A technique to detect and to quantify fasciocutaneous blood vessels in small laboratory animals ex vivo

PURPOSE: A microangiographical technique is described, which allows visualization of small and capillary blood vessels and quantification of fasciocutaneous blood vessels by means of digital computer analysis in very small laboratory animals. MATERIALS AND METHODS: The left carotid artery of 20 nu/nu mice was cannulated (26 gauge) and a mixture of gelatin, bariumsulfate, and green ink was injected according to standardized protocol. Fasciocutaneous blood vessels were visualized by digital mammography and analyzed for vessel length and vessel surface area as standardized units [SU] by computer program. RESULTS: With the described microangiography method, fasciocutaneous blood vessels down to capillary size level can be clearly visualized. Regions of interest (ROIs) can be defined and the containing vascular network quantified. Comparable results may be obtained by calculating the microvascular area index (MAI) and the microvascular length index (MLI), related to the ROIs size. Identical ROIs showed a high reproducibility for measured [SU] < 0.01 +/- 0.0012%. CONCLUSION: Combining microsurgical techniques, pharmacological knowledge, and modern digital image technology, we were able to visualize small and capillary blood vessels even in small laboratory animals. By using our own computer analytical program, quantification of vessels was reliable, highly reproducible, and fast.

New insights into ocular blood flow at very high altitudes

Little is known about the ocular and cerebral blood flow during exposure to increasingly hypoxic conditions at high altitudes. There is evidence that an increase in cerebral blood flow resulting from altered autoregulation constitutes a risk factor for acute mountain sickness (AMS) and high-altitude cerebral edema (HACE) by leading to capillary overperfusion and vasogenic cerebral edema. The retina represents the only part of the central nervous system where capillary blood flow is visible and can be measured by noninvasive means. In this study we aimed to gain insights into retinal and choroidal autoregulatory properties during hypoxia and to correlate circulatory changes to symptoms of AMS and clinical signs of HACE. This observational study was performed within the scope of a high-altitude medical research expedition to Mount Muztagh Ata (7,546 m). Twenty seven participants underwent general and ophthalmic examinations up to a maximal height of 6,800 m. Examinations included fundus photography and measurements of retinal and choroidal blood flow, as well as measurement of arterial oxygen saturation and hematocrit. The initial increase in retinal blood velocity was followed by a decrease despite further ascent, whereas choroidal flow increase occurred later, at even higher altitudes. The sum of all adaptational mechanisms resulted in a stable oxygen delivery to the retina and the choroid. Parameters reflecting the retinal circulation and optic disc swelling correlated well with the occurrence of AMS-related symptoms. We demonstrate that sojourns at high altitudes trigger distinct behavior of retinal and choroidal blood flow. Increase in retinal but not in choroidal blood flow correlated with the occurrence of AMS-related symptoms.

Snake venoms and hemostasis

Snake venoms are complex mixtures of biologically active proteins and peptides. Many of them affect hemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. Based on sequence, these snake venom components have been classified into various families, such as serine proteases, metalloproteinases, C-type lectins, disintegrins and phospholipases. The various members of a particular family act selectively on different blood coagulation factors, blood cells or tissues. For almost every factor involved in coagulation or fibrinolysis there is a venom protein that can activate or inactivate it. Venom proteins affect platelet function by binding or degrading vWF or platelet receptors, activating protease-activated receptors or modulating ADP release and thromboxane A2 formation. Some venom enzymes cleave key basement membrane components and directly affect capillary blood vessels to cause hemorrhaging. L-Amino acid oxidases activate platelets via H2O2 production.

How Noninvasive Haemoglobin Measurement with Pulse CO-Oximetry Can Change Your Practice: An Expert Review

Trauma related haemorrhagic anaemia is rarely diagnosed by physical examination alone but typically includes measurement of blood haemoglobin, one of the most frequently ordered laboratory tests. Recently, noninvasive technologies have been developed that allow haemoglobin to be measured immediately without the need for intravenous access or having to take venous, arterial, or capillary blood. Moreover, with these technologies haemoglobin can be continuously measured in patients with active bleeding, to guide the start and stop of blood transfusions and to detect occult bleeding. Recent studies on the accuracy of the devices showed promising results in terms of accuracy of hemoglobin measurement compared to laboratory determination. The present review gives an overview on the technology itself and reviews the current literature on the subject.

Breathing manoeuvre-dependent changes in myocardial oxygenation in healthy humans.

AIMS CO₂ is an intrinsic vasodilator for cerebral and myocardial blood vessels. Myocardial vasodilation without a parallel increase of the oxygen demand leads to changes in myocardial oxygenation. Because apnoea and hyperventilation modify blood CO₂, we hypothesized that voluntary breathing manoeuvres induce changes in myocardial oxygenation that can be measured by oxygenation-sensitive cardiovascular magnetic resonance (CMR). METHODS AND RESULTS Fourteen healthy volunteers were studied. Eight performed free long breath-hold as well as a 1- and 2-min hyperventilation, whereas six aquatic athletes were studied during a 60-s breath-hold and a free long breath-hold. Signal intensity (SI) changes in T₂*-weighted, steady-state free precession, gradient echo images at 1.5 T were monitored during breathing manoeuvres and compared with changes in capillary blood gases. Breath-holds lasted for 35, 58 and 117 s, and hyperventilation for 60 and 120 s. As expected, capillary pCO₂ decreased significantly during hyperventilation. Capillary pO₂ decreased significantly during the 117-s breath-hold. The breath-holds led to a SI decrease (deoxygenation) in the left ventricular blood pool, while the SI of the myocardium increased by 8.2% (P = 0.04), consistent with an increase in myocardial oxygenation. In contrast, hyperventilation for 120 s, however, resulted in a significant 7.5% decrease in myocardial SI/oxygenation (P = 0.02). Change in capillary pCO₂ was the only independently correlated variable predicting myocardial oxygenation changes during breathing manoeuvres (r = 0.58, P < 0.01). CONCLUSION In healthy individuals, breathing manoeuvres lead to changes in myocardial oxygenation, which appear to be mediated by CO₂. These changes can be monitored in vivo by oxygenation-sensitive CMR and thus, may have value as a diagnostic tool.

Application of Ga(68) -DOTA-exendin-4 PET/CT to localise an occult insulinoma

A 49 year old female presented to our Neuroendocrine Tumour (NET) centre with recurrent severe and disabling hypoglycaemia. She had previously been extensively investigated with a clinical and biochemical diagnosis of endogenous hyperinsulinemic hypoglycaemia although the source of hormonal hypersecretion could not be localised with MRI, EUS and (111) In-Octreotide scans. After extensive discussion the patient opted for blind surgical resection undergoing a pylorus-preserving pancreaticoduodenectomy in December 2010. Histological examination of the resected operative specimen demonstrated a normal pancreas with no evidence of neuroendocrine tumour. Consistent with this, post-surgery her hypoglycaemic symptoms persisted with fasting capillary blood glucose of 2.1-6.0 mmol/l with increasing hypoglycaemia unawareness. Consequently she sought alternative clinical opinions from two European Neuroendocrine Tumour Society (ENETS) Centres of Excellence who investigated her collaboratively. This article is protected by copyright. All rights reserved.

Intussusceptive angiogenesis: pillars against the blood flow

Adaptation of vascular networks to functional demands needs vessel growth, vessel regression and vascular remodelling. Biomechanical forces resulting from blood flow play a key role in these processes. It is well-known that metabolic stimuli, mechanical forces and flow patterns can affect gene expression and remodelling of vascular networks in different ways. For instance, in the sprouting type of angiogenesis related to hypoxia, there is no blood flow in the rising capillary sprout. In contrast, it has been shown that an increase of wall shear stress initiates the splitting type of angiogenesis in skeletal muscle. Otherwise, during development, both sprouting and intussusception act in parallel in building the vascular network, although with differences in spatiotemporal distribution. Thereby, in addition to regulatory molecules, flow dynamics support the patterning and remodelling of the rising vascular tree. Herewith, we present an overview of angiogenic processes with respect to intussusceptive angiogenesis as related to local haemodynamics.

Pulmonary capillary pressure. A review

Pulmonary capillary pressure (Pcap) is the predominant force that drives fluid out of the pulmonary capillaries into the interstitium. Increasing hydrostatic capillary pressure is directly proportional to the lung's transvascular filtration rate, and in the extreme leads to pulmonary edema. In the pulmonary circulation, blood flow arises from the transpulmonary pressure gradient, defined as the difference between pulmonary artery (diastolic) pressure and left atrial pressure. The resistance across the pulmonary vasculature consists of arterial and venous components, which interact with the capacitance of the compliant pulmonary capillaries. In pathological states such as acute respiratory distress syndrome, sepsis, and high altitude or neurogenic lung edema, the longitudinal distribution of the precapillary arterial and the postcapillary venous resistance varies. Subsequently, the relationship between Pcap and pulmonary artery occlusion pressure (PAOP) is greatly variable and Pcap can no longer be predicted from PAOP. In clinical practice, PAOP is commonly used to guide fluid therapy, and Pcap as a hemodynamic target is rarely assessed. This approach is potentially misleading. In the presence of a normal PAOP and an increased pressure gradient between Pcap and PAOP, the tendency for fluid leakage in the capillaries and subsequent edema development may substantially be underestimated. Tho-roughly validated methods have been developed to assess Pcap in humans. At the bedside, measurement of Pcap can easily be determined by analyzing a pressure transient after an acute pulmonary artery occlusion with the balloon of a Swan-Ganz catheter.

Comparison of lactated Ringer's, gelatine and blood resuscitation on intestinal oxygen supply and mucosal tissue oxygen tension in haemorrhagic shock

OBJECTIVES: To evaluate the effects on intestinal oxygen supply, and mucosal tissue oxygen tension during haemorrhage and after fluid resuscitation with either blood (B; n=7), gelatine (G; n=8), or lactated Ringer's solution (R; n=8) in an autoperfused, innervated jejunal segment in anaesthetized pigs. METHODS: To induce haemorrhagic shock, 50% of calculated blood volume was withdrawn. Systemic haemodynamics, mesenteric venous and systemic acid-base and blood gas variables, and lactate measurements were recorded. A flowmeter was used for measuring mesenteric arterial blood flow. Mucosal tissue oxygen tension (PO(2)muc), jejunal microvascular haemoglobin oxygen saturation (HbO(2)) and microvascular blood flow were measured. Measurements were performed at baseline, after haemorrhage and at four 20 min intervals after fluid resuscitation. After haemorrhage, animals were retransfused with blood, gelatine or lactated Ringer's solution until baseline pulmonary capillary wedge pressure was reached. RESULTS: After resuscitation, no significant differences in macrohaemodynamic parameters were observed between groups. Systemic and intestinal lactate concentration was significantly increased in animals receiving lactated Ringer's solution [5.6 (1.1) vs 3.3 (1.1) mmol litre(-1); 5.6 (1.1) vs 3.3 (1.2) mmol litre(-1)]. Oxygen supply to the intestine was impaired in animals receiving lactated Ringer's solution when compared with animals receiving blood. Blood and gelatine resuscitation resulted in higher HbO(2) than with lactated Ringer's resuscitation after haemorrhagic shock [B, 43.8 (10.4)%; G, 34.6 (9.4)%; R, 28.0 (9.3)%]. PO(2)muc was better preserved with gelatine resuscitation when compared with lactated Ringer's or blood resuscitation [20.0 (8.8) vs 13.8 (7.1) mm Hg, 15.2 (7.2) mm Hg, respectively]. CONCLUSION: Blood or gelatine infusion improves mucosal tissue oxygenation of the porcine jejunum after severe haemorrhage when compared with lactated Ringer's solution.

Interferon-beta stabilizes barrier characteristics of the blood-brain barrier in four different species in vitro

BACKGROUND: Blood-brain barrier (BBB) breakdown is an early event in the pathogenesis of multiple sclerosis (MS). In a previous study we have found a direct stabilization of barrier characteristics after treatment of bovine brain capillary endothelial cells (BCECs) with human recombinant interferon-beta-1a (IFN-beta-1a) in an in vitro BBB model. In the present study we examined the effect of human recombinant IFN-beta-1a on the barrier properties of BCECs derived from four different species including humans to predict treatment efficacy of IFN-beta-1a in MS patients. METHODS: We used primary bovine and porcine BCECs, as well as human and murine BCEC cell lines. We investigated the influence of human recombinant IFN-beta-1a on the paracellular permeability for 3H-inulin and 14C-sucrose across monolayers of bovine, human, and murine BCECs. In addition, the transendothelial electrical resistance (TEER) was determined in in vitro systems applying porcine and murine BCECS. RESULTS: We found a stabilizing effect on the barrier characteristics of BCECs after pretreatment with IFN-beta-1a in all applied in vitro models: addition of IFN-beta-1a resulted in a significant decrease of the paracellular permeability across monolayers of human, bovine, and murine BCECs. Furthermore, the TEER was significantly increased after pretreatment of porcine and murine BCECs with IFN-beta-1a. CONCLUSION: Our data suggest that BBB stabilization by IFN-beta-1a may contribute to its beneficial effects in the treatment of MS. A human in vitro BBB model might be useful as bioassay for testing the treatment efficacy of drugs in MS.

The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction

The central nervous system (CNS) is tightly sealed from the changeable milieu of blood by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB). While the BBB is considered to be localized at the level of the endothelial cells within CNS microvessels, the BCSFB is established by choroid plexus epithelial cells. The BBB inhibits the free paracellular diffusion of water-soluble molecules by an elaborate network of complex tight junctions (TJs) that interconnects the endothelial cells. Combined with the absence of fenestrae and an extremely low pinocytotic activity, which inhibit transcellular passage of molecules across the barrier, these morphological peculiarities establish the physical permeability barrier of the BBB. In addition, a functional BBB is manifested by a number of permanently active transport mechanisms, specifically expressed by brain capillary endothelial cells that ensure the transport of nutrients into the CNS and exclusion of blood-borne molecules that could be detrimental to the milieu required for neural transmission. Finally, while the endothelial cells constitute the physical and metabolic barrier per se, interactions with adjacent cellular and acellular layers are prerequisites for barrier function. The fully differentiated BBB consists of a complex system comprising the highly specialized endothelial cells and their underlying basement membrane in which a large number of pericytes are embedded, perivascular antigen-presenting cells, and an ensheathment of astrocytic endfeet and associated parenchymal basement membrane. Endothelial cell morphology, biochemistry, and function thus make these brain microvascular endothelial cells unique and distinguishable from all other endothelial cells in the body. Similar to the endothelial barrier, the morphological correlate of the BCSFB is found at the level of unique apical tight junctions between the choroid plexus epithelial cells inhibiting paracellular diffusion of water-soluble molecules across this barrier. Besides its barrier function, choroid plexus epithelial cells have a secretory function and produce the CSF. The barrier and secretory function of the choroid plexus epithelial cells are maintained by the expression of numerous transport systems allowing the directed transport of ions and nutrients into the CSF and the removal of toxic agents out of the CSF. In the event of CNS pathology, barrier characteristics of the blood-CNS barriers are altered, leading to edema formation and recruitment of inflammatory cells into the CNS. In this review we will describe current knowledge on the cellular and molecular basis of the functional and dysfunctional blood-CNS barriers with focus on CNS autoimmune inflammation.

Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, alpha2PI1-8-VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Development of a method for analysis of ketamine and norketamine enantiomers in equine brain and cerebrospinal fluid by capillary electrophoresis

Ketamine and norketamine are being transported across the blood brain barrier and are also entering from blood into cerebrospinal fluid (CSF). Enantioselective distributions of these compounds in brain and CSF have never been determined. The enantioselective CE based assay previously developed for equine plasma was adapted to the analysis of these compounds in equine brain via use of an acidic pre-extraction of interferences prior to liquid/liquid extraction at alkaline pH. CSF can be treated as plasma. With 100 mg of brain tissue and 0.5 mL of CSF or plasma, assay conditions for up to 30 nmol/g and 6 μM, respectively, of each enantiomer with LOQs of 0.5 nmol/g and 0.1 μM, respectively, were established and the assays were applied to equine samples. CSF and plasma samples analyzed stemmed from anesthetized patient horses and brain, CSF and plasma were obtained from anesthetized horses that were euthanized with an overdose of pentobarbital. Data obtained indicate that ketamine and norketamine enantiomers are penetrating into brain and CSF with those of ketamine being more favorably transported than norketamine, whereas metabolites of norketamine are hindered. More work is required to properly investigate possible stereoselectivities of the ketamine metabolism and transport of metabolites from blood into brain tissue and CSF.

Effects of microbeam radiation therapy on normal and tumoral blood vessels

Microbeam radiation therapy (MRT) is a new form of preclinical radiotherapy using quasi-parallel arrays of synchrotron X-ray microbeams. While the deposition of several hundred Grays in the microbeam paths, the normal brain tissues presents a high tolerance which is accompanied by the permanence of apparently normal vessels. Conversely, the efficiency of MRT on tumor growth control is thought to be related to a preferential damaging of tumor blood vessels. The high resistance of the healthy vascular network was demonstrated in different animal models by in vivo biphoton microscopy, magnetic resonance imaging, and histological studies. While a transient increase in permeability was shown, the structure of the vessels remained intact. The use of a chick chorioallantoic membrane at different stages of development showed that the damages induced by microbeams depend on vessel maturation. In vivo and ultrastructural observations showed negligible effects of microbeams on the mature vasculature at late stages of development; nevertheless a complete destruction of the immature capillary plexus was found in the microbeam paths. The use of MRT in rodent models revealed a preferential effect on tumor vessels. Although no major modification was observed in the vasculature of normal brain tissue, tumors showed a denudation of capillaries accompanied by transient increased permeability followed by reduced tumor perfusion and finally, a decrease in number of tumor vessels. Thus, MRT is a very promising treatment strategy with pronounced tumor control effects most likely based on the anti-vascular effects of MRT.