CatWalk gait analysis in a rat model of multiple sclerosis.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for multiple sclerosis. The characteristic feature of the MOG-EAE model in Brown Norway rats is consistent involvement of the spinal cord resulting in limb paresis. The aim of the study was to investigate whether early subclinical gait abnormalities are present in this animal model and can be detected by CatWalk XT, a fully automated gait analysis system. Furthermore, we investigated the usability of CatWalk system for treatment studies. RESULTS: Our gait analysis showed no preclinical abnormalities in MOG-EAE animals. Nevertheless, we characterized a combination of gait parameters that display a high predictive capacity in regard to disease onset. Our detailed histopathological analysis of the spinal cord revealed that lesion formation starts in the lumbar region and propagates toward the cervical part of the spinal cord during the disease course. In the treatment study, the stabilization of gait parameters under the treatment with methylprednisolone was detected in CatWalk as well as in traditional EAE-scoring system. CONCLUSIONS: The results from CatWalk test indicate no benefit of lab-intensive automated gait system in EAE-model with chronic-progressive disease course as well as in therapeutic studies with pronounced effect on the severity of clinical symptoms. However, due to its quantitative and objective nature this system may display a refined test to detect small but functional relevant changes in regeneration-orientated studies.

Neurodegeneration in Autoimmune Optic Neuritis Is Associated with Altered APP Cleavage in Neurons and Up-Regulation of p53.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS). Histopathological and radiological analysis revealed that neurodegeneration occurs early in the disease course. However, the pathological mechanisms involved in neurodegeneration are poorly understood. Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in Brown Norway rats (BN-rats) is a well-established animal model, especially of the neurodegenerative aspects of MS. Previous studies in this animal model indicated that loss of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve, occurs in the preclinical phase of the disease and is in part independent of overt histopathological changes of the optic nerve. Therefore, the aim of this study was to identify genes which are involved in neuronal cell loss at different disease stages of EAE. Furthermore, genes that are highly specific for autoimmune-driven neurodegeneration were compared to those regulated in RGCs after optic nerve axotomy at corresponding time points. Using laser capture micro dissection we isolated RNA from unfixed RGCs and performed global transcriptome analysis of retinal neurons. In total, we detected 582 genes sequentially expressed in the preclinical phase and 1150 genes in the clinical manifest EAE (P < 0.05, fold-induction >1.5). Furthermore, using ingenuity pathway analysis (IPA), we identified amyloid precursor protein (APP) as a potential upstream regulator of changes in gene expression in the preclinical EAE but neither in clinical EAE, nor at any time point after optic nerve transection. Therefore, the gene pathway analysis lead to the hypothesis that altered cleavage of APP in neurons in the preclinical phase of EAE leads to the enhanced production of APP intracellular domain (AICD), which in turn acts as a transcriptional regulator and thereby initiates an apoptotic signaling cascade via up-regulation of the target gene p53.

First In Vivo Results of a Novel Pediatric Oxygenator with an Integrated Pulsatile Pump.

Extracorporeal membrane oxygenation (ECMO) is a pivotal bridge to recovery for cardiopulmonary failure in children. Besides its life-saving quality, it is often associated with severe system-related complications, such as hemolysis, inflammation, and thromboembolism. Novel oxygenator and pump systems may reduce such ECMO-related complications. The ExMeTrA oxygenator is a newly designed pediatric oxygenator with an integrated pulsatile pump minimizing the priming volume and reducing the surface area of blood contact. The aim of our study was to investigate the feasibility and safety of this new ExMeTrA (expansion mediated transport and accumulation) oxygenator in an animal model. During 6 h of extracorporeal circulation (ECC) in pigs, parameters of the hemostatic system including coagulation, platelets and complement activation, and flow rates were investigated. A nonsignificant trend in C3 consumption, thrombin-antithrombin-III (TAT) complex formation and a slight trend in hemolysis were detected. During the ECC, the blood flow was constantly at 500 ml/min using only flexible silicone tubes inside the oxygenator as pulsatile pump. Our data clearly indicate that the hemostatic markers were only slightly influenced by the ExMeTrA oxygenator. Additionally, the oxygenator showed a constant quality of blood flow. Therefore, this novel pediatric oxygenator shows the potential to be used in pediatric and neonatal support with ECMO.

Beneficial effect of chronic Staphylococcus aureus infection in a model of multiple sclerosis is mediated through the secretion of extracellular adherence protein.

BACKGROUND: Bacterial infections have been assumed to worsen multiple sclerosis (MS) disease symptoms and to lead to increased neurodegeneration. However, the underlying biological mechanisms for these effects are complex and poorly understood. Here, we assessed the disease-modulating effects of chronic infection with Staphylococcus aureus, a common human pathogen, on the clinical course and the extent of neurodegeneration in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. METHODS: To conduct this study, we established a persistent chronic infection in female brown Norway rats by inoculating Staphylococcus aureus (S. aureus) bacteria in a subcutaneously implanted tissue cages. RESULTS: In this study, we observed that the introduction of a localized S. aureus infection during the subclinical phase of EAE induced a chronic systemic inflammatory response, consisting of increased T- and B-cell counts and systemic production of proinflammatory cytokines. Unexpectedly, the S. aureus infection completely prevented the development of clinical EAE, and markedly reduced inflammatory infiltration and demyelination of the optic nerve, while it increased the number of surviving retinal neurons. Using a S. aureus strain that lacked the extracellular adherence protein (Eap), we determined that the extracellular adherence protein is at least partially responsible for the inhibitory effect of S. aureus infection on autoimmune inflammation of the central nervous system. CONCLUSIONS: Our results demonstrate for the first time that chronic infection with S. aureus has a beneficial effect on EAE, indicating a dual role of infection in the pathogenesis of MS. We also showed that secretion of Eap by S. aureus plays a major role in preventing autoimmune inflammation of the CNS. Moreover, we identified Eap as a factor responsible for this protective effect.

CREB in adult neurogenesis--master and partner in the development of adult-born neurons?

The generation of new neurons in the adult brain is modulated by complex stimuli and a broad range of extrinsic signals. It remains a mystery how stem cells and their progeny integrate this wealth of regulatory input to generate a precise number of neurons that matches the physiological needs of the olfactory and hippocampal network. cAMP response element binding protein (CREB)-dependent signalling is controlling essential developmental steps in adult neurogenesis, i.e. survival, maturation and integration of new neurons. Here, we summarize the current knowledge on the function of CREB in adult neurogenesis and discuss the potential of CREB to integrate complex stimuli and to translate these into precise developmental processes in adult neurogenesis. The complex modulation of CREB-signalling may allow the adult neurogenic system to respond to stimuli in a fine-tuned rather than in an on-off fashion.

GABA-cAMP response element-binding protein signaling regulates maturation and survival of newly generated neurons in the adult hippocampus.

Survival and integration of new neurons in the hippocampal circuit are rate-limiting steps in adult hippocampal neurogenesis. Neuronal network activity is a major regulator of these processes, yet little is known about the respective downstream signaling pathways. Here, we investigate the role of cAMP response element-binding protein (CREB) signaling in adult hippocampal neurogenesis. CREB is activated in new granule neurons during a distinct developmental period. Loss of CREB function in a cell-autonomous manner impairs dendritic development, decreases the expression of the neurogenic transcription factor NeuroD and of the neuronal microtubule-associated protein, doublecortin (DCX), and compromises the survival of newborn neurons. In addition, GABA-mediated excitation regulates CREB activation at early developmental stages. Importantly, developmental defects after loss of GABA-mediated excitation can be compensated by enhanced CREB signaling. These results indicate that CREB signaling is a central pathway in adult hippocampal neurogenesis, regulating the development and survival of new hippocampal neurons downstream of GABA-mediated excitation.

alpha1-Adrenergic modulation of synaptic input to Purkinje neurons in rat cerebellar brain slices.

The inhibitory activity in the cerebellar network, as investigated in acute brain slices from 14-20 days old rats, is modulated by alpha1-adrenergic stimulation. The specific alpha1-adrenoceptor agonist phenylephrine (PhE; 10 microM) or the alpha-adrenoceptor agonist 6-fluoronoradrenaline (10 microM) increases the frequency and the amplitude of spontaneous postsynaptic currents (sPSC) in Purkinje neurons. The effects are sensitive to the alpha1-adrenoceptor antagonists prazosin (30 microM) and phentolamine (10 microM). The PhE-induced augmentation is suppressed when phospholipase C is blocked by preincubation with U73122 (10 microM) but is not affected by inhibition of protein kinases with H7 (10 microM) or GF109203X (10 microM). Involvement of intracellular Ca(2+) stores was shown by a reduced PhE effect after blocking of SERCA pumps with cyclopiazonic acid (30 microM) and thapsigargin (1 microM). The persistence of the PhE effect on the frequency of miniature postsynaptic currents, as recorded in presence of tetrodotoxin, indicates a presynaptic localization of the alpha1-adrenoceptors. A block of voltage-gated Ca(2+) channels with nifedipine, verapamil, or omega-conotoxin MVIIC did not suppress the PhE-induced increase of the frequency and amplitude of sPSC. The results suggest that alpha1-adrenoceptors at presynaptic terminals mediate an increase of the spontaneous synaptic inhibition of Purkinje neurons in the cerebellar cortex via release of Ca(2+) from intracellular stores.

Purinergic modulation of synaptic input to Purkinje neurons in rat cerebellar brain slices.

Adenosine triphosphate (ATP) is a cotransmitter and an extracellular neuromodulator in nervous systems, and it influences neural circuits and synaptic strength. We have studied a stimulating effect of ATP (100 micro m) on the synaptic input of Purkinje neurons in acute cerebellar brain slices of juvenile rats (p14-19). Bath application of ATP increased the frequency of spontaneous postsynaptic currents (sPSCs) almost twofold, and increased their amplitude. These effects were fully suppressed by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid (PPADS; 10 microm), or after blocking action potentials with tetrodotoxin (TTX; 0.5 microm), but were not impaired by inhibiting ionotropic, non-NMDA glutamate receptors with 2,3-dioxo-6-nitro-1,2,3,4,-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX; 5 microm). The frequency of sPSCs was reduced by 35% by PPADS and increased by 50% after inhibiting ectonucleotidase with ARL67156 (50 microm), suggesting intrinsic, 'tonic', stimulation of synaptic activity via P2 receptors. The pharmacological profile indicated that the ATP effect was mediated by both P2X and P2Y receptors, most probably of the P2X5- and P2Y(2,4)-like subtypes. The action potential frequency in the inhibitory basket cells was increased by 65%, and decreased in Purkinje neurons by 25%, in the presence of ATP. Our results suggest that ATP continuously modulates the cerebellar circuit by increasing the activity of inhibitory input to Purkinje neurons, and thus decreasing the main cerebellar output activity, which contributes to locomotor coordination.

Rapid detection of subtelomeric deletion/duplication by novel real-time quantitative PCR using SYBR-green dye.

Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, miscarriages, and hematological malignancies. Automated detection of subtle deletions and duplications involving telomeres is essential for high-throughput screening procedures, but impractical when conventional cytogenetic methods are used. Novel real-time PCR quantitative genotyping of subtelomeric amplicons using SYBR-green dye allows high-resolution screening of single copy number gains and losses by their relative quantification against a diploid genome. To assess the applicability of the technique in the screening and diagnosis of subtelomeric imbalances, we describe here a blinded study in which DNA from 20 negative controls and 20 patients with known unbalanced cytogenetic abnormalities involving at least one or more telomeres were analyzed using a novel human subtelomere-specific primer set, producing altogether 86 amplicons, in the SYBR-green I-based real-time quantitative PCR screening approach. Screening of the DNA samples from 20 unrelated controls for copy number polymorphism do not detect any polymorphism in the set of amplicons, but single-copy-number gains and losses were accurately detected by quantitative PCR in all patients, except the copy number alterations of the subtelomeric p-arms of the acrocentric chromosomes in two cases. Furthermore, a detailed mapping of the deletion/translocation breakpoint was demonstrated in two cases by novel real-time PCR "primer-jumping." Because of the simplicity and flexibility of the SYBR-green I-based real-time detection, the primer-set can easily be extended, either to perform further detailed molecular characterization of breakpoints or to include amplicons for the detection and/or analysis of syndromes that are associated with genomic copy number alterations, e.g., deletion/duplication-syndromes and malignant cancers.