Novel method for in vivo hydroxyl radical measurement by microdialysis in fetal sheep brain in utero.

Hydroxyl radical (.OH) is a reactive oxygen species produced during severe hypoxia, asphyxia, or ischemia that can cause cell death resulting in brain damage. Generation of .OH may occur in the fetal brain during asphyxia in utero. The very short half-life of .OH requires use of trapping agents such as salicylic acid or phenylalanine for detection, but their hydroxylated derivatives are either unstable, produced endogenously, or difficult to measure in the small volume of microdialysis samples. In the present study, we used terephthalic acid (TA), hydroxylation of which yields a stable and highly fluorometric isomer (excitation, 326 nm; emission, 432 nm). In vitro studies using .OH generated by the Fenton reaction showed that hydroxylated TA formed quickly (<10 s), was resistant to bleaching (<5% change in fluorescence), and permitted detection of <0.5 pmol .OH. In vivo studies were performed in fetal sheep using microdialysis probes implanted into the parasagittal cortex. The probe was perfused at 2 mul/min with artificial cerebrospinal fluid containing 5 mM TA, and samples were collected every 30 min. Fluorescence measured in 10 mul of dialysate was significantly greater than in the efflux from probes perfused without TA. High-performance liquid chromotography analysis showed that the fluorescence in dialysis samples was entirely due to hydroxylation of TA. Thus this study shows that it is possible to use TA as a trapping agent for detecting low concentrations of .OH both in vitro and in vivo and that low concentrations of .OH are present in fetal brain tissue and fluctuate with time.     

In utero morphological effects of hydroxyurea on the fetal development in Sprague-Dawley rats

In order to investigate in utero morphological effect of hydroxyurea (HU) in Sprague-Dawley rats, HU was intraperitoneally injected to pregnant Sprague-Dawley rats at a dose of 100 or 200 mg/kg/day during the organogenetic period (days 9-12 of gestation). A dose of 200 mg/kg/day induced growth retardation, high mortality and high incidence of malformations, although a dose of 100 mg/kg/day produced no adverse effects in the next generation. In the HU 200 mg/kg/day group the incidence of malformations in pups at 4 days of age was low as compared with that in fetuses and pups at 21 days of age. Increasing perinatal mortality in fetuses and pups due to severe central nervous system (CNS) malformations and disappearance of some cases of ventricular septal defect after delivery were considered as the possible causes to induce difference in malformation rate in various stage of development. Latent effect on the development of CNS malformations was observed between 4 and 21 days of age. There was no sex difference in teratogenic effect. These findings were compared with those in Wistar rats exposed to HU 200 mg/kg/day. The incidence of perinatal malformations and the stillbirths were significantly higher in the Wistar rats as compared with those in the Sprague-Dawley rats. In addition, such morphological effects of HU as the exencephaly, dilatation of lateral ventricle, anophthalmia, cleft palate and micrognathia are less severe in Sprague-Dawley rat fetuses than in Wistar rat fetuses.     

Fructose-1, 6-bisphosphatase in human fetal brain and liver during development

Activity of fructose-1,6-bisphosphatase (EC 3.1.3.11), one of the key gluconeogenic enzymes, was measured in human fetal brain and liver during development. Fructose-1,6-bisphosphatase was distributed throughout the different regions of the brain. In contrast to the partially purified enzyme from the brain, the liver enzyme was dependent on Mg²⁺ for maximal activity, EDTA, citrate, oleate and linoleate were stimulatory, whereas 5′-AMP inhibited the activity of the liver enzyme.     

Regulation of rabbit fetal glycogen: effect of in utero fetal decapitation on the metabolism of glycogen in fetal heart, lung, and liver

To understand the control mechanisms involved in the regulation of fetal glycogen, we have studied the effect of in utero fetal decapitations on glycogen metabolism in rabbit fetal heart, lung, and liver. In utero fetal decapitations were performed between days 18 and 21 of gestation. Two to four fetuses on one side of the horn were decapitated. Fetuses were delivered between days 23 and 26 or between days 28 and 30 of gestation. Fetal heart, lungs, and liver were analyzed for DNA, protein, glycogen, glycogen synthase (I and D forms), glycogen phosphorylase (a and b forms), phosphofructokinase, pyruvate kinase, and lactic dehydrogenase. In fetal heart and lung, no difference was observed in any of the above measurements in the intact and decapitated fetuses. In contrast, fetal liver does not appear to develop the glycogen system as indicated by the very low levels of glycogen (0.02 mg/mg DNA) in decapitated fetuses as compared with intact fetuses (0.4 mg/mg DNA). Similarly the levels of glycogen synthase and phosphorylase were two to three times lower in livers from decapitated fetuses as compared with the livers from intact fetuses. The three enzymes phosphofructokinase, pyruvate kinase, and lactic dehydrogenase were not affected by fetal decapitation in all three tissues. These results indicate that the fetal hypothalamic-pituitary-adrenal (thyroid) axis is not required at least after day 18 of gestation for the normal accumulation and subsequent utilization of glycogen in fetal heart and lungs, while it is an absolute requirement for the development of the fetal liver glycogen system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Taxonomic revision of Tatria Kowalewski, 1904 (Cestoda: Amabiliidae): redescriptions of T. biremis Kowalewski, 1904 and T. minor Kowalewski, 1904, and the description of T. gulyaevi n. sp. from Palaearctic grebes

Two species of Tatria Kowalewski, 1904 are redescribed from grebes in Bulgaria: T. biremis Kowalewski, 1904 (specimens from Podiceps nigricollis) and T. minor Kowalewski, 1904 (specimens from P. cristatus and P. nigricollis). T. mircia Gulyaev, 1990 is synonymised with T. minor. The previous records of T. biremis, T. minor and T. mircia are critically analysed in view of the present results. T. gulyaevi n. sp. is described from P. nigricollis from Bulgaria and the Czech Republic and from an unidentified grebe species from Turkey. Some of the previous records of T. minor and T. biremis are recognised as belonging to T. gulyaevi. One specimen illustrated by Kowalewski (1904) is designated as a lectotype of T. minor in order to stabilise the nomenclatural standing of this species.     

Analysis of expression of CD2, CD3, and T cell antigen receptor molecules during early human fetal thymic development

To define early stages of T cell maturation during human fetal thymic development, we have used mAb reactive with CD2, CD3, and TCR molecules in indirect immunofluorescence assays on a series of early human fetal thymic specimens. Using a technique of quantitating the relative proportions of fluorescent-positive cells present in tissue sections, we found at 8.5 wk of gestational age after arrival of CD7+ T cell precursors into the thymic rudiment, 60% of thymic CD7+ cells were CD2+, 4% were CD3+ and none was TCR-delta+ or TCR beta+. Moreover, cells reactive with anti-CD2 antibodies against T11(2) and T11(3) epitopes of CD2 as well as thymic stromal cells expressing the CD2 ligand, lymphocyte function associated Ag-3, were also present at 8.5 wk. From 9.5 wk to birth TCR beta+ cells increased to include greater than 90% of all CD7+ cells while TCR-delta+ cells fell from a peak of 11% of CD7+ cells at 9.5 wk to 1% of CD7+ cells at birth. These data suggest that epitopes of CD2 molecules are expressed early on during fetal thymic development. Moreover, these data suggest that CD7+, CD2+, cytoplasmic CD3+ T cell precursors in man give rise to both TCR-delta+ T cells as well as to T cells expressing TCR-alpha beta.     

MRI osteitis predicts cartilage damage at the wrist in RA: a three-year prospective 3T MRI study examining cartilage damage

Introduction

Cartilage damage impacts on patient disability in rheumatoid arthritis (RA). The aims of this magnetic resonance imaging (MRI) study were to investigate cartilage damage over three years and determine predictive factors.

Methods

A total of 38 RA patients and 22 controls were enrolled at t = 0 (2009). After 3 years, clinical and MRI data were available in 28 patients and 15 controls. 3T MRI scans were scored for cartilage damage, bone erosion, synovitis and osteitis. A model was developed to predict cartilage damage from baseline parameters.

Results

Inter-reader reliability for the Auckland MRI cartilage score (AMRICS) was high for status scores; intraclass correlation coefficient (ICC), 0.90 (0.81 to 0.95) and moderate for change scores (ICC 0.58 (0.24 to 0.77)). AMRICS scores correlated with the Outcome MEasures in Rheumatoid Arthritis Clinical Trials (OMERACT) MRI joint space narrowing (jsn) and X-Ray (XR) jsn scores (r =0.96, P < 0.0001 and 0.80, P < 0.0001, respectively). AMRICS change scores were greater for RA patients than controls (P = 0.06 and P = 0.04 for the two readers). Using linear regression, baseline MRI cartilage, synovitis and osteitis scores predicted the three-year AMRICS (R² = 0.67, 0.37 and 0.39, respectively). A multiple linear regression model predicted the three-year AMRICS (R² = 0.78). Baseline radial osteitis predicted increased cartilage scores at the radiolunate and radioscaphoid joints, P = 0.0001 and 0.0012, respectively and synovitis at radioulnar, radiocarpal and intercarpal-carpometacarpal joints also influenced three-year cartilage scores (P-values of 0.001, 0.04 and 0.01, respectively).

Conclusions

MRI cartilage damage progression is preceded by osteitis and synovitis but is most influenced by pre-existing cartilage damage suggesting primacy of the cartilage damage pathway in certain patients.     

GdDO3NI,a nitroimidazole-based T 1 MRI contrast agent for imaging tumor hypoxia in vivo

Tumor hypoxia is known to affect sensitivity to radiotherapy and promote development of metastases; therefore, the ability to image tumor hypoxia in vivo could provide useful prognostic information and help tailor therapy. We previously demonstrated in vitro evidence for selective accumulation of a gadolinium tetraazacyclododecanetetraacetic acid monoamide conjugate of 2-nitroimidazole (GdDO3NI), a magnetic resonance imaging T ₁-shortening agent, in hypoxic cells grown in tissue culture. We now report evidence for accumulation of GdDO3NI in hypoxic tumor tissue in vivo. Our data show that GdDO3NI accumulated significantly (p < 0.05) in the central, poorly perfused regions of rat prostate adenocarcinoma AT1 tumors (threefold higher concentration than for the control agent) and showed better clearance from well-perfused regions and complete clearance from the surrounding muscle tissue. Inductively coupled plasma mass spectroscopy confirmed that more GdDO3NI than control agent was retained in the central region and that more GdDO3NI was retained in the central region than at the periphery. These results show the utility of GdDO3NI to image tumor hypoxia and highlight the potential of GdDO3NI for application to image-guided interventions for radiation therapy or hypoxia-activated chemotherapy.     

Comparison of intervertebral disc displacements measured under applied loading with MRI at 3.0 T and 9.4 T

The purpose of this study was to compare displacement behavior of cyclically loaded cadaveric human intervertebral discs as measured noninvasively on a clinical 3.0 T and a research 9.4 T MRI system. Intervertebral discs were cyclically compressed at physiologically relevant levels with the same MRI-compatible loading device in the clinical and research systems. Displacement-encoded imaging was synchronized to cyclic loading to measure displacements under applied loading with MRI (dualMRI). Displacements from the two systems were compared individually using linear regression and, across all specimens, using Bland–Altman analysis. In-plane displacement patterns measured at 3.0 T and 9.4 T were qualitatively comparable and well correlated. Bland–Altman analyses showed that over 90% of displacement values within the intervertebral disc regions of interest lay within the limits of agreement. Measurement of displacement using dualMRI using a 3.0 T clinical system is comparable to that of a 9.4 T research system. Additional refinements of software, technique implementation, and image processing have potential to improve agreement between different MRI systems. Despite differences in MRI systems in this initial implementation, this work demonstrates that dualMRI can be reliably implemented at multiple magnetic field strengths, permitting translation of dualMRI for a variety of applications in the study of tissue and biomaterial biomechanics.     

Nutrient transport systems in brain: 40 years of progress

In the last 40 years, especially with the application of new neurochemical and molecular biologic techniques, there has been an explosive progress in understanding how nutrients are transported across the blood–brain barrier and choroid plexus into brain and CSF, and how nutrient homeostasis in brain is achieved. In most cases, there are separate transporters, or in a few cases, systems that transport related substances (e.g., biotin, lipoic, and pantothenic acids). This review focuses on three crucial nutrients (glucose, ascorbic acid, and folates) for which there is substantial new information including ‘knock down’ and ‘knockout’ models in mice and/or humans. The overall objective is to show that this new knowledge leads not just to a more thorough understanding (e.g., of ‘why’ questions like: why do neurons require up to 10 mM ascorbic acid intracellulary?); but in some cases leads to clinically important predictions that allow treatment of heretofore devastating neurologic disorders.     

Multilineage engraftment with minimal graft-versus-host disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated,sensitized T cells and adult T cell-depleted bone marrow in fetal mice

Although engraftment following in utero stem cell transplantation can readily be achieved, a major limitation is the low level of donor chimerism. We hypothesized that a lack of space for donor cells in the recipient marrow was one of the primary reasons for failure to achieve significant engraftment, and that donor T cells could make space in an allogeneic mismatched setting. We found that 3 x 10(5) C57BL/6 (B6) naive CD3(+) cells coinjected with B6 T cell-depleted bone marrow (TCDBM) into 14- to 15-day-old BALB/c fetuses resulted in multilineage engraftment (median, 68.3%) associated with severe graft-vs-host disease (GvHD; 62 vs 0% with TCDBM alone). When 1.5 x 10(5) CD4(+) or CD8(+) cells were used, low levels of engraftment were seen vs recipients of 1.5 x 10(5) CD3(+) cells (2.4 +/- 1.1 and 6.6 +/- 3.9 vs 20.4 +/- 10.4%, respectively). To test the hypothesis that proliferation of T cells in response to alloantigen resulted in GvHD and increased engraftment, we pretreated naive T cells with photochemical therapy (PCT) using S-59 psoralen and UVA light to prevent proliferation. GvHD was reduced (60-0%), but was also associated with a significant reduction in engrafted donor cells (53.4 +/- 4.2 to 1.7 +/- 0.5%). However, when B6 T cells were sensitized to BALB/c splenocytes, treated with PCT, and coinjected with TCDBM, there was a partial restoration of engraftment (13.3 +/- 2.4% H2Kb(+) cells) with only one of nine animals developing mild to moderate GvHD. In this study we have shown that PCT-treated T cells that are cytotoxic but nonproliferative can provide an engraftment advantage to donor cells, presumably by destroying host hemopoietic cells without causing GvHD.     

Metabolic heterogeneity of the normal human brain: multivariate analysis of <Superscript>1</Superscript>H MRS in vivo spectra acquired at 3T

Introduction

In recent years multivariate projection techniques of data analysis (PCA, PLS-DA) have been increasingly used for detection of complex ¹H MRS derived metabolic signatures in pathologic conditions. However, these techniques have not been applied in the studies of metabolic heterogeneity of the normal human brain.

Objective

In this work we extended current knowledge about regional distribution of metabolites by multivariate analysis of metabolite levels obtained from various cortical and subcortical regions.

Methods

The studied group consisted of 71 volunteers with no neurological disorders. The metabolite levels obtained from short echo time ¹H MRS in vivo spectra were subjected to univariate and multivariate analysis.

Results

The major variance direction in the dataset was dominated by glutamine?+?glutamate, creatine, myo-inositol and was successful in differentiation of the cortical grey matter and cerebellar vermis from the cortical white matter, pons, basal ganglia, hippocampus and thalamus. The projection plane formed by the second and third variance directions was dominated by N-acetylaspartate?+?N-acetylaspartylglutamate, choline and glutamine?+?glutamate variation not explained by the first direction. This plane revealed a huge metabolic contrast between the pons and basal ganglia, differentiation between the cortical grey matter regions and cerebellar vermis as well as biochemical heterogeneity between the regions such as: thalamus, basal ganglia and hippocampus.

Conclusion

Multivariate approach to ¹H MRS data analysis provides an insight into the normal brain biochemistry and is helpful in understanding the regional heterogeneity of the normal brain. Such knowledge is crucial for a proper interpretation of altered metabolic pathways in diseases.