Analysis of 13 C and 14 C labeling in pyruvate and lactate in tumor and blood of lymphoma-bearing mice injected with 13 C- and 14 C-labeled pyruvate.

Measurements of hyperpolarized 13 C label exchange between injected [1-13 C]pyruvate and the endogenous tumor lactate pool can give an apparent first-order rate constant for the exchange. The determination of the isotope flux, however, requires an estimate of the labeled pyruvate concentration in the tumor. This was achieved here by measurement of the tumor uptake of [1-14 C]pyruvate, which showed that <2% of the injected pyruvate reached the tumor site. Multiplication of this estimated labeled pyruvate concentration in the tumor with the apparent first-order rate constant for hyperpolarized 13 C label exchange gave an isotope flux that showed good agreement with a flux determined directly by the injection of non-polarized [3-13 C]pyruvate, rapid excision of the tumor after 30 s and measurement of 13 C-labeled lactate concentrations in tumor extracts. The distribution of labeled lactate between intra- and extracellular compartments and the blood pool was investigated by imaging, by measurement of the labeled lactate concentration in blood and tumor, and by examination of the effects of a gadolinium contrast agent and a lactate transport inhibitor on the intensity of the hyperpolarized [1-13 C]lactate signal. These measurements showed that there was significant export of labeled lactate from the tumor, but that labeled lactate in the blood pool produced by the injection of hyperpolarized [1-13 C]pyruvate showed only relatively low levels of polarization. This study shows that measurements of hyperpolarized 13 C label exchange between pyruvate and lactate in a murine tumor model can provide an estimate of the true isotope flux if the concentration of labeled pyruvate that reaches the tumor can be determined.

Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate.

BACKGROUND: The recent introduction of a dynamic nuclear polarisation technique has permitted noninvasive imaging of tumour cell metabolism in vivo following intravenous administration of (13)C-labelled cell substrates. METHODS: Changes in hyperpolarised [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate metabolism were evaluated in both MDA-MB-231 cells and in implanted MDA-MB-231 tumours following doxorubicin treatment. RESULTS: Treatment of MDA-MB-231 cells resulted in the induction of apoptosis, which was accompanied by a decrease in hyperpolarised (13)C label flux between [1-(13)C]pyruvate and lactate, which was correlated with a decrease in the cellular NAD(H) coenzyme pool. There was also an increase in the rate of fumarate conversion to malate, which accompanied the onset of cellular necrosis. In vivo, the decrease in (13)C label exchange between pyruvate and lactate and the increased flux between fumarate and malate, following drug treatment, were shown to occur in the absence of any detectable change in tumour size. CONCLUSION: We show here that the early responses of a human breast adenocarcinoma tumour model to drug treatment can be followed by administration of both hyperpolarised [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate. These techniques could be used, therefore, in the clinic to detect the early responses of breast tumours to treatment.

Proton exchange as a relaxation mechanism for T1 in the rotating frame in native and immobilized protein solutions.

T1 relaxation in the rotating frame (T1rho) is a sensitive magnetic resonance imaging (MRI) contrast for acute brain insults. Biophysical mechanisms affecting T1rho relaxation rate (R1rho) and R1rho dispersion (dependency of R1rho on the spin-lock field) were studied in protein solutions by varying their chemical environment and pH in native, heat-denatured, and glutaraldehyde (GA) cross-linked samples. Low pH strongly reduced R1rho in heat-denatured phantoms displaying proton resonances from a number of side-chain chemical groups in high-resolution 1H NMR spectra. At pH of 5.5, R1rho dispersion was completely absent. In contrast, in the GA-treated phantoms with very few NMR visible side chain groups, acidic pH showed virtually no effect on R1rho. The present data point to a crucial role of proton exchange on R1rho and R1rho dispersion in immobilized protein solution mimicking tissue relaxation properties.

A model for gene therapy of human hereditary lymphedema.

Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors.

Cerebral T1rho relaxation time increases immediately upon global ischemia in the rat independently of blood glucose and anoxic depolarization.

Time-dependent changes of T1 in the rotating frame (T1rho), diffusion, T2, and magnetization transfer contrast on cardiac arrest-induced global ischemia in rat were investigated. T1rho, as acquired with spin lock amplitudes >0.6 G, started to increase 10-20 sec after cardiac arrest followed by an increase within 3-4 min to a level that was 6-8% greater than in normal brain. The ischemic T1rho response coincided with the drop of water diffusion coefficient in normoglycemic animals. However, unlike the rate of diffusion, the kinetics of T1rho were not affected by either preischemic hypoglycemia or hyperglycemia. Similar to diffusion, the kinetics of anoxic depolarization were dependent on preischemic blood glucose levels. Ischemia caused a reduction in the Hahn spin echo T2 as a result of blood oxygenation level-dependent (BOLD) effect; maximal negative BOLD seen by 40 sec. In the animals injected with an ironoxide particle contrast agent, AMI-227, prior to the insult, both T1rho and T2 immediately increased in concert on induction of ischemia. In contrast to the T1rho and diffusion changes, a much slower change in magnetization transfer contrast was evident over the first 20 min of ischemia. These data demonstrate that T1rho immediately increases following ischemia and that the pathophysiological mechanisms affecting this relaxation time may not directly involve magnetization transfer. The mechanisms prolonging T1rho differ from those affecting water diffusion with respect to their sensitivities to glucose and are apparently independent of membrane depolarization.

HSV-tk gene therapy for human renal cell carcinoma in nude mice.

We have treated Caki-2 human renal cell carcinoma in vivo using herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Both stably transduced Caki-2 tumors, generated using retrovirus-mediated ex vivo HSV-tk gene transfer and direct intratumoral adenovirus-mediated HSV-tk gene transfer of wild type tumors, were tested. Similar treatments with LacZ containing retro- and adenoviruses were used as controls. The outcome was evaluated by imaging the tumors before and after the treatment with magnetic resonance imaging, and using histology, immunocytochemistry, and survival analysis. When implanted orthotopically into nude mouse kidneys, Caki-2 cells formed reproducible cystic papillary kidney carcinomas. In vivo magnetic resonance imaging provided an important tool for the evaluation of tumor growth. Transduction efficiency of wild-type tumors in vivo with adeno-LacZ was 22+/-14%. Significant tumor regression was achieved with direct intratumoral adeno-HSV-tk transduction followed by intraperitoneal ganciclovir (GCV) (P<.001). Also, the treatment of stably transduced Caki-2 tumors with intraperitoneal GCV resulted in a significant treatment response in the HSV-tk group as compared to the LacZ group (P<.009). Increased apoptosis and macrophage infiltrations, reduced proliferation, and degenerative changes were observed in the tumors treated with HSV-tk and GCV. Also, significant prolongation in survival was achieved with adeno-HSV-tk- and GCV-treated mice as compared to the controls. It is concluded that adeno-HSV-tk gene therapy may be useful for the treatment of renal cell carcinoma in vivo.

Use of spin echo T(2) BOLD in assessment of cerebral misery perfusion at 1.5 T.

Inadequate blood supply relative to metabolic demand, a haemodynamic condition termed as misery perfusion, often occurs in conjunction with acute ischaemic stroke. Misery perfusion results in adaptive changes in cerebral physiology including increased cerebral blood volume (CBV) and oxygen extraction ratio (OER) to secure substrate supply for the brain. It has been suggested that the presence of misery perfusion may be an indication of reversible ischaemia, thus detection of this condition may have clinical impact in acute stroke imaging. The ability of single spin echo T(2) to detect misery perfusion in the rat brain at 1.5 T owing to its sensitivity to blood oxygenation level dependent (BOLD) contrast was studied both theoretically and experimentally. Based on the known physiology of misery perfusion, tissue morphometry and blood relaxation data, T(2) behaviour in misery perfusion was simulated. The interpretation of these computations was experimentally assessed by quantifying T(2) in a rat model for cerebral misery perfusion. CBF was quantified with the H(2) clearance method. A drop of CBF from 58+/-8 to 17+/-3 ml/100 g/min in the parieto-frontal cortex caused shortening of T(2) from 66.9+/-0.4 to 64.6+/-0.5 ms. Under these conditions, no change in diffusion MRI was detected. In contrast, the cortex with CBF of 42+/-7 ml/100 g/min showed no change in T(2). Computer simulations accurately predicted these T(2) responses. The present study shows that the acute drop of CBF by 70% causes a negative BOLD that is readily detectable by T(2) MRI at 1.5 T. Thus BOLD may serve as an index of misery perfusion thus revealing viable tissue with increased OER.

Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.

The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.

Interrelations of T(1) and diffusion of water in acute cerebral ischemia of the rat.

Interrelation of T(1) and diffusion of water was studied in rat models of acute global and focal cerebral ischemia. Cortical T(1), as quantified with an inversion recovery method, increased by 4-7% within a few minutes of global ischemia at 4.7 and 9.4 T, but a significantly smaller change was detected at 1.5 T. The initial T(1) change occurred within seconds of cardiac arrest, much earlier than the extensive diffusion drop after 1-2 min. Thus, the initial increase in T(1) upon acute cerebral ischemia is directly caused by cessation of blood flow. In transient middle cerebral artery occlusion (MCAO), prolonged T(1) relaxation was detected within 10 min, with a subsequent increase during the course of ischemia. Spin density did not change during the first hour, showing that T(1) increase was not caused by net accumulation of water. Interestingly, partial recovery of T(1) upon release of MCAO, occurring independent of long-term tissue outcome, was observed only in concert with diffusion recovery.

Early detection of irreversible cerebral ischemia in the rat using dispersion of the magnetic resonance imaging relaxation time, T1rho.

The impact of brain imaging on the assessment of tissue status is likely to increase with the advent of treatment methods for acute cerebral ischemia. Multimodal magnetic resonance imaging (MRI) demonstrates potential for selecting stroke therapy patients by identifying the presence of acute ischemia, delineating the perfusion defect, and excluding hemorrhage. Yet, the identification of tissue subject to reversible or irreversible ischemia has proven to be difficult. Here, the authors show that T1 relaxation time in the rotating frame, so-called T1rho, serves as a sensitive MRI indicator of cerebral ischemia in the rat. The T1rho prolongs within minutes after a drop in the CBF of less than 22 mL 100 g(-1) min(-1). Dependence of T1rho on spin-lock amplitude, termed as T1rho dispersion, increases by approximately 20% on middle cerebral artery (MCA) occlusion, comparable with the magnitude of diffusion reduction. The T1rho dispersion change dynamically increases to be 38% +/- 10% by the first 60 minutes of ischemia in the brain region destined to develop infarction. Following reperfusion after 45 minutes of MCA occlusion, the tissue with elevated T1rho dispersion (yet normal diffusion) develops severe histologically verified neuronal damage; thus, the former parameter unveils an irreversible condition earlier than currently available MRI methods. The T1rho dispersion as a novel MRI index of cerebral ischemia may be useful in determination of the therapeutic window for acute ischemic stroke.

Characterization of a new animal model for human renal cell carcinoma.

BACKGROUND: Human renal cell carcinoma (RCC) is the most common kidney malignancy with significant mortality. Human tumor xenograft models are important tools for cancer research. MATERIALS AND METHODS: We have established and characterized a new animal model for human RCC using Caki-2 cells implanted into the renal subcapsule (RSC) of nude mice. Histology, immunocytochemistry, in situ hybridization and magnetic resonance imaging (MRI) were used to analyze the tumors. RESULTS: The implantations generated reproducible carcinomas which closely resemble human RCC. The tumors showed cystic-papillary structures, rich capillary network and fibro-septa formations. Proliferation varied from 0-5% and from 1-60% in cystic and solid areas, respectively. Apoptosis was less than 1%. Macrophages and other inflammatory cell infiltrations were detected in the tumors. VEGF-A and angiopoietin I were expressed in a small number of cells in large tumors. Tumors did not metastasize outside peritoneal cavity. Survival of the tumor bearing animals was 23 +/- 3 weeks. CONCLUSIONS: It is concluded that Caki-2 carcinomas implanted into renal subcapsule of nude mice resemble human RCC in several aspects and represent a good animal model for studies regarding the pathogenesis and treatment of human RCC.

Graded reduction of cerebral blood flow in rat as detected by the nuclear magnetic resonance relaxation time T2: a theoretical and experimental approach.

The ability of transverse nuclear magnetic resonance relaxation time, T2, to reveal acutely reduced CBF was assessed using magnetic resonance imaging (MRI). Graded reduction of CBF was produced in rats using a modification of Pulsinelli's four-vessel occlusion model. The CBF in cerebral cortex was quantified using the hydrogen clearance method, and both T2 and the trace of the diffusion tensor (Dav = 1/3TraceD) in the adjacent cortical tissue were determined as a function of reduced CBF at 4.7 T. A previously published theory, interrelating cerebral hemodynamic parameters, hemoglobin, and oxygen metabolism with T2, was used to estimate the effects of reduced CBF on cerebral T2. The MRI data show that T2 reduces in a U-shape manner as a function of CBF, reaching a level that is 2.5 to 2.8 milliseconds (5% to 6%) below the control value at CBF, between 15% and 60% of normal. This reduction could be estimated by the theory using the literature values of cerebral blood volume, oxygen extraction ratio, and precapillary oxygen extraction during compromised CBF. Dav dropped with two apparent flow thresholds, so that a small 11% to 17% reduction occurred between CBF values of 16% to 45% of normal, followed by a precipitous collapse by more than 20% at CBF below 15% of normal. The current data show that T2 can be used as an indicator of acute hypoperfusion because of its ability to indicate blood oxygenation level-dependent phenomena on reduced CBF.