Longitudinal brain MRI study in a mouse model of Rett Syndrome and the effects of choline.

Rett Syndrome (RTT), the second most common cause of mental retardation in girls, is associated with mutations of an X-linked gene encoding the transcriptional repressor protein MeCP2. Mecp2(1lox) mutant mice express no functional MeCP2 protein and exhibit behavioral abnormalities similar to those seen in RTT patients. Here we monitor the development of both whole brain and regional volumes between 21 and 42 days of age in this model of RTT using MRI. We see decreases in whole brain volumes in both male and female mutant mice. Cerebellar and ventricular volumes are also decreased in RTT males. Previous work has suggested that perinatal choline supplementation alleviates some of the behavioral deficits in both male and female Mecp2(1lox) mutant mice. Here we show that perinatal choline supplementation also positively affects whole brain volume in heterozygous females, and cerebellar volume in male RTT mice.

Evaluation of therapeutic effectiveness using MR imaging in a rabbit model of anterior uveitis.

Magnetic resonance imaging (MRI) has been used to examine conditions that alter the permeability of the blood-retinal barrier. Our goal was to determine if blood-aqueous barrier permeability could be similarly assessed, because MRI offers the theoretical advantage of providing quantitative data directly from inflamed uveal tissues rather than from the aqueous humor into which the inflammatory reaction spills. As an additional challenge, we sought to use MRI to measure differences between the inflamed uveal tissues of corticosteroid-treated and placebo-treated uveitic eyes. Anterior uveitis was induced in one eye of eight rabbits by subcutaneous injection of Mycobacterium tuberculosis, followed after 10 days with intravitreal challenge. One rabbit of each pair was treated with topical 1% prednisolone acetate while control rabbits were treated with artificial tears. Contrast-enhanced MRI studies were performed prior to uveitis induction, one day after induction and then weekly for at least 2 weeks. MR image data were analyzed to determine percent change in peak enhancement of the ciliary body and anterior chamber. The initial rate of change of enhancement of the anterior chamber was also measured. Extensive contrast agent-induced MR image enhancement of both the anterior chamber and the ciliary processes was measured following the induction of uveitis. More rapid improvement was measured for the 1% prednisolone acetate-treated rabbit eyes (P < 0.001). MR signal enhancement data obtained from the ciliary processes proved to be the most reliable indicator of disease activity in this rabbit model of uveitis. Such data can only be obtained using MRI.

A rapid and sensitive method for the analysis of cyanophycin.

A method has been devised for the quantitative analysis of cyanophycin, based on (1)H nuclear magnetic resonance (NMR) spectroscopy, allowing determination of the nitrogen status of cyanobacteria. Cyanophycin is extracted with minimal washing from small volumes of cells and quantified by integration of the NMR peak attributed to the protons attached to the delta-carbon of arginine. Linear relationships were found between the amount of cyanophycin determined by this method and both known concentrations of cyanophycin solutions and the amount of cyanophycin determined using the standard chemical arginine assay.

NMR study of the metabolic 15N isotopic enrichment of cyanophycin synthesized by the cyanobacterium Synechocystis sp. strain PCC 6308.

1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy has been used to characterize cyanophycin, a multi-l-arginyl-poly-[l-aspartic acid] polypeptide from the cyanobacterium Synechocystis sp. strain PCC 6308. 1H, 13C and 15N chemical shifts and 1JHN and 1JCN coupling constants were measured in isolated 15N-labeled cyanophycin, and showed chemical shift values and J-couplings consistent with the reported polypeptide structure. 15N enrichment levels were determined from the extent of 1H-15N J-coupling in 1H NMR spectra of cyanophycin. Similar experiments using 13C-15N coupling in 13C NMR spectra were not useful in determining enrichment levels.

Two internal pools of soluble polyphosphate in the cyanobacterium Synechocystis sp. strain PCC 6308: an in vivo 31P NMR spectroscopic study.

Two intracellular pools of soluble polyphosphate were identified by in vivo 31P NMR spectroscopy in the cyanobacterium Synechocystis sp. strain PCC 6308. Polyphosphate was present in the cells after growth in sulfur-limited media containing excess phosphate. The presence of polyphosphate was confirmed by transmission electron microscopy and chemical analysis. 31P NMR spectroscopy of whole cells treated with EDTA revealed two pools of mobile polyphosphate. A downfield shift and narrowing of part of the broad polyphosphate resonance was observed after EDTA treatment, suggesting that EDTA binds metal ions normally associated with some of the polyphosphate. Phosphate, but not polyphosphate, leaked out of the cells after this treatment. Addition of magnesium ions caused the downfield shift in the polyphosphate resonance to move back toward its original value. These data show that only part of the cation-complexed polyphosphate is accessible to the added EDTA and suggest that there are two internal fractions of NMR-visible polyphosphate in the cells, only one of which loses its associated cations to EDTA. Spheroplast formation showed that polyphosphate was not present in the periplasm of the cells.

31P NMR identification of metabolites and pH determination in the cyanobacterium Synechocystis sp. PCC 6308.

The identity of a number of phosphorus-containing metabolites present in Synechocystis sp. PCC 6308 has been confirmed by 31P NMR spectroscopy. The presence of D-ribulose 1,5-bisphosphate (RuBP); DL-glyceraldehyde 3-phosphate (GlyP); D(-)3-phosphoglyceric acid (3PGA); D-ribulose 5-phosphate (Ru5P);6-phosphogluconic acid (6PGA); phosphoenolpyruvate (PEP); inorganic phosphate (Pi); uridine diphosphoglucose (UDPG); ADP and ATP were demonstrated by the pH dependence of their 31P NMR chemical shifts in spectra of perchloric acid cell extracts. Intracellular pH of cells was determined to be 7.5-7.7.

Contrast-enhanced magnetic resonance imaging confirmation of an anterior protein pathway in normal rabbit eyes.

PURPOSE: Contrast-enhanced proton magnetic resonance imaging (1H MRI) has been used as a quantitative, noninvasive method to corroborate a pathway for the diffusion of plasma-derived protein into the aqueous humor in the normal rabbit eye. METHODS: T1-weighted magnetic resonance images were produced over 1- to 3-hour periods after the intravenous injection of gadolinium diethylenetriamine-pentaacetic acid. RESULTS: Analysis of the images yielded the time dependence of signal enhancements within the areas of interest. The ciliary body showed an immediate sharp increase, followed by a gradual decrease in signal enhancement with time. Although a gradual increase in signal enhancement was found in the anterior chamber, no significant change occurred in the posterior chamber. A similar MRI experiment with an owl monkey produced parallel, though smaller, signal enhancements in the ciliary body and anterior chamber. Again, however, no significant change was found in the posterior chamber. CONCLUSIONS: These results support and extend those of recent fluorophotometric, tracer-localization, and modeling studies demonstrating that in the normal rabbit and monkey eye, plasma-derived proteins bypass the posterior chamber, entering the anterior chamber directly via the iris root.

A feasibility study of 23Na magnetic resonance imaging of human and rabbit vitreal disorders.

PURPOSE: To assess the clinical feasibility of sodium magnetic resonance imaging for the visualization and characterization of intraocular tissues. METHODS: 23Na magnetic resonance images were obtained of enucleated human eyes and of rabbit eyes in vivo. The magnetic resonance imaging technique used in this study provided slices of < 2 mm thickness and in-plane resolution of < 2 x 2 mm. From each of these slices local values of spin-spin relaxation time (T2*) were calculated from pixel intensities in each of the eight echoes. RESULTS: The images clearly display normal anatomic details of the lens and vitreous humor, and important pathologic details such as intravitreal and subretinal hemorrhages, ocular melanoma, and retinal detachments. Intraocular tissue identifications based on relative spin-spin relaxation time values and pixel intensities correlate with those made by standard diagnostic techniques. CONCLUSIONS: 23Na magnetic resonance imaging may be used for the visualization and characterization of intraocular tissues. Differentiation among vitreous humor, lens, aqueous humor, subretinal fluid, or hemorrhage and tumor may be based on image intensity and/or spin-spin relaxation times.

In vivo sodium chemical shift imaging.

The shift reagents thulium(III) 1,4,7,10-tetraazacyclododecane N,N',N",N"'tetramethylenephosphonate (TmDOTP5-), and dysprosium(III)triethylenetetramine-hexaacetate (DyTTHA3-) are compared in this work for their uses in sodium chemical shift imaging (NaCSI). In a series of experiments using phantoms we evaluated the relative contributions of bulk magnetic susceptibility (BMS) effects and hyperfine shifts to the induced 23Na chemical shift for these two shift reagents. The ratios of BMS effects to hyperfine shifts suggest that TmDOTP5- should be a more effective shift reagent than DyTTHA3- for 23Na NMR spectroscopy as well as NaCSI. The dependence on pH and free Ca2+ concentration of the 23Na NMR frequency shift induced by TmDOTP5- was evaluated. It was found that TmDOTP5- produces good spectral resolution under physiologic conditions. Examples presented from in vivo NaCSI experiments using TmDOTP5- to study diffusion in the posterior chamber of the rabbit eye and to monitor the rate of clearance of aqueous fluid from the anterior chamber demonstrate the effectiveness of this new shift reagent and of the NaCSI technique for in vivo studies.

Dynamic sodium chemical shift imaging for the study of aqueous humor flow.

Ocular images were obtained using sodium chemical shift imaging (CSI) and 1,4,7,10-tetraazacyclododecane-N,N'N",N"'-tetramethylenephospho nate thulium (III) [Tm(DOTP)5-], a paramagnetic chemical shift reagent. After injecting the shift reagent into the anterior chamber of rabbits, serial imaging was done, monitoring the change in chemical shift with time. Sodium CSI produced images of the eye in three dimensions, quantitatively depicting the spatial and temporal changes in the concentration of a paramagnetic tracer substance. The Tm(DOTP)5- is eliminated from the anterior chamber by first-order kinetics with a half-life of 49 min. These data suggest that this substance is eliminated from the anterior chamber at the same rate as aqueous humor is replaced. Sodium CSI shows promise as a valuable technique for monitoring fluid dynamics in the living eye.

An NMR blood test for uveal melanoma?

In a recent article a simple nuclear magnetic resonance (NMR) blood test was suggested for the detection of the presence of cancer. The test's sensitivity to uveal melanoma of both pre- and posttreatment status has been investigated. Cases in this study were 95 patients with uveal melanoma, and controls were 70 participants in an ongoing case control study of retinal eye disease being conducted at the Massachusetts Eye and Ear Infirmary. Proton NMR evaluations at 4.7 T (200 MHz) were performed on plasma obtained from EDTA and citrated blood samples. The average line-width values were calculated from each spectrum. Statistical analysis revealed that mean proton NMR line widths were essentially equal for patients with treated (18.7 Hz) and untreated tumors (18.4 Hz) and for controls (18.5 Hz). Results based on this data set suggest that proton NMR spectroscopy has little predictive power in the detection of uveal melanoma or in the monitoring of therapy.

Magnetic resonance imaging: an emerging technique for the diagnosis of ocular disorders.

With developments in proton and sodium imaging on eyes and ocular lesions, MRI has been rapidly advancing into the field of ophthalmology. Although many intraocular lesions have been T1 and T2 characterized, and MRI may aid in their diagnosis, there remain a few problems with the technique. One is the presence of motion artifacts which sometimes occur with lengthy scanning times. A patient must be imaged on the order of minutes with MRI. In addition ambiguities exist even with the T1 and T2 characterizations, although the specificity surpasses that of CT. Eventually these problems may be solved, but even now proton coupled with sodium imaging and other diagnostic techniques appear promising in the evaluation of ocular disorders.

Magnetic resonance imaging and spectroscopy of intraocular tumors.

Proton magnetic resonance imaging (1H MRI) has emerged as a clinically useful tool for the diagnosis of intraocular tumors. During the last four years 1H MRI characteristics, including spin-lattice relaxation times (T1) and spin-spin relaxation times (T2), have been established for several types of tumors. The introduction of surface coils to the imaging process has significantly improved the quality of intraocular MR images, leading some clinicians to suggest that 1H MR images are preferable to CT scans. Another MRI technique, in which sodium-23 (23Na) is imaged rather than protons, is now under development as tool for intraocular diagnosis. The potential of 23Na MRI depends upon the high concentration and "visibility" of sodium in the vitreous body, and upon the apparent differences in sodium behavior in normal cells vs. tumor cells. The metabolism of normal ocular tissues and intraocular tumors may be probed noninvasively with phosphorus-31 MR spectroscopy (31P MRS). Much progress has been made during the last few years in understanding the appearance of 31P MR spectra of many types of healthy and diseased cells and tissues. Clinical application of this technique to the diagnosis and monitoring of intraocular tumors following conservative treatment will be dependent upon the development of spectroscopy techniques that collect information from the volume of interest (tumor) only.

A perfusion system developed for 31P NMR study of melanoma cells at tissue-like density.

A perfusion culture system has been developed for 31P NMR study of human uveal melanoma metabolism by adapting the Vitafiber I cartridge system (Amicon). 31P NMR spectra collected weekly during periods of up to 10 weeks demonstrated increasing levels of phosphorus metabolites as the anchorage-dependent cells grew to tissue-like density.

Preliminary results on phosphorus-31 nuclear magnetic resonance evaluation of human uveal melanoma in enucleated eyes.

Clinical evaluation of uveal melanomas by nuclear magnetic resonance (NMR) techniques depends on ascertaining how these tumors characteristically appear in NMR images and spectra. The authors have determined NMR characteristics of suspected uveal melanomas by phosphorus-31 (31P) NMR spectroscopy of freshly enucleated human eyes. Nuclear magnetic resonance examination was performed at 8.45 Tesla within 90 minutes after enucleation. Enucleated eyes were maintained at 4 degrees C in tissue culture medium during the 30 minutes required for transport. Nuclear magnetic resonance spectra were obtained within 10 minutes, a clinically acceptable time, using a two-turn 31P surface coil. Spectral parameters included 10-kHz spectral width, 1024 data points, and 0.5-second recycle delay. Phosphorus-31 NMR spectroscopy allowed differentiation of choroidal melanomas from normal ocular structures. Differentiating features include significant peaks in tumor spectra due to the phosphodiesters glycerol 3-phosphoryl ethanolamine (GPE) and glycerol 3-phosphorylcholine (GPC), and the phosphomonoesters phosphorylethanolamine (PE) and phosphorylcholine (PC). These preliminary data are encouraging and suggest that clinical trials at the lower magnetic field strengths available in NMR imaging systems seem feasible and warrant investigation.

Proton and sodium 23 magnetic resonance imaging of human ocular tissues. A model study.

Clinical evaluation of uveal melanomas by magnetic resonance imaging (MRI) techniques depends on developing an understanding of the appearance of these tumors in magnetic resonance (MR) images. We have determined MR characteristics of uveal melanomas by proton (1H) and sodium 23 MRI of freshly enucleated human eyes at 1.5 tesla. The MR images were obtained using two-turn proton and 23Na surface coils, designed to both transmit and receive the radiofrequency signal. Proton MRI techniques included saturation recovery and spin echo; the gradient-recalled echo technique was used for 23Na MRI. Proton and 23Na MR images provide complementary information; contrast between intraocular tumors and vitreous, lens, or subretinal hemorrhage may be varied by using MR pulse sequences that emphasize tissues based on T1, T2, proton, or sodium density values. A combination of proton and 23Na MRI provides differentiation between normal ocular structures and intraocular tumors, as well as associated complications, such as retinal detachments and subretinal hemorrhages.

Proton and phosphorus-31 NMR study of the dependence of diadenosine tetraphosphate conformation on metal ions.

Adenosine 5'-tetraphospho-5'-adenosine (Ap4A) plays a role in cellular metabolism in a wide variety of organisms. Because the divalent cations Mg2+ and Zn2+ are involved in the synthesis and function of Ap4A, the effect of divalent cations on the dinucleotide's conformation is of interest. 1H and 31P chemical shift experiments were carried out as a function of Mg2+ concentration and pH. We propose that Mg2+ stabilizes the unusual ring-stacked conformation of Ap4A at pH greater than 2 by interacting with the beta-phosphates. To further probe conformational effects, stable complexes of Ap4A with Co3+ were studied using 1H and 31P NMR. Co3+ forms two different bidentate complexes with Ap4A, independent of whether the other four octahedral coordination sites are occupied by ammonia or trimethylenediamine. NMR results suggest that in one complex the Co3+ is coordinated to two beta-phosphates and ring stacking is stabilized. In the other complex, Co3+ is coordinated to an alpha-phosphate and its neighboring beta-phosphate and ring stacking is destabilized. These results further support the hypothesis that Mg2+ stabilizes the ring-stacked conformation by interacting symmetrically with the two beta-phosphate groups.

Characterization of human uveal melanoma cells by phosphorus 31 nuclear magnetic resonance spectroscopy.

We performed experiments to determine the potential usefulness of nuclear magnetic resonance spectra in the diagnosis and follow-up of ocular melanoma. High-resolution phosphorus 31 nuclear magnetic resonance spectra at 109.3 MHz were obtained for human uveal melanoma, Greene hamster melanoma, and normal human diploid fibroblast cells. Phosphate metabolites were identified and their concentrations were shown to vary among the different cell lines. Uveal melanoma cells contain unusually high concentrations of the phospholipid metabolite phosphorylcholine and the phosphodiesters glycerol 3-phosphoryl choline and glycerol 3-phosphoryl ethanolamine. Baseline data are thus provided for studies of the effect of various treatment modalities on uveal melanoma. These initial results suggest that the data provided by high-resolution phosphorus 31 nuclear magnetic resonance spectra can provide useful diagnostic and follow-up data with respect to ocular melanoma.