Focal Cerebral Ischemic Injury Research Articles

Dichotomous Effects of Chronic Intermittent Hypoxia on Focal Cerebral Ischemic Injury

Obstructive sleep apnea, a condition associated with chronic intermittent hypoxia (CIH), carries an increased risk of stroke. However, CIH has been reported to either increase or decrease brain injury in models of focal cerebral ischemia. The factors determining the differential effects of CIH on ischemic injury and their mechanisms remain unclear. Here, we tested the hypothesis that the intensity of the hypoxic challenge determines the protective or destructive nature of CIH by modulating mitochondrial resistance to injury. Male C57Bl/6J mice were exposed to CIH with 10% or 6% O2 for ≤35 days and subjected to transient middle cerebral artery occlusion. Motor deficits and infarct volume were assessed 3 days later. Intraischemic cerebral blood flow was measured by laser-Doppler flowmetry and resting cerebral blood flow by arterial spin labeling MRI. Ca2+-induced mitochondrial depolarization and reactive oxygen species production were evaluated in isolated brain mitochondria. We found that 10% CIH is neuroprotective, whereas 6% CIH exacerbates tissue damage. No differences in resting or intraischemic cerebral blood flow were observed between 6% and 10% CIH. However, 10% CIH reduced, whereas 6% CIH increased, mitochondrial reactive oxygen species production and susceptibility to Ca2+-induced depolarizations. The influence of CIH on the ischemic brain is dichotomous and can be attributed, in part, to changes in the mitochondrial susceptibility to injury. The findings highlight a previously unappreciated complexity in the effect of CIH on the brain, which needs to be considered in evaluating the neurological effect of conditions associated with cyclic hypoxia.

Autophagic effect of programmed cell death 5 (PDCD5) after focal cerebral ischemic reperfusion injury in rats

Former studies indicated that programmed cell death 5 (PDCD5) protein could accelerate the process of apoptosis in response to some stimuli in various kinds of cells via the intrinsic or extrinsic pathway. In this study, we aimed to demonstrate for the first time that protein level of PDCD5 are related to autophagic activity after focal ischemic brain injury in rats. One hundred and twenty-five Sprague-Dawley rats (male) were randomly divided into the following groups: Sham operated, Middle Cerebral Artery Occlusion/Reperfusion (MCAO), MCAO+Control siRNA and MCAO+PDCD5 siRNA. Outcome measurements include neurobehavioral outcomes, brain infarct volume, brain water content, BBB disruption, MRI and double fluorescence labeling. Western blot and histopathophysiological techniques were used to measure the expression of PDCD5 and some pro-autophagic proteins such as Beclin 1 and the LC3-II/LC3-I ratio. The study found that decreased PDCD5 expression via intracerebroventricular injection of PDCD5 siRNA significantly improved the neurobehavioral outcome, reduced the infarct ratio, cerebral edema and BBB disruption. These results were associated with decreased expression of Beclin 1 and the LC3-II/LC3-I ratio in the penumbra area. Rapamycin, an inducer of autophagy, partially weakened the effect of PDCD5 siRNA. In conclusion, this study suggested that PDCD5 was a key regulator of autophagy that might play an important role following MCAO injury.

Lycium barbarum Polysaccharide Prevents Focal Cerebral Ischemic Injury by Inhibiting Neuronal Apoptosis in Mice

Aims of the StudyTo investigate the neuroprotective effect of Lycium barbarum polysaccharide (LBP) on focal cerebral ischemic injury in mice and to explore its possible mechanism.Materials and MethodsMale ICR mice were used to make the model of middle cerebral artery occlusion (MCAO) after intragastric administration with LBP (10, 20 and 40 mg/kg) and Nimodipine (0.4 mg/kg) for seven successive days. After 24 h of reperfusion, neurological scores were estimated and infarct volumes were measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Morphological changes in ischemic brains were performed for hematoxylin-eosin (HE) staining. The number of apoptotic neurons was detected by TUNEL staining. The Bax, Bcl-2 protein expression and CytC, Caspase-3, -9 and cleaved PARP-1 activation were investigated by immunofluorescence and western-blot analysis.ResultsLBP (10, 20 and 40 mg/kg) treatment groups significantly reduced infract volume and neurological deficit scores. LBP also relieved neuronal morphological damage and attenuated the neuronal apoptosis. LBP at the dose of 40 mg/kg significantly suppressed overexpression of Bax, CytC, Caspase-3, -9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression.ConclusionsBased on these findings we propose that LBP protects against focal cerebral ischemic injury by attenuating the mitochondrial apoptosis pathway.

Exercise Promotes Axon Regeneration of Newborn Striatonigral and Corticonigral Projection Neurons in Rats after Ischemic Stroke

Newborn striatal neurons induced by middle cerebral artery occlusion (MCAO) can form functional projections targeting into the substantia nigra, which should be very important for the recovery of motor function. Exercise training post-stroke improves motor recovery in clinic patients and increases striatal neurogenesis in experimental animals. This study aimed to investigate the effects of exercise on axon regeneration of newborn projection neurons in adult rat brains following ischemic stroke. Rats were subjected to a transient MCAO to induce focal cerebral ischemic injury, followed by 30 minutes of exercise training daily from 5 to 28 days after MCAO. Motor function was tested using the rotarod test. We used fluorogold (FG) nigral injection to trace striatonigral and corticonigral projection neurons, and green fluorescent protein (GFP)-targeting retroviral vectors combined with FG double labeling (GFP+ -FG+) to detect newborn projection neurons. The results showed that exercise improved the recovery of motor function of rats after MCAO. Meanwhile, exercise also increased the levels of BDNF and VEGF, and reduced Nogo-A in ischemic brain. On this condition, we further found that exercise significantly increased the number of GFP+ -FG+ neurons in the striatum and frontal and parietal cortex ipsilateral to MCAO, suggesting an increase of newborn striatonigral and corticonigral projection neurons by exercise post-stroke. In addition, we found that exercise also increased NeuN+ and FG+ cells in the striatum and frontal and parietal cortex, the ischemic territory, and tyrosine hydroxylase (TH) immunopositive staining cells in the substantia nigra, a region remote from the ischemic territory. Our results provide the first evidence that exercise can effectively enhance the capacity for regeneration of newborn projection neurons in ischemic injured mammalian brains while improving motor function. Our results provide a very important cellular mechanism to illustrate the effectiveness of rehabilitative treatment post-stroke in the clinic.

Oxysophoridine Protects Against Focal Cerebral Ischemic Injury by Inhibiting Oxidative Stress and Apoptosis in Mice

Our previous studies have demonstrated that oxysophoridine (OSR) has protective effects on cerebral neurons damage in vitro induced by oxygen and glucose deprivation. In this study, we further investigated whether OSR could reduce ischemic cerebral injury in vivo and its possible mechanism. Male Institute of cancer research mice were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days, then subjected to brain ischemia induced by the model of middle cerebral artery occlusion. After reperfusion, neurological scores and infarct volume were estimated. Morphological examination of tissues was performed. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Oxidative stress levels were assessed by measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. The expression of various apoptotic markers as Caspase-3, Bax and Bcl-2 were investigated by immunohistochemistry and Western-blot analysis. OSR pretreatment groups significantly reduced infract volume and neurological deficit scores. OSR decreased the percentage of apoptotic neurons, relieved neuronal morphological damage. Moreover, OSR markedly decreased MDA content, and increased SOD, GSH-Px activities. Administration of OSR (250 mg/kg) significantly suppressed overexpression of Caspase-3 and Bax, and increased Bcl-2 expression. These findings indicate that OSR has a protective effect on focal cerebral ischemic injury through antioxidant and anti-apoptotic mechanisms.

Exendin-4 ameliorates renal ischemia-reperfusion injury in the rat

BackgroundGlucagon-like peptide-1 receptor (GLP-1R) activation exerts protective effects against reactive oxygen species by inducing the oxidative defense gene heme oxygenase-1 (HO-1), and provides protection in mice against transient focal cerebral ischemia and ischemia-reperfusion injury in the rat heart. GLP-1R is also expressed in the kidney, but it is unknown whether GLP-1R activation is able to protect against ischemia-reperfusion injury in the rat kidney. Materials and methodsWe used a rat model of renal ischemia-reperfusion injury. The rats were pretreated with the GLP-1R agonist, exendin-4 before reperfusion. We used real-time polymerase chain reaction to evaluate expression of the oxidative defense gene HO-1 and Western blot analysis for HO-1 and GLP-1R. Renal function was assessed at baseline and 24 and 72 h after reperfusion. The kidneys were processed for histologic and morphometric analysis, caspase-3, and ED1 immunohistochemistry at 72 h. The degree of apoptosis of the renal tubular cells was determined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end labeling assays. ResultsExendin-4 pretreatment resulted in GLP-1R activation and upregulation of HO-1. Preconditional activation of GLP-1R significantly improved the serum creatinine levels compared with vehicle (P < 0.05). Furthermore, tissue injury, caspase-3 and ED1 expression, and apoptosis were less severe, as quantified by application of a standardized histologic scoring system in a blinded manner. ConclusionsThese results have demonstrated that preconditional activation of the GLP-1R with exendin-4 in the kidney significantly protected against ischemia-reperfusion injury in rats by increasing HO-1 expression.

Hemopexin induces neuroprotection in the rat subjected to focal cerebral ischemia

BackgroundThe plasma protein hemopexin (HPX) exhibits the highest binding affinity to free heme. In vitro experiments and gene-knock out technique have suggested that HPX may have a neuroprotective effect. However, the expression of HPX in the brain was not well elucidated and its expression after cerebral ischemia-reperfusion injury was also poorly studied. Furthermore, no in vivo data were available on the effect of HPX given centrally on the prognosis of focal cerebral ischemia.ResultsIn the present study, we systematically investigated expression of HPX in normal rat brain by immunofluorescent staining. The results showed that HPX was mainly expressed in vascular system and neurons, as well as in a small portion of astrocytes adjacent to the vessels in normal rat brain. Further, we determined the role of HPX in the process of focal cerebral ischemic injury and explored the effects of HPX treatment in a rat model of transient focal cerebral ischemia. After 2 h’ middle cerebral artery occlusion (MCAO) followed by 24 h’ reperfusion, the expression of HPX was increased in the neurons and astrocytes in the penumbra area, as demonstrated by immunohistochemistry and Western blot techniques. Intracerebroventricular injection of HPX at the onset of reperfusion dose-dependently reduced the infarct volumes and improved measurements of neurological function of the rat subjected to transient focal cerebral ischemia. The neuroprotective effects of HPX sustained for up to 7 days after experiments.ConclusionsOur study provides a new insight into the potential neuroprotective role of HPX as a contributing factor of endogenous protective mechanisms against focal cerebral ischemia injury, and HPX might be developed as a potential agent for treatment of ischemic stroke.

The Effects of Antecedent Exercise on Motor Function Recovery and Brain-derived Neurotrophic Factor Expression after Focal Cerebral Ischemia in Rats.

[Purpose] In the present study, we investigated the effect of antecedent exercise on functional recovery and brain-derived neurotrophic factor (BDNF) expression following focal cerebral ischemia injury. [Subjects] The rat middle cerebral artery occlusion (MCAO) model was employed. Adult male Sprague-Dawley rats were randomly divided into 4 groups. Group I included untreated normal rats (n=10); Group II included untreated rats with focal cerebral ischemia (n=10); Group III included rats that performed treadmill exercise (20 m/min) training after focal cerebral ischemia (n=10); and Group IV included rats that performed antecedent treadmill exercise (20 m/min) training before focal cerebral ischemia (n=10) as well as treadmill exercise after ischemia. At different time points (1, 7, 14, and 21 days) Garcia’s score, and the hippocampal expressions level of BDNF were examined. [Results] In the antecedent exercise group, improvements in the motor behavior index (Garcia’s score) were observed and hippocampal BDNF protein expression levels increased. [Conclusion] These results indicate that antecedent treadmill exercise, before permanent brain ischemia exerts a neuroprotective effect against ischemia brain injury by improving motor performance and increasing the level of BDNF expression. Furthermore, the antecedent treadmill exercise of appropriate intensity is critical for post-stroke rehabilitation.

Tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral ischemia in rats

Tetramethylpyrazine (TMP) has been used to treat ischemic stroke. However, scientific evidence related to its effectiveness or precise modes of neuroprotective action is largely unclear. This study provides evidence of an alternative target for TMP and sheds light on the mechanism of its physiological benefits. We report a global inhibitory effect of TMP on intracerebral cellular inflammatory response in a rat model of permanent cerebral ischemia. TMP exhibited a neuroprotective effect against ischemic deficits by reduction of behavioral disturbance, brain infarction, and edema. The results of immunohistochemistry, enzymatic assay, Western blot, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and flow cytometric analysis revealed that TMP reduced the percentages of activated macrophages/microglia and infiltrative lymphocytes, neutrophils, and macrophages and pro-inflammatory cytokine expression after cerebral ischemia. In parallel with these immunosuppressive phenomena, TMP also attenuated the activities of ischemia-induced inflammation-associated signaling molecules and transcription factors. Another finding in this study was that the anti-inflammatory and neuroprotective effects of TMP were accompanied by a further elevated expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in ipsilateral neurons and macrophages/microglia after cerebral ischemia. Taken together, our results suggest that both the promotion of endogenous defense capacity and the attenuation of the extent and composition percentage of the major cellular inflammatory responses via targeting of macrophages/microglia by elevating Nrf2/HO-1 expression might actively contribute to TMP-mediated neuroprotection against cerebral ischemia.

Baicalin attenuates focal cerebral ischemic reperfusion injury by inhibition of protease-activated receptor-1 and apoptosis

To investigate the neuro-protective effects of baicalin in Wistar rats with focal cerebral ischemic reperfusion injury. Ninety adult male Wistar rats weighing 320-350 g were randomly divided into the following groups (n=5): (a) sham control group; (b) vehicle group, subjected to middle cerebral artery occlusion and received vehicle intraperitoneally; (c-e) baicalin groups, which were subjected to the middle cerebral artery occlusion and treated with baicalin 25, 50 and 100 mg/kg, respectively. The neurological scores were determined at postoperative 1, 3 and 7 d after the treatment. The expression of protease-activated receptor-1 (PAR-1), PAR-1 mRNA and Caspase-3 were determined using Western blot, reverse transcription polymerase chain reaction (RTPCR) analysis and immunohistochemistry, respectively. Significant decrease was noted in the neurological score in the baicalin group compared with that of the vehicle group (P<0.01). Additionally, down-regulation of PAR-1 mRNA, PAR-1 and Caspase-3 was observed in the baicalin groups compared with those obtained from the vehicle group (P<0.01). Compared with the low-dose baicalin group (25 mg/kg), remarkable decrease was noted in neurological score, and the expression of PAR-1 mRNA, PAR-1 as well as Caspase-3 in the high-dose group (P<0.05). Baicalin showed neuro-protective effects in focal cerebral ischemic reperfusion injury through inhibiting the expression of PAR-1 and apoptosis.

Abstract TP429: The Protective and Withdrawal Effects of Atorvastatin on Endothelial Barrier in Cerebral Ischemia and Reperfusion Injury

Background and purpose— Statins can effectively attenuate focal cerebral ischemic injury. However, the role of statins in modulating permeability of endothelial barrier in ischemia-reperfusion (IR) and the impact of statins withdrawal on endothelial barrier during the acute phase of IR are unclear. Methods— Focal cerebral ischemia (2 hours) was created in adult male Sprague-Dawley rats (240 to 270 g) by middle cerebral artery occlusion (MCAO) using a suture. Rats were randomly treated with vehicle, atorvastatin daily for 7 days before MCAO (pretreatment), 3 days after MCAO (post operative treatment), or both 7 days before and 3 days after MCAO (continuous treatment). Brains were harvested at 24 and 72 hours after reperfusion for assays of brain edema (n=5), BBB leakage (n=5), calculations of occludin and claudin-5 positive vessels (n=5) and expression of occludin, claudin-5 and Phosphoinositide 3-kinase (PI3K)-γ (n=5), respectively. Results— 24 hours after reperfusion, pretreatment with atorvastatin significantly reduced BBB leakage (7.55±0.63ng/g versus 9.38±0.77ng/g; P&lt;0.05) and brain edema (83.96%±1.11% versus 86.77%±1.26%; P&lt;0.05), as evidenced by Evans blue extravasation and brain water content measurement. Immunofluorescence assay and western blot revealed atorvastatin significantly increased the expression of occludin and claudin-5 and attenuated IR-induced increased expression of PI3Kγ compared with vehicle treatment. 72 hours after reperfusion, post operative treatment with atorvastatin for 3 days and continuous treatment both attenuated the above IR injuries to endothelial barrier (P&lt;0.05), particularly the latter. However, atorvastatin pretreatment no longer had protective effects on BBB leakage, brain edema and the expression of PI3Kγ (P&lt;0.05), although pretreatment with atorvastain for 7 days increased the expression of occludin and claudin-5 up to 72 hours after reperfusion (P&lt;0.05). Conclusions— These results demonstrate that protective effects of atorvastatin against endothelial barrier breakdown may be related to the up-regulation of occludin and claudin-5 and down-regulation of PI3Kγ. However, there are no withdrawal effects of atorvastain on endothelial barrier during the acute phase of IR (72 hours).

The Permeability of Puerarin Loaded Poly(butylcyanoacrylate) Nanoparticles Coated with Polysorbate 80 on the Blood–Brain Barrier and Its Protective Effect against Cerebral Ischemia/Reperfusion Injury

Puerarin (PUE) is a good candidate for treating stroke, but its low concentration in brain after administration limits its curative efficacy. The aim of the present work was to design and characterize PUE loaded poly(butylcyanoacrylate) nanoparticles (PBCN) coated with polysorbate 80 (Ps 80), and to evaluate the effect of PBCN on the permeability of PUE across the blood-brain barrier (BBB) and the effect of PUE loaded PBCN on the cerebral ischemia/reperfusion injury. PUE loaded PBCN were successfully prepared by anionic polymerization method with the mean particle size of 201.2 nm and the zeta potential of -7.72 mV. The in vitro release behavior of PUE from the nanoparticles showed a biphasic profile manner with an initial burst release followed by a sustained release. The results of pharmacokinetic and biodistribution to brain performed in mice after intravenous administration showed that the drug concentrations in blood and brain for PUE loaded PBCN were both greater than these for the free drug. Moreover, compared with free drug, the vein injection of PUE loaded PBCN exerted the better neuroprotective effect in rats with focal cerebral ischemic injury via significantly decreasing neurological deficit scores, increasing body weight, lowing brain water content, and reducing the infarct volume. The results indicated that this preparation may reduce the total dose required for the stroke therapy with concurrent reduction in dose related toxicity. All these findings suggest that PBCN could enhance the transport of PUE to brain and have a potential as a neuroprotective agent in the focal cerebral ischemic injury.

Ferulic acid regulates the AKT/GSK-3β/CRMP-2 signaling pathway in a middle cerebral artery occlusion animal model

Ferulic acid, a component of the plants Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort, exerts a neuroprotective effect by regulating various signaling pathways. This study showed that ferulic acid treatment prevents the injury-induced increase of collapsin response mediator protein 2 (CRMP-2) in focal cerebral ischemia. Glycogen synthase kinase-3β (GSK-3β) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3β is inactivated by phosphorylation by Akt. This study investigated whether ferulic acid modulates the expression of CRMP-2 and its upstream targets, Akt and GSK-3β, in focal cerebral ischemia. Male rats were treated immediately with ferulic acid (100 mg/kg, i.v.) or vehicle after middle cerebral artery occlusion (MCAO), and then cerebral cortices were collected 24 hr after MCAO. MCAO resulted in decreased levels of phospho-Akt and phospho-GSK-3β, while ferulic acid treatment prevented the decrease in the levels of these proteins. Moreover, phospho-CRMP-2 and CRMP-2 levels increased during MCAO, whereas ferulic acid attenuated these injury-induced increases. These results demonstrate that ferulic acid regulates the Akt/GSK-3β/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury.

Effects of Treadmill Training on Limb Motor Function and Acetylcholinesterase Activity in Rats with Stroke

[Purpose] In the present study, we investigated the effects of treadmill training on limb motor function and acetylcholinesterase activity following focal cerebral ischemia injury. [Methods] Focal cerebral ischemia was examined in adult male Sprague-Dawley rats by using a middle cerebral artery occlusion model. Rats were randomly divided into 3 groups. Group I included untreated normal rats (n=12), Group II included untreated rats with focal cerebral ischemia (n=12), and Group III included rats that performed treadmill exercise (20 m/min) training after focal cerebral ischemia (n=12). We determined the limb placement test score for each rat on days 1,7, 14, and 21; acetylcholinesterase activity in the hippocampus was examined at the end of the experiment. [Results] We observed that the motor behavior index improved in the treadmill group, and hippocampal acetylcholinesterase activity was decreased. [Conclusion] These results indicated that treadmill training after focal cerebral ischemia exerts a neuroprotective effects against ischemic brain injury by improving motor performance and decreasing the levels of acetylcholinesterase activity. Furthermore, these results suggest that treadmill training at an appropriate intensity is critical for post-stroke rehabilitation.

Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke

[Purpose] In the present study, we investigated the effects of antecedent exercise on functional recovery and calpain protein expression following focal cerebral ischemia injury. [Subjects and Methods] The rat middle cerebral artery occlusion model was employed. Adult male Sprague-Dawley rats were randomly divided into 4 groups. Group I comprised untreated normal rats (n=10); Group II comprised untreated rats with focal cerebral ischemia (n=10); Group III comprised rats that performed treadmill exercise (20 m/min) training after focal cerebral ischemia (n=10); and Group IV comprised rats that performed antecedent treadmill exercise (20 m/min) training before focal cerebral ischemia (n=10). At different time points (1, 7, 14, and 21 days), limb placement test score and the levels of calpain protein in the hippocampus were examined. [Results] In the antecedent exercise group, improvements in the motor behavior index (limb placement test) were observed and hippocampal calpain protein levels were decreased. [Conclusion] These results indicated that antecedent treadmill exercise prior to focal cerebral ischemia exerted neuroprotective effects against ischemic brain injury by improving motor performance and decreasing the levels of calpain expression. Furthermore, these results suggest that antecedent treadmill exercise of an appropriate intensity is critical for post-stroke rehabilitation.

Focal cerebral ischemic injury decreases calbindin expression in brain tissue and HT22 cells

Calbindin is a calcium binding protein that controls intracellular calcium levels and has a neuroprotective function against apoptotic stimuli. We investigated the expression of calbindin in ischemic brain injury. Focal cerebral ischemia was induced in male rats by middle cerebral artery occlusion (MCAO) and cerebral cortices were collected 24 h after MCAO. Cerebral ischemia significantly increased infarct volume. RT-PCR and Western blot analyses showed that MCAO injury induced a decrease of calbindin expression. Moreover, immunohistochemical staining showed that the number of calbindin-positive cells decreased in ischemic regions of MCAO-operated animals. In cultured hippocampal-derived cell lines, glutamate exposure increased intracellular Ca2+ concentrations and decreased calbindin expression. Taken together, both in vivo and in vitro results demonstrated decreases of calbindin after neuronal cell injury. These results suggest that decreases of calbindin in ischemic brain injury contribute to neuronal cell death.

WIN55,212-2 protects oligodendrocyte precursor cells in stroke penumbra following permanent focal cerebral ischemia in rats

To explore whether the synthetic cannabinoid receptor agonist WIN55,212-2 could protect oligodendrocyte precursor cells (OPCs) in stroke penumbra, thereby providing neuroprotection following permanent focal cerebral ischemia in rats. Adult male SD rats were subjected to permanent middle cerebral artery occlusion (p-MCAO). The animals were administered WIN55,212-2 at 2 h, and sacrificed at 24 h after the ischemic insult. The infarct volumes and brain swelling were assessed. The expression of cannabinoid receptor type 1 (CB1) in the stroke penumbra was examined using Western blot assay. The pathological changes and proliferation of neural glial antigen 2-positive OPCs (NG2(+) cells) in the stroke penumbra were studied using immunohistochemistry staining. p-MCAO significantly increased the expression of CB1 within the stroke penumbra with the highest level appearing at 2 h following the ischemic insult. Administration of WIN55,212-2 (9 mg/kg, iv) significantly attenuated the brain swelling, and reduced the infarct volume as well as the number of tau-immunoreactive NG2(+) cells (tau-1(+)/NG2(+) cells) in the stroke penumbra. Moreover, WIN55,212-2 significantly promoted the proliferation of NG2(+) cells in the stroke penumbra and in the ipsilateral subventricular zone at 24 h following the ischemic insult. Administration of the selective CB1 antagonist rimonabant (1 mg/kg, iv) partially blocked the effects caused by WIN55,212-2. Tau-1 is expressed in NG2(+) cells following permanent focal cerebral ischemic injury. Treatment with WIN55,212-2 reduces the number of tau-1(+)/NG2(+) cells and promotes NG2(+) cell proliferation in the stroke penumbra, which are mediated partially via CB1 and may contribute to its neuroprotective effects.

Normobaric hyperoxia combined with minocycline provides greater neuroprotection than either alone in transient focal cerebral ischemia

Normobaric hyperoxia (NBO), which maintains penumbral oxygenation, reduces brain injury during cerebral ischemia, and minocycline, a tetracycline derivative, reduces reperfusion injury, including inflammation, apoptosis and matrix metalloproteinases (MMPs) activation. Since they have different mechanisms of action, we hypothesized that combining them would provide greater neuroprotection. To test the hypothesis, we evaluated the neuroprotective effects of the combination of NBO with minocycline. Male Sprague–Dawley rats were exposed to NBO (95% O2) or normoxia (21% O2) during 90-min filament occlusion of the middle cerebral artery, followed by 48h of reperfusion. Minocycline (3mg/kg) or vehicle was intravenously administered to rats 15min after reperfusion onset. Treatment with NBO and minocycline alone resulted in 36% and 30% reductions in infarction volume, respectively. When the two treatments were combined, there was a 68% reduction in infarction volume. The combination therapy also significantly reduced hemispheric swelling, which was absent with monotherapy. In agreement with its greater neuro- and vasoprotection, the combination therapy showed greater inhibitory effects on MMP-2/9 induction, occludin degradation, caspase-3 and -9 activation and apoptosis inducing factor (AIF) induction in ischemic brain tissue. Our results show that NBO plus minocycline effectively reduces brain injury in transient focal cerebral ischemia with protection due to inhibition on MMP-2/9-mediated occludin degradation and attenuation of caspase-dependent and independent apoptotic pathways.

Nicotinamide Restores the Reduction of Parvalbumin in Cerebral Ischemic Injury

The aim of this study investigated whether nicotinamide affects parvalbumin expression in focal cerebral ischemic injury. Rats were treated with vehicle or nicotinamide (500 mg/kg) 2 hr after middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24 hr after MCAO. Nicotinamide significantly decreases the volume of infarct areas in the cerebral cortex. A proteomic approach revealed that MCAO induces decreases of parvalbumin levels, while nicotinamide treatment prevents injury-induced decreases in parvalbumin. RT-PCR and Western blot analyses demonstrated that nicotinamide restores injury-induced decreases in parvalbumin. Moreover, immunohistochemical staining confirmed that the numbers of parvalbumin-positive cells were decreased in vehicle-treated animals with MCAO, and that nicotinamide averted this decrease. In cultured hippocampal cells, nicotinamide treatment prevents the glutamate exposure-induced increase in intracellular Ca(2+) concentration and decrease in parvalbumin expression. These results suggest the fact that the maintenance of parvalbumin expression is mediated to the neuroprotective function of nicotinamide against ischemic brain injury.

Morg1+/− heterozygous mice are protected from experimentally induced focal cerebral ischemia

Focal cerebral ischemia (stroke) and reperfusion injury leads to acute and chronic brain damage. The increase of the hypoxia-inducible transcription factor alpha (HIF-α), an important transcription factor for several genes, may attenuate ischemic brain injury. We recently identified a new WD-repeat protein designated Morg1 (MAPK organizer 1) that interacts with prolyl hydroxylase 3 (PHD3), an important enzyme involved in the regulation of HIF-1α and HIF-2α expression. While homozygous Morg1(-/-) mice are embryonically lethal, heterozygous Morg1(+/-) mice have a normal phenotype. Brain vasculature as well as systolic blood pressure in Morg1(+/-) mice were indistinguishable from wild-type (WT) animals. We show here that Morg1(+/-) mice were partially protected from cerebral ischemia/reperfusion injury in comparison to WT (Morg1(+/+)) animals using the middle cerebral artery occlusion model (MCAO). Morg1(+/-) mice compared with WT animals revealed a significantly reduced infarct volume as detected by Nissl and Map 2 staining despite a similar restriction of blood flow in both mice genotypes as measured by laser Doppler flowmetry. Immunohistochemistry revealed specific Morg1 expression in reactive astrocytes in the ipsilateral (ischemic) hemisphere in Morg1(+/-) and WT mice, especially in the penumbral regions. In the contralateral hemisphere, Morg1 was not detectable. Furthermore, Morg1 mRNA expression was significantly enhanced in the ischemic brain of WT, but not in ischemic brain tissue obtained from Morg1(+/-) animals. However, HIF-1α was expressed with the same intensity in Morg1(+/-) and WT mice with no difference between the ipsilateral and contralateral hemispheres. No positive staining for HIF-2α was found in ischemic (ipsilateral) and non-ischemic (contralateral) brain regions in Morg1(+/+) and Morg1(+/-) mice. Almost no PHD3 staining was found in the contralateral hemispheres of either WT or heterozygous Morg1(+/-) mice. Transcript expression for the HIF1α-dependent genes erythropoietin (Epo) and vascular endothelial growth factor 164 (VEGF 164) were significantly reduced in the ischemic brain from Morg1(+/-) mice. Positive staining for PHD3 in the ipsilateral hemisphere of WT mice was suggested to occur in astrocytes. A compensatory increase in Morg1 expression in astrocytes in the penumbra may negatively influence infarct volume. It appears that these effects are independent of the PHD3-HIF1α axis.