Magnetic labeling of primary murine monocytes using very small superparamagnetic iron oxide nanoparticles.

Due to their very small size, nanoparticles can interact with all cells in the central nervous system. One of the most promising nanoparticle subgroups are very small superparamagnetic iron oxide nanoparticles (VSOP) that are citrate coated for electrostatic stabilization. To determine their influence on murine blood-derived monocytes, which easily enter the injured central nervous system, we applied VSOP and carboxydextran-coated superparamagnetic iron oxide nanoparticles (Resovist). We assessed their impact on the viability, cytokine, and chemokine secretion, as well as iron uptake of murine blood-derived monocytes. We found that (1) the monocytes accumulated VSOP and Resovist, (2) this uptake seemed to be nanoparticle- and time-dependent, (3) the decrease of monocytes viability was treatment-related, (4) VSOP and Resovist incubation did not alter cytokine homeostasis, and (5) overall a 6-hour treatment with 0.75 mM VSOP-R1 was probably sufficient to effectively label monocytes for future experiments. Since homeostasis is not altered, it is safe to label blood-derived monocles with VSOP. VSOP labeled monocytes can be used to study injured central nervous system sites further, for example with drug-carrying VSOP.

Biocompatibility of very small superparamagnetic iron oxide nanoparticles in murine organotypic hippocampal slice cultures and the role of microglia.

Superparamagnetic iron oxide nanoparticles (SPIO) are applied as contrast media for magnetic resonance imaging (MRI) and treatment of neurologic diseases despite the fact that important information concerning their local interactions is still lacking. Due to their small size, SPIO have great potential for magnetically labeling different cell populations, facilitating their MRI tracking in vivo. Before SPIO are applied, however, their effect on cell viability and tissue homoeostasis should be studied thoroughly. We have previously published data showing how citrate-coated very small superparamagnetic iron oxide particles (VSOP) affect primary microglia and neuron cell cultures as well as neuron-glia cocultures. To extend our knowledge of VSOP interactions on the three-dimensional multicellular level, we further examined the influence of two types of coated VSOP (R1 and R2) on murine organotypic hippocampal slice cultures. Our data show that 1) VSOP can penetrate deep tissue layers, 2) long-term VSOP-R2 treatment alters cell viability within the dentate gyrus, 3) during short-term incubation VSOP-R1 and VSOP-R2 comparably modify hippocampal cell viability, 4) VSOP treatment does not affect cytokine homeostasis, 5) microglial depletion decreases VSOP uptake, and 6) microglial depletion plus VSOP treatment increases hippocampal cell death during short-term incubation. These results are in line with our previous findings in cell coculture experiments regarding microglial protection of neurite branching. Thus, we have not only clarified the interaction between VSOP, slice culture, and microglia to a degree but also demonstrated that our model is a promising approach for screening nanoparticles to exclude potential cytotoxic effects.

Studying Axonal Outgrowth and Regeneration of the Corticospinal Tract in Organotypic Slice Cultures.

Studies of axonal outgrowth and regeneration after spinal cord injury are hampered by the complexity of the events involved. Here, we present a simple and improved in vitro approach to investigate outgrowth, regeneration of the corticospinal tract, and intrinsic parenchymal responses. We prepared organotypic co-cultures using explants from the motor cortex of postnatal donor mice ubiquitously expressing green fluorescent protein and cervical spinal cord from wild type pups of the same age. Our data show that: a) motor-cortical outgrowth is already detectable after 1 d in culture and is source specific; b) treatment with neurotrophin-3 and C3 transferase from Clostridium botulinum significantly enhances axonal outgrowth during the course of cultivation; c) outgrowing axons form synaptic connections, as demonstrated by immunohistochemistry and calcium imaging; and d) migrating cells of motor-cortical origin can be reliably identified without previous tracing and are mostly neural precursors that survive and mature in the spinal cord parenchyma. Thus, our model is suitable for screening for candidate substances that enhance outgrowth and regeneration of the corticospinal tract and for studying the role of endogenous neural precursors after lesion induction.

New findings about iron oxide nanoparticles and their different effects on murine primary brain cells.

The physicochemical properties of superparamagnetic iron oxide nanoparticles (SPIOs) enable their application in the diagnostics and therapy of central nervous system diseases. However, since crucial information regarding side effects of particle-cell interactions within the central nervous system is still lacking, we investigated the influence of novel very small iron oxide particles or the clinically approved ferucarbotran or ferumoxytol on the vitality and morphology of brain cells. We exposed primary cell cultures of microglia and hippocampal neurons, as well as neuron-glia cocultures to varying concentrations of SPIOs for 6 and/or 24 hours, respectively. Here, we show that SPIO accumulation by microglia and subsequent morphological alterations strongly depend on the respective nanoparticle type. Microglial viability was severely compromised by high SPIO concentrations, except in the case of ferumoxytol. While ferumoxytol did not cause immediate microglial death, it induced severe morphological alterations and increased degeneration of primary neurons. Additionally, primary neurons clearly degenerated after very small iron oxide particle and ferucarbotran exposure. In neuron-glia cocultures, SPIOs rather stimulated the outgrowth of neuronal processes in a concentration- and particle-dependent manner. We conclude that the influence of SPIOs on brain cells not only depends on the particle type but also on the physiological system they are applied to.

Clonal analysis in recurrent astrocytic, oligoastrocytic and oligodendroglial tumors implicates IDH1- mutation as common tumor initiating event.

BACKGROUND: To investigate the dynamics of inter- and intratumoral molecular alterations during tumor progression in recurrent gliomas. METHODOLOGY/PRINCIPAL FINDINGS: To address intertumoral heterogeneity we investigated non-microdissected tumor tissue of 106 gliomas representing 51 recurrent tumors. To address intratumoral heterogeneity a set of 16 gliomas representing 7 tumor pairs with at least one recurrence, and 4 single mixed gliomas were investigated by microdissection of distinct oligodendroglial and astrocytic tumor components. All tumors and tumor components were analyzed for allelic loss of 1p/19q (LOH 1p/19q), for TP53- mutations and for R132 mutations in the IDH1 gene. The investigation of non-microdissected tumor tissue revealed clonality in 75% (38/51). Aberrant molecular alterations upon recurrence were detected in 25% (13/51). 64% (9/14) of these were novel and associated with tumor progression. Loss of previously detected alterations was observed in 36% (5/14). One tumor pair (1/14; 7%) was significant for both. Intratumoral clonality was detected in 57% (4/7) of the microdissected tumor pairs and in 75% (3/4) of single microdissected tumors. 43% (3/7) of tumor pairs and one single tumor (25%) revealed intratumoral heterogeneity. While intratumoral heterogeneity affected both the TP53- mutational status and the LOH1p/19q status, all tumors with intratumoral heterogeneity shared the R132 IDH1- mutation as a common feature in both their microdissected components. CONCLUSIONS/SIGNIFICANCE: The majority of recurrent gliomas are of monoclonal origin. However, the detection of divertive tumor cell clones in morphological distinct tumor components sharing IDH1- mutations as early event may provide insight into the tumorigenesis of true mixed gliomas.

Migration of monocytes after intracerebral injection.

Recently, we monitored green fluorescent protein (GFP)-expressing monocytes after injection at the entorhinal cortex lesion (ECL) site in mice. We followed their migration out of the central nervous system (CNS) along olfactory nerve fibers penetrating the lamina cribrosa, within the nasal mucosa, and their subsequent appearance within the deep cervical lymph nodes (CLN), with numbers peaking at day 7. This is the same route activated T cells use for reaching the CLN, as we have shown before. Interestingly, GFP cells injected into the brain and subsequently found in the CLN exhibited ramified morphologies, which are typical of microglia and dendritic cells. To gain more insight into immunity and regeneration within the CNS we want to monitor injected monocytes using magnetic resonance imaging (MRI) after labeling with very small superparamagnetic iron oxide particles (VSOP). Due to their small size, nanoparticles have huge potential for magnetic labeling of different cell populations and their MRI tracking in vivo. So far we have verified that incubation with VSOP particles does not alter their migration pattern after ECL.

Migration of monocytes after intracerebral injection at entorhinal cortex lesion site.

The lack of classical lymph vessels within brain tissue complicates immune surveillance of the CNS, and therefore, cellular emigration out of the CNS parenchyma requires alternate pathways. Whereas invasion of blood-derived mononuclear cells and their transformation into ramified, microglia-like cells in areas of axonal degeneration across an intact BBB have been demonstrated, it still remained unclear whether these cells reside permanently, undergo apoptosis, or leave the brain to present antigen in lymphoid organs. With the use of ECL of mice and injection of GFP-expressing monocytes, we followed the appearance of injected cells in spleen and LNs and the migratory pathways in whole-head histological sections. Monocytes migrated from the lesion site to deep CLNs, peaking in number at Day 7, but they were virtually absent in spleen and in superficial CLNs and inguinal LNs until Day 21 after lesion/injection. In whole-head sections, GFP monocytes were found attached to the olfactory nerves and located within the nasal mucosa at 48 hpi. Thus, monocytes are capable of migrating from lesioned brain areas to deep CLNs and use the cribriform plate as an exit route.

Complications in percutaneous vertebroplasty associated with puncture or cement leakage.

Due to the minimally invasive character and excellent clinical outcome of percutaneous vertebroplasty (PVP), the procedure is being performed in greatly increasing numbers. While PVP has a low complication rate in general, severe complications can occur. We focus on the imaging appearance of complications of PVP associated with puncture or cement leakage--from harmless to life-threatening.

Bilateral basal ganglia infarction following resection of a right parietal parasagittal dural tumour requiring sinus repair -- an atypical complication of an atypical venous drainage.

The case of a 68-year old woman who underwent surgery for a right parietal parasagittal dural tumour is reported. In the preoperative angiography the straight sinus failed to show. During tumour resection the lateral wall of the parietal superior sagittal sinus was opened and reconstructed. Postoperatively, the patient remained unresponsive. A computerized tomography scan revealed bilateral infarction of the basal ganglia. Angiography showed an occlusion of the superior sagittal sinus with hypervolaemia of the deep venous system. The patient did not recover. We conclude that patency of the superficial venous drainage system can be mandatory for drainage of the basal ganglia in cases with deep venous obstruction. To our knowledge, this is an extremely rare complication of neurosurgery in cortical supratentorial areas.

Intracavitary chemotherapy (paclitaxel/carboplatin liquid crystalline cubic phases) for recurrent glioblastoma -- clinical observations.

Human malignant brain tumors have a poor prognosis in spite of surgery and radiation therapy. Cubic phases consist of curved biocontinuous lipid bilayers, separating two congruent networks of water channels. Used as a host for cytotoxic drugs, the gel-like matrix can easily be applied to the walls of a surgical resection cavity. For human glioblastoma recurrences, the feasibility, safety, and short-term effects of a surgical intracavitary application of paclitaxel and carboplatin encapsulated by liquid crystalline cubic phases are examined in a pilot study. A total of 12 patients with a recurrence of a glioblastoma multiforme underwent re-resection and received an intracavitary application of paclitaxel and carboplatin cubic phases in different dosages. Six of the patients received more than 15 mg paclitaxel and suffered from moderate to severe brain edema, while the remaining patients received only a total of 15 mg paclitaxel. In the latter group, brain edema was markedly reduced and dealt medically. Intracavitary chemotherapy in recurrent glioblastoma using cubic phases is feasible and safe, yet the clinical benefit remains to be examined in a clinical phase II study.

Local chemotherapy of F98 rat glioblastoma with paclitaxel and carboplatin embedded in liquid crystalline cubic phases.

Implanted drug carrier systems for retarded chemotherapy against gliomas are mainly based upon polymers containing nitrosoureas. The authors have developed an intracavitary carrier system of biodegradable liquid crystalline cubic phases encapsulating carboplatin and paclitaxel and studied it for release kinetics, antitumor activity, and survival prolongation. A total of 61 Fisher rats with F98 tumors were divided into six treatment groups at day 12 post-inoculation, receiving either no treatment, surgery with partial tumor resection, or partial resection with implantation of cubic phases containing either paclitaxel and carboplatin, paclitaxel alone, carboplatin alone, or no drug. Animals were killed for tumor size analysis at day 21 post-inoculation (n=28) or were included in survival studies (n=33). Additional 12 animals received a paclitaxel/carboplatin application and were killed at different time intervals (6 h, 24 h, 48 h, 5 d, 7 d, 10 d post-agent application) for in vivo diffusion studies. Animals from the paclitaxel/carboplatin group showed a significantly smaller tumor (mean 3.25 mm2+/-SD 1.79 mm2) than animals from the control group (15.30+/-5.86 mm2; P=0.0031), animals having received the empty matrix (11.62+/-6.66 mm2; P=0.0241), and animals after tumor resection without implantation (20.87+/-3.56 mm2; P

Glioblastoma-induced attraction of endogenous neural precursor cells is associated with improved survival.

Neural precursor cells contribute to adult neurogenesis and to limited attempts of brain repair after injury. Here we report that in a murine experimental glioblastoma model, endogenous neural precursors migrate from the subventricular zone toward the tumor and surround it. The association of endogenous precursors with syngenic tumor grafts was observed, after injecting red fluorescent protein-labeled G261 cells into the caudate-putamen of transgenic mice, which express green fluorescent protein under a promoter for nestin (nestin-GFP). Fourteen days after inoculation, the nestin-GFP cells surrounded the tumors in several cell layers and expressed markers of early noncommitted and committed precursors. Nestin-GFP cells were further identified by a characteristic membrane current pattern as recorded in acute brain slices. 5-bromo-2-deoxyuridine labeling and dye tracing experiments revealed that the tumor-associated precursors originated from the subventricular zone. Moreover, in cultured explants from the subventricular zone, the neural precursors showed extensive tropism for glioblastomas. Tumor-induced endogenous precursor cell accumulation decreased with age of the recipient; this correlated with increased tumor size and shorter survival times in aged mice. Coinjection of glioblastoma cells with neural precursors improved the survival time of old mice to a level similar to that in young mice. Coculture experiments showed that neural precursors suppressed the rapid increase in tumor cell number, which is characteristic of glioblastoma, and induced glioblastoma cell apoptosis. Our results indicate that tumor cells attract endogenous precursor cells; the presence of precursor cells is antitumorigenic; and this cellular interaction decreases with aging.

Case history: multicentric glioma with involvement of the optic chiasm.

The case is reported of a man, aged 68, with a right-sided temporal glioblastoma multiform and a left sided chiasmal anaplastic glioma, as well as an occipital tumor, presumably of glial nature. The patient had a complete prostatectomy of adenocarcinoma a year before. The coincidence of multicentric gliomas and prostate cancer is briefly discussed.

Genetic signature of oligoastrocytomas correlates with tumor location and denotes distinct molecular subsets.

Oligoastrocytomas are heterogeneous tumors that have molecular features that overlap with either oligodendrogliomas or astrocytomas. Differences in the frequency of chromosomal losses of 1p and 19q in oligodendrogliomas are related to tumor location, with a low rate of allelic loss in tumors of the temporal and a high rate in tumors of the frontal, parietal, and occipital lobes. To test the possibility of regional molecular heterogeneity in oligoastrocytoma, we examined a series of 203 gliomas including 68 oligoastrocytomas and two control groups of 73 oligodendrogliomas and 62 astrocytomas for allelic losses of chromosomal arms 1p and 19q, and TP53 mutations, and compared these data with tumor localization. Common molecular alterations were found in oligodendrogliomas and oligoastrocytomas arising in extratemporal sites. In respect to the molecular parameters analyzed, temporal oligoastrocytomas were either indistinguishable from astrocytoma or similar to temporal oligodendrogliomas. Oligodendroglial neoplasms can thus be separated into three molecular subsets, two of which include lesions with the morphological features of oligodendrogliomas and oligoastrocytomas and one resembling temporal oligoastrocytoma. Molecular subclassification thus unifies previous findings about prognosis, behavior, response to therapy, genotype, and location in oligodendroglial tumors.