Comparison of T2 and T2*-weighted MR molecular imaging of a mouse model of glioma.

BACKGROUND: Standard MRI has been used for high-grade gliomas detection, albeit with limited success as it does not provide sufficient specificity and sensitivity to detect complex tumor structure. Therefore targeted contrast agents based on iron oxide, that shorten mostly T2 relaxation time, have been recently applied. However pulse sequences for molecular imaging in animal models of gliomas have not been yet fully studied. The aim of this study was therefore to compare contrast-to-noise ratio (CNR) and explain its origin using spin-echo (SE), gradient echo (GE), GE with flow compensation (GEFC) as well as susceptibility weighted imaging (SWI) in T2 and T2* contrast-enhanced molecular MRI of glioma. METHODS: A mouse model was used. U87MGdEGFRvIII cells (U87MG), derived from a human tumor, were injected intracerebrally. A 9.4 T MRI system was used and MR imaging was performed on the 10 day after the inoculation of the tumor. The CNR was measured prior, 20 min, 2 hrs and 24 hrs post intravenous tail administration of glioma targeted paramagnetic nanoparticles (NPs) using SE, SWI, GE and GEFC pulse sequences. RESULTS: The results showed significant differences in CNR among all pulse sequences prior injection. GEFC provided higher CNR post contrast agent injection when compared to GE and SE. Post injection CNR was the highest with SWI and significantly different from any other pulse sequence. CONCLUSIONS: Molecular MR imaging using targeted contrast agents can enhance the detection of glioma cells at 9.4 T if the optimal pulse sequence is used. Hence, the use of flow compensated pulse sequences, beside SWI, should to be considered in the molecular imaging studies.

Infrared optical imaging of matrix metalloproteinases (MMPs) up regulation following ischemia reperfusion is ameliorated by hypothermia.

BACKGROUND: We investigated the use of a new MMP activatable probe MMPSense™ 750 FAST (MMPSense750) for in-vivo visualization of early MMP activity in ischemic stroke. Following middle cerebral artery occlusion (MCAO) optical imaging was performed. Near-infrared (NIR) fluorescent images of MMPSense activation were acquired using an Olympus fluorescent microscope, 1.25 x objective, a CCD camera and an appropriate filter cube for detecting the activated probe with peak excitation and emission at 749 and 775 nm, respectively. Images were acquired starting at 2 or 24 hours after reperfusion over the ipsilateral and contralateral cortex before and for 3 hours after, MMPSense750 was injected. RESULTS: Increased intensities ipsilaterally were observed following MMPSense750 injection with ischemic injury but not in sham animals. There were significant ipsilateral and contralateral differences at 15 minutes (P <0.05) in early ischemic reperfusion and at time 0 in 24 hours post ischemia (P <0.05) which persisted at 180 minutes in both these groups (P <0.01), but not following sham surgery. The increase in ipsilateral signal intensity was attenuated by hypothermia. These observations corresponded with a significant increase in the total MMP-9 protein levels, 5 and 24 hours following ischemia reperfusion (P <0.05) and their reduction by hypothermia. CONCLUSIONS: Matrix-metalloproteinase upregulation in ischemia reperfusion can be imaged acutely in-vivo with NIRF using MMPSense750. Hypothermia attenuated both the optical increase in intensity after MMPSense750 and the increase in MMP-9 protein expression supporting the proof of concept that NIRF imaging using MMPSense can be used to assess potential therapeutic strategies for stroke treatment.

Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke.

BACKGROUND: The link between early blood- brain barrier (BBB) breakdown and endothelial cell activation in acute stroke remain poorly defined. We hypothesized that P-selectin, a mediator of the early phase of leukocyte recruitment in acute ischemia is also a major contributor to early BBB dysfunction following stroke. This was investigated by examining the relationship between BBB alterations following transient ischemic stroke and expression of cellular adhesion molecule P-selectin using a combination of magnetic resonance molecular imaging (MRMI), intravital microscopy and immunohistochemistry. MRMI was performed using the contrast, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated to Sialyl Lewis X (Slex) where the latter is known to bind to activated endothelium via E- or P selectins. Middle cerebral artery occlusion was induced in male C57/BL 6 wild-type (WT) mice and P-selectin-knockout (KO) mice. At 24 hours following middle cerebral artery occlusion, T1 maps were acquired prior to and following contrast injection. In addition to measuring P- and E-selectin expression in brain homogenates, alterations in BBB function were determined immunohistochemically by assessing the extravasation of immunoglobulin G (IgG) or staining for polymorphonuclear (PMN) leukocytes. In vivo assessment of BBB dysfunction was also investigated optically using intravital microscopy of the pial circulation following the injection of Fluorescein Isothiocyanate (FITC)-dextran (MW 2000 kDa). RESULTS: MRI confirmed similar infarct sizes and T1 values at 24 hours following stroke for both WT and KO animals. However, the blood to brain transfer constant for Gd DTPA (Kgd) demonstrated greater tissue extravasation of Gd DTPA in WT animals than KO mice (P < 0.03). In the P selectin KO mice, Delta T1 stroke -Delta T1 contralateral control cortex, decreased significantly in the Gd-DTPA(sLeX) group compared to Gd-DTPA, indicative of sLeX mediated accumulation of the targeted contrast agent. Regarding BBB function, in the P-selectin KO mice compared to WT control mice, there was an attenuation in the extravasation of IgG (P < 0.001), a trend for decreased FITC extravasation and less infiltration of PMN leukocytes (P < 0.001) thereby supporting the observed increase in Kgd permeability in stroke brain of WT compared to KO mice. CONCLUSION: P-selectin expression contributes to enhanced BBB dysfunction at 24 hours after transient focal cerebral ischemia.

Transient hypertension concurrent with forepaw stimulation enhances functional MRI responsiveness in infarct and peri-infarct regions.

Although functional magnetic resonance imaging (fMRI) is gaining use as a tool to assess cerebral recovery following various insults, the effects of potential confounders such as hypertension are poorly defined. We hypothesized that after stroke, transient hypertension during an fMRI study could produce a detected activation unrelated to neuronal activity within the infarct. Thus, the effect of norepinephrine induced increases in blood pressure (BP) on the fMRI response to forepaw stimulation were investigated in controls or 1 week after transient middle cerebral artery occlusion in rats. Images were smoothed spatially and voxels correlating to either forepaw stimulation or the change in BP time courses were analyzed. Transient hypertension increased the signal intensity and numbers of voxels correlating to the BP time courses within and adjacent to the ischemic infarct and these exceeded the response in the contralateral hemisphere or in controls. With left paw stimulation at normotension, there was a loss of activation in right sensory-motor cortex -- a region with necrosis and disruption of cerebral vessels. As BP increased left paw stimulation also resulted in the detection of activation in the infarcted sensory-motor cortex and peri-infarct regions. Thus, BP changes synchronous with tasks in fMRI studies can result in MR signal changes consistent with a loss of cerebral blood flow (CBF) autoregulation rather than neuronal activation in necrotic brain. After stroke, the use of stressful tasks associated with BP changes in fMRI studies should be limited or the BP change should be considered as a potential source of MR signal changes.

Glucagon- and secretin-related peptides differentially alter ocular growth and the development of form-deprivation myopia in chicks.

PURPOSE: Exogenous glucagon inhibits the induction of myopia in chicks, but the endogenous peptide and receptor that regulate eye growth are unknown. The purpose of this study was to determine which peptides and receptors in the glucagon-secretin family play a role in the control of ocular growth. METHODS: The effect of intravitreally injected peptides on the development of form-deprivation (FD) myopia and on the growth of eyes with unrestricted vision was determined by refraction and A-scan ultrasonography. Chicks received three injections, one every 48 hours, of secretin-related peptides (porcine secretin, human peptide histidine-isoleucine-amide-27, vasoactive intestinal peptide [VIP], VIP fragment 6-28, or pituitary adenylate cyclase-activating polypeptide; 10(-8)-10(-4) M in 20 microL) or five injections of proglucagon-derived peptides (human glucagon, oxyntomodulin, miniglucagon, or glucagon-like peptide [GLP]-2 or chicken GLP-1). Immunohistochemistry was used to detect proglucagon-derived peptides in the eye. RESULTS: Secretin-related peptides had no effect on FD myopia, whereas some proglucagon-derived peptides did. Both glucagon and oxyntomodulin dose-dependently inhibited development of myopia, primarily by inhibition of vitreous chamber elongation (EC(50) = 10(-4) M and 10(-5.5) M, respectively). GLP-1 increased deprivation-induced myopic refractive error by altering anterior chamber development. None of the peptides significantly affected refractive error in eyes with unrestricted vision, although changes in anterior and posterior eye growth were observed in response to glucagon, oxyntomodulin, GLP-1, and miniglucagon. Immunoreactivity for miniglucagon and GLP-1 was colocalized in glucagon-immunoreactive amacrine cells. CONCLUSIONS: Prevention of experimental myopia by exogenous glucagon is mediated by receptors selective for glucagon and oxyntomodulin, indicating that glucagon-like peptides and receptors are probable endogenous retinal regulators of the development of myopia.

Glucagon receptor agonists and antagonists affect the growth of the chick eye: a role for glucagonergic regulation of emmetropization?

PURPOSE: In chicks, plus defocus retards eye growth, thickens the choroid, and activates glucagonergic amacrine cells, probably releasing glucagon. Glucagon receptor antagonists (expected to inhibit compensation to plus defocus) and agonists (expected to block myopia induction by form deprivation) were administered to eyes of chicks, to test the hypothesis that glucagon mediates the induction of changes in eye growth by plus defocus. METHODS: Seven-day-old (P7) chick eyes were injected intravitreally with peptides at concentrations of approximately 10(-9) to 10(-5) M in 20 microL (injection volume). The glucagon-receptor antagonists [des-His(1),des- Phe(6),Glu(9)]-glucagon-NH(2) (des- Phe(6)-antagonist) and [des-His(1),Glu(9)]-glucagon-NH(2) (Phe(6)-antagonist) were administered daily for 4 to 5 days to plus-defocused eyes. Agonists (porcine glucagon-[1-29] and [Lys(17,18),Glu(21)]-glucagon-NH(2)) were monocularly administered daily for 5 days to form-deprived eyes. The contralateral eye remained open and received saline. After treatment, eyes were refracted, measured, and examined for histologic changes. RESULTS: The Phe(6)-antagonist at 10(-5) M (in the syringe) inhibited changes in both refractive error and axial length compensation induced by +7-D lens wear; however, des-Phe(6)-antagonist (10(-5) M) had weak, inconsistent effects and did not antagonize the action of exogenous glucagon. Glucagon prevented ocular elongation and myopia and induced choroidal thickening in form-deprived eyes. [Lys(17,18),Glu(21)]-glucagon-NH(2) had little effect at 10(-7) M, but at 10(-6) to 10(-5) M altered rod structure and inhibited eye growth. CONCLUSIONS: Exogenous glucagon inhibited the growth of form-deprived eyes, whereas Phe(6)-antagonist inhibited compensation to plus defocus, as might be expected if glucagon is an endogenous mediator of emmetropization. The reason for the failure of des-Phe(6)-antagonist to counteract the effects of exogenous glucagon requires further investigation.

Very Mild Hypothermia (35°C) Postischemia Reduces Infarct Volume and Blood/Brain Barrier Breakdown Following tPA Treatment in the Mouse.

Reperfusion therapies for stroke diminish in effectiveness and safety as time to treatment increases. Hypothermia neuroprotection for stroke is established, but its clinical translation has been hampered by uncertainties regarding optimal temperature and complications associated with moderate hypothermia. Also, hypothermia targeting temperatures of 32-33°C is associated with clinical and logistical problems related to induction and adverse side effects. We hypothesized that ischemic damage and tPA-exacerbated blood/brain barrier (BBB) breakdown produced following 30 minutes of middle cerebral artery occlusion and either 1 hour of saline or tPA infusion would be reduced by treatment with very mild cooling of 1.5°C for 48 hours followed by 24 hours of gradual rewarming. Infarct volume was reduced by 29.6% (p<0.001) and 41.9% (p<0.001) in hypothermic-tPA (Hypo_tPA)-treated and hypothermic-saline (Hypo_Sal)-treated animals compared to normothermic-tPA (Norm_tPA) and saline (Norm_Sal)-treated animals, respectively. Hypothermia also reduced IgG extravasation in tPA-treated, but not saline-treated groups compared to their normothermic controls (p<0.001). The ipsilateral-contralateral changes in optical density for IgG extravasation were 18.4% greater in the Norm_tPA than Norm_Sal (p<0.001) group. The ipsilateral-contralateral changes in optical density for IgG extravasation were reduced by 17.8% (p<0.001) in the Hypo_tPA compared to Norm_tPA group. No significant mean difference in IgG extravasation was seen between Hypo_tPA and Hypo_Sal groups (p>0.05). Very modest hypothermia to reduce the BBB breakdown could improve the availability and safety of reperfusion treatments for stroke.

The acrylamide content of smokeless tobacco products.

BACKGROUND: There is considerable interest from a regulatory and public health perspective in harmful and potentially harmful constituents in tobacco products, including smokeless tobacco products (STPs). A wide range of commercial STPs from the US and Sweden, representing 80-90 % of the 2010 market share for all the major STP categories in these two countries, were analysed for the IARC Group 2A carcinogen acrylamide. These STPs comprised the following styles: Swedish loose and portion snus, US snus, chewing tobacco, moist snuff, dry snuff, soft pellet, hard pellet and plug. RESULTS: Acrylamide was detected in all the products tested and quantified in all but one product. Concentrations ranged from 62 to 666 ng/g wet weight basis (WWB). The average levels of acrylamide (WWB) by type of STP were not significantly different (p > 0.05) except for US snus which had, on average, greater levels but with a very wide range of individual levels according to the manufacturer. Acrylamide levels in STPs were significantly and positively correlated with pH, but not with levels of either reducing sugars or ammonia nitrogen. Levels of acrylamide increased by sixfold or more (on a dry weight basis) during manufacture of a snus sample and then decreased during subsequent storage for up to 22 weeks. Acrylamide generation in tobacco generally appears to occur at lower temperatures, but longer time scales than found with food production. CONCLUSIONS: Acrylamide is a common contaminant of STPs, formed through heat treatment of tobacco. Our data show that exposure to acrylamide from consumption of STPs is small compared with exposure from food consumption or cigarette smoking.

Cellular correlates of longitudinal diffusion tensor imaging of axonal degeneration following hypoxic-ischemic cerebral infarction in neonatal rats.

Ischemically damaged brain can be accompanied by secondary degeneration of associated axonal connections e.g. Wallerian degeneration. Diffusion tensor imaging (DTI) is widely used to investigate axonal injury but the cellular correlates of many of the degenerative changes remain speculative. We investigated the relationship of DTI of directly damaged cerebral cortex and secondary axonal degeneration in the cerebral peduncle with cellular alterations in pan-axonal neurofilament staining, myelination, reactive astrocytes, activation of microglia/macrophages and neuronal cell death. DTI measures (axial, radial and mean diffusivity, and fractional anisotropy (FA)) were acquired at hyperacute (3 h), acute (1 and 2 d) and chronic (1 and 4 week) times after transient cerebral hypoxia with unilateral ischemia in neonatal rats. The tissue pathology underlying ischemic and degenerative responses had a complex relationship with DTI parameters. DTI changes at hyperacute and subacute times were smaller in magnitude and tended to be transient and/or delayed in cerebral peduncle compared to cerebral cortex. In cerebral peduncle by 1 d post-insult, there were reductions in neurofilament staining corresponding with decreases in parallel diffusivity which were more sensitive than mean diffusivity in detecting axonal changes. Ipsilesional reductions in FA within cerebral peduncle were robust in detecting both early and chronic degenerative responses. At one or four weeks post-insult, radial diffusivity was increased ipsilaterally in the cerebral peduncle corresponding to pathological evidence of a lack of ontogenic myelination in this region. The detailed differences in progression and magnitude of DTI and histological changes reported provide a reference for identifying the potential contribution of various cellular responses to FA, and, parallel, radial, and mean diffusivity.

Molecular susceptibility weighted imaging of the glioma rim in a mouse model.

BACKGROUND: Glioma is the most common and most difficult to treat brain cancer. Despite many efforts treatment, efficacy remains low. As neurosurgical removal is the standard procedure for glioma, a method, allowing for both early detection and exact determination of the location, size and extent of the tumor, could improve a patient's positive response to therapy. NEW METHOD: We propose application of susceptibility weighted molecular magnetic resonance imaging using, targeted contrast agents, based on superparamagnetic iron oxide nanoparticles, for imaging of the, glioma rim, namely brain-tumor interface. Iron oxide attached to the targeted cells increases, susceptibility differences at the boundary between tumor and normal tissue, providing the opportunity, to utilize susceptibility weighted imaging for improved tumor delineation. We investigated potential, enhancement of the tumor-brain contrast, including tumor core and rim when using susceptibility, weighted MRI for molecular imaging of glioma. RESULTS: There were significant differences in contrast-to-noise ratio before, 12 and 120min after contrast, agent injection between standard gradient echo pulse sequence and susceptibility weighted molecular, magnetic resonance imaging for the core-brain, tumor rim-core and tumor rim-brain areas. COMPARISON WITH EXISTING METHODS: Currently, the most common MRI contrast agent used for glioma diagnosis is a non-specific, gadolinium-based agent providing T1-weighted enhancement. Susceptibility-weighted magnetic, resonance imaging is much less efficient when no targeted superparamagnetic contrast agents are, used. CONCLUSION: The improved determination of glioma extent provided by SWI offers an important new tool for, diagnosis and surgical planning.

Approaches for the design of reduced toxicant emission cigarettes.

Cigarette smoking causes serious diseases through frequent and prolonged exposure to toxicants. Technologies are being developed to reduce smokers' toxicant exposure, including filter adsorbents, tobacco treatments and substitutes. This study examined the effect of modifications to filter ventilation, variations in cigarette circumference and active charcoal filter length and loading, as well as combinations of these features in a reduced-toxicant prototype (RTP) cigarette, on the yields of toxicants in cigarette smoke. An air-dilution mechanism, called split-tipping, was developed in which a band of porous paper in the centre of the filter tipping functions to minimise the loss of effective filter ventilation that occurs at the high flow rates encountered during human-smoking, and to facilitate the diffusional loss of volatile toxicants. As compared with conventional filter ventilation cigarettes, split-tipping reduced tar and volatile smoke constituent emissions under high flow rate machine-smoking conditions, most notably for products with a 1-mg ISO tar yield. Furthermore, mouth level exposure (MLE) to tar and nicotine was reduced among smokers of 1-mg ISO tar cigarettes in comparison to smokers of cigarettes with traditional filter ventilation. For higher ISO tar level cigarettes, however, there were no significant reductions in MLE. Smaller cigarette circumferences reduced sidestream toxicant yields and modified the balance of mainstream smoke chemistry with reduced levels of aromatic amines and benzo[a]pyrene but increased yields of formaldehyde. Smaller circumference cigarettes also had lower mainstream yields of volatile toxicants. Longer cigarette filters containing increased levels of high-activity carbon (HAC) showed reduced machine-smoking yields of volatile toxicants: with up to 97% removal for some volatile toxicants at higher HAC loadings. Split-tipping was combined with optimal filter length and cigarette circumference in an RTP cigarette that gave significantly lower mainstream (up to ~90%) and sidestream (predominately 20%-60%) smoke yields of numerous toxicants as compared with a commercial comparator cigarette under machine-smoking conditions. Significantly lower mainstream and sidestream smoke toxicant yields were observed for an RTP cigarette comprising several toxicant reducing technologies; these observations warrant further evaluation in clinical studies where real-world relevance can be tested using biomarkers of exposure and physiological effect.

Functional magnetic resonance imaging within the rat spinal cord following peripheral nerve injury.

Functional magnetic resonance imaging (fMRI) was used to detect the effects of graded peripheral nerve injury at the spinal level. Graded peripheral nerve injury in rats was accomplished by transection of nerves entering the spinal cord at the L3 and L4 levels of the spinal cord segments. Electrical stimulation of the hindpaw was used to elicit activity within the spinal cord. The stimulation experimental paradigm consisted of 62 functional images, 5 slices each, with a total of 3 rest and 2 stimulation periods. A 9.4 T MRI system and a quadrature volume rf coil covering the lumbar spinal cord were used for the fMRI study. Sets of fast spin echo images were acquired repeatedly following sham preparatory surgery under control conditions and in rats following sham surgery (pre nerve cut), followed by L3 nerve and then L4 nerve section. In rats with sham surgery, there was a significant activation within the dorsal horn of slices corresponding to L3 and L4 spinal cord segments. Following section of the L3 nerve, there was a reduction in the number of active voxels in the L3 and L4 spinal cord segments. The activation was reduced further by sectioning of the L4 nerve. Thus, following an increasing loss of axonal connections to the spinal cord, there was a decreasing number of active voxels within the spinal cord. The results demonstrate that spinal fMRI in the rat has sufficient sensitivity to detect within the spinal cord the effects of a graded reduction in peripheral connectivity.