Microfluidic interactions between red blood cells and drug carriers by image analysis techniques

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.Blood is a complex biological fluid composed of deformable cells and platelets suspended in plasma, a protein-rich liquid. The peculiar nature of blood needs to be considered when designing a drug delivery strategy based on systemically administered carriers. Here, we report on an in vitro fluid dynamic investigation of the influence of the microcapillary flow of red blood cells (RBCs) on micron sized carriers by high speed imaging methods. The experiments were carried out in a 50μm diameter glass capillary that mimicked the hydrodynamic conditions of human microcirculation. Spherical μ particles (μ-Ps), with sizes ranging between 0.5 and 3μm, were tested. Images of the flowing RBCs and μ-Ps were acquired by a highspeed/ high-magnification microscopy. The transport and distribution of rigid particles in a suspension of RBCs under shear flow were followed for: i) the migration of RBCs towards the vessel centerline due to their deformability; ii) the cross-flow migration of μ-Ps towards the vessel wall due to their hydrodynamic interactions with RBCs; iii) the radial distribution of μ-Ps in the presence of RBCs. This study suggests that the therapeutic efficacy of μ-Ps could be ultimately affected by their interactions with the flowing RBCs in the vasculature

Levosimendan in Sepsis.

Commentum to "Levosimendan for the Prevention of Acute Organ Dysfunction in Sepsis

Protocol TOP-Study (tacrolimus organ perfusion): a prospective randomized multicenter trial to reduce ischemia reperfusion injury in transplantation of marginal liver grafts with an "ex vivo" tacrolimus perfusion

Background: Critical organ shortage results in the utilization of extended donor criteria (EDC) liver grafts. These marginal liver grafts are prone to increased ischemia reperfusion injury (IRI) which may contribute to deteriorated graft function and survival. Experimental data have shown that the calcineurin inhibitor tacrolimus exerts protective effects on hepatic IRI when applied intravenously or directly as a hepatic rinse. Therefore, the aim of the present study is to examine the effects of an ex vivo tacrolimus perfusion on IRI in transplantation of EDC liver grafts. Methods/Design: The TOP-Study (tacrolimus organ perfusion) is a randomized multicenter trial comparing the ex vivo tacrolimus perfusion of marginal liver grafts with placebo. We hypothesize that a tacrolimus rinse reduces IRI, potentially improving organ survival following transplantation of EDC livers. The study includes livers with two or more EDC, according to Eurotransplant International Foundation’s definition of EDC livers. Prior to implantation, livers randomized to the treatment group are rinsed with tacrolimus at a concentration of 20 ng/ml in 1000 ml Custodiol solution and in the placebo group with Custodiol alone. The primary endpoint is the maximum serum alanine transamninase (ALT) level within the first 48 hours after surgery; however, the study design also includes a 1-year observation period following transplantation. The TOP-Study is an investigator-initiated trial sponsored by the University of Munich Hospital. Seven other German transplant centers are participating (Berlin, Frankfurt, Heidelberg, Mainz, Münster, Regensburg, Tübingen) and aim to include a total of 86 patients. Discussion: Tacrolimus organ perfusion represents a promising strategy to reduce hepatic IRI following the transplantation of marginal liver grafts. This treatment may help to improve the function of EDC grafts and therefore safely expand the donor pool in light of critical organ shortage. Trial register: EudraCT number: 2010-021333-31, ClinicalTrials.gov identifier: NCT0156409

Angiotensin II and VEGF are Involved in Angiogenesis Induced by Short-Term Exercise Training

Results from our laboratory have suggested a pathway involving angiotensin II type 1 (AT1) receptors and vascular endothelial growth factor (VEGF) in angiogenesis induced by electrical stimulation. The present study investigated if similar mechanisms underlie the angiogenesis induced by short-term exercise training. Seven days before training and throughout the training period, male Sprague-Dawley rats received either captopril or losartan in their drinking water. Rats underwent a 3-day treadmill training protocol. The tibialis anterior and gastrocnemius muscles were harvested under anesthesia and lightly fixed in formalin (vessel density) or frozen in liquid nitrogen (VEGF expression). In controls, treadmill training resulted in a significant increase in vessel density in all muscles studied. However, the angiogenesis induced by exercise was completely blocked by either losartan or captopril. Western blot analysis showed that VEGF expression was increased in the exercised control group, and both losartan and captopril blocked this increase. The role of VEGF was directly confirmed using a VEGF-neutralizing antibody. These results confirm the role of angiotensin II and VEGF in angiogenesis induced by exercise

A model of oxygen dynamics in the cerebral microvasculature and the effects of morphology on flow and metabolism

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.The cerebral microvasculature plays a vital role in adequately supplying blood to the brain. Determining the health of the cerebral microvasculature is important during pathological conditions, such as stroke and dementia. Recent studies have shown the complex behaviour of cerebral metabolic rate with transit time distribution. In this paper, we extend a recently developed technique to solve for residue function and transit time distribution in an existing physiologically accurate model of the cerebral microvasculature to calculate cerebral metabolism. We present the mathematical theory based on solving the mass transport equation followed by results of the simulations. It is found that oxygen extraction fraction and cerebral metabolic rate are dependent on both mean and heterogeneity of the transit time distribution. For changes in cerebral blood flow, a positive correlation can be observed between mean transit time and oxygen extraction fraction, and a negative correlation between mean transit time and metabolic rate of oxygen. The metabolic rate is thus affected more significantly by cerebral blood flow than oxygen extraction fraction. A negative correlation can also be observed between transit time heterogeneity and the metabolic rate of oxygen for a constant cerebral blood flow. The heterogeneity of the transit time distribution also has an effect on the response of oxygen extraction fraction and cerebral metabolic rate to sudden changes. These results provide information on the role of the cerebral microvasculature and its effects on flow and metabolism. They thus open up the possibility of obtaining additional valuable clinical information for diagnosing and treating cerebrovascular diseases

Non-equivalence of antibiotic generic drugs and risk for intensive care patients

Background: The underlying axiom in applying generic drugs is the equivalence of their active ingredient with the (usually more expensive) innovator product, an all-embracing statement with the insidious result that physicians assume that the generic products have been subjected to the same rigorous testing regimens as the brand-name products. The present paper presents novel experimental data on an investigator-blinded comparison between the innovator imipenem antibiotic, and a number of its generics. Methods: Particulate matter contamination of each group was visualized by means of a membrane filter method. Functional studies in an animal model–the dorsal skinfold chamber technique in mice-designed to simulate the state of microcirculatory dysfunction in intensive care patients was performed, in order to assess the influence of the particulate matter of each group on the functional capillary density of the striated skin muscle, after their intravenous injection. Results: The results showed massive particulate contamination of the generics, in a size range relevant for impacting the microcirculation. The particulate contamination contributed in some generic groups to a significant shutdown of tissue perfusion. Conclusion: The presented data underscore the need to raise the regulatory barriers for the entry of generics to the market, well beyond the simplistic proof of “bioequivalence”, which in no measure deals with the essential questions of quality and patient safety. If generics are used, they should be tested by a filter technique and optical microscopy, to ensure the absence especially of small particulate contaminants and their purity

The Role of Sex Hormones in Inducing Maternal Uterine Remodeling and Vasodilation During Pregnancy

Uterine vascular adaptations such as vessel growth and vasodilation are needed to facilitate the more than 10-fold increase of uteroplacental blood flow (UPBF) during pregnancy. Adverse adaptations may result in pregnancy complications such as preeclampsia and intrauterine growth restriction. Pregnancy milieu, placentation and the attendant change in wall shear stress are major regulators of uterine vascular adaptation. In this study, we aimed at delineating : (1) the contribution of these regulators in vascular remodeling and (2) the effects of pregnancy milieu (estrogen and progesterone) alone and in combination with wall shear stress on the vascular reactivity. Using Sprague Dawley rats as the animal model, three surgical methods were utilized: (1) unilateral oviductal ligation (OHL) that restricts pregnancy to one uterine horn; (2) cervical-end main uterine artery and vein ligation (VL) that alters the hemodynamic pattern of the UPBF and wall shear stress; and (3) ovariectomy (OVX) with the implant of estrogen + progesterone pellet (0.5 and 100 mg, respectively). A segment of ovarian-end main uterine artery from each uterine horn was dissected, cannulated, and pressurized in an arteriograph system. Lumen diameters in response to phenylephrine (vasoconstrictor) and acetylcholine (vasodilator) were measured. Passive lumen diameters, wall thickness, vessel cross-sectional area, and distensibility were also measured under a microscope. Significant remodeling was seen in OVX rats in response to hormone replacement (p=0.0457); however, the extent of remodeling did not reach that seen in the nonpregnant horn of OHL rats. No significant change in wall thickness, cross-sectional area or wall: lumen ratio was found in OVX (+pellet), compared to OVX (-pellet) rats. Estrogen + progesterone had no significant effect on the sensitivity to phenylephrine or acetylcholine. In conclusion, estrogen + progesterone does have a significant effect on vascular remodeling. The presence of other factors, such as placentation, likely augment this process

Homocysteine-induced peripheral microcirculation dysfunction in zebrafish and its attenuation by L-arginine

Elevated blood homocysteine (Hcy) level is frequently observed in aged individuals and those with age-related vascular diseases. However, its effect on peripheral microcirculation is still not fully understood. Using in vivo zebrafish model, the degree of Hcy-induced peripheral microcirculation dysfunction is assessed in this study with a proposed dimensionless velocity parameter (V) over bar (CV)/(V) over bar (PCV), where (V) over bar (CV) and (V) over bar (PCV) represent the peripheral microcirculation perfusion and the systemic perfusion levels, respectively. The ratio of the peripheral microcirculation perfusion to the systemic perfusion is largely decreased due to peripheral accumulation of neutrophils, while the systemic perfusion is relatively preserved by increased blood supply from subintestinal vein. Pretreatment with L-arginine attenuates the effects of Hcy on peripheral microcirculation and reduces the peripheral accumulation of neutrophils. Given its convenience, high reproducibility of the observation site, non-invasiveness, and the ease of drug treatment, the present zebrafish model with the proposed parameters will be used as a useful drug screening platform for investigating the pathophysiology of Hcy-induced microvascular diseases.111Ysciescopu

Design of a bioreactor to study the role of red blood cells in the transport of nitric oxide in the microcirculation

Nitric oxide (NO) plays an important role in physiological functions like vasodilation, neurotransmission, and inhibition of platelet aggregation. The endothelium-derived NO diffuses into the vascular lumen where it interacts with flowing blood as well as the smooth muscles where it modulates vascular tone. However, uncertainty exists on how NO escapes the rapid scavenging by hemoglobin (Hb) and reaches smooth muscles. Several proposed hypotheses include 1) a reduced reaction rate of NO with Hb contained inside red blood cells (RBCs) and 2) NO preservation in the bound form of s-nitrosohemoglobin or nitrite. The mechanism and magnitude of reduction of NO reaction rate with Hb contained inside RBCs are not well established. In this study, an in vitro experimental system was designed to expose stirred RBC suspension to physiologically relevant NO flux. NO-RBC interactions were studied by measuring the reaction products, nitrite and total NOx, using chemiluminescence method. We studied the effect of increasing hematocrit from 5% to 45% on NO-RBC interaction under oxygenated condition. Results show that the system maintained a steady state in the bioreactor and could be easily modified to control NO delivery flux. An increase in product concentration was observed by increasing the hematocrit from 5% to 45%. The study is clinically important as the understanding of molecular interaction of NO with Hb in RBCs and mode of NO transport in microcirculation may provide therapeutic opportunities in the biomedical field in areas as diverse as sickle cell anemia, septic shock, hypoxic pulmonary vasoconstriction, and blood substitutes

Replacing the Transfusion of 1-2 Units of Blood with Plasma Expanders that Increase Oxygen Delivery Capacity: Evidence from Experimental Studies.

At least a third of the blood supply in the world is used to transfuse 1-2 units of packed red blood cells for each intervention and most clinical trials of blood substitutes have been carried out at this level of oxygen carrying capacity (OCC) restoration. However, the increase of oxygenation achieved is marginal or none at all for molecular hemoglobin (Hb) products, due to their lingering vasoactivity. This has provided the impetus for the development of "oxygen therapeutics" using Hb-based molecules that have high oxygen affinity and target delivery of oxygen to anoxic areas. However it is still unclear how these oxygen carriers counteract or mitigate the functional effects of anemia due to obstruction, vasoconstriction and under-perfusion. Indeed, they are administered as a low dosage/low volume therapeutic Hb (subsequently further diluted in the circulatory pool) and hence induce extremely small OCC changes. Hyperviscous plasma expanders provide an alternative to oxygen therapeutics by increasing the oxygen delivery capacity (ODC); in anemia they induce supra-perfusion and increase tissue perfusion (flow) by as much as 50%. Polyethylene glycol conjugate albumin (PEG-Alb) accomplishes this by enhancing the shear thinning behavior of diluted blood, which increases microvascular endothelial shear stress, causes vasodilation and lowering peripheral vascular resistance thus facilitating cardiac function. Induction of supra-perfusion takes advantage of the fact that ODC is the product of OCC and blood flow and hence can be maintained by increasing either or both. Animal studies suggest that this approach may save a considerable fraction of the blood supply. It has an additional benefit of enhancing tissue clearance of toxic metabolites