Maria C. Marciano

Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation.

Erythropoietin prevents in vitro glutamate-induced neuronal death and could play a role in the central nervous system. We investigated the in vivo effects of recombinant human erythropoietin after intraperitoneal (i.p.; 25-100 U) or intracerebroventricular (i.c.v.; 0.25-25 U) administration on survival, brain malonildialdehyde (MDA) levels, brain edema, hippocampal neuronal death and brain nitric oxide (NO) synthesis after bilateral carotid occlusion (5 min), followed by reperfusion in the Mongolian gerbil. Peripheral posttreatment with recombinant human erythropoietin reduced postischemic MDA levels, brain edema and increased survival. Either peripheral or i.c.v. posttreatment with recombinant human erythropoietin significantly reduced hippocampal CA1 neuronal loss, observed 7 days after the ischemic event. Increase of nitrite and nitrate (as an index of NO formation) in the hippocampus, as observed after ischemia, was reduced in animals treated with recombinant human erythropoietin. These data suggest that in vivo recombinant human erythropoietin effects on brain ischemic injury could be due to inhibition of NO overproduction.

Erythropoietin prevents cognition impairment induced by transient brain ischemia in gerbils.

Erythropoietin has recently been studied for its role in the central nervous system (CNS). It has been shown to exert neuroprotective effects in different models of brain injury. We studied whether neuroprotective effects assessed from the reduction of neuronal loss after transient brain ischemia are associated to the preservation of learning ability. Recombinant human erythropoietin (0.5-25 U) was injected in the lateral cerebral ventricle of gerbils that are subjected to temporary (3 min) bilateral carotid occlusion. Post-ischemic histological evaluation of CA1 area neuronal loss and passive avoidance test were performed. Treatment with recombinant human erythropoietin significantly reduced delayed neuronal death in the CA1 area of the hippocampus and prevented cognition impairment in the passive avoidance test. These data indicate that recombinant human erythropoietin neuroprotective effects in brain ischemia are associated with the preservation of learning function.

Neuroprotective effects of Ginkgo biloba extract in brain ischemia are mediated by inhibition of nitric oxide synthesis

We studied the effects of pre-treatment (15 days) with oral administration of Ginkgo biloba extract (Ph-Gb 37.5-150 mg/kg) on brain malonildialdehyde (MDA), brain edema, brain nitrite and nitrate and delayed neuronal death following transient cerebral ischemia in the Mongolian gerbil. Survival was not modified, however, pre-treatment with Ginkgo biloba significantly and in a dose-dependent way reduced post-ischemic brain MDA levels and post-ischemic brain edema. Delayed neuronal death in the CA1 of the hippocampus was attenuated by the highest dose of the extract. Increase of nitrite and nitrate was observed after cerebral ischemia in the hippocampus and it was dose-dependently reduced in animals pretreated with Ph-Gb, thus suggesting that neuroprotective effects of Ginkgo biloba may be due to an inhibitory action on nitric oxide formation.

Enhancement of expression of vascular endothelial growth factor after adeno-associated virus gene transfer is associated with improvement of brain ischemia injury in the gerbil.

Angiogenesis induced by growth factors may represent a rational therapy for patients with stroke. Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis and VEGF expression is enhanced in the post-ischemic brain. VEGF induced by brain hypoxia can lead to the growth of new vessels and may represent a natural protective mechanism improving survival after stroke. In the light of these findings we investigated changes of VEGF expression in different brain regions after intracerebroventricular injection of adeno-associated virus transferring gene for VEGF (rAAV-VEGF) in the gerbil, and after transient brain ischemic injury, we studied the effects of rAAV-VEGF injection on survival, brain edema, delayed neuronal death in the CA1 area and learning ability. Treatment with rAAV-VEGF 6 days or 12 days before ischemia significantly improves survival, brain edema and CA1 delayed neuronal death and post-ischemic learning evaluated by passive avoidance test. Animals treated with rAAV-VEGF showed in the thalamus and the cortex, a significant positive immunostaining for VEGF similar to those subjected to brain ischemia and not treated with rAAV-VEGF. These data represent a further contribution to a possible employment of gene therapy by using rAAV-VEGF in brain ischemia and indicate that thalamus and cortex may be targets for neuroprotective effects of VEGF.

Inhibition of nitric oxide formation reduces voluntary ethanol consumption in the rat

Brain nitric oxide is involved in the mechanisms that regulate ingestive behaviour. To test whether this compound plays a role in alcohol preference, we studied the effects of different doses ofNG-nitro-L-arginine (L-NO arg), an inhibitor of nitric oxide synthase (NOS), on voluntary consumption of ethanol and on blood alcohol levels produced by a single intraperitoneal dose of alcohol in the rat. L-NO arg produced a significant and dose-dependent reduction of ethanol intake (P<0.001) without influencing total fluid consumption or feeding behaviour. L-NO arg did not influence the kinetics of alcohol. Our data show that inhibition of nitric oxide formation accompanies reduction of ethanol intake and suggest a possible role for nitric oxide in ethanol self-administration.

Transglutaminase activity and transglutaminase mRNA transcripts in gerbil brain ischemia

Brain injury of the ischemia/reperfusion type induces neuronal damage, mainly by excitatory amino acid release, intracellular Ca(2+) overload and reactive oxygen species production. We have previously demonstrated that glutamate exposure increased transglutaminase activity and transglutaminase 2 expression in cultured cerebellar granule cells and astrocytes. The aim of this study is to evaluate changes in transglutaminase activity and expression using a gerbil model of global cerebral ischemia. Moreover, the distribution and amounts of different transglutaminase isoforms were examined. Transglutaminase activity was measured by incorporation of [(3)H]putrescine into dimethylcasein throughout 48 h of reperfusion following a 3 min occlusion. Compared to sham-operated brains, significant increases were found in the ischemic hippocampus at 24 h of reperfusion, while minor changes were observed in the cortex. RT-PCR demonstrated the presence of significant mRNA amounts of transglutaminase 2 and transglutaminase 1, both in the hippocampus and the cerebral cortex, while low levels were found for transglutaminase 3 transcripts. Interestingly, transglutaminase 2 and transglutaminase 1 mRNAs were 4-fold and 2-fold increased, respectively, in the ischemic hippocampus after 24 h of reperfusion. Western blot analysis of transglutaminase 2 expression confirmed a strong up-regulation in the ischemic hippocampus. However, it is possible to hypothesize that different expression rates of transglutaminase isoforms may be dependent on different responsiveness of their transcription regulatory elements to intracellular calcium overload following excitotoxic cell injury. Our results suggest that increases in transglutaminases may be part of the tissue stress response in global brain ischemia.

MULTIPLE ACTIONS OF THE COUMARINE DERIVATIVE CLORICROMENE AND ITS PROTECTIVE EFFECTS ON ISCHEMIC BRAIN INJURY

The effects of different doses (0.25, 0.5, 1 and 2 mg/kg/i.p.) of cloricromene, a coumarine derivative, have been investigated on brain malondialdehyde levels, brain edema, myeloperoxidase activity, survival, locomotor hyperactivity and hippocampal neuronal loss following transient cerebral ischemia induced by temporary bilateral carotid occlusion in the Mongolian gerbil. Cloricromene reduced brain lipid peroxidation, measured through the evaluation of malondialdehyde (−82.9% with the highest dose), and the formation of postischemic brain edema, evaluated by water content. The increase in myeloperoxidase activity observed in the hippocampus of postischemic animals was also reduced: 0.7±0.3 U × 10−3 vs. 3.3±0.3 U × 10−3/g tissue. The same treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischemia and reperfusion. Histological observations of the pyramidal layer of CAI showed a reduction of neuronal loss in animals that received the drug before occlusion but not in those that were treated after the occlusion. These results show that cloricromene, a drug with multiple actions, improves brain injury induced by transient cerebral ischemia.

Cloricromene antagonizes antidipsogenic effects induced by endotoxin, but not by TNFα, in the rat

Abstract Intravenous (640 μg/kg) or intracerebroventricular (0.5 and 1 μg) injection of Escherichia coli endotoxin (LPS) causes inhibition of water intake induced by 24 hour period of water deprivation in the rat. Tumor necrosis factor α (TNF-α; 20 and 40 ng/rat) given into the lateral cerebral ventricle (i.c.v.) causes effects similar to those observed after LPS. Cloricromene, given either intravenously (1 and 2 mg/kg) or i.c.v. (250 and 500 ng), abolished the antidipsogenic effect induced by LPS (administered both i.v. and i.c.v.). Cloricromene (2 mg/kg, i.v. or 500 ng/rat, i.c.v.), on the contrary, did not modify the antidipsogenic effects induced by TNF-α. These data indicate that peripherally injected cloricromene (as well as that i.c.v. injected) antagonizes the effects of mediators of LPS on sites regulating thirst and suggest that cloricromenes action may be due to inhibition of brain TNF-α formation induced by LPS.

Effects of water deprivation and angiotensin II intracerebroventricular administration on brain nitric oxide synthase activity

Intracranial administration of L-arginine causes a reduction of the water intake induced by water deprivation or by intracerebroventricular (i.c.v.) injection of angiotensin II (angiotensin II), through the release of nitric oxide (NO) in the central nervous system. We studied the effects of i.c.v. angiotensin II (120 ng/rat) in association with i.c.v. L-arginine (2.5-10 microg/rat) on blood pressure. We also studied the effects of both peripheral and central angiotensin II injection (1.5-6 mg kg(-1) i.p. and 30-120 ng rat(-1) i.c.v., respectively) on NO synthase activity in the cortex, diencephalon and brainstem, after water deprivation (24 h), conditions producing activation of the renin-angiotensin system. L-arginine dose dependently antagonized the increase in blood pressure induced by i.c.v. angiotensin II (P < 0.001). Peripheral administration of angiotensin II produced a dose-dependent reduction of NO synthase activity in the brainstem and cortex (P < 0.001), but not in the diencephalon. Water deprivation produced similar effects on brain NO synthase activity. Angiotensin II i.c.v. injection caused NO synthase activity reduction in all brain regions studied (P < 0.001). Our findings suggest that NO and angiotensin II could play opposite roles in brain regulation of blood pressure and drinking behaviour.

INTERLEUKIN-1 INHIBITS DRINKING BEHAVIOUR THROUGH PROSTAGLANDINS, BUT NOT BY NITRIC OXIDE FORMATION

Interleukin-1 beta (IL-1 beta) causes inhibition of drinking behaviour. Moreover it induces formation of prostaglandins (PGs) and nitric oxide (NO). Both PGs and NO are able to inhibit drinking stimulated by water deprivation or by intracerebroventricular (i.c.v.) administration of angiotensin II. In this study, we studied in the preoptic area (POA) the possible role of PGs and NO in the antidipsogenic action induced by IL-1 beta. IL-1 beta was injected in the lateral cerebral ventricle (i.c.v.) (2.5, 10, 20, and 40 ng/rat) or into POA (0.625, 1.25, 2.5, and 10 ng/rat). L-arginine (12.5, 25, 50, and 100 ng/rat), the precursor of NO, or NG-nitro-L-arginine methyl ester (L-NAME) (25, 50, and 100 ng/rat), an inhibitor of nitric oxide synthase (NOS), were injected only into POA. Drinking behaviour was induced by water deprivation (24 h). IL-1 beta injected either i.c.v. or into POA caused dose dependent inhibition of drinking. In the POA a treatment with acetylsalicylic acid (ASA) (33, 66, and 135 micrograms/rat), but not with L-NAME, antagonized the inhibition of drinking behaviour induced by the highest doses of IL-1 beta in the POA. In the POA, a treatment with ASA or L-NAME antagonized the inhibition of drinking behaviour caused by injection of the highest doses of L-arginine. Our data suggest that the central inhibition of drinking behaviour of IL-1 beta is mediated through the formation of PGs, but not NO, in the POA.