Polymer-brush-afforded SPIO Nanoparticles Show a Unique Biodistribution and MR Imaging Contrast in Mouse Organs.

INTRODUCTION: To investigate the biodistribution and retention properties of the new super paramagnetic iron oxide (new SPIO: mean hydrodynamic diameter, 100 nm) nanoparticles, which have concentrated polymer brushes in the outer shell and are difficult for phagocytes to absorb, and to compare the new SPIO with clinically approved SPIO (Resovist: mean hydrodynamic diameter, 57 nm). MATERIALS AND METHODS: 16 male C57BL/6N mice were divided in two groups according to the administered SPIO (n = 8 for each group; intravenous injection does, 0.1 ml). In vivo magnetic resonance imaging (MRI) was performed before and one hour, one day, one week and four weeks after SPIO administration by two dimensional-the fast low angle shot (2D-FLASH) sequence at 11.7T. Ex vivo high-resolution images of fixed organs were also obtained by (2D-FLASH). After the ex vivo MRI, organs were sectioned and evaluated histologically to confirm the biodistribution of each particle precisely. RESULTS: The new SPIO was taken up in small amounts by liver Kupffer cells and showed a unique in vivo MRI contrast pattern in the kidneys, where the signal intensity decreased substantially in the boundaries between cortex and outer medulla and between outer and inner medulla. We found many round dark spots in the cortex by ex vivo MRI in both groups. Resovist could be detected almost in the cortex. The shapes of the dark spots were similar to those observed in the new SPIO group. Transmission electron microscopy revealed that Resovist and the new SPIO accumulated in different cells of glomeruli, that is, endothelial and mesangial cells, respectively. CONCLUSION: The new SPIO was taken up in small amounts by liver tissue and showed a unique MRI contrast pattern in the kidney. The SPIO were found in the mesangial cells of renal corpuscles. Our results indicate that the new SPIO may be potentially be used as a new contrast agent for evaluation of kidney function as well as immunune function.

From cartoon to real time MRI: in vivo monitoring of phagocyte migration in mouse brain.

Recent studies have demonstrated that immune cells play an important role in the pathogenesis of many neurological conditions. Immune cells constantly survey the brain microvasculature for irregularities in levels of factors that signal homeostasis. Immune responses are initiated when necessary, resulting in mobilisation of the microglial cells resident in the central nervous system (CNS) and/or of infiltrating peripheral cells. However, little is known about the kinetics of immune cells in healthy and diseased CNS, because it is difficult to perform long-term visualisation of cell motility in live tissue with minimal invasion. Here, we describe highly sensitive in vivo MRI techniques for sequential monitoring of cell migration in the CNS at the single-cell level. We show that MRI combined with intravenous administration of super-paramagnetic particles of iron oxide (SPIO) can be used to monitor the transmigration of peripheral phagocytes into healthy or LPS-treated mouse brains. We also demonstrate dynamic cell migration in live animal brains with time-lapse MRI videos. Time-lapse MRI was used to visualise and track cells with low motility in a control mouse brain. High-sensitivity MRI cell tracking using SPIO offers new insights into immune cell kinetics in the brain and the mechanisms of CNS homeostasis.

Early pathological alterations of lower lumbar cords detected by ultrahigh-field MRI in a mouse multiple sclerosis model.

Magnetic resonance imaging (MRI) is widely employed for the diagnosis of multiple sclerosis (MS). However, sometimes, the lesions found by MRI do not correlate with the neurological impairments observed in MS patients. We recently showed autoreactive T cells accumulate in the fifth lumbar cord (L5) to pass the blood-brain barrier and cause inflammation in the central nervous system of experimental autoimmune encephalomyelitis (EAE) mice, an MS model. We here investigated this early event using ultrahigh-field MRI. T2-weighted image signals, which conform to the water content, increased in L4 and L5 during the development of EAE. At the same time, the sizes of L4 and L5 changed. Moreover, angiographic images of MRI showed branch positions of the blood vessels in the lower lumbar cords were significantly altered. Interestingly, EAE mice showed occluded and thickened vessels, particularly during the peak phase, followed by reperfusion in the remission phase. Additionally, demyelination regions of some MS patients had increased lactic acid content, suggesting the presence of ischemic events. These results suggest that inflammation-mediated alterations in the lower lumbar cord change the homeostasis of the spinal cord and demonstrate that ultrahigh-field MRI enables the detection of previously invisible pathological alterations in EAE.

Constitutive lymphocyte transmigration across the basal lamina of high endothelial venules is regulated by the autotaxin/lysophosphatidic acid axis.

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC's effect was abrogated by ATX inhibition, whereas LPA's effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte-HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.

Arginine vasopressin receptor antagonists (vaptans): pharmacological tools and potential therapeutic agents.

Arginine vasopressin (AVP) attracted attention as a potentially important neurohormonal mediator of the heart failure (HF) syndrome and hyponatremic states in humans because AVP influences renal handling of free water, vasoconstriction and myocyte biology through activation of V2 and V1(a) receptors. Current research is exploring V2- and dual V1(a)/V2 receptor antagonism for the treatment of hyponatremia, as well as for the congestion and edema associated with chronic HF, because vasopressin receptor antagonists might offer benefits in comparison with conventional loop diuretics. The purpose of this review is to update the current status of experimental and clinical studies with available vasopressin receptor antagonists (conivaptan and tolvaptan) and their potential role in the treatment of HF and hyponatremia of multiple causes.

Gd3+-functionalized near-infrared quantum dots for in vivo dual modal (fluorescence/magnetic resonance) imaging.

Gd(3+)-functionalized near-infrared emitting quantum dots were synthesized as a dual modal contrast agent for in vivo fluorescence imaging and magnetic resonance imaging.

Preparation and characterization of highly fluorescent, glutathione-coated near infrared quantum dots for in vivo fluorescence imaging.

Fluorescent probes that emit in the near-infrared (NIR, 700-1,300 nm) region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs) have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH)-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs) were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell) QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4). The GSH-QDs (800 nm emission) were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer), and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM), the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIR-fluorescence imaging of a lymph node in a mouse is presented.

Mechanotransduction in leukocyte activation: a review.

We review recent evidence which suggests that leukocytes in the circulation and in the tissue may readily respond to physiological levels of fluid shear stress in the range between about 1 and 10 dyn/cm 2, a range that is below the level to achieve a significant passive, viscoelastic response. The response of activated neutrophilic leukocytes to fluid shear consists of a rapid retraction of lamellipodia with membrane detachment from integrin binding sites. In contrast, a subgroup of non-activated neutrophils may project pseudopods after exposure to fluid shear stress. The evidence suggests that G-protein coupled receptor downregulation by fluid shear with concomitant downregulation of Rac-related small GTPases and depolymerization of F-actin serves to retract the lamellipodia in conjunction with proteolytic cleavage of beta 2 integrin to facilitate membrane detachment. Furthermore, there exists a mechanism to up- and down-regulate the fluid shear-response, which involves nitric oxide and the second messenger cyclic guanosine monophosphate (cGMP). Many physiological activities of circulating leukocytes are under the influence of fluid shear stress, including transendothelial migration of lymphocytes. We describe a disease model with chronic hypertension that suffers from an attenuated fluid shear-response with far reaching implications for microvascular blood flow.

Blood flow regulation in the cerebral microvasculature with an arcadal network: a numerical simulation.

Blood flow regulation in the cerebral microvasculature with an arcadal network was investigated using a numerical simulation. A mathematical model for blood flow in the arcadal network, based on in vivo data of cat cerebral microvasculature and flow velocity was developed. The network model consists of 45 vessel segments and 25 branching points. To simulate microvascular response to blood flow, non-reactive (solid), cerebral arteriole-like, or skeletal muscle arteriole-like responses to wall shear stress were taken into account. Numerical calculation was carried out in the flow condition where the inlet (arterial) pressure was changed from 60 to 120 mmHg. Flow-rate in each efferent vessel and the mean flow-rate over all efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The simulation demonstrated the wall shear stress-induced vasodilation in the arcadal network worked to maintain the blood flow at a constant level with pressure variable in a wide range. It is suggested that an individual microvessel (segment) should join in the regulatory process of flow, interacting with other microvessels (cooperative regulation).

Microembolic flow disturbances in the cerebral microvasculature with an arcadal network: a numerical simulation.

Flow disturbance due to microembolism in the cerebral microvasculature with an arcadal network was studied by a numerical simulation. A mathematical model for flow in the arcadal network was developed, based on in vivo data of cat cerebral microvasculature and flow velocity. The network model consisted of 45 vessel segments, and 25 branching points. To simulate microvascular responses to blood flow, the following three types of responses to wall shear stress were considered; non-reactive (solid-like), cerebral arteriole, and skeletal muscle arteriole-like responses. The numerical calculation was carried out in the condition where a feeding arteriole was occlused. Flow changes in efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The present simulation has demonstrated that blood flow in efferent vessels was influenced by the topology of the vascular network and the response pattern in single vessels. The arcadal structure of arterioles might be most effective in response to flow disturbances in efferent vessels.

De-activation of neutrophils in suspension by fluid shear stress: a requirement for erythrocytes.

Leukocyte de-activation in response to a mechanical stimulus may be an important mechanism to reduce inflammation in the circulation and cardiovascular complications. We examine here a specific form of leukocyte activation in the form of pseudopod projection, a process that is important during cell spreading and migration, but if it occurs in circulating leukocytes, may also lead to their entrapment in the microvascular network. Fresh neutrophils were activated with fMLP, suspended without adhesion to endothelium, and sheared in a cone-and-plate device while both shear stress and shear rate were measured. A fraction of the activated neutrophils retracted their pseudopods under the influence of fluid shear and returned to round shape. Pseudopod retraction was observed only in the presence of erythrocytes (at shear stresses up to approximately 25 dyn/cm(2)). At a constant hematocrit and increasing plasma viscosities with addition of macromolecules, the number of de-activated neutrophils scaled with shear stress and less so with shear rate. We examined a biochemical and rheological role of erythrocytes during shear de-activation of neutrophils. Addition of superoxide dismutase (SOD) in phosphate buffer served to enhance neutrophil de-activation by fluid shear. Replacement of erythrocytes by solid microspheres (5.4 mum) to simulate the particle properties of the erythrocytes, did not serve to enhance neutrophil de-activation unless in the presence of SOD. At higher shear stresses without erythrocytes (38-77 dyn/cm(2)), we also observed neutrophil de-activation but only in the presence of SOD. These results suggest that erythrocytes play an important role in neutrophil de-activation by reducing the superoxide level in plasma. Shear stress, rather than shear rate, is the key determinant that regulates neutrophil de-activation.

Microvascular hemodynamic responses to arteriovenous shunting in rat limb.

Autologus veins have been used clinically as a bypass conduit for reconstruction of small arteries, but there are few data available for microvascular response to arteriovenous (AV) shunting. This study was aimed to evaluate microvascular hemodynamic changes induced by creating AV anastomosis in rat hindlimb. Using intravital fluorescence videomicroscopy, we measured velocities of red blood cells (RBCs) flowing in the microvascular network in the control state, in the occlusion state where the superficial femoral artery (SFA) was occluded, and in the AV shunting state where the AV anastomosis was opened after occlusion of SFA. RBC velocities were measured in 155 capillaries of 6 rats using a dual window method and a frame-by-frame technique. The mean velocity and the coefficient of variation were 0.61 mm/sec and 0.90 in the control state, 0.34 mm/sec and 1.30 in the occlusion state, 0.83 mm/sec and 1.24 in the AV shunting state, respectively. These indicated that hemodynamic heterogeneity among capillaries increased with decrease in mean velocity following the arterial occlusion, while the AV shunting augmented the heterogeneity with increase in mean velocity. Capillaries with low perfusion (<0.1 mm/sec) or high perfusion (>1.0 mm/sec) were 5.8% or 20.6%, 29.6 or 5.2%, and 22.6 or 30.3% out of all measured capillaries in the control, occlusion and AV shunting conditions, respectively. In conclusion, AV shunting increased capillary perfusion and also its spatial heterogeneity, preferentially inducing high velocity in the microvasculature.

Heterogeneity of capillary flow in the retrograde microcirculation induced in rat limb by arteriovenous shunting.

Arteriovenous (AV) fistulas have been used clinically for improving adjunctive bypass patency. Such AV shunting induces retrograde flow in the microvascular network, which may induce microvascular remodeling and angiogenesis at the chronic phase. This paper was aimed to examine heterogeneity of blood flow among capillaries in the retrograde microcirculation induced by AV shunting. An AV anastomosis was created in rat hind limb. Using a dual window method or frame-by-frame technique on the fluorescence microscopic video images, we measured velocities of red blood cells (RBCs) flowing in the capillary network in three flow conditions: control (normal flow), arterial occlusion, and AV shunting (retrograde flow). For each flow condition, RBC velocities were obtained in 155 capillaries of 6 rats. By classifying all the capillaries into four groups based on the levels of RBC velocity in the occlusion state, we evaluated the mean velocities, coefficient of variation (CV), and histograms for each group of capillaries. The mean velocity and CV in each group changed significantly from the control to AV shunting states. Especially, most significant changes appeared in capillary groups where the superficial femoral artery or its collateral arteries might have a direct influence. Though the AV shunting improved capillary perfusion in the mean level, major parts of capillaries still remained at low perfusion.

Cell morphological changes in venous remodeling induced by arteriovenous grafting in rat limb.

Vascular remodeling induced in rat limb by arteriovenous (AV) shunting was investigated by evaluating changes in vascular diameter and cell morphology. In Wistar rats, a vein graft was implanted in situ in the hind limb. Flow-rate in the grafted vein was assessed by measuring flow in the common femoral artery using an ultrasonic flowmeter. Nuclei and actin filaments of the venous wall were stained with propidium iodine and phalloidine-FITC, and the samples were observed using confocal laser microscopy. The grafted veins became circular in cross-section with increase in diameter during two weeks after AV shunting. Owing to the increase in diameter, the estimated wall shear stress was not increased so much as the flow-rate. The confocal laser microscopic observation showed that endothelial cells (ECs) and smooth muscle cells (SMCs) in the grafted veins were either aligned well (2 out of 8 samples), or ECs were denudated and SMCs were disrupted (in 6 out of 8 samples). The cell density of ECs was unchanged from the control level. In conclusion, the grafted vein was remodeled with morphological changes in ECs and SMCs during 2 weeks after AV shunting.

Capillary angiogenesis and remodeling induced in rat limb by arteriovenous shunting.

Capillary angiogenesis and remodeling induced by arteriovenous (AV) shunting in rat hind limb was investigated by evaluating changes in capillary density and diameter in the skeletal muscle subject to retrograde flow and high pressure. Wistar rats were used, and an AV anastomosis was created in the hind limb. Two weeks after AV shunting, the microvasculature in the limb was visualized by GS-lectine, and the samples were observed using confocal laser microscopy. The capillary density were increased by approximately 150% for small vessels (<13 microm in diameter) under retrograde flow condition, but no change appeared for large vessels (>13 microm in diameter). The capillary diameters were not significantly different between control and chronic condition. In conclusion, retrograde flow produced by AV shunting increased capillary density but it did not change the capillary diameter significantly.