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Barclay Morrison - One of the best experts on this subject based on the ideXlab platform.
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isolated primary blast inhibits long term potentiation in organotypic Hippocampal Slice cultures
Journal of Neurotrauma, 2016Co-Authors: Edward W Vogel, Gwen Brink Effgen, Tapan P Patel, David F Meaney, Cameron Dale R Bass, Barclay MorrisonAbstract:Abstract Over the last 13 years, traumatic brain injury (TBI) has affected over 230,000 U.S. service members through the conflicts in Iraq and Afghanistan, mostly as a result of exposure to blast events. Blast-induced TBI (bTBI) is multi-phasic, with the penetrating and inertia-driven phases having been extensively studied. The effects of primary blast injury, caused by the shockwave interacting with the brain, remain unclear. Earlier in vivo studies in mice and rats have reported mixed results for primary blast effects on behavior and memory. Using a previously developed shock tube and in vitro sample receiver, we investigated the effect of isolated primary blast on the electrophysiological function of rat organotypic Hippocampal Slice cultures (OHSC). We found that pure primary blast exposure inhibited long-term potentiation (LTP), the electrophysiological correlate of memory, with a threshold between 9 and 39 kPa·ms impulse. This deficit occurred well below a previously identified threshold for cell de...
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isolated primary blast alters neuronal function with minimal cell death in organotypic Hippocampal Slice cultures
Journal of Neurotrauma, 2014Co-Authors: Gwen Brink Effgen, David F Meaney, Edward Vogel, Kimberly A Lynch, Ayelet Lobel, Cameron Dale Bass, Barclay MorrisonAbstract:Abstract An increasing number of U.S. soldiers are diagnosed with traumatic brain injury (TBI) subsequent to exposure to blast. In the field, blast injury biomechanics are highly complex and multi-phasic. The pathobiology caused by exposure to some of these phases in isolation, such as penetrating or inertially driven injuries, has been investigated extensively. However, it is unclear whether the primary component of blast, a shock wave, is capable of causing pathology on its own. Previous in vivo studies in the rodent and pig have demonstrated that it is difficult to deliver a primary blast (i.e., shock wave only) without rapid head accelerations and potentially confounding effects of inertially driven TBI. We have previously developed a well-characterized shock tube and custom in vitro receiver for exposing organotypic Hippocampal Slice cultures to pure primary blast. In this study, isolated primary blast induced minimal Hippocampal cell death (on average, below 14% in any region of interest), even for ...
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gpr30 activation is neither necessary nor sufficient for acute neuroprotection by 17β estradiol after an ischemic injury in organotypic Hippocampal Slice cultures
Brain Research, 2014Co-Authors: Michael R Lamprecht, Barclay MorrisonAbstract:Abstract In this study, we investigated the role of GPR30 in 17β-estradiol- (E2) mediated neuroprotection after an ischemic injury in an organotypic Hippocampal Slice culture (OHSC) model. We report that after oxygen–glucose deprivation (OGD), a physiological concentration of 100 pM E2 provided the greatest significant reduction in cell death while supra-physiological levels were less effective. The canonical estrogen receptor (ER) inhibitor ICI 182,780 completely abrogated the therapeutic effect of E2 while the GPR30 antagonist G−15 effected a slight but not significant reduction in neuroprotection. Only supra-physiological levels of E2 led to significantly increased phosphorylation of Akt and Erk which are well known downstream effects of GPR30 activation. We conclude that GPR30 activation may facilitate acute E2 mediated neuroprotection after OGD, but is neither necessary nor sufficient.
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gpr30 activation is neither necessary nor sufficient for acute neuroprotection by 17β estradiol after an ischemic injury in organotypic Hippocampal Slice cultures
Brain Research, 2014Co-Authors: Michael R Lamprecht, Barclay MorrisonAbstract:Abstract In this study, we investigated the role of GPR30 in 17β-estradiol- (E2) mediated neuroprotection after an ischemic injury in an organotypic Hippocampal Slice culture (OHSC) model. We report that after oxygen–glucose deprivation (OGD), a physiological concentration of 100 pM E2 provided the greatest significant reduction in cell death while supra-physiological levels were less effective. The canonical estrogen receptor (ER) inhibitor ICI 182,780 completely abrogated the therapeutic effect of E2 while the GPR30 antagonist G−15 effected a slight but not significant reduction in neuroprotection. Only supra-physiological levels of E2 led to significantly increased phosphorylation of Akt and Erk which are well known downstream effects of GPR30 activation. We conclude that GPR30 activation may facilitate acute E2 mediated neuroprotection after OGD, but is neither necessary nor sufficient.
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neuroprotection by genipin against reactive oxygen and reactive nitrogen species mediated injury in organotypic Hippocampal Slice cultures
Brain Research, 2014Co-Authors: Rebecca H Hughes, David I. Shreiber, Victoria A Silva, Ijaz Ahmed, Barclay MorrisonAbstract:Genipin, the multipotent ingredient in Gardenia jasmenoides fruit extract (GFE), may be an effective candidate for treatment following stroke or traumatic brain injury (TBI). Secondary injury includes damage mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can alter the biological function of key cellular structures and eventually lead to cell death. In this work, we studied the neuroprotective potential of genipin against damage stemming from ROS and RNS production in organotypic Hippocampal Slice cultures (OHSC), as well as its potential as a direct free radical scavenger. A 50 mM dose of genipin provided significant protection against tert-butyl hydroperoxide (tBHP), a damaging organic peroxide. This dosage of genipin significantly reduced cell death at 48 h compared to vehicle control (0.1% DMSO) when administered 0, 1, 6, and 24 h after addition of tBHP. Similarly, genipin significantly reduced cell death at 48 hw hen administered 0, 1, 2, and 6ha fter addition of rotenone, which generates reactive oxygen species via a more physiologically relevant mechanism. Furthermore, genipin significantly reduced both cell death and nitrite levels at 24 h caused by S-nitroso-N- acetylpenicillamine (SNAP), a direct nitric oxide (NO) donor, and successfully quenched 1,1- Diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, suggesting that genipin may act as a direct free radical scavenger. Our encouraging findings suggest that genipin should be tested in animal models of CNS injury with a significant component of ROS- and RNS-mediated damage, such as TBI and stroke, to assess its in vivo efficacy.
Jens Zimmer - One of the best experts on this subject based on the ideXlab platform.
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comparison of neuroprotective effects of erythropoietin epo and carbamylerythropoietin cepo against ischemia like oxygen glucose deprivation ogd and nmda excitotoxicity in mouse Hippocampal Slice cultures
Experimental Neurology, 2007Co-Authors: Maria Montero, Jens Noraberg, Frantz Rom Poulsen, Agnete Kirkeby, Johan Van Beek, Marcel Leist, Jens ZimmerAbstract:In addition to its well-known hematopoietic effects, erythropoietin (EPO) also has neuroprotective properties. However, hematopoietic side effects are unwanted for neuroprotection, underlining the need for EPO-like compounds with selective neuroprotective actions. One such compound, devoid of hematopoietic bioactivity, is the chemically modified, EPO-derivative carbamylerythropoietin (CEPO). For comparison of the neuroprotective effects of CEPO and EPO, we subjected organotypic Hippocampal Slice cultures to oxygen–glucose deprivation (OGD) or N-methyl-D-aspartate (NMDA) excitotoxicity. Hippocampal Slice cultures were pretreated for 24 h with 100 IU/ml EPO (=26 nM) or 26 nM CEPO before OGD or NMDA lesioning. Exposure to EPO and CEPO continued during OGD and for the next 24 h until histology, as well as during the 24 h exposure to NMDA. Neuronal cell death was quantified by cellular uptake of propidium iodide (PI), recorded before the start of OGD and NMDA exposure and 24 h after. In cultures exposed to OGD or NMDA, CEPO reduced PI uptake by 49±3 or 35±8%, respectively, compared to lesion-only controls. EPO reduced PI uptake by 33±5 and 15±8%, respectively, in the OGD and NMDA exposed cultures. To elucidate a possible mechanism involved in EPO and CEPO neuroprotection against OGD, the integrity of α-II-spectrin cytoskeletal protein was studied. Both EPO and CEPO significantly reduced formation of spectrin cleavage products in the OGD model. We conclude that CEPO is at least as efficient neuroprotectant as EPO when excitotoxicity is modeled in mouse Hippocampal Slice cultures.
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Organotypic Hippocampal Slice cultures for studies of brain damage, neuroprotection and neurorepair.
Current Drug Target -CNS & Neurological Disorders, 2005Co-Authors: Jens Noraberg, Bjarne Winther Kristensen, Frantz Rom Poulsen, Morten Blaabjerg, Christian Bonde, Maria Montero, Morten Meyer, Jan Bert Gramsbergen, Jens ZimmerAbstract:Slices of developing brain tissue can be grown for several weeks as socalled organotypic Slice cultures. Here we summarize and review studies using Hippocampal Slice cultures to investigate mechanisms and treatment strategies for the neurodegenerative disorders like stroke (cerebral ischemia), Alzheimer's disease (AD) and epilepsia. Studies of non-excitotox ic neurotoxic compounds and the experimental use of Slice cultures in studies of HIV neurotoxicity, traumatic brain injury (TBI) and neurogenesis are included. For cerebral ischemia, experimental models with oxygen-glucose deprivation (OGD) and exposure to glutamate receptor agonists (excitotoxins) are reviewed. For epilepsia, focus is on induction of seizures with effects on neuronal loss, axonal sprouting and neurogenesis. For Alzheimer's disease, the review centers on the use of beta-amyloid (Abeta) in different models, while the section on repair is focused on neurogenesis and cell migration. The culturing techniques, set-up of models, and analytical tools, including markers for neurodegeneration, like the fluorescent dye propidium iodide (PI), are reviewed and discussed. Comparisons are made between Hippocampal Slice cultures and other in vitro models using dispersed cell cultures, experimental in vivo models, and in some instances, clinical trials. New techniques including Slice culturing of Hippocampal tissue from transgenic mice as well as more mature brain tissue, and Slice cultures coupled to microelectrode arrays (MEAs), on-line biosensor monitoring, and time-lapse fluorescence microscopy are also presented.
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modulator effects of interleukin 1beta and tumor necrosis factor alpha on ampa induced excitotoxicity in mouse organotypic Hippocampal Slice cultures
The Journal of Neuroscience, 2005Co-Authors: Liliana Bernardino, Frantz Rom Poulsen, Sara Xapelli, Ana P Silva, Birthe Jakobsen, Catarina R Oliveira, Annamaria Vezzani, Joao O Malva, Jens ZimmerAbstract:The inflammatory cytokines interleukin-1β and tumor necrosis factor-α (TNF-α) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects of these cytokines on neuronal death caused by exposure of mouse organotypic Hippocampal Slice cultures to toxic concentrations of AMPA. Either potentiation of excitotoxicity or neuroprotection was observed, depending on the concentration of the cytokines and the timing of exposure. A relatively high concentration of mouse recombinant TNF-α (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-α to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol. By using TNF-α receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-α involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed by macrophage antigen-1 and bromodeoxyuridine immunohistochemistry, suggesting a functional recruitment of cytokine-producing cells at sites of neurodegeneration. Together, these findings are relevant for understanding the role of proinflammatory cytokines and microglia activation in acute and chronic excitotoxic conditions.
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ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen glucose deprivation of Hippocampal Slice cultures
Neuroscience, 2005Co-Authors: Christian Bonde, Jens Noraberg, Helle Noer, Jens ZimmerAbstract:Organotypic Hippocampal Slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen-glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on Hippocampal Slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen-glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal Slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen-glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen-glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen-glucose deprivation induced necrotic cell death only. Addition of 10 microM of the N-methyl-D-aspartate glutamate receptor antagonist MK-801, and 20 microM of the non-N-methyl-D-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline to the culture medium confirmed that both N-methyl-D-aspartate and non-N-methyl-D-aspartate ionotropic glutamate receptors were involved in the oxygen-glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the DL-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of DL-threo-beta-benzyloxyaspartate in concentrations of 25 microM or more to otherwise untreated Hippocampal Slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen-glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10 microM) dose of DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation (but not during the next 48 h after oxygen-glucose deprivation) significantly reduced the oxygen-glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the Slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen-glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen-glucose deprivation-induced necrotic cell death in Hippocampal Slice cultures, which have proven to be a feasible tool in experimental studies on this topic.
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The GABAA receptor agonist THIP is neuroprotective in organotypic Hippocampal Slice cultures
Brain Research, 2003Co-Authors: Bjarne Winther Kristensen, Jens Noraberg, Jens ZimmerAbstract:The potential neuroprotective effects of the GABA(A) receptor agonists THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and muscimol, and the selective GluR5 kainate receptor agonist ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid), which activates GABAergic interneurons, were examined in Hippocampal Slice cultures exposed to N-methyl-D-aspartate (NMDA). The NMDA-induced excitotoxicity was quantified by densitometric measurements of propidium iodide (PI) uptake. THIP (100-1000 microM) was neuroprotective in Slice cultures co-exposed to NMDA (10 microM) for 48 h, while muscimol (100-1000 microM) and ATPA (1-3 microM) were without effect. The results demonstrate that direct GABA(A) agonism can mediate neuroprotection in the hippocampus in vitro as previously suggested in vivo.
Uwe Heinemann - One of the best experts on this subject based on the ideXlab platform.
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primary afterdischarge in organotypic Hippocampal Slice cultures effects of standard antiepileptic drugs
Epilepsia, 2012Co-Authors: Klaus Albus, Abdul Wahab, Uwe HeinemannAbstract:Summary Purpose: In the hippocampus intense high frequency electrical stimulation induces a long-lasting rhythmic synchronization (primary afterdischarge). In order to examine the suitability of primary afterdischarges (PADs) in organotypic Hippocampal Slice cultures (OHSCs) as an in vitro model of evoked seizures, we have worked out in detail the sensitivity of PADs to standard antiepileptic drugs (AEDs) and compared the necessary concentrations to those that were effective in animal models of partial and generalized tonic–clonic seizures. Methods: OHSCs were prepared according to the interface culture method from 8 to 11-day-old Wistar rats. A PAD in CA1 was elicited by stimulating the stratum radiatum with an intensity of two times that required to elicit a maximal population spike. The effects of carbamazepine, phenytoin, valproic acid, phenobarbital, diazepam, and ethosuximide on the duration and on frequency properties of PADs and the tonic-like and clonic-like subdivisions of PADs were determined, and as a measure of the AEDs potency half maximal effective concentration (EC50) values were calculated from concentration–response curves. Key Finding: Carbamazepine, phenytoin, valproic acid, phenobarbital, and diazepam reduced the durations of PADs and tonic-like and clonic-like subdivisions of PADs. The effects were concentration dependent and reversible. Ethosuximide was ineffective. The effects on subdivisions of PADs differed between AEDs. Carbamazepine and phenytoin shortened the tonic-like and clonic-like subdivisions at similar proportions, whereas phenobarbital, diazepam, and valproic acid preferentially shortened the clonic-like subdivision. Diazepam at low concentrations increased the duration of tonic-like subdivisions, an effect not seen with the other AEDs. The suppressive effects of AEDs on frequency properties of tonic-like and clonic-like subdivisions were variable and observed only at higher concentrations. Significance: Carbamazepine and phenytoin were more effective in the PAD test in OHSCs than in the maximal electroshock and kindled seizures tests. The effectiveness of phenobarbital, diazepam, and valproic acid in the PAD test matched their effectiveness in the MES test and—with the exception of valproic acid and diazepam—in kindled seizures tests. Valproic acid was less effective in OHSCs than in the kindled seizure tests, and diazepam was more (generalized seizures) or less (focal seizures and afterdischarge durations) effective in this animal model than in OHSCs. We conclude that the PAD test in OHSCs is a suitable in vitro model of evoked seizures. The model could serve as an initial screen to identify the most promising leads for further evaluation and characterization in in vivo models of efficacy and toxicity.
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Effects of γ-aminobutyric acid (GABA) agonists and a GABA uptake inhibitor on pharmacoresistant seizure like events in organotypic Hippocampal Slice cultures
Epilepsy Research, 2009Co-Authors: Abdul Wahab, Uwe Heinemann, Klaus AlbusAbstract:Summary Purpose Seizure like events (SLEs) induced by low magnesium or 4-aminopyridine in organotypic Hippocampal Slice cultures (OHSCs) are resistant to standard antiepileptic drugs including phenobarbital, and 1,4-benzodiazepines [Albus, K., Wahab, A., Heinemann, U., 2008. Standard antiepileptic drugs fail to block epileptiform activity in rat organotypic Hippocampal Slice cultures. Br. J. Pharmacol. 154, 709–724]. The present study was undertaken in order to test the effects of other compounds on SLEs in OHSCs that enhance GABA-mediated actions. Methods Six to 12 days old Wistar rats were used to cultivate OHSCs according to the interface method [Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182]. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering the magnesium concentration. The effects of GABA A agonists muscimol and isoguvacine, the GABA B agonist baclofen, the GABA uptake blocker nipecotic acid and the neurosteroid alfaxalone on induction and ongoing SLEs were analyzed. Results Low magnesium induced SLEs were dose dependently suppressed by the GABA A receptor agonists muscimol, isoguvacine and alfaxalone and by the GABA uptake inhibitor nipecotic acid whereas the GABA B receptor agonist baclofen attenuated but did not suppress SLE. Discussion Our findings demonstrate that in OHSCs GABA has an inhibitory effect on SLEs. Proconvulsant effects of GABA agonists on spontaneous neuronal activity and seizure like activity were never observed. Our findings exclude a possible contribution of impaired/altered GABA-ergic mechanisms based on immaturity of receptors and/or low receptor density to seizure susceptibility and pharmacoresistance in OHSCs.
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standard antiepileptic drugs fail to block epileptiform activity in rat organotypic Hippocampal Slice cultures
British Journal of Pharmacology, 2008Co-Authors: Klaus Albus, Abdul Wahab, Uwe HeinemannAbstract:Background and purpose: Earlier studies had demonstrated that tonic–clonic seizure-like events (SLEs) resembling electrographic correlates of limbic seizures in animals and humans can be induced in organotypic Hippocampal Slice cultures (OHSCs). We have explored OHSCs for their suitability to serve as in vitro models of limbic seizures for studying seizure mechanisms and screening new antiepileptic compounds. Experimental approach: OHSCs were cultivated according to the interface method. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering magnesium concentration or by applying the potassium channel blocker 4-aminopyridine. The effects of standard antiepileptic drugs (AEDs), carbamazepine, phenytoin, valproic acid, clonazepam, diazepam and phenobarbital sodium on SLEs were analysed. Key results: In more than 93% of OHSCs, AEDs did not prevent the induction of SLEs or stop ongoing seizure activity even when toxic concentrations were applied. This pharmacoresistance was independent of the method of seizure provocation, postnatal age at explantation (P2–P10) and cultivation time in vitro (2 months). SLEs were reversibly blocked by glutamate antagonists or the GABAA-agonist muscimol. Conclusions and implications: We present a simple to establish in vitro model of tonic–clonic SLEs that is a priori pharmacoresistant and thus has an advantage over animal models of pharmacoresistant seizures in which responders and non-responders can be sorted out only after an experiment. OHSCs could be suitable for exploring mechanisms of pharmacoresistant seizures and be used for the identification of new anticonvulsive compounds eventually effective in drug refractory epilepsy. British Journal of Pharmacology (2008) 154, 709–724; doi:10.1038/bjp.2008.112; published online 14 April 2008
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mitochondrial calcium ion and membrane potential transients follow the pattern of epileptiform discharges in Hippocampal Slice cultures
The Journal of Neuroscience, 2005Co-Authors: Richard Kovacs, Uwe Heinemann, Julianna Kardos, Oliver KannAbstract:Emerging evidence suggests that mitochondrial dysfunction contributes to the pathophysiology of epilepsy. Recurrent mitochondrial Ca2+ ion load during seizures might act on mitochondrial membrane potential (DeltaPsim) and proton motive force. By using electrophysiology and confocal laser-scanning microscopy, we investigated the effects of epileptiform activity, as induced by low-Mg2+ ion perfusion in Hippocampal Slice cultures, on changes in DeltaPsim and in mitochondrial Ca2+ ion concentration ([Ca2+]m). The mitochondrial compartment was identified by monitoring DeltaPsim in the soma and dendrites of patched CA3 pyramidal cells using the mitochondria-specific voltage-sensitive dye rhodamine-123 (Rh-123). Interictal activity was accompanied by localized mitochondrial depolarization that was restricted to a few mitochondria in small dendrites. In contrast, robust Rh-123 release into the cytosol was observed during seizure-like events (SLEs), indicating simultaneous depolarization of mitochondria. This was critically dependent on Ca2+ ion uptake and extrusion, because inhibition of the mitochondrial Ca2+ ion uniporter by Ru360 and the mitochondrial Na+/Ca2+ ion exchanger by 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one but not the inhibitor of mitochondrial permeability transition pore, cyclosporin A, decreased the SLE-associated mitochondrial depolarization. The Ca2+ ion dependence of simultaneous mitochondrial depolarization suggested enhanced Ca2+ ion cycling across mitochondrial membranes during epileptiform activity. Indeed, [Ca2+]m fluctuated during interictal activity in single dendrites, and these fluctuations spread over the entire mitochondrial compartment during SLEs, as revealed using mitochondria-specific dyes (rhod-2 and rhod-ff) and spatial frequency-based image analysis. These findings strengthen the hypothesis that epileptic activity results in Ca2+ ion-dependent changes in mitochondrial function that might contribute to the neuronal injury during epilepsy.
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free radical mediated cell damage after experimental status epilepticus in Hippocampal Slice cultures
Journal of Neurophysiology, 2002Co-Authors: Richard Kovacs, Sebastian Schuchmann, Siegrun Gabriel, Oliver Kann, Julianna Kardos, Uwe HeinemannAbstract:Generation of free radicals may have a key role in the nerve cell damage induced by prolonged or frequently recurring convulsions (status epilepticus). Mitochondrial function may also be altered due to production of free radicals during seizures. We therefore studied changes in field potentials (fp) together with measurements of extracellular, intracellular, and intramitochondrial calcium concentration ([Ca(2+)]e, [Ca(2+)]i, and [Ca(2+)]m, respectively), mitochondrial membrane potential (deltapsi), NAD(P)H auto-fluorescence, and dihydroethidium (HEt) fluorescence in Hippocampal Slice cultures by means of simultaneous electrophysiological and microfluorimetric measurements. As reported previously, each seizure-like event (SLE) resulted in mitochondrial depolarization associated with a delayed rise in oxidation of HEt to ethidum, presumably indicating ROS production. We show here that repeated SLEs led to a decline in intracellular and intramitochondrial Ca(2+) signals despite unaltered Ca(2+) influx. Also, mitochondrial depolarization and the NAD(P)H signal became smaller during recurring SLEs. By contrast, the ethidium fluorescence rises remained constant or even increased from SLE to SLE. After about 15 SLEs, activity changed to continuous afterdischarges with steady depolarization of mitochondrial membranes. Staining with a cell death marker, propidium iodide, indicated widespread cell damage after 2 h of recurring SLEs. The free radical scavenger, alpha-tocopherol, protected the Slice cultures against this damage and also reduced the ongoing impairment of NAD(P)H production. These findings suggest involvement of reactive oxygen species (ROS) of mitochondrial origin in the epileptic cell damage and that free radical scavenging may prevent status epilepticus-induced cell loss.
Klaus Albus - One of the best experts on this subject based on the ideXlab platform.
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primary afterdischarge in organotypic Hippocampal Slice cultures effects of standard antiepileptic drugs
Epilepsia, 2012Co-Authors: Klaus Albus, Abdul Wahab, Uwe HeinemannAbstract:Summary Purpose: In the hippocampus intense high frequency electrical stimulation induces a long-lasting rhythmic synchronization (primary afterdischarge). In order to examine the suitability of primary afterdischarges (PADs) in organotypic Hippocampal Slice cultures (OHSCs) as an in vitro model of evoked seizures, we have worked out in detail the sensitivity of PADs to standard antiepileptic drugs (AEDs) and compared the necessary concentrations to those that were effective in animal models of partial and generalized tonic–clonic seizures. Methods: OHSCs were prepared according to the interface culture method from 8 to 11-day-old Wistar rats. A PAD in CA1 was elicited by stimulating the stratum radiatum with an intensity of two times that required to elicit a maximal population spike. The effects of carbamazepine, phenytoin, valproic acid, phenobarbital, diazepam, and ethosuximide on the duration and on frequency properties of PADs and the tonic-like and clonic-like subdivisions of PADs were determined, and as a measure of the AEDs potency half maximal effective concentration (EC50) values were calculated from concentration–response curves. Key Finding: Carbamazepine, phenytoin, valproic acid, phenobarbital, and diazepam reduced the durations of PADs and tonic-like and clonic-like subdivisions of PADs. The effects were concentration dependent and reversible. Ethosuximide was ineffective. The effects on subdivisions of PADs differed between AEDs. Carbamazepine and phenytoin shortened the tonic-like and clonic-like subdivisions at similar proportions, whereas phenobarbital, diazepam, and valproic acid preferentially shortened the clonic-like subdivision. Diazepam at low concentrations increased the duration of tonic-like subdivisions, an effect not seen with the other AEDs. The suppressive effects of AEDs on frequency properties of tonic-like and clonic-like subdivisions were variable and observed only at higher concentrations. Significance: Carbamazepine and phenytoin were more effective in the PAD test in OHSCs than in the maximal electroshock and kindled seizures tests. The effectiveness of phenobarbital, diazepam, and valproic acid in the PAD test matched their effectiveness in the MES test and—with the exception of valproic acid and diazepam—in kindled seizures tests. Valproic acid was less effective in OHSCs than in the kindled seizure tests, and diazepam was more (generalized seizures) or less (focal seizures and afterdischarge durations) effective in this animal model than in OHSCs. We conclude that the PAD test in OHSCs is a suitable in vitro model of evoked seizures. The model could serve as an initial screen to identify the most promising leads for further evaluation and characterization in in vivo models of efficacy and toxicity.
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Effects of γ-aminobutyric acid (GABA) agonists and a GABA uptake inhibitor on pharmacoresistant seizure like events in organotypic Hippocampal Slice cultures
Epilepsy Research, 2009Co-Authors: Abdul Wahab, Uwe Heinemann, Klaus AlbusAbstract:Summary Purpose Seizure like events (SLEs) induced by low magnesium or 4-aminopyridine in organotypic Hippocampal Slice cultures (OHSCs) are resistant to standard antiepileptic drugs including phenobarbital, and 1,4-benzodiazepines [Albus, K., Wahab, A., Heinemann, U., 2008. Standard antiepileptic drugs fail to block epileptiform activity in rat organotypic Hippocampal Slice cultures. Br. J. Pharmacol. 154, 709–724]. The present study was undertaken in order to test the effects of other compounds on SLEs in OHSCs that enhance GABA-mediated actions. Methods Six to 12 days old Wistar rats were used to cultivate OHSCs according to the interface method [Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182]. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering the magnesium concentration. The effects of GABA A agonists muscimol and isoguvacine, the GABA B agonist baclofen, the GABA uptake blocker nipecotic acid and the neurosteroid alfaxalone on induction and ongoing SLEs were analyzed. Results Low magnesium induced SLEs were dose dependently suppressed by the GABA A receptor agonists muscimol, isoguvacine and alfaxalone and by the GABA uptake inhibitor nipecotic acid whereas the GABA B receptor agonist baclofen attenuated but did not suppress SLE. Discussion Our findings demonstrate that in OHSCs GABA has an inhibitory effect on SLEs. Proconvulsant effects of GABA agonists on spontaneous neuronal activity and seizure like activity were never observed. Our findings exclude a possible contribution of impaired/altered GABA-ergic mechanisms based on immaturity of receptors and/or low receptor density to seizure susceptibility and pharmacoresistance in OHSCs.
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standard antiepileptic drugs fail to block epileptiform activity in rat organotypic Hippocampal Slice cultures
British Journal of Pharmacology, 2008Co-Authors: Klaus Albus, Abdul Wahab, Uwe HeinemannAbstract:Background and purpose: Earlier studies had demonstrated that tonic–clonic seizure-like events (SLEs) resembling electrographic correlates of limbic seizures in animals and humans can be induced in organotypic Hippocampal Slice cultures (OHSCs). We have explored OHSCs for their suitability to serve as in vitro models of limbic seizures for studying seizure mechanisms and screening new antiepileptic compounds. Experimental approach: OHSCs were cultivated according to the interface method. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering magnesium concentration or by applying the potassium channel blocker 4-aminopyridine. The effects of standard antiepileptic drugs (AEDs), carbamazepine, phenytoin, valproic acid, clonazepam, diazepam and phenobarbital sodium on SLEs were analysed. Key results: In more than 93% of OHSCs, AEDs did not prevent the induction of SLEs or stop ongoing seizure activity even when toxic concentrations were applied. This pharmacoresistance was independent of the method of seizure provocation, postnatal age at explantation (P2–P10) and cultivation time in vitro (2 months). SLEs were reversibly blocked by glutamate antagonists or the GABAA-agonist muscimol. Conclusions and implications: We present a simple to establish in vitro model of tonic–clonic SLEs that is a priori pharmacoresistant and thus has an advantage over animal models of pharmacoresistant seizures in which responders and non-responders can be sorted out only after an experiment. OHSCs could be suitable for exploring mechanisms of pharmacoresistant seizures and be used for the identification of new anticonvulsive compounds eventually effective in drug refractory epilepsy. British Journal of Pharmacology (2008) 154, 709–724; doi:10.1038/bjp.2008.112; published online 14 April 2008
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primary afterdischarge in organotypic Hippocampal Slice cultures effects of standard antiepileptic drugs
Epilepsia, 2012Co-Authors: Klaus Albus, Abdul Wahab, Uwe HeinemannAbstract:Summary Purpose: In the hippocampus intense high frequency electrical stimulation induces a long-lasting rhythmic synchronization (primary afterdischarge). In order to examine the suitability of primary afterdischarges (PADs) in organotypic Hippocampal Slice cultures (OHSCs) as an in vitro model of evoked seizures, we have worked out in detail the sensitivity of PADs to standard antiepileptic drugs (AEDs) and compared the necessary concentrations to those that were effective in animal models of partial and generalized tonic–clonic seizures. Methods: OHSCs were prepared according to the interface culture method from 8 to 11-day-old Wistar rats. A PAD in CA1 was elicited by stimulating the stratum radiatum with an intensity of two times that required to elicit a maximal population spike. The effects of carbamazepine, phenytoin, valproic acid, phenobarbital, diazepam, and ethosuximide on the duration and on frequency properties of PADs and the tonic-like and clonic-like subdivisions of PADs were determined, and as a measure of the AEDs potency half maximal effective concentration (EC50) values were calculated from concentration–response curves. Key Finding: Carbamazepine, phenytoin, valproic acid, phenobarbital, and diazepam reduced the durations of PADs and tonic-like and clonic-like subdivisions of PADs. The effects were concentration dependent and reversible. Ethosuximide was ineffective. The effects on subdivisions of PADs differed between AEDs. Carbamazepine and phenytoin shortened the tonic-like and clonic-like subdivisions at similar proportions, whereas phenobarbital, diazepam, and valproic acid preferentially shortened the clonic-like subdivision. Diazepam at low concentrations increased the duration of tonic-like subdivisions, an effect not seen with the other AEDs. The suppressive effects of AEDs on frequency properties of tonic-like and clonic-like subdivisions were variable and observed only at higher concentrations. Significance: Carbamazepine and phenytoin were more effective in the PAD test in OHSCs than in the maximal electroshock and kindled seizures tests. The effectiveness of phenobarbital, diazepam, and valproic acid in the PAD test matched their effectiveness in the MES test and—with the exception of valproic acid and diazepam—in kindled seizures tests. Valproic acid was less effective in OHSCs than in the kindled seizure tests, and diazepam was more (generalized seizures) or less (focal seizures and afterdischarge durations) effective in this animal model than in OHSCs. We conclude that the PAD test in OHSCs is a suitable in vitro model of evoked seizures. The model could serve as an initial screen to identify the most promising leads for further evaluation and characterization in in vivo models of efficacy and toxicity.
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Effects of γ-aminobutyric acid (GABA) agonists and a GABA uptake inhibitor on pharmacoresistant seizure like events in organotypic Hippocampal Slice cultures
Epilepsy Research, 2009Co-Authors: Abdul Wahab, Uwe Heinemann, Klaus AlbusAbstract:Summary Purpose Seizure like events (SLEs) induced by low magnesium or 4-aminopyridine in organotypic Hippocampal Slice cultures (OHSCs) are resistant to standard antiepileptic drugs including phenobarbital, and 1,4-benzodiazepines [Albus, K., Wahab, A., Heinemann, U., 2008. Standard antiepileptic drugs fail to block epileptiform activity in rat organotypic Hippocampal Slice cultures. Br. J. Pharmacol. 154, 709–724]. The present study was undertaken in order to test the effects of other compounds on SLEs in OHSCs that enhance GABA-mediated actions. Methods Six to 12 days old Wistar rats were used to cultivate OHSCs according to the interface method [Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182]. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering the magnesium concentration. The effects of GABA A agonists muscimol and isoguvacine, the GABA B agonist baclofen, the GABA uptake blocker nipecotic acid and the neurosteroid alfaxalone on induction and ongoing SLEs were analyzed. Results Low magnesium induced SLEs were dose dependently suppressed by the GABA A receptor agonists muscimol, isoguvacine and alfaxalone and by the GABA uptake inhibitor nipecotic acid whereas the GABA B receptor agonist baclofen attenuated but did not suppress SLE. Discussion Our findings demonstrate that in OHSCs GABA has an inhibitory effect on SLEs. Proconvulsant effects of GABA agonists on spontaneous neuronal activity and seizure like activity were never observed. Our findings exclude a possible contribution of impaired/altered GABA-ergic mechanisms based on immaturity of receptors and/or low receptor density to seizure susceptibility and pharmacoresistance in OHSCs.
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standard antiepileptic drugs fail to block epileptiform activity in rat organotypic Hippocampal Slice cultures
British Journal of Pharmacology, 2008Co-Authors: Klaus Albus, Abdul Wahab, Uwe HeinemannAbstract:Background and purpose: Earlier studies had demonstrated that tonic–clonic seizure-like events (SLEs) resembling electrographic correlates of limbic seizures in animals and humans can be induced in organotypic Hippocampal Slice cultures (OHSCs). We have explored OHSCs for their suitability to serve as in vitro models of limbic seizures for studying seizure mechanisms and screening new antiepileptic compounds. Experimental approach: OHSCs were cultivated according to the interface method. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering magnesium concentration or by applying the potassium channel blocker 4-aminopyridine. The effects of standard antiepileptic drugs (AEDs), carbamazepine, phenytoin, valproic acid, clonazepam, diazepam and phenobarbital sodium on SLEs were analysed. Key results: In more than 93% of OHSCs, AEDs did not prevent the induction of SLEs or stop ongoing seizure activity even when toxic concentrations were applied. This pharmacoresistance was independent of the method of seizure provocation, postnatal age at explantation (P2–P10) and cultivation time in vitro (2 months). SLEs were reversibly blocked by glutamate antagonists or the GABAA-agonist muscimol. Conclusions and implications: We present a simple to establish in vitro model of tonic–clonic SLEs that is a priori pharmacoresistant and thus has an advantage over animal models of pharmacoresistant seizures in which responders and non-responders can be sorted out only after an experiment. OHSCs could be suitable for exploring mechanisms of pharmacoresistant seizures and be used for the identification of new anticonvulsive compounds eventually effective in drug refractory epilepsy. British Journal of Pharmacology (2008) 154, 709–724; doi:10.1038/bjp.2008.112; published online 14 April 2008