Antisense inhibition of beta(1)-adrenergic receptor mRNA in a single dose produces a profound and prolonged reduction in high blood pressure in spontaneously hypertensive rats.

BACKGROUND: beta-Blockers are the first line of therapy for hypertension. However, they are associated with side effects because of central nervous system (CNS) effects and beta(2)-adrenergic antagonism. To overcome these problems and provide a long-term beta(1)-blockade, antisense oligonucleotides against rat beta(1)-adrenergic receptor (beta(1)-AR) mRNA (beta(1)-AS-ODN) were designed and tested for the ability to inhibit cardiac beta(1)-ARs as well as lower blood pressure in spontaneously hypertensive rats (SHRs). METHODS AND RESULTS: Radioligand binding assay showed that a single intravenous injection of beta(1)-AS-ODN delivered in cationic liposomes significantly decreased cardiac beta(1)-AR density by 30% to 50% for 18 days (P<0.01), with no effect on beta(2)-ARs. This was accompanied by marked attenuation of beta(1)-AR-mediated positive inotropic response in isolated perfused hearts in vitro (P<0.02) and in conscious SHRs monitored by telemetry in vivo (P<0.02). Furthermore, the blood pressure of SHRs was reduced for 20 days, with a 38 mm Hg maximum drop. Heart rate was not significantly decreased. Quantitative autoradiography was performed to assess beta(1)-AS-ODN effects on the CNS, which demonstrated no changes in beta(1)-ARs in brain, in contrast to a significant reduction in heart and kidney (P<0.05). For comparison with beta-blockers, the effects of atenolol on cardiovascular hemodynamics were examined, which lowered blood pressure for only 10 hours and elicited appreciable bradycardia in SHRs. CONCLUSIONS: These results indicate that beta(1)-AS-ODN, a novel approach to specific beta(1)-blockade, has advantages over currently used beta-blockers in providing a profound and prolonged reduction in blood pressure without affecting heart rate, beta(2)-ARs, and the CNS. Diminished cardiac contractility resulting from less beta(1)-AR expression contributes to the antihypertensive effect.

Effect of cell density and HLA-DR incompatibility on T-cell proliferation and forkhead box P3 expression in human mixed lymphocyte reaction.

BACKGROUND: The proliferation rates of human T cells in vitro are affected by some factors such as initial T-cell number, dose of stimulating cells, and duration of culture. The transcription factor forkhead box P3 (FoxP3) has been used to identify regulatory T cells in humans and is thought to correlate with tolerance to allogeneic organ transplant. Thus, it is important to optimize conditions to expand FoxP3 cell proliferation to improve engraftment of allogeneic organ transplants. METHODS: We studied proliferative responses and FoxP3 expression in divided T cells with the use of flow cytometric analysis of Ki-67 in culture of different concentrations of responding cells (6 × 10(6), 4 × 10(6), 2 × 10(6), 1 × 10(6), and 0.5 × 10(6)cells/mL), different types of stimulating cells (lymphocytes and low density cells), and different numbers of HLA mismatches. RESULTS: The proportion of CD3(+) cells, CD4(+)CD25(+) cells, and CD4(+)CD25(+)FoxP3(+) cells among mononuclear cells were highest at initial cell concentration of 2 × 10(6) responder cells/mL with lymphocytes as stimulators at day-5 mixed lymphocyte reaction (MLR). They were highest at a concentration of 4 × 10(6) responder cells/mL with low density cells as stimulators. The recovery (%), proportion of CD3(+) cells, CD4(+)CD25(+) cells, and CD4(+)CD25(+)FoxP3(+) cells with 2 HLA-DR incompatibility were significantly higher than those of 1 HLA-DR incompatibility at day-5 MLR. CONCLUSIONS: Initial cell concentration and HLA-DR incompatibility can affect the generation of FoxP3+ T cells in human MLR. These factors could be considered for efficient generation of Tregs for clinical trials in the future.

Estrogens decrease reperfusion-associated cortical ischemic damage: an MRI analysis in a transient focal ischemia model.

BACKGROUND AND PURPOSE: Early identification of irreversible cerebral ischemia is critical in defining strategies that influence neuronal survival after stroke. We used MRI to investigate the effects of 17beta-estradiol (E2) on the temporal evolution of focal ischemia. METHODS: Female rats were ovariectomized and divided into 1 of 2 groups: ovariectomy alone (OVX; n=4) or ovariectomy with estrogen replacement (OVX+E2; n=3). Both groups were then subjected to 1-hour middle cerebral artery occlusion (MCAO), with the use of a standardized endovascular monofilament model, followed by reperfusion. Sequential diffusion-weighted (DWI) and T2-weighted (T2WI) MRI were obtained during and after the MCAO. In separate groups of animals (n=5 for OVX and OVX+E2), cerebral blood flow (CBF) was measured by laser-Doppler methods before, during, and after occlusion. RESULTS: DWI detected similar lesion characteristics during MCAO in both groups. In the OVX group, lesion size did not change during reperfusion, but the signal intensity ratio increased early and stabilized during the latter stages. In contrast, DWI lesion size decreased during reperfusion in OVX+E2 rats by 50% to 60% (P<0.05), a size reduction almost exclusively limited to cortical regions. During MCAO, the signal intensity ratio in OVX+E2 rats was reduced compared with OVX rats. Reperfusion further attenuated the signal intensity ratio in cortical but not subcortical regions (P<0.05 versus OVX). T2WI revealed no lesions in either group during MCAO, but it detected lesion sizes similar to that of DWI during reperfusion. Furthermore, similar patterns and magnitudes of estrogen treatment-related decrease in lesion size were noted after reperfusion. T2WI demonstrated less intense signal intensity ratio changes in both groups compared with DWI. There were no differences in CBF between groups either during occlusion, early reperfusion, or 1 day after reperfusion. CONCLUSIONS: This study strongly suggests that estrogens selectively protect cortical tissue from ischemic damage during MCAO and that this protection is exerted during both the occlusion and reperfusion phases of ischemia and does not involve an estrogen-related change in CBF.

Nuclear magnetic resonance imaging measurements of water diffusion in the perfused hippocampal slice during N-methyl-D-aspartate-induced excitotoxicity.

Significant changes in the apparent diffusion coefficient of water are observed in nuclear magnetic resonance images of patients with acute ischemic stroke. However, the underlying mechanisms of these apparent diffusion coefficient changes are still unresolved. To analyse possible mechanisms, this study applies nuclear magnetic resonance imaging on a 14.1 Tesla narrow-bore magnet to quantitatively study water diffusion in individually perfused brain slices following exposure to N-methyl-D-aspartate excitotoxicity. The results indicate that brain slices have at least two distinct diffusing water compartments with apparent diffusion coefficients of 0.96+/-0.10x10(-3) mm2/s and 0.06+/-0.01x10(-3) mm2/s. When excitotoxicity was induced with N-methyl-D-aspartate, there was a significant decrease in the fraction of the fast diffusing water component in the slices (P<0.001). However, neither apparent diffusion coefficient changed significantly. Prior treatment with dizocilpine maleate (MK-801) depressed the effects of N-methyl-D-aspartate (P<0.01, ANOVA). The results demonstrate brain slice compartmental changes resulting from direct receptor stimulation and provide evidence for tissue water redistribution as an important mechanism for changes in apparent diffusion coefficient seen in clinical magnetic resonance imaging. The brain slice preparation affords a well-controlled method to study the mechanisms of tissue nuclear magnetic resonance contrast, bridging the gap between basic nuclear magnetic resonance studies and clinical magnetic resonance imaging. The brain slice model also offers a new way to test the utility of potential anti-stroke drugs using high field nuclear magnetic resonance imaging.

In vivo dynamics and distribution of intracerebroventricularly administered gadodiamide, visualized by magnetic resonance imaging.

Direct injections into the cerebroventricles have been extensively utilized in neurophysiological studies. Mapping the distribution of injectate after intracerebroventricular injection has been made only by post mortem analysis, and the dynamic distribution of injectate within the brain has not been well characterized. In this report, we apply contrast-enhanced magnetic resonance imaging to study the pharmacokinetics and extent of non-ionic gadodiamide transport into brain tissue in vivo after intracerebroventricular administration. The results indicate that intracerebroventricular injectate travels quickly throughout the ventricular system from the lateral ventricular site of injection to the fourth ventricle and foramina of Luschka and Magendie within 2 min. After this, the signal intensity begins to increase in the periventricular and paraventricular brain parenchyma. Contrast enhancement is visible 2 mm into the brain tissue from the ventricles. Quantitative analysis of the data shows that the transport of gadodiamide across the ependymal layer that lines the cerebrospinal fluid space characterized a rate constant of 0.066+/-0.017 min(-1). These results provide a better understanding of chemical transport and diffusion following direct injection into the cerebroventricles. They provide information on the in vivo dynamics of injectate after intracerebroventricular administration, and show that contrast enhanced magnetic resonance imaging may be used to more precisely define the target sites of chemicals after intracerebroventricular administration into the brain.

Probing intracellular dynamics in living cells with near-field optics.

Near-field optics (NFO) overcomes the diffraction limit of light microscopes and permits visualization of single molecules. However, despite numerous applications of NFO in the physical sciences, there is still a paucity of applications in the neurosciences. In this work, the authors have developed NFO probes to image intracellular dynamic processes in living cells. This is the first time a NFO probe has been inserted inside a living cell to deliver light to a spatially controlled region for optical measurements and to record cellular responses to external stimuli. Two different optical detection systems (CCD camera and avalanche photon detection) were developed to monitor cellular responses to drug administration in two different cell types. NG108-15 neuroblastoma cells and vascular smooth muscle cells (VSMC) were penetrated with NFO probes. Intracellular Ca2+ increases post drug stimulation were detected by NFO probes. The cells were loaded with either fura-2/AM or fluo-3/AM calcium dyes. VSMC were stimulated with angiotensin II, resulting in a precise area of intracellular Ca2+ increase. Different response profiles of Ca2+ increases were observed after ionomycin and bradykinin administration in NG108-15 cells. Responsive heterogeneities due to ionomycin among different cells of the same type were recorded. The results show that NFO probes make possible real-time visualization of intracellular events. With refinement, intracellular NFO probes offer the potential of probing cell function with fast temporal and excellent spatial resolutions.

Losartan potassium, a nonpeptide antagonist of angiotensin II, chronically administered p.o. does not readily cross the blood-brain barrier.

Recently several novel nonpeptide antagonists of angiotensin II (Ang II) have been identified. One of these, losartan potassium (formerly DuP 753) was developed as an orally active and highly selective antagonist for Ang II. As it is inhibited by sulfhydryl agents, it is specific for the AT1 receptor subtype. Since Ang II has both central and peripheral effects, we investigated whether losartan, given p.o. chronically, crosses the blood-brain barrier. The effects of chronic administration of losartan orally (p.o.) at 3 mg/kg per day for three days on the dipsogenic and pressor responses to a pre-established dose of Ang II i.v.t. (50 ng) were studied. Three series of experiments were carried out using conscious normotensive Sprague-Dawley rats. The rats were injected with Ang II intraventricularly (i.v.t.) before and after treatment of losartan p.o. and blood pressure and drinking responses measured. The experiments established that 3 mg/kg losartan p.o. for 3 days antagonized pressor effects of Ang II intravenously (i.v.), but did not antagonize the pressor or drinking effects of Ang II i.v.t. Daily water intake significantly increased with chronic losartan p.o.. Since chronic administration of losartan p.o. was able to block the effects of Ang II i.v. but had no effect on Ang II i.v.t. we conclude that losartan potassium does not readily cross the blood-brain barrier using this dose regimen.

Native epitopes of human IgA.

We have studied the epitopes of the human IgA molecule by using 27 monoclonal antibodies in two simple gel diffusion methods. By testing pairs of monoclonal antibodies for coprecipitation of an IgA myeloma protein, we have clearly identified sterically independent epitopes on the molecule. By testing the non-coprecipitating monoclonal antibodies with soluble IgA-monoclonal antibody immune complexes, we have identified the overlapping epitopes. Eleven proto-epitopes were mapped on the IgA molecule. Since we have not used solid-phase methods, we have presumably identified native epitopes of the IgA molecule.

Structure and dynamics of transmembrane signaling by the Escherichia coli aspartate receptor.

The structure of the cytosolic extension of the first transmembrane region (TM1) of the Escherichia coli aspartate receptor (residues 3, 4, and 5) and conformational changes within that region have been characterized by targeted cross-linking studies and by measurement of the effect of aspartate binding on cross-linking and methylation rates and compared with the periplasmic extension of the same helix. These experiments show that (1) the cytosolic extension of TM1 is helical, with residues 4 and 4' closest together at the dimer interface; (2) the helix is more solvent-exposed at the cytosolic side of the membrane than on the periplasmic side; and (3) aspartate binding enhances the rate of cross-linking at Cys 4, and the resulting cross-linked receptor displays aspartate-induced transmembrane increases in methylation by the cytoplasmic methylase (the CheR protein). We conclude that aspartate induces a conformational change that does not involve large intersubunit movements that lead to an increase in distance between the cytosolic ends of the first membrane-spanning helices; rather, the motion involved is largely contained within individual subunits, possibly resulting in a small movement between positions 4 and 4'.

MRI measurement of cell volume fraction in the perfused rat hippocampal slice.

T(1)-weighted NMR imaging of the isolated perfused rat hippo-campal slice was used to estimate cell volume fraction. Eight brain slices were studied in artificial cerebrospinal fluid (aCSF) using a 600 MHz narrow bore spectrometer and a home built perfusion chamber. Cell volume fraction was calculated as 1 - f(ECS), where f(ECS) is the distribution volume of gadodiamide in the slice. This was determined by measuring the T(1) of the slice before and after perfusion with gadodiamde. A mean cell volume fraction of 0.66 +/- 0. 04 was estimated. The addition of 60 mM mannitol to three of the brain slices produced a 26% decrease in the cell volume fraction. The technique affords a simple means of estimating cell volume fraction and can be extended to produce images reflecting cell density. Magn Reson Med 42:603-607, 1999.

NMR microscopy--beginnings and new directions.

In this paper we briefly review the origins of NMR microscopy, and in the spirit of the Sir Peter Mansfield Symposium of which this presentation was a part, point out especially Sir Mansfield and his co-workers contributions in this area. We then review some recent studies applying magnetic resonance (MR) microscopy focusing on our own contributions in these regards, in particular with reference to imaging of single neurons and more recent microimaging studies on isolated perfused brain slices. Finally we briefly describe recent preliminary studies on the feasibility of spectroscopic experiments that may be performed at the single cell level, further illustrating the growing scope and potential of magnetic resonance imaging (MRI) in general as a tool for examining biological systems non-invasively.

MR imaging measurement of compartmental water diffusion in perfused heart slices.

Myocardial tissue slices were isolated from the left ventricular free wall (7 slices) and left ventricular papillary muscle (3 slices) of New Zealand White male rabbits (n = 4) and were subsequently superfused with a modified St. Thomas' Hospital cardioplegic solution at 19 degrees C. The diffusion-weighted images were obtained with a 600-MHz nuclear magnetic resonance spectrometer using diffusion gradient b-values that ranged from 166 to 6,408 s/mm(2); the apparent diffusion coefficient of water in the tissues were subsequently calculated. All of the tissue samples that were studied exhibited nonmonoexponential diffusion. Data from seven slices were mathematically fitted by a biexponential expression with a fast diffusion component of 0.72 +/- 0.07 x 10(-3) mm(2)/s, and a slow diffusion component of 0.060 +/- 0.033 x 10(-3) mm(2)/s. The fast component dominated the calculated apparent diffusion coefficient of the tissue, composed of 82 +/- 3% of the overall diffusion-dependent signal decay. Thus myocardial tissue exhibits characteristics consistent with multiple compartments of diffusion. This work has important implications for myocardial diffusion tensor imaging, as well as the changes in diffusion that have been reported following myocardial ischemia.

A role for CaMKII in T cell memory.

In order to study the role of calcium/calmodulin kinase II (CaMKII) in T cells, we generated transgenic mice expressing CaMKIIgammaB* (T287D), a partially calcium-independent mutant of CaMKIIgammaB. In these mice, the size of the thymus was increased 1.5- to 2-fold, at least in part due to an increase in the lifespan of double-positive (DP) thymocytes. More importantly, there was an increase in the number of T cells in the secondary lymphoid organs that had acquired an antigen-dependent memory phenotype. These T cells were bonafide memory cells as assessed by a variety of criteria. In addition, T cells from wild-type mice acquired calcium-independent CaMKII activity after several rounds of antigen-stimulated division. We propose that CaMKII controls a distinct process of activation-induced cellular differentiation.

Two-component diffusion tensor MRI of isolated perfused hearts.

Nonmonoexponential MR diffusion decay behavior has been observed at high diffusion-weighting strengths for cell aggregates and tissues, including the myocardium; however, implications for myocardial MR diffusion tensor imaging are largely unknown. In this study, a slow-exchange-limit, two-component diffusion tensor model was fitted to diffusion-weighted images obtained in isolated, perfused rat hearts. Results indicate that there are at least two distinct components of anisotropic diffusion, characterized by a "fast" component whose principal diffusivity is comparable to that of the perfusate, and a highly anisotropic "slow" component. It is speculated that the two components correspond to tissue compartments and have a general agreement with the orientations of anisotropy, or fiber orientations, in the myocardium. Moreover, consideration of previous studies of myocardial diffusion suggests that the presently observed fast component may likely be dominated by diffusion in the vascular space, whereas the slow component may include the intracellular and interstitial compartments. The implications of the results for myocardial fiber orientation mapping and limitations of the current two-component model used are also discussed.

MR microscopy of perfused brain slices.

To study the origins of signal changes in clinical MRI we have previously studied isolated single neuronal cells by MR microscopy. To account for the extracellular environment of the cells, we have developed a prototype perfusion chamber for MR microimaging of perfused rat hippocampal brain slices. To demonstrate the utility of this model, brain slices were initially perfused in isotonic solutions and then subjected to osmotic perturbations via perfusate exchange with 20% hypertonic and 20% hypotonic solutions. In diffusion weighted images, signal intensity changes of +16(sigma(n-1) = 11)% (hypotonic) and -26(sigma(n-1) = 10)% (hypertonic) were observed. No significant variation in response was observed across the slice when several subregions were examined. These observations are consistent with the view that contrast changes are driven primarily by changes in the intra- and extracellular compartmentation of water. This is the first report of MR microimaging of the isolated brain slice. The technique will enable the correlation of MR microimaging measurements with microscopic changes using other modalities and techniques to provide a better understanding of signals in clinical MRI.

The effect of ouabain on water diffusion in the rat hippocampal slice measured by high resolution NMR imaging.

High resolution NMR imaging of the isolated perfused rat hippocampal slice was used to quantitate ADC changes following ouabain-induced cell swelling. Hippocampal slices were studied in artificial cerebrospinal fluid and then in ouabain using a 600-MHz narrow bore spectrometer and a home-built perfusion chamber. The brain slices demonstrated biexponential diffusion behavior. After perfusion with 1 mMouabain, there was an increase in the fraction of slowly diffusing water. The ADCs of the two fractions did not change. These data support the hypothesis that the decrease in the ADC of brain water following an ischemic attack is caused by cell swelling. The relative amplitudes of the two diffusing fractions do not match the expected ratio of intracellular and extracellular fractions. This discrepancy may be principally due to the difference in T2 relaxation rates of the two compartments.