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.     

Characterizing Photothrombotic Distal Middle Cerebral Artery Occlusion and YAG Laser-Induced Reperfusion Model in the Izumo Strain of Spontaneously Hypertensive Rats

No study has systematically studied the relevance of original Izumo strain of spontaneously hypertensive rats (SHR/Izm) as a stroke model. Furthermore, both SHR/Izm and stroke-prone SHR/Izm (SHRSP/Izm) are commercially available, and recent progress in genetic studies allowed us to use several congenic strains of rats constructed with SHR/Izm and SHRSP/Izm as the genetic background strains. A total of 166 male SHR/Izm and 17 male SHRSP/Izm were subjected to photothrombotic middle cerebral artery (MCA) occlusion with or without YAG laser-induced reperfusion. The pattern of distal MCA was recorded. Infarct volumes were determined with 2,3,5-triphenyltetrazolium chloride. At 24 or 48 h after MCA occlusion, infarct volumes in the permanent occlusion and 2-h occlusion groups (88 ± 22 [SD] and 87 ± 25 mm³, respectively) were significantly larger than that in the 1-h occlusion group (45 ± 14 mm³), indicating the presence of sizeable zone of penumbra. Infarct size in SHRSP/Izm determined at 24 h after MCA occlusion was fairly large (124.0 ± 34.8 mm³, n = 10). Infarct volume in SHR/Izm with simple distal MCA was 76 ± 19 mm³, which was significantly smaller than 95 ± 22 mm³ in the other SHR/Izm with more branching MCA. These data suggest that this stroke model in SHR/Izm is useful in the preclinical testing of stroke therapies and elucidating the pathophysiology of cerebral ischemia/reperfusion.     

Delayed Administration of Parecoxib, a Specific COX-2 Inhibitor, Attenuated Postischemic Neuronal Apoptosis by Phosphorylation Akt and GSK-3β

Parecoxib is a recently described novel COX-2 inhibitor whose functional significance and neuroprotective mechanisms remain elusive. Therefore, in this study, we aimed to investigate whether delayed administration of parecoxib inhibited mitochondria-mediated neuronal apoptosis induced by ischemic reperfusion injury via phosphorylating Akt and its downstream target protein, glycogen synthase kinase 3β (GSK-3β). Adult male Sprague–Dawley rats were administered parecoxib (10 or 30 mg kg⁻¹, IP) or isotonic saline twice a day starting 24 h after middle cerebral artery occlusion (MCAO) for three consecutive days. Cerebral infarct volume, apoptotic neuron, caspase-3 immunoreactivity and the protein expression of p-Akt, p-GSK-3β and Cytochrome C in cerebral ischemic cortex were evaluated at 96 h after reperfusion. Parecoxib significantly diminished infarct volume and attenuated neuron apoptosis in a dose-independent manner, compared with MCAO group alone. Increased p-Akt and p-GSK-3β was observed in the ischemic penumbra of parecoxib group after stroke. Moreover, parecoxib also reduced the release of Cytochrome C from mitochondrial into cytosol and attenuated the caspase-3 immunoreactivity in the penumbra. Taken together, these results suggested that parecoxib ameliorated postischemic mitochondria-mediated neuronal apoptosis induced by focal cerebral ischemia in rats and this neuroprotective potential is involved in phosphorylation of Akt and GSK-3β.     

Early ischemic preconditioning against focal transient and permanent brain ischemia in rats: role of collateral circulation

We hypothesize that early ischemic preconditioning (IPC) can afford protection against focal brief and prolonged cerebral ischemia with subsequent reperfusion as well as permanent brain ischemia in rats by amelioration of regional cerebral blood flow. Adult male Wistar rats (n=97) were subjected to transient (30 and 60 minutes) and permanent middle cerebral artery (MCA) occlusion. IPC protocol consisted of two episodes of 5-min common carotid artery occlusion + 5-min reperfusion prior to test ischemia either followed by 48 hours of reperfusion or not. Triphenyltetrazolium chloride and Evans blue were used for delineation of infarct size and anatomical area at risk (comprises ischemic penumbra and ischemic core), respectively. Blood flow in the MCA vascular bed was measured with use of Doppler ultrasound. The IPC resulted in significant infarct size limitation in both transient and permanent MCA occlusion. Importantly, IPC caused significant reduction of area at risk after 30 min of focal ischemia as compared to controls [med(min-max) 11.4% (3.59-2 0.35%) vs. 2.47% (0.8-9.31%), p = 0.018] but it failed to influence area at risk after 5 min of ischemia [med(min-max) 7.61% (6.32-10.87%) vs. 8.2% (4.87-9.65%), p > 0.05]. No differences in blood flow were found between IPC and control groups using Doppler ultrasound. This is suggestive of the fact that IPC does not really influence blood flow in the large cerebral arteries such as MCA but it might have some effect on smaller arteries. It seems that, along with well established cytoprotective effects of IPC, IPC-mediated reduction of area at risk by means of improvement in local cerebral blood flow may contribute to infarct size limitation after focal transient and permanent brain ischemia in rats.     

Cornel Iridoid Glycoside Inhibits Inflammation and Apoptosis in Brains of Rats with Focal Cerebral Ischemia

The capacity of cornel iridoid glycoside (CIG) to suppress the manifestations of ischemic stroke was investigated. CIG was administered to rats by the intragastric route once daily for 7 days. Focal cerebral ischemia was induced by 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. In non-treated rats large infarct areas were observed within 24 h of reperfusion. Examination of the ischemic cerebral cortex revealed microglia and astrocyte activation, increased interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) concentrations, increased DNA fragmentation in the ischemia penumbra, elevated Bax expression, increased caspase-3 cleavage, and decreased Bcl-2 expression. Pretreatment with CIG decreased the infarct area, DNA fragmentation, IL-1β and TNF-α concentrations, microglia and astrocyte activation, Bax expression, and caspase-3 cleavage while increasing Bcl-2 expression. CIG exerts anti-neuroinflammatory and anti-apoptotic effects which should prove beneficial for prevention or treatment of stroke.     

Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient focal cerebral ischemia in mice

The present observations examined the hypothesis that the iNOS expression in the ischemic penumbra after a transient focal ischemic insult is involved in the recruitment of penumbra into infarction. The middle cerebral artery in mice was occluded for 2 h by an intraluminal filament and then recirculated. The measurement of iNOS activity, iNOS protein formation and NO concentration in the ischemic core and penumbra, and the determination of infarct volume were performed at 6, 12, 24 and 48 h after reperfusion. iNOS protein and iNOS enzymatic activity appeared at 6 h, peaked at 24 h, and declined at 48 h in the penumbra after reperfusion. iNOS protein was not detectable in contralateral area and in sham-operated brains. The time course of iNOS protein, enzymatic activity and NO concentration in the penumbra but not in the core matched the process of infarct maturation. Treatment with iNOS inhibitor aminoguanidine (100 mg.kg(-1), i.p.) at 6 and 12 h after reperfusion inhibited iNOS activity by 88.0 +/- 10.4% and reduced NO concentration by 48.5 +/- 8.3% in the penumbra, and lessened infarct size by 48.8 +/- 7.2%. The iNOS activity and NO level in the core were not affected by the administration of aminoguanidine. These results suggest that iNOS expression in the ischemic penumbra is involved in the recruitment of penumbra into infarction and thereby contributing to the enlargement of infarct.     

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.     

Nitric oxide down-regulates caveolin-1 expression in rat brains during focal cerebral ischemia and reperfusion injury

As a signalling molecule of the integral membrane protein family, caveolin participates in cellular signal transduction via interaction with other signalling molecules. The nature of interaction between nitric oxide (NO) and caveolin in the brain, however, remains largely unknown. In this study we investigated the role(s) of NO in regulating caveolin-1 expression in rat ischemic brains with middle cerebral artery occlusion (MCAO). Exposure to 1 h ischemia induced the increases in neuronal nitric oxide synthase (nNOS) and NO concentration with concurrent down-regulation of caveolin-1 expression in the ischemic core of rat brains. Subsequent 24 h or more reperfusion time led to an increase in inducible NOS (iNOS) expression and NO production, as well as a decline of caveolin-1 protein at the core and penumbra of the ischemic brain. Afterwards, NOS inhibitors and an NO donor were utilized to clarify the link between NO production and caveolin-1 expression in the rats with 1 h ischemia plus 24 h reperfusion. N(G)-nitro-l-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), N(6)-(1-iminoethyl)-lysine (NIL, an iNOS inhibitor), and 7-nitroindazole (7-NI, a nNOS inhibitor) prevented the loss of caveolin-1 in the core and penumbra of the ischemic brain, whereas l-N(5)-(1-iminoethyl)-ornithine (L-NIO, an endothelial NOS inhibitor) showed less effect than the other NOS inhibitors. S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) down-regulated the expression of caveolin-1 protein in normal and ischemic brains. These results, when taken together, suggest that NO modulates the expression of caveolin-1 in the brain and that the loss of caveolin-1 is associated with NO production in the ischemic brain.     

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.     

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.     

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.     

Sesamin attenuates behavioral,biochemical and histological alterations induced by reversible middle cerebral artery occlusion in the rats

Restoration of blood flow to an ischemic brain region is associated with generation of reactive oxygen species (ROS) with consequent reperfusion injury. ROS cause lipid peroxidation, protein oxidation, and DNA damage, all of which are deleterious to cells. So diminishing the production of free radicals and scavenging them may be a successful therapeutic strategy for the protection of brain tissue in cerebral stroke. The present study investigated the neuroprotective effect of sesamin (Sn) to reduce brain injury after middle cerebral artery occlusion (MCAO). The middle cerebral artery (MCA) of adult male Wistar rat was occluded for 2 h and reperfused for 22 h. Sesamin is the most abundant lignan in sesame seed oil is a potent antioxidant. Sesamin (30 mg/kg) was given orally twice, 30 min before the onset of ischemia and 12 h after reperfusion. The initial investigations revealed that sesamin reduced the neurological deficits in terms of behavior and reduced the level of thiobarbituric acid reactive species (TBARS), and protein carbonyl (PC) in the different areas of the brain when compared with the MCAO group. A significantly depleted level of glutathione and its dependent enzymes (glutathione peroxidase [GPx] and glutathione reductase [GR]) in MCAO group were protected significantly in MCAO group treated with sesamin. The present study suggests that sesamin may be able to attenuate the ischemic cell death and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the rat brain. Thus, sesamin may be a potential compound in stroke therapy.     

Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke

In vitro nitric oxide (NO) regulates calpain and caspase-3 activation, and in vivo neuronal nitric oxide synthase (nNOS), calpain and caspase-3 participate in the ischemic brain injury. Our objective was to investigate whether nNOS was involved in the ischemic brain injury through activating calpain and caspase-3 during experimental stroke. Rats received 1-h ischemia by intraluminant filament, and then reperfused for 23 h (R 23 h). nNOS inhibitor 7-nitroindozale (7-NI, 50 mg/kg) was administrated intraperitoneally 5 min before ischemia. Our data showed that treatment with 7-NI markedly reduced neurological deficits, the brain swelling, and the infarct volume at R 23 h. Enzyme studies revealed significant suppression of the activities of m-calpain and caspase-3 in penumbra and core, and the activities of μ-calpain in penumbra, but not in core, in 7-NI-treated rats versus vehicle-treated rats. Western blot analysis demonstrated that 7-NI markedly increased the levels of MAP-2 and spectrin in penumbra and core compared with vehicle-treated rats. Histopathological studies displayed that 7-NI significantly reduced the necrotic cell death in penumbra and core, and apoptotic cell death in penumbra, but not in core. These data demonstrate the involvement of NO produced by nNOS in the ischemic neuronal injury through affecting the activation of calpain and caspase-3 in penumbra and core after experimental stroke, which provides a new perspective on possible mechanisms of action of nNOS inhibition in cerebral ischemia.     

Recovery from Edema and of Protein Synthesis Differs Between the Cortex and Caudate Following Transient Focal Cerebral Ischemia in Rats

Postischemic recovery from brain edema and of protein synthesis was examined following 1 h of middle cerebral artery (MCA) occlusion in rats. Recovery from brain edema and of protein synthesis showed a good correlation until 7 days after reperfusion in each area (cerebral cortex or lateral caudate) in the occluded MCA side. However, regional differences in the above types of recovery in the cortex and in the lateral caudate were found for the first time in this experiment. A profound inhibition of protein synthesis and formation of brain edema began sooner in the lateral caudate than in the cortex and continued long after reperfusion. Grades of cerebral blood flow during ischemia and the early period of reperfusion were almost the same in the two regions. Therefore, the regional differences in the above recoveries may not be due to the difference in the blood flow during ischemia and reperfusion, but may be partly attributable to the imbalance of excitatory and inhibitory innervation in the above two areas of the brain, may be due to a distinctive response to ischemic stress, and may be caused also by the potentiative effect of free arachidonate on the excitotoxic mechanism.     

Changes in the level of glial fibrillary acidic protein (GFAP) after mild and severe focal cerebral ischemia

In the present study, we examined the temporal and spatial expression profiles of GFAP mRNA and protein in a focal cerebral ischemia model with ischemic injury confined to the cerebral cortex in the right middle cerebral artery (MCA) territory. Northern blot analysis showed a respective 5.5-fold and 7.2-fold increase in the GFAP mRNA in the ischemic right MCA cortex in rats subjected to 30-min (mild) or 60-min (severe) ischemia followed by 72-hr reperfusion. The GFAP mRNA signal remained elevated up to 2-week reperfusion. Interestingly, increased GFAP mRNA signal was clearly demonstrated for the first time in the left MCA cortex. A significant 1.5-fold and 5-fold increase was observed after 72-hr reperfusion following mild and severe ischemia, respectively. However, unlike the ischemic right MCA cortex, this induction was transient in the non-ischemic left MCA counterpart. In situ hybridization studies further revealed characteristic spatial induction profile following mild vs. severe ischemia. In mild ischemia, following 24-hr reperfusion, increase in GFAP mRNA was observed mainly within the ischemic right MCA cortex. Following 72-hr reperfusion, GFAP mRNA signal was observed in virtually the entire ischemic cortex, particularly the amygdala region, then gradually reduced and restricted to right MCA territory and subcortical thalamic nucleus following 2-week reperfusion. On the other hand, in severe ischemia, following 24-hr reperfusion increased GFAP mRNA signal was observed in area surrounding right MCA territory (infarct region) and outer cortical layers within the right MCA territory. Following 72-hr reperfusion, no signal was detected within right MCA cortex; however, increased GFAP signal was detected throughout the remaining ipsilateral cortex and subcortical region, as well as the contralateral cerebral cortex. GFAP mRNA signals then gradually reduced its intensity and was restricted to area surrounding necrosis and ipsilateral thalamic nucleus following 2-week reperfusion. GFAP-like immunoreactivity was also detected in area expressing GFAP mRNA. It is very likely that de novo synthesis was responsible for this increase. In summary, increased GFAP signal was noted in both ipsilateral and contralateral cerebral following mild and severe ischemia. Although the temporal induction profile for mild vs. severe ischemia was similar, the spatial induction profile was different. The mechanism leading to this differential induction and their physiological and functional significance are not clear at present. It is very likely that some local factors may involve, nevertheless, the detailed mechanisms remain to be fully explored.     

The obligatory role of COX-2 expression for induction of HO-1 in ischemic preconditioned rat brain

The molecular mechanisms of preconditioning-induced ischemic tolerance (PCIT) have yet to be elucidated. We investigated whether minimal expression levels of COX-2 induced by preconditioning trigger HO-1, thereby inducing the synthesis of cytoprotective proteins. We show that both COX-2 and HO-1 are induced in rat brains subjected to preconditioning by middle cerebral artery (MCA) occlusion for 10 min followed by different amounts of reperfusion time (1-24 h). Although preconditioning significantly reduced the brain infarct size against severe ischemia (24 h MCA occlusion), pretreatment with the COX-2-selective inhibitor rofecoxib increased infarct size and abolished PCIT-induced COX-2 and HO-1 expression in vivo. We also found that PGE₂ increased the phosphorylation of Akt, which was significantly inhibited by the PI3 kinase inhibitor LY294002. Taken together, we conclude that the kinetic changes in COX-2 induction during the reperfusion period following preconditioning may be important for ischemic tolerance.     

A surgical model of permanent and transient middle cerebral artery stroke in the sheep

Background

Animal models are essential to study the pathophysiological changes associated with focal occlusive stroke and to investigate novel therapies. Currently used rodent models have yielded little clinical success, however large animal models may provide a more suitable alternative to improve clinical translation. We sought to develop a model of acute proximal middle cerebral artery (MCA) ischemic stroke in sheep, including both permanent occlusion and transient occlusion with reperfusion.

Materials and Methods

18 adult male and female Merino sheep were randomly allocated to one of three groups (n = 6/gp): 1) sham surgery; 2) permanent proximal MCA occlusion (MCAO); or 3) temporary MCAO with aneurysm clip. All animals had invasive arterial blood pressure, intracranial pressure and brain tissue oxygen monitoring. At 4 h following vessel occlusion or sham surgery animals were killed by perfusion fixation. Brains were processed for histopathological examination and infarct area determination. 6 further animals were randomized to either permanent (n = 3) or temporary MCAO (n = 3) and then had magnetic resonance imaging (MRI) at 4 h after MCAO.

Results

Evidence of ischemic injury in an MCA distribution was seen in all stroke animals. The ischemic lesion area was significantly larger after permanent (28.8%) compared with temporary MCAO (14.6%). Sham animals demonstrated no evidence of ischemic injury. There was a significant reduction in brain tissue oxygen partial pressure after permanent vessel occlusion between 30 and 210 mins after MCAO. MRI at 4 h demonstrated complete proximal MCA occlusion in the permanent MCAO animals with a diffusion deficit involving the whole right MCA territory, whereas temporary MCAO animals demonstrated MRA evidence of flow within the right MCA and smaller predominantly cortical diffusion deficits.

Conclusions

Proximal MCAO can be achieved in an ovine model of stroke via a surgical approach. Permanent occlusion creates larger infarct volumes, however aneurysm clip application allows for reperfusion.     

成年鼠缺血性脑损伤诱导nestin的表达

应用免疫组化和免疫荧光双标技术结合激光共聚焦扫描显微镜,观察缺血性脑损伤后脑内nestin的表达及其细胞类型。实验观察结果为,再灌后1天,在缺血中心区可见nestin阳性突起;再灌后3天和1周时,除缺血中心区外,周边I、Ⅱ、Ⅲ区均有nestin阳性突起主要与GFAP共存;2周时,nestin阳性突起变粗、变长,并与NSE的共存明显增多。上述研究结果提示,脑缺血可诱导大鼠脑缺血区域表达nestin,该表达可能与神经细胞的修复有关。     

Inhibition of Autophagy Contributes to Ischemic Postconditioning-Induced Neuroprotection against Focal Cerebral Ischemia in Rats

Background

Ischemic postconditioning (IPOC), or relief of ischemia in a stuttered manner, has emerged as an innovative treatment strategy to reduce programmed cell death, attenuate ischemic injuries, and improve neurological outcomes. However, the mechanisms involved have not been completely elucidated. Recent studies indicate that autophagy is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. This study aims to determine the role of autophagy in IPOC-induced neuroprotection against focal cerebral ischemia in rats.

Methodology/Principal Findings

A focal cerebral ischemic model with permanent middle cerebral artery (MCA) occlusion plus transient common carotid artery (CCA) occlusion was established. The autophagosomes and the expressions of LC3/Beclin 1/p62 were evaluated for their contribution to the activation of autophagy. We found that autophagy was markedly induced with the upregulation of LC3/Beclin 1 and downregulation of p62 in the penumbra at various time intervals following ischemia. IPOC, performed at the onset of reperfusion, reduced infarct size, mitigated brain edema, inhibited the induction of LC3/Beclin 1 and reversed the reduction of p62 simultaneously. Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effects induced by IPOC. Conversely, autophagy inhibitor 3-methyladenine (3-MA) attenuated the ischemic insults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.

Conclusions/Significance

The present study suggests that inhibition of the autophagic pathway plays a key role in IPOC-induced neuroprotection against focal cerebral ischemia. Thus, pharmacological inhibition of autophagy may provide a novel therapeutic strategy for the treatment of stroke.     

Spatio-temporal properties of 5-lipoxygenase expression and activation in the brain after focal cerebral ischemia in rats

The role of 5-lipoxygenase (5-LOX) in brain injury after cerebral ischemia has been reported; however, the spatio-temporal properties of 5-LOX expression and the enzymatic activation are unclear. To determine these properties, we observed post-ischemic 5-LOX changes from 3 h to 14 days after reperfusion in rats with transient focal cerebral ischemia induced by 30 min of middle cerebral artery occlusion. We found that the expression of 5-LOX, both mRNA and protein, was increased in the ischemic core 12-24 h after reperfusion, and in the boundary zone adjacent to the ischemic core 7-14 days after reperfusion. The increased 5-LOX was primarily localized in the neurons in the ischemic core at 24 h, but in the proliferated astrocytes in the boundary zone 14 days after reperfusion. As 5-LOX metabolites, the level of cysteinyl-leukotrienes in the ischemic brain was substantially increased 3 h to 24 h, near control at 3 days, and moderately increased again 7 days after reperfusion; whereas the level of LTB(4) was increased mildly 3 h but substantially 7-14 days after reperfusion. Thus, we conclude that 5-LOX expression and the enzymatic activity are increased after focal cerebral ischemia, and spatio-temporally involved in neuron injury in the acute phase and astrocyte proliferation in the late phase.