Avaliação estereológica glomerular em rins de ratos após isquemia-reperfusão por clampeamento arteriovenoso e arterial

A técnica de isquemia-reperfusão tem sido utilizada em cirurgias conservadoras do rim como a nefrectomia parcial e em transplantes renais. Para se realizar a isquemia pode-se fazer o bloqueio do fluxo sanguíneo da artéria renal ou o bloqueio simultâneo da artéria e da veia renal. O evento isquêmico acarreta em dano celular ao rim principalmente pelo estresse oxidativo local e a liberação de radicais livres assim como o aumento da resposta inflamatória. Diversos autores verificaram lesão renal após a isquemia-reperfusão, porém, apenas testes funcionais foram realizados até o momento. Os autores que tentaram avaliar a lesão morfológica do rim apenas fizeram a quantificação de escores subjetivos. O nosso objetivo é avaliar por quantificação estereológica o dano causado pela isquemia-reperfusão comparando o clampeamento somente arterial com o clampeamento arteriovenoso. Utilizamos 24 ratos wistar, machos, de quatro meses de idade. Os animais foram divididos em três grupos: o grupo Sham (n=8), o grupo de clampeamento somente da artéria renal (n=8) e o grupo de clampeamento simultâneo da artéria e da veia renal (n=8). Os animais foram submetidos a laparotomia mediana. Os animais do grupo Sham permaneceram os 60 minutos anestesiados mas sem obstrução do fluxo sanguíneo de seus vasos renais. Os animais do grupo de clampeamento arterial foram submetidos à clampeamento de sua arterial renal esquerda por 60 minutos e os animais do grupo de clampeamento arterial e venoso tiveram seus vasos renais esquerdos clampeados simultaneamente e em bloco pelo mesmo tempo. Após os 60 minutos os animais foram suturados e mantidos por 30 dias em caixas próprias sendo mortos por sobredose anestésica após decorrido esse tempo. Os rins foram coletados e mantidos em solução de formalina tamponada e posteriormente processados para análise histológica e estereológica. Foram analisados a densidade volumétrica (Vv) dos glomérulos, o número de glomérulos/mm3(Nv) e o volume glomerular médio (VGM). A Vv e Nv se encontrou reduzida nos rins esquerdos submetidos à isquemia mas foi somente significativa nos animais do grupo de clampeamento arterial e venoso. Mesmo usando o rato como modelo animal experimental, a partir de nossos resultados recomendamos o uso do clampeamento somente arterial nos casos em que mínina lesão ao rim é imperiosa.

An extended computational model of the circulatory system for designing ventricular assist devices.

An extended computational model of the circulatory system has been developed to predict blood flow in the presence of ventricular assist devices (VADs). A novel VAD, placed in the descending aorta, intended to offload the left ventricle (LV) and augment renal perfusion is being studied. For this application, a better understanding of the global hemodynamic response of the VAD, in essence an electrically driven pump, and the cardiovascular system is necessary. To meet this need, a model has been established as a nonlinear, lumped-parameter electrical analog, and simulated results under different states [healthy, congestive heart failure (CHF), and postinsertion of VAD] are presented. The systemic circulation is separated into five compartments and the descending aorta is composed of three components to accurately yield the system response of each section before and after the insertion of the VAD. Delays in valve closing time and blood inertia in the aorta were introduced to deliver a more realistic model. Pump governing equations and optimization are based on fundamental theories of turbomachines and can serve as a practical initial design point for rotary blood pumps. The model's results closely mimic established parameters for the circulatory system and confirm the feasibility of the intra-aortic VAD concept. This computational model can be linked with models of the pump motor to provide a valuable tool for innovative VAD design.

beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice.

Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and Gi/Go proteins, here we dissected the roles of G proteins and the adaptor proteins, beta-arrestins, in nicotinic acid-induced signaling and physiological responses. In a human cell line-based signaling assay, nicotinic acid stimulation led to pertussis toxin-sensitive lowering of cAMP, recruitment of beta-arrestins to the cell membrane, an activating conformational change in beta-arrestin, and beta-arrestin-dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of beta-arrestin1 to activated cytosolic phospholipase A2 as well as beta-arrestin1-dependent activation of cytosolic phospholipase A2 and release of arachidonate, the precursor of prostaglandin D2 and the vasodilator responsible for the flushing response. Moreover, beta-arrestin1-null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by beta-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein-biased ligands that activate GPR109A in a beta-arrestin-independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.

Magnetic Resonance Imaging Biomarkers of Renal Structure and Function

The kidney's major role in filtration depends on its high blood flow, concentrating mechanisms, and biochemical activation. The kidney's greatest strengths also lead to vulnerability for drug-induced nephrotoxicity and other renal injuries. The current standard to diagnose renal injuries is with a percutaneous renal biopsy, which can be biased and insufficient. In one particular case, biopsy of a kidney with renal cell carcinoma can actually initiate metastasis. Tools that are sensitive and specific to detect renal disease early are essential, especially noninvasive diagnostic imaging. While other imaging modalities (ultrasound and x-ray/CT) have their unique advantages and disadvantages, MRI has superb soft tissue contrast without ionizing radiation. More importantly, there is a richness of contrast mechanisms in MRI that has yet to be explored and applied to study renal disease.

The focus of this work is to advance preclinical imaging tools to study the structure and function of the renal system. Studies were conducted in normal and disease models to understand general renal physiology as well as pathophysiology. This dissertation is separated into two parts--the first is the identification of renal architecture with ex vivo MRI; the second is the characterization of renal dynamics and function with in vivo MRI. High resolution ex vivo imaging provided several opportunities including: 1) identification of fine renal structures, 2) implementation of different contrast mechanisms with several pulse sequences and reconstruction methods, 3) development of image-processing tools to extract regions and structures, and 4) understanding of the nephron structures that create MR contrast and that are important for renal physiology. The ex vivo studies allowed for understanding and translation to in vivo studies. While the structure of this dissertation is organized by individual projects, the goal is singular: to develop magnetic resonance imaging biomarkers for renal system.

The work presented here includes three ex vivo studies and two in vivo studies:

1) Magnetic resonance histology of age-related nephropathy in sprague dawley.

2) Quantitative susceptibility mapping of kidney inflammation and fibrosis in type 1 angiotensin receptor-deficient mice.

3) Susceptibility tensor imaging of the kidney and its microstructural underpinnings.

4) 4D MRI of renal function in the developing mouse.

5) 4D MRI of polycystic kidneys in rapamycin treated Glis3-deficient mice.

Snap-shot multispectral imaging of vascular dynamics in a mouse window-chamber model.

Understanding tumor vascular dynamics through parameters such as blood flow and oxygenation can yield insight into tumor biology and therapeutic response. Hyperspectral microscopy enables optical detection of hemoglobin saturation or blood velocity by either acquiring multiple images that are spectrally distinct or by rapid acquisition at a single wavelength over time. However, the serial acquisition of spectral images over time prevents the ability to monitor rapid changes in vascular dynamics and cannot monitor concurrent changes in oxygenation and flow rate. Here, we introduce snap shot-multispectral imaging (SS-MSI) for use in imaging the microvasculature in mouse dorsal-window chambers. By spatially multiplexing spectral information into a single-image capture, simultaneous acquisition of dynamic hemoglobin saturation and blood flow over time is achieved down to the capillary level and provides an improved optical tool for monitoring rapid in vivo vascular dynamics.

Anti-hypotensive treatment and endothelin blockade synergistically antagonize exercise fatigue in rats under simulated high altitude.

Rapid ascent to high altitude causes illness and fatigue, and there is a demand for effective acute treatments to alleviate such effects. We hypothesized that increased oxygen delivery to the tissue using a combination of a hypertensive agent and an endothelin receptor A antagonist drugs would limit exercise-induced fatigue at simulated high altitude. Our data showed that the combination of 0.1 mg/kg ambrisentan with either 20 mg/kg ephedrine or 10 mg/kg methylphenidate significantly improved exercise duration in rats at simulated altitude of 4,267 m, whereas the individual compounds did not. In normoxic, anesthetized rats, ephedrine alone and in combination with ambrisentan increased heart rate, peripheral blood flow, carotid and pulmonary arterial pressures, breathing rate, and vastus lateralis muscle oxygenation, but under inspired hypoxia, only the combination treatment significantly enhanced muscle oxygenation. Our results suggest that sympathomimetic agents combined with endothelin-A receptor blockers offset altitude-induced fatigue in rats by synergistically increasing the delivery rate of oxygen to hypoxic muscle by concomitantly augmenting perfusion pressure and improving capillary conductance in the skeletal muscle. Our findings might therefore serve as a basis to develop an effective treatment to prevent high-altitude illness and fatigue in humans.

A generic time and space accurate numerical approach to closely coupled fluid-structure interaction problems

Fluid structure interaction, as applied to flexible structures, has wide application in diverse areas such as flutter in aircraft, flow in elastic pipes and blood vessels and extrusion of metals through dies. However a comprehensive computational model of these multi-physics phenomena is a considerable challenge. Until recently work in this area focused on one phenomenon and represented the behaviour of the other more simply even to the extent in metal forming, for example, that the deformation of the die is totally ignored. More recently, strategies for solving the full coupling between the fluid and soild mechanics behaviour have developed. Conventionally, the computational modelling of fluid structure interaction is problematical since computational fluid dynamics (CFD) is solved using finite volume (FV) methods and computational structural mechanics (CSM) is based entirely on finite element (FE) methods. In the past the concurrent, but rather disparate, development paths for the finite element and finite volume methods have resulted in numerical software tools for CFD and CSM that are different in almost every respect. Hence, progress is frustrated in modelling the emerging multi-physics problem of fluid structure interaction in a consistent manner. Unless the fluid-structure coupling is either one way, very weak or both, transferring and filtering data from one mesh and solution procedure to another may lead to significant problems in computational convergence. Using a novel three phase technique the full interaction between the fluid and the dynamic structural response are represented. The procedure is demonstrated on some challenging applications in complex three dimensional geometries involving aircraft flutter, metal forming and blood flow in arteries.

A Quantitative and Mechanistic Assessment of Activated Thrombin-Activatable Fibrinolysis Inhibitor and its Role in Pathological Bleeding and Thrombosis

The coagulation and fibrinolytic systems are linked by the thrombin-thrombomodulin complex which regulates each system through activation of protein C and TAFI, respectively. We have used novel assays and techniques to study the enzymology and biochemistry of TAFI and TAFIa, to measure TAFI activation in hemophilia A and protein C deficiency and to determine if enhancing TAFI activation can improve hemostasis in hemophilic plasma and whole blood. We show that TAFIa not TAFI attenuates fibrinolysis in vitro and this is supported by a relatively high catalytic efficiency (16.41μM-1s-1) of plasminogen binding site removal from fibrin degradation products (FDPs) by TAFIa. Since the catalytic efficiency of TAFIa in removing these sites is ~60-fold higher than that for inflammatory mediators such as bradykinin it is likely that FDPs are a physiological substrate of TAFIa. The high catalytic efficiency is primarily a result of a low Km which can be explained by a novel mechanism where TAFIa forms a binary complex with plasminogen and is recruited to the surface of FDPs. The low Km also suggests that TAFIa would effectively cleave lysines from FDPs during the early stages of fibrinolysis (i.e. at low concentrations of FDPs). Since individuals with hemophilia suffer from premature fibrinolysis as a result of insufficient TAFI activation we quantified TAFI activation in whole blood from hemophilic subjects. Both the rate of activation and the area under the TAFI activation time course (termed TAFIa potential) was determined to be reduced in hemophilia A and the TAFIa potential was significantly and inversely correlated with the clinical bleeding iii phenotype. Using a novel therapeutic strategy, we used soluble thrombomodulin to increase TAFI activation which improved the clot lysis time in factor VIII deficient human plasma and hemophilic dog plasma as well as hemophilic dog blood. Finally, we briefly show in a biochemical case study that TAFI activation is enhanced in protein C deficiency and when afflicted individuals are placed on Warfarin anticoagulant therapy, TAFI activation is reduced. Since TAFIa stabilizes blood clots, this suggests that reducing TAFI activation or inhibiting TAFIa may help restore blood flow in vessels with pathological thrombosis.

No evidence of endothelial dysfunction in patients with Alzheimer's disease

In view of accumulating evidence of vascular pathology in Alzheimer's disease (AD), we tested the hypothesis that AD patients have impaired endothelial function. This was assessed using the technique of strain-gauge venous occlusion plethysmography, which measures forearm blood flow (FBF). Intra-arterial (brachial) infusion of acetylcholine (ACh) and sodium nitroprusside (SNP) was used to assess local endothelial dependent and independent responses, respectively. There was no difference in the basal FBF of patients and controls. ACh and SNP caused dose-related increases in FBF from baseline, but no difference was recorded between the AD and control group. This study provides no evidence of endothelial dysfunction in the systemic circulation of patients with AD.

Higher brain neurons succumb to acute stroke-like insult while lower brain neurons strongly resist

Pyramidal neurons (PyNs) in ‘higher’ brain are highly susceptible to acute stroke injury yet ‘lower’ brain regions better survive global ischemia, presumably because of better residual blood flow. Here we show that projection neurons in ‘lower’ brain regions of hypothalamus and brainstem intrinsically resist acute stroke-like injury independent of blood flow in the brain slice. In contrast `higher` projection neurons in neocortex, hippocampus, striatum and thalamus are highly susceptible. In live brain slices from rat deprived of oxygen and glucose (OGD), we imaged anoxic depolarization (AD) as it propagates through these regions. AD, the initial electrophysiological event of stroke, is a depolarizing front that drains residual energy in compromised gray matter. The extent of AD reliably determines ensuing damage in higher brain, but using whole-cell recordings we found that all CNS neurons do not generate a robust AD. Higher neurons generate strong AD and show no functional recovery in contrast to neurons in hypothalamus and brainstem that generate a weak and gradual AD. Most dramatically, lower neurons recover their membrane potential, input resistance and spike amplitude when oxygen and glucose is restored, while higher neurons do not. Following OGD, new recordings could be acquired in all lower (but not higher) brain regions, with some neurons even withstanding multiple OGD exposure. Two-photon laser scanning microscopy confirmed neuroprotection in lower, but not higher gray matter. Specifically pyramidal neurons swell and lose their dendritic spines post-OGD, whereas neurons in hypothalamus and brainstem display no such injury. Exposure to the Na+/K+ ATPase inhibitor ouabain (100 μM), induces depolarization similar to OGD in all cell types tested. Moreover, elevated [K+]o evokes spreading depression (SD), a milder version of AD, in higher brain but not hypothalamus or brainstem so weak AD correlates with the inability to generate SD. In summary, overriding the Na+/K+ pump using OGD, ouabain or elevated [K+]o evokes steep and robust depolarization of higher gray matter. We show that this important regional difference can be largely accounted for by the intrinsic properties of the resident neurons and that Na+/K+ ATPase pump efficiency is a major determining factor generating strong or weak spreading depolarizations.

Altered pericyte-endothelial relations in the rat retina during aging: Implications for vessel stability

Mural cells (smooth muscle cells and pericytes) regulate blood flow and contribute to vessel stability. We examined whether mural cell changes accompany age-related alterations in the microvasculature of the central nervous system. The retinas of young adult and aged Wistar rats were subjected to immunohistofluorescence analysis of a-smooth muscle actin (SMA), caldesmon, calponin, desmin, and NG2 to identify mural cells. The vasculature was visualized by lectin histochemistry or perfusion of horse-radish peroxidase, and vessel walls were examined by electron microscopy. The early stage of aging was characterized by changes in peripheral retinal capillaries, including vessel broadening, thickening of the basement membrane, an altered length and orientation of desmin filaments in pericytes, a more widespread SMA distribution and changes in a subset of pre-arteriolar sphincters. In the later stages of aging, loss of capillary patency, aneurysms, distorted vessels, and foci of angiogenesis were apparent, especially in the peripheral deep vascular plexus. The capillary changes are consistent with impaired vascular autoregulation and may result in reduced pericyte-endothelial cell contact, destabilizing the capillaries and rendering them susceptible to angiogenic stimuli and endothelial cell loss as well as impairing the exchange of metabolites required for optimal neuronal function. This metabolic uncoupling leads to reactivation of

Calcium signaling in ocular arterioles

Local control of blood flow to the photoreceptors and associated neurons in the retina is largely achieved through changes in tone within the choroidal and retinal arterioles. This is primarily achieved through changes in [Ca2+] within the smooth muscle of these vessels, which regulates cell contraction and vascular constriction. Here we review some aspects of the cell physiology involved in these Ca2+-signaling processes, with particular emphasis on the molecular mechanisms involved. Ca2+-influx across the plasma membrane can occur via a variety of Ca2+-channels, including voltage-operated, store-operated, and receptor-operated channels. Ca2+ may also be released from intracellular stores via RyR-, or IP3R-gated channels in the SR membrane. Using high-speed confocal Ca2+-imaging, we have also demonstrated that the resulting signals are far from homogeneous, with spontaneous activity in retinal arterioles being characterized by both localized Ca2+-sparks and more global Ca2+-waves and oscillations. These signals may be specifically and differentially targeted, for example, to Ca2+-sensitive ion channels (stimulus-excitation coupling), or pathways regulating contraction (stimulus-contraction coupling). Exploring the role of changes in such targeting in disease states will provide exciting opportunities for future research.

Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.

Control of ocular blood flow occurs predominantly at the level of the retinal and choroidal arterioles. The present article provides an overview of the Ca2 + handling mechanisms and plasmalemmal ion channels involved in the regulation of retinal and choroidal arteriolar smooth muscle tone. Increases in global intracellular free Ca2 + ([Ca2 +]i) involve multiple mechanisms, including agonist-dependent release of Ca2 + from intracellular stores through activation of the inositol trisphosphate (IP3) pathway. Ca2 + enters by voltage-dependent L-type Ca2 + channels and novel dihydropyridine-sensitive store-operated nonselective cation channels. Ca2 + extrusion is mediated by plasmalemmal Ca2 +-ATPases and through Na+/Ca2+ exchange. Local Ca2 + transients (Ca2 + sparks) play an important excitatory role, acting as the building blocks for more global Ca2 + signals that can initiate vasoconstriction. K+ and Cl- channels may also affect cell function by modulating membrane potential. The precise contribution of each of these mechanisms to the regulation of retinal and choroidal perfusion in vivo warrants future investigation.

Tissue factor expression on monocyte subpopulations during normal pregnancy.

Pregnancy is often referred to as a hypercoaguable state due to changes in the haemostatic system. Tissue factor (TF) is the initiator of blood clotting in vivo. The effect of pregnancy on monocyte TF expression was determined in a longitudinal case control study, (89 pregnant, 39 non-pregnant). Using whole blood flow cytometry and CD14 as a monocyte marker, TF expression was measured on all CD14 positive, CD14Bright and CD14Dim cells. TF expression was significantly lower in pregnant women than in non-pregnant control subjects, on all CD14 positive cells at 20 and 35 weeks, on CD14Bright cells at 12 and 35 weeks and on CD14Dim cells at 20 weeks. Additionally, we report that a higher percentage of CD14Dim than CD14Bright cells express TF. These results suggest that, in order to maintain homeostasis in haemostasis in an otherwise hypercoaguable state, monocyte TF expression is reduced during normal pregnancy.