Renal cortex microperfusion evaluated by laser speckle contrast imaging in an ex vivo perfused kidney model-A proof-of-concept study.

BACKGROUND: Validated quantitative biomarkers for assessment of renal graft function during normothermic machine perfusion (NMP) conditions are lacking. The aim of this project was to quantify cortex microperfusion during ex vivo kidney perfusion using laser speckle contrast imaging (LSCI), and to evaluate the sensitivity of LSCI when measuring different levels of renal perfusion. Furthermore, we aimed to introduce LSCI measurements during NMP in differentially damaged kidneys. METHODS: Eleven porcine kidneys were nephrectomized and perfused ex vivo. Cortex microperfusion was simultaneously monitored using LSCI. First, a flow experiment examined the relationship between changes in delivered renal flow and corresponding changes in LSCI-derived cortex microperfusion. Second, renal cortical perfusion was reduced stepwise by introducing a microembolization model. Finally, LSCI was applied for measuring renal cortex microperfusion in kidneys exposed to minimal damage or 2 h warm ischemia (WI). RESULTS: Cortex microperfusion was calculated from the LSCI-obtained data. The flow experiment resulted in relatively minor changes in cortex microperfusion compared to the pump-induced changes in total renal flow. Based on stepwise injections of microspheres, we observed different levels of cortex microperfusion that correlated with administrated microsphere dosages (r2 = 0.95-0.99). We found no difference in LSCI measured cortex microperfusion between the kidneys exposed to minimal damage (renal cortex blood flow index, rcBFI = 2090-2600) and 2 h WI (rcBFI = 2189-2540). CONCLUSIONS: Based on this preliminary study, we demonstrated the feasibility of LSCI in quantifying cortex microperfusion during ex vivo perfusion. Furthermore, based on LSCI-measurements, cortical microperfusion was similar in kidneys exposed to minimal and 2 h WI.

Ultra-low-dose non-contrast CT and CT angiography can be used interchangeably for assessing maximal abdominal aortic diameter.

Background: Routine CT scans may increasingly be used to document normal aortic size and to detect incidental abdominal aortic aneurysms. Purpose: To determine whether ultra-low-dose non-contrast CT (ULDNC-CT) can be used instead of the gold standard CT angiography (CTA) for assessment of maximal abdominal aortic diameter. Materials and Methods: This retrospective study included 50 patients who underwent CTA and a normal-dose non-contrast CT for suspected renal artery stenosis. ULDNC-CT datasets were generated from the normal-dose non-contrast CT datasets using a simulation technique. Using the centerline technique, radiology consultants (n = 4) and residents (n = 3) determined maximal abdominal aortic diameter. The limits of agreement with the mean (LOAM) was used to access observer agreement. LOAM represents how much a measurement by a single observer may plausibly deviate from the mean of all observers on the specific subject. Results: Observers completed 1400 measurements encompassing repeated CTA and ULDNC-CT measurements. The mean diameter was 24.0 and 25.0 mm for CTA and ULDNC-CT, respectively, yielding a significant but minor mean difference of 1.0 mm. The 95% LOAM reproducibility was similar for CTA and ULDNC-CT (2.3 vs 2.3 mm). In addition, the 95% LOAM and mean diameters were similar for CTA and ULDNC-CT when observers were grouped as consultants and residents. Conclusions: Ultra-low-dose non-contrast CT exhibited similar accuracy and reproducibility of measurements compared with CTA for assessing maximal abdominal aortic diameter supporting that ULDNC-CT can be used interchangeably with CTA in the lower range of aortic sizes.

Local Pressure Drives Low-Density Lipoprotein Accumulation and Coronary Atherosclerosis in Hypertensive Minipigs.

BACKGROUND: The mechanisms by which hypertension accelerates coronary artery disease are poorly understood. Patients with hypertension often have confounding humoral changes, and to date, no experimental models have allowed analysis of the isolated effect of pressure on atherosclerosis in a setting that recapitulates the dimensions and biomechanics of human coronary arteries. OBJECTIVES: This study sought to analyze the effect of pressure on coronary atherosclerosis and explore the underlying mechanisms. METHODS: Using inflatable suprarenal aortic cuffs, we increased mean arterial pressure by >30 mm Hg in the cephalad body part of wild-type and hypercholesterolemic proprotein convertase subtilisin kexin type 9 (PCSK9)D374Y Yucatan minipigs for >1 year. Caudal pressures remained normal. RESULTS: Under hypercholesterolemic conditions in PCSK9D374Y transgenic minipigs, cephalad hypertension accelerated coronary atherosclerosis to almost 5-fold with consistent development of fibroatheromas that were sufficiently large to cause stenosis on computed tomography angiography. This was caused by local pressure forces, because vascular beds shielded from hypertension, but exposed to the same humoral factors, showed no changes in lesion formation. The same experiment was conducted under normocholesterolemic conditions in wild-type minipigs to examine the underlying mechanisms. Hypertension produced clear changes in the arterial proteome with increased abundance of mechanical strength proteins and reduced levels of infiltrating plasma macromolecules. This was paralleled by increased smooth muscle cells and increased intimal accumulation of low-density lipoproteins in the coronary arteries. CONCLUSIONS: Increased pressure per se facilitates coronary atherosclerosis. Our data indicate that restructuring of the artery to match increased tensile forces in hypertension alters the passage of macromolecules and leads to increased intimal accumulation of low-density lipoproteins.

18Fluorodeoxyglucose Accumulation in Arterial Tissues Determined by PET Signal Analysis.

BACKGROUND: Arterial 18fluorodeoxyglucose (FDG) positron emission tomography (PET) is considered a measure of atherosclerotic plaque macrophages and is used for quantification of disease activity in clinical trials, but the distribution profile of FDG across macrophages and other arterial cells has not been fully clarified. OBJECTIVES: The purpose of this study was to analyze FDG uptake in different arterial tissues and their contribution to PET signal in normal and atherosclerotic arteries. METHODS: Wild-type and D374Y-PCSK9 transgenic Yucatan minipigs were fed a high-fat, high-cholesterol diet to induce atherosclerosis and subjected to a clinical FDG-PET and computed tomography scan protocol. Volumes of arterial media, intima/lesion, macrophage-rich, and hypoxic tissues were measured in serial histological sections. Distributions of FDG in macrophages and other arterial tissues were quantified using modeling of the in vivo PET signal. In separate transgenic minipigs, the intra-arterial localization of FDG was determined directly by autoradiography. RESULTS: Arterial FDG-PET signal appearance and intensity were similar to human imaging. The modeling approach showed high accuracy in describing the FDG-PET signal and revealed comparable FDG accumulation in macrophages and other arterial tissues, including medial smooth muscle cells. These findings were verified directly by autoradiography of normal and atherosclerotic arteries. CONCLUSIONS: FDG is taken up comparably in macrophage-rich and -poor arterial tissues in minipigs. This offers a mechanistic explanation to a growing number of observations in clinical imaging studies that have been difficult to reconcile with macrophage-selective FDG uptake.

Quantitative applications in positron emission tomography achieved through signal modelling.

Positron emission tomography (PET) is a valuable tool in medical imaging, but it provides limited quantitative utility in a number of important applications, such as mapping of tracer accumulation in small tissues and quantitative assessment of factors affecting tracer uptake. We aimed to develop a quantification approach based on signal modelling, to address the above limitations. Our signal modelling approach allows for a comprehensive description of target and background signals. We used in silico simulations to exemplify the quantitative utility of signal modelling in a number of applications and conducted scans of standardized PET phantoms to validate our computer simulation algorithms. The simulations showed that the modelling approach allows applications not supported by current techniques, such as estimation of activity fractions of sub-resolution small tissues and accurate quantification of the effect of biological factors, such as hypoxia, on tracer accumulation. There was strong agreement between the simulation data and actual scans of phantoms, providing support for the validity of the simulation algorithms. We conclude that the presented signal modelling approach may provide a framework for image analysis that can improve and expand the quantitative capacity of PET imaging.

Tissue volume and activity mapping using total intensity projection of PET/CT images.

Autoradiography using phosphor imaging screens is often used to characterize tissue distribution of positron emission tomography (PET) radiotracers. PET tracers emit positrons with limited penetration range, and valid quantitative autoradiography can therefore only be achieved in thin tissue slices. However, in some settings, quantitative tracer profiling in thick tissues is required. Our aim was to develop a reliable method for this purpose. In this paper, we present a method based on total intensity projections (TIPs) of PET and computed tomography (CT) images. We show theoretically and experimentally that tissue total activity and tissue volume maps can be derived from the TIPs of PET and CT images, respectively. We also show that these maps are free of signal displacement artifacts in the direction of projection. To demonstrate the utility of the approach, we obtain and compare TIP-based maps and autoradiography of ex-vivo atherosclerotic minipig aortas following in-vivo injection of 18F-fluorodeoxyglucose. We show that autoradiography of the thick aortas yields distorted results due to positron range effects, whereas TIP-mapping is free from such bias. The TIP-based maps may, thus, provide a low-resolution alternative to autoradiography, when tracer accumulation profiling in thick tissues is required.

Diet-Induced Abdominal Obesity, Metabolic Changes, and Atherosclerosis in Hypercholesterolemic Minipigs.

BACKGROUND: Obesity and metabolic syndrome (MetS) are major risk factors for atherosclerotic diseases; however, a causal link remains elusive. Animal models resembling human MetS and its complications, while important, are scarce. We aimed at developing a porcine model of human MetS. METHODS: Forty pigs with familial hypercholesterolemia were fed a high fat + fructose diet for 30 weeks. Metabolic assessments and subcutaneous fat biopsies were obtained at 18 and 30 weeks, and fat distribution was assessed by CT-scans. Postmortem, macrophage density, and phenotype in fat tissues were quantified along with atherosclerotic burden. RESULTS: During the experiment, we observed a >4-fold in body weight, a significant but small increase in fasting glucose (4.1 mmol/L), insulin (3.1 mU/L), triglycerides (0.5 mmol/L), and HDL cholesterol (2.6 mmol/L). Subcutaneous fat correlated with insulin resistance, but intra-abdominal fat correlated inversely with insulin resistance and LDL cholesterol. More inflammatory macrophages were found in visceral versus subcutaneous fat, and inflammation decreased in subcutaneous fat over time. CONCLUSIONS: MetS based on human criteria was not achieved. Surprisingly, visceral fat seemed part of a healthier metabolic and inflammatory profile. These results differ from human findings, and further research is needed to understand the relationship between obesity and MetS in porcine models.

Treatment with a human recombinant monoclonal IgG antibody against oxidized LDL in atherosclerosis-prone pigs reduces cathepsin S in coronary lesions.

BACKGROUND: Immunization with oxidized LDL (oxLDL) reduces atherosclerosis in rodents. We tested the hypothesis that treatment with a human recombinant monoclonal antibody against oxLDL will reduce the burden or composition of atherosclerotic lesions in hypercholesterolemic minipigs. METHODS AND RESULTS: Thirty-eight hypercholesterolemic minipigs with defective LDL receptors were injected with an oxLDL antibody or placebo weekly for 12weeks. An 18F-fluorodeoxyglucose positron emission tomography (FDG PET) scan (n=9) was performed before inclusion and after 3months of treatment. Blood samples were obtained prior to each injection. Following the last injection all animals were sacrificed, and the heart, aorta, and iliac arteries were removed. The left anterior descending coronary artery was sectioned at 5mm intervals for quantitative and qualitative assessments of atherosclerosis, including immunohistochemical phenotyping of macrophages using a pan-macrophage marker (CD68) and markers for putative pro-atherogenic (cathepsin S) and atheroprotective (CD163) macrophages. Aorta, right coronary artery, and left iliac artery were stained en face with Sudan IV and the amount of atherosclerosis quantified. There was no effect of treatment on plasma lipid profile, vascular FDG-PET signal or the amount of atherosclerosis in any of the examined arteries. However, immunostaining of coronary lesions revealed reduced cathepsin S positivity in the treated group compared with placebo (4.8% versus 8.2% of intima area, p=0.03) with no difference in CD68 or CD163 positivity. CONCLUSIONS: In hypercholesterolemic minipigs, treatment with a human recombinant monoclonal antibody against oxLDL reduced cathepsin S in coronary lesions without any effect on the burden of atherosclerosis or aortic FDG-PET signal.

Diabetes with poor glycaemic control does not promote atherosclerosis in genetically modified hypercholesterolaemic minipigs.

AIMS/HYPOTHESIS: Diabetes is associated with an increased risk of atherosclerotic cardiovascular disease, but whether there is a direct and independent role for impaired glucose control in atherogenesis remains uncertain. We investigated whether diabetes with poor glycaemic control would accelerate atherogenesis in a novel pig model of atherosclerosis, the D374Y-PCSK9(+) transgenic minipig. METHODS: Nineteen minipigs were fed a cholesterol-enriched, high-fat diet; ten of these pigs were injected with streptozotocin to generate a model of diabetes. Restricted feeding was implemented to control the pigs' weight gain and cholesterol intake. After 49 weeks of high-fat feeding, the major arteries were harvested for a detailed analysis of the plaque burden and histological plaque type. RESULTS: Stable hyperglycaemia was achieved in the diabetic minipigs, while the plasma total and LDL-cholesterol and creatinine levels were unaffected. Diabetes failed to increase atherosclerosis in any of the vessels examined. The plaque burden in the aorta and right coronary artery was comparable between the groups, and was even reduced in the left anterior descending (LAD) coronary and iliofemoral arteries in the diabetic pigs compared with the controls. The distribution of plaque types and the collagen and macrophage contents were similar between the groups, except for a reduced infiltration of macrophages in the LAD arteries of the diabetic pigs. CONCLUSIONS/INTERPRETATION: Poorly controlled diabetes with no alterations in plasma cholesterol or creatinine concentrations did not augment the plaque burden or promote the development of more advanced lesions in this large-animal model of human-like atherosclerosis. This is consistent with clinical studies in patients with type 1 diabetes, indicating that hyperglycaemia per se is not an independent promoter of atherosclerotic disease, but that other diabetes-associated risk factors are important.

Relaxation of porcine retinal arterioles exposed to hypercholesterolemia in vivo is modified by hepatic LDL-receptor deficiency and diabetes mellitus.

Metabolic disturbances in diabetes mellitus include changes in the type and concentration of lipids in the blood plasma which may contribute to the development of diabetic retinopathy. This disease is characterized by changes in retinal blood flow secondary to changes in the tone of retinal arterioles which is regulated by compounds such as adenosine, adenosine triphosphate (ATP), the glutamate agonist N-methyl-d-aspartate (NMDA) and prostaglandin E2 (PGE2). However, the relation between increased plasma low density lipoprotein (LDL) and tone regulation in retinal resistance vessels has not been studied in detail. Twelve male and nine female Yucatan minipigs overexpressing a gain-of-function mutant (D374Y) of the human gene PCSK9 that blocks LDL transport into the liver and twelve wild-type males were studied. The animals were fed a cholesterol rich diet from the age of 60 days, followed by induction of diabetes mellitus in twelve of the transgenic animals. The animals were sacrificed at a mean age of 51 weeks (range 26-60 weeks), followed by inspection and histological examination of retinal vessels, and examination of the changes in vascular tone induced by adenosine, ATP, NMDA and PGE2. In the transgenic pigs without diabetes mellitus ATP-induced relaxation was reduced in isolated arterioles, and a whitish infiltration in an arteriole was observed in 4/8 (50%) of the animals, whereas these changes were not found in the other groups. Histological examination of one of the infiltrations showed staining with Oil Red O representing foamy cells sub-endothelially in the vascular wall indicating atheromatosis. Adenosine, ATP and PGE2 induced a significant concentration-dependent relaxation of retinal arterioles in all groups. The presence of perivascular retinal tissue had no effect on the relaxing effect of adenosine, but increased the relaxing effect of ATP and PGE2 in the two transgenic animal groups, whereas NMDA had no significant effect on vascular tone in any of the groups. Relaxation of porcine retinal arterioles exposed to hypercholesterolemia in vivo is modified by hepatic LDL-receptor deficiency and diabetes mellitus. This suggests that transgenic animal models are suitable for studying the influence of systemic diseases on retinal vascular function.

Familial hypercholesterolemia and atherosclerosis in cloned minipigs created by DNA transposition of a human PCSK9 gain-of-function mutant.

Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

Mouth-to-mouth ventilation is superior to mouth-to-pocket mask and bag-valve-mask ventilation during lifeguard CPR: a randomized study.

AIM: The quality of cardiopulmonary resuscitation (CPR) is a crucial determinant of outcome following cardiac arrest. Interruptions in chest compressions are detrimental. We aimed to compare the effect of mouth-to-mouth ventilation (MMV), mouth-to-pocket mask ventilation (MPV) and bag-valve-mask ventilation (BMV) on the quality of CPR. MATERIALS AND METHODS: Surf lifeguards in active service were included in the study. Each surf lifeguard was randomized to perform three sessions of single-rescuer CPR using each of the three ventilation techniques (MMV, MPV and BMV) separated by 5 min of rest. Data were obtained from a resuscitation manikin and video recordings. RESULTS: A total of 60 surf lifeguards were included (67% male, 33% female, mean age 25 years). Interruptions in chest compressions were significantly reduced by MMV (8.9 ± 1.6 s) when compared to MPV (10.7 ± 3.0 s, P < 0.001) and BMV (12.5 ± 3.5s, P < 0.001). Significantly more effective ventilations (visible chest rise) were delivered using MMV (91%) when compared to MPV (79%, P < 0.001) and BMV (59%, P < 0.001). The inspiratory time was longer during MMV (0.7 ± 0.2 s) and MPV (0.7 ± 0.2s, P < 0.001 for both) compared to BMV (0.5 ± 0.2s). Tidal volumes were significantly lower using BMV (0.4 ± 0.2L) compared to MMV (0.6 ± 0.2L, P < 0.001) and MPV (0.6 ± 0.3 L, P < 0.001), whereas no differences were observed when comparing MMV and MPV. CONCLUSION: MMV reduces interruptions in chest compressions and produces a higher proportion of effective ventilations during lifeguard CPR. This suggests that CPR quality is improved using MMV compared to MPV and BMV.

T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles.

Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium-induced constriction. Further, constriction by the thromboxane analogue U46619 was significantly inhibited by mibefradil at a concentration specific for T-type channels. Using PCR, we found that two channel subtypes, Ca(v)3.1 and Ca(v)3.2, were expressed in microdissected efferent arterioles. Ca(v)3.1 was found by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Ca(v)3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response. Low concentrations of nickel, an agent that blocks Ca(v)3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles.

Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse.

Adenosine can induce vasodilatation and vasoconstriction of the renal afferent arteriole of the mouse. We determined here its direct effect on efferent arterioles of mouse kidneys. Using isolated-perfused cortical efferent arterioles, we measured changes in luminal diameter in response to adenosine. Extraluminal application of adenosine and cyclohexyladenosine had no effect on the luminal diameter. When the vessels were constricted by the thromboxane mimetic U46619, application of adenosine and 5'-N-ethylcarboxamido-adenosine dilated the efferent arterioles in a dose-dependent manner. We also found that the adenosine-induced vasodilatation was inhibited by the A(2)-specific receptor blocker 3,7-dimethyl-1-propargylxanthine. In the presence of this inhibitor, adenosine failed to alter the basal vessel diameter of quiescent efferent arterioles. Using primer-specific polymerase chain reaction we found that the adenosine A(1), A(2a), A(2b), and A(3) receptors were expressed in microdissected mouse efferent arterioles. We conclude that adenosine dilates the efferent arteriole using the A(2) receptor subtype at concentrations compatible with activation of the A(2b) receptor.