células de medula óssea MSC/AJ em relação ao controle em 96 h, porém sem diferença significativa, o que poderia estar relacionado a proteínas que inibem a transcrição, como a Forkhead box protein P2 Foxp2. Este mesmo campo aumentou a viabilidade celular em relação ao baseline para todos os tempos experimentais, o que poderia estar relacionado a proteínas relacionadas à ligação ao Ca+2. Esses mecanismos, entretanto, precisam ser estudados mais profundamente para que possam ser comprovados ou não. Já a exposição ao CME levou a uma tendência à diminuição da proliferação e viabilidade celular em relação ao grupo controle, embora sem diferenças significativas, provavelmente por conta do tamanho amostral e tempo de avaliação (24 h).(AU)

Inserted in the transdisciplinarity paradigm, the present work was developed by steps with the following aims: a) To build a device of non-magnetic metal to hold permanent magnets for the generation of a Static Magnetic Field (SMF) or a Compensated Magnetic Field (CMF); b) To expose mesenchimal cells to the SMF and to CMF or to none of the fields (control); c) To analyze the influence of these fields on cell viability and cell proliferation and in the case where it occurred alteration in at least one of these parameters, to use proteomics as a tool for the comprehension of the involved mechanisms. The device was built in stainless steel, able to generate two kinds of Magnetic Fields: Compesated (CMF) with an intensity of nearly zero mT and Static (SMF) with a mean intensity of 165 mT. These fields were applied to bone marrow mesenchimal cell cultures from AJ mice (MSC/AJ), for 0, 24, 48, 72 and 96 h (CMF) and 24 h (SMF) periods. The effects on the proliferation and viability were assessed by tripan blue dying and manual counting of the cells. Proteomics was done for the experiments with CMF, aiming to describe the involved proteins on found alterations. The exposition to CMF tends to reduce the bone marrow cell proliferation of MSC/AJ in relation to control in 96 h, but with no significant difference, which may be related to proteins that inhibit the transcription, like Forkhead box protein P2 Foxp2. This very field raised the cell viability in relation to the baseline for all the experimental times that could be related to proteins connected to Ca2+ binding. However, these mechanisms need more experiments, so they can be confirmed or not. The exposition to the SMF tends to decrease both cell proliferation and viability in relation to the control group, although with no significant difference, probably because of the sample number and the exposition time (24h).(AU)

Is the combination of fat grafts and platelet rich plasma effective in rats?

ABSTRACT PURPOSE: To investigate if the association of fat grafts and platelet-rich plasma (PRP) improves graft viability in female rats. METHODS: This is an experimental, randomized and blinded study, which involved 47 rats. Fat was harvested from the inguinal region and grafted to the cranial region. The experimental group consisted of PRP-enriched fat grafts (n=22) whilst the control group consisted of fat graft only (n=25). After a 100-day period, the animals were euthanised and the fat grafts were analyzed using scores from 0 (absent) to 4 (abundant), in optical microscopy by two independent and blinded pathologists. RESULTS: Regarding fat graft cell viability, the PRP group scored moderate/abundant in 63% of cases and the fat graft only group scored absent/slight in 72% of cases (p=0.03). The PRP group also presented lower fat necrosis scores when compared to the fat graft only group (p=0.03). Tumors (dermoid cysts) within the fat grafts were observed in three animals in which the grafts were mixed with PRP. CONCLUSION: Platelet-rich plasma improves the viability and integration of fat grafts in rats, but more studies are needed to fully understand the exact mechanisms that lead to this improvement and assess the safety of the method for use in humans.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Viabilidad de Ovocitos Vitrificados y Madurados in Vitro de Gata Doméstica Adulta (Felis catus) en Estación Reproductiva / Vitrified and in Vitro Matured Oocytes Viability from Adult Housecat (Felis catus) in Reproductive Season

La vitrificación de ovocitos de mamíferos es considerada una técnica en experimentación. La supervivencia de los ovocitos es extremadamente variable, según las técnicas utilizadas e influida por una serie de condiciones. El objetivo de éste trabajo fue determinar los efectos de la vitrificación en ovocitos de gata domestica adulta en estación reproductiva y madurados in vitro. Se obtuvieron 33 ovarios correspondientes a hembras de más de un año en buen estado nutricional y sin tratamiento hormonal, ovariectomizadas en domicilio del propietario por profesional veterinario. Los ovarios fueron trasladados al laboratorio y se fragmentaron por microdisección bajo microscopio estereoscópico en caja de Petri con solución de buffer fosfato salino modificado con suero de ternero inactivado y antibióticos para la obtención, evaluación y selección de los complejos cúmulo-ovocitos (CCOs) de buena calidad. Se vitrificaron 506 CCOs en pajuelas de 0.25 ml con 10-12 ovocitos cada una y almacenados por 30 días en nitrógeno líquido (N2) a -196 C. Posteriormente se desvitrificaron las pajuelas, recuperándose 320 CCOs, los que fueron madurados in vitro. Transcurrido ese tiempo se evaluaron los CCOs, descartándose 50 por presentar signos de degeneración. En los 270 restantes se observó buena expansión del cúmulo, citoplasma uniforme y oscuro e integridad de la zona pelúcida. A estos últimos se los dividió en dos grupos similares para evaluar la viabilidad, a uno se lo coloreó con metil tiazol tetrazolio y al otro con Azul Tripán. En ambos se constató un resultado positivo para la viabilidad de los CCOs. El análisis de los resultados nos permite concluir que la vitrificación comprometió la integridad de un importante número de CCOs aunque más del 50% respondió en cultivo favorablemente, mostrando signos de viabilidad esperados. Estos resultados hacen necesarios la profundización en el mejoramiento del protocolo para incrementar el porcentaje de viabilidad.

Vitrification of mammalian oocytes is a technique considered in experimentation. Oocyte survival is extremely variable, according to the techniques used and influenced by a number of conditions. The objective of this study was to determine the effects of adult domestic cat oocyte matured in vitro and vitrified in breeding season. We obtained 33 ovaries from housecats in good nutritional status without hormonal treatment, ovariectomized by a veterinary professional at home. The ovaries were transported to the laboratory and fragmented by microdissection under a stereoscopic microscope in a petri dish with modified buffer saline phosphate solution, inactivated calf serum and antibiotics to the collection, evaluation and selection of good quality cumulus-oocyte complexes (COCs). 506 COCs were vitrified in 0.25 ml straws each with 10­12 oocytes and stored for 30 days in liquid nitrogen (N2) at -196 °C. Then the straws were thawed, recovering 320 CCOs, which were matured in vitro. After this time the COCs were evaluated, discarding 50 which showed signs of degeneration. In the remaining 270 COCs good cumulus expansion was observed and also uniformly dark cytoplasm and integrity of the zona pellucida. The latter were divided into two similar groups to assess the feasibility; one was stained with Methylthiazblyl tetrazolium and the other with Trypan Blue. Both tested positive for the viability of the CCOs was found. The analysis of the results allow us to conclude that vitrification compromised the integrity of a large number of CCOs although more than 50% responded favorably in culture, showing signs of expected viability. According to these results it is necessary to further improve the protocol to increase the percentage of viability.

Influence of the magnetic field on microorganisms in the oral cavity

Since the beginning of their lives, all living organisms are exposed to the influence of geomagnetic fields. Objectives : With respect to the positive effects that magnetic fields have on human tissues, especially the bactericidal effect, this investigation aimed to assess their influence on the reduction of oral microorganisms. Material and Methods : In order to obtain adequate specimens of dental plaque deposit, microbes such as Streptococcus parasanguinis, Staphylococcus epidermidis, Rhodococcus equi and Candida albicans were isolated from the human mouth. To establish the intensity of microbial growth on the basis of the modified optical density (OD) of agar turbidimetry assay, microbial count and spectrophotometry were applied. The study was carried out with two microbial concentrations (1 and 10 CFU/ml) after periods of incubation of 24 and 48 h and using micromagnets. Results : A positive effect of the magnetic field, resulting in the reduction of dental plaque microbes in vitro, was found. In the first 24 hours of exposure to the magnetic field, the number of all isolated microbes was significantly reduced. The most potent influence of magnets and the most intensified reduction after 24 hours were evident in Candida albicans colonies. The decrease in the influence of magnets on microbes in vitro was also detected. Conclusions : Magnets reduce the number of microbes and might be recommended as a supplement in therapy for reduced periodontal tissues. This is important because periodontal tissues that are in good conditions provide prolonged support to the oral tissues under partial and supradental denture. .

Repeated microendoscopic discectomy for recurrent lumbar disk herniation

OBJECTIVES: To explore the microendoscopic discectomy technique and inclusion criteria for the treatment of recurrent lumbar disc herniation and to supply feasible criteria and technical notes to avoid complications and to increase the therapeutic effect. METHODS: A consecutive series of 25 patients who underwent posterior microendoscopic discectomy for recurrent lumbar disc herniation were included. The inclusion criteria were as follows: no severe pain in the lumbar region, no lumbar instability observed by flexion-extension radiography and no intervertebral discitis or endplate damage observed by magnetic resonance imaging. All patients were diagnosed by clinical manifestations and imaging examinations. RESULTS: Follow-up visits were carried out in all cases. Complications, such as nerve injuries, were not observed. The follow-up outcomes were graded using the MacNab criteria. A grade of excellent was given to 12 patients, good to 12 patients and fair to 1 patient. A grade of excellent or good occurred in 96% of cases. One patient relapsed 3 months after surgery and then underwent lumbar interbody fusion and inner fixation. The numerical rating scale of preoperative leg pain was 7.4± 1.5, whereas it decreased to 2.1±0.8 at 7 days after surgery. The preoperative Oswestry disability index of lumbar function was 57.5±10.0, whereas it was 26.0±8.5 at 7 days after surgery. CONCLUSION: In these cases, microendoscopic discectomy was able to achieve satisfactory clinical results. Furthermore, it has advantages over other methods because of its smaller incision, reduced bleeding and more efficient recovery. .

Estudo do potencial antimicrobiano e citotóxico da espécie Pouteria venosa (Sapotaceae) / Study of antimicrobial and cytotoxic potential of Pouteria venosa species (Sapotaceae)

RESUMO Estudo experimental in vitro que objetivou investigar o potencial antimicrobiano e citotóxico de quatro frações e um extrato etanólico da espécie Pouteria venosa usada como planta medicinal. A atividade antimicrobiana foi determinada pelos testes de sensibilidade microbiana, como o método de difusão em disco e o método da microdiluição em caldo, para a determinação da Concentração Inibitória Mínima (CIM). Obteve-se a avaliação da citotoxicidade por meio do método colorimétrico do Metiltetrazolium. No estudo da atividade antimicrobiana, os principais resultados foram obtidos contra Staphylococus aureus para a fração AcOEt das cascas do caule, CIM de 125 µg/mL; Streptococcus pneumoniae e Proteus mirabilis para a fração AcOEt das cascas do caule, CIMde250 µg/mL; Staphylococus epidermidis, Pseudomonas aeruginosa para a fração AcOEt das folhas e cascas do caule, CIM de 250 µg/mL. Todas as amostras foram inativas para os fungos testados. A fração AcOEt das cascas do caule foi considerada atóxica, podendo ser utilizada em testes pré-clínicos in vivo

ABSTRACT Study of antimicrobial and cytotoxic potential of Pouteria venosa species (Sapotaceae). This experimental in vitro study aimed to investigate the antimicrobial and cytotoxic potential of four fractions and one ethanolic extract of the specie Pouteria venosa used as a medicinal plant. The antimicrobial activity was determined by microbial sensitivity tests, as the method of disk diffusion and the broth microdilution method for determination of Minimum Inhibitory Concentration (MIC). The evaluation of the cytotoxicity was obtained by the Metiltetrazolium colorimetric method. In the antimicrobial activity research, the main results were obtained against the Staphylococcus aureus for the AcOEt fraction of the stem bark MIC 125 µg/mL, Streptococcus pneumoniae and Proteus mirabilis for the AcOEt fraction from the stem bark, CIM 250 µg/mL, Staphylococcus epidermidis, Pseudomonas aeruginosa to the AcOEt fraction of the leaves and stem bark, MIC 250 µg/mL. All samples did not react for the fungi tested. The AcOEt fraction of the stem bark was considered non-toxic and can be used at in vivo pre-clinical testing

Inquérito Brasileiro de Diálise Crônica 2013 - Análise das tendências entre 2011 e 2013 / Brazilian Chronic Dialysis Survey 2013 - Trend analysis between 2011 and 2013

Introdução: Dados nacionais sobre diálise crônica têm tido impacto no planejamento do tratamento. Objetivo: Apresentar dados do inquérito da Sociedade Brasileira de Nefrologia sobre os pacientes com doença renal crônica em tratamento dialítico em julho de 2013 e comparar com dados de 2011- 12. Métodos: Levantamento de dados de unidades de diálise do país. A coleta de dados foi feita utilizando questionário preenchido on-line pelas unidades de diálise. Resultados: Trezentos e trinta e quatro (51%) unidades responderam ao inquérito. Em julho de 2013, o número total estimado de pacientes em diálise foi de 100.397. As estimativas nacionais das taxas de prevalência e de incidência de tratamento dialítico foram de 499 (variação: 284 na região Norte e 622 na Sul) e 170 pacientes por milhão da população, respectivamente. O número estimado de pacientes que iniciaram tratamento em 2013 foi 34.161. A taxa anual de mortalidade bruta foi de 17,9%. Dos pacientes prevalentes, 31,4% tinham idade ≥ 65 anos, 90,8% estavam em hemodiálise e 9,2% em diálise peritoneal, 31.351 (31,2%) estavam em fila de espera para transplante, 30% tinham diabetes, 17% tinham PTH > 600 pg/ml e 23% hemoglobina < 10 g/dl. Cateter venoso era usado como acesso em 15,4% dos pacientes em hemodiálise. Conclusão: O número absoluto de pacientes em diálise tem aumentado 3% ao ano nos últimos 3 anos. As taxas de prevalência e incidência de pacientes em diálise ficaram estáveis, e a taxa de mortalidade tendeu a diminuir em relação a 2012. Houve tendência a melhor controle da anemia e dos níveis de PTH. .

Introduction: National chronic dialysis data have had impact in the treatment planning. Objective: To report data of the annual survey of the Brazilian Society of Nephrology about chronic kidney disease patients on dialysis in July 2013 and compare with 2011-12. Methods: A survey based on data of dialysis units from the whole country. The data collection was performed by using a questionnaire filled out on-line by the dialysis units. Results: Three hundred thirty four (51%) of the dialysis units in the country answered the questionnaire. In July 2013, the total estimated number of patients on dialysis was 100,397. The estimated prevalence and incidence rates of chronic maintenance dialysis were 449 (range: 284 in the North region and 622 in the South) and 170 patients per million population, respectively. The estimated number of new patients starting dialysis in 2013 was 34,161. The annual gross mortality rate was 17.9%. For prevalent patients, 31.4% were aged 65 years or older, 90.8% were on hemodialysis and 9.2% on peritoneal dialysis, 31,351 (31.2%) were on a waiting list of renal transplant, 30% were diabetics, 17% had PTH levels > 600 pg/ml and 23% hemoglobin < 10 g/ dl. A venous catheter was the vascular access for 15.4% of the hemodialysis patients. Conclusion: The absolute number of patients on dialysis has increased 3% per year. The prevalence and incidence rates of patients on dialysis leveled off, while the mortality rate tended to decrease compared with 2012. There was a trend towards a better control of the anemia and PTH levels. .

Rapid flow cytometry based cytotoxicity assay for evaluation of NK cell function.

Assessment of natural killer cells (NK-cell) cytotoxicity is used not only in research settings but is also important in diagnosis of various diseases. NK-cell cytotoxicity assays are based on measurement of target cells killed by cytotoxic cells analyzed either by chromium (51Cr) release assay or flow cytometry. Both these methods use peripheral blood mononuclear cells (PBMC) or pure NK-cell population and hence require large volume of blood sample which is difficult to obtain in pediatric patients and patients with cytopenia. Hence, a flow cytometric assay was designed to determine NK cell activity using whole blood, eliminating the need for isolation of PBMCs or pure NK cells. This assay is based on a dual fluorescent staining of target cells (K562 cell line). The DIOC18 dye labeled K562 cells are incubated with whole blood and then counterstained with 7-AAD enabling the measurement of dead target cell and then percent cytotoxicity is calculated. This study compared the NK cell cytotoxicity using PBMC and whole blood in clinically relevant samples. There was no significant difference between two assays in the measurement of lytic activity or in reproducibility in the repeated samplings of healthy individuals. The whole blood assay required less volume of blood and also less processing time as compared to PBMC assay. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis expected to have low NK-cell activity. This assay is rapid, sensitive and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK-cell function.