The studying of differences of a tumoral cell from normal, in particular features of its duplication, represents to rational therapy of tumours. We propose a new approach to the prolifirative activity of tumours cells of cns with the developing brain edema. Studying changes of brain substances at tumours astrocyter, s lines, we recognised that tumours changes of its structural elements can influence. We worked on two groups of patients-operating and non operating. In group operating and non operating close and on a distance from a tumour, it was observed changes of nervous cells. Changes in grey substance of a brain, it was observed near to all tumour. These changes were more significant. Changes in cerebral water compartments were assessed by diffusion-weighted MRI with determination of the apparent diffusion coefficient (ADC). There were several lines of evidence indicating that changes in the properties of the peripheral nervous system. CT scans showed massive cerebral edema and development of obstructive hydrocephalus. With a help of a method tutoradiography appeared possible appilication and reception most complete estimations of these positions, is especial on an experimental material and on cultures of a fabrics. At the same time, we observed that the phagocytic figure and total number of cycling immune complexes decreased evidently. Marked alteration of quantitative correlations of T- and B-lymphocytes were observed. Due to the increase of peripheral blood lymphocytes with simultaneous evident reduction of the monocyte count (by 28%).
309. The role of C-Jun N-terminal kinase in a mouse model of intracerebral hemorrhage
D. Michel-Monigadon1, V. Mottier2, C. Bonny3 and L. Hirt1
1Laboratory of Neurology, Centre Hospitalier Universitaire Vaudois; 2Institut de Biologie et de Morphologie, Université de Lausanne; 3Xigen Pharmaceuticals, Lausanne, Switzerland
Background: Intracerebral hemorrhage (ICH) is a subtype of stroke characterized by a haematoma within the brain parenchyma resulting from blood vessel rupture and with a poor outcome. In ICH, the blood entry into the brain triggers toxicity resulting in a substantial loss of neurons and an inflammatory response. At the same time, blood-brain barrier (BBB) disruption increases water content (edema) leading to growing intracranial pressure, which in turn worsens neurological outcome. Although the clinical presentation is similar in ischemic and hemorrhagic stroke, the treatment is different and the stroke type needs to be determined beforehand by imaging which delays the therapy.
C-Jun N-terminal kinases (JNKs) are a family of kinases activated in response to stress stimuli and involved in several pathways such as apoptosis. Specific inhibition of JNK by a TAT-coupled peptide (XG-102) mediates strong neuroprotection in several models of ischemic stroke in rodents. Recently, we have observed that the JNK pathway is also activated in a mouse model of ICH, raising the question of the efficacy of XG-102 in this model.
Method: ICH was induced in the mouse by intrastriatal injection of bacterial collagenase (0,1 U). Three hours after surgery, animals received an intravenous injection of 100 μg/kg of XG-102. The neurological outcome was assessed everyday until sacrifice using a score (from 0 to 9) based on 3 behavioral tests performed daily until sacrifice. Then, mice were sacrificed at 6 h, 24 h, 48 h, and 5d after ICH and histological studies performed.
Results: The first 24 h after surgery are critical in our ICH mice model, and we have observed that XG-102 significantly improves neurological outcome at this time point (mean score: 1,8+1.4 for treated group versus 3,4+1.8 for control group, P<0.01). Analysis of the lesion volume revealed a significant decrease of the lesion area in the treated group at 48 h (29+11 mm3 in the treated group versus 39+5 mm3 in the control group, P = 0.04). XG-102 mainly inhibits the edema component of the lesion. Indeed, a significant inhibition of the brain swelling was observed in treated animals at 48 h (14%+13% versus 26+9% in the control group, P = 0.04) and 5d (−0.3%+4.5% versus 5.1+3.6% in the control group, P = 0.01).
Conclusions: Inhibition of the JNK pathway by XG-102 appears to lead to several beneficial effects. We can show here a significant inhibition of the cerebral edema in the ICH model providing a further beneficial effect of the XG-102 treatment, in addition to the neuroprotection previously described in the ischemic model. This result is of interest because currently, clinical treatment for brain edema is limited. Importantly, the beneficial effects observed with XG-102 in models of both stroke types open the possibility to rapidly treat stroke patients before identifying the stroke subtype by imaging. This will save time which is precious for stroke outcome.
428. The effects of pioglitazone treatment on the regulations of aquaporin-4 and adiponectin in oletf rats
T. Shimazu1, K. Takayanagi2, H. Hasegawa2, R. Suge3, M. Ninomiya4, J. Asakura2, Y. Kato4, M. Yamashiro2, T. Mitarai2 and N. Araki4
1Neurology, Saitama Neuropsychiatric Institute; 2Nephrology and Hypertension, Saitama Medical School, Medical Center; 3Physiology; 4Neurology, Saitama Medical University, Saitama, Japan
Objectives: Aquaporin (AQP), especially AQP-4 has been seen to be involved not only in the brain edema formation,1 but also in several disorders including trauma and cerebral ischemia. In addition, several preliminary studies have demonstrated that AQP-4 may be concerning with learning ability in rats with cognitive impairment. Recently, it is recognized that peroxisome-proliferators-activated receptor gamma (PPAR gamma) agonists improve disability of cognitive function in patients with early Alzheimer disease2 and stroke in diabetes.3 We were interested in the possible effect of pioglitazone (PPAR-gamma agonist) on the expression of AQP-4 in deabetic brain. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an outbred strain of Long Evans Tokushima Otsuka rat (LETO), lacks CCK-1 receptor and is an animal model of type-2 diabetes. It has been reported that the OLETF was selectively impaired learning ability, depending on types of learning.
Methods: OLETF and LETO (22 weeks age, male, n = 5 each) were used as subjects. Half of each group was administrated pioglitazone (20 mg/kg/day) orally. All subjects were sacrificed at 24-weeks old, and brains were removed. Plasma adiponectin levels were measured after 14 days of treatment.
Brains were subdivided into four different parts, cortex, cerebellum, hippocampus and striatum. These samples were applied to RNA extraction or immunostaining. Total RNA was extracted by GITC method. Synthesized single strand cDNA was applied to real time PCR amplification with TaqMan probes. Results were analyzed by DCT method.
Results: AQP-4 expression in cortex of OLETF was greater than that of LETO, while effect of pioglitazone was not significant. In hippocampus, AQP-4 expression of OLETF was significantly increased comparing to LETO. Administration of pioglitazone attenuated the increase in AQP-4 expression in OLETF. Plasma adiponectin levels significant increased in OLETF rats with pioglitazone treatment.
Conclusions: These results indicate that in the OLETF rats pioglitazone has an important role in the diabetes cognitive function treatment through the regulation of AQP-4 expression and adiponectin. In hippocampus the AQP-4 expression of OLETF may be related to be increased circulating adiponectin level in response to pioglitazone treatment.
817. Involvement of mitogen-activated protein kinase pathways in expression of aquaporin-4 in rat cortical astrocytes after oxygen glucose deprivation
C. Nito1,2, M. Ueda1, P.H. Chan2 and Y. Katayama1
1Department of Internal Medicine, Divisions of Neurology, Nephrology, and Rheumatology, Nippon Medical School, Tokyo, Japan; 2Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University Medical School, Stanford, California, USA
Objectives: Brain edema is a key determinant of morbidity and mortality following ischemic brain injury. Aquaporin-4 (AQP4) plays an important role in water transport in the central nervous system and is highly expressed in astrocytes. However, the AQP4 regulatory mechanisms under pathological conditions like ischemia are unknown. In this study, we investigated whether the mitogen-activated protein kinases (MAPKs), which are involved in changes in osmolality,1,2 may mediate AQP4 expression in models of rat cortical astrocytes after oxygen-glucose deprivation (OGD), an in vitro ischemic-like condition. We hypothesized that AQP4 expression is regulated by a MAPK signaling pathway, which is affected by ischemic conditions.
Methods: Astrocyte cultures were prepared from the cerebral cortices of postnatal Sprague-Dawley rats. Cells were pretreated with selective inhibitors of ERK1/2 (PD98059), JNK (SP600125), p38 (SB203580), or DMSO (control) prior to OGD injury. AQP4 protein levels were measured by Western blot after 6 h of OGD. To analyze the contribution of MAPKs after OGD, ERK1/2, JNK, and p38 were examined by measuring levels of phosphorylation and activation on immunoblotting. Cell death was determined after 24 h of recovery in the normoxic incubator using a lactate dehydrogenase assay.3
Results: p38 MAPK and JNK were activated in response to OGD. Increased AQP4 protein levels in astrocytes were observed after OGD and 2 h of reperfusion, after which they decreased temporarily, as detected by immunoblotting. AQP4 was significantly upregulated at 16 h compared with the control. Phosphorylation of all three MAPKs was significantly decreased by OGD and increased during reoxygenation. Treatment with SB203580 and SP600125 attenuated the increase in AQP4 to normal levels, and only SB203580 significantly decreased cell death (P< 0.05).
Conclusions: These results suggest that p38 MAPK positively regulates AQP4 expression in astrocytes after ischemic-like injury. We suggest that p38 MAPK signaling can be a molecular target for regulating brain edema formation under ischemic conditions.
884. Chemical preconditioning ameliorates post-ischemic brain edema by aquaporin-4 downregulation
A. Hoshi, T. Yamamoto, T. Kumagai, K. Shimizu, Y. Sugiura and Y. Ugawa
Neurology, Fukushima Medical University, Fukushima, Japan
Objectives: Aquaporin-4 (AQP4) is abundantly expressed in the astrocyte foot processes at the blood-brain barrier. In general, it has been believed that AQP4 plays an important role in the generation of cytotoxic edema under ischemic stroke.1 So far, we have shown that 3-nitropropionic acid (3-NP) of mitochondrial inhibitor was one of tolerance-inducers to ischemia, and astrocytic glutamine synthetase (GS) was induced by the chemical preconditioning.2,3 Using this model, we investigated whether the AQP4 or GS expression is affected by 3-NP preconditioning. The effectiveness of the chemical preconditioning on the post-ischemic edema was also studied.
Methods: Male Sprague-Dawley rats were used in all experiments. Three groups of animals were studied: control, preconditioned at two different stages. The animals were preconditioned by intraperitoneal injection with 10 mg/kg of 3-NPA at tolerance-inducible stage (TS: 24 to 48 h) or non tolerance-inducible stage (NTS: 96 h) before middle cerebral artery (MCA) occlusion.3 The control group was injected with physiological saline. All of the animals were sacrificed 4 days after MCA occlusion for the measurement of the cerebral hemisphere swelling. The cerebral hemisphere swelling was analyzed by measuring the percentage of hemispheric enlargement (HE) [(ipsilateral hemisphere volume−contralateral hemisphere volume)/contralateral hemisphere volume] × 100, which represents the amount of edema formation. In addition, single or double immunofluorescence analysis of AQP4 and GS was conducted after the each 3-NP treatment and vehicle applied. Fluorescence intensities of AQP4 were analyzed quantitatively by image analysis software (Lumina vision, Mitani Corp., Japan).
Results: The HE was significantly reduced in TS group compared with the control group (P<0.01) or NTS group (P<0.05). Temporal profiles of AQP4 immunoreactivity in the cortex and striatum revealed that the AQP4 expression was downregulated by 3-NP preconditioning at the tolerance-inducible period but not at another period. On the contrary, GS immunoreactivity was enhanced by the preconditioning at any periods (in particular tolerance-induced) as our previous report.3
Conclusions: 3-NP preconditioning may attenuate post-ischemic brain edema by AQP4 downregulation. Inhibition of AQP4 by pharmacologic agents would offer a new therapeutic option for some forms of cerebral edema.
References
1.
TaniguchiMYamashitaT. Brain Res Mol Brain Res2000;78:131–137.
2.
WatsonGSCholertonBA. The American Journal of Geriatric Psychiatry2005;13:950–58.
3.
WicoxRBousserMG. Stroke2007;38:865–73.
4.
ArimaHYamamotoNSobueKUmenishiFTadaTKatsuyaHAsaiK. Hyperosmolar mannitol stimulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes. J Biol Chem2003;278:44525–34.
5.
HanJLeeJ-DBibbsLUlevitchRJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science1994;265:808–11.
6.
KohJYChoiDW. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods1987;20:83–90.
7.
ManleyGTFujimuraMMaTNoshitaNFilizFBollenAWChanPVerkmanAS. Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med2000;6:159–63.
8.
HoshiANakaharaTOgataMYamamotoT. The critical threshold of 3-nitropropionic acid-induced ischemic tolerance in the rat. Brain Res2005;1050:33–39.
9.
HoshiANakaharaTKayamaHYamamotoT. Ischemic tolerance in chemical preconditioning: Possible role of astrocytic glutamine synthetase buffering glutamate-mediated neurotoxicity. J Neurosci Res2006;84:130–41.
