RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury.

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia-reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia-reperfusion injury model, the high-dose TNF model, and in A20(-/-) mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia-reperfusion injury model and no benefit in A20(-/-) mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

Dynamic contrast-enhanced magnetic resonance imaging as a surrogate marker of tumor response to anti-angiogenic therapy in a xenograft model of glioblastoma multiforme.

PURPOSE: To evaluate the effects of a neutralizing anti-vascular endothelial growth factor (anti-VEGF) antibody on tumor microvascular permeability, a proposed indicator of angiogenesis, and tumor growth in a rodent malignant glioma model. MATERIALS AND METHODS: A dynamic contrast-enhanced magnetic resonance imaging (MRI) technique, permitting noninvasive in vivo and in situ assessment of potential therapeutic effects, was used to measure tumor microvascular characteristics and volumes. U-87, a cell line derived from a human glioblastoma multiforme, was implanted orthotopically into brains of athymic homozygous nude rats. RESULTS: Treatment with the monoclonal antibody A4.6.1, specific for VEGF, significantly inhibited tumor microvascular permeability (6.1 +/- 3.6 mL min(-1)100 cc(-1)), compared to the control, saline-treated tumors (28.6 +/- 8.6 mL min(-1)100 cc(-1)), and significantly suppressed tumor growth (P <.05). CONCLUSION: Findings demonstrate that tumor vascular permeability and tumor growth can be inhibited by neutralization of endogenous VEGF and suggest that angiogenesis with the maintenance of endothelial hyperpermeability requires the presence of VEGF within the tissue microenvironment. Changes in tumor vessel permeability and tumor volumes as measured by contrast-enhanced MRI provide an assay that could prove useful for clinical monitoring of anti-angiogenic therapies in brain tumors.

Dobutamine stress cine-MRI of cardiac function in the hearts of adult cardiomyocyte-specific VEGF knockout mice.

A mouse model of non-necrotic vascular deficiency in the adult heart was studied using cine-magnetic resonance imaging (MRI) and other techniques. The mice lacked cardiomyocyte-derived vascular endothelial growth factor (VEGF) following a targeted knockout in the ventricular cardiomyocytes. Quantitative endothelial labeling showed that the capillary density was significantly reduced in the hearts of knockout mice. Gene expression patterns suggested that they were hypoxic. Semiautomated MR image analysis was employed to obtain both global and regional measurements of left ventricular function at 10 or more time points through the cardiac cycle. MRI measurements showed a marked reduction in ejection fraction both at rest and under low- and high-dose dobutamine stress. Regional wall thickness, thickening, and displacement were all attenuated in the knockout mice. A prolonged high-dose dobutamine challenge was monitored by MRI. A maximal response was sustained for 90 minutes, suggesting that it did not depend on endogenous glycogen stores.

Increased binding activity at an antioxidant-responsive element in the metallothionein-1 promoter and rapid induction of metallothionein-1 and -2 in response to cerebral ischemia and reperfusion.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are potentially involved in zinc homeostasis and free radical scavenging. The expression pattern of MT-1 and the binding activity of various MT-1 promoter elements were investigated after mild focal cerebral ischemia in the rat. Transient focal ischemia was induced by occluding both common carotid arteries and the right middle cerebral artery for 30 min. By the use of real-time quantitative PCR, a 10-fold increase in MT-1 and -2 mRNA levels was found in the cortex 24 hr after reperfusion. In situ hybridization and immunocytochemistry showed a rapid increase in MT-1 and -2 mRNA and MT protein in endothelial cells of microvessels at 6 hr after reperfusion, followed by an increased expression in astrocytes of the infarcted cortex at 24 hr after reperfusion. The early increase in MT expression preceded an increase in cerebral edema measured with T2-weighted magnetic resonance imaging. Gel shift assays were performed on nuclear extracts prepared from cortices before and at 6 and 24 hr after reperfusion. Increased binding activity was found at an antioxidant/electrophilic response element (ARE) sequence in the MT-1 promoter at 6 hr with a lower and variable binding activity at 24 hr after reperfusion. Constitutive binding activity was found for Sp1 and a metal response element in the MT-1 promoter that did not increase after ischemia and reperfusion. This study suggests a role of ARE-binding proteins in inducing cerebral MT-1 expression and implicates MT-1 as one of the early detoxifying genes in an endogenous defense response to cerebral ischemia and reperfusion.

VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain.

VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8-12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion-related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.

High-resolution functional magnetic resonance imaging of the rat brain: mapping changes in cerebral blood volume using iron oxide contrast media.

Contrast-enhanced magnetic resonance imaging was used to produce high-resolution activation maps reflecting local changes in cerebral blood volume after a simple sensory stimulus. Activation of the forelimb region of the somatosensory cortex was performed in alpha-chloralose-anaesthetized rats with an electrical stimulus (5 V, 3 Hz) delivered through needle electrodes placed subcutaneously on the left forelimb. A gradient echo magnetic resonance imaging sequence, sensitive to changes in the relative amount of deoxyhemoglobin within the cerebral vasculature, produced a 4.05%+/-1.69% increase in signal intensity. This effect was enhanced with an injection of an intravascular iron oxide contrast agent (Combidex, Advanced Magnetics), resulting in a 9.11%+/-1.52% decrease in signal intensity.

Magnetic resonance imaging detects suppression of tumor vascular permeability after administration of antibody to vascular endothelial growth factor.

Macromolecular contrast medium-enhanced magnetic resonance imaging (MRI) and tumor-volume measurements were applied to monitor the effects of anti-vascular endothelial growth factor (anti-VEGF) antibody on microvascular characteristics and tumor growth of MDA-MB-435 human breast cancer cells implanted in nude rats. Administration of anti-VEGF antibody (three 1 mg doses at 3-day intervals) induced significant reductions in tumor growth rates (p < 0.05) and in MRI-assayed microvascular permeabilities (p < 0.05). Results of the study were consistent with previous observations that new microvessels formed in response to angiogenesis are hyperpermeable, and with the hypothesis that hyperpermeability is a mechanistic element in angiogenesis. Variations in tumor-vessel hyperpermeability can be measured by contrast-enhanced MRI, which may prove useful for assessing antiangiogenesis therapy.

Assessing tumor angiogenesis using macromolecular MR imaging contrast media.

MRI enhanced with a macromolecular contrast medium (MMCM) has previously been shown to estimate tumor microvascular characteristics that correlate closely with histologic microvascular density, an established surrogate of tumor angiogenesis. A similar MMCM-enhanced MRI technique has now been used to investigate the acute tumor microvascular effects of antibody-mediated inhibition of vascular endothelial growth factor (VEGF), a well-studied and potent angiogenesis stimulator. Athymic rats xenografted with a human breast carcinoma (MDA-MB-435) were imaged after administration of albumin-gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA30) using a heavily T1-weighted three dimensional-spoiled gradient-refocused acquisition in a steady-state pulse sequence before and 24 hours after treatment with anti-VEGF antibody (single dose of 1 mg). Changes in longitudinal relaxivity (delta R1) were analyzed using a bidirectional two-compartment kinetic model to estimate tumor fractional blood volume (fBV) and permeability surface area product (PS). Data showed a significant decrease (P < 0.05) of tumor PS with respect to macromolecular contrast medium at 24 hours after treatment with anti-VEGF antibody. No significant change was observed in fBV. Suppression of tumor microvascular permeability induced by anti-VEGF antibody can be detected and quantified by MMCM-enhanced MRI. MRI grading of tumor angiogenesis and monitoring of anti-angiogenesis interventions could find wide clinical application.

q-Space imaging of the brain.

q-Space imaging (Callaghan, J. Magn. Reson. 88, 493 (1990)) has been used to obtain mouse brain water displacement profiles. These profiles take the form of a unidirectional incoherent-displacement probability density distribution. Two groups of mice were studied, a normal group and one in which surgery had been performed to reduce the supply of blood to the forebrain. In the normal group the incoherent displacement of water was reduced postmortem. Four of the surgically treated mice yielded displacement profiles that resembled those obtained postmortem; the remaining two were near normal. This study demonstrates the feasibility of in vivo q-space imaging. The displacement profile changes that occur subsequent to an interruption of the forebrain blood supply are consistent with the hyperintensity changes seen in diffusion-weighted imaging.

Diffusion-weighted imaging studies of cerebral ischemia in gerbils. Potential relevance to energy failure.

BACKGROUND AND PURPOSE: Diffusion-weighted magnetic resonance imaging has been shown to be particularly suited to the study of the acute phase of cerebral ischemia in animal models. The studies reported in this paper were undertaken to determine whether this technique is sensitive to the known ischemic thresholds for cerebral tissue energy failure and disturbance of membrane ion gradients. METHODS: Diffusion-weighted images of the gerbil brain were acquired under two sets of experimental conditions: as a function of cerebral blood flow after controlled graded occlusion of the common carotid arteries (partial ischemia), as a function of time following complete bilateral carotid artery occlusion (severe global ischemia), and on deocclusion after 60 minutes of ischemia. RESULTS: During partial cerebral ischemia, the diffusion-weighted images remained unchanged until the cerebral blood flow was reduced to 15-20 ml.100 g-1.min-1 and below, when image intensity increased as the cerebral blood flow was lowered further. This is similar to the critical flow threshold for maintenance of tissue high-energy metabolites and ion homeostasis. After the onset of severe global cerebral ischemia, diffusion-weighted image intensity increased gradually after a delay of approximately 2.5 minutes, consistent with complete loss of tissue adenosine triphosphate and with the time course of increase in extracellular potassium. This hyperintensity decreased on deocclusion following 60 minutes of ischemia. CONCLUSIONS: The data suggest that diffusion-weighted imaging is sensitive to the disruption of tissue energy metabolism or a consequence of this disruption. This raises the possibility of imaging energy failure noninvasively. In humans, this could have potential in visualizing brain regions where energy metabolism is impaired, particularly during the acute phase following stroke.

Cell and tissue responses of a murine tumour to phthalocyanine-mediated photodynamic therapy.

Mice bearing a subcutaneously growing tumour (Colo 26) were injected intravenously with the photosensitiser chloroaluminum sulphonated phthalocyanine (5 mg/kg) 24 h prior to irradiating the tumour with laser light (675 nm; 50mW, 100 J/tumour). Energy status of the tumour, as assessed by the loss of high energy phosphates in the 31P-nuclear magnetic resonance spectra, was altered dramatically following treatment, such that the ATP fell to undetectable levels within 1 h of light irradiation. However, assessment of the clonogenic capacity of neoplastic cells isolated from dissociated tumours showed that these rapid changes in cellular metabolism were not reflected in similar rapid changes in cell viability. Reductions in clonogenic capacity, which fell to less than 0.1% of control values at 24h postirradiation, closely mirrored those resulting from the cessation of vascular perfusion. Evaluation of tumour blood flow, using the technique of hydrogen washout, showed that the treatment protocol evoked a gradual and selective reduction in flow within the tumour resulting in complete vascular stasis by approximately 5 h after treatment. The results indicate that while chloroaluminum sulphonated phthalocyanine-mediated photodynamic therapy caused early metabolic damage in neoplastic cells, loss of viability paralleled the induction of complete inhibition of vascular flow in the tumour.

Normal and abnormal white matter tracts shown by MR imaging using directional diffusion weighted sequences.

A pulsed magnetic field gradient spin echo technique was used to study the brain of two volunteers and eight patients. The pulsed gradients were applied both perpendicular and parallel to the image slice. Striking changes in signal intensity were demonstrated in white matter depending on the direction in which pulsed gradients were applied. These effects enabled specific white matter tracts to be identified depending on the direction of their fibres. Abnormalities were also demonstrated in these tracts in patients with a variety of diseases, including cases where only minor abnormalities were seen with conventional, highly T2-weighted sequences. The effects were attributed to anisotropically restricted diffusion within white matter. The technique may have application in a wide range of neurological disease and result in better localisation of lesions and improved detection of disease.

Proton MR spectroscopy of intracranial tumours: in vivo and in vitro studies.

Proton magnetic resonance spectroscopy (1H MRS) was used to investigate intracranial tumours in vitro and in vivo. Biopsy specimens were studied from 47 patients, 11 of whom were also examined in vivo. Analysis was based on the signals from N-acetylaspartate (NAA), phosphocreatine plus creatine (Cr), choline-containing compounds (Cho), alanine (Ala), and lactate. Biopsy data from 26 astrocytomas showed that the NAA/Cr ratio differs significantly in all grades from its value in normal white matter and that the Cho/Cr ratio differs significantly in grade IV tumours from its value in the other grades. Meningiomas have an unusually high Ala/Cr ratio. Spectra obtained in vivo are consistent with in vitro results from the same patients, and their lactate signal provides additional information about abnormal metabolism. We conclude that 1H MRS has a clear role in the diagnosis and biochemical assessment of intracranial tumours and in the evaluation and monitoring of therapy.

Identification of tumor hemorrhage in an animal model using spin echoes and gradient echoes.

We report here how magnetic resonance imaging can be used to gain definitive information about tissue pathology by the combined use of spin-echo and gradient-echo sequences. We also show how artifacts arising from respiratory motion can be eliminated by using a simple respiratory gating technique.

Brain metabolites as 1H NMR markers of neuronal and glial disorders.

1H NMR spectroscopy of human brain in vivo can be used to detect a number of cerebral metabolites including N-acetylaspartate, creatine + phosphocreatine and choline-containing compounds. We have used 1H NMR spectroscopy to analyse these signals in (i) biopsy material from both normal human brain and astrocytomas, and (ii) primary astrocyte cultures. On the basis of this analysis, we conclude that in vivo 1H NMR spectroscopy could play an important clinical role in the non-invasive assessment of neuronal degeneration and proliferation of non-neuronal cells.