Heike Franke

Institut für Pharmakologie und Toxikologie der Bundeswehr, München, Bavaria, Germany

Are you Heike Franke?

Claim your profile

Publications (97)337.56 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y1R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y1R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPβS (P2Y1,12,13R agonist). The observed stimulatory potential of ADPβS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y1R knockout (P2Y1R(-/-)) mice, the ADP®S-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y1R. Using wortmannin and PD98059 we further show that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPβS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y1R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration. Copyright © 2015. Published by Elsevier Ltd.
    Neuropharmacology 02/2015; · 4.82 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mesenchymal stem cells (MSCs) have been identified as promising candidates for neuroregenerative cell therapies. However, the impact of different isolation procedures on the functional and regenerative characteristics of MSC populations has not been studied thoroughly. To quantify these differences, we directly compared classically isolated bulk bone marrow-derived MSCs (bulk BM-MSCs) to the subpopulation Sca-1+Lin-CD45--derived MSCs- (SL45-MSCs), isolated by FACS from bulk BM-cell suspensions. Both populations were analyzed with respect to functional readouts, i.e. frequency of fibroblast colony forming units (CFU-f), general morphology, and expression of stem cell markers. The SL45-MSC population is characterized by greater morphological homogeneity, higher CFU-f frequency, and significantly increased nestin expression compared to bulk BM-MSCs. We further quantified the potential of both cell populations to enhance neuronal fiber growth, using an ex vivo model of organotypic brain slice co-cultures of the mesocortical dopaminergic projection system. The MSC populations were cultivated underneath the slice co-cultures without direct contact using a transwell system. After cultivation, the fiber density in the border region between the two brain slices was quantified. While both populations significantly enhanced fiber outgrowth as compared to controls, purified SL45-MSCs stimulated fiber growth to a larger degree. Subsequently, we analyzed the expression of different growth factors in both cell populations. The results show a significantly higher expression of BDNF and FGF2 in the SL45-MSCs population. Altogether, we conclude that MSC preparations enriched for primary MSCs promote neuronal regeneration and axonal regrowth, an effect that may be mediated by a higher BDNF secretion.
    Stem Cells and Development 11/2014; · 4.20 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: International Journal of Developmental Neuroscience j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i j d e v n e u Nimodipine enhances neurite outgrowth in dopaminergic brain slice co-cultures a b s t r a c t Calcium ions (Ca 2+) play important roles in neuroplasticity and the regeneration of nerves. Intracellular Ca 2+ concentrations are regulated by Ca 2+ channels, among them L-type voltage-gated Ca 2+ channels, which are inhibited by dihydropyridines like nimodipine. The purpose of this study was to investigate the effect of nimodipine on neurite growth during development and regeneration. As an appropriate model to study neurite growth, we chose organotypic brain slice co-cultures of the mesocortical dopaminergic projection system, consisting of the ventral tegmental area/substantia nigra and the prefrontal cortex from neonatal rat brains. Quantification of the density of the newly built neurites in the border region (region between the two cultivated slices) of the co-cultures revealed a growth promoting effect of nimodipine at concentrations of 0.1 ␮M and 1 ␮M that was even more pronounced than the effect of the growth factor NGF. This beneficial effect was absent when 10 ␮M nimodipine were applied. Toxicological tests revealed that the application of nimodipine at this higher concentration slightly induced caspase 3 activation in the cortical part of the co-cultures, but did neither affect the amount of lactate dehydrogenase release or propidium iodide uptake nor the ratio of bax/bcl-2. Furthermore, the expression levels of different genes were quantified after nimodipine treatment. The expression of Ca 2+ binding proteins, immediate early genes, glial fibrillary acidic protein, and myelin components did not change significantly after treatment, indicating that the regulation of their expression is not primarily involved in the observed nimodipine mediated neurite growth. In summary, this study revealed for the first time a neurite growth promoting effect of nimodipine in the mesocortical dopaminergic projection system that is highly dependent on the applied concentrations.
    International Journal of Developmental Neuroscience 11/2014; 40(40):1-11. · 2.92 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The availability of markers able to provide an insight in protein changes in the central nervous system after fatal traumatic brain injury (TBI) is limited. The present study reports on the semi-quantitative assessments of the immunopositive neuroglial cells (both astrocytes and oligodendrocytes) and neurons for S100 protein (S100) as well as neuronal specific enolase (NSE) in the cerebral cortex, hippocampus and cerebellum with regard to the survival time and the cause of death. Brain tissues of 47 autopsy cases with TBI (survival times between several minutes and 34 days) and 10 age- and gender matched controls (natural deaths) were examined. TBI cases were grouped according to their survival time in acute, subacute and delayed death after brain injury (ABI, n = 25; SBI, n = 18; DBI, n =4). There were no significant changes in the percentages of S100-stained astrocytes between TBI and control cases. The percentages of S100-positive oligodendrocytes in the pericontusional zone (PCZ) in cases with SBI were significantly lower than in controls (p < 0.05) and in ABI (p < 0.05). In the hippocampus, S100-positive oligodendrocytes were significantly lower in cases with ABI and SBI (both p < 0.05) compared to control cases. It is of particular interest that there were also S100-positive neurons in the PCZ and hippocampus in TBI cases > 2 h survival, but not in ABI cases or controls. The percentages of NSE-positive neurons in the hippocampus were likewise significantly lower in cases with ABI compared to controls (p < 0.05) but increased in cases with SBI in PCZ (p < 0.05). In conclusion, the present findings emphasize that S100 and NSE-immunopositivity might be useful for detecting the cause and process of death due to TBI. Further, S100-positivity in neurons may be helpful to estimate the survival time of fatal injuries in legal medicine.
    Journal of neurotrauma. 09/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: We hypothesize that cortical ATP and ADP accumulating in the extracellular space, e.g. during prolonged network activity, contribute to a decline in cognitive performance in particular via stimulation of the G protein-coupled P2Y1 receptor (P2Y1R) subtype. Here, we report first evidence on P2Y1R-mediated control of cognitive functioning in rats using bilateral microinfusions of the selective agonist MRS2365 into medial prefrontal cortex (mPFC). MRS2365 attenuated prepulse inhibition of the acoustic startle reflex while having no impact on startle amplitude. Stimulation of P2Y1Rs deteriorated performance accuracy in the delayed non-matching to position task in a delay dependent manner and increased the rate of magazine entries consistent with both working memory disturbances and impaired impulse control. Further, MRS2365 significantly impaired performance in the reversal learning task. These effects might be related to MRS2365-evoked increase of dopamine observed by microdialysis to be short-lasting in mPFC and long-lasting in the nucleus accumbens. P2Y1Rs were identified on pyramidal cells and parvalbumin-positive interneurons, but not on tyrosine hydroxylase-positive fibers, which argues for an indirect activation of dopaminergic afferents in the cortex by MRS2365. Collectively, these results suggest that activation of P2Y1Rs in the mPFC impairs inhibitory control and behavioral flexibility mediated by increased mesocorticolimbic activity and local disinhibition.Neuropsychopharmacology accepted article preview online, 15 July 2014; doi:10.1038/npp.2014.173.
    Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 07/2014; · 8.68 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background And PurposeIt is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane.Experimental ApproachA molecular model of the pain-relevant human hP2X3 receptor was used to identify amino acid pairs which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP.Key ResultsA series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP), due to the formation of spontaneous inter-subunit disulfide bonds. Dithiothreitol (DTT), by reducing these bonds, reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit crosslinks in cysteine double mutants and in addition, confirmed their correct trimeric assembly and cell surface expression.Conclusions And ImplicationsIn conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.
    British Journal of Pharmacology 07/2014; · 5.07 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole-cell patch clamp recordings from SG spinal cord slices documented that in a low Ca(2+) /no Mg(2+) (low X(2+) ) external medium adenosine triphosphate (ATP)/dibenzoyl-ATP, Bz-ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X(2+) -containing bath medium. The effect of Bz-ATP was antagonized by the selective P2X7 receptor antagonist A-438079. Neuronal, but not astrocytic Bz-ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP-5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H2 O2 potentiated the effect of Bz-ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP-5 and CNQX almost abolished the effect of Bz-ATP, H2 O2 was inactive. A Bz-ATP-dependent and A-438079-antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7-immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA- and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions. GLIA 2014.
    Glia 06/2014; · 5.47 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Unveiling the roles of distinct cell types in brain response to insults is a partially unsolved challenge and a key issue for new neuroreparative approaches. In vivo models are not able to dissect the contribution of residential microglia and infiltrating blood-borne monocytes/macrophages, which are fundamentally undistinguishable; conversely, cultured cells lack original tissue anatomical and functional complexity, which profoundly alters reactivity. Here, we tested whether rodent organotypic co-cultures from mesencephalic ventral tegmental area/substantia nigra and prefrontal cortex (VTA/SN-PFC) represent a suitable model to study changes induced by oxygen/glucose deprivation and reperfusion (OGD/R). OGD/R induced cytotoxicity to both VTA/SN and PFC slices, with higher VTA/SN susceptibility. Neurons were highly affected, with astrocytes and oligodendrocytes undergoing very mild damage. Marked reactive astrogliosis was also evident. Notably, OGD/R triggered the activation of CD68-expressing microglia and increased expression of Ym1 and Arg1, two markers of "alternatively" activated beneficial microglia. Treatment with two well-known neuroprotective drugs, the anticonvulsant agent valproic acid and the purinergic P2-antagonist PPADS, prevented neuronal damage. Thus, VTA/SN-PFC cultures are an integrated model to investigate OGD/R-induced effects on distinct cells and easily screen neuroprotective agents. The model is particularly adequate to dissect the microglia phenotypic shift in the lack of a functional vascular compartment.
    Neurochemistry International 01/2014; · 2.65 Impact Factor
  • Heike Franke, Peter Illes
    [Show abstract] [Hide abstract]
    ABSTRACT: Acute brain injury and neurodegenerative disorders may result in astroglial activation. Astrocytes are able to determine the progression and outcome of these neuropathologies in a beneficial or detrimental way. Nucleotides, e.g. adenosine 5'-triphosphate (ATP), released after acute or chronic neuronal injury, are important mediators of glial activation and astrogliosis.Acute injury may cause significant changes in ATP balance, resulting in (1) a decline of intracellular ATP levels and (2) an increase in extracellular ATP concentrations via efflux from the intracellular space. The released ATP may have trophic effects, but can also act as a proinflammatory mediator or cytotoxic factor, inducing necrosis/apoptosis as a universal "danger" signal. Furthermore, ATP, primarily released from astrocytes, is a means of communication between neurons, glial cells, and intracerebral blood vessels.Astrocytes express a heterogeneous battery of purinergic ionotropic and metabotropic receptors (P2XRs and P2YRs, respectively) to respond to extracellular nucleotides.In this chapter, we summarize the contemporary knowledge on the pathological potential of P2Rs in relation to changes of astrocytic functions, determined by distinct molecular signaling cascades, in a variety of diseases. We discuss specific aspects of reactive astrogliosis, with respect to the involvement of prominent receptor subtypes, such as the P2X7 and P2Y1/2Rs. Examples of purinergic signaling of microglia, oligodendrocytes, and blood vessels under pathophysiological conditions will also be presented.The understanding of the pathological potential of purinergic signaling in "controlling and fine-tuning" of astrocytic responses is important for identifying possible therapeutic principles to treat acute and chronic central nervous system diseases.
    Advances in neurobiology. 01/2014; 11:213-56.
  • Claudia Heine, Heike Franke
    [Show abstract] [Hide abstract]
    ABSTRACT: Organotypic slice co-cultures are suitable tools to study axonal regeneration and development (growth or regrowth) of different projection systems of the CNS under ex vivo conditions.In this chapter, we describe in detail the reconstruction of the mesocortical and nigrostriatal dopaminergic projection system culturing tissue slices from the ventral tegmental area/substantia nigra (VTA/SN) with the prefrontal cortex (PFC) or the striatum (STR). The protocol includes the detailed slice preparation and incubation. Moreover, different application possibilities of the ex vivo model are mentioned; as an example, the substance treatment procedure and biocytin tracing are described to reveal the effect of applied substances on fiber outgrowth.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1162:97-111. · 1.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The adenosine 5'-triphosphate (ATP)-gated P2X7 receptor is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. The P2X7 senses high extracellular ATP concentrations and seems to be implicated in a wide range of cellular functions as well as pathophysiological processes, including immune responses and inflammation, release of gliotransmitters and cytokines, cancer cell growth or development of neurodegenerative diseases. In the present study, we identified natural compounds and analogues that can block or sensitize the ATP (1 mM)-induced Ca(2+) response using a HEK293 cell line stably expressing human P2X7 and fluorometric imaging plate reader technology. For instance, teniposide potently blocked the human P2X7 at sub-miromolar concentrations, but not human P2X4 or rat P2X2. A marked block of ATP-induced Ca(2+) entry and Yo-Pro-1 uptake was also observed in human A375 melanoma cells and mouse microglial cells, both expressing P2X7. On the other hand, agelasine (AGL) and garcinolic acid (GA) facilitated the P2X7 response to ATP in all three cell populations. GA also enhanced the YO-PRO-1 uptake, whereas AGL did not affect the ATP-stimulated intracellular accumulation of this dye. According to the pathophysiological role of P2X7 in various diseases, selective modulators may have potential for further development, e.g. as neuroprotective or antineoplastic drugs.
    Purinergic Signalling 10/2013; · 2.64 Impact Factor
  • Heike Franke, Peter Illes
    [Show abstract] [Hide abstract]
    ABSTRACT: Acute and chronic damage to the central nervous system (CNS) releases large quantities of ATP. Whereas the ATP concentration in the extracellular space is normally in the micromolar range, under these conditions it increases to millimolar levels. Ligand-gated cationic channels termed P2X receptors (7 mammalian subtypes), and G protein-coupled P2Y receptors (8 mammalian subtypes) are located at astrocytes, as confirmed by the measurement of the respective mRNA and protein. Activation of both the P2X7 and P2Y1,2 subtypes identified at astrocytes initiates astrogliosis isolating damaged brain areas from surrounding healthy cells and synthesizing neurotrophins and pleotrophins that participate in neuronal recovery. Astrocytes are considered as cells of high plasticity which may alter their properties in a culture medium. Therefore, recent work concentrates on investigating nucleotide effects at in situ (acute brain slices) and in vivo astrocytes. A wealth of data relates to the involvement of purinergic mechanisms in astrogliosis induced by acute CNS injury such as mechanical trauma and hypoxia/ischemia. The released ATP may act within minutes as an excitotoxic molecule; at a longer time-scale within days it causes neuroinflammation. These effects sum up as necrosis/apoptosis on the one hand and proliferation on the other. Although the role of nucleotides in chronic neurodegenerative illnesses is not quite clear, it appears that they aggravate the consequences of the primary disease. Epilepsy and neuropathic pain are also associated with the release of ATP and a pathologic glia-neuron interaction leading to astrogliosis and cell death. In view of these considerations, P2 receptor antagonists may open new therapeutic vistas in all forms of acute and chronic CNS damage.
    Neuroscience Letters 10/2013; · 2.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients' post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.
    Purinergic Signalling 06/2013; 9(3). · 2.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Postmortem analysis of relevant biomarkers might aid in characterizing causes of death and survival times in legal medicine. However, there are still no sufficiently established results of practical postmortem biochemical investigations in cases of traumatic brain injury (TBI). The two biomarkers S100B and neuronal specific enolase (NSE) could be of special interest. Therefore, the aim of the present study was to investigate changes in their postmortem levels for further determination of brain damage in TBI. In 17 cases of TBI (average age: 58 years) and in 23 controls with different causes of death (average age: 59 years), serum and cerebrospinal fluid (CSF) samples were analyzed with a chemiluminescence immunoassay for marker expression. An increase in serum S100B as well as a subsequent decrease after survival times > 4 days were detected in TBI cases (p < 0.01). CSF NSE values > 6,000 ng/ml and CSF S100B levels > 10,000 ng/ml seem to indicate a TBI survival time of at least 15 minutes (p < 0.01). It will be of particular interest that CSF S100B levels (p < 0.01) and serum S100B levels (p < 0.05) as well as CSF NSE values (p < 0.01) were significantly higher in TBI cases in comparison to the controls, especially when compared to fatal non-head injuries. In conclusion, the present findings emphasize that S100B and NSE are useful markers also in postmortem biochemistry in cases of suspected TBI. Furthermore, S100B may be helpful to estimate the survival time of fatal injuries in legal medicine.
    Journal of neurotrauma 06/2013; · 4.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Neurogenesis requires the balance between the proliferation of newly formed progenitor cells and subsequent death of surplus cells. RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptor mRNA and immunoreactivity in cultured neural progenitor cells (NPCs) prepared from the adult mouse subventricular zone (SVZ). Whole-cell patch-clamp recordings showed a marked potentiation of the inward current responses both to ATP and the prototypic P2X7 receptor agonist dibenzoyl-ATP (Bz-ATP) at low Ca(2+) and zero Mg(2+) concentrations in the bath medium. The Bz-ATP-induced currents reversed their polarity near 0 mV; in NPCs prepared from P2X7(-/-) mice, Bz-ATP failed to elicit membrane currents. The general P2X/P2Y receptor antagonist PPADS and the P2X7 selective antagonists Brilliant Blue G and A-438079 strongly depressed the effect of Bz-ATP. Long-lasting application of Bz-ATP induced an initial current, which slowly increased to a steady-state response. In combination with the determination of YO-PRO uptake, these experiments suggest the dilation of a receptor-channel and/or the recruitment of a dye-uptake pathway. Ca(2+)-imaging by means of Fura-2 revealed that in a Mg(2+)-deficient bath medium Bz-ATP causes [Ca(2+)]i transients fully depending on the presence of external Ca(2+). The MTT test indicated a concentration-dependent decrease in cell viability by Bz-ATP treatment. Correspondingly, Bz-ATP led to an increase in active caspase 3 immunoreactivity, indicating a P2X7-controlled apoptosis. In acute SVZ brain slices of transgenic Tg(nestin/EGFP) mice, patch-clamp recordings identified P2X7 receptors at NPCs with pharmacological properties identical to those of their cultured counterparts. We suggest that the apoptotic/necrotic P2X7 receptors at NPCs may be of particular relevance during pathological conditions which lead to increased ATP release and thus could counterbalance the ensuing excessive cell proliferation.
    Neuropharmacology 05/2013; · 4.82 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Drug addiction is a chronic, relapsing disease caused by neurochemical and molecular changes in the brain. In this human autopsy study qualitative and quantitative changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the hippocampus of 26 lethally intoxicated drug addicts and 35 matched controls are described. The morphological characterization of these cells reflected alterations representative for astrogliosis. But, neither quantification of GFAP-positive cells nor the Western blot analysis indicated statistical significant differences between drug fatalities versus controls. However, by semi-quantitative scoring a significant shift towards higher numbers of activated astrocytes in the drug group was detected. To assess morphological changes quantitatively, graph-based representations of astrocyte morphology were obtained from single cell images captured by confocal laser scanning microscopy. Their underlying structures were used to quantify changes in astroglial fibers in an automated fashion. This morphometric analysis yielded significant differences between the investigated groups for four different measures of fiber characteristics (Euclidean distance, graph distance, number of graph elements, fiber skeleton distance), indicating that e.g. astrocytes in drug addicts on average exhibit significant elongation of fiber structures as well as two fold increase in GFAP-positive fibers as compared with those in controls. In conclusion, the present data show characteristic differences in morphology of hippocampal astrocytes in drug addicts versus controls and further supports the involvement of astrocytes in human pathophysiology of drug addiction. The automated quantification of astrocyte morphologies provides a novel, testable way to assess the fiber structures in a quantitative manner as opposed to standard, qualitative descriptions.
    Brain research 01/2013; 1500:72-87. · 2.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gene expression of specific brain biomarkers offers the possibility to shed light on the diffi-cult molecular pathways of traumatic brain injury (TBI) and may be useful to estimate the age of trauma. Gene expression rates of cerebellar injuries are not yet sufficiently established. In 12 cases of TBI including a pathological change in cerebellum (mean age 42 years) with known survival times ranging from an immediate death up to 96 hours, brain tissue samples from different brain regions were analyzed with real-time PCR for expression of Caspase-3, TrkB, S100B and GFAP mRNA. The pH was measured to gain information about a possible correlation to RNA degradation. For comparison corresponding brain regions were arranged from control samples died from sudden death. We found a correlation between pH and the degradation of RNA in samples from the contra-lateral site, where the samples with degraded RNA have a lower pH (p < 0.05). For short sur-vival times, the expression changes of Caspase-3 (p < 0.05) and also the expression changes of TrkB (p < 0.1) in the cerebellum show a significant increase compared to the controls. The cerebellar gene expression changes seem to occur much faster and stronger compared to the other investigated regions, in particular the cerebral trauma site. These findings could make the cerebellum to an important target to study the expression changes after TBI.
    Journal of neurotrauma 10/2012; · 4.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: CNS reparative-medicine therapeutic strategies need answers on the putative recapitulation of the basic rules leading to mammalian CNS development. To achieve this aim, we focus on the regeneration of functional connections in the mesocorticolimbic dopaminergic system. We used organotypic slice cocultures of ventral tegmental area/substantia nigra (VTA/SN) and prefrontal cortex (PFC) on a multielectrode array (MEA) platform to record spikes and local field potentials. The spontaneously growing synaptically based bidirectional bursting activity was followed from 2 to 28 days in vitro (DIV). A statistical analysis of excitatory and inhibitory neurons properties of the physiological firing activity demonstrated a remarkable, exponentially increasing maturation with a time constant of about 5-7 DIV. Immunohistochemistry demonstrated that the ratio of excitatory/inhibitory neurons (3:1) was in line with the functional results obtained. Exemplary pharmacology suggested that GABA(A) receptors were able to exert phasic and tonic inhibition typical of an adulthood network. Moreover, dopamine D2 receptor inactivation was equally inhibitory both on the spontaneous neuronal activity recorded by MEA and on patch-clamp electrophysiology in PFC pyramidal neurons. These results demonstrate that axon growth cones reach synaptic targets up to full functionality and that organotypic cocultures of the VTA/SN-PFC perfectly model their newly born dopaminergic, glutamatergic and GABAergic neuronal circuitries.
    Cerebral Cortex 09/2012; · 8.31 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The development of appropriate models assessing the potential of substances for regeneration of neuronal circuits is of great importance. Here, we present procedures to analyze effects of substances on fiber outgrowth based on organotypic slice co-cultures of the nigrostriatal dopaminergic system in combination with biocytin tracing and tyrosine hydroxylase labeling and subsequent automated image quantification. Selected phosphodiesterase inhibitors (PDE-Is) were studied to identify their potential growth-promoting capacities. Immunohistochemical methods were used to visualize developing fibers in the border region between ventral tegmental area/substantia nigra co-cultivated with the striatum as well as the cellular expression of PDE2A and PDE10. The quantification shows a significant increase of fiber density in the border region induced by PDE2-Is (BAY60-7550; ND7001), comparable with the potential of the nerve growth factor and in contrast to PDE10-I (MP-10). Analysis of tyrosine hydroxylase-positive fibers indicated a significant increase after treatment with BAY60-7550 and nerve growth factor in relation to dimethyl sulfoxide. Additionally, a dose-dependent increase of intracellular cGMP levels in response to the applied PDE2-Is in PDE2-transfected HEK293 cells was found. In summary, our findings show that PDE2-Is are able to significantly promote axonal outgrowth in organotypic slice co-cultures, which are a suitable model to assess growth-related effects in neuro(re)generation.
    Neurosignals 08/2012; · 4.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Astrocytes are fundamental for central nervous system (CNS) physiology and are the fulcrum of neurological diseases. Astroglial cells control development of the nervous system, regulate synaptogenesis, maturation, maintenance and plasticity of synapses and are central for nervous system homeostasis. Astroglial reactions determine progression and outcome of many neuropathologies and are critical for regeneration and remodelling of neural circuits following trauma, stroke, ischaemia or neurodegenerative disorders. They secrete multiple neurotransmitters and neurohormones to communicate with neurones, microglia and the vascular walls of capillaries. Signalling through release of ATP is the most widespread mean of communication between astrocytes and other types of neural cells. ATP serves as a fast excitatory neurotransmitter and has pronounced long-term (trophic) roles in cell proliferation, growth, and development. During pathology, ATP is released from damaged cells and acts both as a cytotoxic factor and a proinflammatory mediator, being a universal "danger" signal. In this review, we summarise contemporary knowledge on the role of purinergic receptors (P2Rs) in a variety of diseases in relation to changes of astrocytic functions and nucleotide signalling. We have focussed on the role of the ionotropic P2X and metabotropic P2YRs working alone or in concert to modify the release of neurotransmitters, to activate signalling cascades and to change the expression levels of ion channels and protein kinases. All these effects are of great importance for the initiation, progression and maintenance of astrogliosis-the conserved and ubiquitous glial defensive reaction to CNS pathologies. We highlighted specific aspects of reactive astrogliosis, especially with respect to the involvement of the P2X(7) and P2Y(1)R subtypes. Reactive astrogliosis exerts both beneficial and detrimental effects in a context-specific manner determined by distinct molecular signalling cascades. Understanding the role of purinergic signalling in astrocytes is critical to identifying new therapeutic principles to treat acute and chronic neurological diseases.
    Purinergic Signalling 05/2012; 8(3):629-57. · 2.64 Impact Factor

Publication Stats

2k Citations
337.56 Total Impact Points

Institutions

  • 2014
    • Institut für Pharmakologie und Toxikologie der Bundeswehr
      München, Bavaria, Germany
  • 1997–2014
    • University of Leipzig
      • • Rudolf Boehm Institute of Pharmacology and Toxicology
      • • Institut für Pharmakologie, Pharmazie und Toxikologie
      Leipzig, Saxony, Germany
  • 2011
    • Friedrich-Alexander Universität Erlangen-Nürnberg
      • Department of Neurology
      Erlangen, Bavaria, Germany
  • 1999
    • Forschungszentrum Jülich
      Jülich, North Rhine-Westphalia, Germany