Relapse to cocaine seeking increases activity-regulated gene expression differentially in the prefrontal cortex of abstinent rats.

RATIONALE: Alterations in the activity of the prefrontal and orbitofrontal cortices of cocaine addicts have been linked with re-exposure to cocaine-associated stimuli. OBJECTIVES: Using an animal model of relapse to cocaine seeking, the present study investigated the expression patterns of four different activity-regulated genes within prefrontal cortical brain regions after 22 h or 15 days of abstinence during context-induced relapse. MATERIALS AND METHODS: Rats self-administered cocaine or received yoked-saline for 2 h/day for 10 days followed by 22 h or 2 weeks of abstinence when they were re-exposed to the self-administration chamber with or without levers available to press for 1 h. Brains were harvested and sections through the prefrontal cortex were processed for in situ hybridization using radioactive oligonucleotide probes encoding c-fos, zif/268, arc, and bdnf. RESULTS: Re-exposure to the chamber in which rats previously self-administered cocaine but not saline, regardless of lever availability, increased the expression of all genes in the medial prefrontal and orbitofrontal cortices at both time points with one exception: bdnf mRNA was significantly increased in the medial prefrontal cortex at 22 h only if levers previously associated with cocaine delivery were available to press. Furthermore, re-exposure of rats to the chambers in which they received yoked saline enhanced both zif/268 and arc expression selectively in the orbitofrontal cortex after 15 days of abstinence. CONCLUSIONS: These results support convergent evidence that cocaine-induced changes in the prefrontal cortex are important in regulating drug seeking following abstinence and may provide additional insight into the molecular mechanisms involved in these processes.

Orthogonal time-of-flight mass spectrometry of an ion beam with a broad kinetic energy profile.

A combined experimental and modeling effort is undertaken to assess a detection system composed of an orthogonal extraction time-of-flight (TOF) mass spectrometer coupled to a continuous ion source emitting an ion beam with kinetic energy of several hundred eV. The continuous ion source comprises an electrospray capillary system employing an undiluted ionic liquid emitting directly into vacuum. The resulting ion beam consists of ions with kinetic energy distributions of width greater than a hundred of eV and mass-to-charge (m/q) ratios ranging from 111 to 500 000 amu/q. In particular, the investigation aims to demonstrate the kinetic energy resolution along the ion beam axis (axial) of orthogonally extracted ions in measurements of the axial kinetic energy-specific mass spectrum, mass flow rate, and total ion current. The described instrument is capable of simultaneous measurement of a broad m/q range in a single acquisition cycle with approximately 25 eV/q axial kinetic energy resolution. Mass resolutions of ∼340 (M/ΔM, FWHM) were obtained for ions at m/q = 1974. Comparison of the orthogonally extracted TOF mass spectrum to mass flow and ion current measurements obtained with a quartz-crystal microbalance and Faraday cup, respectively, shows reasonable numeric agreement and qualitative agreement in the trend as a function of energy defect.

Elevated reactive oxygen species and antioxidant enzyme activities in animal and cellular models of Parkinson's disease.

The dopaminergic neurotoxin N-methyl,4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) causes a syndrome in primates and humans which mimics Parkinson's disease (PD) in clinical, pathological, and biochemical findings, including diminished activity of complex I in the mitochondrial electron transport chain. Reduced complex I activity is found in sporadic PD and can be transferred through mitochondrial DNA, suggesting a mitochondrial genetic etiology. We now show that MPTP treatment of mice and N-methylpyridinium (MPP+) exposure of human SH-SY5Y neuroblastoma cells increases oxygen free radical production and antioxidant enzyme activities. Cybrid cells created by transfer of PD mitochondria exhibit similar characteristics; however, PD cybrids' antioxidant enzyme activities are not further increased by MPP+ exposure, as are the activities in control cybrids. PD mitochondrial cybrids are subject to metabolic and oxidative stresses similar to MPTP parkinsonism and provide a model to determine mechanisms of oxidative damage and cell death in PD.

Significance of renewal asymmetry in bone scans: experience in 795 cases.

A retrospective study of 795 consecutive bone scans employing either 18F or 99mTc-pyrophosphate to evaluate the diagnostic value of renal asymmetry in such scans has been carried out. It is concluded that asymmetric renal images in bone scans convey relatively specific information regarding renal pathology, especially in the 99mTc-pyrophosphate studies.

Gallium-67 and magnetic resonance imaging in aortic root abscess.

Perivascular abscess in the aortic root is a serious complication of infective endocarditis and is recognized infrequently with noninvasive techniques. This report describes a patient with sepsis who was imaged with 67Ga and magnetic resonance (MR) in order to locate a site of infection. The 67Ga image showed increased activity in the substernal region. Magnetic resonance correctly detected the multilocular aortic root abscess, which had extended into the interatrial septum. The combination of the sensitive 67Ga with the high-resolution MR image provided a useful method to locate aortic root abscesses and to identify contiguous spread.

Noninvasive estimation of pulmonary arterial pressure by analysis of pulmonary blood-flow distribution.

To determine whether a correlation exists between pulmonary arterial (PA) pressure (Pa) and the distribution of pulmonary blood flow, this distribution was measured in four upright dogs in the control state and during intravenous infusions of epinephrine or prostaglandin F2 alpha. During suspension of respiration, 15 mCi of Xe-133 were injected intravenously, and perfusion and equilibration lung images were recorded with a scintillation camera. The procedure was performed several times on each dog, with and without pharmacological elevation of PA pressure by 5 to 50 cm H2O. For each scintigram, the relative blood flow per unit ventilated lung volume (F) was plotted against centimeters above the hilum (h). Pulmonary arterial pressure was derived from each curve, assuming the relation F = B(Pa - hD)2, where B = constant and D = specific gravity of blood. Calculated PA pressure correlated strongly (r = 0.83) with measured PA pressure, suggesting a possible means of noninvasive estimation of PA pressure.

Radiation exposure and protection in cardiac catheterization laboratories.

Cardiac catheterization with angiography can deliver the greatest dose of x-radiation of any diagnostic medical examination. The physicians and technologists in the angiography room receive low-level scattered radiation over a period of months to decades. Although the radiobiology is complex, the physicians who perform cardiac catheterization should be familiar with the potential genetic and somatic effects of radiation and with the methods to reduce or eliminate x-ray exposure. The aim of radiation protection criteria is to reduce the risk of cancer death to less than the fatality risk for other occupations regarded as safe. This report is a review of the literature relating to radiation exposure and protection in cardiac catheterization laboratories. Catheterization personnel have control over the time duration of exposure, placement of technologists, shielding, location of equipment and monitoring of dose received.

Assessment of drug intervention on the ischemic myocardium: serial imaging and measurement with computerized tomography.

Computerized tomography was evaluated as a technique for imaging and measuring the effect of an intervention on acutely ischemic myocardium. Because cell edema occurs with acute myocardial ischemia and decreases the X-ray attenuation coefficients (tissue density) of myocardium, computerized tomographic images were used to quantitate the effect of hyperosmotic mannitol on ischemia-induced edema. Canine hearts were arrested and scanned after (1) temporary occlusion of the proximal circumflex artery followed by reflow of blood, or (2) continued occlusion of the distal left anterior descending coronary artery. X-ray attenuation values (Hounsfield units) were linearly related to tissue wet/dry weight ratios (r = 0.87, P less than 0.001). After 2 hours of occlusion of the left anterior descending coronary artery the hearts that received mannitol manifested a significant reduction (P less than 0.05) in the volume of left ventricular wall involved with edema. Although the area of edema measured with computerized tomography tended to be smaller in the hearts treated with mannitol than in untreated hearts subjected to a 6 hour occlusion of the left anterior descending coronary artery, the size of the lesion was variable and did not differ significantly from that in untreated hearts. With either short periods of circumflex arterial occlusion followed by blood reflow or with 2 or 6 hours of prolonged occlusion of the left anterior descending coronary artery, the difference in mean attenuation coefficients between the ischemic and nonischemic areas of myocardium in mannitol-treated and untreated hearts was significantly less. These results indicate that computerized tomography in the arrested heart can detect and quantitate the lesion of early acute myocardial ischemia and can quantitate the effect of drug intervention.

Definition of cardiac structures using computerized tomography in isolated arrested and beating canine hearts.

Computerized axial tomography of the brain is a revolutionary noninvasive technique that has provided remarkable resolution of intracerebral structures. This study was undertaken to determine the potential of computerized tomography for defining the anatomy of 40 arrested and 9 beating (ejecting left heart preparation) canine hearts. Using an EMI cranial unit, scanning was performed in hearts under in vitro conditions with a lactated Ringer's interface. The tomographic scans obtained were later compared with comparable 8 mm thick sections of the heart. At physiologic levels of hematocrit (range 36 to 45%) external structures were well defined, but because of the lack of a differential between cavitary and myocardial densities, it was not possible to distinguish intracardiac structures. With very small amounts of iodinated contrast material (remotely administered in the case of the beating hearts) or with anemia (hematocrit less than 33%), the right and left ventricular cavities became clearly visible in both horizontal and longitudinal tomographic scans. It was possible to distinguish readily the boundaries of the ventricular and atrial cavities, the papillary muscles, the major trabeculae and the aorta. Rhythmic motion of the beating heart, suspended in its pericardium, did not eliminate structural definition. Thus, computerized tomography provides a new approach to the definition of cardiac structure with a relatively high degree of resolution. These observations point to the potential usefulness of this noninvasive technique for the evluation of both cardiac function and ventricular wall abnormalities.

Method for estimating right ventricular volume by planes applicable to cross-sectional echocardiography: correlation with angiographic formulas.

Right ventricular (RV) volumes determined by echocardiography were compared with those measured using established angiographic formulas. RV cast displacement volumes were first correlated with data derived from radiographic images of the casts corresponding to standard angiographic RV views. Four established angiographic formulas (Ferlinz, Boak, Fisher and Thilenius) correlated well with cast volume, with the corrected prism method of Fisher showing a best fit (r = 0.98, y = 1.1 + 0.9 x, standard error of the estimate = 3.6). Cast volumes calculated using our echocardiographic formula were then examined relative to the volumes derived from radiographic images of the RV casts. Volumes calculated using the corrected area-length Thilenius formula correlated best with those obtained using our derived 2-dimensional echocardiographic formula (r = 0.96, y = 4.6 + 1.0 x, standard error of the estimate = 6.8). These data confirm that volume calculated using the suggested optimal echocardiographic formula correlates well with volume obtained using derived angiographic data. Accordingly, confirmation in humans by the use of angiography is a rational step.

Effects of radiographic contrast material on cultured myocardial cells.

Radiographic contrast material in doses commonly used in clinical practice produces major alterations in cardiac rhythm. In order to separate the direct cellular effect of the constituents of contrast material from the indirect actions of neural and hormonal mediators, a tissue culture technique was developed using newborn rat heart cells. The rate of contraction of both individual cells and of a syncytium of cells decreased and became briefly asystolic after contrast material was added to the media, then recovered with varying degrees of arrhythmia. Cells in culture less than one week showed significantly fewer arrhythmias and less recurrent asystole after contract material than older cells (P less than 0.05). Cells pretreated with pharmacologic amounts of atropine (0.08 microgram/ml), ouabain (0.02 microgram/ml), lidocaine (16 microgram/ml), and quinidine (20 microgram/ml) continued to demonstrate the initial asystole, although quinidine and lidocaine diminished the frequency of arrhythmias in the recovery stage. Hyperosmolar dextrose produced asystole, as did Renografin-60, Hypaque-50, and Conray only at concentrations greater than 1000 mOsm, suggesting that an osmolarity threshold may exist for the production of asystole. Less fibrillation occurred when the sodium and calcium ionic concentrations of contrast material were adjusted to that of plasma, although the arrhythmias could not be eliminated. Renografin-60, containing the greatest amount of calcium-chelating agents, produced a significantly greater degree of terminal asystole (P less than 0.05), which was reversible upon addition of CaCl2. Thus, contrast material produces a decrease in the rate of contraction in cultured cardiac cells in the absence of neural and hormonal mediators. The evoked arrhythmias are dependent upon the osmolality and ionic sodium and calcium concentrations.

Computerized tomography of ischemic myocardium: quantitation of extent and severity of edema in an in vitro canine model.

The capacity of computerized tomography to assess myocardial edema resulting from acute coronary occlusion was investigated in 19 arrested and 2 beating canine hearts. Edematous myocardium was consistently detected as areas of decreased attenuation values. The magnitude of the decrease in attenuation value was linearly related to the severity of the edema. The anatomic definition and tomographic information in the arrested hearts provided the capability for localization and quantitation of the extent of the lesion. The results of these in vitro investigations indicate that CT scanning is a technique which will contribute important pathophysiologic information about the evolution of myocardial ischemia.

Two-dimensional electrophoresis and mass spectrometric identification of mitochondrial proteins from an SH-SY5Y neuroblastoma cell line.

To probe the mitochondrial involvement in neurodegenerative processes, we have generated a high-resolution map of the mitochondrial proteome from a human neuroblastoma SH-SY5Y cell line that has been used for creating cytoplasmic hybrid cell systems. Two mitochondrial preparations were evaluated using two-dimensional (2D) gel electrophoresis and mass spectrometry; one obtained from differential centrifugation and the other by a multiple-step percoll/metrizamide gradient. The 2D gel maps prepared from these mitochondrial fractions separated over 300 distinct spots as visualized by colloidal Coomassie blue (CCB), or closer to 400 proteins with silver staining. The most abundant proteins identified in the mitochondrial fraction prepared by differential centrifugation were those of mitochondrial, cytoplasmic, and endoplasmic reticulum origin. Proteins obtained using the more intensive two-step gradient method were almost exclusively known to be associated with mitochondria. From this latter preparation, 84 of the most abundant gel spots were analyzed, out of which 61 proteins were identified. The absence of many membrane-associated proteins known to be associated with the mitochondrion and the limited number of total proteins observed in the 2D gel maps suggest that the majority of mitochondrial proteins are not being detected under these separation and staining conditions. An insoluble pellet obtained after solubilization of the mitochondrial fraction prepared with the percoll/metrizamide gradient was boiled in sodium dodecylsulfate (SDS) and separated by 1D sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This separation yielded some additional proteins, many of which are likely membrane-associated. These studies form the basis for the analysis of differential protein expression in cybrid cellular models of neurodegenerative disorders and in affected tissue from diseased states.

Intratympanic injection of sodium arsanilate (atoxyl) solution results in postural changes consistent with changes described for labyrinthectomized rats.

Intratympanic injections of sodium arsanilate (atoxyl) have been shown to produce vestibular dysfunction in the rat. Unilateral and bilateral dysfunction can be distinguished by changes in the animals' postures. These changes are consistent with changes described for unilateral or bilateral labyrinthectomized rats. The intratympanic injection technique offers a simple yet effective alternative to labyrinthectomy.

Use of cytoplasmic hybrid cell lines for elucidating the role of mitochondrial dysfunction in Alzheimer's disease and Parkinson's disease.

There is substantial evidence of mitochondrial defects in neurodegenerative disorders such as Alzheimer's and Parkinson's diseases (AD and PD). We have probed the molecular implications of mitochondrial dysfunction in these diseases by transferring mitochondria from platelets obtained from disease and control donors into mitochondrial DNA-depleted recipient neuron-based cells (rho 0 cells). This process creates cytoplasmic hybrid (cybrid) cells where the mitochondrial DNA (mtDNA) from the donor is expressed in the nuclear and cellular background of the host rho 0 cell. Differences in phenotype between disease and control groups can thus be attributed to the exogenous mitochondria and mtDNA. Key methodological issues relating to this approach were addressed by demonstrating that recipient rho 0 cells have < 1 mtDNA copy/cell, and that exclusive repopulation with donor mtDNA occurs in cybrid cells. Further, we describe that sampling of heterogeneous cell populations is a valid approach for cybrid analysis. Our studies show that the focal respiratory chain defects reported in platelets of AD and PD cybrids can be recapitulated in AD and PD cybrids. In addition, both AD and PD cybrids display increased oxidative stress and perturbations in calcium homeostasis. These data suggest that the transfer of a mtDNA defect from disease donor platelets is the likely cause of the cybrid biochemical phenotype, and highlight the potential value of these cell lines as cellular disease models.

Recovery of muscle transfers replacing the total plantar flexor muscle group in rats.

In rats, combinations of plantar flexor muscles representing approximately 20, 40, 60, and 80% of the mass of the total plantar flexor group were transferred orthotopically in the absence of synergistic muscles and allowed to recover for 120 days. We hypothesized that, compared with their individual control values for structural and functional variables, the transfers would display a hierarchical array of deficits, proportional to their initial mass and, consequently, inversely proportional to the relative load on the transfers. Surprisingly, compared with their individual control values, each muscle transfer displayed deficits of 30-40% in muscle mass, total fiber cross-sectional area, and maximum isometric force, with the exception of the smallest transfer, the plantaris (PLN) muscle, which recovered 100% of its control value for each of these variables. Therefore, except for the PLN transfer, the muscle transfers studied displayed deficits similar in magnitude to those reported for muscles transferred in the presence of synergistic muscles. The greater recovery of the PLN transfer was attributed to the relatively large requirement for force production imposed on this transfer due to the average force requirements of the total plantar flexor group.

Recovery of medial gastrocnemius muscle grafts in rats: implications for the plantar flexor group.

Medial gastrocnemius (MGN) muscles were grafted in 18 rats and evaluated at 60, 90, and 120 days after the operation. Our purpose was to investigate the degree of recovery of the vascularized MGN grafts and the entire plantar flexor muscle group. Compared with control values, muscle mass and maximum force of MGN grafts were decreased by 33 and 38% at 60 days, 22 and 32% at 90 days, and 13 and 15% at 120 days. At 60 and 90 days, the deficits in maximum force for the entire plantar flexor muscle group, including the graft, were 29 and 17%, respectively. No difference was observed at 120 days. At 60 days, the deficit in the total mass of the plantar flexor group was 14% compared with control values, but by 90 days no deficit was observed. The restoration of normal plantar flexor group structure and function indicates that the degree of recovery attained by MGN grafts, although not complete, was sufficient to ensure that the performance of the total muscle group was not compromised.

Imaging pericardial disease.

Pericardial disease has a variety of presentations depending upon whether the pericardium is absent, is thickened from inflammation or tumor, or constrains fluid under pressure, thereby restricting cardiac filling and output. The choice of an imaging modality is influenced by a number of factors: the anticipated presence of calcium, cardiac motion, spatial and contrast resolution required, adequate imaging window into the mediastinum, contrast media requirements, and clinical situation. Although many of the imaging modalities discussed herein have common features, some offer unique attributes, namely motion analysis, fat and water discrimination, and calcium detection. The complexity of many pericardial problems plus the risk of pericardial surgery not uncommonly require a multimodality imaging approach for complete definition.

Assessing the adequacy of myocardial perfusion in man: anatomic and functional techniques.

The dynamic nature of the coronary vasculature and its stenoses frequently requires more than a subjective anatomic visualization to evaluate potential myocardial ischemia. Although coronary arteriography can assess stenoses of no hemodynamic importance (less than 20 per cent diameter reduction) and lesions that severely impair blood flow (greater than 80 per cent diameter reduction). Most coronary stenoses fall between these two extremes. Quantitative arteriography and radioisotope perfusion studies can improve the accuracy of predicting myocardial ischemia.

Cardiac magnetic resonance imaging: the Massachusetts General Hospital experience.

The combination of gated cardiac imaging with the magnetic resonance parameters of flow void and tissue characterization forms the basis for a powerful noninvasive technique for imaging the cardiovascular system. Images constructed in planes orthogonal to the axis of the heart allow cardiac structures to be reproducibly detected in serial studies and measured. The data acquisition techniques used at Massachusetts General Hospital are presented, along with examples of their application in a variety of cardiac diseases. Future prospects are illustrated with flow-encoded images and with surface coil techniques.