Cell Axons Research Articles

Dye Labeling Retinal Ganglion Cell Axons in Live Xenopus Tadpoles

Individual neurons in the developing nervous system can be visualized by the targeted delivery of a fluorophore. In this article, we describe a method for introducing a fluorescent dye via iontophoresis into retinal ganglion cell (RGC) axons in albino Xenopus laevis tadpoles. Iontophoresis is the enhanced permeation of molecules across biological membranes under the influence of an electrical field. Lipophilic dyes such as DiI are well suited to this method--being insoluble in the aqueous environment of the eye, they precipitate instantaneously, and only cells in contact with the dye crystal are labeled as the dye diffuses through the plasma membrane. A dissection stereomicroscope is used to allow a wide range of approach angles for the micropipette. The goal is to introduce a small bolus of dye into the neural retina where the ganglion cell somata are located and the axons course, with the expectation that it will be taken up by a small enough number of axons to allow individual cells to be distinguished. Because RGC axons will typically be imaged in the tectum far from the injection site, a relatively large injection can be made, increasing the probability of labeling axons without obscuring their visualization at the target. This approach is particularly useful under conditions in which it might be too difficult to perform juxtacellular electroporation because of limited visibility or access.

Erratum to: Microglial TIR-domain-containing adapter-inducing interferon-β (TRIF) deficiency promotes retinal ganglion cell survival and axon regeneration via nuclear factor-κB

Erratum to: Microglial TIR-domain-containing adapter-inducing interferon-β (TRIF) deficiency promotes retinal ganglion cell survival and axon regeneration via nuclear factor-κB

Abstract 4933: Functional properties of PPFIA1, located at the 11q13 amplification region, in epithelial cancer cells.

Abstract PPFIA1 (liprin α1) is a member of the liprin (LAR protein-tyrosine phosphatase-interacting protein) family and is located at the 11q13 amplification region, which is one of the most common amplicons in multiple epithelial cancers. In squamous cell carcinoma of head and neck (HNSCC), this amplification occurs in 30-50% of the cases. In epithelial cancers, the size and structure of 11q13 amplicon is complex and consists of multiple amplicon cores that differ between different tumor types. In HNSCC, the amplicon core contains 10 genes that are amplified in cases with 11q13 amplification. This region contains well-established oncogenes, such as CCND1 and CTTN (cortactin), yet the role of several genes in this region is unknown. Our aim is to study the role of PPFIA1, which has recently been found to be involved in cell motility, modeling of the cell edge dynamics and axon guidance. PPFIA1 is amplified and overexpressed in several epithelial cancers, but the role of it is controversial. PPFIA1 is required for breast and cervical cancer cell invasion, but in contrast depletion of it results in increased invasion of HNSCC cells. We have overexpressed liprin α1 and cortactin in HNSCC cells with no 11q13 amplification and related phenotypes have been analysed in in vitro assays. Migration assays using matrigel coated inserts have shown that overexpression of cortactin increases invasive properties of the HNSCC cells with no 11q13 amplicon whereas liprin α1 has an opposite effect. We have also shown that liprin α1 localizes with cortactin in same vesicular structures, but they do not show physical interaction. Our aim is now to explore the molecular mechanisms behind these findings to assess the role of liprin α1 in vesicular transport and cell motility. We are also carrying out immunoprecipitation and mass spectrometry analyses to identify novel interaction partners for this protein. Our study will provide novel information on the potential specialized functions of liprin α1 in different epithelial cancers, which is likely to improve our understanding on the functional role of 11q13 amplification in malignant potential of cancer cells. Citation Format: Henna Pehkonen, Tatiana Lepikhova, Riku Louhimo, Nina Peitsaro, Misa Imai, Rafael Martinez, Juha Kleftsröm, Sampsa Hautaniemi, Outi Monni. Functional properties of PPFIA1, located at the 11q13 amplification region, in epithelial cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4933. doi:10.1158/1538-7445.AM2013-4933

Intravitreal Injection of Triamcinolone Acetonide into Healthy Rabbit Eyes Alters Retinal Function and Morphology

Aim: To study the effects of intravitreally injected triamcinolone acetonide (TA) and/or its preservative benzyl alcohol (BA) in healthy rabbit retina.Methods: Forty-eight rabbits (aged 4 months, body weight ≈3 kg) were randomized into four groups (n = 12). They were examined with electroretinography (ERG) prior to drug exposure, and then injected intravitreally with a combination of TA and BA, TA without BA, BA alone or a balanced saline solution (BSS). The electroretinograms were assessed 1 week and 7 weeks post-injection. The rabbits were euthanized and the sectioned retinas were studied. Immunohistochemical analysis was performed on rods, cones, rod bipolar cells, horizontal cells, amacrine cells and Müller cells.Results: Rabbits injected with BA showed a significantly lower rod-mediated b-wave amplitude than the controls 1 week after injection. TA-injected rabbits demonstrated significantly higher a- and b-wave amplitudes in the total retinal response than the controls 1 week post-injection. The rabbits injected with TA + BA demonstrated a significantly higher b-wave amplitude in the total retinal response than the controls 1 week after injection. The significantly higher a-wave amplitude in the total retinal response remained in the TA-injected rabbits 7 weeks after injection. Immunohistochemistry revealed that protein kinase C alpha (PKC α) was down-regulated in both the perikarya and the axons of bipolar cells in histological sections from rabbit retina injected with TA + BA, BA and TA.Conclusions: Intravitreal injection of the preservative BA reduces the isolated rod-mediated retinal response in the rabbit, transiently and selectively. Intravitreal injection of TA increases the total retinal response in the rabbit up to seven weeks after injection. The effects observed are not only limited to retinal function, but also include changes in the expression of PKC α in rod bipolar cells, indicating drug-related interference with normal retinal physiology in the healthy rabbit eye.

Distribution of methionine-enkephalin in the minipig brainstem

We have studied the distribution of immunoreactive cell bodies and axons are containing methionine-enkephalin in the minipig brainstem. Immunoreactive axons were widely distributed, whereas the distribution of perikarya was less widespread. A high or moderate density of axons containing methionine-enkephalin were found from rostral to caudal levels in the substantia nigra, nucleus interpeduncularis, nucleus reticularis tegmenti pontis, nucleus dorsalis raphae, nucleus centralis raphae, nuclei dorsalis and ventralis tegmenti of Gudden, locus ceruleus, nucleus sensorius principalis nervi trigemini, nucleus cuneatus externalis, nucleus tractus solitarius, nuclei vestibularis inferior and medialis, nucleus ambiguus, nucleus olivaris inferior and in the nucleus tractus spinalis nervi trigemini. Immunoreactive perikarya were observed in the nuclei centralis and dorsalis raphae, nucleus motorius nervi trigemini, nucleus centralis superior, nucleus nervi facialis, nuclei parabrachialis medialis and lateralis, nucleus ventralis raphae, nucleus reticularis lateralis and in the formatio reticularis. We have also described the presence of perikarya containing methionine-enkephalin in the nuclei nervi abducens, ruber, nervi oculomotorius and nervi trochlearis. These results suggest that in the minipig the pentapeptide may be involved in many physiological functions (for example, proprioceptive and nociceptive information; motor, respiratory and cardiovascular mechanisms).

P53 Inactivation leads to enhancement of tyrosine hydroxylase biosynthesis in brain dopaminergic neurons

In this paper we address a possibility of interaction among protein 53, members of the ERK1/2 signaling pathway, and the CREB transcription factor in regulation of activity of dopaminergic neurons. Nerve cells in the substantia nigra and zona incerta have been studied in control rats and in rats with pifithrin-alpha, a transcriptional inhibitor of p53, administered intraperitoneally. We have shown that p53 inhibition leads to an increased tyrosine hydroxylase content both in cell bodies and axon terminals. The CREB transcription factor activity is also enhanced in brain dopaminergic neurons. However, there have been observed no significant differences in phospho-ERK1/2 kinase content between cell bodies from pifithrin-alpha injected group and the control. It has been shown therefore that p53 exerts an inhibitory effect on tyrosine hydroxylase biosynthesis which may be mediated by the CREB.

Biolistic Loading of Voltage-Sensitive Dyes into Cells in Rat Brain Slices for Optical Recording of Neuron Activity

Neuron electrical activity was recorded using an optical method in which the probes were widely used monomolecular voltage-sensitive dyes. Current methods of loading voltage-sensitive dyes into nervous tissue have serious limitations, severely restricting their areas of use (insolubility in water, the need for toxic organic solvents, and the toxicity of the voltage-sensitive dyes themselves at high concentrations). We have developed a new method of loading voltage-sensitive dyes by “shooting” dye-coated gold microparticles into living brain slices. Three-dimensional reconstruction of nervous tissue fluorescence with a scanning confocal microscope showed that after loading with dye by this method, it propagated along the cell membrane, completely staining only one target cell with its processes, without spreading to surrounding cells via the intercellular fluid or through cell contacts. This method of staining neurons can be used for the optical recording of the electrical activity of individual neurons and to analyze the distribution of electric voltages across the compartments of excitable cells (axons and dendrites).

Address event representation neuromorphics to epiretinal colour vision

Presented is a methodology of conversion of a conventional colour measure, e.g. 24-bit colour, to a neuromorphic representation for use with epiretinal prostheses. The conversion implemented, for a 4 by 4 image, within a field programmable gate array outputs three groups of timing signals. These three groups correspond to red (R), green (G) and blue (B) colour channels. In future work the channels will input to a retinal implant stimulator, via a receiver chip housed over the earlobe of a prosthesis recipient, to form biphasic pulses to drive electrodes stimulating the axons of colour specific retinal ganglion cells. A test setup was used to encode the hue pulses back to intensity to display test images on a VGA monitor for validation.

Chemical Neurotransmission from Retinal Ganglion Cells to Superior Colliculus Neurons under Conditions of Long-Lasting Neighboring Co-Culturing

Axons of retinal ganglion cells (RGCs) form the optic tract (OT), whose fibers transmit impulsation from the retina to subcortical visual centers. We studied the properties of chemical neurotransmission at the level of retinal projections to the above centers using our original in vitro model of neighboring coculturing of dissociated retinal cells and neurons of the superior colliculus (SC). The proposed model, as compared with recently developed similar methods, allowed us to stimulate clearly identified separate RGC axons and to record postsynaptic currents (PSCs) from neurons of the SC synaptically connected with these axons. Our experiments showed that most of these contacts are excitatory; in 12 of 15 cases, we recorded evoked excitatory PSCs (eEPSCs) in SC neurons, while inhibitory synaptic contacts between RGCs and neurons of the SC were significantly more rare. Excitatory neurotransmission was mediated by glutamate release and activation of postsynaptic NMDA and non-NMDA receptors. We found that non-NMDA receptors play the main functional role in transmission of visual signals through well-formed synaptic connections. Inhibitory synaptic transmission is mediated by activation of GABAA receptors on the postsynaptic membrane of SC neurons.

Myelinated nerve bundles developed on the plano‐concave fibers containing nerve conduit

Morphologically and chemically modified plano-concave fibers (PCFs) are designed as a unit of guided channels for supporting Schwann cells to facilitate mass transport and promote nerve regeneration. The surface-modified PCFs are imprinted with linearly patterned grooves (LPGs) to guide adherent Schwann cell elongation and axon extension. After being cocultured with PC12 neuron-like cells, Schwann cells differentiate into the myelinated type and interact with PC12 axons. The myelinated axons aggregate as a linear bundle and extend along the direction of LPGs on a PCF. The cross section of a myelin structure is examined using a transmission electron microscope. The PCFs can potentially bridge gaps in injured nerves, improving the therapeutic efficacy of nerve regeneration.

Neuroanatomical technique for studying long axonal projections in the central nervous system: combined axonal staining and pre-labeling in parasagittal gerbil brain slices

A method is described for studying the morphological features of extensive axonal projections within the central nervous system of the gerbil, Meriones anguiculatus. Potentially long descending axonal projections between the auditory thalamus and lower brainstem were used as a model. The inferior colliculus (IC) in the tectum was injected in vivo with a fluorescent retrograde tracer, Fluoro-Gold, to label cells in the medial geniculate body (MGB) that had descending projections to the IC, and cells in the superior olivary complex (SOC) that had ascending projections to the IC. Another fluorescent retrograde tracer, fast blue, was injected into the cochlea to label olivocochlear (OC) cells in the SOC. Inferomedially curved parasagittal slices containing ipsilateral auditory cell groups from the thalamus to the brainstem were cut and descending axons of the pre-labeled MGB cells were traced anterogradely with Biocytin. After visualizing histologically the injected Biocytin, discretely labeled IC-projecting axons of the MGB cells were traced including their collaterals that extended further into the SOC. In the SOC, these axons terminated on pre-labeled cells including OC cells. The combination of anterograde and retrograde tracing in the slice preparations described here demonstrated extensive descending axonal projections from the thalamus to their targets in the lower brainstem that had known ascending/descending projections within the auditory system.

Abnormal Mitochondrial Trafficking and Cytoskeletal Organization in a Human Induced Pluripotent Stem Cell and a Mouse Model of Charcot-Marie-Tooth Disease Type 2E (IN7-2.001)

Objective: To characterize human neuronal cells derived from induced pluripotent stem cells of a patient with CMT2E and a mouse model of the same mutation. Background Induced pluripotent stem cells (iPS) offer an alternative human model for the study of neurogenetic conditions. Previous studies have demonstrated the feasibility of generating and differentiating iPS cells from patients with neurological diseases into functional neurons and glia. This approach could be particularly useful in the pathophysiological study of distinct forms of inherited neuropathies. Charcot-Marie-Tooth disease type 2E (CMT2E) is caused by heterozygous point mutations in the NEFL gene, which encodes the intermediate filament neurofilament light chain, an essential component of the neuronal cytoskeleton. Design/Methods: We developed iPS cell lines from skin fibroblasts of a patient with a N98S NFL point mutation. Knockin mice carrying the same N98S mutation were also generated. Following a differentiation protocol for generating spinal cord motor neurons, we obtained human CMT2E neuronal cultures consisting of at least 50% ISLET1 immunoreactive cells. These neuronal cultures were characterized by time-lapse microscopy and axonal mitochondrial kinetics was analyzed. Results: Immunostaining for intermediate filaments revealed an increased content of these proteins in the neuronal body of CMT2E spinal cord motor neurons. Immunostaining of both the spinal cord and brain of a N98S mouse shows abundant NFL positive aggregates in the cell bodies and axons of the motor neurons, as well as in the cerebellum. Multiple filamentous spheroid-like structures were also observed in the brain stem of the mice. An increased proportion of paused mitochondria was found in axons from human CMT2E neurons, compared to an age-matched control. Conclusions: This study demonstrates disease relevant phenotypes in human CMT2E motor neurons that were supported by a mouse model of the same mutation, demonstrating the potential of iPS cell technology to model neurogenetic diseases. Supported by: M.S. was funded by a Rare Disease Clinical Research Network Fellowship from the National Institutes of Health. This study was also supported by iPierian Inc. Disclosure: Dr. Saporta has nothing to disclose. Dr. Volfson has received personal compensation for activities with iPierian Inc. Dr. Volfson has received research support from iPierian Inc. Dr. Liem has nothing to disclose. Dr. Shy has nothing to disclose. Dr. Liem has nothing to disclose. Dr. Dimos has received personal compensation for activities with iPierian Inc. as an employee.Dr. Dimos has received research support from iPierian Inc.

Subthreshold somatic voltage in neocortical pyramidal cells can control whether spikes propagate from the axonal plexus to axon terminals: a model study

There is suggestive evidence that pyramidal cell axons in neocortex may be coupled by gap junctions into an "axonal plexus" capable of generating very fast oscillations (VFOs) with frequencies exceeding 80 Hz. It is not obvious, however, how a pyramidal cell in such a network could control its output when action potentials are free to propagate from the axons of other pyramidal cells into its own axon. We address this problem by means of simulations based on three-dimensional reconstructions of pyramidal cells from rat somatosensory cortex. We show that somatic depolarization enables propagation via gap junctions into the initial segment and main axon, while somatic hyperpolarization disables it. We show further that somatic voltage cannot effectively control action potential propagation through gap junctions on minor collaterals; action potentials may therefore propagate freely from such collaterals regardless of somatic voltage. In previous work, VFOs are all but abolished during the hyperpolarization phase of slow oscillations induced by anesthesia in vivo. This finding constrains the density of gap junctions on collaterals in our model and suggests that axonal sprouting due to cortical lesions may result in abnormally high gap junction density on collaterals, leading in turn to excessive VFO activity and hence to epilepsy via kindling.

Phosphorylation and Cleavage of the Family of Collapsin Response Mediator Proteins May Play a Central Role in Neurodegeneration after CNS Trauma

The family of the collapsin response mediator proteins (CRMPs) plays a significant physiological role in neuronal cell bodies and axons within the integrated mammalian central nervous system (CNS). Trauma-induced damage to the CNS results in variable degrees of axonal degeneration, and this may lead to neuronal cell death in key grey matter regions. Site-specific phosphorylation of certain CRMPs has been associated with trauma-induced axonal degeneration. Moreover, recent data implicate the pro-apoptotic, calcium-dependent protease calpain as a key initiator of CRMP cleavage. The primary cleavage product of injury-induced neuronal calpain activation is a C-terminus truncated 55- to 58-kDa form of CRMP, which may exert its effects within the cytoplasm and axonal core, or alternatively through its translocation into the nucleus, initiating neuronal cell death. The precise structure of cleaved CRMP has yet to be elucidated, as is the reason for nuclear translocation. Once the crystal structure of the cytoplasmic and nuclear-translocated forms of CRMPs is determined, a greater molecular understanding of why these forms can initiate neurodegeneration following CNS injury will be established. Such information will be particularly informative in the design of inhibitors of specific protein-protein interaction sites between cleaved CRMP and vital cytosolic or nuclear molecules.

Embryonic rat dorsal root ganglia organotypic culture: a morphometric model to test neurotoxicology

Neurotoxicity is a common dose-limiting side-effect of several drugs (Cavaletti et al., 2008). So far a validated test method to screen drugs neurotoxicity does not exist, therefore in this interdepartment study we have analyzed the effectiveness of a morphometric neurotoxicty assessment model. Drug neurotoxicity evaluation is based on embryonic rat dorsal root ganglia (DRG) organotypic culture. DRG primary sensory neurons are the principal target of drugs neurotoxic action. In fact, primary sensory neurons lie outside the blood-nerve barrier and are supplied by capillaries with fenestrated walls. Moreover, the axons of these cells are among the longest of the entire nervous system and, therefore, are more susceptible to any agent that interferes with the energy metabolism or the structural basis of axonal transport. In particular, in this interdepartment study, the interference of the under study neurotoxic compound with NGF-induced neurite elongation is analysed. The effectiveness and reproducibility of this model, even if commonly used to test drugs, has not yet been demonstrated. In order to assess the validity of this in vitro model, antineoplastic drugs known to be in clinical use and in animal models neurotoxic (paclitaxel and oxaliplatin) or not dangerous (cyclophosphamide and 5-Fluorouracil) have been tested. DRGs explanted from E15 rat embryos have been treated for 24h with drugs concentrations comparable to those achievable in vivo. The length of the longest neurite of each DRG has been measured by ImageJ program. Experiments have been performed by three different blinded researchers in two different laboratories. Mean and standard deviation of each experiment were obtained, subsequently the mean value and standard deviation of the three independent experiments for each researcher were calculated. Data obtained by the three researchers in two different laboratories resulted statistically comparable and no significant differences were detected (One Way Anova analysis of variance and Tukey post test; p<0.05). This interdepartment in vitro study, therefore, indicates that a purely morphometric model represents a reliable tool to study drug neurotoxicity, permitting to make prediction of neurotoxic effects on humans because the concentrations tested are the same to which DRG are exposed during clinical use.

&lt;I&gt;Drosophila&lt;/I&gt; spen protein: generation of ployclonal antibodies, functional analysis and tissue-specific expression

The spen family of proteins participates in various biological processes. It is involved in neuronal cell fate, survival and axon guidance, and cell cycle regulation. Recent studies showed the Drosophila spen gene was required for Wnt-dependent signaling in the eye, wing and leg. However, the genetic role and biological function in Drosophila remain largely unclear. A Drosophila C-terminal fragment of spen was cloned and expresed in E. coli. The purified 6×His-spen protein was injected into SD rat to generate polyclonal antibodies. Subcellular localization and tissue-specific expression of spen protein were analysed by immunostaining and histoimmunochemistry. The results indicated that spen protein was localized in the nucleus and expressed at high levels in brain, fat body, hemocyte, gut and salivary gland. To assay the function of mutant hemocytes in vivo, wild-type and spen mutant larvae were infected with fluorescent microspheres. Wild-type hemocytes showed a strong fluorescence signal from the phagocytosed microspheres; however, spen mutant had a weak fluorescence signal, indicating that the mutant hemocytes were defective in the uptake of the microspheres.

P4‐423: RGS‐14(414) gene delivery into brain area V2 induces both the recovery and prevention of recognition memory in ageing and Alzheimer's disease

neurogenesis and maturation of newly generated neurons in the brain of subjects with AD and delay or prevent the progression of the disease is stimulation of endogenous neurotrophic factors. Neurotrophic factors a family of structurally related, secreted proteins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), ciliary neurotrophic factor (CNTF), NT-3, and NT4/5, are small proteins that provide trophic events in neuronal cells. These secreted neurotrophic factors act by binding to the specific cell surface receptors that signal the neuron to survive. Methods: Neurotrophic factors are important in the developing and mature nervous system. They influence cell proliferation, survival, differentiation, migration, axon and dendritic growth and synaptic plasticity. They play critical roles in complex behaviors including anxiety, depression and learning. They can be given exogenously as pure proteins or through gene therapy to prevent nerve cell death caused by various insults including nerve injury, brain trauma, and exposure to toxins. Results: The high potency of neurotrophic factors, as shown by many experimental studies, makes them a rational candidate therapeutic agent in neurodegenerative disease. Because the administration of exogenous neurotrophic factors is time consuming and expensive for the most of patients and also it needs to gene delivery, intense care considerations and probably it’s side effects, therefore it seems that the manipulation of endogenous neurotrophic factors is suitable treatment for AD.Conclusions: It is concluded that the manipulation and stimulation of endogenous neurotrophic factors is a promising therapeutic strategy in Alzheimer’s disease.

Selecting synaptic partners: GRASPing the role of UNC-6/netrin

Forming synaptic connections of the appropriate strength between specific neurons is crucial for constructing neural circuits to control behavior. A recent paper in Neural Development describes the use of a synapse-specific label in Caenorhabditis elegans to implicate local UNC-6/netrin signaling in this developmental process. Thus, as well as their well known roles in cell migration and axon guidance, UNC-6/netrin signals distinguish an appropriate synaptic partner from other potential targets.See Research article: http://www.neuraldevelopment.com/content/6/1/28

Abstract 1197: EphA3 kinase ablation induces glioblastoma differentiation and prevents tumor progression

Abstract Purpose: Eph receptors constitute the largest sub-family of receptor tyrosine kinases and interact with membrane-bound ligands termed ephrins. Eph and ephrins have many vital functions including cell adhesion, migration and axon guidance. Eph and ephrins have been found to be aberrantly expressed in many malignancies including brain tumor. The purpose of this study was to investigate EphA3 receptor function in the most common and aggressive form of brain tumor, Glioblastoma (GBM). Methods: Gene expression was investigated by Q-PCR, IHC and flow cytometry in high grade glioma (HGG) surgical specimens and primary derived serum free cell cultures. Targeted reduction of Eph expression was performed using both a constitutive and inducible shRNA system. Murine in-vivo studies were performed using both subcutaneous and orthotopic ‘intracranial’ xenografts in immuno-compromised animals. Signaling pathways were assessed by western blotting. Results: To establish whether the receptor tyrosine kinase EphA3 was over expressed in HGG we assessed 12 normal human brain specimens, 56 HGG specimens and 26 HGG primary cultures. EphA3 mRNA expression was negligible in normal brain while 30% of clinical specimens and 46% of primary cultured tumor cells expressed EphA3. EphA3 protein was also detected in HGG clinical specimens using IHC. To further investigate EphA3 function the receptor was down regulated using shRNA in an EphA3+ GBM neurosphere cell line. Constitutive and inducible down regulation of the EphA3 receptor resulted in initiation of neuronal and glial cell differentiation following activation of the ERK/MAPK pathway. A reduction in stem/progenitor cell proliferation was also observed following EphA3 knockdown by shRNA (46%) or by alternately inhibiting EphA3 function using soluble ephrin A5-Fc (33%). CFSE division tracking identified slower cell division in populations in which EphA3 signaling was attenuated. In-vivo studies were performed using a NOD/SCID mouse subcutaneous and intracranial xenograft model. Results highlighted a marked reduction in tumor formation in the EphA3 knockdown as opposed to control tumors. Subcutaneous control tumors formed with a median survival of 66 days while EphA3 knockdown animals survived beyond 100 days (p&amp;lt;0.05). Similar to the subcutaneous xenograft model a marked lack of intracranial tumor formation was observed when EphA3 was neutralized. Control mice formed large well vascularized invasive tumors with a median survival of 62 days. All EphA3 knockdown animals were free of tumor following autopsy at 145 days when the experiment was terminated (p&amp;lt;0.05). Importantly a mutant EphA3 rescue of the knockdown culture returned the tumorigenic potential of these cells. Conclusions: We propose EphA3, in part, regulates cancer stem cell self renewal and cell division rate in GBM and could prove a potential therapeutic target and marker of brain tumor initiating stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2011-1197

Endothelin receptors A and B are expressed in distinct cellular compartments of rat hippocampus following global ischemia: an immunocytochemical study

Objectives: The syntheses of endothelin receptors A and B were previously shown to be upregulated in rat dorsal hippocampus after traumatic brain injury. Here we characterize endothelin receptor A and endothelin receptor B cellular distribution in hippocampus after permanent global brain ischemia and their possible association to nerve cell injury.Methods: Twenty-minute global ischemia was induced using the Pulsinelli’s four-vessel occlusion in conjunction with systemic hypovolemia in male rats. Endothelin receptor A and endothelin receptor B immunoreactivities from sham-operated and ischemic rats were assessed qualitatively in dentate gyrus, Cornu Ammonis, and hilus regions of the hippocampus. Quantitative immunoreactivity measurements were also obtained by optical densitometry.Results: In sham-operated control hippocampus, endothelin receptor A immunoreactivity was absent in nerve cell bodies but strongly expressed in the mossy fiber pathway (axons of dentate gyrus granule cells). After ischemia endothelin receptor A immunoreactivity in the same regions was reduced by 40-50% from control. In contrast, endothelin receptor B immunoreactivity in control hippocampus was widely distributed in pyramidal neurons, granule cells and glial cells, this immunoreactivity increasing by approximately 25-30% after ischemia.Discussion: Endothelin receptor A’s marked decrease in mossy fibers after ischemia may contribute to glutamate release from mossy fiber terminals, thus enhancing excitotoxic effects on their Cornu Ammonis synaptic targets. Additionally, endothelin receptor B increased expression in neurons and glia could be related to a more generalized activation of survival mechanisms involving elements of the neurovascular unit.