RESUMO
Diabetes is among the most frequently diagnosed endocrine disorder in dogs and its prevalence continues to increase. Medical management of this pathology is lifelong and challenging because of the numerous serious complications. A therapy based on the use of autologous viable insulin-producing cells to replace the lost ß cell mass would be very advantageous. A protocol to enable the epigenetic conversion of canine dermal fibroblasts, obtained from a skin biopsy, into insulin-producing cells (EpiCC) is described in the present manuscript. Cells were briefly exposed to the DNA methyltransferase inhibitor 5-azacytidine (5-aza-CR) in order to increase their plasticity. This was followed by a three-step differentiation protocol that directed the cells towards the pancreatic lineage. After 36 days, 38 ± 6.1% of the treated fibroblasts were converted into EpiCC that expressed insulin mRNA and protein. Furthermore, EpiCC were able to release insulin into the medium in response to an increased glucose concentration. This is the first evidence that generating a renewable autologous, functional source of insulin-secreting cells is possible in the dog. This procedure represents a novel and promising potential therapy for diabetes in dogs.
Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Epigênese Genética , Fibroblastos/citologia , Células Secretoras de Insulina/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Cães , Cultura Primária de Células , Pele/citologia , Pele/crescimento & desenvolvimentoRESUMO
A combined physical-chemical protocol for whole full-thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non-ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole-organ mechanical response to a physiological-like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content < 50 ng/mg dry tissue weight. Furthermore, the collagen network and elastic fibres distribution were preserved in the acellular ECM, which exhibited suitable biomechanical properties in the perspective of its future use as an implant for bladder augmentation.
Assuntos
Matriz Extracelular/metabolismo , Engenharia Tecidual/métodos , Bexiga Urinária/citologia , Bexiga Urinária/fisiologia , Animais , Fenômenos Biomecânicos , DNA/metabolismo , Matriz Extracelular/ultraestrutura , Masculino , RNA/metabolismo , Coelhos , Coloração e Rotulagem , Suporte de CargaRESUMO
Detailed knowledge of the vascular anatomy is important for improving surgical approaches to the liver. Twelve canine livers were skeletonized to describe the anatomy of their portal and hepatic veins in details. Our data suggest that the liver can be divided into two sections, three divisions, seven lobes and two to four sub-lobes. This differs from the classic division into four lobes, four sub-lobes and two processes. The right section was perfused by the right portal branch and drained by independent hepatic veins, while most of the left section, perfused by the left portal branch, was drained by the main hepatic vein deriving from the middle and the left hepatic vein confluence. Part of the right medial lobe, and in some cases the papillary process of the caudate lobe, drained directly into the caudal vena cava. A proper right hepatic vein draining blood from more than one lobe was never observed. Portal connections between the quadrate and the left medial lobe were frequently recorded. Two sub-lobes with different portal blood supply and venous drainage could be identified in the right lateral (33.3% of cases) and the left lateral (100% of cases) lobes. From our results, the classic nomenclature of the liver lobes does not reflect their vascularization. Based on similarities between canine lobes and human segments, a new nomenclature is possible and may be less confounding in surgical settings.
Assuntos
Cães/anatomia & histologia , Veias Hepáticas/anatomia & histologia , Fígado/cirurgia , Veia Porta/anatomia & histologia , Animais , Cães/cirurgia , Feminino , Humanos , Fígado/anatomia & histologia , Fígado/irrigação sanguínea , Masculino , Veia Cava Inferior/anatomia & histologiaRESUMO
Cardiovascular disease is the leading cause of death in developed countries and is one of the leading causes of disease burden in developing countries. Therapies have markedly increased survival in several categories of patients, nonetheless mortality still remains high. For this reason high hopes are associated with recent developments in stem cell biology and regenerative medicine that promise to replace damaged or lost cardiac muscle with healthy tissue, and thus to dramatically improve the quality of life and survival in patients with various cardiomyopathies. Much of our insight into the molecular and cellular basis of cardiovascular biology comes from small animal models, particularly mice. However, significant differences exist with regard to several cardiac characteristics when mice are compared with humans. For this reason, large animal models like dog, sheep and pig have a well established role in cardiac research. A distinct characteristic of cardiac stem cells is that they can either be endogenous or derive from outside the heart itself; they can originate as the natural course of their differentiation programme (e.g., embryonic stem cells) or can be the result of specific inductive conditions (e.g., mesenchymal stem cells). In this review we will summarize the current knowledge on the kind of heart-related stem cells currently available in large animal species and their relevance to human studies as pre-clinical models.
Assuntos
Cardiopatias/cirurgia , Miocárdio/citologia , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Células-Tronco/fisiologia , AnimaisRESUMO
The pharmacokinetics and intramuscular (IM) bioavailability of flumequine (15 mgkg(-1)) were investigated in healthy pigs and the findings related to published minimal inhibitory concentrations (MICs) for susceptible bacteria of animal origin, and to experimentally determined MICs for susceptible strains of porcine origin. We found MICs for Escherichia coli, Salmonella spp., Pasteurella spp. and Bordetella spp. in the range 0.5 to >64 microg mL(-1) isolated from infected pigs in the Forli area of Italy; only the Pasteurella multocida strains were sensitive (MIC(90)=0.5 microg mL(-1)). After intravenous (IV) injection, flumequine was slowly distributed and eliminated (t(1/2lambda(1))1.40+/-0.16 h and t(1/2lambda(2))6.35+/-1.69 h). The distribution volume at steady state (V(dss)) was 752.59+/-84.03 mL kg(-1) and clearance (Cl(B)) was 237.19+/-17.88 mL kg(-1)h(-1). After IM administration, peak serum concentration (4.99+/-0.92 microg mL(-1)) was reached between the 2nd and the 3rd hour. The results on MIC of isolated bacteria, although only indicative, suggest that the efficacy of flumequine on Gram-negative bacteria may be impaired by the emergence of less sensitive or resistant strains.
Assuntos
Fluoroquinolonas/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Suínos/metabolismo , Animais , Área Sob a Curva , Estudos Cross-Over , Escherichia coli/efeitos dos fármacos , Feminino , Fluoroquinolonas/administração & dosagem , Fluoroquinolonas/sangue , Fluoroquinolonas/farmacocinética , Injeções Intramusculares/veterinária , Injeções Intravenosas/veterinária , Masculino , Testes de Sensibilidade Microbiana , Pasteurella/efeitos dos fármacos , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/veterinária , Salmonella/efeitos dos fármacos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/microbiologiaRESUMO
Knowledge of the mechanical behaviour of immature tracheae is crucial in order to understand the effects exerted on central airways by ventilatory treatments, particularly of Total Liquid Ventilation. In this study, a combined experimental and computational approach was adopted to investigate the compliance and particularly collapsibility of preterm lamb tracheae in the range of pressure likely applied during Total Liquid Ventilation (-30 to 30 cmH2O). Tracheal samples of preterm lambs (n = 5; gestational age 120-130 days) were tested by altering transmural pressure from -30 to 30 cmH2O. Inflation (Si) and collapsing (Sc) compliance values were calculated in the ranges 0 to 10 cmH2O and -10 to 0 cmH2O, respectively. During the tests, an asymmetric behaviour of the DeltaV/V0 vs. P curves at positive and negative pressure was observed, with mean Si = 0.013 cmH2O(-1) and Sc = 0.053 cmH2O(-1). A different deformed configuration of the sample regions was observed, depending on the posterior shape of cartilaginous ring. A three-dimensional finite-element structural model of a single tracheal ring, based on histology measurements of the tested samples was developed. The model was parameterised in order to represent rings belonging to three different tracheal regions (craniad, median, caudal) and numerical analyses replicating the collapse test conditions were performed to evaluate the ring collapsibility at pressures between 0 and -30 cmH2O. Simulation results were compared to experimental data to verify the model's reliability. The best model predictions occurred at pressures -30 to -10 cmH2O. In this range, a model composed of median rings best interpreted the experimental data, with a maximum error of 2.7%; a model composed of an equal combination of all rings yielded an error of 12.6%.
Assuntos
Feto/fisiologia , Traqueia/fisiologia , Animais , Fenômenos Biomecânicos , Complacência (Medida de Distensibilidade) , Feminino , Feto/anatomia & histologia , Idade Gestacional , Técnicas In Vitro , Ventilação Líquida , Modelos Anatômicos , Modelos Biológicos , Gravidez , Pressão , Ovinos , Traqueia/anatomia & histologiaRESUMO
Knowledge of immature tracheae mechanical behavior is fundamental in understanding the effects exerted on the upper airways by tidal liquid ventilation (TLV). Particularly, negative pressure can take place along the airways during expiration, which can cause airway collapse and flow limitation; therefore, representing a critical issue in preterm infant patients, whose airways are less stiff than adult ones. In this study, we investigated the expiratory pressure drop vs flow relationship of isolated preterm lamb tracheal samples to determine their hydraulic resistance, collapse pressure and collapse flow rate; a liquid flow through the samples was obtained by applying negative pressure at the outlet (cephalad) extremity of the tra-cheal sample, while keeping the inlet (caudal) extremity at atmospheric pressure. Histological analyzes were performed on the tracheal samples after each test session, in order to examine the morphological structure of the tracheal wall. Flow resistance tests demonstrated progressive lumen narrowing at increasing pressure drop (∆P=P in -P out ). The flow rate increased with ∆P un-til a plateau was reached, and then decreased, describing the onset of a collapse phenomenon; however, complete occlusion was not reached. The tracheal samples demonstrated a similar behavior to that of a Starling resistor during the collapse phase: when a critical ∆P was reached, collapse was observed starting at the outlet region, which was subjected to the greatest negative pressure, then propagating towards the caudal direction. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 177-82).
RESUMO
The aim of our research is to develop a laryngeal tissue augmentation technique that will be useful in treating glottal and neoglottal insufficiency. In a previous study the feasibility of insertion of tubular expanded poly-tetrafluoroethylene (e-PTFE) tissue implants into the larynx of a porcine model was demonstrated. In this paper we report the clinical, endoscopic, computed tomography (CT) and magnetic resonance image (MRI) and histopathological follow-up findings. These results demonstrate host tolerance of the implant over the period of study (six months) with little tissue reactivity to tubular e-PTFE implants in the porcine model. The tubular shape of the implant enhances stabilization by the rapid and complete host tissue penetration of the implant's interstices, making tubular e-PTFE a potentially useful soft tissue augmentation material for laryngeal incompetence.
Assuntos
Laringe/cirurgia , Politetrafluoretileno , Próteses e Implantes , Paralisia das Pregas Vocais/cirurgia , Animais , Modelos Animais de Doenças , Laringoscopia , Laringe/diagnóstico por imagem , Laringe/patologia , Imageamento por Ressonância Magnética , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Período Pós-Operatório , Implantação de Prótese/métodos , Suínos , Tomografia Computadorizada por Raios XRESUMO
The problem of the therapy of glottal and neoglottal insufficiency is still unsolved. The surgical techniques and the materials employed up until now are not completely free from possible partial or total failures, early or late complications, or the need of a second operation. The objective of the study is to introduce a new thyroplasty technique of inserting tubular expanded polytetrafluoroethylene (e-PTFE) into the larynx of an animal model, with the purpose of using it next in human laryngeal tissue augmentation. Seven 30 kg pigs had tubular e-PTFE implanted under endoscopic control into a vocal fold or laryngeal vestibule. The implants were secured by suture to the thyroid cartilage. Short-term results demonstrate the ease and effectiveness of this mini-invasive implant surgical technique. Long-term follow-up is underway in anticipation of applying this technique to human laryngeal tissue augmentation.
Assuntos
Glote , Implantes Experimentais , Doenças da Laringe/cirurgia , Laringoscopia/métodos , Implantação de Prótese/métodos , Animais , Humanos , Laringe Artificial , Politetrafluoretileno/uso terapêutico , SuínosRESUMO
OBJECTIVE: To describe an anatomic and surgical approach to the efferent parasympathetic branches of the pterygopalatine ganglia in sheep, with particular reference to the ethmoidal nerve and innervation of nasal and cerebral blood vessels. ANIMALS: 12 adult sheep used for monolateral (n = 7) or bilateral (n = 5) ethmoidal neurectomy; 2 sheep used for angiography (1 live sheep for digital subtraction angiography, 1 embalmed cadaver for injection studies); and 5 embalmed cadavers, 4 frozen specimens, and 2 dry skulls used for dissection, x-rays, and computed tomographic (CT) or magnetic resonance (MR) scans. PROCEDURE: Transverse (coronal) MR scans, transverse, sagittal, and dorsal CT scans, radiography, angiography, photographic images, and dissections of embalmed material were used to study the topographic anatomy of the temporal and pterygopalatine fossae of the head. RESULTS: Images were stored, then compared with photographs of frozen sections from the same or a similar specimen to plan a surgical approach to the ethmoidal nerve. Mono- and bilateral experimental ethmoidal neurectomies were performed, allowing characterization of a safe and reliable method. The series of pterygopalatine ganglia typical of this species was localized, dissected, and analyzed for topographic relations. CONCLUSIONS: From the results, a new approach to the efferent branches of the pterygopalatine ganglia (ethmoidal nerve) for experimental parasympathectomy of the cerebral and nasal circle is proposed. This experimental approach could be used for studies involving thermoregulation of the face, and in experimental control of blood flow in the nasal cavity and rostral part of the brain.
Assuntos
Seio Etmoidal/inervação , Gânglios Parassimpáticos/anatomia & histologia , Ovinos/anatomia & histologia , Angiografia Digital/veterinária , Animais , Encéfalo/anatomia & histologia , Encéfalo/irrigação sanguínea , Encéfalo/fisiologia , Gânglios Parassimpáticos/fisiologia , Imageamento por Ressonância Magnética/veterinária , Cavidade Nasal/irrigação sanguínea , Cavidade Nasal/inervação , Órbita/irrigação sanguínea , Estudos Prospectivos , Ovinos/cirurgia , Tomografia Computadorizada por Raios X/veterináriaRESUMO
To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3-5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 +/- 1.15 versus 122.83 +/- 0.74 microns). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [35S]methionine and [35S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence.
Assuntos
Envelhecimento/fisiologia , Oócitos/crescimento & desenvolvimento , Animais , Bovinos , Separação Celular/veterinária , Metabolismo Energético/fisiologia , Feminino , Meiose/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Biossíntese de ProteínasRESUMO
After a major search of the more recent literature, the Authors present a critical examination of guidelines now in use for the surgical treatment in utero of numerous fetal abnormalities. Specifically, they underline not only ethical and deontological but also technical principles relating to this particular surgery. They are also involved in examining the depth all the more common surgical procedures used in different pathologies and in suggesting new lines of future development.