Electrochemical failure of the brain cortex is more deleterious when it is accompanied by low perfusion.

BACKGROUND AND PURPOSE: Clinical and experimental evidence suggests that spreading depolarization facilitates neuronal injury when its duration exceeds a certain time point, termed commitment point. We here investigated whether this commitment point is shifted to an earlier period, when spreading depolarization is accompanied by a perfusion deficit. METHODS: Electrophysiological and cerebral blood flow changes were studied in a rat cranial window model followed by histological and immunohistochemical analyses of cortical damage. RESULTS: In group 1, brain topical application of artificial cerebrospinal fluid (ACSF) with high K(+) concentration ([K(+)](ACSF)) for 1 hour allowed us to induce a depolarizing event of fixed duration with cerebral blood flow fluctuations around the baseline (short-lasting initial hypoperfusions followed by hyperemia). In group 2, coapplication of the NO-scavenger hemoglobin ([Hb](ACSF)) with high [K(+)](ACSF) caused a depolarizing event of similar duration, to which a severe perfusion deficit was coupled (=spreading ischemia). In group 3, intravenous coadministration of the L-type calcium channel antagonist nimodipine with brain topical application of high [K(+)](ACSF)/[Hb](ACSF) caused spreading ischemia to revert to spreading hyperemia. Whereas scattered neuronal injury occurred in the superficial cortical layers in the window areas of groups 1 and 3, necrosis of all layers with partial loss of the tissue texture and microglial activation were observed in group 2. CONCLUSIONS: The results suggest that electrochemical failure of the cortex is more deleterious when it is accompanied by low perfusion. Thus, the commitment point of the cortex is not a universal value but depends on additional factors, such as the level of perfusion.

Visualization of Connexin 43-positive cells of glioma and the periglioma zone by means of intravenously injected monoclonal antibodies.

The selectivity of monoclonal antibodies against the E2 extracellular fragment of connexin 43 (Cx43) for a glioma focus was studied in in vivo experiments on animals with intracranial C6 glioma. Antibodies labeled with two alternative labels, the radioisotope (125)I and the fluorophore Alexa 660, were intravenously injected to rats with 18-day gliomas. Seventy-two hours after injection, (125)I-labeled antibodies accumulated in the hemisphere where the glioma was located to a concentration of 0.27 ± 0.01% of the injected dose per gram of wet weight, which exceeded their accumulation in the liver, spleen, and other organs. Fluorescent-labeled antibodies against the Cx43 fragment E2 specifically visualized cells in the peritumoral astroglial bank (a zone of active invasion of glioma cells). Double immunofluorescent visualization using antibodies against the Cx43 fragment E2 and glial fibrillar acidic protein (GFAP) showed that only a small proportion of the cells that bound the antibodies injected into the blood circulation were reactive astrocytes, whereas most of these cells were GFAP-negative and morphologically corresponded to astroblasts. These results suggest that antibodies against the extracellular Cx43 fragment E2 can be used for targeted transport of diagnostic and therapeutic drugs to the peritumoral invasion zone of high-grade gliomas.

Gene Encoding Chitinase 3-Like 1 Protein (CHI3L1) is a Putative Oncogene.

An important task in understanding oncogenesis is the identification of those genes whose copy number and expression increase during tumorigenesis. Previously, in an effort to identify genes which could be used as molecular markers for glial tumors, we compared gene expression in glioblastoma to the normal brain cells. Among the genes with the most pronounced increased expression in tumors there was CHI3L1, encoding the secreted chitinase 3-like 1 protein (also known as HC gp-39 or YKL-40). Expression of CHI3L1 was found increased significantly in various tumors in comparison with corresponding normal tissues. Here we show that CHI3L1 can decrease the doubling time of 293 cells. We have also demonstrated that CHI3L1 allows the anchorage-independent growth in soft agar and, in addition, stable CHI3L1 expression made 293 cells tumorigenic: these cells stimulate the initiation of tumors after their xenograft transplantation into the Wistar rat brains. Thus, the overexpression of CHI3L1 is likely to be critical in the development of some tumors and when we gain more information about mechanisms of CHI3L1 oncogenicity, it could be used as one of the potential targets for anticancer therapy.

Neuroprotective effects of the beta-carboline abecarnil studied in cultured cortical neurons and organotypic retinal cultures.

Presently there is no neuroprotective pharmacological treatment of proven clinical safety and efficacy available. The purpose of this study was to investigate whether the beta-carboline, abecarnil (Abe), which has already passed clinical phase III trials in patients with anxiety disorders, is neuroprotective in in vitro models of cerebral ischemia or excitotoxicity. Abe (100 nM) protected cultured cortical neurons when applied 20 min before or 20 min after combined oxygen glucose deprivation (OGD). Furthermore, cultured cortical neurons were protected from NMDA excitotoxicity when Abe (100 nM) was administered 20 min before or concurrent with 100 microM NMDA. In contrast, in adult rat organotypic retinal cultures, Abe failed to protect retinal ganglion cells (RGCs) against glutamate (Glu) excitotoxicity. Thus, although our data demonstrate that Abe is a potential neuroprotectant in cultured neurons, the lack of effect in an organotypical model of Glu toxicity indicates that further study is required before Abe might be considered for human neuroprotection trials.

Endothelin-1-induced spreading depression in rats is associated with a microarea of selective neuronal necrosis.

Two different theories of migraine aura exist: In the vascular theory of Wolff, intracerebral vasoconstriction causes migraine aura via energy deficiency, whereas in the neuronal theory of Leão and Morison, spreading depression (SD) initiates the aura. Recently, it has been shown that the cerebrovascular constrictor endothelin-1 (ET-1) elicits SD when applied to the cortical surface, a finding that could provide a bridge between the vascular and the neuronal theories of migraine aura. Several arguments support the notion that ET-1-induced SD results from local vasoconstriction, but definite proof is missing. If ET-1 induces SD via vasoconstriction/ischemia, then neuronal damage is likely to occur, contrasting with the fact that SD in the otherwise normal cortex is not associated with any lesion. To test this hypothesis, we have performed a comprehensive histologic study of the effects of ET-1 when applied topically to the cerebral cortex of halothane-anesthetized rats. Our assessment included histologic stainings and immunohistochemistry for glial fibrillary acidic protein, heat shock protein 70, and transferase dUTP nick-end labeling assay. During ET-1 application, we recorded (i) subarachnoid direct current (DC) electroencephalogram, (ii) local cerebral blood flow by laser-Doppler flowmetry, and (iii) changes of oxyhemoglobin and deoxyhemoglobin by spectroscopy. At an ET-1 concentration of 1 muM, at which only 6 of 12 animals generated SD, a microarea with selective neuronal death was found only in those animals demonstrating SD. In another five selected animals, which had not shown SD in response to ET-1, SD was triggered at a second cranial window by KCl and propagated from there to the window exposed to ET-1. This treatment also resulted in a microarea of neuronal damage. In contrast, SD invading from outside did not induce neuronal damage in the absence of ET-1 (n = 4) or in the presence of ET-1 if ET-1 was coapplied with BQ-123, an ET(A) receptor antagonist (n = 4). In conclusion, SD in presence of ET-1 induced a microarea of selective neuronal necrosis no matter where the SD originated. This effect of ET-1 appears to be mediated by the ET(A) receptor.

Roller culture of free-floating retinal slices: a new system of organotypic cultures of adult rat retina.

No experimental system exists to date for the in vitro study of retinal ganglion cell populations in a three-dimensional organotypic tissue environment. Here, we describe such a novel method for roller cultivation of adult retinas. Retinas of adult (1-3 months old) rats were cut into rectangular slices of approximately 1 mm(2). Free-floating slices were cultured on a horizontal rotating roller drum (50-60 rpm) in a dry incubator at 36.5 degrees C. During the first days of cultivation, primary flat retinal slices changed their configuration and transformed into ball-shaped tissue spheres (retinal bodies). Histological and immunocytochemical studies showed that the outer wall of the retinal bodies was formed by cell and fibre layers typical of mature retina with photoreceptors located on the outside. Initially, retinal bodies contained an inner cavity which later was completely obliterated and filled with glial cells, sprouting nerve fibres, and vascular structures. This culture system was further developed into a robust model of glutamate-induced neurotoxicity. Using a novel culture method of adult rat retina, preservation of the three-dimensional organotypic retinal cytoarchitecture was achieved, including survival of neurons in the ganglion cell layer and sprouting of nerve fibres of the axotomized retinal ganglion cells. This novel culture model promises to facilitate studies of retinal physiology and pathology.

Preventive antibacterial treatment improves the general medical and neurological outcome in a mouse model of stroke.

BACKGROUND AND PURPOSE: Epidemiological studies have demonstrated a high incidence of infections after severe stroke and their prominent role in morbidity and mortality in stroke patients. In a mouse model, it has been shown recently that stroke is coupled with severe and long-lasting immunosuppression, which is responsible for the development of spontaneous systemic infections. Here, we investigated in the same model the effects of preventive antibiotic treatment on survival and functional outcome of experimental stroke. METHODS: Mice were subjected to experimental stroke by occlusion of the middle cerebral artery (MCAO) for 60 minutes. A group of mice received moxifloxacin (6x100 mg/kg body weight every 2 hours over 12 hours) either immediately or 12 hours after MCAO. Control animals received the vector only. Behavior, neurological deficit, fever, survival, and body weight were monitored over 14 days. In a subgroup, infarct volume was measured 4 days after MCAO. Microbiological assessment was based on cultures of lung tissue, blood, and feces of animals 3 days after stroke. For a dose-response study, moxifloxacin was given immediately after MCAO in different doses and at different time points. RESULTS: Microbiological analyses of blood and lung tissue demonstrated high bacterial burden, mainly Escherichia coli, 3 days after stroke. Accordingly, we observed clinical and histological signs of septicemia and pneumonia. Moxifloxacin prevented the development of infections and fever, significantly reduced mortality, and improved neurological outcome. CONCLUSIONS: Preventive antibiotic treatment may be an important new therapeutical approach to improve outcome in patients with severe stroke.

Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation.

Infections are a leading cause of death in stroke patients. In a mouse model of focal cerebral ischemia, we tested the hypothesis that a stroke-induced immunodeficiency increases the susceptibility to bacterial infections. 3 d after ischemia, all animals developed spontaneous septicemia and pneumonia. Stroke induced an extensive apoptotic loss of lymphocytes and a shift from T helper cell (Th)1 to Th2 cytokine production. Adoptive transfer of T and natural killer cells from wild-type mice, but not from interferon (IFN)-gamma-deficient mice, or administration of IFN-gamma at day 1 after stroke greatly decreased the bacterial burden. Importantly, the defective IFN-gamma response and the occurrence of bacterial infections were prevented by blocking the sympathetic nervous system but not the hypothalamo-pituitary-adrenal axis. Furthermore, administration of the beta-adrenoreceptor blocker propranolol drastically reduced mortality after stroke. These data suggest that a catecholamine-mediated defect in early lymphocyte activation is the key factor in the impaired antibacterial immune response after stroke.

Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin.

BACKGROUND AND PURPOSE: Cellular response to hypoxia is mainly controlled by hypoxia-inducible factor 1 (HIF-1). The HIF-1 target gene erythropoietin (EPO) has been described as neuroprotective. Thus, we hypothesize EPO to be an essential mediator of protection in hypoxic preconditioning. METHODS: We randomized Sv129 mice into groups for different pretreatments, different hypoxia-ischemia intervals, or different durations of ischemia. For hypoxic preconditioning, the animals were exposed to a hypoxic gas mixture (8% O2 and 92% N2) for 30, 60, 180, 300, or 360 minutes. At 0, 24, 48, 72, or 144 hours later, we performed middle cerebral artery occlusion and allowed reperfusion after 30, 45, 60, or 120 minutes, or occlusion was left to be permanent. We studied EPO gene expression in brain tissue with a real-time reverse transcriptase-polymerase chain reaction and measured HIF-1 DNA-binding activity with an electrophoretic mobility shift assay. To block endogenously produced EPO, we instilled soluble EPO receptor into the cerebral ventricle. RESULTS: Hypoxic preconditioning for 180 or 300 minutes induced relative tolerance to transient focal cerebral ischemia, as evidenced by a reduction of infarct volumes to 75% or 54% of the control, respectively. Hypoxic pretreatment was effective only when applied 48 or 72 hours before middle cerebral artery occlusion. Sixty minutes after hypoxia, we found a marked activation of HIF-1 DNA-binding activity and a 7-fold induction of EPO transcription. Infusion of soluble EPO receptor significantly reduced the protective effect of hypoxic pretreatment by 40%. CONCLUSIONS: Endogenously produced EPO is an essential mediator of ischemic preconditioning.