Publications by authors named "Jörg Mey"

45 Publications

Retinoic acid increases phagocytosis of myelin by macrophages.

J Cell Physiol 2021 May 9;236(5):3929-3945. Epub 2020 Nov 9.

Laboratorio Regeneración Neuronal e Inmunidad Innata, Hospital Nacional de Parapléjicos, Toledo, Spain.

Traumatic injuries of the central nervous system (CNS) are followed by the accumulation of cellular debris including proteins and lipids from myelinated fiber tracts. Insufficient phagocytic clearance of myelin debris influences the pathological process because it induces inflammation and blocks axonal regeneration. We investigated whether ligands of nuclear receptor families retinoic acid receptors (RARs), retinoid X receptors, peroxisome proliferator-activated receptors, lipid X receptors, and farnesoid X receptors increase myelin phagocytosis by murine bone marrow-derived macrophages and Raw264.7 cells. Using in vitro assays with 3,3'-dioctadecyloxacarbocyanine perchlorate- and pHrodo-labeled myelin we found that the transcriptional activator all-trans retinoic acid (RA)enhanced endocytosis of myelin involving the induction of tissue transglutaminase-2. The RAR-dependent increase of phagocytosis was not associated with changes in gene expression of receptors FcγR1, FcγR2b, FcγR3, TREM2, DAP12, CR3, or MerTK. The combination of RA and myelin exposure significantly reduced the expression of M1 marker genes inducible nitric oxide synthase and interleukin-1β and increased expression of transmembrane proteins CD36 and ABC-A1, which are involved in lipid transport and metabolism. The present results suggest an additional mechanism for therapeutic applications of RA after CNS trauma. It remains to be studied whether endogenous RA-signaling regulates phagocytosis in vivo.
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http://dx.doi.org/10.1002/jcp.30137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984038PMC
May 2021

Expression patterns of chloride transporters in the auditory brainstem of developing chicken.

Hear Res 2020 08 3;393:108013. Epub 2020 Jun 3.

Department for Zoology and Animal Physiology, RWTH Aachen University, Aachen, Germany.

GABAergic transmission changes from depolarization to hyperpolarization in most vertebrate brain regions during development. By contrast, in the auditory brainstem of chicken a depolarizing effect of GABA persists after hatching. Since auditory brainstem neurons that receive GABAergic input have a Cl reversal potential above resting membrane potential, a specifically tuned activity of Cl transporters is likely. We here present a developmental study of the expression patterns of several members of the SLC12 family (NKCC1, NKCC2, KCC1, KCC2, KCC4, CCC6, CCC9) and of AE3 at developmental ages E7, E10, E12, E15, E17, and P1 with quantitative RT-PCR. NKCC2 and CCC9 were not detected in auditory brainstem (positive control: kidney). KCC1, CCC6 and AE3 were expressed, but not regulated, while NKCC1, KCC2 and KCC4 were regulated. The expression of the latter transporters increased, with KCC2 exhibiting the strongest expression at all time points. Biochemical analysis of the protein expression of NKCC1, KCC2 and KCC4 corroborated the findings on the mRNA level. All three transporters showed a localization at the outer rim of the cells, with NKCC1 and KCC2 expressed in neurons, and KCC4 predominantly in glia. The comparison of the published chloride reversal potential and expression of transporter proteins suggest strong differences in the efficiency of the three transporters. Further, the strong KCC2 expression could reflect a role in the structural development of auditory brainstem synapses that might lead to changes in the physiological properties.
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http://dx.doi.org/10.1016/j.heares.2020.108013DOI Listing
August 2020

Treatment of rats with spinal cord injury using human bone marrow-derived stromal cells prepared by negative selection.

J Biomed Sci 2020 Feb 18;27(1):35. Epub 2020 Feb 18.

Hospital Nacional de Parapléjicos, c/Finca la Peraleda, 45071, Toledo, Spain.

Background: Spinal cord injury (SCI) is a highly debilitating pathology without curative treatment. One of the most promising disease modifying strategies consists in the implantation of stem cells to reduce inflammation and promote neural regeneration. In the present study we tested a new human bone marrow-derived stromal cell preparation (bmSC) as a therapy of SCI.

Methods: Spinal cord contusion injury was induced in adult male rats at thoracic level T9/T10 using the Infinite Horizon impactor. One hour after lesion the animals were treated with a sub-occipital injection of human bmSC into the cisterna magna. No immune suppression was used. One dose of bmSC consisted, on average, of 2.3 million non-manipulated cells in 100 μL suspension, which was processed out of fresh human bone marrow from the iliac crest of healthy volunteers. Treatment efficacy was compared with intraperitoneal injections of methylprednisolone (MP) and saline. The recovery of motor functions was assessed during a surveillance period of nine weeks. Adverse events as well as general health, weight and urodynamic functions were monitored daily. After this time, the animals were perfused, and the spinal cord tissue was investigated histologically.

Results: Rats treated with bmSC did not reject the human implants and showed no sign of sickness behavior or neuropathic pain. Compared to MP treatment, animals displayed better recovery of their SCI-induced motor deficits. There were no significant differences in the recovery of bladder control between groups. Histological analysis at ten weeks after SCI revealed no differences in tissue sparing and astrogliosis, however, bmSC treatment was accompanied with reduced axonal degeneration in the dorsal ascending fiber tracts, lower Iba1-immunoreactivity (IR) close to the lesion site and reduced apoptosis in the ventral grey matter. Neuroinflammation, as evidenced by CD68-IR, was significantly reduced in the MP-treated group.

Conclusions: Human bmSC that were prepared by negative selection without expansion in culture have neuroprotective properties after SCI. Given the effect size on motor function, implantation in the acute phase was not sufficient to induce spinal cord repair. Due to their immune modulatory properties, allogeneic implants of bmSC can be used in combinatorial therapies of SCI.
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http://dx.doi.org/10.1186/s12929-020-00629-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026953PMC
February 2020

Vibratory movements in contests between females of the feather-legged spider (Uloborus plumipes).

Zoology (Jena) 2017 12 1;125:87-93. Epub 2017 Sep 1.

Institut für Biologie II (Zoologie), RWTH Aachen University, Worringerweg 3, D-52074 Aachen, Germany; Hospital Nacional de Parapléjicos, SESCAM, Finca la Peraleda s/n, E-45071 Toledo, Spain. Electronic address:

Females of the feather-legged spider Uloborus plumipes invade, and compete for, each other's orb webs. In the context of these competitive interactions the question arose how the spiders communicate. Since substrate-borne vibrations are the most important component of the sensory environment of web-building spiders, we investigated vibratory movements that might serve as signals of communication. Three behaviors were found to be associated with female-female contests and to cause propagating vibrations in the spider webs: thread pulling, abdominal trembling, and web shaking. While thread pulling and abdominal trembling were also observed when prey insects were caught in the webs, web shaking occurred only in response to the presence of a competing conspecific. Caused by flexing of the first legs and a vigorous rotary movement of the opisthosoma, web shaking creates a short burst of strong oscillations of the orb web. This behavior always elicited a behavioral reaction by the competitor and may serve as an intraspecific signal in the mutual assessment of competing spiders. We suggest that web shaking communicates resource holding potential in U. plumipes.
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http://dx.doi.org/10.1016/j.zool.2017.08.009DOI Listing
December 2017

Cell-free artificial implants of electrospun fibres in a three-dimensional gelatin matrix support sciatic nerve regeneration in vivo.

J Tissue Eng Regen Med 2017 12 27;11(12):3289-3304. Epub 2017 Jan 27.

EURON Graduate School of Neuroscience, Maastricht University, the Netherlands.

Surgical repair of larger peripheral nerve lesions requires the use of autologous nerve grafts. At present, clinical alternatives to avoid nerve transplantation consist of empty tubes, which are only suitable for the repair over short distances and have limited success. We developed a cell-free, three-dimensional scaffold for axonal guidance in long-distance nerve repair. Sub-micron scale fibres of biodegradable poly-ε-caprolactone (PCL) and collagen/PCL (c/PCL) blends were incorporated in a gelatin matrix and inserted in collagen tubes. The conduits were tested by replacing 15-mm-long segments of rat sciatic nerves in vivo. Biocompatibility of the implants and nerve regeneration were assessed histologically, with electromyography and with behavioural tests for motor functions. Functional repair was achieved in all animals with autologous transplants, in 12 of 13 rats that received artificial implants with an internal structure and in half of the animals with empty nerve conduits. In rats with implants containing c/PCL fibres, the extent of recovery (compound muscle action potentials, motor functions of the hind limbs) was superior to animals that had received empty implants, but not as good as with autologous nerve transplantation. Schwann cell migration and axonal regeneration were observed in all artificial implants, and muscular atrophy was reduced in comparison with animals that had received no implants. The present design represents a significant step towards cell-free, artificial nerve bridges that can replace autologous nerve transplants in the clinic. Copyright © 2017 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2237DOI Listing
December 2017

Fighting for the web: competition between female feather-legged spiders (Uloborus plumipes).

Zoology (Jena) 2017 04 14;121:10-17. Epub 2016 Dec 14.

Institut für Biologie II (Zoologie), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany; Hospital Nacional de Parapléjicos, SESCAM, Finca la Peraleda s/n, 45071 Toledo, Spain. Electronic address:

Most spider species are solitary, and among the few social interactions among them, resource competition between females has received little attention. We discovered that females of the feather-legged spider Uloborus plumipes invade the orb webs of conspecifics and compete for webs. Following observations in the wild, intruder-defender interactions were studied in a terrarium and in controlled laboratory experiments. We found that contests for orb webs occurred spontaneously between adult females. Competitive interactions in U. plumipes were characterized by an escalation of ritualized behaviors. In 27% of the contests the winner was determined by interactions at a distance, which involved behaviors that caused vibratory signaling on the web. The remaining interactions escalated to physical contact, and in 78% of these a fight occurred between the contestants. Using multivariate logistic regression we determined the factors that predicted the outcome of the contests: (i) Web ownership did not give the defender a competitive advantage. (ii) The difference in physical size between the competing spiders was the most important predictor for the outcome of web contests. (iii) Independent of body size, the display of certain behaviors, specifically the ability to reach the hub before the contestant and the frequency of attacks, increased the probability of winning. (iv) Winning or losing a fight did not affect the chances of winning subsequent contests. The interactions reported here provide a promising approach to investigate communication in spiders and to test theoretical models of intraspecific competition.
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http://dx.doi.org/10.1016/j.zool.2016.12.002DOI Listing
April 2017

Characterisation of cell-substrate interactions between Schwann cells and three-dimensional fibrin hydrogels containing orientated nanofibre topographical cues.

Eur J Neurosci 2016 Feb 18;43(3):376-87. Epub 2015 Aug 18.

Institute of Neuropathology, Uniklinik RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany.

The generation of complex three-dimensional bioengineered scaffolds that are capable of mimicking the molecular and topographical cues of the extracellular matrix found in native tissues is a field of expanding research. The systematic development of such scaffolds requires the characterisation of cell behaviour in response to the individual components of the scaffold. In the present investigation, we studied cell-substrate interactions between purified populations of Schwann cells and three-dimensional fibrin hydrogel scaffolds, in the presence or absence of multiple layers of highly orientated electrospun polycaprolactone nanofibres. Embedded Schwann cells remained viable within the fibrin hydrogel for up to 7 days (the longest time studied); however, cell behaviour in the hydrogel was somewhat different to that observed on the two-dimensional fibrin substrate: Schwann cells failed to proliferate in the fibrin hydrogel, whereas cell numbers increased steadily on the two-dimensional fibrin substrate. Schwann cells within the fibrin hydrogel developed complex process branching patterns, but, when presented with orientated nanofibres, showed a strong tendency to redistribute themselves onto the nanofibres, where they extended long processes that followed the longitudinal orientation of the nanofibres. The process length along nanofibre-containing fibrin hydrogel reached near-maximal levels (for the present experimental conditions) as early as 1 day after culturing. The ability of this three-dimensional, extracellular matrix-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggest that it may be an appropriate device design for the bridging of experimental lesions of the peripheral nervous system.
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http://dx.doi.org/10.1111/ejn.13026DOI Listing
February 2016

Is retinoic acid a signal for nerve regeneration in insects?

Neural Regen Res 2015 Jun;10(6):901-3

Hospital Nacional de Parapléjicos, Toledo, Spain ; Euron Graduate School of Neuroscience, Maastricht, Netherlands.

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http://dx.doi.org/10.4103/1673-5374.158349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498350PMC
June 2015

Retinoic acid as a survival factor in neuronal development of the grasshopper, Locusta migratoria.

Cell Tissue Res 2014 Nov 9;358(2):303-12. Epub 2014 Aug 9.

Institut für Biologie II (Zoologie), RWTH Aachen University, Worringer Weg 3, 52074, Aachen, Germany.

Based on experience with cell cultures of adult insect neurons, we develop a serum-free culture system for embryonic locust neurons. Influences of trophic substances on survival and neurite outgrowth of developing neurons are investigated. For the first time, a positive trophic effect of 9-cis retinoic acid (9-cis RA) was shown in vitro on embryonic neurons of an insect. We observed longer cell survival of 50 % developmental stage neurons in cultures supplemented with 0.3 nM 9-cis RA. Furthermore, an influence on neuron morphology was revealed, as the addition of 9-cis RA to cell culture medium led to an increase in the number of neurites per cell. Although an RA receptor gene, LmRXR (Locusta migratoria retinoid X receptor), was expressed in the central nervous system throughout development, the influence of 9-cis RA on neuronal survival and outgrowth was restricted to 50 % stage embryonic cells.
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http://dx.doi.org/10.1007/s00441-014-1957-yDOI Listing
November 2014

Three-dimensional configuration of orientated fibers as guidance structures for cell migration and axonal growth.

J Biomed Mater Res B Appl Biomater 2014 Feb 16;102(2):356-65. Epub 2013 Sep 16.

Institut für Biologie II, RWTH Aachen, Germany; EURON Graduate School of Neuroscience, Maastricht University, Netherlands.

Peripheral nerve injuries can be surgically repaired by suturing the transected nerve stumps or, in case of larger lesions, by the transplantation of an autologous nerve graft. To avoid donor site morbidity, the development of artificial implants is desired. Clinically, hollow conduits have been used for this purpose but are inferior to the autograft because they lack internal guidance cues for Schwann cells and regenerating axons. In this article, we describe the design of a three-dimensional (3D) scaffold consisting of parallel fibers embedded in a collagen matrix. For this purpose, an electrospinning device was developed to produce and manipulate a 3D array of aligned poly(ɛ-caprolactone) (PCL) microfibers. This fiber array was then incorporated into biodegradable PCL tubes to serve as artificial nerve bridges. Using primary cultures of embryonic chicken dorsal root ganglia, we show that PCL microfibers in the 3D matrix of our composite scaffold guide the direction of Schwann cell migration and axonal growth.
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http://dx.doi.org/10.1002/jbm.b.33014DOI Listing
February 2014

Spinal cord injury induced changes of nuclear receptors PPARα and LXRβ and modulation with oleic acid/albumin treatment.

Brain Res 2013 Oct 16;1535:89-105. Epub 2013 Aug 16.

Laboratorio Regeneración Nerviosa, Hospital Nacional de Parapléjicos, Servicio de Salud de Castilla la Mancha, Toledo, Spain.

In previous studies with animal models of spinal cord injury (SCI) pharmacological activation of peroxisome proliferator activated receptors (PPAR) and liver X receptors (LXR) were used to reduce tissue damage and promote behavioral recovery in animal models. We have studied the endogenous expression of the transcription factors PPARα and LXRβ in the chronic stage after SCI in rats. The immunohistochemical investigation revealed a long lasting increase in the level of PPARα in white matter in the vicinity of the lesion site. The source of this signal was identified in a subpopulation of astrocytes outside of the glial scar area. Intrathecal injections of oleic acid/albumin reduced the lesion-induced PPARα immunoreactivity. In addition, ependymal cells displayed a prominent PPARα signal in the non-injured spinal cord, and continued to express the receptor as they proliferated and migrated within the damaged tissue. The nuclear receptor LXRβ was detected at similar levels after SCI as in sham operated animals. We found high levels of immunoreactivity in the gray matter, while in the white matter it was present in subpopulations of astrocytes and oligodendrocytes. Macrophages that had accumulated within the center of the lesion contained LXRβ in their cell nuclei. Possible endogenous functions of PPARα and LXRβ after SCI are discussed, specifically the control of fatty acid and cholesterol metabolism and the regulation of inflammatory reactions.
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http://dx.doi.org/10.1016/j.brainres.2013.08.022DOI Listing
October 2013

Expression of retinoid X receptor β is induced in astrocytes during corpus callosum demyelination.

J Chem Neuroanat 2012 Mar 28;43(2):120-32. Epub 2012 Jan 28.

Institut für Biologie II, RWTH Aachen, Germany.

The experimental activation of retinoid receptors reduces pathological symptoms in animal models of multiple sclerosis. In order to assess the involvement of endogenous retinoid signaling during the process of demyelination we investigated retinoic acid synthesizing enzymes and nuclear receptors using the mouse model of cuprizone toxicity. The initiation of myelin degradation in the corpus callosum was accompanied with a local increase of retinaldehyde dehydrogenase (RALDH) immunoreactivity. On the level of receptors we observed a striking increase in protein expression of the retinoid X receptor (RXR)-β in the affected corpus callosum. The RXRβ immunoreactivity appeared exclusively in astrocytes, where it reached a maximum at five weeks of treatment, following the RALDH response. In the cerebral cortex and basal ganglia of affected mice RXRβ was also observed in neurons. Among nuclear receptor antigens RARα showed a cuprizone associated increase in the corpus callosum. Quantitative RT-PCR revealed strong basal expression of RXRβ and a significant, over 20-fold upregulation of the peroxisome proliferator-activated receptor-γ during demyelination. The results indicate that compensatory mechanisms during central demyelination may engage nuclear receptor dimers with an RXRβ partner.
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http://dx.doi.org/10.1016/j.jchemneu.2012.01.002DOI Listing
March 2012

Functional recovery, serotonergic sprouting, and endogenous progenitor fates in response to delayed environmental enrichment after spinal cord injury.

J Neurotrauma 2012 Feb 19;29(3):514-27. Epub 2011 Dec 19.

Department of Anesthesiology, Maastricht University Medical Center, Maastricht, The Netherlands.

Environmental enrichment (EE) is a way to induce voluntary locomotor training that positively affects locomotor recovery after acute spinal cord injury (SCI). The beneficial effect on SCI outcome is thought to be based on enhanced plasticity in motor pathways, triggered by locomotor-specific sensory feedback to the spinal cord circuitry for locomotion (central pattern generators [CPGs]). In view of chronic SCI, we tested the hypothesis that EE improves motor outcome after SCI in the rat when started after a clinically relevant delay of 3 weeks. At the CPG level (i.e., the spinal L1-L2 level), where EE-related sensory feedback is processed, two key mechanisms of anatomical plasticity were examined: (1) serotonergic innervation, and (2) survival and differentiation of spinal cord progenitor cells. Delayed EE improved interlimb coordination, which was associated with an increased serotonergic innervation of the ventro-lateral grey matter within the L1-L2 segments. Although spinal cord progenitor cells were found to differentiate into both neurons and glial cells, EE did not affect their survival. These results show that EE induces a substantial improvement of motor outcome after SCI when commenced after a clinically-relevant delay. Increased serotonergic innervation of the lumbar CPG area is therefore suggested to play an important role in the EE-induced recovery of interlimb coordination.
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http://dx.doi.org/10.1089/neu.2011.1949DOI Listing
February 2012

Inflammatory cytokine release of astrocytes in vitro is reduced by all-trans retinoic acid.

J Neuroimmunol 2010 Dec 9;229(1-2):169-79. Epub 2010 Sep 9.

Institut für Biologie II, RWTH Aachen University, Aachen, Germany.

In the central nervous system inflammation is mediated by microglia and astrocytes. To investigate its regulation, murine astrocyte cultures were treated with bacterial lipopolysaccharides (LPS) and analyzed with Affymetrix gene array, qRT-PCR and ELISA. Cells responded to LPS with a strong upregulation of pro-inflammatory cytokines and chemokines. Treatment with the transcriptional activator retinoic acid (RA) suppressed mRNA expression and protein release of several important cytokines (IL-1β 4%, IL-6 21%, TNFα 30%, IL-12p40 42%, and IL-12p35/p40 27%; p<0.01). The data are consistent with the hypothesis that all-trans RA takes part in endogenous anti-inflammatory feedback loops in the CNS.
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http://dx.doi.org/10.1016/j.jneuroim.2010.08.005DOI Listing
December 2010

Functionalization of electrospun poly(ε-caprolactone) fibers with the extracellular matrix-derived peptide GRGDS improves guidance of schwann cell migration and axonal growth.

Tissue Eng Part A 2011 Feb 21;17(3-4):475-86. Epub 2010 Oct 21.

Institut für Biologie II, RWTH Aachen, Aachen, Germany.

The best available treatment of peripheral nerve lesions involves transplantation of an autologous nerve. This approach, however, entails sensory deficits at the donor site and requires additional surgery. Such limitations have motivated the search for a bioengineering solution to design artificial implants. For this purpose we are producing orientated biodegradable microfibers of poly(ε-caprolactone) (PCL) with electrospinning. The present study describes the functionalization of these electrospun fibers with biologically active peptides to produce guidance structures for Schwann cell migration and axonal regeneration. For the chemical modification PCL was blended with star-shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) (PCL/sPEG) as a covalent linker for the peptide GRGDS, derived from extracellular matrix proteins. To test biological functions of electrospun fibers, Schwann cell migration and axonal growth from dorsal root ganglia explants were investigated with time lapse video microscopy. Migrating Schwann cells as well as growing sensory axons closely followed the electrospun fibers with occasional leaps between adjacent fibers. Cell migration was characterized by frequent changes in velocity and direction reversals. Comparison of substrates showed that functionalized fibers caused more Schwann cells to move out of the explants, supported faster cell migration and axonal growth than the nonfunctional fibers. Using inhibitors of intracellular signaling kinases, we found that these biological effects required activation of the phosphatidyl inositol-3-kinase pathway. Since sPEG-containing fibers also showed low levels of nonspecific protein adsorption, which is desirable in the context of artificial implant design, the peptide modification of fibers appears to provide good substrates for nerve repair.
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http://dx.doi.org/10.1089/ten.TEA.2010.0369DOI Listing
February 2011

Repetitive intrathecal VEGF(165) treatment has limited therapeutic effects after spinal cord injury in the rat.

J Neurotrauma 2010 Oct;27(10):1781-91

Department of Anesthesiology, School for Mental Health and Neuroscience, Maastricht University Medical Center, Maastricht, The Netherlands.

Neuropathic pain and motor deficits are detrimental consequences of injury to the spinal cord. In experimental settings, numerous neuroprotective agents are being explored for their therapeutic benefits. Vascular endothelial growth factor (VEGF) is an interesting candidate molecule in this respect since it is not only associated with angiogenesis, but also with neuroprotection and neurite growth. Other investigators have reported improved motor outcomes following intraparenchymal VEGF treatment. Here we demonstrate the therapeutic effects of daily intrathecal treatment of the contused thoracic rat spinal cord with the 165-isoform of VEGF during the first week after injury. We show that VEGF treatment resulted in a statistically significant attenuation of mechanical, but not thermal, hypersensitivity of the hindpaws, while motor deficits remained unaffected. Tissue sparing was also unchanged by VEGF treatment. Microglial responses at the lumbar spinal cord, which have been linked with spinal cord injury-induced hypersensitivity, were found to be unaffected by VEGF treatment. We conclude that repetitive intrathecal VEGF delivery has limited therapeutic effects on spinal cord injury outcome.
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http://dx.doi.org/10.1089/neu.2010.1484DOI Listing
October 2010

Functionalization of electrospun fibers of poly(epsilon-caprolactone) with star shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) for neuronal cell guidance.

J Mater Sci Mater Med 2010 Sep 22;21(9):2637-51. Epub 2010 Jun 22.

Institute of Technical and Macromolecular Chemistry and DWI e.V., RWTH Aachen University, Pauwelsstr. 8, 52056, Aachen, Germany.

Microfibers produced with electrospinning have recently been used in tissue engineering. In the development of artificial implants for nerve regeneration they are of particular interest as guidance structures for cell migration and axonal growth. Using electrospinning we produced parallel-orientated biocompatible fibers in the submicron range consisting of poly(epsilon-caprolactone) (PCL) and star shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) (sPEG). Addition of the bioactive peptide sequence glycine-arginine-glycine-aspartate-serine (GRGDS) or the extracellular matrix protein fibronectin to the electrospinning solution resulted in functionalized fibers. Surface characteristics and biological properties of functionalized and non-functionalised fibers were investigated. Polymer solutions and electrospinning process parameters were varied to obtain high quality orientated fibers. A polymer mixture containing high molecular weight PCL, PCL-diol, and sPEG permitted a chemical reaction between hydroxyl groups of the diol and isocyanante groups of the sPEG. Surface analysis demonstrated that sPEG at the fiber surface minimized protein adhesion. In vitro experiments using dorsal root ganglia explants showed that the cell repellent property of pure PCL/sPEG fibers was overcome by functionalization either with GRGDS peptide or fibronectin. In this way cell migration and axonal outgrowth along fibers were significantly increased. Thus, functionalized electrospun PCL/sPEG fibers, while preventing non-specific protein adsorption, are a suitable substrate for biological and medical applications.
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http://dx.doi.org/10.1007/s10856-010-4112-7DOI Listing
September 2010

Inflammatory chemokine release of astrocytes in vitro is reduced by all-trans retinoic acid.

J Neurochem 2010 Sep 16;114(5):1511-26. Epub 2010 Jun 16.

Institut für Biologie II, RWTH Aachen University, Germany.

The production of chemokines by astrocytes constitutes an important component of neuroinflammatory processes in the brain. As the transcriptional activator retinoic acid (RA), used for chemotherapy and dermatological applications, exerts anti-inflammatory effects on monocytes and lymphocytes, we have tested whether the physiologically occurring isomer, all-trans RA, affects chemokine expression by astrocytes. Under control conditions, primary cultures of murine cortical astrocytes expressed no or very low levels of CCL and CXCL chemokines. After treatment with bacterial lipopolysaccharides to simulate inflammation in vitro, we detected a strong increase in the release of CCL2 (to > 4 ng/mL in cell culture supernatant), CCL3 (> 20 ng/mL), CCL5 (> 25 ng/mL), CXCL1 (> 30 ng/mL) and CXCL2 (> 20 ng/mL). Although simultaneous exposure to RA did not significantly affect this response, 12 h pre-treatment with 0.1 microM all-trans RA strongly suppressed mRNA expression and protein release of all chemokines. The anti-inflammatory activity of RA engaged RA and retinoid X receptors and correlated with a decreased expression of the lipopolysaccharides co-receptor CD14. A minor reduction of nuclear NF-kappaB was observed but not significant, activation of Jun amino-terminal kinase, p38 and signal transducer and activator of transcription 3 were not altered by RA. The results suggest that retinoids should be further investigated as candidates for the treatment of neuroinflammation.
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http://dx.doi.org/10.1111/j.1471-4159.2010.06867.xDOI Listing
September 2010

Increase of Kv3.1b expression in avian auditory brainstem neurons correlates with synaptogenesis in vivo and in vitro.

Brain Res 2009 Dec 18;1302:64-75. Epub 2009 Sep 18.

Institute of Biology 2, RWTH Aachen University, Aachen, Germany.

In the auditory system voltage-activated currents mediated by potassium channels Kv1.1 and Kv3.1b and their interaction with sodium inward currents play a crucial role for computational function. However, it is unresolved how these potassium channels are developmentally regulated. We have therefore combined a biochemical investigation of Kv1.1 and Kv3.1b protein expression with electrophysiological recordings of membrane currents to characterize neuronal differentiation in the auditory brain stem of the chick. Differentiation in vitro was compared with cells prepared from corresponding embryonic stages in vivo. Using a computer model based on the empirical data we were then able to predict physiological properties of developing auditory brain stem neurons. In vivo Kv3.1b expression increased strongly between E10 and E14, a time of functional synaptogenesis in the auditory brainstem. We also found this increase of expression in vitro, again coinciding with synaptogenesis in the cultures. Whole-cell patch recordings revealed a corresponding increase of the (Kv3.1-like) high threshold potassium current. In contrast, Kv1.1 protein expression failed to increase in vitro, and changes in (Kv1.1-like) low threshold potassium current with time in culture were not significant. Electrophysiological recordings revealed that sodium inward currents increased with cultivation time. Thus, our data suggest that Kv3.1b expression occurs with the onset of functional synaptogenesis, while a different signal, absent from cultures of dissociated auditory brain stem, is needed for Kv1.1 expression. A biophysical model constructed with parameters from our recordings was used to investigate the functional impact of the currents mediated by these channels. We found that during development both high and low threshold potassium currents need to be increased in a concerted manner with the sodium conductance for the neurons to exhibit fast and phasic action potential firing and a narrow time window of coincidence detection.
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http://dx.doi.org/10.1016/j.brainres.2009.09.046DOI Listing
December 2009

Human neural cell interactions with orientated electrospun nanofibers in vitro.

Nanomedicine (Lond) 2009 Jan;4(1):11-30

Institute for Neuropathology, Medical Faculty, RWTH Aachen University, Germany.

Aim: Electrospun nanofibers represent potent guidance substrates for nervous tissue repair. Development of nanofiber-based scaffolds for CNS repair requires, as a first step, an understanding of appropriate neural cell type-substrate interactions.

Materials & Methods: Astrocyte-nanofiber interactions (e.g., adhesion, proliferation, process extension and migration) were studied by comparing human neural progenitor-derived astrocytes (hNP-ACs) and a human astrocytoma cell line (U373) with aligned polycaprolactone (PCL) nanofibers or blended (25% type I collagen/75% PCL) nanofibers. Neuron-nanofiber interactions were assessed using a differentiated human neuroblastoma cell line (SH-SY5Y).

Results & Discussion: U373 cells and hNP-AC showed similar process alignment and length when associated with PCL or Type I collagen/PCL nanofibers. Cell adhesion and migration by hNP-AC were clearly improved by functionalization of nanofiber surfaces with type I collagen. Functionalized nanofibers had no such effect on U373 cells. Another clear difference between the U373 cells and hNP-AC interactions with the nanofiber substrate was proliferation; the cell line demonstrating strong proliferation, whereas the hNP-AC line showed no proliferation on either type of nanofiber. Long axonal growth (up to 600 microm in length) of SH-SY5Y neurons followed the orientation of both types of nanofibers even though adhesion of the processes to the fibers was poor.

Conclusion: The use of cell lines is of only limited predictive value when studying cell-substrate interactions but both morphology and alignment of human astrocytes were affected profoundly by nanofibers. Nanofiber surface functionalization with collagen significantly improved hNP-AC adhesion and migration. Alternative forms of functionalization may be required for optimal axon-nanofiber interactions.
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http://dx.doi.org/10.2217/17435889.4.1.11DOI Listing
January 2009

Deposition of electrospun fibers on reactive substrates for in vitro investigations.

Tissue Eng Part C Methods 2009 Mar;15(1):77-85

Institute for Technical and Macromolecular Chemistry and DWI e.V. RWTH Aachen , Aachen, Germany.

Recent in vitro studies with electrospun nanofibers have used a range of techniques. The in vitro system presented in this article describes electrospun fibers deposited onto chemically reactive substrates to provide fiber adherence and surface chemistry control of the substrate. Fibers of poly(epsilon-caprolactone) (PCL) or of a blend of PCL and collagen type I (C/PCL) were electrospun directly onto collectors coated with isocyanate-terminated star (polyethylene glycol) (sPEG). Alternatively, parallel electrospun fibers were collected on dual collectors in "dilute" quantities and transferred onto sPEG-coated substrates. The initial reactive nature of the substrates allows the collection of very few fibers, which adhere well during frequent washes. Furthermore, the sPEG layer transforms into protein-repellent substrates with the additional potential to include specific cellular mediators such as glycine-arginine-glycine-aspartate-serine (GRGDS) peptides to promote cell adhesion. Therefore, the fiber and substrate chemistry can be modified independently, which is particularly useful for in vitro studies of guided migrating cells. In the present work, dissociated cells of dorsal root ganglia seeded onto the substrates were investigated to assess the influence of different combinations of fiber material, fiber orientation, and surface functionalization. Cell adhesion was observed predominantly on the nanofibers, except when the sPEG layer on the substrate contained GRGDS. On the cell-repellent sPEG substrates, neurites were aligned in direct contact with parallel C/PCL fibers and less so with PCL fibers. In contrast, neurite alignment showed less guidance effect with C/PCL electrospun fibers on the GRGDS/sPEG-coated substrates. Therefore, the combination of oriented biologically active fibers on cell-repellent surfaces enhanced the guidance of such cells. These reactive substrate systems provide a multitude of in vitro combinations for providing cells with specific mediators and, in turn, defining the optimum environment of regenerating devices for in vivo studies.
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http://dx.doi.org/10.1089/ten.tec.2008.0324DOI Listing
March 2009

RAR/RXR and PPAR/RXR signaling in neurological and psychiatric diseases.

Prog Neurobiol 2008 Aug 4;85(4):433-51. Epub 2008 May 4.

Institute of Biology II, RWTH Aachen, Aachen, Germany.

Retinoids are important signals in brain development. They regulate gene transcription by binding to retinoic acid receptors (RAR) and, as was discovered recently, a peroxisome proliferator-activated receptor (PPAR). Traditional ligands of PPAR are best known for their functions in lipid metabolism and inflammation. RAR and PPAR are ligand-activated transcription factors, which share members of the retinoid X receptor (RXR) family as heterodimeric partners. Both signal transduction pathways have recently been implicated in the progression of neurodegenerative and psychiatric diseases. Since inflammatory processes contribute to various neurodegenerative diseases, the anti-inflammatory activity of retinoids and PPARgamma agonists recommends them as potential therapeutic targets. In addition, genetic linkage studies, transgenic mouse models and experiments with vitamin A deprivation provide evidence that retinoic acid signaling is directly involved in physiology and pathology of motoneurons, of the basal ganglia and of cognitive functions. The activation of PPAR/RXR and RAR/RXR transcription factors has therefore been proposed as a therapeutic strategy in disorders of the central nervous system.
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http://dx.doi.org/10.1016/j.pneurobio.2008.04.006DOI Listing
August 2008

Regulation of RALDH-1, RALDH-3 and CYP26A1 by transcription factors cVax/Vax2 and Tbx5 in the embryonic chick retina.

Int J Dev Neurosci 2008 Aug 14;26(5):435-45. Epub 2008 Mar 14.

Institut für Biologie II, RWTH Aachen, Kopernikusstrasse 16, Aachen, Germany.

In vertebrate development, polarity of the dorsal-ventral eye axis is determined by the spatially restricted expression of transcription factors cVax/Vax2 in the ventral retina and Tbx5 in the dorsal retina. In addition, an early gradient of retinoic acid (RA) has been suggested to be involved in this process. Using retroviral transfection of the early eye anlage of chick embryos we investigated whether cVax/Vax2 and Tbx5 regulate the expression of enzymes that determine the retinal distribution of RA. Transfection of cVax/Vax2 completely abolished the spatial expression pattern of the RA synthesizing enzyme RALDH-1, of the RA degrading enzyme Cyp26A1 and caused an ectopic expression of RALDH-3 in the dorsal retina. Transfection of Tbx5 did not significantly alter the distribution of RALDH-1 and RALDH-3 but caused additional patches of Cyp26 outside of its normal domain of expression. The distribution of RA was experimentally changed either by ectopic expression of Cyp26A1, or by applications of RA. These manipulations did not affect the distribution of cVax/Vax2 or interfere with development of the retinotectal projection from dorsal retinal ganglion cells. The results show that in the chick retina the compartmentalized expression of RA metabolizing enzymes requires the spatially restricted expression of the transcription factors cVax/Vax2 and Tbx5. On the other hand, RA may not be decisive for the correct retinal projection toward the optic tectum.
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http://dx.doi.org/10.1016/j.ijdevneu.2008.03.003DOI Listing
August 2008

Anti-inflammatory effect of retinoic acid on prostaglandin synthesis in cultured cortical astrocytes.

J Neurochem 2008 Jul 1;106(1):320-32. Epub 2008 Jul 1.

Institut für Biologie II, RWTH Aachen, Germany.

Prostanoids are important mediators of inflammation and pain signaling. Although it is now well accepted that astrocytes participate in inflammatory reactions in the CNS, the molecular regulation of this activity is still largely unknown. Specifically, the regulation of prostanoid synthesis by this type of glia remains to be resolved. Recent evidence suggests that the transcriptional regulator retinoic acid (RA) is involved in regulation of the immune response. We have investigated the expression pattern of the enzymes that catalyze prostanoid and leukotriene synthesis in cultured cortical astrocytes, their stimulation by lipopolysaccharides (LPS) and their regulation by RA. The data indicate that astrocytes are an important source of prostaglandins (PGs) and that RA reduces their inflammatory biosynthesis. LPS treatment induced the expression of enzymes for the production of arachidonic acid and PGs but caused down-regulation of a PG degrading enzyme and of leukotriene synthesizing enzymes that compete with PG synthesis. Consequently, the secretion of the PGE(2) was highly increased after LPS exposure. RA counteracted the inflammatory regulation of cyclooxygenase (COX)-2 mRNA and protein in astrocytes and thereby reduced the synthesis of PGE(2) by approximately 60%. In the absence of LPS, RA enhanced the expression of COX-1 mRNA. In conclusion, RA might be effective in suppressing inflammatory processes in the brain by inhibiting PG synthesis.
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http://dx.doi.org/10.1111/j.1471-4159.2008.05395.xDOI Listing
July 2008

Expression of enzymes involved in the prostanoid metabolism by cortical astrocytes after LPS-induced inflammation.

J Mol Neurosci 2008 Feb 3;34(2):177-85. Epub 2008 Jan 3.

Institute of Neuroanatomy, Medical Clinic, RWTH, Aachen, Germany.

Neuroinflammatory processes are a common epiphenomenon for a number of neurological and neurodegenerative diseases. Besides microglia, astrocytes are implicated in brain inflammation in response to harmful stimuli and pathological processes. Bacterial endotoxins can induce the synthesis and release of proinflammatory mediators, i.e., cytokines and chemokines, by astroglia. In this study, we have investigated the effect of lipopolysaccharide (LPS) treatment on the expression of enzymes of prostanoid synthesis and degradation in cultured mouse cortical astrocytes using an Affymetrix Gene Chip array, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and an enzyme-immunosorbent assay. LPS treatment induced an upregulation of enzymes responsible for prostaglandin E2 synthesis, a downregulation of enzymes that catalyzes prostaglandin E2 (PGE2) degradation and production of proinflammatory leukotrienes. Changes in enzyme expression were accompanied by a highly significant increase in extracellular PGE2. Our data demonstrate that astrocytes are directly involved in the complex regulation of proinflammatory prostanoids in the CNS under pathological processes, thus being of potential interest as targets for therapeutical interventions. Further studies are required to unravel the different roles and interactions between astroglia and other cells of the brain-intrinsic innate immune system during inflammation.
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http://dx.doi.org/10.1007/s12031-007-9028-4DOI Listing
February 2008

RAR/RXR and PPAR/RXR Signaling in Spinal Cord Injury.

PPAR Res 2007 ;2007:29275

Institute of Biology II, RWTH Aachen University, 52056 Aachen, Germany.

The retinoid acid receptors (RAR) and peroxisome proliferator-activated receptors (PPAR) have been implicated in the regulation of inflammatory reactions. Both receptor families contain ligand-activated transcription factors which form heterodimers with retinoid X receptors (RXR). We review data that imply RAR/RXR and PPAR/RXR pathways in physiological reactions after spinal cord injury. Experiments show how RAR signaling may improve axonal regeneration and modulate reactions of glia cells. While anti-inflammatory properties of PPAR are well documented in the periphery, their possible roles in the central nervous system have only recently become evident. Due to its anti-inflammatory function this transcription factor family promises to be a useful target after spinal cord or brain lesions.
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http://dx.doi.org/10.1155/2007/29275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950239PMC
July 2011

Retinoic acid enhances Erk phosphorylation in the chick retina.

Neurosci Lett 2007 Oct 6;426(1):18-22. Epub 2007 Aug 6.

Institut für Biologie II, RWTH-Aachen, Kopernikusstrasse 16, D-52074 Aachen, Germany.

The transcriptional activator retinoic acid (RA) is a regulator of neural development and regeneration. Synergistic effects with brain-derived neurotrophic factor suggested that RA influences neurotrophin signaling. To test this hypothesis RA was administered systemically to E17 chick embryos, and retinas were prepared 12h and 24h later to measure mRNA or protein expression. While there was no significant influence on activation of Akt, CREB and STAT-3, RA-treatment caused elevated levels of Erk-phosphorylation, a kinase involved in Trk signaling. A small but significant increase in the expression of TrkB mRNA and protein was observed but no significant change in TrkA, TrkC and p75 expression.
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http://dx.doi.org/10.1016/j.neulet.2007.07.039DOI Listing
October 2007

Distribution of the cellular retinoic acid binding protein CRABP-I in the developing chick optic tectum.

Brain Res 2007 Sep 27;1168:21-31. Epub 2007 Jul 27.

Institut für Biologie II, RWTH Aachen, Kopernikusstrasse 16, 52056 Aachen, Germany.

Vitamin A is a major morphogen for the visual system. Most of its effects are mediated by retinoic acid (RA), whose developmental functions include pattern formation, neuronal differentiation and possibly axonal guidance. Although RA has been suggested to regulate development of the retina and its central projection, little is known about the distribution of retinoid receptors and binding proteins in the optic tectum, which in birds is the direct target of most retinofugal axons. We investigated the spatial and temporal distribution of the cellular retinoic acid binding protein-I (CRABP-I) in the chick midbrain. While the precise role of CRABP-I is still unknown, this is an intracellular transport protein for RA, which tends to be expressed in cells that are responsive to retinoic acid. Our data show immunoreactivity of CRABP-I in the tectal anlage at E2.5 and during the entire period of embryonic development. It was found in differentiating neurons of the generative zone, in migrating cells of the prospective stratum griseum et fibrosum superficiale and in mature neurons in this layer. In addition, we detected retinoid receptors RARalpha, RARbeta, RXRalpha, RXRbeta and RXRgamma in the developing tectum. Cell culture experiments demonstrate CRABP-I expression in a subpopulation of tectal neurons as they differentiate in vitro. These results are consistent with a regulatory role of RA in tectal neurogenesis and physiology.
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http://dx.doi.org/10.1016/j.brainres.2007.06.089DOI Listing
September 2007

Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend.

Biomaterials 2007 Jul 19;28(19):3012-25. Epub 2007 Mar 19.

Institut für Biologie II, RWTH Aachen, Aachen, Germany.

Our long-term goal is to develop an artificial implant as a conduit for axonal regeneration after peripheral nerve injury. In this study, biodegradable, aligned poly-epsilon-caprolactone (PCL) and collagen/PCL (C/PCL) nanofibers designed as guidance structures were produced by electrospinning and tested in cell culture assays. We compared fibers of 100% PCL with fibers consisting of a 25:75% C/PCL blend. To test their biocompatibility, assays of cell adhesion, survival, migration, effects on cell morphology, axonal growth and axonal guidance were performed. Both types of eletrospun fibers supported oriented neurite outgrowth and glial migration from dorsal root ganglia (DRG) explants. Schwann cell migration, neurite orientation, and process formation of Schwann cells, fibroblasts and olfactory ensheathing cells were improved on C/PCL fibers, when compared to pure PCL fibers. While the velocity of neurite elongation from DRG explants was higher on PCL fibers, analysis of isolated sensory neurons showed significantly better axonal guidance by the C/PCL material. The data demonstrate that electrospun fibers composed of a collagen and PCL blend represent a suitable substrate for supporting cell proliferation, process outgrowth and migration and as such would be a good material for artificial nerve implants.
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http://dx.doi.org/10.1016/j.biomaterials.2007.03.009DOI Listing
July 2007

Neuronal differentiation of the early embryonic auditory hindbrain of the chicken in primary culture.

Eur J Neurosci 2007 Feb;25(4):974-84

Institute of Biology 2, RWTH Aachen, D-52074 Aachen, Germany.

Neurons in the auditory hindbrain pathway of the chicken are physiologically and morphologically highly specialized. It remains unclear to what extent independent differentiation vs. activity-dependent mechanisms determines the development of this system. To address this question we established a primary culture system of the early auditory hindbrain neurons. Primary cultures of neurons from nucleus magnocellularis and nucleus laminaris were prepared from embryonic day 6.5 chicken. These cells developed in culture under serum-free conditions for up to 15 days. Immunocytochemical staining and whole-cell patch recordings were used to characterize the development of the neurons. A stable expression of the calcium-binding protein calretinin, which serves as a characteristic marker of the auditory pathway, was found at all stages. A voltage-gated potassium channel (Kv3.1b) with a specific function in the auditory system was also expressed after about 1 week in culture. Electrophysiological recordings showed a general maturation of the neuronal phenotype as reflected by an increase in the mean resting membrane potential, a decrease in the mean input resistance as well as a maturation of action potential parameters. Four groups of neurons that generate action potentials could be distinguished. One of these showed the phasic firing pattern of auditory brainstem neurons known from slice preparations. In older cultures we demonstrated functional synaptogenesis in vitro by recording postsynaptic activity elicited by extracellular stimulation and styryl dye loading of vesicles. Thus, isolated neurons from the auditory region of the avian brainstem differentiate to specific neuronal subtypes and autonomously develop synaptic connections in vitro.
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http://dx.doi.org/10.1111/j.1460-9568.2007.05343.xDOI Listing
February 2007