endothelin-1 and Hypothermia

Stroke is an important cause of morbidity and mortality and few therapies exist thus far. Mild hypothermia (33°C) is a promising neuroprotective strategy to improve outcome after ischemic stroke. However, its complete mechanism of action has not yet been fully elaborated. This study is the first to investigate whether this neuroprotection occurs through modulation of the neuroinflammatory response after stroke in a time-dependent manner.. The Endothelin-1 (Et-1) model was used to elicit a transient focal cerebral ischemia in male Wistar rats. In this model, the core and penumbra of the insult are represented by the striatum and the cortex respectively. We assessed the effects of 2 hours of hypothermia, started 20 minutes after Et-1 injection on neurological outcome and infarct volume. Furthermore, pro- and anti-inflammatory cytokine expression was determined using ELISA. Microgliosis and astrogliosis were investigated using CD-68 and GFAP staining respectively. All parameters were determined 8, 24, 72 hours and 1 week after the administration of Et-1.. Et-1 infusion caused neurological deficit and a reproducible infarct size which increased up to 3 days after the insult. Both parameters were significantly reduced by hypothermia. The strongest reduction in infarct volume with hypothermia, at 3 days, corresponded with increased microglial activation. Reducing the brain temperature affected the stroke induced increase in interleukin-1β and tumor necrosis factor α in the striatum, 8 hours after its induction, but not at later time points. Transforming growth factor β increased as a function of time after the Et-1-induced insult and was not influenced by cooling. Hypothermia reduced astrogliosis at 1 and 3 days after stroke onset.. The beneficial effects of hypothermia after stroke on infarct volume and functional outcome coincide with a time-dependent modulation of the cytokine expression and gliosis.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cytokines; Endothelin-1; Gliosis; Hypothermia; Interleukin-1beta; Ischemic Attack, Transient; Male; Rats; Rats, Wistar; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Previously we showed that treatment with mild hypothermia (34 degrees C for 2 h) after a focal cerebral infarct was neuroprotective by reducing apoptosis in the penumbra (cortex), but not in the core (striatum) of the infarct. In this study we examined whether administration of N-acetyl-aspartyl-glutamate (NAAG) in combination with mild hypothermia could improve striatal neuroprotection in the endothelin-1 rat model. NAAG (10 mg/kg i.p.) was injected under normothermic (37 degrees C) or mild hypothermic conditions, either 40 min before or 20 min after the insult. NAAG reduced caspase 3 immunoreactivity in the striatum, irrespective of the time of administration and brain temperature. This neuroprotective effect could be explained, at least partially, by decreased nitric oxide synthase activity in the striatum and was blocked by the group II metabotropic glutamate receptor antagonist, LY341495. Hypothermia applied together with NAAG reduced both cortical and striatal caspase 3 immunoreactivity, as well as the overall ischaemic damage in these areas. However, no pronounced improvement was seen in total damaged brain volume. Extracellular glutamate levels did not correlate with the observed protection, whatever treatment protocol was applied. We conclude that treatment with NAAG causes the same degree of neuroprotection as treatment with hypothermia. Combination of the two treatments, although reducing apoptosis, does not considerably improve ischaemic damage.

Topics: Amino Acids; Animals; Blood-Brain Barrier; Brain Ischemia; Dipeptides; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Endothelin-1; Hypothermia; Male; Microdialysis; Neuroprotective Agents; Rats; Rats, Wistar; Time Factors; Xanthenes

Previously, we showed that treatment with resuscitative, post-ischaemic mild hypothermia (34 degrees C for 2 h) reduced apoptosis in the penumbra (cortex), but not in the core (striatum) of an endothelin-1 (Et-1)-induced focal cerebral infarct in the anaesthetized rat. Therefore, the purpose of this study was to investigate by which pathways resuscitative mild hypothermia exerts its neuroprotective effect in this model. The amino acids glutamate, serine, glutamine, alanine, taurine, arginine and the NO-related compound citrulline were sampled from the striatum and cortex of the ischaemic hemisphere using in vivo microdialysis. The in vivo salicylate trapping method was applied for monitoring hydroxyl radical formation via 2,3 dihydroxybenzoic acid (2,3 DHBA) detection. Caspase-3, neuronal nitric oxide synthase (nNOS) immunoreactivity and the volume of ischaemic damage were determined 24 h after the insult. In both the striatum and the cortex, Et-1-induced increases in glutamate, taurine and alanine were refractory to mild hypothermia. However, mild hypothermia significantly attenuated the ischaemia-induced 2,3 DHBA levels and the nNOS immunoreactivity in the cortex, but not in the striatum. These observations were associated with a decreased caspase-3 immunoreactivity. These results suggest that mild hypothermia exerts its neuroprotective effect in the penumbra partially by reducing nNOS activity and thereby preventing oxidative stress. Furthermore, we confirm our previous findings that the neuroprotective effect of resuscitative hypothermia is not mediated by changes in ischaemia-induced amino acid release as they could not be associated with the ischaemia-induced damage in the Et-1 rat model.

Topics: Amino Acids; Animals; Apoptosis; Body Temperature; Brain Ischemia; Caspase 3; Caspases; Cerebral Cortex; Cerebral Infarction; Endothelin-1; Enzyme Inhibitors; Extracellular Space; Hydroxyl Radical; Hypothermia; Immunohistochemistry; Male; Microdialysis; Neostriatum; NG-Nitroarginine Methyl Ester; Oxidants; Rats; Rats, Wistar

The aim of the present study was to investigate whether endothelin-1 (ET-1) in cerebral arteries is inhibited by the new, non-peptidergic ET(A) receptor antagonist Ro 61-1790 and, if it is, whether that inhibition reduces the lesion volume induced by cold injury in the parietal cortex. In vitro experiments were performed by measuring the isometric contractions of the rat middle cerebral and basilar arteries. A cold lesion was induced in vivo by the application of a pre-cooled (-78 degrees C) copper cylinder (diameter 3 mm) to the intact dura of rats for 6 s. After 24 h, lesion volume was determined by the triphenyltetrazolium method. In vitro, ET-1 (10(-12) - 3x10(-7) M) caused a dose-dependent contraction under resting conditions in the middle cerebral and basilar arteries of control rats. Ro 61-1790 (3x10(-9) M, 10(-7) M) shifted the concentration-effect curves for ET-1 in a parallel fashion (Emax unaltered). Post-treatment with Ro 61-1790 (10(-7)-10(-5) M) also inhibited the prior contraction elicited by ET-1 (3x10(-9) M) significantly. In vitro ET-1 application 3 h after the intracerebroventricular in vivo administration of Ro 61-1790 showed that the antagonist had reached the arteries and was bound to their ET(A) receptors. Intracerebroventricular pre-treatment of Ro 61-1790 reduced significantly the lesion volume by 23% after the injury. We conclude that ET-1 is involved in the development of secondary brain damage and that intracerebroventricular treatment with Ro 61-1790 reduces the size of the brain lesion caused by cold injury.

Topics: Animals; Basilar Artery; Cold Temperature; Dioxanes; Endothelin Receptor Antagonists; Endothelin-1; Hypothermia; Injections, Intraventricular; Male; Middle Cerebral Artery; Parietal Lobe; Pyridines; Pyrimidines; Rats; Rats, Wistar; Receptor, Endothelin A; Sulfonamides; Tetrazoles; Vasoconstriction