Kohsuke Hagisawa

Thrombus-targeted perfluorocarbon-containing liposomal bubbles for enhancement of ultrasonic thrombolysis: in vitro and in vivo study.

External low‐frequency ultrasound (USD) in combination with microbubbles has been reported to recanalize thrombotically occluded arteries in animal models.

Enhancement of ultrasonic thrombus imaging using novel liposomal bubbles targeting activated platelet glycoprotein IIb/IIIa complex—in vitro and in vivo study

BACKGROUND We developed perfluorocarbon gas-containing bubble liposomes (BL) with Arg-Gly-Asp (RGD) sequence-containing peptides, which bind to activated platelet glycoprotein IIb/IIIa complexes. The aim of this study was to examine the enhancing effects in ultrasonic thrombus imaging using these targeted BL in vitro and in vivo. METHODS Liposomes composed of phosphatidylcholine and cholesterol were manufactured, and RGD peptide was attached by a covalent coupling reaction. Sonication was used to conjugate liposomes and perfluorocarbon gas, which formed targeted BL. In vitro, targeted BL were mixed with whole blood, which was allowed to coagulate while being shaken and rotated. In vivo, we administered targeted BL to 10 rabbits with acute thrombotic occlusions in the ilio-femoral artery. Thrombi were imaged using a 7.5-9 MHz linear transducer and a conventional ultrasound machine, and by scanning electron microscopy. Ultrasound images were digitized, and mean pixel gray-scale level (black = 0, white = 255) was measured. RESULTS In vitro, mean pixel gray-scale level of the thrombi in targeted BL group was significantly higher than in control and non-targeted BL groups (93 ± 26 vs. 58 ± 16, 48 ± 9, p = 0.002, n = 10). Scanning electron microscopy revealed large amounts of targeted BL attached to the thrombi. In vivo, mean pixel gray-scale level of the thrombi with targeted BL was significantly higher (33.2 ± 6.4 vs. 24.8 ± 8.5, p = 0.0051, n = 10) than that before targeted BL administration. CONCLUSIONS Perfluorocarbon gas-containing BL with RGD peptide represent a novel echo contrast agent, which can markedly enhance ultrasonic thrombus imaging in vitro and in vivo, and may be useful for noninvasively diagnosing acute thrombotic vessel occlusion.

Fibrinogen γ‐chain peptide‐coated, ADP‐encapsulated liposomes rescue thrombocytopenic rabbits from non‐compressible liver hemorrhage

Summary.  Background:  We developed a fibrinogen γ‐chain (dodecapeptide HHLGGAKQAGDV [H12])‐coated, ADP‐encapsulated liposome (H12‐[ADP]‐liposome) that accumulates at bleeding sites via interaction with activated platelets via glycoprotein IIb–IIIa and augments platelet aggregation by releasing ADP.

Treatment with fibrinogen γ-chain peptide-coated, adenosine 5′-diphosphate-encapsulated liposomes as an infusible hemostatic agent against active liver bleeding in rabbits with acute thrombocytopenia

We evaluated the hemostatic efficacy of H12‐(adenosine 5′‐diphosphate [ADP])‐liposomes in the setting of active liver bleeding in rabbits with dilutional thrombocytopenia after massive transfusion.

Sympathetic Activity in Dahl Salt-Sensitive Hypertension - Why is aNitric Oxide-Induced Inhibitory System Up-Regulated in the SympatheticCenter?

In essential hypertension, peripheral sympathetic nerve activity is generally thought to be increased regardless of salt sensitivity or insensitivity. Recent reports suggest that the cause may be abnormal central nervous system enhancement. However, other several reports have shown that a central sympathetic inhibitory system, the neuronal nitric oxide synthase system, may be strongly enhanced in salt-sensitive hypertensive Dahl rats, an animal model of salt-sensitive hypertension. These two facts lead to questions what happens finally in peripheral sympathetic activity and what is the relationship between sympathetic nerves and hypertension. In this review, we will show evidences for enhancement of central sympathetic inhibitory system, putative cause for up-regulation of central neuronal nitric oxide synthase system, and a role of its function, then lastly we consider the relationship between hypertension and sympathetic nerves in a rat model, with a focus on salt-sensitive hypertension.

Novel therapeutic use of polysaccharide nanosheets for arachnoid plasty and enhancement of venous tensile strength in rat microneurosurgery.

Subdural effusion sometimes occurs during neurosurgery after opening the Sylvian fissure, due to cerebrospinal fluid (CSF) leakage from the torn arachnoid membrane. Unexpected bleeding from the fragile bridging veins may also result from brain retraction. Neurosurgeons must always watch carefully for these complications during surgery. To prevent such complications, we have attempted the clinical application of a polysaccharide nanosheet that is semi-absorbent and has a potent physical adhesive strength to investigate its therapeutic utility for arachnoid plasty and enhancement of bridging vein tensile strength in Sprague-Dawley rats. The use of overlapping nanosheets completely prevented CSF leakage from injured arachnoid membranes in the cerebral cortex. No inflammatory infiltration was observed on the cerebral surface after 6 months of follow up. In addition, the use of nanosheet bandages significantly reinforced venous tensile strength. This reinforcement increased with the number of overlaid nanosheets. We report that polysaccharide nanosheets can be used for arachnoid plasty without chemical bonding agents and for reinforcement of venous tensile strength in rat vessels. Nanosheets may be an effective neurosurgical tool.

Development of the Liposomes Entrapped Ultrasound Imaging Gas (“Bubble Liposomes”) as Novel Gene Delivery Carriers

Recently, microbubbles and ultrasound have been investigated with a view to improving the transfection efficiency of nonviral delivery systems for gene by cavitation. However, microbubbles had some problems in terms of stability and targeting ability. To solve these problems, we paid attention to liposomes that had many advantages such as stable and safe in vivo and easy to modify targeting ligand. Previously, we have represented that liposomes are good drug and gene delivery carriers. In addition, we developed that the liposomes (“Bubble liposomes”) were entrapped with perfluoropropane known as ultrasound imaging gas. In this study, we assessed about feasibility of “Bubble liposomes” as gene delivery tool utilized cavitation by ultrasound irradiation. “Bubble liposomes” could effectively deliver plasmid DNA to cells by combination of ultrasound irradiation without cyototoxicity. This result suggested that “Bubble liposomes” might be a new class of tool for gene delivery.

Development of diagnostic system for atherosclerosis based on intrinsic fluorescence using multispectral imaging

Composition of atherosclerotic arterial walls is rich in lipids such as cholesterol; unlike normal arterial walls. In this study, we aimed to utilize this difference to diagnose atherosclerosis via multispectral imaging, which allows for identification of fluorescence originating from the substance in the arterial wall. The inner surface of extracted arteries (rabbit abdominal aorta, human coronary aorta) was illuminated by an excitation light and multispectral fluorescence images were obtained. The fluorescence spectra in atherosclerotic sites were shown to be different from those in normal sites. A ratio of fluorescence intensity at a wavelength of two significant differences was then calculated for each pixel and ratio images were reconstructed. As a result, we succeeded in “disease mapping”, by which atherosclerotic sites can be discriminated from normal sites. The differences in fluorescence spectra may be attributed to the differences in fluorophores contained in the intima/media of the artery.

0919. Effect of catecholamine immediately after blast lung injury caused by laser-induced shock wave in a mouse model

The physical damage inflicted by blast waves is called primary blast injury, and lungs are vulnerable to blast waves [1]. Blast lung injuries (BLI) can be extremely critical during the super-acute phase, and hypotension is supposed to be the main cause of death (1) , but its etiology has not been elucidated. Recent studies have demonstrated that hypotension is mediated by the absence of vasoconstriction [2]. However, research investigated the effectiveness of catecholamine for BLI during the super-acute phase was not identified.

High blood pressure enhances brain stem neuronal nitric oxide synthase activity in Dahl salt-sensitive rats

The aims of the present study were to determine the mechanism underlying enhanced neuronal nitric oxide synthase (nNOS) activity in the brain of hypertensive Dahl salt‐sensitive (DSS) rats and the consequences of enhanced nNOS activity. Male DSS rats were fed either a regular (0.4% NaCl) or high‐salt (8% NaCl) diet, with or without 0.25% nifedipine, for 4 weeks. The effects of nifedipine, which lowers blood pressure peripherally, on central nNOS were determined by measuring nNOS activity, as well as the number of nNOS‐positive neurons in the brain stem and diencephalon. The effects of chronic (12 days) infusion of 7 μg (0.5 μL/h, i.c.v.) S‐methyl‐l‐thiocitrulline (SMTC; a stereoselective competitive nNOS inhibitor) on mean arterial pressure were assessed in conscious DSS rats using a radiotelemetry system. In addition, the number of central nNOS‐positive neurons was compared between DSS and salt‐insensitive Sprague‐Dawley rats. Normalization of blood pressure by nifedipine attenuated the increase in nNOS activity in the brain stem of DSS rats. Chronic i.c.v. infusion of SMTC further enhanced hypertension in DSS rats. Feeding of a high‐salt diet increased nNOS‐positive neurons in the lateral parabrachial nucleus, rostral ventrolateral medulla and nucleus tractus solitarius of DSS compared with Sprague‐Dawley rats, whereas nNOS‐positive neurons in the paraventricular nucleus remained downregulated in DSS rats. The results of the present study suggest that hypertension, rather than a high‐salt diet, increases central nNOS activity in hypertensive DSS rats to buffer high blood pressure. However, this compensatory response may be insufficient to relieve salt‐induced hypertension.