Novel Development of Magnetic Resonance Imaging to Quantify the Structural Anatomic Growth of Diverse Organs in Adult and Mutant Zebrafish.

Zebrafish (Danio rerio) is a widely used vertebrate animal for modeling genetic diseases by targeted editing strategies followed by gross phenotypic and biomarker characterization. While larval transparency permits microscopic detection of anatomical defects, histological adult screening for organ-level defects remains invasive, tedious, inefficient, and subject to technical artifact. Here, we describe a noninvasive magnetic resonance imaging (MRI) approach to systematically screen adult zebrafish for anatomical growth defects. An anatomical atlas of wild-type (WT) zebrafish at 5-31 months post-fertilization was created by ex vivo MRI with a 9.4 T magnet. Volumetric growth over time was measured of animals and major organs, including the brain, spinal cord, heart, eyes, optic nerve, ear, liver, kidneys, and swim bladder. Subsequently, surf1-/-, fbxl4-/-, and opa1+/- mitochondrial disease mutant adult zebrafish were quantitatively studied to compare organ volumes with age-matched WT zebrafish. Results demonstrated that MRI enabled noninvasive, high-resolution, rapid screening of mutant adult zebrafish for overall and organ-specific growth abnormalities. Detailed volumetric analyses of three mitochondrial disease mutants delineated specific organ differences, including significantly increased brain growth in surf1-/- and opa1+/-, and marginally significant decreased heart and spinal cord volumes in surf1-/- mutants. This is interesting as we know neurological involvement can be seen in SURF1-/- patients with ataxia, dystonia, and lesions in basal ganglia, as well as in OPA1+/- patients with spasticity, ataxia, and hyperreflexia indicative of neuropathology. Similarly, cardiomyopathy is a known sequelae of cardiac pathology in patients with SURF1-/--related disease. Future studies will define MRI signaling patterns of organ dysfunction to further delineate specific pathology.

Deficits in Seizure Threshold and Other Behaviors in Adult Mice without Gross Neuroanatomic Injury after Late Gestation Transient Prenatal Hypoxia.

Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain.

Imaging of a high concentration of iron labeled cells with positive contrast in a rat knee.

PURPOSE: The sweep imaging with Fourier transformation (SWIFT) imaging technique has been shown to provide positive contrast from diluted cell suspensions labeled with super-paramagnetic iron oxide (SPIO) in a tissue, as an alternative to T2*-weighted imaging. Here we demonstrate a variation of the SWIFT technique that yields a hyperintense signal from a concentrated cell suspension. The proposed technique provides minimal background signal from host tissue and facilitates visualization of injected cells. METHODS: The proton resonance frequency and linewidth were determined for SPIO solutions of different concentrations. The original SWIFT sequence was modified and a dual saturation Gaussian shape RF pulse with ~200 Hz bandwidth was incorporated into the acquisition protocol to suppress host tissue and fat signals. This modification of the original acquisition protocol permits the detection of a hyperintense signal from grafted cells with minimal background signal from the host tissue. RESULTS: SPIO particles not only induce broadening of NMR line-width but also an initiate proton resonance frequency shift. This shift is linearly proportional to the concentration of the iron oxide particles and induced by the bulk magnetic susceptibility of SPIOs. The shift of the resonance frequency of iron labeled cells allowed us effectively suppress the host tissues with saturation RF pulse to improve MRI detection of grafted cells. CONCLUSIONS: Iron oxide particles increase the resonance frequency of water proton signal. This shift permitted us to add the tissue/fat saturation RF pulse into the original SWIFT acquisition protocol and detect distinct hyperintense signals from grafted cells with minimal background signal from the host tissue.

Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration.

Human diseases are often caused by loss of somatic cells that are incapable of re-entering the cell cycle for regenerative repair. Here, we report a combination of cell-cycle regulators that induce stable cytokinesis in adult post-mitotic cells. We screened cell-cycle regulators expressed in proliferating fetal cardiomyocytes and found that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome-mediated degradation of the protein products. In vivo lineage tracing revealed that 15%-20% of adult cardiomyocytes expressing the four factors underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Chemical inhibition of Tgf-ß and Wee1 made CDK1 and cyclin B dispensable. These findings reveal a discrete combination of genes that can efficiently unlock the proliferative potential in cells that have terminally exited the cell cycle.

Antisecretory Factor-Mediated Inhibition of Cell Volume Dynamics Produces Antitumor Activity in Glioblastoma.

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AF's ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777-90. ©2018 AACR.

Modic type 1 change is an autoimmune response that requires a proinflammatory milieu provided by the 'Modic disc'.

BACKGROUND CONTEXT: Modic changes (MCs) are magnetic resonance imaging (MRI) evidence of inflammatory and fibrotic vertebral bone marrow lesions that associate with adjacent disc degeneration and end plate damage. Although MC etiology is uncertain, historical data suggest a linkage to an autoimmune response of bone marrow triggered by the nucleus pulposus (NP). PURPOSE: The aim of this study was to test whether bone marrow has an autoimmune response to NP cells that is amplified by an inflammatory milieu and ultimately leads to MC development in vivo. We hypothesized that an inflammatory co-stimulus is required for bone marrow/NP crosstalk to stimulate MC. STUDY DESIGN: This is an in-vitro cell co-culture study plus in-vivo experiments in rat caudal vertebrae. METHODS: In in-vitro study, bone marrow mononuclear cells (BMNCs) and NP cells (NPCs) from rats were co-cultured with and without interleukin (IL)-1α stimulation. Cell viability (n=3) of BMNCs and NPCs and gene expression (n=7) were analyzed. In in-vivo study, proinflammatory lipopolysaccharide (LPS) and control disc nucleus surrogates (NP micromass pellets) were generated in vitro from rat NPCs and implanted into rat tail vertebrae, and the response was compared with sham surgery (n=12 each). Tissue changes were investigated with T1w and T2w MRI (7T), histology, and immunohistochemistry (tumor necrosis factor, CD3) 1 (n=6) and 2 weeks (n=6) after implantation. RESULTS: BMNC/NPC co-culture significantly increased lymphocyte viability (42%-69%, p<.05) and reduced NPC viability (96%-88%, p<.001), indicating immunogenicity of NPC. However, IL-1α was required to cause significant transcriptional upregulation of IL-1, IL-6, IL-10, and tropomyosin receptor kinase A. Therefore, an inflammatory activation is required to amplify the immune response. Immunogenicity of the NP was corroborated in vivo by CD3 cell accumulation around LPS and control disc surrogates at Day 7. However, only the LPS disc surrogate group demonstrated infiltration of CD3 cells at Day 14. Furthermore, end plate defects (p<.05, LPS: n=4/6, Ctrl: n=0/6, sham: n=0/6) and MC1-like MRI changes (T2w hyperintensity, p<.05) were only seen with LPS disc surrogates. CONCLUSIONS: NPCs are immunogenic but cannot trigger MC without an additional proinflammatory stimulus. Our data suggest that MC requires end plate defects that allow marrow/NPC co-mingling plus an adjacent inflammatory "MC disc" that can amplify the immune response.

Quantification of Propionic Acid in the Bovine Spinal Disk After Infection of the Tissue With Propionibacteria acnes Bacteria.

STUDY DESIGN: Research. OBJECTIVE: The goal of this study was to investigate whether Propionibacteria acnes infection of the intervertebral disc can be detected noninvasively by nuclear magnetic resonance (NMR) spectroscopy. SUMMARY OF BACKGROUND DATA: Microbiological studies of surgical samples suggest that a significant subpopulation of back pain patients may have occult disc infection with P. acnes bacteria. This hypothesis is further supported by a double-blind clinical trial showing that back pain patients with Modic type 1 changes may respond to antibiotic treatment. Because significant side effects are associated with antibiotic treatment, there is a need for a noninvasive method to detect whether specific discs in back pain patients are infected with P acnes bacteria. METHODS: P. acnes bacteria were obtained from human patients. NMR detection of a propionic acid (PA) in the bacteria extracts was conducted on 500 MHz high-resolution spectrometer, whereas in vivo NMR spectroscopy of an isolated bovine disk tissue infected with P. acnes was conducted on 7 T magnetic resonance imaging scanner. RESULTS: NMR spectra of P. acnes metabolites revealed a distinct NMR signal with identical chemical shits (1.05 and 2.18 ppm) as PA (a primary P. acne metabolite). The 1.05 ppm signal does not overlap with other bacteria metabolites, and its intensity increases linearly with P. acnes concentration. Bovine disks injected with P. acnes bacteria revealed a very distinct NMR signal at 1.05 ppm, which linearly increased with P. acnes concentration. CONCLUSION: The 1.05 ppm NMR signal from PA can be used as a marker of P. acnes infection of discs. This signal does not overlap with other disc metabolites and linearly depends on P. acnes concentration. Consequently, NMR spectroscopy may provide a noninvasive method to detect disc infection in the clinical setting. LEVEL OF EVIDENCE: N/A.

Heterogeneous drug penetrance of veliparib and carboplatin measured in triple negative breast tumors.

BACKGROUND: Poly(ADP-ribose) polymerase inhibitors (PARPi), coupled to a DNA damaging agent is a promising approach to treating triple negative breast cancer (TNBC). However, not all patients respond; we hypothesize that non-response in some patients may be due to insufficient drug penetration. As a first step to testing this hypothesis, we quantified and visualized veliparib and carboplatin penetration in mouse xenograft TNBCs and patient blood samples. METHODS: MDA-MB-231, HCC70 or MDA-MB-436 human TNBC cells were implanted in 41 beige SCID mice. Low dose (20 mg/kg) or high dose (60 mg/kg) veliparib was given three times daily for three days, with carboplatin (60 mg/kg) administered twice. In addition, blood samples were analyzed from 19 patients from a phase 1 study of carboplatin + PARPi talazoparib. Veliparib and carboplatin was quantified using liquid chromatography-mass spectrometry (LC-MS). Veliparib tissue penetration was visualized using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) and platinum adducts (covalent nuclear DNA-binding) were quantified using inductively coupled plasma-mass spectrometry (ICP-MS). Pharmacokinetic modeling and Pearson's correlation were used to explore associations between concentrations in plasma, tumor cells and peripheral blood mononuclear cells (PBMCs). RESULTS: Veliparib penetration in xenograft tumors was highly heterogeneous between and within tumors. Only 35% (CI 95% 26-44%), 74% (40-97%) and 46% (9-37%) of veliparib observed in plasma penetrated into MDA-MB-231, HCC70 and MDA-MB-436 cell-based xenografts, respectively. Within tumors, penetration heterogeneity was larger with the 60 mg/kg compared to the 20 mg/kg dose (RSD 155% versus 255%, P = 0.001). These tumor concentrations were predicted similar to clinical dosing levels, but predicted tumor concentrations were below half maximal concentration values as threshold of response. Xenograft veliparib concentrations correlated positively with platinum adduct formation (R 2 = 0.657), but no PARPi-platinum interaction was observed in patients' PBMCs. Platinum adduct formation was significantly higher in five gBRCA carriers (ratio of platinum in DNA in PBMCs/plasma 0.64% (IQR 0.60-1.16%) compared to nine non-carriers (ratio 0.29% (IQR 0.21-0.66%, P < 0.0001). CONCLUSIONS: PARPi/platinum tumor penetration can be measured by MALDI-MSI and ICP-MS in PBMCs and fresh frozen, OCT embedded core needle biopsies. Large variability in platinum adduct formation and spatial heterogeneity in veliparib distribution may lead to insufficient drug exposure in select cell populations.

Positive contrast from cells labeled with iron oxide nanoparticles: Quantitation of imaging data.

PURPOSE: Conventional T2 -weighted MRI produces a hypointense signal from iron-labeled cells, which renders quantification unfeasible. We tested a SWeep Imaging with Fourier Transformation (SWIFT) MRI pulse sequence to generate a quantifiable hyperintense signal from iron-labeled cells. METHODS: Mesenchymal stem cells (MSCs) were labeled with different concentrations of iron oxide particles and examined for cell viability, proliferation, and differentiation. The SWIFT sequence was optimized to detect and quantify the amount of iron in the muscle tissue after injection of iron oxide solution and iron-labeled MSCs. RESULTS: The incubation of MSCs with iron oxide and low concentration of poly-L-lysine mixture resulted in an internalization of up to 22 pg of iron per cell with no adverse effect on MSCs. Phantom experiments showed a dependence of SWIFT signal intensity on the excitation flip angle. The hyperintense signal from iron-labeled cells or solutions was detected, and an amount of the iron oxide in the tissue was quantified with the variable flip angle method. CONCLUSIONS: The SWIFT sequence can produce a quantifiable hyperintense MRI signal from iron-labeled cells. The graft of 18 x 106 cells was detectable for 19 days after injection and the amount of iron was quantifiable. The proposed protocol simplifies the detection and provides a means to quantify cell numbers. Magn Reson Med 78:1900-1910, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming.

BACKGROUND: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. METHODS: We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. RESULTS: We found that a combination of the transforming growth factor-ß inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-ß and WNT inhibition and was achieved most efficiently with GMT plus myocardin. CONCLUSIONS: Transforming growth factor-ß and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.

Propionibacterium acnes infected intervertebral discs cause vertebral bone marrow lesions consistent with Modic changes.

Modic type I change (MC1) are vertebral bone marrow lesions adjacent to degenerated discs that are specific for discogenic low back pain. The etiopathogenesis is unknown, but occult discitis, in particular with Propionibacteria acnes (P. acnes), has been suggested as a possible etiology. If true, antibiotic therapy should be considered for patients with MC1. However, this hypothesis is controversial. While some studies report up to 40% infection rate in herniated discs, others fail to detect infected discs and attribute reports of positive cultures to contamination during sampling procedure. Irrespective of the clinical controversy, whether it is biologically plausible for P. acnes to cause MC1 has never been investigated. Therefore, the objective of this study was to test if P. acnes can proliferate within discs and cause reactive changes in the adjacent bone marrow. P. acnes was aseptically isolated from a symptomatic human L4/5 disc with MC1 and injected into rat tail discs. We demonstrate proliferation of P. acnes and up-regulation of IL-1 and IL-6 within three days of inoculation. At day-7, disc degeneration was apparent along with fibrotic endplate erosion. TNF-α immunoreactivity was enhanced within the effected endplates along with cellular infiltrates. The bone marrow appeared normal. At day-14, endplates and trabecular bone close to the disc were almost completely resorbed and fibrotic tissue extended into the bone marrow. T-cells and TNF-α immunoreactivity were identified at the disc/marrow junction. On MRI, bone marrow showed MC1-like changes. In conclusion, P. acnes proliferate within the disc, induce degeneration, and cause MC1-like changes in the adjacent bone marrow. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1447-1455, 2016.

Kartogenin treatment prevented joint degeneration in a rodent model of osteoarthritis: A pilot study.

Osteoarthritis (OA) is a major degenerative joint disease characterized by progressive loss of articular cartilage, synovitis, subchondral bone changes, and osteophyte formation. Currently there is no treatment for OA except temporary pain relief and end-stage joint replacement surgery. We performed a pilot study to determine the effect of kartogenin (KGN, a small molecule) on both cartilage and subchondral bone in a rat model of OA using multimodal imaging techniques. OA was induced in rats (OA and KGN treatment group) by anterior cruciate ligament transection (ACLT) surgery in the right knee joint. Sham surgery was performed on the right knee joint of control group rats. KGN group rats received weekly intra-articular injection of 125 µM KGN 1 week after surgery until week 12. All rats underwent in vivo magnetic resonance imaging (MRI) at 3, 6, and 12 weeks after surgery. Quantitative MR relaxation measures (T1ρ and T2 ) were determined to evaluate changes in articular cartilage. Cartilage and bone turnover markers (COMP and CTX-I) were determined at baseline, 3, 6, and 12 weeks. Animals were sacrificed at week 12 and the knee joints were removed for micro-computed tomography (micro-CT) and histology. KGN treatment significantly lowered the T1ρ and T2 relaxation times indicating decreased cartilage degradation. KGN treatment significantly decreased COMP and CTX-I levels indicating decreased cartilage and bone turnover rate. KGN treatment also prevented subchondral bone changes in the ACLT rat model of OA. Thus, kartogenin is a potential drug to prevent joint deterioration in post-traumatic OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1780-1789, 2016.

Magnetic resonance spectroscopy of the occipital cortex and the cerebellar vermis distinguishes individual cats affected with alpha-mannosidosis from normal cats.

A genetic deficiency of lysosomal alpha-mannosidase causes the lysosomal storage disease alpha-mannosidosis (AMD), in which oligosaccharide accumulation occurs in neurons and glia. The purpose of this study was to evaluate the role of magnetic resonance spectroscopy (MRS) in detecting the oligosaccharide accumulation in AMD. Five cats with AMD and eight age-matched normal cats underwent in vivo MRS studies with a single voxel short echo time (20 ms) STEAM spectroscopy sequence on a 4.7T magnet. Two voxels were studied in each cat, from the cerebellar vermis and the occipital cortex. Metabolites of brain samples from these regions were extracted with perchloric acid and analyzed by high resolution NMR spectroscopy. A significantly elevated unresolved resonance signal between 3.4 and 4. ppm was observed in the cerebellar vermis and occipital cortex of all AMD cats, which was absent in normal cats. This resonance was shown to be from carbohydrate moieties by high resolution NMR of tissue extracts. Resonances from the Glc-NAc group (1.8-2.2 ppm) along with anomeric proton signals (4.6-5.4 ppm) from undigested oligosaccharides were also observed in the extract spectra from AMD cats. This MRS spectral pattern may be a useful biomarker for AMD diagnosis as well as for assessing responses to therapy.

Transplantation and magnetic resonance imaging of canine neural progenitor cell grafts in the postnatal dog brain.

Cellular transplantation in the form of bone marrow has been one of the primary treatments of many lysosomal storage diseases (LSDs). Although bone marrow transplantation can help central nervous system manifestations in some cases, it has little impact in many LSD patients. Canine models of neurogenetic LSDs provide the opportunity for modeling central nervous system transplantation strategies in brains that more closely approximate the size and architectural complexity of the brains of children. Canine olfactory bulb-derived neural progenitor cells (NPCs) isolated from dog brains were expanded ex vivo and implanted into the caudate nucleus/thalamus or cortex of allogeneic dogs. Canine olfactory bulb-derived NPCs labeled with micron-sized superparamagnetic iron oxide particles were detected by magnetic resonance imaging both in vivo and postmortem. Grafts expressed markers of NPCs (i.e. nestin and glial fibrillary acidic protein), but not the neuronal markers Map2ab or beta-tubulin III. The NPCs were from dogs with the LSD mucopolysaccharidosis VII, which is caused by a deficiency of beta-glucuronidase. When mucopolysaccharidosis VII canine olfactory bulb-NPCs that were genetically corrected with a lentivirus vector ex vivo were transplanted into mucopolysaccharidosis VII recipient brains, they were detected histologically by beta-glucuronidase expression in areas identified by antemortem magnetic resonance imaging tracking. These results demonstrate the potential for ex vivo stem cell-based gene therapy and noninvasive tracking of therapeutic grafts in vivo.

Magnetic resonance imaging detects differences in migration between primary and immortalized neural stem cells.

RATIONALE AND OBJECTIVES: The study was performed to evaluate the effect of magnetic resonance imaging (MRI) contrast agent (super paramagnetic iron oxide [SPIO]) on differentiation and migration of primary murine neural stem cells (NSCs) in comparison to a neural stem cell line (C17.2). Because detection of labeled cells depends on the concentration of SPIO particles per imaging voxel, the study was performed at various concentrations of SPIO particles to determine the concentration that could be used for in vivo detection of small clusters of grafted cells. MATERIALS AND METHODS: Murine primary NSCs or C17.2 cells were labeled with different concentrations of SPIO particles (0, 25, 100, and 250 microg Fe/mL) and in vitro assays were performed to assess cell differentiation. In vivo MRI was performed 7 weeks after neonatal transplantation of labeled cells to evaluate the difference in migration capability of the two cell populations. RESULTS: Both the primary NSCs and the C17.2 cells differentiated to similar number of neurons (Map2ab-positive cells). Similar patterns of engraftment of C17.2 cells were seen in transplanted mice regardless of the SPIO concentration used. In vivo MRI detection of grafted primary and C17.2 cells was only possible when cells were incubated with 100 microg/mL or higher concentration of SPIO. Extensive migration of C17.2 cells throughout the brain was observed, whereas the migration of the primary NSCs was more restricted. CONCLUSIONS: Engraftment of primary NSCs can be detected noninvasively by in vivo MRI, and the presence of SPIO particles do not affect the viability, differentiation, or engraftment pattern of the donor cells.

The therapeutic mechanisms of ranpirnase-induced enhancement of radiation response on A549 human lung cancer.

AIM: The goal of this study was to investigate the therapeutic potential of combining radiation therapy and cytotoxic RNase, ranpirnase (ONCONASE; ONC), in human lung tumor models in vitro and in vivo. As translational implications, the non-invasive monitoring response to individual therapy with ONC was also investigated to determine the underlying therapeutic mechanisms. MATERIALS AND METHODS: A clonogenic survival assay was used to measure the effect of ONC and radiation on A549 human non-small cell lung carcinoma (NSCLC) cells. H&E staining, TUNEL staining and caspase-3-antibody labeling were used for in vivo analysis of apoptosis. A growth-delay assay was applied to detect the therapeutic potential of ONC as a radiation sensitizer in vivo. ONC-induced changes in blood flow and biochemical metabolites were measured by various noninvasive dynamic contrast enhanced magnetic resonance imaging (DCE MRI), non-localized 1H magnetic resonance spectroscopy (MRS), and near-infrared spectroscopy (NIRS) methods. RESULTS: ONC at 5-10 microg/ml sensitized the radiation response of A549 tumor cells in vitro. Remarkable increases in ONC-induced apoptosis in vivo were observed in caspase-3 antibody labeling and TUNEL staining assays. ONC significantly increased the radiation-induced tumor growth delay of A549 tumors. It was observed, when using a DCE MRI method, that there were significant increases in K(trans) values at the rim of tumor regions at 1.5 h post-injection of ONC. When using non-localized 1H MRS, an approximately 20% decrease in lactate levels with ONC was found. CONCLUSION: ONC may be a new and promising drug in the treatment of NSCLC as a radiation therapy enhancer.

Structure-specific patterns of neural stem cell engraftment after transplantation in the adult mouse brain.

Transplantation of neural stem cells (NSCs) may be useful for delivering exogenous gene products to the diseased CNS. When NSCs are transplanted into the developing mouse brain, they can migrate extensively and differentiate into cells appropriate to the sites of engraftment, in response to the normal signals directing endogenous cells to their appropriate fates. Much of the prior work on NSC migration in the adult brain has examined directed migration within or toward focal areas of injury such as ischemia, brain tumors, or 6-hydroxydopamine (6-OHDA) lesions. However, treatment of many genetic disorders that affect the CNS will require widespread dissemination of the donor cells in the postnatal brain, because the lesions are typically distributed globally. We therefore tested the ability of NSCs to migrate in the unlesioned adult mouse brain after stereotaxic transplantation into several structures including the cortex and hippocampus. NSC engraftment was monitored in live animals by magnetic resonance imaging (MRI) after superparamagnetic iron oxide (SPIO) labeling of cells. Histological studies demonstrated that the cells engrafted in significantly different patterns within different regions of the brain. In the cerebral cortex, donor cells migrated in all directions from the injection site. The cells maintained an immature phenotype and cortical migration was enhanced by trypsin treatment of the cells, indicating a role for cell surface proteins. In the hippocampus, overall cell survival and migration were lower but there was evidence of neuronal differentiation. In the thalamus, the transplanted cells remained in a consolidated mass at the site of injection. These variations in pattern of engraftment should be taken into account when designing treatment approaches in nonlesion models of neurologic disease.

EPR characterization of ubisemiquinones and iron-sulfur cluster N2, central components of the energy coupling in the NADH-ubiquinone oxidoreductase (complex I) in situ.

The proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron-sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q1) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQNf (fast-relaxing component), SQNS (slow-relaxing), and SQNx (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQNf, because of its high sensitivities to DeltamicroH+ and to specific complex I inhibitors (rotenone and piericidin A) in a unique manner. Rotenone inhibits the forward electron transfer reaction more strongly than the reverse reaction, while piericidine A inhibits both reactions with a similar potency. Rotenone quenched the SQNf signal at a much lower concentration than that required to quench the slower relaxing components (SQNs and SQNx). A close correlation was shown between the line shape alteration of the g// = 2.05 signal of the cluster N2 and the quenching of the SQNf signal, using two different experimental approaches: (1) changing the DeltamicroH+ poise by the oligomycin titration which decreases proton leak across the SMP membrane; (2) inhibiting the reverse electron transfer with different concentrations of rotenone. These new experimental results further strengthen our earlier proposal that a direct spin-coupling occurs between SQNf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex .