Capillary Fraction Research Articles

Some features of intrauterine development of capillaries in the head and neck area

BACKROUND: The development of capillaries in human embryogenesis is a multi-stage process influenced by genetic factors and signaling pathways, which is of interest in studying the mechanisms of vascular bed formation in the prenatal period of human development.
 AIM: To study the development of capillaries in the embryonic and early fetal periods and determine the morphological prere­quisites leading to the formation of developmental defects.
 MATERIAL AND METHODS: The biomaterial of 50 embryos and fetuses from 4 to 12 weeks was studied. A histological and electron microscopic examination of the specimens was performed in the axial plane at the level of both jaws and neck. The volume fraction of capillaries and muscle fibers in the structure of the sternocleidomastoid muscle was determined. Data on the volume fraction of each component were presented as median and interquartile range. Quantitative analysis was performed using the Kruskal–Wallis and Mann–Whitney methods with Bonferroni correction.
 RESULTS: At the 4th week, a capillary network began to develop from the mesenchyme, which took on a complete form by the 8th–10th week. The proportion of capillaries increased from the 6th to the 10th week, and by the 12th week it decreased. A statistically significant pattern of changes in the ratio of the proportion of capillaries in muscles with the volume fraction of muscle fibers itself was revealed. As the mass of the muscle fibers themselves increased, the proportion of capillaries in it decreased significantly from 1.92 (1.77; 2)% at 4–6 weeks of embryogenesis to 0.25 (0.23; 0.26)% at 10–12 weeks.
 CONCLUSION: The critical period for the development of capillaries is the intrauterine period from the 4th to the 12th week of development, when in the structure of muscles as an organ, the vascular component first prevails over the muscular component itself, and then sharply decreases.

Development of a method for isolating brain capillaries from a single neonatal mouse brain and comparison of proteomic profiles between neonatal and adult brain capillaries

BackgroundThe functions and protein expressions of the blood–brain barrier are changed throughout brain development following birth. This study aimed to develop a method to isolate brain capillaries from a single frozen neonatal mouse brain and elucidate the enrichment of brain capillaries by quantitative proteomic analysis. We further compared the expression profile of proteins between neonatal and adult brain capillary fractions.MethodsThe brain capillary fraction was prepared by the optimized method from a single frozen mouse neonatal brain on postnatal day 7. The brain capillary fractions and brain lysates were digested by trypsin and analyzed by liquid chromatography-mass spectrometry for quantitative proteomics.ResultsBy optimizing the isolation method, we observed brain capillaries in the fraction prepared from a single neonatal mouse brain (nBC fraction). A protein amount of 31.5 μg, which is enough for proteomic analysis, was recovered from the nBC fraction. By proteomics analysis, the brain capillary selective proteins, including Abcb1a/Mdr1, Slc2a1/Glut1, Claudin-5, and Pecam-1, were found to be concentrated > 13.4-fold more in nBC fractions than in whole brain lysates. The marker proteins for neurons and astrocytes were not concentrated in nBC fractions, while those of pericytes and microglia were concentrated. Compared to adult mouse brain capillary fractions (aBC fractions), the expressions of Abcb1a/Mdr1a, Abcc4/Mrp4, and Slc2a1/Glut1 were significantly lower in nBC fractions than in aBC fractions, whereas those of Slc1a4/Asct1, Slc1a5/Asct2, Slc7a1/Cat1, and Slc16a1/Mct1 were significantly higher. Amino acid transporters, Slc38a5/Snat5, showed the greatest nBC-to-aBC ratio among transporters (9.83-fold). Network analysis of proteins expressed differentially between nBC and aBC fractions revealed that the proteins with terms related to the extracellular matrix were enriched.ConclusionsWe succeeded in isolating brain capillaries from a single frozen brain of a neonatal mouse at postnatal day 7. Proteomic analysis revealed the differential expression in brain capillaries between neonatal and adult mice. Specifically, amino acid transporters, including Slc1a5/Asct2 and Slc38a5/Snat5, were found to be induced in neonatal brain capillaries. The present isolation method will promote the study of the function and expression of the neonatal blood–brain barrier.

Influence of MDR1 gene polymorphism (2677G>T) on expression and function of P-glycoprotein at the blood-brain barrier: utilizing novel P-glycoprotein humanized mice with mutation.

P-glycoprotein, the encoded product of the MDR1 / ABCB1 gene in humans, is expressed in numerous tissues including brain capillary endothelial cells and restricts the distribution of xenobiotics into the brain as an efflux pump. Although a large number of single nucleotide polymorphisms in the MDR1 gene have been identified, the influence of the nonsynonymous 2677G>T/A single nucleotide polymorphism on P-glycoprotein at the blood-brain barrier has remained unclear. In the present study, we developed a novel P-glycoprotein humanized mouse line carrying the 2677G>T mutation by utilizing a mouse artificial chromosome vector constructed by genetic engineering technology and we evaluated the influence of 2677G>T on the expression and function of P-glycoprotein at the blood-brain barrier in vivo . The results of this study showed that the introduction of the 2677G>T mutation does not alter the expression levels of P-glycoprotein protein in the brain capillary fraction. On the other hand, the brain penetration of verapamil, a representative substrate of P-glycoprotein, was increased by the introduction of the 2677G>T mutation. These results suggested that the 2677G>T single nucleotide polymorphism may attenuate the function of P-glycoprotein, resulting in increased brain penetration of P-glycoprotein substrates, without altering the expression levels of P-glycoprotein protein in the blood-brain barrier. This mutant mouse line is a useful model for elucidating the influence of an MDR1 gene single nucleotide polymorphism on the expression and function of P-glycoprotein at the blood-brain barrier.

Diurnal Changes in Protein Expression at the Blood-Brain Barrier in Mice.

Circadian rhythms influence the transport function of the blood-brain barrier (BBB) and peripheral organs. However, the influence of circadian rhythms on protein expression in the BBB remains to be completely elucidated. Therefore, we aimed to investigate diurnal changes in protein expression in the mouse BBB using quantitative proteomics. Quantitative proteomics showed that the expression of 67, 10, and 20 proteins in the isolated mouse brain capillary fraction changed significantly at zeitgeber time (ZT) 6, 12, and 18, respectively, compared to ZT0. Among them, the levels of 44 proteins were significantly increased at ZT6 and then returned to the same level as ZT0 at ZT12 and ZT18. Gene ontology analysis indicated that the proteins significantly increased at ZT6 were majorly related to translation. The brain capillary endothelial cell-selective proteins sepiapterin reductase and vascular endothelial growth factor receptor 2 showed diurnal variation. In contrast, the expression of ABC transporters, SLC transporters, and receptors associated with receptor-mediated transcytosis, and tight junction proteins did not change within a day. The present findings demonstrated that protein expression related to transport function and physical barrier at the BBB was maintained throughout the day, although the proteins involved in some biological processes exhibited diurnal variation at the BBB.

Effect of the Thermal Insulation Cover Curing on Temperature Rise and Early-Age Strength of Concrete.

In the present paper, the effects of thermal insulation cover curing on the rise in temperature of concrete, and further on the mechanical properties of the concrete are measured experimentally. In the experiments, polyurethane foam boards of 100 mm thickness were used for thermal insulation, and three concrete strength grades of C30, C50 and C80, were used as concrete samples. Conventional standard curing, and two heat curing methods with a constant temperature of 40 °C and 60 °C were used for comparison. The pore structure of the cement paste subject to the different curing procedures, including insulation cover curing, heat curing and standard curing, was experimentally measured using mercury intrusion porosimetry. The test results show that rate of increase in compressive strength compared with standard curing is 22–34%, 16–26%, and 23–67%, respectively, for C30, C50, C80 concrete after being subject to 1 to 3 days thermal insulation cover curing in the initial period after concrete casting. As expected, initial heat curing of concrete will result in a reduction in the long-term strength of the concrete. At 28 days, the strength reduction rate compared with standard curing due to the insulation cover curing is 3.1–5.9%, 0.6–3.0%, 0–3.2%, respectively, for C30, C50, C80 concrete. By contrast, the compressive strength reduction compared with standard curing at 28 days is 8.6–10.5%, 8.6–9.1%, 4.7–5.6%, respectively, for C30, C50, C80 concrete after being subject to a constant heat curing of 40 °C and 60 °C in the initial period after concrete casting. Measurement of the pore structure of the paste subject to different curing procedures initially after casting shows that rising curing temperature leads to coarser pores, especially an increase in capillary pore fraction. Among the four curing methods used in the present study, the effect of insulation cover curing and low temperature (40 °C) heat curing on the capillary pore content is small, while the effect of high temperature (60 °C) heat curing is significant.

Human kidney graft survival correlates with structural parameters in baseline biopsies: a quantitative observational cohort study with more than 14 years' follow-up.

This prospective cohort study evaluates associations between structural and ultrastructural parameters in baseline biopsies from human kidney transplants and long-term graft survival after more than 14 years' follow-up. Baseline kidney graft biopsies were obtained prospectively from 54 consecutive patients receiving a kidney transplant at a single institution. Quantitative measurements were performed on the baseline biopsies by computer-assisted light microscopy and electron microscopy. Stereology-based techniques estimated the fraction of interstitial tissue, the volume of glomeruli, mesangial fraction, and basement membrane thickness of glomerular capillaries. The fraction of occluded glomeruli and scores according to the Banff classification were achieved. Kidney graft survival was analyzed by Kaplan-Meier estimates and Cox regression. Association to long-term kidney function was also analyzed. The long-term surviving kidney transplants were characterized at implantation by less arteriolar hyaline thickening (P < 0.001) and less interstitial fibrosis (P = 0.001), as well as a lower fraction of occluded glomeruli (P = 0.004) and lower glomerular volume (P = 0.03). At the latest follow-up, eGFR was decreased by 12 ml/min/1.73 m2 per unit increase in the score for arteriolar hyalinosis at implantation (P = 0.02), and eGFR was decreased by 19 ml/min/1.73 m2 per 106 μm3 increase in glomerular volume at baseline (P = 0.03). The unbiased Cavalieri estimate of glomerular volume and the ultrastructural parameters are the first to be evaluated in a cohort study with prospective follow-up for more than 14 years. The study shows that baseline biopsies from human kidney grafts contain extraordinary long-term prognostic information, and it highlights the importance of these intrinsic graft factors.

Perfusion controls muscle glucose uptake by altering the rate of glucose dispersion in vivo.

These studies test, using intravital microscopy (IVM), the hypotheses that perfusion effects on insulin-stimulated muscle glucose uptake (MGU) are 1) capillary recruitment independent and 2) mediated through the dispersion of glucose rather than insulin. For experiment 1, capillary perfusion was visualized before and after intravenous insulin. No capillary recruitment was observed. For experiment 2, mice were treated with vasoactive compounds (sodium nitroprusside, hyaluronidase, and lipopolysaccharide), and dispersion of fluorophores approximating insulin size (10-kDa dextran) and glucose (2-NBDG) was measured using IVM. Subsequently, insulin and 2[14C]deoxyglucose were injected and muscle phospho-2[14C]deoxyglucose (2[C14]DG) accumulation was used as an index of MGU. Flow velocity and 2-NBDG dispersion, but not perfused surface area or 10-kDa dextran dispersion, predicted phospho-2[14C]DG accumulation. For experiment 3, microspheres of the same size and number as are used for contrast-enhanced ultrasound (CEU) studies of capillary recruitment were visualized using IVM. Due to their low concentration, microspheres were present in only a small fraction of blood-perfused capillaries. Microsphere-perfused blood volume correlated to flow velocity. These findings suggest that 1) flow velocity rather than capillary recruitment controls microvascular contributions to MGU, 2) glucose dispersion is more predictive of MGU than dispersion of insulin-sized molecules, and 3) CEU measures regional flow velocity rather than capillary recruitment.

Blood\u2013brain and blood\u2013cerebrospinal fluid barrier permeability in spontaneously hypertensive rats

BackgroundHypertension is an important risk factor for cerebrovascular disease, including stroke and dementia. Both in humans and animal models of hypertension, neuropathological features such as brain atrophy and oedema have been reported. We hypothesised that cerebrovascular damage resulting from chronic hypertension would manifest itself in a more permeable blood–brain barrier and blood–cerebrospinal fluid barrier. In addition, more leaky barriers could potentially contribute to an enhanced interstitial fluid and cerebrospinal fluid formation, which could, in turn, lead to an elevated intracranial pressure.MethodsTo study this, we monitored intracranial pressure and estimated the cerebrospinal fluid production rate in spontaneously hypertensive (SHR) and normotensive rats (Wistar Kyoto, WKY) at 10 months of age. Blood–brain barrier and blood–cerebrospinal fluid barrier integrity was determined by measuring the leakage of fluorescein from the circulation into the brain and cerebrospinal fluid compartment. Prior to sacrifice, a fluorescently labelled lectin was injected into the bloodstream to visualise the vasculature and subsequently study a number of specific vascular characteristics in six different brain regions.ResultsBlood and brain fluorescein levels were not different between the two strains. However, cerebrospinal fluid fluorescein levels were significantly lower in SHR. This could not be explained by a difference in cerebrospinal fluid turnover, as cerebrospinal fluid production rates were similar in SHR and WKY, but may relate to a larger ventricular volume in the hypertensive strain. Also, intracranial pressure was not different between SHR and WKY. Morphometric analysis of capillary volume fraction, number of branches, capillary diameter, and total length did not reveal differences between SHR and WKY.ConclusionIn conclusion, we found no evidence for blood–brain barrier or blood–cerebrospinal fluid barrier leakage to a small solute, fluorescein, in rats with established hypertension.

Administration of an antagonist of P2X7 receptor to EAE rats prevents a decrease of expression of claudin-5 in cerebral capillaries

Purinergic P2X receptors, when activated under pathological conditions, participate in induction of the inflammatory response and/or cell death. Both neuroinflammation and neurodegeneration represent hallmarks of multiple sclerosis (MS), an autoimmune disease of the central nervous system. In the current study, we examined whether P2X7R is expressed in brain microvasculature of rats subjected to experimental autoimmune encephalomyelitis (EAE) and explore possible relationships with blood-brain barrier (BBB) protein—claudin-5 after administration of P2X7R antagonist—Brilliant Blue G (BBG). Capillary fraction isolated from control and EAE rat brains was subjected to immunohistochemical and Western blot analyses. We document the presence of P2X7R in brain capillaries isolated from brain tissue of EAE rats. P2X7R is found to be localized on the abluminal surface of the microvessels and is co-expressed with PDGFβR, a marker of pericytes. We also show over-expression of this receptor in isolated capillaries during the course of EAE, which is temporally correlated with a lower protein level of PDGFβR, as well as claudin-5, a tight junction-building protein. Administration of a P2X7R antagonist to the immunized rats significantly reduced clinical signs of EAE and enhances protein expression of both claudin-5 and PDGFβR. These results indicate that P2X7 receptor located on pericytes may contribute to pathological mechanisms operated during EAE in cerebral microvessels influencing the BBB integrity.

Characterization of P-Glycoprotein Humanized Mice Generated by Chromosome Engineering Technology: Its Utility for Prediction of Drug Distribution to the Brain in Humans.

P-glycoprotein (P-gp), encoded by the MDR1 gene in humans and by the Mdr1a/1b genes in rodents, is expressed in numerous tissues and performs as an efflux pump to limit the distribution and absorption of many drugs. Owing to species differences of P-gp between humans and rodents, it is difficult to predict the impact of P-gp on pharmacokinetics and the tissue distribution of P-gp substrates in humans from the results of animal experiments. Therefore, we generated a novel P-gp humanized mouse model by using a mouse artificial chromosome (MAC) vector [designated human MDR1-MAC (hMDR1-MAC) mice]. The results showed that hMDR1 mRNA was expressed in various tissues of hMDR1-MAC mice. Furthermore, the expression of human P-gp was detected in the brain capillary fraction and plasma membrane fraction of intestinal epithelial cells isolated from hMDR1-MAC mice, although the expression levels of intestinal P-gp were extremely low. Thus, we evaluated the function of human P-gp at the blood-brain barrier of hMDR1-MAC mice. The brain-to-plasma ratios of P-gp substrates in hMDR1-MAC mice were much lower than those in Mdr1a/1b-knockout mice, and the brain-to-plasma ratio of paclitaxel was significantly increased by pretreatment with a P-gp inhibitor in hMDR1-MAC mice. These results indicated that the hMDR1-MAC mice are the first P-gp humanized mice expressing functional human P-gp at the blood-brain barrier. This mouse is a promising model with which to evaluate species differences of P-gp between humans and mice in vivo and to estimate the brain distribution of drugs in humans while taking into account species differences of P-gp.

Correction function for accuracy improvement of the Composite Smeared Finite Element for diffusive transport in biological tissue systems

Modeling of drug transport within capillaries and tissue remains a challenge, especially in tumors and cancers where the capillary network exhibits extremely irregular geometry. Recently introduced Composite Smeared Finite Element (CSFE) provides a new methodology of modeling complex convective and diffusive transport in the capillary–tissue system. The basic idea in the formulation of CSFE is in dividing the FE into capillary and tissue domain, coupled by 1D connectivity elements at each node. Mass transport in capillaries is smeared into continuous fields of pressure and concentration by introducing the corresponding Darcy and diffusion tensors. Despite theoretically correct foundation, there are still differences in the overall mass transport to (and from) tissue when comparing smeared model and a true 3D model. The differences arise from the fact that the smeared model cannot take into account the detailed non-uniform pressure and concentration distribution in the vicinity of capillaries. We introduced a field of correction function for diffusivity through the capillary walls of smeared models, in order to have the same mass accumulation in tissue as in case of true 3D models. The parameters of the numerically determined correction function are as follows: ratio of thickness and diameter of capillary wall, ratio of diffusion coefficient in capillary wall and surrounding tissue; and volume fraction of capillaries within tissue domain. Partitioning at the capillary wall–blood interface can also be included. It was shown that the correction function is applicable to complex configurations of capillary networks, providing improved accuracy of our robust smeared models in computer simulations of real transport problems, such as in tumors or human organs.

Measurement of the perfusion fraction in brain tumors with intravoxel incoherent motion MR imaging: validation with histopathological vascular density in meningiomas.

To evaluate the quantification performance of the perfusion fraction (f) measured with intravoxel incoherent motion (IVIM) MR imaging in a comparison with the histological vascular density in meningiomas. 29 consecutive patients with meningioma (59.0 ± 16.8 years old, 8 males and 21 females) who underwent a subsequent surgical resection were examined with both IVIM imaging and a histopathological analysis. IVIM imaging was conducted using a single-shot SE-EPI sequence with 13 b-factors (0, 10, 20, 30, 50, 80, 100, 200, 300, 400, 600, 800, 1000 s mm-2) at 3T. The perfusion fraction (f) was calculated by fitting the IVIM bi-exponential model. The 90-percentile f-value in the tumor region-of-interest (ROI) was defined as the maximum f-value (f-max). Histopathological vascular density (%Vessel) was measured on CD31-immunostainted histopathological specimens. The correlation and agreement between the f-values and %Vessel was assessed. The f-max (15.5 ± 5.5%) showed excellent agreement [intraclass correlation coefficient (ICC) = 0.754] and a significant correlation (r = 0.69, p < 0.0001) with the %Vessel (12.9 ± 9.4%) of the tumors. The Bland-Altman plot analysis showed excellent agreement between the f-max and %Vessel (bias, -2.6%; 95% limits of agreement, from -16.0 to 10.8%). The f-max was not significantly different among the histological subtypes of meningioma. An excellent agreement and a significant correlation were observed between the f-values and %Vessel. The f-value can be used as a noninvasive quantitative imaging measure to directly assess the vascular volume fraction in brain tumors. Advances in knowledge: The f-value measured by IVIM imaging showed a significant correlation and an excellent agreement with the histological vascular density in the meningiomas. The f-value can be used as a noninvasive and quantitative imaging measure to directly assess the volume fraction of capillaries in brain tumors.

Large-scale identification of human cerebrovascular proteins: Inter-tissue and intracerebral vascular protein diversity.

The human cerebrovascular system is responsible for regulating demand-dependent perfusion and maintaining the blood-brain barrier (BBB). In addition, defects in the human cerebrovasculature lead to stroke, intracerebral hemorrhage, vascular malformations, and vascular cognitive impairment. The objective of this study was to discover new proteins of the human cerebrovascular system using expression data from the Human Protein Atlas, a large-scale project which allows public access to immunohistochemical analysis of human tissues. We screened 20,158 proteins in the HPA and identified 346 expression patterns correlating to blood vessels in human brain. Independent experiments showed that 51/52 of these distributions could be experimentally replicated across different brain samples. Some proteins (40%) demonstrated endothelial cell (EC)-enriched expression, while others were expressed primarily in vascular smooth muscle cells (VSMC; 18%); 39% of these proteins were expressed in both cell types. Most brain EC markers were tissue oligospecific; that is, they were expressed in endothelia in an average of 4.8 out of 9 organs examined. Although most markers expressed in endothelial cells of the brain were present in all cerebral capillaries, a significant number (21%) were expressed only in a fraction of brain capillaries within each brain sample. Among proteins found in cerebral VSMC, virtually all were also expressed in peripheral VSMC and in non-vascular smooth muscle cells (SMC). Only one was potentially brain specific: VHL (Von Hippel-Lindau tumor suppressor). HRC (histidine rich calcium binding protein) and VHL were restricted to VSMC and not found in non-vascular tissues such as uterus or gut. In conclusion, we define a set of brain vascular proteins that could be relevant to understanding the unique physiology and pathophysiology of the human cerebrovasculature. This set of proteins defines inter-organ molecular differences in the vasculature and confirms the broad heterogeneity of vascular cells within the brain.

Uptake and metabolism of sulphated steroids by the blood-brain barrier in the adult male rat.

Little is known about the origin of the neuroactive steroids dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PregS) in the brain or of their subsequent metabolism. Using rat brain perfusion in situ, we have found 3H‐PregS to enter more rapidly than 3H‐DHEAS and both to undergo extensive (> 50%) desulphation within 0.5 min of uptake. Enzyme activity for the steroid sulphatase catalysing this deconjugation was enriched in the capillary fraction of the blood–brain barrier and its mRNA expressed in cultures of rat brain endothelial cells and astrocytes. Although permeability measurements suggested a net efflux, addition of the efflux inhibitors GF120918 and/or MK571 to the perfusate reduced rather than enhanced the uptake of 3H‐DHEAS and 3H‐PregS; a further reduction was seen upon the addition of unlabelled steroid sulphate, suggesting a saturable uptake transporter. Analysis of brain fractions after 0.5 min perfusion with the 3H‐steroid sulphates showed no further metabolism of PregS beyond the liberation of free steroid pregnenolone. By contrast, DHEAS underwent 17‐hydroxylation to form androstenediol in both the steroid sulphate and the free steroid fractions, with some additional formation of androstenedione in the latter. Our results indicate a gain of free steroid from circulating steroid sulphates as hormone precursors at the blood–brain barrier, with implications for ageing, neurogenesis, neuronal survival, learning and memory.

Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences

The shear rheology of an emulsion of viscous drops in the presence of finite inertia is investigated using direct numerical simulation. In the absence of inertia, emulsions display a non-Newtonian rheology with positive first and negative second normal stress differences. However, recently it was discovered that a small amount of drop-level inertia alters their signs – the first normal stress difference becomes negative and the second one becomes positive, each in a small range of capillary numbers (Li &amp; Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal was shown numerically and analytically, but only in the limit of a dilute emulsion where drop–drop interactions were neglected. Here, we compute the rheology of a density- and viscosity-matched emulsion, accounting for the interactions in the volume fraction range of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheological properties (effective shear viscosity and first and second normal stress differences) in the Stokes limit match well with previous theoretical (Choi–Schowalter in the dilute limit) and simulated results (for concentrated systems) using the boundary element method. The two distinct components of the rheology arising from the interfacial stresses at the drop surface and the perturbative Reynolds stresses are investigated as functions of the drop Reynolds number, capillary number and volume fraction. The sign change is caused by the increasing drop inclination in the presence of inertia, which in turn directly affects the interfacial stresses. Increase of the volume fraction or capillary number increases the critical Reynolds number for sign reversals due to enhanced alignment of the drops with the flow directions. The effect of increasing the volume fraction on the rheology is explained by relating it to interactions and specifically to the contact pair-distribution function computed from the simulation. The excess stresses are seen to show an approximately linear behaviour with the Reynolds number in the range of 0.1–5, while with the capillary number and volume fraction, the variation is weakly quadratic.

Simulations on the gelling process of particle suspension systems forin-situpreparing porous materials in a capillary

The gelling process of particle suspension in a capillary which is crucial for in-situ preparing small size foam products has been simulated with an off-lattice diffusion limited cluster aggregation (DLCA) model by the three-dimensional Monte Carlo simulations. The effects of the model parameters, such as the interaction between capillary wall and particles, particle volume fraction, capillary size etc. on the density distribution of the system have been fully explored. And the aggregation kinetics process over a broad range of volume fractions and interactions have also been discussed. The results show that the geometric constraint of capillary can be analogous to a weak repulsive interaction between capillary wall and particles. And we found that as the capillary size or particle volume fraction increase, particle concentration distribution will be more uniform with other parameters constant. Porous network with relatively uniform density distribution can be also obtained through controlling the interaction between capillary wall and particles. In addition, by analyzing the aggregation kinetics process, we found that the attraction of capillary wall dramatically reduces the probability of gelation in the small-scale capillary. The obtained results will be of great importance in controlling the density distribution of porous materials prepared by in-situ methods.

Stalled cerebral capillary blood flow in mouse models of essential thrombocythemia and polycythemia vera revealed by in vivo two‐photon imaging

Essential thrombocythemia (ET) and polycythemia vera (PV) are myeloproliferative neoplasms (MPNs) that share the JAK2(V617F) mutation in hematopoietic stem cells, leading to excessive production of predominantly platelets in ET, and predominantly red blood cells (RBCs) in PV. The major cause of morbidity and mortality in PV and ET is thrombosis, including cerebrovascular occlusive disease. To identify the effect of excessive blood cells on cerebral microcirculation in ET and PV. We used two-photon excited fluorescence microscopy to examine cerebral blood flow in transgenic mouse models that mimic MPNs. We found that flow was 'stalled' in an elevated fraction of brain capillaries in ET (18%), PV (27%), mixed MPN (14%) and secondary (non-MPN) erythrocytosis (27%) mice, as compared with controls (3%). The fraction of capillaries with stalled flow increased when the hematocrit value exceeded 55% in PV mice, and the majority of stalled vessels contained only stationary RBCs. In contrast, the majority of stalls in ET mice were caused by platelet aggregates. Stalls had a median persistence time of 0.5 and 1h in ET and PV mice, respectively. Our findings shed new light on potential mechanisms of neurological problems in patients with MPNs.

P16.33 * AN IN SILICO ESTIMATION OF THE PHARMACOKINETIC PROFILE AND THE DISPOSITION OF GD-DTPA IN BRAIN TUMOR LESIONS OF DIFFERENT VASCULATURE THROUGH PBPK MODELS

Brain tumor lesions (BTL), i.e. high grade gliomas, are known to have a prominence of vasculature, which is promoted through hypoxia mechanisms and differential expression of vascular endothelial growth factor (VEGF). Imaging techniques such as dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) with intra-venous (i.v.) administration of a Gd-based contrast agent (GBCA) are successfully used for the diagnosis and characterization of the BTLs. Tracer kinetics plays an important role in DCE-MRI by assessing the vessel leakage through estimation of the transfer and disposition of GBCAs in a lesion. Physiologically-based pharmacokinetic modeling (PBPK) represents a well-documented approach to estimate in silico the disposition of pharmacologic agents in the body. In this work, we sought to i)present a whole-body PBPK approach in order to estimate the PK profile of Gd-DTPA (Gadopentetic acid, Magnevist®) and ii)evaluate the impact of vascular fraction of tracer's extravascular-extracellular disposition in a simulated BTL. PK profile was assessed through the application of Simcyp® simulator platform and the generation of whole-body PBPK model. BTL was introduced as an additional compartment (∼5% of total brain weight) with tissue characteristics matching those of a brain tumor. In silico clinical trials (ISCTs) were designed by integrating literature data for a virtual population of patients with cancer for Simcyp® and Gd-DTPA properties. The ISCTs were generated for a representative individual and concentrations were estimated for 15 minutes following i.v. bolus injection of Gd-DTPA (0.1 mmol/kg). Given the simulated BTL's size, all parameters were kept constant except for capillary fraction in order to evaluate the impact of vasculature. RESULTS: from the whole-body PBPK simulations estimate the maximum plasma concentration of Gd-DTPA to be 3.0 mM whereas the intracranial blood concentration to be 1.7 mM. Regarding the simulated BTL, concentrations varied between 1.5-1.7 mM for the extravascular-extracellular space following modulation of tissue percentage in capillaries. Finally, zero concentrations are predicted for brain parenchyma and intracellular tissues due to the presence of blood-brain barrier and the absence of cellular intake, transfer or passive mediated, for Gd-DTPA, respectively. The presented in silico approach was capable of assessing the PK profiles of Gd-DTPA and especially its disposition in the extravascular-extracellular space of a putative BTL. The need for exploitation of the imaging techniques with studies correlating image analysis with clinical outcome and in silico tools is of great interest. The incorporation of PBPK models along with in silico tools for tumor growth and/or clinical data could reveal novel approaches for improving DCE-MRI techniques applied in diagnosis and treatment protocols (i.e. novel anti-VEGF treatments).

Skeletal muscle capillary function: contemporary observations and novel hypotheses.

The capillary bed constitutes a vast surface that facilitates exchange of O2, substrates and metabolites between blood and organs. In contracting skeletal muscle, capillary blood flow and O2 diffusing capacity, as well as O2 flux, may increase two orders of magnitude above resting values. Chronic diseases, such as heart failure and diabetes, and also sepsis impair these processes, leading to compromised energetic, metabolic and, ultimately, contractile function. Among researchers seeking to understand blood-myocyte exchange in health and the basis for dysfunction in disease, there is a fundamental disconnect between microcirculation specialists and many physiologists and physiologist clinicians. While the former observe capillaries and capillary function directly (muscle intravital microscopy), the latter generally use indirect methodologies (e.g. post-mortem tissue analysis, 1-methyl xanthine, contrast-enhanced ultrasound, permeability-surface area product) and interpret their findings based upon August Krogh's observations made nearly a century ago. 'Kroghian' theory holds that only a small fraction of capillaries support red blood cell (RBC) flux in resting muscle, leaving the vast majority to be 'recruited' (i.e. to initiate RBC flux) during contractions, which would constitute the basis for increasing surface area for capillary exchange and reducing capillary-mitochondrial diffusion distances. Experimental techniques each have their strengths and weaknesses, and often the correct or complete answer to a problem emerges from integration across multiple technologies. Today, Krogh's entrenched 'capillary recruitment' hypothesis is challenged by direct observations of capillaries in contracting muscle, which is something that he and his colleagues could not do. Moreover, in the peer-reviewed scientific literature, application of a range of contemporary physiological technologies, including intravital microscopy of contracting muscle, magnetic resonance, near-infrared spectroscopy and phosphorescence quenching, combined with elegant in situ and in vivo models, suggest that the role of the capillary bed, at least in contracting muscle, is subserved without the necessity for de novo capillary recruitment of previously non-flowing capillaries. When viewed within the context of the capillary recruitment hypothesis, this evidence casts serious doubt on the interpretation of those data that are based upon Kroghian theory and indirect methodologies. Thus, today a wealth of evidence calls for a radical revision of blood-muscle exchange theory to one in which most capillaries support RBC flux at rest and, during contractions, capillary surface area is 'recruited' along the length of previously flowing capillaries. This occurs, in part, by elevating capillary haematocrit and extending the length of the capillary available for blood-myocyte exchange (i.e. longitudinal recruitment). Our understanding of blood-myocyte O2 and substrate/metabolite exchange in health and the mechanistic basis for dysfunction in disease demands no less.

Localization of organic anion transporting polypeptide (Oatp) 1a4 and Oatp1c1 at the rat blood-retinal barrier.

BackgroundOrganic anion transporting polypeptide (Oatp) transporters at the blood–brain barrier (BBB) and the blood-retinal barrier (BRB), which consists of retinal capillary endothelial cells and retinal pigment epithelial cells, are major determinants of the control of anionic drugs into the brain and retina. Although Oatp1a4 (Slco1a4) and Oatp1c1 (Slco1c1) are known to be expressed in the abluminal and luminal membrane of the rat BBB and Oatp1a4 is known to be expressed at the BRB, the expression and localization of Oatp1c1 at the BRB and subcellular localization of Oatp1a4 at the BRB have received little attention. Therefore, the purpose of present study was to determine the cellular and subcellular localization of Oatp1a4 and 1c1 at the BRB.MethodsWe used guinea pig polyclonal antibodies to Oatp1a4 and 1c1 for immunoblot and immunohistochemical analysis to determine their cellular and subcellular distributions in the rat retina. We compared these distributions with those of the glucose transporter 1 (GLUT1/Slc2a1). Whole brain, brain capillary fractions and kidney were used as control.ResultsOatp1a4 and 1c1 immunoreactivities were detected in the rat retinal capillaries and co-localized with GLUT1, suggesting that both proteins are located on the abluminal and luminal membrane of the retinal capillary endothelial cells. Oatp1a4 and 1c1 immunoreactivities were preferentially detected on the apical and basolateral membrane of rat retinal pigment epithelial cells, respectively, suggesting that Oatp1a4 and 1c1 are localized on the apical membrane and the basolateral membrane of the retinal pigment epithelial cells, respectively.ConclusionOatp1a4 and 1c1 are present at the BRB and contribute to the transcellular transport of amphipathic organic anions across the BRB.