CC BY
19
Section CELLULAR NEUROSCIENCE
2006). We checked EEG state before and after blocks of random single (110% MT) and paired (90% - 110% MT, 2 or 10 ms IPI (intracortical facilitation (ICF) and short-interval intracortical inhibition (SICI)); 4 - 8 sec between single or paired pulses) pulse stimulation (about 300 total stimuli) by calculation of Long-Range Temporal Correlations (LRTC, Linken-kaer-Hansen et al., 2001; Nikulin et al., 2012) in EEG . LRTC in the alpha frequency range were calculated with Detrend-ed Fluctuation Analysis applied to multichannel EEG recordings.
LRTC in the alpha frequency range showed moderate intra-class correlation between the two rest sessions thus indicating a relative stability of the temporal dynamics between the sessions. Topographically, intra-class correlation was least pronounced over the stimulated areas thus demonstrating a potential long-lasting offline effect of TMS on neuronal activity. This was further confirmed by showing that there was a positive correlation between the magnitude of ICF and the magnitude of LRTC in 8-13 Hz range. Importantly, such correlation was regionally specific demonstrating strongest values over sensorimotor areas where TMS was applied. Our data suggests single and paired pulses TMS can have a previously unobserved long lasting effect on the temporal dynamics of neuronal oscillations and can be considered as offline.
References
1. Terao Y, Ugawa Y(2006),' Studying higher cerebral functions by transcranial magnetic stimulation.' Suppl Clin Neuro-physiol., vol. 59, pp.9-17.
2. Linkenkaer-Hansen, K. (2001), 'Long-range temporal correlations and scaling behavior in human brain oscillations.' Journal of Neuroscience, vol. 21, no. 4, pp. 1370-1377
3. Nikulin, V. (2012), 'Attenuation of long-range temporal correlations in the amplitude dynamics of alpha and beta neuronal oscillations in patients with schizophrenia.' NeuroImage, vol. 61 no. 1, pp. 162-169
OM&P
Role of the Oligodendrocyte Lineage in Acute CNS Trauma
Frank Kirchhoff *
Molecular Physiology, Center for Integrative Physiology and Molecular Medicine, Homburg, Germany. * Presenting e-mail: [email protected]
Acute brain injuries activate signaling cascades essential for scar formation. Here, we report that acute lesions associated with a disruption of the blood-brain barrier (BBB) trigger re-programming of the oligodendrocyte lineage. Differentiated oligodendrocytes and their precursor cells can generate another neuroglial cell type: astrocytes. By in vivo 2P-LSM analysis we followed oligodendrocytes after injury in PLP-DsRed/GFAP-EGFP transgenic mice. Adjacent to the lesion site, oligodendrocytes first turned into an intermediate cell stage with astro- and oligodendroglial gene expression properties (AO cells). Subsequently, portions of AO cells differentiated into astrocytes, while others stayed in the oligodendrocyte lineage. In split-Cre mice, AO cells showed a clear glia-restricted differentiation potential that also depended on local cues. At the lesion higher expression levels of glial differentiation factors were detected. And indeed, local injection of IL-6 promoted the formation of AO cells. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma.
Molecular Changes in Penumbra After Focal Photothrombotic Stroke in the Rat Cerebral Cortex
A.B. Uzdensky* and S.V. Demyanenko
Southern Federal University, Rostov-on-Don, Russia. * Presenting e-mail: [email protected]
Aims
Ischemic stroke is a leading cause of human disability and mortality. Vascular occlusion and energy deficit rapidly, for a few minutes induce cerebral infarct. The cell damage propagates from the infarct core to surrounding tissues (penumbra). Acute cell necrosis inside the infarct core cannot be prevented, but tissue damage in penumbra develops slower,
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OM&P
Section CELLULAR NEUROSCIENCE
during several hours, and this "therapeutic window" provides time for neuroprotection. However, the efficient neuroprotective drugs for stroke pharmacotherapy are still not found. Therefore, deeper studies of biochemical mechanisms that regulate neurodegeneration and neuroprotection in penumbra are necessary for development of new approaches to stroke treatment.
Methods
We studied morphological and biochemical consequences of the focal photothrombotic infarct (PTI) in the rat cerebral cortex induced by the local laser irradiation (532 nm diode laser, 64 mW/cm2, 03 mm, 30 min) after i.v. administration of Bengal Rose (20 mg/kg). This hydrophilic photosensitizer does not cross the blood brain barrier and accumulates in microvessels. The following laser irradiation induces focal occlusion of microvessels. In this stroke model location, size, and degree of the injury are well controlled and reproducible. Preliminary histological and ultrastructural studies showed that PTI core was surrounded by 1.5-2 mm penumbra. To characterize changes in expression of neuronal and signaling proteins in penumbra, we used proteomic microarrays "The Panorama Antibody Array - Neurobiology" (Sig-ma-Aldrich). Each microarray contains 448 microdroplets with immobilized antibodies against 144 neuronal proteins and their isoforms and epitops (totally 224 spots in duplicate). At 1, 4 or 24 hours after PTI, the rat cortex was extracted, and the penumbra ring was excised. The symmetrical piece from the non-irradiated contralateral cortex was used as a control. After homogenization, the samples were incubated with fluorochromes Cy3 or Cy5. In another set, these samples were stained oppositely, by Cy5 and Cy3, respectively. Such swapped staining provides verification of results and compensation of a bias in binding of Cy3 or Cy5 dyes to protein samples. This provides the double test and full control of the experiment. Two microarrays were incubated with these labeled samples. After washing and drying the microarrays were scanned on the GenePix 4100A Microarray Scanner (Molecular Devices, USA) at 532 and 635 nm (fluorescence maximums of Cy3 and Cy5, respectively). The fluorescence images of the antibody microarrays were normalized (ratio-based normalization). The median fluorescence value determined over all spot pixels was used for estimation of the protein content in each spot, and the ratios of the experimental to control values characterized the difference in the protein levels between photothrombotic and untreated cortical tissue. The standard statistical treatment based on Student's t-test was used. The cut-off level was 30%.
Results
The histological and ultrastructural studies showed edema and degeneration of neurons, glia and capillaries in the PTI core. Morphological changes decreased gradually across the penumbra. The ultrastructure of the penumbra region adjacent to the PTI core was similar to that inside the core, but at a distance of 1.5-2 mm it looked almost normal except edema around some vessels and neurons. The proteomic study showed that the changes in protein expression in penumbra were maximal 4 hours after PTI (upregulation of 22 proteins and downregulation of 21 proteins) as compared with 1 hour (18 and 6 proteins) and 24 hours reperfusion intervals (10 and 9 proteins, respectively).
Diverse cellular subsystems were involved in the complex response of the penumbra tissue 4 hours after PTI: signal transduction pathways including protein kinase Ba/GSK-3, protein kinase C and its pi and |2 isoforms, Wnt/p-catenin (axini, GSK-3, FRAT1), Notch/NUMB, DYRK1A, TDP43; axon outgrowth and guidance (NAV-3, CRMP2, PKC|32); intercellular interactions and tissue integrity (N-cadherin, PMP22); cytoskeleton elements (neurofilament 68, neurofilaments, doublecortin); vesicular trafficking (syntaxin-8, TMP21, Munc-18-3, synip, ALS2, VILIP1, syntaxin, synapto-physin, synaptotagmin); biosynthesis of neuromediators (tryptophan hydroxylase, monoamine oxidase B, glutamate decarboxylase, tyrosine hydroxylase, DOPA decarboxylase, dopamine transporter); ubiquitin-mediated proteolysis (ubiquilin-1, UCHL1); mitochondria quality control (Pink1, parkin, HtrA2); miscellaneous proteins (LRP1, prion protein, p-amyloid). Additional immunohistochemical and Western blot studies of expression of monoamine oxidase B, UCHL1, DYRK1A, and Munc-18-3 confirmed the proteomic data.
Conclusions
The obtained data on changes in expression of more than 40 proteins that participate in various cellular subsystems provided the integral view on the cellular and tissue responses in the penumbra to photothrombotic infarct. They are involved either in neurodegeneration, or in neuroprotection. These changes were most expressed 4 hours after photo-thrombotic impact and reduced at the next day. Some of these proteins may serve as potential targets for ischemic stroke therapy.
Acknowledgements
The work was supported by the Russian Scientific Foundation (grant 14-15-00068). A.B. Uzdensky was supported by the Ministry of Education and Science of Russian Federation (grant "Science organization" 790).
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