Efeitos macrovasculares, microvasculares e interação leucócito-endotélio, na endotoxemia experimental após o uso de Dobutamina com e sem ressuscitação volêmica

Na sepse, o mecanismo desencadeador de morte é a disfunção múltipla de órgãos e sistemas. Com isso a microcirculação é considerada o motor na patogênese da sepse. A perfusão microcirculatória representa um dos principais objetivos para melhorar as taxas de sobrevida. Uma vez reconhecida a síndrome séptica, o protocolo clínico estabelece o uso de fluidoterapia com salina, de forma vigorosa na primeira hora e seguida de suporte inotrópico com Dobutamina. A partir daí foi levantada a hipótese das drogas β-agonistas serem relevantes na recuperação da microcirculação, antes mesmo de seu conhecido papel na recuperação do choque cardiogênico. Assim, estudar o papel da Dobutamina, um β-agonista, na resposta adrenérgica em situação de sepse se faz necessário e urgente e o entendimento de sua ação, associada à reposição volêmica, foi objeto deste estudo. Foram usados no presente estudo, 78 hamsters, induzida a endotoxemia com LPS (2mg/kg/de massa de peso corporal) e divididos em 9 grupos: controle (n=10), endotóxico(n=10), endotóxico tratados com Dobutamina na dose de 5 e 15 μg /kg/min (n=10), Isoproterenol(n=10), ressuscitação volêmica (n=10) e ressuscitação volêmica associada à Dobutamina 5 (n=10) e 15 μg/kg/min (n=4) e Isoproterenol (n=4). Foram comparados os resultados de recuperação da densidade capilar funcional ao longo do tempo entre os grupos, e obteve-se resultado estatisticamente significativo no grupo em que se usa Dobutamina de 5μg/kg/min associada à ressuscitação volêmica p< 0,05. Em conclusão este estudo mostra que o papel da ressuscitação volêmica é crucial na resposta da microcirculação para melhorar a densidade capilar funcional, que a velocidade da hemácia capilar tem relação direta com a melhora na perfusão tecidual e que a associação de recuperação volêmica com solução salina e Dobutamina na dose de 5 μg /kg /min melhora significativamente sua resposta e melhora a perfusão.

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.

Tissue viability imaging of skin microcirculation following exposure to whole body cryotherapy (-110°C) and cold water immersion (8°C)

Cryotherapy is currently used in various clinical, rehabilitative, and sporting settings. However, very little is known regarding the impact of cooling on the microcirculatory response. Objectives: The present study sought to examine the influence of two commonly employed modalities of cryotherapy, whole body cryotherapy (WBC; -110°C) and cold water immersion(CWI; 8±1°C), on skin microcirculation in the mid- thigh region. Methods: The skin area examined was a 3 × 3 cm located between the most anterior aspect of the inguinal fold and the patella. Following 10 minutes of rest, 5 healthy, active males were exposed to either WBC for 3 minutes or CWI for 5 minutes in a randomised order. Volunteers lay supine for five minutes after treatment, in order to monitor the variation of red blood cell (RBC) concentration in the region of interest for a duration of 40 minutes. Microcirculation response was assessed using a non-invasive, portable instrument known as a Tissue Viability imaging system. After a minimum of seven days, the protocol was repeated. Subjective assessment of the volunteer’s thermal comfort and thermal sensation was also recorded. Results: RBC was altered following exposure to both WBC and CWI but appeared to stabilise approximately 35 minutes after treatments. Both WBC and CWI affected thermal sensation (p < 0.05); however no betweengroup differences in thermal comfort or sensation were recorded (p > 0.05). Conclusions: As both WBC and CWI altered RBC, further study is necessary to examine the mechanism for this alteration during whole body cooling.

The effects of slip velocity at a membrane surface on blood flow in the microcirculation

Closed-form solutions are presented for blood flow in the microcirculation by taking into account the influence of slip velocity at the membrane surface. In this study, the convective inertia force is neglected in comparison with that of blood viscosity on the basis of the smallness of the Reynolds number of the flow in microcirculation. The permeability property of the blood vessel is based on the well known Starling's hypothesis [11]. The effects of slip coefficient on the velocity and pressure fields are clearly depicted.

A couple stress model of blood flow in the microcirculation

A simple mathematical model depicting blood flow in the capillary is developed with an emphasis on the permeability property of the blood vessel based on Starling's hypothesis. In this study the effect of inertia has been neglected in comparison with the viscosity on the basis of the smallness of the Reynolds number of the flow in the capillary. The capillary blood vessel is approximated by a circular cylindrical tube with a permeable wall. The blood is represented by a couple stress fluid. With such an ideal model the velocity and pressure fields are determined. It is shown that an increase in the couple stress parameter increases the resistance to the flow and thereby decreases the volume rate flow. A comparison of the results with those of the Newtonian case has also been made.

Carotid Stenosis, Endarterectomy, and the Brain : Brain microcirculation, diffusion, and cognitive function before and after carotid endarterectomy in patients with a high-grade carotid stenosis

Carotid atherosclerotic disease is a major cause of stroke, but it may remain clinically asymptomatic. The factors that turn the asymptomatic plaque into a symptomatic one are not fully understood, neither are the subtle effects that a high-grade carotid stenosis may have on the brain. The purpose of this study was to evaluate brain microcirculation, diffusion, and cognitive performance in patients with a high-grade stenosis in carotid artery, clinically either symptomatic or asymptomatic, undergoing carotid endarterectomy (CEA). We wanted to find out whether the stenoses are associated with diffusion or perfusion abnormalities of the brain or variation in the cognitive functioning of the patients, and to what extent the potential findings are affected by CEA, and compare the clinically symptomatic and asymptomatic subjects as well as strictly healthy controls. Coagulation and fibrinolytic parameters were compared with the rate microembolic signals (MES) in transcranial Doppler (TCD) and the macroscopic appearance of stenosing plaques in surgery. Patients (n=92) underwent CEA within the study. Blood samples pertaining to coagulation and fibrinolysis were collected before CEA, and the subjects underwent repeated TCD monitoring for MES. A subpopulation (n= 46) underwent MR imaging and repeated neuropsychological examination (preoperative, as well 4 and 100 days after CEA). In MRI, the average apparent diffusion coefficients were higher in the ipsilateral white matter (WM), and altough the interhemispheric difference was abolished by CEA, the levels remained higher than in controls. Symptomatic stenoses were associated with more sluggish perfusion especially in WM, and lower pulsatility of flow in TCD. All patients had poorer cognitive performance than healthy controls. Cognitive functions improved as expected by learning effect despite transient postoperative worsening in a few subjects. Improvement was greater in patients with deepest hypoperfusion, primarily in executive functions. Symptomatic stenoses were associated with higher hematocrit and tissue plasminogen activator antigen levels, as well as higher rate of MES and ulcerated plaques, and better postoperative improvement of vasoreactivity and pulsatility. In light of the findings, carotid stenosis is associated with differences in brain diffusion, perfusion, and cognition. The effect on diffusion in the ipsilateral WM, partially reversible by CEA, may be associated with WM degeneration. Asymptomatic and symptomatic subpopulations differ from each other in terms of hemodynamic adaptation and in their vascular physiological response to removal of stenosis. Although CEA may be associated with a transient cognitive decline, a true improvement of cognitive performance by CEA is possible in patients with the most pronounced perfusion deficits. Mediators of fibrinolysis and unfavourable hemorheology may contribute to the development of a symptomatic disease in patients with a high-grade stenosis.

Citation Analysis of Theses/Dissertations for a Marine Biological Laboratory

A basic analysis of sources, dates, authors for an Environmental Science Laboratory based at waters edge. (22 powerpoint slides)

Selective loss of vascular smooth muscle cells in the retinal microcirculation of diabetic dogs

This study was undertaken to further characterise the fine structural changes occurring in the retinal circulation in early diabetes. The eyes of eight alloxan/streptozotocin and three spontaneously diabetic dogs were examined by trypsin digest and electron microscopy after durations of diabetes of between 1 and 7 years. Basement membrane (BM) thickening in the retinal capillaries was the only obvious fine structural change identified during the first 3 years of diabetes and was established within 1 year of induction. Widespread pericyte loss was noted after 4 years of diabetes and was paralleled by loss of smooth muscle (SM) cells, in the retinal arterioles. SM cell loss was most obvious in the smaller arterioles of the central retina. No microaneurysms were noted in the experimental diabetic dogs with up to 5 years' duration of diabetes but were widespread in a spontaneously diabetic animal at 7 years. This study has shown that SM cell loss, a hitherto unrecognised feature of diabetic microangiopathy, accompanies pericyte loss in the retinal circulation of diabetic dogs.

Endocytosis in the retinal and choroidal microcirculation

The endocytosis of horseradish peroxidase (HRP) by the vascular cells of retinal and choroidal blood vessels was compared in immersion and perfusion fixed eyes from individual rats. The mechanisms of endocytosis of HRP appeared identical in both retinal and choroidal vessels. The bulk of internalised tracer occurred in macropinosomes 300-400 nm in diameter. Tracer was localised to a 20-30 nm layer on the internal aspect of the limiting membrane. This layer was coincident with the glycocalyx of the luminal plasma membrane as revealed by ruthenium redosmium tetroxide staining. Horseradish peroxidase was also internalised by a small scattered population of vesicles (100-130 nm in diameter). The size of these vesicles suggested that they may have arisen from clathrin coated regions of the plasma membrane. It is suggested that the endocytosis of HRP in retinal and choroidal vascular endothelium occurs as a function of plasma membrane recycling. Horseradish peroxidase may also be internalised as a 'contaminant' of the glycocalyx in coated pits involved in receptor mediated endocytosis. The smooth 80 nm plasmalemmal caveolae of the retinal and choroidal vascular endothelial cells did not appear to participate either in absorptive endocytosis or vesicular transport.

Intracoronary Delivery of Human Mesenchymal/Stromal Stem Cells: Insights from Coronary Microcirculation Invasive Assessment in a Swine Model

BACKGROUND: Mesenchymal stem/stromal cells have unique properties favorable to their use in clinical practice and have been studied for cardiac repair. However, these cells are larger than coronary microvessels and there is controversy about the risk of embolization and microinfarctions, which could jeopardize the safety and efficacy of intracoronary route for their delivery. The index of microcirculatory resistance (IMR) is an invasive method for quantitatively assessing the coronary microcirculation status. OBJECTIVES: To examine heart microcirculation after intracoronary injection of mesenchymal stem/stromal cells with the index of microcirculatory resistance. METHODS: Healthy swine were randomized to receive by intracoronary route either 30x106 MSC or the same solution with no cells (1% human albumin/PBS) (placebo). Blinded operators took coronary pressure and flow measurements, prior to intracoronary infusion and at 5 and 30 minutes post-delivery. Coronary flow reserve (CFR) and the IMR were compared between groups. RESULTS: CFR and IMR were done with a variance within the 3 transit time measurements of 6% at rest and 11% at maximal hyperemia. After intracoronary infusion there were no significant differences in CFR. The IMR was significantly higher in MSC-injected animals (at 30 minutes, 14.2U vs. 8.8U, p = 0.02) and intragroup analysis showed a significant increase of 112% from baseline to 30 minutes after cell infusion, although no electrocardiographic changes or clinical deterioration were noted. CONCLUSION: Overall, this study provides definitive evidence of microcirculatory disruption upon intracoronary administration of mesenchymal stem/stromal cells, in a large animal model closely resembling human cardiac physiology, function and anatomy.