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.

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.

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.

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.

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.

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.

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.

Probable Alzheimer disease: diagnosis with proton MR spectroscopy.

PURPOSE: To distinguish probable Alzheimer disease (AD) from other dementias (ODs) and normality in the elderly. MATERIALS AND METHODS: A double-blind trial of proton magnetic resonance (MR) spectroscopy was performed, principally in gray matter, in the occipital cortex of 114 patients with dementia (AD [n = 65], OD [n = 39], or frontal lobe dementia [FLD] [n = 10]), 98 patients without dementia, and 32 healthy control subjects. RESULTS: Reduced levels of N-acetylaspartate (NAA) (P < .0005) and increased levels of myo-inositol (MI) (P < .0005) characterize AD. Patients with OD had significantly reduced levels of NAA (P < .01) but normal levels of MI (P [vs AD] < .0005). When MI/NAA was used, AD was distinguished from normality with 83% sensitivity and 98% specificity. When MI/creatine was used, OD was distinguished from AD and FLD with a negative predictive rate of 80%, sensitivity of 82%, and specificity of 64%. CONCLUSION: Hydrogen-1 MR spectroscopy enables identification of mild to moderate AD with a specificity and sensitivity that suggest clinical utility.

Subclinical hepatic encephalopathy: proton MR spectroscopic abnormalities.

PURPOSE: To determine whether hydrogen-1 magnetic resonance (MR) spectroscopy of the brain allows detection of subclinical hepatic encephalopathy (SCHE). MATERIALS AND METHODS: In a double-blind study, overt hepatic encephalopathy (HE) and SCHE (defined with clinical and neuropsychiatric tests) were compared by means of H-1 MR spectroscopic criteria--reduction in cerebral myo-inositol (< 2 standard deviations [SDs] from normal) and choline (< 2 SDs from normal) with or without increased cerebral glutamine (> 1 SD from normal)--in 20 patients with cirrhosis. RESULTS: Concordance between MR spectroscopic and neuropsychiatric test results was 94% (kappa = 0.84). MR spectroscopy allowed diagnosis of SCHE in nine of nine patients (100%) and of HE in seven of eight (88%). Myo-inositol depletion alone had 80%-85% sensitivity for detection of HE and SCHE. CONCLUSION: H-1 MR spectroscopy allows accurate diagnosis of SCHE, and the results suggest an important role for myo-inositol in psychomotor and visuopractic functions.

Automated single-voxel proton MRS: technical development and multisite verification.

To improve clinical utility, an integrated method has been developed to automatically acquire and process single-voxel in vivo proton spectra on a 1.5 T clinical scanner. This method includes automated adjustment of linear shims using a very rapid modified simplex method, automated water suppression, and applies a water referencing scheme to correct for phase and residual eddy current effects. No operator intervention is required for the acquisition and processing of these pure-absorption spectra. This method was tested in a preliminary multisite trial to determine intersite and intrasite variability of metabolite ratio measurements. In a sample of over 100 examinations, the standard deviation of the ratios NAA:Cr, Cho:Cr, and ml:Cr were found to be under 15% when using this method, a substantially narrower range than has been found in studies relying on manual adjustment of the instrument and/or manual processing. This result indicates that automated setting of acquisition and processing parameters is of critical importance in the clinical application of in vivo spectroscopy.

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.

Alzheimer disease: depiction of increased cerebral myo-inositol with proton MR spectroscopy.

To define altered metabolites in the brain of patients with probable Alzheimer disease (AD) of two brain regions, localized in vivo hydrogen-1 magnetic resonance (MR) spectroscopy was performed with a short echo time (30 msec) in 11 elderly patients and 10 healthy age-matched subjects. The patients had mild to moderate dementia, assessed with standard neuropsychological tests. Two abnormalities in the patients' cerebral cortex were defined: When compared with healthy subjects, the patients showed a 22% increase (P = .005) (approximately equal to 1.5 mmol/kg) in myo-inositol (MI) and an 11% decrease (P = .005) in residues of N-acetyl (NA), a putative neuronal marker. The elevation of MI in patients with mild to moderate AD suggests that abnormalities in the inositol polyphosphate messenger pathway occur early in the natural history of AD. The combination of high MI and low NA at examination with H-1 MR spectroscopy shows promise as an early diagnostic test for AD.

Brain phenylalanine concentration in the management of adults with phenylketonuria.

Diagnosis by newborn screening and the implementation of a phenylalanine-restricted diet have resulted in normal neurological development in approximately 10,000 persons with phenylketonuria (PKU) in the United States. While it is accepted that a phenylalanine-restricted diet is necessary in childhood, the recommended concentration of phenylalanine in the blood varies. Clinicians now must make recommendations for adults with PKU who probably tolerate higher levels of phenylalanine than children. This factor, quality of life issues, the expense of the diet, and varying genetic and socioeconomic backgrounds, make the choice of dietary recommendations difficult. Molecular analysis of the mutations in PKU has provided insight but has not resulted in clear recommendations for phenylalanine concentration in the blood. Magnetic resonance imaging has provided the recognition that white-matter changes are present in PKU. However, owing to poor correlation of white-matter changes with clinical factors, analysis of white-matter changes has not proved useful. We hypothesize that measurement of brain phenylalanine directly will aid in clinical decision making. Twenty-one subjects with PKU had blood and brain phenylalanine measured simultaneously. Fifteen were randomly selected, 2 were examined for clinical reasons and 4 exceptional patients were chosen because they had maintained high IQs, despite having high historic blood concentrations and having been off the diet for at least 10 years. The correlation of blood and brain phenylalanine is in general poor. However, the four exceptional patients all had relatively low concentrations of phenylalanine in their brains compared to their blood. We suggest that their good clinical status, despite high historic blood levels, is due to their comparatively low brain levels of phenylalanine. We further suggest that measurement of brain phenylalanine concentration is useful in the management of PKU patients.

Added value of automated clinical proton MR spectroscopy of the brain.

OBJECTIVE: A trial was conducted to establish the added diagnostic value of an automated proton MR spectroscopy (MRS) examination (PROBE). MATERIALS AND METHODS: The PROBE and MRS were compared for metabolite ratios of normal controls and 21 patients. In addition, PROBE was performed in either the occipital cortex (gray matter) or the parietal cortex (white matter) or, more rarely, within the confines of a focal lesion identified on MRI, using a GE Signa 1.5 T whole-body scanner, in 112 patients undergoing routine brain MRI. The trial was conducted in three different MR centers to establish percentage of positive findings with MRI vs. MRI plus MRS. RESULTS: Cerebral metabolite ratios (N-acetylaspartate/creatine, choline/creatine, myo-inositol/creatine) obtained by PROBE and MRS were similar. Metabolite profiles in dementia, head trauma, herpes encephalitis, hepatic and hypoxic encephalopathy, stroke, and tumor were identified using PROBE. The PROBE technique increased the number of positive findings ("added value") achieved by MRI; the added value was 28, 21, and 93% for the three trial sites. CONCLUSION: With only minor variations, PROBE reproduces the cerebral metabolite patterns obtained with MRS. It significantly increases the diagnostic yield of routine neuroimaging and might be incorporated as a standard sequence in a cost-effective manner.