18. Induction of LC3 and gabarap in motor neurons after transient spinal cord ischemia in rabbits
H. Baba1, M. Sakurai2, K. Abe3 and R. Tominaga1
1Department of Cardiovascular Surgery, Kyushu University Gaduate School of Medicine, Fukuoka; 2Department of Cardiovascular Surgery, National Hospital Organization Sendai Medical Center, Sendai; 3Department of Neurology, Okayama University Graduate School of Medicine, Okayama, Japan
Background and aims: Spinal cord injury is considered to be related to a vulnerability of spinal motor neurons to ischemia. However, the mechanisms underlying this vulnerability are not fully understood.
Methods: We used a rabbit spinal cord ischemia model with use of a balloon catheter. The spinal cord was removed at 8 h, 1, 2, or 7 days after 15 min of transient ischemia, and histological changes were examined with hematoxylin-eosin staining. Western blot analysis for LC3 and GABARAP, temporal profiles of LC3 and GABARAP immunoreactivity, and double-label fluorescence immunocytochemical studies were performed.
Purpose: We previously reported that spinal motor neurons might be lost by programmed cell death, and investigated the role of autophagy at motor neuron as a potential mechanism of neuronal death by immunohistochemical analysis for microtubule-associated protein light chain3 (LC3) and γ-aminobutyric-acid type-A-receptor-associated protein (GABARAP).
Results: In the ischemia group, the majority of motor neurons were preserved until 2 days after reperfusion, but were selectively lost at 7 days. Western blot analysis demonstrated slight immunoreactivity for LC3 and GABARAP in the sham-operated spinal cords. In contrast, in the ischemia group LC3 and GABARAP immunoreactivity became apparent at 8 h after reperfusion. LC3 was strongly induced up to 2 days, but GABARAP was decreased at 2 days after reperfusion. At 8 h after reperfusion, co-labeling of LC3 and GABARAP was observed in the same motor neurons that eventually died.
Conclusion: This study demonstrated that immunoreactivities for both LC3 and GABARAP were induced in the same motor neurons which eventually die. These results suggest that autophagy was induced in motor neurons by transient spinal cord ischemia in rabbits.
72. Sexual steroids of a brain and sexual features of conditioned-reflected activity
V. Sashkov
Institute of Age Physiology, Moscow, Russia
Sexual steroids render potent influence on morphology and functions of brain. It is supposed, that sexual hormones can be involved in a regulation of behavior process, learning and memory both formed in gonads, and synthesized in brain. According to it to understand the role of sexual steroids in a conditioned-reflected activity we need to find out their level in separate structures of brain during the learning process and the decrease process of conditional reflex.
Researches were executed on adult rats' males and females Vistar's line. A conditioned reflex was developed on the basis of an electrodermal reinforcement. Testing of conservation of a conditioned reflex was held in 24 h, and during the next 5 days was observed its decrease. During all stages of conditioned-reflected activity the amount of testosteron and estradiol in blood plasma, hypothalamus, hippocampus, amygdale, cingular and frontal corex were found.
The carried out results have shown, that learning to a conditioned reflex of adult rats hasn't sexual dimorphism, however the decrease of a skill of males occurs rather quickly, and talking about rats it happens females gradually during five days of the tested period. Results of the correlation analysis have shown the selectivity of involving sexual hormones in mechanisms of conditioned-reflected activity rats' males and females.
Definition of sexual steroids level of during forming of a conditioned reflex has revealed the increasing of the male rats' testosterone value in all investigated structures of brain at its constant level in blood plasma. During female rats' training to a conditioned reflex the level of testosterone raises in blood plasma, hypothalamus, hippocampus, amygdale, cingular and frontal corex. When a conditioned reflex decreases, the level of testosterone of both rats' gender it is reduced in blood plasma and in all investigated structures of brain.
The level of estradiole during the males rats' training to a conditioned reflex is raised in blood plasma and amygdale. During female training to the conditioned reflex, the level of estradiole remains constant in blood plasma, but it grows in amygdale. In decrease process a conditioned reflex the males rats' level of estradiole is reduced in blood plasma, hypothalamus and hippocampus. During decrease of conditioned reflex females rats have a reduced concentration of estradiole in blood plasma, hypothalamus and amygdale and raised amount of estradiole in cingular corex and hippocampus.
The revealed rising of testosterone value in hypothalamus, hippocampus, amygdale, cingular and frontal corex according to its constant plasma level that males have during formation of conditioned reflex specifies gives us an opportunity to suppose that this steroid can be formed in brain. The increase of estradiole concentration in amygdale female during training to a conditioned reflex, and also in hippocampus and cingular corex at its decrease also testifies to a cerebral parentage of these quantities of the hormone. It is possible, that except for gonadal sexual hormones penetrating through a blood-brain barrier, androgens at males and estrogens at females, having a cerebral parentage, can provide optimum neurochemistry environment in brain, modulating processes of learning and memory.
639. MAPK signalling pathway regulate cerebrovascular receptor expression in human cerebral arteries
S. Ansar1,2, E. Nilsson1, O. Nilsson3, H. Säveland3 and L. Edvinsson1,2
1Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund, Sweden; 2Clinical Experimental Research, Glostrup Research Institute, Glostrup University Hospital, Glostrup, Denmark; 3Neurosurgery, Lund University Hospital, Lund, Sweden
Objectives: Stroke is a serious disease and the third leading cause of death worldwide. During the last two decades substantial efforts have been made to understand the intracellular mechanisms involved in ischemia-induced cerebral damage and to develop drugs that protect the brain from damage once a stroke has occurred. However, despite extensive research, few therapies have been proven effective in the clinic so far.
Cerebral ischemia is multifactorial and we have before demonstrated upregulation of contractile endothelin-1 (ET), angiotensin (Ang II) and 5-hydroxytryptamine (5-HT) receptors in the major cerebral arteries from experimental focal and global ischemia, via enhanced transcription and translation. Previous studies have shown that inhibitors of the mitogen-activated protein kinases (MAPK) can diminish the ischemic area and contractile cerebrovascular receptors after experimental cerebral ischemia. Here we hypotheses that there is an upregulation of contractile cerebrovascular receptors after 48 h of organ culture in human cerebral arteries and this upregulation occur via the MAPK ERK1/2 pathway and can be inhibited by the MEK1/2 inhibitor U0126.
Methods: Human cerebral arteries were obtained from patients undergoing intracranial tumor surgery. The vessels were divided into segments and incubated for 48 h with or without the MEK1/2 inhibitor U0126. The vessels were then examined by using in vitro pharmacological methods. The contractile responses to ET-1 (ETA and ETB receptor), AngII, 5-carboxamidotryptamine (5-CT) and U46619 (Thromboxane (TP) receptor agonist) were investigated.
Results: After organ culture of the cerebral arteries the contractile responses of the ET-1, AngII and TP receptors were enhanced in comparison with data for fresh human arteries. However, 5-CT induced decreased contractile responses after organ culture as compared to fresh arteries. Incubation with the MEK1/2 inhibitor U0126 diminished the maximum contraction elicited by application of ET-1, Ang II and U46619 in human cerebral arteries considerably compared to control (organ culture). In addition, the MEK1/2 inhibitor decreased the contractile response to 5-CT. Statistical analyses were performed with Kruskal-Wallis non-parametric test with Dunn's post-hoc test, where P<0.05 was considered significant. Data are expressed as mean±s.e.m.
Conclusion: This is the first study to demonstrate that there is a clear association between human cerebrovascular receptor upregulation via transcription involving activation of the MAPK pathway after organ culture. Inhibition of the MAPK pathways attenuated the vasoconstriction mediated by ET, AT and TP receptors in human cerebral arteries after organ culture. These results indicate that MAPK inhibition is a novel target for treatment of cerebrovascular disorders.
644. Unraveling TLR-4 mediated signal transduction pathways activated in glia and the inhibitory actions of unfractioned heparin
R. Gorina1, A. Chamorro2 and A.M. Planas1
1Cerebral Ischemia and Neurodegeneration, IIBB (Institute of Biomedical Research)-CSIC -IDIBAPS; 2Stroke Unit, Neurology Service, Hospital Clinic, Barcelona, Spain
Objectives: Inflammation is regarded as a target for the treatment of stroke.
1
Heparin exerts beneficial effects in brain ischemia2,3 that can be in part mediated by its anti-inflammatory properties.
4
Innate immune responses mediated by toll-like receptor-4 (TLR-4) are involved in ischemic brain damage.5,6 In this study we examined the putative anti-inflammatory effect of heparin following activation of TLR-4.
Methods: Astrocyte-enriched cultures from rat cortex were exposed to the TLR-4 agonist LPS in the presence or absence of heparin. Activation of signal transduction pathways was examined, and small interference RNA (siRNA), blocking antibodies, or inhibitory drugs were used to demonstrate the involvement of various molecules.
Results: LPS induced the expression of inflammatory mediators, such as vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNFα), interferon-gamma inducible protein-10 (IP-10) and monocyte chemoattractant protein-1(MCP-1). Activation of TLR-4 by LPS induced nuclear factor-kappa B (NFkB) activation within the first hour, as demonstrated by proteasome-dependent IkBalpha degradation and nuclear translocation of the p65 subunit of the NFkB complex. In addition, LPS caused phosphorylation (Tyr701) of signal transductor and activator of transcription-1 (Stat1), but this effect was delayed for 2 h. Then, activation of Stat-1 dependent gene transcription was observed (e.g. suppressors of cytokine signalling-1 (SOCS-1) and IL-15 mRNA). LPS-induced pStat1 was mediated by Jak1; it was dependent on previous activation of NFkB, on new protein synthesis, and on release of a soluble factor to the medium. Heparin did not prevent NFkB activation or the expression of several target genes induced by LPS, in spite that this has been described in other cell types.
7
However, heparin strongly inhibited the Jak1/Stat1 pathway. In addition, LPS induced a rapid activation of extracellular signal regulated kinase-1/2 (ERK1/2) that was partially inhibited by heparin, it was independent of NFkB and on new protein synthesis, but it was fully prevented by antioxidants. The Stat1 activation after LPS reported above was also dependent on ERK1/2 phosphorylation. We have evidences supporting that signaling through TNFα after LPS is involved in ERK1/2 activation, and that heparin interferes with this process.
Conclusions: The results show that oxidative stress induced by LPS caused early ERK1/2 phosphorylation that indirectly participated in delayed Stat1 tyrosine phosphorylation, and heparin interferes with these processes. Therefore, TLR-4 activation induces a complex pattern of intracellular and extracellular signaling that is, at least in part, cell-type dependent, and that heparin can interfere with the inflammatory cascade and specifically attenuate certain proinflammatory responses.
Acknowledgement: Supported by the Spanish Ministry of Education and Science (CICYT) and by the European Community's Seventh Framework Programme (FP7/2007-201, grant no 201024, European Stroke Network).
712. Nitric oxide production induced by hypoxia modulates HIF-1 alpha expression in cultured astrocytes through interaction with superoxide
Q. Chen, W. Liu, X. Sun and K. Liu
College of Pharmacy, University of New Mexico, Albuquerque, New Mexico, USA
Objectives: Ischemic stroke results in cerebral tissue hypoxia, and increased expression of hypoxia-inducible factor (HIF) is critically implicated in ischemic tissue damage. Understanding the mechanisms of HIF-1alpha regulation has been an important research focus.
1
Nitric oxide (NO) and superoxide generation are increased under hypoxia/ischemic conditions,2,3 and each of them has been independently shown to regulate HIF-1alpha expression under several pathological conditions. In this study, we investigated the cross-affects of these two radicals on the expression of HIF-1 alpha in hypoxic astrocytes.
Methods: Immediately after the change of oxygen-free medium, astrocytes were placed into a humidity and air-tight chamber, which was flushed with 5%CO2/95%N2 for 15 min, then sealed and kept under 37°C for another 145 min. NO concentration in the conditioned medium was measured with the inNO-T nitric oxide measurement system using the amino-700 sensor; intracellular superoxide production was analyzed by fluorescence staining; HIF-1 alpha expression was measured with western blot.
Results: 2 hr-hypoxia remarkably increased HIF-1alpha protein levels in astrocytes, which was accompanied by increased NO and superoxide production. Decreasing superoxide with NAC, NADPH oxidase inhibitor (DPI), or SOD mimetic (MnTMPyP) decreased hypoxia-induced HIF-1 alpha protein expression, and increased NO production. NO synthase (NOS) inhibitor L-NAME alone inhibited superoxide generation as well as hypoxia-induced HIF-1alpha protein expression, however, it increased HIF-1apha protein in the presence of superoxide scavengers. Moreover, NO scavenger PTIO increased hypoxia-induced HIF-1 alpha protein expression and superoxide production. Incubation of astrocytes with peroxynitrite, the product of NO and superoxide reaction, did not change the expression of HIF-1 alpha protein expression under hypoxia condition.
Conclusion: These results suggest that superoxide critically contributes to hypoxia-induced HIF-1alpha protein stabilization under hypoxic condition. More importantly, our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for limiting the over-reaction to hypoxia stress by inhibiting HIF-1alpha protein expression.
Keywords: Hypoxia, nitric oxide, superoxide, hypoxia-inducible factor-1 alpha.
