Atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in ischemic stroke

Statins have recently been shown to exert neuronal protection in ischemic stroke. Reactive oxygen species, specifically superoxide formed during the early phase of reperfusion, augment neuronal injury. NADPH oxidase is a key enzyme for superoxide production. The present study tested the hypothesis that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. Transient focal ischemia was created in halothane-anesthetized adult male Sprague-Dawley rats (250-300 g) by middle cerebral artery occlusion (MCAO). Atorvastatin (Lipitor, 10 mg/kg sc) was administered three times before MCAO. Infarct volume was measured by triphenyltetrazolium chloride staining. NADPH oxidase enzymatic activity and superoxide levels were quantified in the ischemic core and penumbral regions by lucigenin (5 microM)-enhanced chemiluminescence. Expression of NADPH oxidase membrane subunit gp91(phox) and membrane-translocated subunit p47(phox) and small GTPase Rac-1 was analyzed by Western blot. NADPH oxidase activity and superoxide levels increased after reperfusion and peaked within 2 h of reperfusion in the penumbra, but not in the ischemic core, in MCAO rats. Atorvastatin pretreatment prevented these increases, blunted expression of membrane subunit gp91(phox), and prevented translocation of cytoplasmic subunit p47(phox) to the membrane in the penumbra 2 h after reperfusion. Consequently, cerebral infarct volume was significantly reduced in atorvastatin-treated compared with nontreated MCAO rats 24 h after reperfusion. These results indicate that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia.     

Biochemical and Molecular Characteristics of the Brain with Developing Cerebral Infarction

1. We review the biochemical and molecular changes in brain with developing cerebral infarction, based on recent findings in experimental focal cerebral ischemia.2. Occlusion of a cerebral artery produces focal ischemia with a gradual decline of blood flow, differentiating a severely ischemic core where infarct develops rapidly and an area peripheral to the core where the blood flow reduction is moderate (called penumbra). Neuronal injury in the penumbra is essentially reversible but only for several hours. The penumbra area tolerates a longer duration of ischemia than the core and may be salvageable by pharmacological agents such as glutamate antagonists or prompt reperfusion.3. Upon reperfusion, brain cells alter their genomic properties so that protein synthesis becomes restricted to a small number of proteins such as stress proteins. Induction of the stress response is considered to be a rescue program to help to mitigate neuronal injury and to endow the cells with resistance to subsequent ischemic stress. The challenge now is to determine how the neuroprotection conferred by prior sublethal ischemia is achieved so that rational strategies can be developed to detect and manipulate gene expression in brain cells vulnerable to ischemia.4. Expansion of infarction may be caused by an apoptotic mechanism. Investigation of apoptosis may also help in designing novel molecular strategies to prevent ischemic cell death.5. Ischemia/reperfusion injury is accompanied by inflammatory reactions induced by neutrophils and monocytes/macrophages infiltrated and accumulated in ischemic areas. When the role of the inflammatory/immune systems in ischemic brain injury is revealed, new therapeutic targets and agents will emerge to complement and synergize with pharmacological intervention directed against glutamate and Ca²⁺ neurotoxicity.     

Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats

The aim of this study was to investigate the role of apoptosis or necrosis in the development of delayed infarct, and the relationship between the level of XIAP gene, caspase-3 activation and ischemic cell death following transient focal cerebral ischemia. Adult male Sprague-Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 50 min and reperfusion for 0.5, 4, 8, 24 h, 3, 7, 14 days. On TTC-stained coronal sections, delayed infarct was observed to develop in the whole MCA territory, especially in frontoparietal cortex after ischemia. Near total infarct was shown in striatum 24 h after MCAo, while delayed infarct was evident in the cortex. By day 3, the infarct had progressively expanded to the nearly whole area of the frontoparietal cortex. Flow cytometric analysis of Annexin-V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that MCAo dominantly induced necrosis in ischemic core, striatum. Apoptosis contributed to delayed infarct and cell death in the border zone, dorsolateral cortex and hippocampus. The time-course of caspase-3 activation was consistent with the changes of apoptosis and infarct following MCAo. Further RT-PCR experiments indicated that there was a biphasic regulation of XIAP in time- and region-dependent manner after ischemia. In the infarct core (striatum), following a transient and slight increase during 0.5 h to 4 h post-MCAo, expression of XIAP mRNA markedly decreased. On the other hand, a longer and larger upregulation of XIAP was observed at early time points in border zone (0.5 to 8 h, in dorsolateral cortex; 0.5 to 24 h in hippocampus), then the level of XIAP reduced. A negative correlation was observed between apoptosis and regulation of XIAP gene in these regions. Our findings suggest a possible association between expression of XIAP gene, apoptosis and delayed infarct following ischemia.     

Characterization of neuronal cell death in normal and diabetic rats following exprimental focal cerebral ischemia.

We have studied the forms of cell death following ischemia/reperfusion, and the influence of diabetes mellitus (DM) as an additional factor. Based on the models of diabetes and middle cerebral artery occlusion (MCAO), characteristics of cell death after ischemia/reperfusion were evaluated synthetically by different methods: pathology, FCM, TUNEL and DNA agarose electrophoresis. The results showed that the occurrence of cerebral injury after ischemia/reperfusion was accompanied by cell necrosis and cell apoptosis. Cell apoptosis was mainly located in the ischemic penumbral (IP) zone around the densely ischemic focus. The ischemic core was characterized by cell necrosis. At the same time, the results showed that the process of ischemic cerebral injury worsened by DM was related to inducing cell apoptosis in IP and mid zone. In conclusion, there existed not only cell apoptosis but cell necrosis in brain damage following focal cerebral ischemia/reperfusion and showed a close, internal relationship between them. Brain damage following cerebral ischemia/reperfusion was worsened distinctly under diabetic conditions.     

Striatal Protection Induced by Lesioning the Substantia Nigra of Rats Subjected to Focal Ischemia

Unilateral 6-hydroxydopamine lesion of the substantia nigra reduced the volume of striatal necrosis and suppressed the increase in extracellular glutamate concentration in the striatum induced by middle cerebral artery occlusion in rats. These results indicate that the dopaminergic nigrostriatal pathway is highly involved in the vulnerability of the striatum to ischemia and suggest that glutamate-dopamine interactions may play a key role in the striatal ischemic insult.     

Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract: Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16–72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.     

Neuroprotective effect of N-acetyl-aspartyl-glutamate in combination with mild hypothermia in the endothelin-1 rat model of focal cerebral ischaemia

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.     

The Involvement of Upregulation and Translocation of Phospho-Rb in Early Neuronal Apoptosis Following Focal Cerebral Ischemia in Rats

The aim of this study was to investigate the temporal and spatial relationship between phospho-Rb (ser 795) and neuronal apoptotic death in rats subjected to transient focal cerebral ischemia. We found increased phosphorylation of Rb and translocation from neuronal nucleus to cytoplasm in the penumbra zone at 12 h, 1 day, 3 days and 7 days after middle cerebral artery occlusion (MCAO)/reperfusion, compared with sham-operated controls. At 12 h and 1 day, phospho-Rb appeared to be colocalizated with TUNEL staining in neurons, but staining was not colocalizated at 3 days and 7 days. These results demonstrated that cytoplasmic translocation of phospho-Rb from nucleus of neurons occurs in potential apoptotic neurons in the early stages of ischemia/reperfusion, suggesting that the Rb pathway may only be involved in early neuronal apoptosis and may be not an apoptotic signal in the late stages of transient cerebral ischemia. Ying Yu and Xiang Luo contributed equally to this work.     

Apelin-13 Inhibits Apoptosis of Cortical Neurons Following Brain Ischemic Reperfusion Injury in a Transient Model of Focal Cerebral Ischemia

Stroke is the third leading cause of death world-wide, affecting 15 million people annually. Diminished blood supply to the brain cells is the main cause of damage following stroke. When focal ischemia occurs, the core of brain tissue influenced by reduced blood supply undergoes necrotic cell death. The adipocytokine Apelin is a peptide that was isolated from a bovine stomach for the first time. This peptide and its receptor are abundantly expressed in the nervous and cardiovascular systems. According to previous studies, Apelin-13 protects cardiomyocytes from ischemic injury and apoptosis. In addition, this peptide has neuroprotective effect on hippocampal and cultured mouse cortical neurons against NMDA receptor-mediated excitotoxicity as well as cortical neurons from ischemic injury. The present study was conducted to determine whether Apelin-13 inhibits apoptosis in the ischemic penumbra in transient focal cerebral ischemia. Focal cerebral ischemia was induced in male Wistar rats by 60 min middle cerebral artery occlusion (MCAO) using a filament method, followed by 23-h reperfusion. Saline as a vehicle and Apelin-13 at doses of 50 and 100 μg were injected intracerebro-ventriculary (ICV) at the beginning of ischemia. Apoptosis and neurological dysfunction were assessed 24-h after MCAO. Our results indicated that administration of Apelin-13 at doses of 50 and 100 μg ICV markedly reduced apoptosis by decreasing positive TUNEL cells (P < 0.001). In addition, Apelin-13 at doses of 100 μg significantly change neurological dysfunction (P < 0.05). Our findings demonstrate that treatment by Apelin-13 exerts its protective effects in ischemic models via blocking programmed cell-death. We suggest that Apelin-13 might be a promising therapeutic target for stroke, although more researches are necessary to take into account the potential therapeutic effects of Apelin-13 in stroke patients.     

Anti-inflammatory mechanism of taurine against ischemic stroke is related to down-regulation of PARP and NF-κB

Taurine is reported to reduce tissue damage induced by inflammation and to protect the brain against experimental stroke. The objective of this study was to investigate whether taurine reduced ischemic brain damage through suppressing inflammation related to poly (ADP-ribose) polymerase (PARP) and nuclear factor-kappaB (NF-κB) in a rat model of stroke. Rats received 2 h ischemia by intraluminal filament and were then reperfused. Taurine (50 mg/kg) was administered intravenously 1 h after ischemia. Treatment with taurine markedly reduced neurological deficits, lessened brain swelling, attenuated cell death, and decreased the infarct volume 72 h after ischemia. Our data showed the up-regulation of PARP and NF-κB p65 in cytosolic fractions in the core and nuclear fractions in the penumbra and core, and the increases in the nuclear poly (ADP-ribose) levels and the decreases in the intracellular NAD⁺ levels in the penumbra and core at 22 h of reperfusion; these changes were reversed by taurine. Moreover, taurine significantly reduced the levels of tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase, and intracellular adhesion molecule-1, lessened the activities of myeloperoxidase and attenuated the infiltration of neutrophils in the penumbra and core at 22 h of reperfusion. These data demonstrate that suppressing the inflammatory reaction related to PARP and NF-κB-driven expression of inflammatory mediators may be one mechanism of taurine against ischemic stroke.     

Immune-related GTPase M (IRGM1) regulates neuronal autophagy in a mouse model of stroke

Autophagy is an important cellular recycling mechanism through self-digestion in responses to cellular stress such as starvation. Studies have shown that autophagy is involved in maintaining the homeostasis of the neural system during stroke. However, molecular mechanisms underlying neuronal autophagy in ischemic stroke remain poorly understood. Previously, we and others have shown that immune-related GTPase M (IRGM; termed IRGM1 in the mouse nomenclature) can regulate the survival of immune cells through autophagy in response to infections and autoimmune conditions. Here, using a permanent middle cerebral artery occlusion (pMCAO) mouse model, we found that IRGM1 was upregulated in the ischemic side of the brain, which was accompanied by a significant autophagic response. In contrast, neuronal autophagy was almost complete lost in Irgm1 knockout (KO) mice after pMCAO induction. In addition, the infarct volume in the Irgm1-KO pMCAO mice was significantly increased as compared to wild-type mice. Histological studies suggested that, at the early stage (within 24 h) of ischemia, the IRGM1-dependent autophagic response is associated with a protection of neurons from necrosis in the ischemic core but a promotion of neuronal apoptosis in the penumbra area. These data demonstrate a novel role of IRGM1 in regulating neuronal autophagy and survival during ischemic stroke.     

Dopamine induces apoptosis in young, but not in neonatal, neurons via Ca2+-dependent signal

Dopamine signaling plays a major role in regulation of neuronal apoptosis. During the postnatal period, dopamine signaling is known to be dramatically changed in the striatum. However, because it is difficult to culture neurons after birth, little is known about developmental changes in dopamine-mediated apoptosis. To examine such changes, we established the method of primary culture of striatal neurons from 2- to 3-wk-old (young) mice. Dopamine, via D(1)-like receptors, induced apoptosis in young, but not neonatal, striatal neurons, suggesting that the effect of dopamine on apoptosis changed with development. In contrast, although isoproterenol (Iso), a beta-adrenergic receptor agonist, increased cAMP production to a greater degree than dopamine, Iso did not increase apoptosis in striatal neurons from young and neonatal mice, suggesting a minor role of cAMP in dopamine-mediated apoptosis. Next, we examined the effect of dopamine on Ca(2+) signaling. Dopamine, but not Iso, markedly increased intracellular Ca(2+) in striatal neurons from young mice, and Ca(2+)-chelating agents abolished dopamine-induced apoptosis, suggesting that Ca(2+) played a major role in the dopamine-mediated apoptosis pathway. In contrast, dopamine failed to increase intracellular Ca(2+) in neonatal neurons, and the expression of PLC, which can increase intracellular Ca(2+) via D(1)-like receptor activation, was significantly greater in young than in neonatal striatal neurons. These data suggest that the developmental change in dopamine-mediated Ca(2+) signaling was responsible for differences between young and neonatal striatum in induction of apoptosis. Furthermore, the culture of young striatal neurons is feasible and may provide a new tool for developmental studies.     

Immune-related GTPase M (IRGM1) regulates neuronal autophagy in a mouse model of stroke

Autophagy is an important cellular recycling mechanism through self-digestion in responses to cellular stress such as starvation. Studies have shown that autophagy is involved in maintaining the homeostasis of the neural system during stroke. However, molecular mechanisms underlying neuronal autophagy in ischemic stroke remain poorly understood. Previously, we and others have shown that immune-related GTPase M (IRGM; termed IRGM1 in the mouse nomenclature) can regulate the survival of immune cells through autophagy in response to infections and autoimmune conditions. Here, using a permanent middle cerebral artery occlusion (pMCAO) mouse model, we found that IRGM1 was upregulated in the ischemic side of the brain, which was accompanied by a significant autophagic response. In contrast, neuronal autophagy was almost complete lost in Irgm1 knockout (KO) mice after pMCAO induction. In addition, the infarct volume in the Irgm1-KO pMCAO mice was significantly increased as compared to wild-type mice. Histological studies suggested that, at the early stage (within 24 h) of ischemia, the IRGM1-dependent autophagic response is associated with a protection of neurons from necrosis in the ischemic core but a promotion of neuronal apoptosis in the penumbra area. These data demonstrate a novel role of IRGM1 in regulating neuronal autophagy and survival during ischemic stroke.     

Glutamate and GABA metabolism in transient and permanent middle cerebral artery occlusion in rat: importance of astrocytes for neuronal survival

The aim of the present study was to identify the distinguishing metabolic characteristics of brain tissue salvaged by reperfusion following focal cerebral ischemia. Rats were subjected to 120 min of middle cerebral artery occlusion followed by 120 min of reperfusion. The rats received an intravenous bolus injection of [1-(13)C]glucose plus [1,2-(13)C]acetate. Subsequently two brain regions considered to represent penumbra and ischemic core, i.e. the frontoparietal cortex and the lateral caudoputamen plus lower parietal cortex, respectively, were analyzed with (13)C NMRS and HPLC. The results demonstrated four metabolic events that distinguished the reperfused penumbra from the ischemic core. (1) Improved astrocytic metabolism demonstrated by increased amounts of [4,5-(13)C]glutamine and improved acetate oxidation. (2) Neuronal mitochondrial activity was better preserved although the flux of glucose via pyruvate dehydrogenase into the tricarboxylic acid (TCA) cycle in glutamatergic and GABAergic neurons was halved. However, NAA content was at control level. (3) Glutamatergic and GABAergic neurons used relatively more astrocytic metabolites derived from the pyruvate carboxylase pathway. (4) Lactate synthesis was not increased despite decreased glucose metabolism in the TCA cycle via pyruvate dehydrogenase. In the ischemic core both neuronal and astrocytic TCA cycle activity declined significantly despite reperfusion. The utilization of astrocytic precursors originating from the pyruvate carboxylase pathway was markedly reduced compared the pyruvate dehydrogenase pathway in glutamate, and completely stopped in GABA. The NAA level fell significantly and lactate accumulated. The results demonstrate that preservation of astrocytic metabolism is essential for neuronal survival and a predictor for recovery.     

Differential Changes in Pyridoxine 5′-Phosphate Oxidase Immunoreactivity and Protein Levels in the Somatosensory Cortex and Striatum of the Ischemic Gerbil Brain

Pyridoxal 5'-phosphate (PLP) is an important cofactor in a wide range of biochemical reactions, such as the metabolism of various amino acids, including GABA. PLP is synthesized by the oxidation of pyridoxine 5'-phosphate (PNP), which is catalyzed by PNP oxidase (PNPO). We observed the changes in cresyl violet-positive neurons, PNPO immunoreactivity and PNPO protein levels in the somatosensory cortex and striatum in gerbils after 5 min of transient forebrain ischemia. Cresyl violet-positive neurons showed condensed cytoplasm in the somatosensory cortex and lateral part of the striatum at 2 days after ischemia/reperfusion. PNPO immunoreactivity began to increase in neurons in layers III and V at 3 h after ischemia/reperfusion and this immunoreactivity was significantly increased at 12 h after ischemia/reperfusion. Thereafter, PNPO immunoreactivity decreased with time after ischemia/reperfusion. PNPO-immunoreactive neurons were only slightly detected in the lateral part of the striatum at 12 h after ischemia/reperfusion. In addition, the PNPO protein levels in both the somatosensory cortex and striatum homogenates peaked at 12 h after ischemia/reperfusion. These results indicate that PNPO is significantly increased in the ischemic somatosensory cortex and lateral part of the striatum, and this change in the level of PNPO may be associated with the neuronal damage induced by ischemia.     

Targeting autophagy to prevent neonatal stroke damage

Cell death due to cerebral ischemia has been attributed to necrosis and apoptosis, but autophagic mechanisms have recently been implicated as well. Using rats exposed to neonatal focal cerebral ischemia, we have shown that lysosomal and autophagic activities are increased in ischemic neurons, and have obtained strong neuroprotection by post-ischemic inhibition of autophagy.     

Temporal Profile of Astrocytes and Changes of Oligodendrocyte-Based Myelin Following Middle Cerebral Artery Occlusion in Diabetic and Non-diabetic Rats

The long-term impacts of cerebral ischemia and diabetic ischemia on astrocytes and oligodendrocytes have not been defined. The objective of this study is to define profile of astrocyte and changes of myelin in diabetic and non-diabetic rats subjected to focal ischemia.Focal cerebral ischemia of 30-min duration was induced in streptozotocin-induced diabetic and vehicle-injected normoglycemic rats. The brains were harvested for immunohistochemistry of glial fibrillary acidic protein (GFAP) and 2'', 3''-cyclic nucleotide 3''-phosphodiesterase (CNPase) at various reperfusion endpoints ranging from 30 min up to 28 days. The results showed that activate astrocytes were observed after 30 min and peaked at 3 h to 1 day after reperfusion in ischemic penumbra, and peaked at 7 days of reperfusion in ischemic core. Diabetes inhibited the activation of astrocytes in ischemic hemisphere. Demyelination occurred after 30 min of reperfusion in ischemic core and peaked at 1 day. Diabetes caused more severe demyelination compared with non-diabetic rats. Remyelination started at 7 days and completed at 14 and 28 days in ischemic region. Diabetes inhibited the remyelination processes. It is concluded that ischemia activates astrocytes and induces demyelination. Diabetes inhibits the activation of astrocytes, exacerbates the demyelination and delays the remyelination processes. These may contribute to the detrimental effects of hyperglycemia on ischemic brain damage.     

Characteristic Changes of Astrocyte and Microglia in Rat Striatum Induced by 3-NP and MCAO

Our previous studies had confirmed that both 3-NP and MCAO induced the behavioral defect as well as striatal neuronal injury and loss in experimental rats. This study aimed to examine different response forms of striatal astrocyte and microglia in 3-NP and MCAO rat models. The present results showed that the immunoreaction for GFAP was extremely weak in the lesioned core of striatum, but in the transition zone of 3-NP model and the penumbra zone of MCAO model, GFAP+ cells showed strong hypertrophic and proliferative changes. Statistical analysis for the number, size and integral optical density (IOD) of GFAP+ cells showed significant differences when compared with their controls and compared between the core and the transition zone or the penumbra zone, respectively, but no differences between the 3-NP and MCAO groups. However, Iba-1+ cells showed obvious hypertrophy and proliferation in the injured striatum in the 3-NP and the MCAO models, especially in the transition zone of 3-NP model and the penumbra zone of MCAO model. These Iba-1+ cells displayed two characteristic forms as branching cells with thick processes and amoeboid cells with thin processes. Statistical analysis showed that the number, size and IOD of Iba-1+ cells were significantly increased in the cores and the transition zone of 3-NP group and the penumbra zone of MCAO group than that of the controls, and the immune response of Iba-1 was stronger in the MCAO group than in the 3-NP group. The present results suggested that characteristic responses of astrocyte and microglia in the 3-NP and the MCAO models display their different effects on the pathological process of brain injury.     

Nicotinamide Attenuates Focal Ischemic Brain Injury in Rats: With Special Reference to Changes in Nicotinamide and NAD+ Levels in Ischemic Core and Penumbra

We investigated the neuroprotective action of nicotinamide in focal ischemia. Male spontaneously hypertensive rats (5–7 months old) were subjected to photothrombotic occlusion of the right distal middle cerebral artery (MCA). Either nicotinamide (125 or 250 mg/kg) or vehicle was injected IV before MCA occlusion. Changes in the cerebral blood flow (CBF) were monitored using laser-Doppler flowmetry, and infarct volumes were determined with TTC staining 3 days after MCA occlusion. In another set of experiments, the brain nicotinamide and nicotinamide adenine dinucleotide (NAD⁺) levels were analyzed by HPLC using the frozen samples dissected from the regions corresponding to the ischemic core and penumbra. In the 250-mg/kg nicotinamide group, the ischemic CBF was significantly increased compared to that the untreated group, and the infarct volumes were substantially attenuated (–36%). On the other hand, the ischemic CBF in the 125 mg/kg nicotinamide group was not significantly different from the untreated CBF, however, the infarct volumes were substantially attenuated (–38%). Cerebral ischemia per se did not affect the concentrations of nicotinamide and NAD⁺ both in the penumbra and ischemic core. In the nicotinamide groups, the brain nicotinamide levels increased significantly in all areas examined, and brain NAD⁺ levels increased in the penumbra but not in the ischemic core. Increased brain levels of nicotinamide are considered to be primarily important for neuroprotection against ischemia, and the protective action may be partly mediated through the increased NAD⁺ in the penumbra.     

ATP extracellular concentrations are increased in the rat striatum during in vivo ischemia

Interest is growing in the role of adenosine triphosphate (ATP) on P2 receptors during hypoxic/ischemic events in the brain. However, there is no direct evidence of an increase in extracellular ATP levels during cerebral ischemia in vivo. The aim of the present study was to evaluate ATP outflow from the rat striatum by the microdialysis technique associated with focal cerebral ischemia in vivo by intraluminal occlusion of the right middle cerebral artery (MCA). Between 1 and 4h after ischemia, rats showed a clear turning behavior contralateral to the ischemic side. Twenty-four hour after MCA occlusion, ischemic rats had definite neurological deficit and striatal and cortical damage. The ATP concentration (mean+/-S.E.M.) in the striatum of normoxic rats (n = 8) was 3.10+/-0.34 nM. During 220 min after MCA occlusion, the extracellular ATP levels significantly increased two-fold, being 5.90+/-0.61 nM (p < 0.01 versus normoxic level). ATP outflow showed a tendency to increase over time during the 220 min of ischemia. Since extracellular ATP is rapidly metabolized to adenosine, we also assessed ATP outflow in the presence of the ecto-5'-nucleotidase inhibitor, alpha,beta-methylene-adenosine diphosphate (AOPCP, 1 mM) directly perfused into the striatum. The ATP concentration in normoxic rats (n = 8) was increased three-fold in the presence of the ecto-5'-nucleotidase inhibitor (9.57+/-0.26 nM). During 220 min of ischemia, extracellular ATP levels significantly increased 1.3-fold in AOPCP-treated rats (12.62+/-0.65 nM, p < 0.01 versus normoxic level). The present study confirms that ATP is continuously released in the brain and demonstrates for the first time that ATP outflow increases during ischemia in vivo. These results confirm that ATP may be an important mediator in brain ischemia.