Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration.

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation.

Neural stem cell-mediated CE/CPT-11 enzyme/prodrug therapy in transgenic mouse model of intracerebellar medulloblastoma.

Medulloblastoma is a heterogeneous diffuse neoplasm that can be highly disseminated, and is the most common malignant childhood brain tumor. Although multimodal treatments have improved survival rates for patients with medulloblastoma, these tumors are associated with high morbidity and mortality. New treatment strategies are urgently needed to improve cure rates and, importantly, to spare normal brain tissue from neurotoxicity and patients from life-long cognitive and functional deficits associated with current therapies. In numerous preclinical brain tumor models, neural stem cells (NSCs) have shown great promise as delivery vehicles for therapeutic genes. Here, we have used an established, genetically modified human NSC line (HB1.F3.CD) to deliver carboxylesterase (CE) to cerebellar tumor foci and locally activate the prodrug camptothecin-11 (CPT-11) (Irinotecan) to the potent topoisomerase I inhibitor SN-38. HB1.F3.CD NSC tumor tropism, intratumoral distribution and therapeutic efficacy were investigated in clinically relevant experimental models. Magnetic resonance imaging was used for in vivo tracking of iron nanoparticle-labeled NSCs, and to assess the therapeutic efficacy of CE-expressing HB1.F3.CD cells. As compared with controls, a significant decrease in tumor growth rate was seen in mice that received both NSCs and CPT-11 as their treatment regimen. Thus, this study provides proof-of-concept for NSC-mediated CE/CPT-11 treatment of medulloblastoma, and serves as a foundation for further studies toward potential clinical application.

Corticotomy-/osteotomy-assisted tooth movement microCTs differ.

Corticotomy-assisted and osteotomy-assisted tooth movement involves surgical incisions through the alveolar bone. To ascertain whether teeth move by distraction osteogenesis or by regional accelerated phenomenon (RAP), we randomly assigned 30 Sprague-Dawley rats to one of 5 experimental groups: corticotomy alone, corticotomy-assisted tooth movement, osteotomy alone, osteotomy-assisted tooth movement, or tooth movement alone. Each animal was imaged by microtomography immediately after surgery, after 21 days, and after 2 months. After 21 days, regional accelerated phenomenon was observed in the alveolar bone of the corticotomy-treated animals and distraction osteogenesis in the osteotomy-assisted tooth movement animals. Pixel count data were analyzed by nested ANOVA for 5 experimental groups, split-mouth controls, 3 levels along the root, and 5 sites per level. The most demineralized sites after 21 days differed for each of the experimental groups. Our study indicates that osteotomies and corticotomies induce different alveolar bone reactions, which can be exploited for tooth movement.

In vivo visualization of gene expression using magnetic resonance imaging.

High-resolution in vivo imaging of gene expression is not possible in opaque animals by existing techniques. Here we present a new approach for obtaining such images by magnetic resonance imaging (MRI) using an MRI contrast agent that can indicate reporter gene expression in living animals. We have prepared MRI contrast agents in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a substrate that can be removed by enzymatic cleavage. Following cleavage, the paramagnetic ion can interact directly with water protons to increase the MR signal. Here, we report an agent where galactopyranose is the blocking group. This group renders the MRI contrast agent sensitive to expression of the commonly used marker gene, beta-galactosidase. To cellular resolution, regions of higher intensity in the MR image correlate with regions expressing marker enzyme. These results offer the promise of in vivo mapping of gene expression in transgenic animals and validate a general approach for constructing a family of MRI contrast agents that respond to biological activity.

Analysis of glioblastoma tumor coverage by oncolytic virus-loaded neural stem cells using MRI-based tracking and histological reconstruction.

In preclinical studies, neural stem cell (NSC)-based delivery of oncolytic virus has shown great promise in the treatment of malignant glioma. Ensuring the success of this therapy will require critical evaluation of the spatial distribution of virus after NSC transplantation. In this study, the patient-derived GBM43 human glioma line was established in the brain of athymic nude mice, followed by the administration of NSCs loaded with conditionally replicating oncolytic adenovirus (NSC-CRAd-S-pk7). We determined the tumor coverage potential of oncolytic adenovirus by examining NSC distribution using magnetic resonance (MR) imaging and by three-dimensional reconstruction from ex vivo tissue specimens. We demonstrate that unmodified NSCs and NSC-CRAd-S-pk7 exhibit a similar distribution pattern with most prominent localization occurring at the tumor margins. We were further able to visualize the accumulation of these cells at tumor sites via T2-weighted MR imaging as well as the spread of viral particles using immunofluorescence. Our analyses reveal that a single administration of oncolytic virus-loaded NSCs allows for up to 31% coverage of intracranial tumors. Such results provide valuable insights into the therapeutic potential of this novel viral delivery platform.

Magnetic resonance imaging for the evaluation of a novel metastatic orthotopic model of human neuroblastoma in immunodeficient mice.

Neuroblastoma is the second most common solid tumor in children. So far few tumor models for this cancer have been reported in mice. We have created a murine tumor model for studying human neuroblastoma based on surgical orthotopic implantation in scid mice. Small fragments of subcutaneous tumors of SK-N-BE(2) human neuroblastoma cells expressing enhanced green fluorescent protein were surgically implanted near the left adrenal gland of scid mice. One hundred percent of the animals (n = 21) successfully implanted developed a large retroperitoneal tumor and became moribund between 22 and 57 days after implantation (mean survival time = 41 days). At the time of sacrifice the presence of bone marrow metastasis was detected by RT-PCR for green fluorescent protein in 95% of the cases. The growth of small tumor implants could be easily visualized and quantified by surveillance MR imaging, with a resolution of 117 x 117 x 750 microm in two orthogonal planes allowing accurate volume measurements, as well as assessment of necrosis and tissue invasion. This novel model should be a valuable tool to study the biology and therapeutic approaches to neuroblastoma.

Quantitative proton-decoupled 31P MRS and 1H MRS in the evaluation of Huntington's and Parkinson's diseases.

OBJECTIVE: To determine cerebral energy status in patients with Huntington's disease (HD) and Parkinson's disease (PD). METHODS: The study included 15 patients with DNA-proven, symptomatic HD and five patients with medically treated, idiopathic PD, all of whom were candidates for neurotransplant treatment, as well as 20 age-related normal subjects. Quantitative noninvasive, MRI-guided proton MRS was performed of single volumes in putamen of basal ganglia (BG), occipital gray matter, and posterior parietal white matter; in addition, quantitative phosphorus and proton-decoupled phosphorus MRS of superior biparietal white and gray matter was done. Outcome measures were quantitative metabolite ratios and millimolar concentrations of neuronal and glial markers, creatine (Cr) and adenosine triphosphate (ATP), and intracellular pH. RESULTS: In volume-corrected control BG (10.46 +/- 0.37 mM), [Cr] was 29% (p < 0.05) higher than in control gray matter (8.10 +/- 1.04 mM). In HD and PD, energy metabolism was not abnormal in the four cerebral locations measured by MRS. No increase in cerebral lactate or decrease in phosphocreatine and ATP was detected. Small, systematic abnormalities in N-acetylaspartate (NAA, decreased), Cr (decreased), choline-containing compounds (Cho, increased), and myoinositol (mI, increased) were demonstrable in all patient's individually and in summed spectra but were insufficient to make diagnosis possible in the individual patient. CONCLUSION: Previously described failure of global energy metabolism in HD was not confirmed. However, quantitative 1-hydrogen MRS and decoupled 31-phosphorus MRS are sensitive to +/-10% alterations in key cerebral metabolites, and may be of value in noninvasive monitoring of appropriate therapies.

High resolution magnetic resonance imaging of the brain in the dy/dy mouse with merosin-deficient congenital muscular dystrophy.

Magnetic resonance imaging (MRI) abnormalities in the cerebral white matter are a consistent feature of merosin-deficient human congenital muscular dystrophy, a disease caused by a primary defect in the expression of the laminin alpha2 chain of merosin. To investigate the relationship between imaging changes and merosin deficiency we undertook a MRI study in the dy/dy mouse, an animal model for this form of human congenital muscular dystrophy. High resolution in vivo imaging was performed on anaesthetized animals (two homozygous dy/dy mutants and two heterozygous dy/DY controls, aged 2.5 months) in a dedicated 11.7T magnetic resonance imaging scanner. T(1) and T(2) weighted images were normal in all mice and white matter changes were not seen at a stage of maturity when MRI changes are already very striking in human patients. Cerebral MRI abnormalities do not appear to be a feature of dy/dy mice, despite the virtual absence of merosin expression in the dy/dy mouse brain. Possible causes for this absence of MRI changes, and implications for the pathogenesis of the MRI changes in humans are reviewed.

Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice.

In vivo bioluminescence imaging is becoming increasingly popular. Quantification of bioluminescence signals requires knowledge of the variability and reproducibility of this technique. The objective of this study was to analyze the time course of luminescent signal emitted from firefly luciferase-expressing tumors in two locations, following luciferin injection and at different times after tumor cell implantation. Knowledge of the kinetics of the bioluminescent signals is required for the reliable quantification and comparison of signal during longitudinal studies. The kinetics of bioluminescence was evaluated in orthotopic and heterotopic brain tumors in mice using a human brain tumor cell line constitutively expressing luciferase. Tumor cells were implanted in the brains and flanks of the animals, and whole-body images revealing tumor location were obtained. Tumor burden was monitored over time by the quantitation of photon emission. The magnitude of bioluminescence measured in vivo varied with time after the injection of luciferin, as well as with dose, which necessitated that the comparison of the quantitative results take into consideration the time after injection. Heterotopic and orthotopic tumors exhibited significantly different time courses; however, time after implantation as characterized by kinetic studies performed on days 4 and 14 after cell implantation revealed no significant differences in orthotopic tumors. Future quantitative longitudinal studies must take into account the differences in the kinetics of different models.

Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA:colloidal gold by hep G2 cells.

Previously, we have identified a membrane-mediated uptake and translocation of 1,3,5(10)-estratrien-3, 17beta-diol 6-(O-carboxymethyl)oxime:(125)I-labeled BSA (E6(125)I-BSA) in vivo in immature female rat liver from the plasma membrane (P3 fraction) to the mitochondria and/or lysosomes (P2 fraction). To further investigate this unique effect, current experiments have involved the use of 1,3,5(10)-estratrien-3, 17beta-diol 17-hemisuccinate:( 125)I-BSA (E17(125)I-BSA) to demonstrate the presence of binding sites and translocation of the ligand in human hepatoblastoma (Hep G2) cells. In addition, an estrogen-BSA:colloidal gold conjugate, E17 BSA:Au, was used to directly visualize this uptake in Hep G2 cells. Hep G2 cells displayed high-affinity, stereospecific binding of E17(125)I-BSA. This same ligand was also translocated from the P3 fraction to the P2 fraction. In contrast, (125! )I-BSA was minimally removed from the culture medium. Electron micrographs of Hep G2 cells labeled with E17 BSA:Au demonstrated uptake of this ligand by clathrin-coated pits, indicative of receptor-mediated endocytosis. Furthermore, this ligand was also found in larger vesicles and multivesicular bodies, suggesting the involvement of the compartment of uncoupling of receptor and ligand (CURL), but never in the nucleus. As early as 30 min post-exposure, the ligand could be viewed in organelles, many of which had vesiculated interiors, resembling rounded, vesiculated mitochondria. Labeled BSA was detected mainly in the extracellular compartment, with few multivesicular bodies containing the labeled BSA. The translocation of E17 BSA:Au was virtually eliminated by 100 nM unlabeled E17 BSA or free 17beta-estradiol, but not 17alpha-E6 BSA, 17alpha-estradiol or P6 BSA, and also by exposure of the cells to reduced temperature. These experiments are the first t! o visually demonstrate membrane binding and specific uptake of an estrogen-containing ligand while allowing the intracellular structures responsible to be seen. Furthermore, they identify a potentially new pathway of receptor-mediated endocytosis; namely, the shuttling of estrogens to the mitochondria, in addition to the classical lysosomal pathway.

Evaluation of automated MR spectroscopy: application in Alzheimer disease.

In a trial involving 21 patients with dementia and 3 healthy control subjects, a comparison between the major cerebral metabolite ratios obtained with an established manually optimized proton MR spectroscopic examination and those obtained with an automated proton MR spectroscopic procedure shows that the two techniques provide very comparable results.

Scyllo-inositol depletion in hepatic encephalopathy.

Cerebral myo-inositol depletion is found in patients with hepatic encephalopathy and can be implicated in the pathogenesis of hepatic encephalopathy. We measured scyllo-inositol, a stereoisomer of myo-inositol, in brain extracts from patients dying in hepatic coma using HPLC and high resolution 1H MRS. The cerebral scyllo-inositol concentration, determined by both methods, in patients without hepatic encephalopathy was 0.41 +/- 0.11 mmol/kg wet weight. It decreased by 73% and 76%, respectively, as measured by HPLC and 1H MRS, in patients with hepatic encephalopathy. These findings indicate that myo-inositol depletion in patients with hepatic encephalopathy is not due to enhanced conversion of myo-inositol to scyllo-inositol or inhibition of myo-inositol transport by scyllo-inositol, but rather to the reduced biosynthesis or transport of both inositols.

Rapid uptake and binding of estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA by female rat liver.

To investigate potential membrane-mediated responses to estrogen, a membrane-impermeant, radioiodinated, steroid-BSA conjugate--estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA (17beta-E-6-125I-BSA)--or related steroid conjugates, or 125I-BSA was injected into female Sprague-Dawley rats, and tissues were collected at varying times postinjection. The liver, adrenal, and spleen displayed the most prominent uptake of 17beta-E-6-125I-BSA, reaching a maximum of 43 times blood levels in sonicated liver samples at 5 min postinjection, but no uptake of 125I-BSA. Isolation of liver membranes by differential centrifugation showed that over 50% of recovered radioactivity was in association with microsomes and plasmalemma (P3 fraction) at 30 sec postinjection. By 60 min postinjection, over 75% of recovered radioactivity was in association with mitochondrial and lysosomal membranes (P2 fraction), and less than 10% remained in the P3 fraction. In vitro competition assays demonstrated two binding sites in liver P3 fractions. The spleen and liver also showed saturable binding in vivo. These data suggest the presence of at least one membrane-binding protein for estrogen in liver, adrenal, and spleen. Initial studies of affinity-purified liver P3 fractions using ligand blots indicated the presence of two binding proteins. These potential membrane estrogen-binding proteins may be involved in a very rapid shuttling of estrogen from the plasmalemma to mitochondria and lysosomes.

Ester and ketone components of aggregation pheromone ofDrosophila hydei (Diptera: Drosophilidae).

Existence of an aggregation pheromone inDrosophila hydei was demonstrated by laboratory bioassay. The pheromone was produced by mature males, but both sexes responded. The nonpolar components consisted of three esters: the methyl, ethyl, and 1-methylethyl (isopropyl) esters of 2-methyl-(E)-2 butenoic (tiglic) acid, and two ketones: 2-tridecanone and 2-pentadecanone. The ketones and esters alone were only minimally active in the laboratory bioassay, but 2-tridecanone was highly synergistic with each of the esters, mixtures attracting 3-60 times more flies than the single components. 2-Pentadecanone was less active, but it did cause significant increases in activity when added to synthetic mixtures. The nonpolar portion of an extract of mature males and an equivalent mixture of the synthetic components were not significantly different in bioassay. Neither the esters nor the ketones were detected in sexually immature males or in females of any age. In extracts of mature males, ethyl tiglate was usually the most abundant ester component, with a mean of 8 ± 5 (SD) ng/male. The absolute and relative levels of the other esters were more variable. The mean level of methyl ketones in the extracts was 122 ± 106 (SD) ng/male, of which 85-93% was 2-tridecanone.

Abnormal cerebral metabolite concentrations in patients with probable Alzheimer disease.

To establish whether recently described abnormalities of peak ratios are the result of changes in metabolite concentrations, quantitative 1H magnetic resonance spectroscopy was performed in 10 patients with Alzheimer disease (AD) and seven normal elderly. CSF volumes, metabolite T1 and T2 relaxation rates, ratios and concentrations of N-acetyl residues, creatine, choline residues, myo-inositol, glutamine plus glutamate (Glx), and glucose were obtained. Difference spectroscopy and quantitative assays showed a 50% increase in myo-inositol (6.4-9.8 mM; P < 0.005) and a decrease in N-acetyl in occipital gray matter. A reduction in beta, gamma-Glx and a significant increase in intracerebral [glucose], greater than attributable to CSF, were defined. Choline concentration increased with age, but was not elevated above normal in AD patients. These findings indicate the need for quantitative 1H MRS to substantiate metabolite ratios. The increased myo-inositol concentration in AD is demonstrated by these studies.

Blood-brain phenylalanine relationships in persons with phenylketonuria.

OBJECTIVES: Clinicians caring for persons with phenylketonuria (PKU) have been perplexed by the occasional normal individual with the classical biochemical profile consistent with the diagnosis of PKU. Usually untreated subjects with the biochemical profile of blood phenylalanine (Phe) levels >1200 micromol/L are severely mentally retarded and may have neurological findings. Preliminary reports have recently appeared suggesting that low brain Phe levels, in comparison with elevated blood Phe levels, account for the occurrence of these occasional unaffected individuals with the biochemical profile consistent with PKU. METHOD: Magnetic resonance imaging/magnetic resonance spectroscopy was used to measure brain Phe content compared with simultaneously obtained blood Phe levels determined on the amino acid analyzer. This comparison was obtained in 5 normal non-PKU persons, 4 carriers of the gene causing PKU, and in 29 individuals with the proven form of the disorder. RESULTS: Blood-brain measurements in 5 normal persons ranged from.051 to.081 mmol/L, with a mean of.058 mmol/L. Their simultaneously measured brain levels of Phe ranged from.002 to.15 mmol/L, with a mean of.09 mmol/L. Similar measurements were obtained in 4 carriers of the gene causing PKU. Their blood levels varied between.068 and.109 mmol/L, with a mean of.091 mmol/L and simultaneously obtained brain levels of Phe varied between.06 and.21 mmol/L, with a mean of.11 mmol/L. Twenty subjects with a mean IQ of 104 exhibited a mean blood level of 1.428 mmol/L and a simultaneous mean brain level of.23 mmol/L, whereas 9 persons with a mean IQ of 98.7 exhibited a mean blood Phe level of 1.424 and a mean brain Phe level of.64 mmol/L. The correlation between blood and brain levels was not significant. CONCLUSION: In usual cases, intellectually normal persons who have never been treated but who have a biochemical profile consistent with classical PKU exhibit lower brain levels of Phe. Such individuals are exceptional and may not need the vigorous restriction of their blood Phe levels that is required by the majority of persons with PKU.

Danger of high-protein dietary supplements to persons with hyperphenylalaninaemia.

A 16-year-old adolescent with mild hyperphenylalaninaemia was given a high-protein 'body building' supplement twice daily, causing headaches, decreased school performance and mild depression. All symptoms disappeared after cessation of the supplement. The phenylalanine hydroxylase mutation H170D/IVS1nt5G>T was found to be responsive to tetrahydrobiopterin with significant decrease in blood phenylalanine concentration and increase in tyrosine blood content. A brain phenylalanine level of 0.5 mmol/L was initially documented, which decreased to the normal carrier range of 0.2 mmol/L within one month of discontinuance of the protein supplement. At present, the patient is on a normal diet without phenylalanine restriction.

Decrease in cerebral inositols in rats and humans.

Rats with portacaval shunts and humans with hepatic encephalopathy show severe myo-inositol depletion in the brain. The portacaval-shunted rat may therefore be a useful model for the investigation of neurochemical pathways containing myoinositol, which are modulated not only in hepatic encephalopathy but also in diabetes mellitus and Alzheimer's disease.

Neuropsychological outcome of subjects participating in the PKU adult collaborative study: a preliminary review.

Adult subjects with classical phenylketonuria (PKU) who were diagnosed and treated neonatally participated in this long-term follow-up study. Twenty-four subjects received neuropsychological (NP) assessment and a subset received magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) to identify: (1) pattern of cognitive dysfunction; (2) effect of high blood phenylalanine (Phe) level at time of cognitive testing; and (3) treatment variables that may be associated with cognitive difficulties in adulthood. All subjects had average IQ except one subject in the borderline range. Diet was initiated by the 15th day of life. All subjects except one were on diet until age 6 years (mean years of treatment = 15). Blood Phe levels at cognitive testing ranged from 157 to 1713 micromol/L (mean = 1038); 11 subjects had levels < 1000 micromol/L and 13 subjects had levels >1000 micromol/L. Results suggest that adults with early-treated PKU demonstrate specific cognitive deficits, a number of which are associated with the frontal and temporal area of the brain. Deficits were noted in several domains including executive functioning, attention, verbal memory, expressive naming and verbal fluency. Self-report measures of depression and anxiety were generally in the normal/mild range. The group with a Phe level > 1000 micromol/L scored lower than the group with Phe level < 1000 micromol/L on measures of focused attention, verbal fluency, reaction time, verbal recognition memory, visual memory and naming. Tests of cognitive functioning were often correlated with measures of treatment during childhood rather than with Phe level at the time of cognitive testing. Subjects with abnormal MRI scored significantly lower on two cognitive tests (Trails A and CVLT Recognition Memory). We found no significant correlation between current brain Phe level obtained through MRS (n = 10) and neuropsychological functioning. Future longitudinal investigation with a larger sample size will assist in clarifying the aetiology of neuropsychological deficits and association with treatment history.