Assuntos
Internato e Residência , Radiologia , Humanos , Radiologia/educação , Radiografia , EscolaridadeRESUMO
Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose-basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.
Assuntos
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Neovascularização Fisiológica , Oxigênio/administração & dosagem , Tela Subcutânea/irrigação sanguínea , Administração por Inalação , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Sobrevivência de Enxerto , Masculino , Oxigênio/metabolismo , Ratos , Ratos Endogâmicos LewRESUMO
The safety and effectiveness of islet transplantation has been proven through world-wide trials. However, acute and chronic islet loss has hindered the ultimate objective of becoming a widely used treatment option for type 1 diabetes. A large islet loss is attributed, in part, to the liver being a less-than-optimal site for transplantation. Over half of the transplanted islets are destroyed shortly after transplantation due to direct exposure to blood and non-specific inflammation. Successfully engrafted islets are continuously exposed to the liver micro-environment, a unique immune system, low oxygen tension, toxins and high glucose, which is toxic to islets, leading to premature islet dysfunction/death. Investigations have continued to search for alternate sites to transplant islets that provide a better environment for prolonged function and survival. This article gathers courses and conditions that lead to islet loss, from organ procurement through islet transplantation, with special emphasis on hypoxia, oxidative stress, and antigen non-specific inflammation, and reviews strategies using pharmacological agents that have shown effectiveness in protecting islets, including a new treatment approach utilizing siRNA. Pharmacological agents that support islet survival and promote ß-cell proliferation are also included. Treatment of donor pancreata and/or islets with these agents should increase the effectiveness of islets transplanted into extrahepatic sites. Furthermore, the development of methods designed to release these agents over an extended period, will further increase their efficacy. This requires the combined efforts of both islet transplant biologists and bioengineers.
Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Fígado/cirurgia , Transplante Heterotópico/métodos , Indutores da Angiogênese/farmacologia , Indutores da Angiogênese/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Hipóxia Celular/efeitos dos fármacos , Microambiente Celular , Avaliação Pré-Clínica de Medicamentos , Técnicas de Silenciamento de Genes , Peptídeo 1 Semelhante ao Glucagon/agonistas , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Glucose/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Ilhotas Pancreáticas/efeitos dos fármacos , Fígado/citologia , Fígado/imunologia , Fígado/metabolismo , Camundongos , Especificidade de Órgãos , Estresse Oxidativo/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Inibidores Teciduais de Metaloproteinases/farmacologia , Inibidores Teciduais de Metaloproteinases/uso terapêutico , Obtenção de Tecidos e Órgãos/métodosRESUMO
AIMS: To investigate the efficacy and safety of the dipeptidyl peptidase-4 inhibitor linagliptin in patients with Type 2 diabetes mellitus inadequately controlled by a combination of metformin and pioglitazone. METHODS: This was a multi-centre, phase 3, randomized, double-blind, placebo-controlled study comparing linagliptin 5 mg once daily (n = 183) and placebo (n = 89) as add-on to metformin and pioglitazone. The primary endpoint was the change from baseline in glycated haemoglobin (HbA1c ) after 24 weeks. RESULTS: The placebo-corrected adjusted mean (se) change in HbA1c from baseline to 24 weeks was -6 (1) mmol/mol [-0.57 (0.13)%] (P < 0.0001). In patients with baseline HbA1c ≥ 53 mmol/mol (7.0%), 32.4% of patients in the linagliptin group and 13.8% in the placebo group achieved HbA1c < 53 mmol/mol (7.0%) (odds ratio 2.94; P = 0.0033). The placebo-corrected adjusted mean (se) change from baseline in fasting plasma glucose at week 24 was -0.57 (0.26) mmol/l [-10.4 (4.7) mg/dl] (P = 0.0280). The incidence of serious adverse events was 2.2% with linagliptin and 3.4% with placebo. Investigator-defined hypoglycaemia occurred in 5.5% of the linagliptin group and 5.6% of the placebo group. No meaningful changes in mean body weight were noted for either group. CONCLUSIONS: Linagliptin as add-on therapy to metformin and pioglitazone produced significant and clinically meaningful improvements in glycaemic control, without an additional risk of hypoglycaemia or weight gain (Clinical Trials Registry No: NCT 00996658).
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Purinas/uso terapêutico , Quinazolinas/uso terapêutico , Tiazolidinedionas/uso terapêutico , Adulto , Idoso , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Hipoglicemia/induzido quimicamente , Linagliptina , Masculino , Pessoa de Meia-Idade , Pioglitazona , Falha de Tratamento , Resultado do Tratamento , Aumento de PesoRESUMO
BACKGROUND: Providing smoking cessation programmes through workplaces is an effective method of assisting employees to quit smoking; however, few employers provide such services, and achieving long-term success remains challenging. AIMS: To evaluate the effectiveness of a workplace-based tailored smoking cessation programme that combined telephone-based counselling with group behaviour therapy sessions in helping employees to quit. METHODS: A smoking cessation programme was offered to employees of a large corporation that is respons ible for the passenger rail network in New South Wales (NSW), Australia. Two hundred and thirty participants enrolled in the programme, which offered telephone-based coaching and group sessions designed around cognitive behavioural therapy principles. One hundred and eight participants (47%) completed the 6 month follow-up assessment. RESULTS: Of the estimated 2850 smokers in the organization, 8% (230) registered for the smoking cessation programme, with 77% (176) participating in telephone-based coaching and/or group sessions. Intention-to-treat analysis indicated 22% of participants achieved 7 day point prevalence abstinence and 10% achieved 3 month prolonged abstinence at the 6 month follow-up. Over 75% of those still smoking at follow-up reported intentions to quit in the next 6 months. Psychological distress was also significantly lower at 6 month follow-up. Participants reported high levels of satisfaction with the programme. CONCLUSIONS: The smoking cessation programme successfully assisted employees to quit smoking. Unique aspects of the programme such as continuity of care were valued by participants and may have contributed to the programme's success.
Assuntos
Terapia Comportamental/métodos , Aconselhamento/métodos , Abandono do Hábito de Fumar/métodos , Prevenção do Hábito de Fumar , Local de Trabalho , Humanos , New South Wales , Satisfação do Paciente , Avaliação de Programas e Projetos de Saúde , Psicoterapia de Grupo , Telefone , Resultado do TratamentoRESUMO
AIMS/HYPOTHESIS: TNF-α plays important roles in the pathogenesis of type 1 and type 2 diabetes mellitus. In light of this, we examined the involvement of a pro-apoptotic gene, BBC3 (also known as PUMA), in TNF-α-mediated beta cell dysfunction and destruction in human islets. METHODS: Human islets were exposed in vitro to TNF-α alone or in combination with IFN-γ. Gene expression was assessed by RT-PCR using a set of single islets. Protein abundance and cellular localisation of BBC3 were assessed by immunoblot and immunohistochemistry. A marginal number of islets were transplanted into diabetic NODscid mice to correlate in vivo islet function with BBC3 expression. RESULTS: BBC3 and IL8 mRNA were upregulated in TNF-α-stimulated islets in a dose-dependent manner and enhanced through addition of IFN-γ, but not upregulated by IFN-γ alone. Immunohistochemistry revealed that TNF-α in combination with IFN-γ upregulated basal BBC3 abundance in the cytoplasm of beta cells along with the perinuclear clustering of mitochondria partially co-localised with BBC3. TNF-α alone did not induce beta cell death, but did abrogate preproinsulin precursor mRNA synthesis in response to high glucose stimulation, which was inversely associated with upregulation of BBC3 mRNA expression by TNF-α. Higher BBC3 mRNA expression in islets correlated with decreased graft function in vivo. CONCLUSIONS/INTERPRETATION: These results suggest that BBC3 mRNA can serve as a molecular marker to detect early TNF-α-induced beta cell stress and may help identify islet-protective compounds for the treatment of diabetes.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Morte Celular/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Idoso , Animais , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Interferon gama/farmacologia , Interleucina-8/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/genética , Adulto JovemRESUMO
BACKGROUND: In colorectal cancer (CRC), tumour microsatellite instability (MSI) status and CpG island methylator phenotype (CIMP) status are indicators of patient outcome, but the molecular events that give rise to these outcomes remain largely unknown. Wnt5a is a critical regulator of non-canonical Wnt activity and promoter hypermethylation of this gene has emerging prognostic roles in CRC; however the frequency and prognostic significance of this epigenetic event have not been explored in the context of colorectal tumour subtype. Consequently, we investigated the frequency and prognostic significance of Wnt5a methylation in a large cohort of MSI-stratified CRCs. METHODS: Methylation was quantified in a large cohort of 1232 colorectal carcinomas from two clinically distinct populations from Canada. Associations were examined between methylation status and clinicopathlogical features, including tumour MSI status, BRAF V600E mutation, and patient survival. RESULTS: In Ontario, Wnt5a methylation was strongly associated with MSI tumours after adjustment for age, sex, and tumour location (odds ratio (OR)=4.2, 95% confidence interval (CI)=2.4-7.4, P<10(-6)) and with BRAF V600E mutation, a marker of CIMP (OR=12.3, 95% CI=6.9-21.7, P<10(-17)), but was not associated with patient survival. Concordant results were obtained in Newfoundland. CONCLUSION: Methylation of Wnt5a is associated with distinct tumour subtypes, strengthening the evidence of an epigenetic-mediated Wnt bias in CRC.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Instabilidade de Microssatélites , Mutação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética , Adulto , Idoso , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Wnt-5aRESUMO
We have studied brain stem cells in the ewe brain that project to the bed nucleus of the stria terminalis (BNST) and determined if these cells are activated by estradiol-17beta. This would predicate an indirect role in the estradiol-17beta regulation of gonadotropin releasing hormone (GnRH) cells, since these receive input from the BNST. Ovariectomized ewes received 50 mug estradiol-17beta benzoate (i.m.) 1 h prior to brain collection, so that activated cells could be identified by Fos immunohistochemistry. Retrograde tracer (FluoroGold; FG), was injected into the three divisions of the BNST and labeled cells were mapped to the A1 and A2 regions and the parabrachial nucleus (PBN) of the brain stem. With FG injection into the dorsal and lateral BNST, all FG-containing cells in the caudal A1 and 45% of those in A2 stained for dopamine-beta-hydroxylase (DBH), indicating noradrenergic type. No FG-labelled cells in the PBN were DBH-positive. In A1 and A2 respectively, 42% and 46% of FG-labelled cells were Fos-positive, with no double-labeling in cells of the PBN. In ewes receiving FG injections into the ventral BNST, estrogen receptor (ER)alpha-immunoreactive nuclei were found in 82% of A1-FG labeled and 38% of A2-FG labeled cells. No FG-labelled cells of the PBN were ERalpha-positive. Anterograde tracing from A1 with microruby injection identified projections to the PBN, BNST and preoptic area (POA). Thus, A1 and A2 noradrenergic neurons project to the BNST in the ewe brain, express ERalpha and are activated by estradiol-17beta. These noradrenergic, estrogen-responsive cells may provide indirect input to GnRH cells, via the BNST.
Assuntos
Tronco Encefálico/efeitos dos fármacos , Estradiol/análogos & derivados , Estrogênios/farmacologia , Receptores Adrenérgicos/metabolismo , Núcleos Septais/efeitos dos fármacos , Animais , Tronco Encefálico/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Imuno-Histoquímica , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Marcadores do Trato Nervoso , Ovariectomia , Área Pré-Óptica/efeitos dos fármacos , Área Pré-Óptica/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Núcleos Septais/metabolismo , OvinosRESUMO
Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (DeltaOCR(glc)) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of DeltaOCR(glc) obtained at both institutions were compared. Islet specificity was determined by comparing DeltaOCR(glc) in islet and nonislet tissue. The DeltaOCR(glc) ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (DeltaOCR(glc) was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The DeltaOCR(glc) was statistically independent of percent beta cells, demonstrating that DeltaOCR(glc) is governed more by islet quality than by islet composition. The DeltaOCR(glc), but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing DeltaOCR(glc) as a measurement of islet quality for human islet transplantation.
Assuntos
Glucose/fisiologia , Transplante das Ilhotas Pancreáticas/normas , Ilhotas Pancreáticas/metabolismo , Consumo de Oxigênio/fisiologia , Animais , Células Cultivadas , Humanos , Hiperglicemia/etiologia , Hiperglicemia/metabolismo , Hiperglicemia/prevenção & controle , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. beta Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucose-stimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.
Assuntos
Criopreservação/métodos , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Preservação de Órgãos/métodos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Peptídeo C/metabolismo , Contagem de Células , Sobrevivência Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/cirurgia , Glucose/farmacologia , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.
Assuntos
Transplante de Medula Óssea/métodos , Quimera , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Tolerância Imunológica/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Transplante de Células-Tronco Mesenquimais , Animais , Células Cultivadas , Diabetes Mellitus Experimental/cirurgia , Diabetes Mellitus Tipo 1/terapia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Doença Enxerto-Hospedeiro/imunologia , Terapia de Imunossupressão , Transplante das Ilhotas Pancreáticas/patologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Tolerância ao Transplante/imunologia , Transplante Homólogo/imunologiaRESUMO
The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.
Assuntos
Ilhotas Pancreáticas/citologia , Células-Tronco Multipotentes/citologia , Pâncreas/citologia , Adulto , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Meios de Cultura Livres de Soro , Humanos , Coleta de Tecidos e Órgãos/métodosRESUMO
The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.
Assuntos
Criopreservação/métodos , Ilhotas Pancreáticas/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular , Ativação Enzimática , HumanosRESUMO
BACKGROUND: Resident work hours may impact patient care. We hypothesized that "call-associated" acute sleep deprivation has no effect on technical dexterity as measured on a minimally invasive surgery trainer, virtual reality (MIST VR) surgical simulator. METHODS: Thirty-five surgical residents were prospectively evaluated pre-call (rested), on-call (rested), and post-call (acutely sleep deprived). Participants completed questionnaires regarding sleep hours and level of fatigue. Technical skill was assessed using the MIST VR. Speed, errors, and economy of motion were automatically recorded by the MIST VR computer simulator. Data were analyzed by paired Student t test and analysis of variance. RESULTS: Estimated hours of sleep and subjective indicators of fatigue were different between rested and sleep-deprived residents. The number of errors and time to complete all tasks increased at the post-call assessment. CONCLUSIONS: Resident work schedules lead to sleep deprivation and fatigue. Call-associated sleep deprivation and fatigue are associated with increased technical errors in the performance of simulated laparoscopic surgical skills.
Assuntos
Competência Clínica , Cirurgia Geral , Internato e Residência , Laparoscopia , Privação do Sono , Análise e Desempenho de Tarefas , Adulto , Feminino , Cirurgia Geral/educação , Humanos , Masculino , Admissão e Escalonamento de PessoalRESUMO
REASONS FOR PERFORMING STUDY: Increased mucin gene expression may be an important cause of mucus accumulation observed in recurrent airway obstruction (RAO)-affected horses. To date, however, no mucin gene sequences are available for the horse. OBJECTIVES: To identify equine homologues of gel-forming mucins and investigate their expression at different airway generations of healthy and RAO-affected horses. METHODS: Two equine homologues were identified by cloning and sequencing fragments of equine (eq)MUC5AC and eqMUC2. RESULTS: Semiquantitative RT-PCR on RNA from airways (generations 1, 5, 10, 15; small airways and parenchyma), stomach (glandular), and colon revealed that eqMUC5AC is expressed in equine stomach and in all of the airway samples. In contrast, eqMUC2 steady-state mRNA levels were detected in colon and very faintly in stomach, but not in airway tissue. EqMUC5AC expression was also compared to that of ZO-1, a tight junction protein, and eqMUC5AC/ZO-1 ratios were higher in RAO-affected compared to control horses at all airway generations. CONCLUSIONS: That eqMUC5AC is expressed in horse airways, but any expression of MUC2 is undetectable and unlikely to be of physiological consequence. POTENTIAL RELEVANCE: EqMUC5AC up-regulation may be a primary mechanism responsible for mucus hypersecretion and accumulation in RAO.
Assuntos
Obstrução das Vias Respiratórias/veterinária , Doenças dos Cavalos/metabolismo , Mucinas/genética , Muco/metabolismo , Obstrução das Vias Respiratórias/etiologia , Obstrução das Vias Respiratórias/metabolismo , Animais , Feminino , Expressão Gênica , Cavalos , Pulmão/metabolismo , Masculino , Mucina-5AC , Mucina-2 , Mucinas/metabolismo , RNA Mensageiro/metabolismo , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Homologia de Sequência do Ácido Nucleico , Traqueia/metabolismoRESUMO
Oestrogen produces a positive feedback effect on the secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) when implanted into the ventromedial/arcuate nucleus of the ovariectomized (OVX) ewe. This has led to the belief that it is in this area of the hypothalamus that oestrogen causes the preovulatory surge in GnRH/LH. To date, however, the cell types that are integral to this response have not been identified. The present study aimed to examine cellular responsiveness to oestrogen in this region of the brain using Fos immunohistochemistry and further aimed to determine the cell type that shows an acute response to oestrogen. OVX ewes (n = 4-6 per group) were given i.m. injections of oestradiol benzoate or oil (vehicle) and were killed 1-6 h later. Brains were perfused for immunohistochemistry. The number of cells in the arcuate nucleus which were immunopositive for Fos was greater (two- to fourfold) in the oestradiol benzoate-treated OVX ewes (n = 5) 1 h after injection. The number of Fos-positive cells in the ventromedial hypothalamic nucleus was 10-fold greater in the oestradiol benzoate-treated ewes 1 h after injection. Because there were high levels of Fos-immunoreactive cells in oil-treated ewes, we repeated the experiment with i.v. injection of 50 microg oestrogen or vehicle (n = 5). With this latter procedure, we found that oestrogen injection caused a significant increase in the number of Fos immunoreactive cells in the arcuate nucleus within 1 h, but there was no response in the ventromedial hypothalamus. To further characterize the types of cells that might respond to oestrogen, we double-labelled cells for Fos and either adrenocorticotropin hormone, neuropeptide Y or tyrosine hydroxylase (a marker for dopaminergic cells). These cell types could account for less than 30% of the total number of cells that were Fos-positive and oestrogen treatment did not cause an increase in the Fos labelling of any of these types of cell. These data show that oestrogen activates cells of the arcuate/ventromedial hypothalamus within 1 h of injection and that this response could relate to the feedback effects of this gonadal hormone. The majority of cells that produce Fos following oestrogen injection are of unknown phenotype. The data further suggest that induction of cells of the ventromedial hypothalamic nucleus require more prolonged oestrogen stimulus than cells of the arcuate nucleus.
Assuntos
Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Estrogênios/farmacologia , Núcleo Hipotalâmico Ventromedial/citologia , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos , Hormônio Adrenocorticotrópico/análise , Animais , Núcleo Arqueado do Hipotálamo/fisiologia , Retroalimentação/fisiologia , Feminino , Hormônio Liberador de Gonadotropina/fisiologia , Imuno-Histoquímica , Injeções Intramusculares , Injeções Intravenosas , Neurônios/química , Neurônios/efeitos dos fármacos , Neuropeptídeo Y/análise , Ovariectomia , Proteínas Proto-Oncogênicas c-fos/análise , Ovinos , Tirosina 3-Mono-Oxigenase/análise , Núcleo Hipotalâmico Ventromedial/fisiologiaRESUMO
This study aimed to determine whether cells in the region of the arcuate and ventromedial hypothalamic nuclei (ARC/VMH) project to the gonadotropin-releasing hormone (GnRH) cells in the preoptic area (POA) and diagonal band of Broca (dbB) of the female sheep brain. An anterograde tracer, biotinylated dextran amine (BDA), was injected (70 nl) into the ARC/VMH (n=7) and the brains were perfused 3 weeks later. BDA terminals were mainly found in the dbB, POA and bed nucleus of stria terminalis (BNST). In order to determine the extent of input to GnRH neurons, we performed immunocytochemistry on the same sections with a GnRH antibody and examined close association of GnRH-immunoreactive (GnRH-IR) neurons (cell bodies and proximal dendrites) with BDA terminals. Of 223 GnRH-IR neurons that were examined, only three (1.3%) had BDA terminals in close proximity. Neither was close proximity observed between BDA terminals and GnRH-IR fibres. Injection of BDA into the BNST (n=6) showed terminals in POA, but only one of 273 GnRH-IR cells examined had BDA terminals in close proximity and no GnRH-IR fibres had BDA terminals in close proximity. Our results suggest that (1) although there are projections from the VMH/ARC to the dbB, POA and BNST, an interneuron or chain of interneurons is required for input to the GnRH neurones; (2) any input to GnRH neurons from the BNST involves at least one interneuron. The identity of these interneurons remains to be determined. Thus, input to the GnRH neurons from the estrogen receptor-rich area of ARC/VMH and from the BNST is not direct.
Assuntos
Núcleo Arqueado do Hipotálamo/citologia , Biotina/análogos & derivados , Hormônio Liberador de Gonadotropina/metabolismo , Área Pré-Óptica/citologia , Terminações Pré-Sinápticas/ultraestrutura , Núcleos Septais/citologia , Ovinos/anatomia & histologia , Núcleo Hipotalâmico Ventromedial/citologia , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Biotina/farmacocinética , Dextranos/farmacocinética , Feminino , Corantes Fluorescentes/farmacocinética , Imuno-Histoquímica , Vias Neurais/citologia , Vias Neurais/metabolismo , Área Pré-Óptica/metabolismo , Terminações Pré-Sinápticas/metabolismo , Núcleos Septais/metabolismo , Ovinos/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismoRESUMO
We measured the levels of progesterone receptor (PR) mRNA in the hypothalamus and preoptic area (POA) of the ewe across the oestrous cycle. Perfusion-fixed hypothalamic tissue was collected from sheep killed during the luteal and follicular phases and during behavioural oestrus. Blood samples taken at the time of tissue collection verified that the oestrous ewes were undergoing a preovulatory luteinizing hormone (LH) surge. Matched sections were taken from the POA, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and arcuate nucleus of each animal. In situ hybridization was performed using a sheep specific, (35)S-labelled riboprobe for PR and semiquantitative image analysis was conducted on emulsion-dipped slides. The number of silver grains per cell was greater in the VMN and arcuate nucleus of oestrous ewes than in luteal and follicular phase ewes; there was no cyclic variation in the level of PR mRNA expression in the cells of the POA and PeVN. The number of labelled cells per mm2 in the VMN was higher in the oestrous ewes than in luteal phase and follicular phase ewes. The number of labelled cells in the PeVN was also higher in the oestrous ewes than in follicular phase ewes, but there was no cyclic variation in the POA and arcuate nucleus. In the ewe, the onset of behavioural oestrus corresponds to the onset of the preovulatory LH surge and increased PR mRNA expression at this time is likely to be due to the high concentrations of circulating oestrogen that precede this period.
Assuntos
Estro/fisiologia , Área Pré-Óptica/fisiologia , Receptores de Progesterona/genética , Animais , Núcleo Arqueado do Hipotálamo/fisiologia , Feminino , Expressão Gênica/fisiologia , Hormônio Liberador de Gonadotropina/fisiologia , Hibridização In Situ , Núcleo Hipotalâmico Paraventricular/fisiologia , RNA Mensageiro/análise , Ovinos , Núcleo Hipotalâmico Ventromedial/fisiologiaRESUMO
Previous studies have shown that there is a population of noradrenergic cells in the caudal A1 field of the brainstem of the ewe that contain oestrogen receptors and project to the preoptic area, where gonadotrophin releasing hormone (GnRH) neurones are located. There are some discrepancies in the literature regarding the extent of this projection and the location of the cells in the A1 region. The issue has been a focus of attention because the positive feedback response to oestrogen that causes the ovulatory GnRH/luteinizing hormone surge may originate from this brainstem region. The aim of the present study was to determine the extent of the projections to the preoptic area and to determine whether the caudal A1 cells are activated by oestrogen. Eleven ovariectomized ewes received an injection of the retrograde tracer FluoroGold into the preoptic hypothalamus and four of these also received an i.m. injection of oestrogen 2 h before tissue collection. A further three sheep received i.m. oil injections to act as controls for those receiving oestrogen. Dopamine-beta-hydroxylase (DBH)-positive, retrogradely labelled cells were found within the A1 field in sheep that received preoptic FluoroGold injections. Cells in the vicinity of the A2 and A6 fields, that were retrogradely labelled with FluoroGold, were not DBH-positive. Thus, cells in the A1 field provide a direct noradrenergic projection to the preoptic area and may be involved in the control of the secretion of GnRH in this species. Cells that project to the preoptic hypothalamus from more rostrally located areas of the brainstem are not noradrenergic. In the animals that received oestrogen, double-labelling immunohistochemistry was performed throughout the A1 field for FluoroGold, DBH and Fos. DBH cells of the A1 field expressed Fos only in the oestrogen-treated animals and not in the oil-treated animals. There was a decline in the number of DBH cells that were retrogradely labelled from the caudal region of A1 towards obex. There was a similar gradient in the number of cells that were double-labelled for Fos and FluoroGold. We conclude that there is a population of noradrenergic cells in the caudal A1 field that project to the preoptic area; this is a larger group of cells than previously reported. Oestrogen elicits an acute Fos response in these cells, which may be involved in the time-delayed positive feedback response on GnRH cells. The caudal-to-rostral gradient in the labelling with FluoroGold and Fos in DBH-positive cells is similar to that seen previously for oestrogen receptor in DBH-positive cells in the A1 field.
Assuntos
Tronco Encefálico/fisiologia , Estrogênios/farmacologia , Norepinefrina/fisiologia , Área Pré-Óptica/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estilbamidinas , Transmissão Sináptica/fisiologia , Animais , Tronco Encefálico/citologia , Tronco Encefálico/efeitos dos fármacos , Dopamina beta-Hidroxilase/metabolismo , Feminino , Corantes Fluorescentes , Neurônios/metabolismo , Neurônios/fisiologia , OvariectomiaRESUMO
Non-centrosymmetric structures are promoted through the use of perfluoroaromatics containing structure-directing CN...X (X = Br, I) supramolecular synthons.