Neurologische Klinik und Poliklinik
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Institute
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- Sahin Lab, F.M. Kirby Neurobiology Center Boston Children’s Hospital, Department of Neurology, Harvard Medical School (1)
- Wurzburg Fabry Center for Interdisciplinary Therapy (FAZIT), Wurzburg, Germany (1)
- Würzburg Fabry Center for Interdisciplinary Therapy (FAZIT), University of Würzburg, Würzburg, Germany (1)
Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events. In order to examine injury processes associated with TBI, a number of rodent models to induce brain trauma have been described. However, none of these models covers the entire spectrum of events that might occur in TBI. Here we provide a thorough methodological description of a straightforward closed head weight drop mouse model to assess brain injuries close to the clinical conditions of human TBI.
Background: Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear. Methodology/Principal Findings: We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patient’s amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. Conclusion/Significance: The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region.
Background: The role of serotonin (5-hydroxytrptamine, 5-HT) in the modulation of pain has been widely studied. Previous work led to the hypothesis that 5-hydroxyindolacetic acid (5-HIAA), a main metabolite of serotonin, might by itself influence pain thresholds. Results: In the present study, we investigated the role of 5-HIAA in inflammatory pain induced by intraplantar injection of complete Freund’s adjuvant (CFA) into the hind paw of mice. Wild-type mice were compared to mice deficient of the 5-HT transporter (5-HTT-/- mice) using behavioral tests for hyperalgesia and high-performance liquid chromatography (HPLC) to determine tissue levels of 5-HIAA. Wild-type mice reproducibly developed thermal hyperalgesia and paw edema for 5 days after CFA injection. 5-HTT-/- mice treated with CFA had reduced thermal hyperalgesia on day 1 after CFA injection and normal responses to heat hereafter. The 5-HIAA levels in spinal cord and sciatic nerve as measured with HPLC were lower in 5-HTT-/- mice than in wild-type mice after CFA injection. Pretreatment of wild-type mice with intraperitoneal injection of para-chlorophenylalanine (p-CPA), a serotonin synthesis inhibitor, resulted in depletion of the 5-HIAA content in spinal cord and sciatic nerve and decrease in thermal hyperalgesia in CFA injected mice. The application of exogenous 5-HIAA resulted in potentiation of thermal hyperalgesia induced by CFA in 5-HTT-/- mice and in wild-type mice pretreated with p- CPA, but not in wild-type mice without p-CPA pretreatment. Further, methysergide, a broad-spectrum serotonin receptor antagonist, had no effect on 5-HIAA-induced potentiation of thermal hyperalgesia in CFA-treated wildtype mice. Conclusion: Taken together, the present results suggest that 5-HIAA plays an important role in modulating peripheral thermal hyperalgesia in CFA induced inflammation, probably via a non-serotonin receptor mechanism.
The calpain inhibitor MDL-28710 blocks the early local pro-inflammatory cytokine gene expression in mice after chronic constriction nerve injury (CCI). Onehundred- thirteen wild type mice of C57Bl/6J background received CCI of the right sciatic nerve. Mechanical paw withdrawal thresholds and thermal withdrawal latencies were investigated at baseline and at 1, 3, and 7 days after CCI. Three application regimens were used for MDL-28170: a) single injection 40 min before CCI; b) serial injections of MDL- 28170 40 min before and up to day three after CCI; c) sustained application via intraperitoneal osmotic pumps. The control animals received the vehicle DMSO/PEG 400. The tolerable dose of MDL-28170 for mice was 30 mg/kg body weight, higher doses were lethal within the first hours after application. Mechanical withdrawal thresholds and thermal withdrawal latencies were reduced after CCI and did not normalize after single or serial injections, nor with application of MDL-28170 via osmotic pumps. Although the calpain inhibitor MDL-28170 inhibits the early local cytokine upregulation in the sciatic nerve after CCI, pain behavior is not altered. This finding implies that local cytokine upregulation after nerve injury alone is only one factor in the induction and maintenance of neuropathic pain.
Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice
(2010)
Background: Although it has been largely demonstrated that nitric oxide synthase (NOS), a key enzyme for nitric oxide (NO) production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Results: Intraperitoneal (i.p.) pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor), aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor), L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor), significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl.) injection of complete Freund’s adjuvant (CFA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1b), and interleukin-10 (IL-10) gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1b. The increase of the antiinflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO) mice had lower gene expression of TNF, IL-1b, and IL-10 following CFA, overall corroborating the inhibitor data. Conclusion: These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.
Interleukin-18 (IL-18) is a proinflammatory cytokine of the interleukin-1 family which is upregulated after cerebral ischemia. The functional role of IL-18 in cerebral ischemia is unknown. In the present study, we compared infarct size in IL-18 knock-out and wild-type mice 24 hours and 48 hours after 1-hour transient middle cerebral artery occlusion (tMCAO). Moreover, the functional outcome was evaluated in a modified Bederson score, foot fault test and grip test. There were no significant differences in infarct size or functional outcome tests between wild-type and IL-18 knock-out mice. These data indicate that the early inflammatory response to cerebral ischemia does not involve IL-18, in contrast to other interleukin-1 family members such as interleukin-1.
BACKGROUND: Recently, members of the two-pore domain potassium channel family (K2P channels) could be shown to be involved in mechanisms contributing to neuronal damage after cerebral ischemia. K2P3.1-/- animals showed larger infarct volumes and a worse functional outcome following experimentally induced ischemic stroke. Here, we question the role of the closely related K2P channel K2P9.1. METHODS: We combine electrophysiological recordings in brain-slice preparations of wildtype and K2P9.1-/- mice with an in vivo model of cerebral ischemia (transient middle cerebral artery occlusion (tMCAO)) to depict a functional impact of K2P9.1 in stroke formation. RESULTS: Patch-clamp recordings reveal that currents mediated through K2P9.1 can be obtained in slice preparations of the dorsal lateral geniculate nucleus (dLGN) as a model of central nervous relay neurons. Current characteristics are indicative of K2P9.1 as they display an increase upon removal of extracellular divalent cations, an outward rectification and a reversal potential close to the potassium equilibrium potential. Lowering extracellular pH values from 7.35 to 6.0 showed comparable current reductions in neurons from wildtype and K2P9.1-/- mice (68.31 +/- 9.80% and 69.92 +/- 11.65%, respectively). These results could be translated in an in vivo model of cerebral ischemia where infarct volumes and functional outcomes showed a none significant tendency towards smaller infarct volumes in K2P9.1-/- animals compared to wildtype mice 24 hours after 60 min of tMCAO induction (60.50 +/- 17.31 mm3 and 47.10 +/- 19.26 mm3, respectively). CONCLUSIONS: Together with findings from earlier studies on K2P2.1-/- and K2P3.1-/- mice, the results of the present study on K2P9.1-/- mice indicate a differential contribution of K2P channel subtypes to the diverse and complex in vivo effects in rodent models of cerebral ischemia.
Transgenic mice bred on C57Bl/6 or Sv/129 genetic background are frequently used in stroke research. It is well established that variations in cerebrovascular anatomy and hemodynamics can influence stroke outcome in different inbred mouse lines. We compared stroke development in C57Bl/6 and Sv/129 mice in the widely used model of transient middle cerebral artery occlusion (tMCAO) by multimodal ultra-high field magnetic resonance imaging (MRI). C57Bl/6 and Sv/129 mice underwent 60 min of tMCAO and were analyzed by MRI 2 h and 24 h afterwards. Structural and functional images were registered to a standard anatomical template. Probability maps of infarction were rendered by automated segmentation from quantitative T2-relaxometric images. Whole-brain segmentation of infarction was accomplished manually on high-resolution T2-weighted (T2-w) RARE images. Cerebral perfusion (cerebral blood flow, CBF) was measured quantitatively by modified continuous arterial-spin-labeling (CASL) and apparent diffusion coefficients (ADC) by spin-echo diffusion-weighted imaging (DWI). Probabilities of cortical (95.1% ± 3.1 vs. 92.1% ± 2.5; p > 0.05) and subcortical (100% vs. 100%; p > 0.05) infarctions at 24 h were similar in both groups as was the whole-brain volumetric extent of cerebral infarction. In addition, CBF and ADC values did not differ between C57Bl/6 and Sv/129 mice at any time point or region of interest. The C57Bl/6 and Sv/129 genetic background is no major confounding factor of infarct size and cerebral perfusion in the tMCAO model.
Background: Anticoagulation is an important means to prevent from acute ischemic stroke but is associated with a significant risk of severe hemorrhages. Previous studies have shown that blood coagulation factor XII (FXII)- deficient mice are protected from pathological thrombus formation during cerebral ischemia without bearing an increased bleeding tendency. Hence, pharmacological blockade of FXII might be a promising and safe approach to prevent acute ischemic stroke and possibly other thromboembolic disorders but pharmacological inhibitors selective over FXII are still lacking. In the present study we investigated the efficacy of COU254, a novel nonpeptidic 3-carboxamide-coumarin that selectively blocks FXII activity, on stroke development and post stroke functional outcome in mice. Methods: C57Bl/6 mice were treated with COU254 (40 mg/kg i.p.) or vehicle and subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 h infarct volumes were determined from 2,3,5-Triphenyltetrazoliumchloride(TTC)-stained brain sections and functional scores were assessed. Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus formation within the infarcted brain areas was analyzed by immunoblot. Results: Infarct volumes and functional outcomes on day 1 after tMCAO did not significantly differ between COU254 pre-treated mice or untreated controls (p > 0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. COU254 treatment did not prevent intracerebral fibrin(ogen) formation. Conclusions: COU254 at the given concentration of 40 mg/kg failed to demonstrate efficacy in acute ischemic stroke in this preliminary study. Further preclinical evaluation of 3-carboxamide-coumarins is needed before the antithrombotic potential of this novel class of FXII inhibitors can be finally judged.