Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.

Interactions of tumor cells with lymphatic vessels are of paramount importance for tumor progression, however, the underlying molecular mechanisms are poorly understood. Whereas enlarged lymphatic vessels are frequently observed at the periphery of malignant melanomas, it has remained unclear whether intratumoral lymphangiogenesis occurs within these tumors. Here, we demonstrate the presence of intratumoral lymphatics and enlargement of lymphatic vessels at the tumor periphery in vascular endothelial growth factor (VEGF)-C-overexpressing human melanomas transplanted onto nude mice. VEGF-C expression also resulted in enhanced tumor angiogenesis, indicating a coordinated regulation of lymphangiogenesis and angiogenesis in melanoma progression. The specific biological effects of VEGF-C were critically dependent on its proteolytic processing in vivo. Furthermore, VEGF-C induced chemotaxis of macrophages in vitro and in vivo, revealing a potential function of VEGF-C as an immunomodulator. Taken together, our results identify VEGF-C as multifunctional factor involved in regulating tumor lymphangiogenesis, angiogenesis, and immune response.

Time course of nodal enhancement with CT X-ray nanoparticle contrast agents: effect of particle size and chemical structure.

RATIONALE AND OBJECTIVES: Levels of CT enhancement in rabbit lymph nodes were followed with time after subcutaneous injection of four iodinated, insoluble nanoparticle contrast agents to provide experimental support for the hypothesis that clearance of these agents is related to the chemical structure of the agent itself. The impact of particle size was also studied. METHODS: Subcutaneous injections (2 x 0.25 mL) were made in the dorsum of rabbit paws with 15% suspensions of four nanoparticle contrast agents. Images were obtained at 4, 10, 24, 48, and 72 hours and 5, 7, and 14 days after injection. Average attenuation (in Hounsfield units [HU]), node volume, and total iodine uptake were estimated from the CT scans for each lymph node at each time point. RESULTS: All the agents provided adequate enhancement of both the popliteal and axillary lymph nodes of the rabbit (ie, > delta100 HU). Lymph node volume appears to be related to the persistence of enhancement, with long-lived agents demonstrating the greatest increase in size. The rate of clearance from the lymph nodes is related to the structure of the agent. CONCLUSIONS: Clearance of insoluble, iodinated nanoparticle contrast agents from lymph nodes can be modulated by changes in the structure of the agent itself. Using the same agent, smaller particles deliver material to the lymph nodes more quickly and clear more quickly.

An adaptive microwave phased array for targeted heating of deep tumours in intact breast: animal study results.

It has previously been reported in phantoms, that an adaptive radiofrequency phased array can generate deep focused heating distributions without overheating the skin and superficial healthy tissues. The present study involves adaptive microwave phased array hyperthermia tests in animals (rabbits) with and without tumours. The design of the adaptive phased array as applied to the treatment of tumours in intact breast, is described. The adaptive phased array concept uses breast compression and dual-opposing 915 MHz air-cooled waveguide applicators with electronic phase shifters and electric-field feedback, to focus automatically by computer control the microwave radiation in deep tissue. Temperature measurements for a clinical adaptive phased array hyperthermia system demonstrate tissue heating at depth with reduced skin heating.

Enhancement of fluid filtration across tumor vessels: implication for delivery of macromolecules.

Cancer therapies using genes and other macromolecules might realize their full clinical potential if they could be delivered to tumor tissue in optimal quantities. Unfortunately, the compromised circulation within tumors poses a formidable resistance to adequate and uniform penetration of these agents. Previously, we have proposed elevated interstitial fluid pressure (IFP) as a major physiological barrier to delivery of macromolecules. Here we postulate that modulation of tumor microvascular pressure (MVP) and associated changes in IFP would enhance macromolecular delivery into a solid tumor. To test our hypothesis, we altered tumor MVP by either periodic injection or continuous infusion of angiotensin II (AII) and measured the resulting changes in IFP and uptake of macromolecules. We used the nicotinyl hydrazine derivative of human polyclonal IgG (HYNIC-IgG) as a nonspecific macromolecule and CC49 antibody as a specific macromolecule. We found that both chronic and periodic modulation of tumor MVP enhances transvascular fluid filtration, leading to a 40% increase in total uptake of the specific antibody within 4 hr of its administration. Conversely, neither continuous nor periodic infusion of AII induced any increase in uptake of nonspecific antibodies. Strategies to improve delivery of macromolecules and limitations of this approach are identified.

Lymph node extraction of radiopaque nanoparticulates in the rabbit as measured in vivo with CT.

RATIONALE AND OBJECTIVES: The purpose of this study was to estimate in vivo extraction of lymphographic material in the popliteal node of the rabbit. MATERIALS AND METHODS: Serial quantitative computed tomography (CT) of target tissues in four legs of two rabbits was performed after subcutaneous injection of an improved lymphographic contrast agent. Massage was used as a lymphotrophic intervention. RESULTS: At 15 minutes, the mean change in Hounsfield units measured 815 in the popliteal node, 219 in afferent lymphatic vessels, and 127 in efferent lymphatic vessels. The nodal extraction of nanoparticulates from the lymph was approximately 55%. Nodal massage allowed the amount of nanoparticulate remaining in sinusoidal lymph to be estimated. CONCLUSION: Functional CT performed with timed studies, proper radiopaque materials, and physiologic interventions can depict in vivo lymphatic physiology under minimally invasive conditions.

CT visualization of blood pool in rats by using long-circulating, iodine-containing micelles.

RATIONALE AND OBJECTIVES: Small, long-circulating particulate carriers of contrast agents, such as micelles, are potentially useful in computed tomographic (CT) blood-pool imaging. An iodine-containing amphiphilic block-copolymer consisting of iodine-substituted poly-L-lysine (MPEG-iodolysine) forms micelles in an aqueous solution. The biodistribution and CT depiction of these radiopaque micelles were therefore studied in rats. MATERIALS AND METHODS: MPEG-iodolysine micelles were synthesized and injected into rats via the tail vein at a dose of 170 mg iodine per kilogram. Three animals were used, and tissue enhancement was followed on serial CT scans. RESULTS: MPEG-iodolysine block-copolymer forms particulates with an average diameter of 80 nm and an iodine content of 33.8%. After intravenous injection into rats, the agent produced noticeable and sustained enhancement of the blood pool (aorta and heart), liver, and spleen for least 3 hours. CONCLUSION: In rats, MPEG-iodolysine micelles were a long-lived blood-pool contrast agent useful for CT.

Changes in vascularization of human breast cancer xenografts responding to antiestrogen therapy.

To elucidate the previously suggested vascular effect(s) of antiestrogen therapy, we studied the effect of estrogen withdrawal and tamoxifen on 1) vascular resistance, 2) glucose and oxygen consumption, and 3) vascular density in a perfused breast cancer line (ZR75-1). Furthermore, we examined ZR75-1 tumors by functional CT-scanning (fCT) to determine changes in parameters related to tumor capillary transfer constants and vascular volume fraction in response to antiestrogenic manipulations. The vascular resistance decreased significantly from 42.7 to 20.8 mmHg x min x g x ml(-1) (P< .03) on day 9 after estrogen withdrawal, but not after 9 days of tamoxifen treatment. The estrogen-depleted tumors were significantly smaller than controls on day 9. There was no difference in nutrient consumption or vascular density in any of the experimental groups compared to controls. fCT showed an increase (P < .03) in vascular volume fraction during tumor growth, and this parameter was significantly lower after estrogen withdrawal when compared to controls (P < .05). Vascular resistance correlated with tumor size (R = 0.7, P < .0001), indicating that vascular resistance increases during tumor growth. The changes in vascular parameters after estrogen withdrawal indicate a vascular remodeling effect. This inhibition of vascular development by hormone deprivation may have important implications for future planning of multimodal treatment regimens.

Indicator measurement in tissues: CT with iohexol versus storage-phosphor autoradiography with carbon-14-labeled inulin.

RATIONALE AND OBJECTIVES: Computed tomography (CT) provides accurate measurement of blood iodine concentration in vivo, as well as in phantoms simulating tissue; however, its ability to measure radiopaque agents in biologic tissues in comparison with a standard technique does not seem to have been demonstrated. To validate the performance of CT imaging for quantification of contrast media in a variety of biologic tissues in vivo, a comparison between CT imaging with an iodinated contrast agent (iohexol) and the reference tracer quantification technique (storage-phosphor autoradiography with carbon-14-labeled inulin) was performed. MATERIALS AND METHODS: Six New Zealand White rabbits were injected intravenously with a cocktail of iohexol and C-14-labeled inulin at different dose ratios and sacrificed shortly after injection to arrest blood flow at different stages of tissue tracer distribution. One rabbit received no iohexol-inulin mixture and provided baseline data. Liver, spleen, kidneys, testis, and heart were excised and rapidly frozen. Each organ was scanned with CT (1-mm contiguous sections) to determine tissue iodine distribution. Twenty-micrometer tissue slices were made in the same planes in which the CT images had been acquired, and storage-phosphor screen autoradiography was performed to quantify C-14-labeled inulin distribution. RESULTS: Digital image analysis of CT images and autoradiograms was performed on spatially matched regions, and resultant tracer concentrations were compared. Tracer concentrations were highly correlated, with resultant R2 values exceeding 0.9 in all tissues. CONCLUSION: The highly correlated results for iodinated tracer quantification in tissues for CT versus those obtained with the reference technique validate the performance of CT as an accurate means of measuring concentration of radiopaque agent in tissue, independent of tracer dose.

YM872, a highly water-soluble AMPA receptor antagonist, preserves the hemodynamic penumbra and reduces brain injury after permanent focal ischemia in rats.

BACKGROUND AND PURPOSE: We recently described an image analysis technique based on the temporal correlation mapping (TCM) of injected contrast agents that can be used to distinguish the hemodynamic core and hemodynamic penumbra after focal ischemia. In this study we used this technique for the first time to investigate the effects of the water-soluble AMPA receptor antagonist YM872 in permanent focal ischemia. METHODS: Fischer 344 rats were subjected to permanent occlusion of the middle cerebral artery. Approximately 30 minutes after ischemia, functional CT images were collected with the use of a dynamic scanning protocol with bolus injections of nonionic contrast agent iohexol (1 mL/kg). TCM analysis defined the distributions of hemodynamic core and hemodynamic penumbra. Cerebral perfusion indices were calculated on the basis of the area under the first-pass transit curves. One hour after ischemia, animals were randomly treated with YM872 (n=8, 20 mg/kg per hour over 4 hours) or normal saline (n=10). Twenty-four hours later, neurological deficits were evaluated, and conventional CT and triphenyltetrazolium chloride staining were used to define volumes of ischemic damage. RESULTS: At 24 hours after ischemia, hypodense lesions were visible on conventional CT scans that were highly correlated with triphenyltetrazolium chloride lesion volumes. YM872 improved neurological deficits and reduced volumes of ischemic damage in cortex (90+/-14 versus 170+/-16 mm3 in controls) but not striatum (57+/-14 versus 79+/-6 mm3 in controls). Comparison of early TCM images with conventional CT scans of ischemic injury showed that the hemodynamic core was always damaged in all rats. In controls, 54% of the tissue within the hemodynamic penumbra evolved into ischemic damage compared with 24% in YM872-treated rats. Furthermore, the perfusion index corresponding to the ischemic damage threshold was significantly reduced by YM872 (28+/-2% versus 37+/-2% in controls). CONCLUSIONS: These results indicate that YM872 is a neuroprotective compound that ameliorates the deterioration of the hemodynamic penumbra after focal ischemia.

Massage-induced release of subcutaneously injected liposome-encapsulated drugs to the blood.

Liposome-based, externally regulated drug delivery system is described in which liposome-encapsulated bioactive molecules can be delivered into the blood in response to simple mechanical action. Without any mechanical stimulation, subcutaneously injected 200 mm liposomes are usually trapped in the interstitial for prolonged time. However, upon lymphotropic stimulation (such as manual massage of the injection site), the liposomes can be mobilized into the blood via lymphatic pathway. Up to 40% of the injection dose can be delivered to the blood via lymphatic pathway from the injection site at the rabbit's front paw dorsum during 5 min manual massage cycle. Using vasoconstricting hormone angiotensin II as liposome-encapsulated pharmacological marker, we demonstrated that physiological response to encapsulated drug (average blood pressure increase) can also induced and modulated by massage. Massage itself was found to have no effect on the blood pressure. Modification of liposome surface with polyethylene glycol was found to increase blood localization of the liposome-encapsulated drug presumably due to decreasing the uptake of the drug carrier by lymph node macrophages. Pressure-dependent gaps between lymphatic capillary endothelial cells are thought to play the role of the size discrimination device allowing larger particulates into the lymphatics and, eventually into the blood after increase of interstitial pressure caused by injection site massage.

Assessment of cerebral perfusion and arterial anatomy in hyperacute stroke with three-dimensional functional CT: early clinical results.

PURPOSE: Our purpose was to determine the clinical feasibility of quantitative three-dimensional functional CT in patients with hyperacute stroke. METHODS: Twenty-two patients who underwent clinically indicated CT angiography were studied: nine patients had no stroke, eight had mature stroke, and five had hyperacute stroke (less than 3 hours since ictus). Maps were obtained of perfused cerebral blood volume (PBV), and CT angiograms were generated by using standard techniques. RESULTS: Normal PBV values (mean +/- SEM) were 4.6 +/- 0.15% in the gray matter, 1.75 +/- 0.09% in the white matter, 2.91 +/- 0.20% in the cerebellum, 3.18 +/- 0.10% in the caudate, 2.84 +/- 0.23% in the putamen, 2.92 +/- 0.29% in the thalamus, and 1.66 +/- 0.03% in the brain stem. For patients with mature stroke, ischemic changes were visible on noncontrast, contrast-enhanced, and PBV scans. In patients with hyperacute stroke, ischemic changes were either absent or subtle before contrast administration, but became apparent on contrast-enhanced scans. Quantitative PBV maps confirmed reduced regional perfusion. CT angiograms in the hyperacute group showed occlusion of vessels in locations appropriate to the PBV deficits seen. CONCLUSION: Quantitative three-dimensional functional CT is feasible for patients with hyperacute stroke. It is performed by using helical CT techniques, and yields measures of cerebrovascular physiological function, which are useful in this patient population.

Bacteraemia during direct laryngoscopy and endotracheal intubation: a study using a multiple culture, large volume technique.

Bacteraemia secondary to orotracheal intubation has been reported to occur in 0-5.3% of patients. Bacteraemia detection is dependent upon several factors including the volume of blood per culture and the number of cultures. Prior studies used small volumes of blood and one or two cultures, and may therefore have underestimated the incidence of bacteraemia. Sixty-two adult patients who underwent direct laryngoscopy and endotracheal intubation were studied. Baseline blood cultures were sterile in all patients. After intubation, four blood cultures were obtained in ten minutes, with 10 ml being evenly divided between aerobic and anaerobic media. Two patients (3.2%) became bacteraemic. This is a lower incidence than occurs in association with other procedures for which The American Heart Association does not recommend administration of prophylactic antibiotics. Therefore, prophylactic antibiotics are not recommended prior to direct laryngoscopy. However, when a prophylactic antibiotic is administered prior to surgery, it would be best to administer the antibiotic prior to direct laryngoscopy and intubation.

MRI measurements of water diffusion and cerebral perfusion: their relationship in a rat model of focal cerebral ischemia.

The aim of this study was to examine the quantitative relationship between changes in apparent diffusion coefficient (ADC) and transverse relaxivity (delta R2*) measurements of relative perfusion deficits within the gradients of a focal ischemic insult. Sixty minutes after permanent occlusion of the middle cerebral artery, rats (n = 7) were subjected to spin echo diffusion-weighted scans followed by fast low-angle shot (FLASH) perfusion-sensitive scans. Diffusion-weighted images showed clear ischemic lesions in the affected basal ganglia and cortex. Ischemic deficits were demonstrated as a decrease in first-pass transit of injected boluses of gadodiamide. ADC maps were generated and regions of interest (ROIs) were obtained to span the range of ADC reductions from the lesion center or core to the periphery or penumbra. Corresponding ROIs from the bolus injection images were used to calculate perfusion indexes relative to contralateral levels as ratios of delta R2* integrals and ratios of delta R2* peak values. In all animals, the degree of ADC reductions was related to the degree of delta R2* perfusion deficits, ranging from severe ischemia in the core of the lesion to intermediate and moderate changes toward the lesion periphery. In the ischemic periphery, ADC reductions were linearly correlated with delta R2* peak ratios. However, no significant correlation was found between ADC reductions and delta R2* integral ratios. These data suggest that magnetic resonance measurements of ADC and delta R2* peak ratios can be used to quantitatively assess the variable gradients in focal ischemia, including potentiallyn critical areas at risk in the ischemic periphery.

Block-copolymer of polyethylene glycol and polylysine as a carrier of organic iodine: design of long-circulating particulate contrast medium for X-ray computed tomography.

In order to obtain small, polymer-stabilized particulate carriers for organic iodine to serve as a contrast agent for X-ray computed tomography (CT) an attempt was made to design a carrier based on polymeric micelles. Here we describe the synthesis of an iodine-containing amphiphilic block-copolymer which can micellize in aqueous solutions. The two blocks of the copolymer consisted of methoxypoly(ethyleneglycol) and poly[epsilon,N-(triiodobenzoyl)-L-lysine]. Upon dispersion in water, the block copolymer formed particles with average diameter 80 nm and iodine content up to 44.7%. The particles start to dissociate to the individual polymeric chains in the concentration range of 0.05-0.5 microM in water at 23 degrees C. Upon intravenous injection at 250 mg of iodine/kg (570 mg of the agent/kg) in rabbits the medium demonstrated exceptional 24 hr half-life in the blood substantiating corona/core structure of the particles with PEG chains protecting the iodine-containing core. The possible use of these particulates as contrast medium for X-ray computed tomography is discussed.

Measurement of cerebral blood volume with subtraction three-dimensional functional CT.

PURPOSE: To implement a three-dimensional subtraction functional CT technique to permit rapid quantitative mapping of regional cerebral blood volume (CBV). METHODS: The 3-D functional CT technique was implemented in a rabbit model using normal and ischemic animals. Two spiral data acquisitions were performed, one before and one during biphasic administration of contrast material. CBV maps were then produced on a voxel-by-voxel basis through the whole brain. RESULTS: The average normal CBV was 3.3 +/- 0.4 mL/100 g (n = 7), and the regional values were 4.5 +/- 0.6 mL/100 g for cortical gray matter, 2.5 +/- 0.6 mL/100 g for white matter, and 3.7 +/- 0.4 mL/100 g for the basal ganglia. The CBVs in ischemic regions were 1.5 +/- 0.4 mL/100 g, 0.7 +/- 0.7 mL/100 g, and 1.8 +/- 0.9 mL/100 g, respectively. CONCLUSION: Subtraction 3-D functional CT is a fast, potentially cost-effective method with which to assess whole-brain CBV. Because the data collected in 3-D functional CT imaging also can be used to produce large-vessel angiograms, its use in a clinical setting can provide a multiparametric study of cerebrovascular abnormalities that encompasses both large and small vessel circulations for patients being examined for stroke.