Cells In Penumbra Research Articles

Hypoxia-Inducible Factor-1α Promotes Neuroregeneration and Angiogenesis after Cerebral Ischemia

To promote neuroregeneration and angiogenesis is a therapeutic target for the treatment of stroke. Hypoxia-inducible factor-1α (HIF-1α) has pleiotropic effects on neurogenesis, angiogenesis and neuroprotection in central nervous system. In this study we investigated whether HIF-1α can increase the proliferation of ischemia induced neural stem cells in SVZ and neuroregeneration in penumbra. The angiogenesis in penumbra was also investigated. Transient middle cerebral artery occlusion (tMCAO) rat model was used in this study. Rats were divided into 3 groups, NS group, vehicle group and HIF-lα group. In study we found that rats with HIF-1α treatment had better behavioral recovery at day7, 14, 21 and 28 (p<0.05). HIF-lα treatment increased the number of ischemia induced endogenetic NSCs in SVZ obviously (p<0.01). HIF-1α also increased newborn neurons and glial cells in penumbra on the 28 d (p<0.01). Angiogenesis in penumbra was promoted by HIF-lα treatment(p<0.01). In conclusion, our results indicate that modulate HIF-1α after ischemia may be a therapeutic target for the treatment of ischemic stroke through increasing neuroregeneration and angiogenesis.

Abstract W MP94: Neuroprotective Effect of Isosteviol Sodium on Focal Cerebral Ischemia in Rats

Background &amp; Objective: Isosteviol is a molecule derived from Steviaside which has been used as sweetener worldwide. In this study, sodium salt of isosteviol (STVNA) was given i.v. in rats hours after the transient or permanent middle cerebral artery occlusion (tMCAO or pMCAO) to investigate its therapeutic neuroprotective effects. Methods: In male Sprague-Dawley rats 2 hours tMCAO with reperfusion or pMCAO was induced and ischemia were confirmed by a laser doppler flowmetry simultaneously. In dosage study, animals were divided into 6 groups: sham, vehicle, or treatment with STVNA at dosage of 1, 5, 10mg•kg-1 or Edaravone 1 hour before the onset of reperfusion. In therapeutic time window study, animals were divided into 5 groups: sham, vehicle, STVNA (10mg•kg-1) at 0, 2 or 4 hours after reperfusion. In pMCAO study, animals were divided into 5 groups: sham, vehicle or STVNA (10mg•kg-1) at 1, 2 or 4 hours after ischemia. Rats were assessed for neurobehavioral deficits after 24 hours and sacrificed for infarct volume quantitation and histology evaluation. Proteomic analysis of the penumbra area in some rats used a Snaps G2x MS-TOF system. Results: In dosage study, the infarct volume of STVNA 10mg•kg-1 group was significantly less compared either with the vehicle group (22±2% vs 41±5%, p&lt; 0.01) or with the Edaravone group (22±2% vs 30±3%, p&lt; 0.05 ). The therapeutic window study shows that STVNA treated at 4h after reperfusion still has significant effects than vehicle group (32±4% vs 41±5%, p&lt;0.05). In pMCAO study, the infarct volume of STVNA at 4h still decreased comparing the vehicle group(29±5% vs 50±6%, p&lt; 0.05).In all STVNA treated groups the neurobehavioral deficits were significantly improved, and there are more restored NeuN-labeled neurons and alleviated TUNEL positive cells in penumbra in comparing with the vehicle group. Proteomic analysis indicates that proteins involved in various inflammations associated signal pathways were dramatically increased by tMCAO, and then were greatly reduced after treated with STVNA. Conclusions: STVNA exhibited remarkable neuroprotective effects when administered 4 hours after pMCAO or 4 hours after reperfusion of tMCAO. Since STVNA has low systemic toxicity, it may be a better alternative for the treatment of stroke.

Expression of arginyl-tRNA synthetase in rats with focal cerebral ischemia

Aminoacyl-tRNA syntheses (AARS) can catalyze the adenosine triphosphate (ATP)-dependent acylation of their cognate tRNA(s) with a specific amino acid. They can be seen as an index to reflect the energy metabolic rate of ischemic brain cells in ischemic penumbra. This study examined the relationship between arginyl-tRNA synthetase (ArgRS), one of the AARS, and cerebral ischemia in rats. The model of middle cerebral artery occlusion (MCAO) was established in rats. The expression levels of ArgRS protein and mRNA were detected in rat brain tissues at different time points following MCAO by Western blotting and RT-PCR, respectively. The results showed that the MCAO model was successfully established. Western blotting and RT-PCR analysis revealed that the ArgRS protein and mRNA were expressed in brain cells in both ischemic and normal penumbra tissues. The expression levels of ArgRS protein and mRNA peaked at 6 h after MCAO and decreased gradually. At 24 h, the expression levels of ArgRs protein and mRNA in ischemic penumbral tissues were lower than those in normal tissues. The expression levels of ArgRS mRNA and protein in ischemic penumbra varied with ischemic time, suggesting that the energy metabolism of brain cells in penumbra changed dynamically after ischemia to ensure the endogenous self-protection of the body. The brain oxygen supply should be improved as soon as possible, especially within 6-12 h after ischemia, so as to meet the demand for energy metabolism in ischemic penumbra and make sure the cell structure remains stable.

Neuroprotective effect of s-methylisothiourea in transient focal cerebral ischemia in rat

Over production of NO by nitric oxide synthase (NOS) in the brain parenchyma has been demonstrated to contribute to tissue damage. NO may be toxic by formation of peroxinitrite after a reaction between NO and superoxide appears to be one of the major pathways leading to cell death. Of three types of NOS, nNOS is neurotoxic in early and iNOS in late stage of transient cerebral ischemia (TFCI), while eNOS is neuroprotective in all stages. We examined the neuroprotective effect of a preferential iNOS inhibitor s-methylisothiourea (SMT) at 0, 8, 24 and 48 h as multiple injections (30 and 100 mg/kg, i.p.) in ischemia and reperfusion injury in a rat model of middle cerebral artery occlusion (2 h) and reperfusion (72 h). After 2 h of ischemia and 72 h of reperfusion, animals were sacrificed for studying the infarct volume, brain edema and apoptosis and neuro-behavioral abnormality was assessed at 24, 48 and 72 h of reperfusion. SMT reduced significantly the infarct volume, neuro-behavioral abnormality, brain edema, number of apoptotic cells in penumbra and NOx levels in plasma and brain both at 30 and 100 mg/kg in dose-dependent manner. The amount of peroxynitrite measured by rhodamine assay was significantly reduced by SMT, as compared to control group. SMT protected Neuro 2a cells against sodium azide-induced damage. It is concluded that, SMT may possibly targeting both constitutive as well as inducible NOS at varying time interval to elicit neuroprotection in TFCI rats.

Neuroprotective effect of 5,7,3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-O-galloyl)-β- d-glucopyranoside, a polyphenolic compound in focal cerebral ischemia in rat

Ischemia/reperfusion injury ends up in the cascade of excitotoxic stimulation of superoxide and nitric oxide formation leading to the generation of highly reactive products, including peroxinitrite and hydroxyl radical, which are capable of damaging lipids, proteins and DNA. Several polyphenolic compounds scavenge the radicals and protect from injury. 5,7,3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-O-galloyl)-β- d-glucopyranoside (AP1), a polyphenolic compound, isolated from Anogeissus pendula Edgew was tested for its neuroprotective effect in transient focal cerebral ischemia in rats. Transient focal cerebral ischemia was produced by middle cerebral artery occlusion for 2 h for studying infarct volume, brain edema, apoptosis and oxidative stress. AP1 was tested for in vitro protection from glutamate and hydrogen peroxide-induced damage to Neuro-2a cells by MTT assay. It was also tested for its in vitro antioxidant, lipid peroxidation inhibition, NO scavenging and cyclooxygenase inhibitory activities. AP1 treatment (30 mg/kg i.p.) before reperfusion injury (0 h) significantly reduced the infarct volume, cerebral edema, number of apoptotic cells in penumbra and neurobehavioural abnormality score and lipid peroxidation, protein carbonyl levels and total thiols in brain. Increased catalase activity and NOx levels in ischemic animals were significantly reduced by AP1 treatment. AP1 (3 µg/ml) protected Neuro-2a cells to H 2O 2 and glutamate-induced damage. In in vitro studies, AP1 was found to possess reducing and NO scavenging activities. It also reduced lipid peroxidation and inhibited cyclooxygenase activity (cyclooxygenase-1 and cyclooxygenase-2). AP1 can be used as a neuroprotective agent in stroke as it reduced apoptosis and found to be a good antioxidant and anti-inflammatory compound.

Preconditioning as a method of neuroprotection in a model of brain infarct

Preconditioning of ischemic and hypoxic type was investigated as a method of protecting brain against acute ischemic injury. The preconditioning methods were applied to experimental rats 24 h before the time when local brain infarct was done by middle cerebral artery occlusion (MCAO). It was found that the ischemic and hypoxic preconditioning resulted in three general morphological changes: 1) the size of infarct zone was reduced by 2.2–3.8 times compared with rats that had not been treated with the preconditioning before MCAO; 2) the preconditioning treatment retained the number of living neurons in penumbra at the level of control rats, while without the preconditioning neuronal count in the penumbra after MCAO was 29% lower; 3) the number of glial cells in penumbra was increased after MCAO by 38% compared with the control level, and continued to increase under the preconditioning treatment up to 60%, that suggests an important role of neuroglia in neuroprotection. Selective blockers of ATP2dependant K+2channels (52hydroxydecanoate and glibenclamide) completely abolished the neuroprotective effects of the preconditioning.

Involvement of Acidic Fibroblast Growth Factor in Spinal Cord Injury Repair Processes Revealed by a Proteomics Approach

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.

Calpain activity and expression increased in activated glial and inflammatory cells in penumbra of spinal cord injury lesion

Calpain activity and expression increased in activated glial and inflammatory cells in penumbra of spinal cord injury lesion

Calpain activity and expression increased in activated glial and inflammatory cells in penumbra of spinal cord injury lesion.

Following traumatic injury of the spinal cord, cells adjacent to the lesion are subject to ischemic cell death as a result of vascular disruption and secondary inflammatory responses. Proteases such as calcium-activated neutral proteinase (calpain) have been implicated in axon and myelin destruction following injury since they degrade structural proteins in the axon-myelin unit. To examine the role of calpain in cell death following spinal cord injury (SCI), calpain activity and translational expression were evaluated using Western blotting techniques. Calpain activity (as measured by specific substrate degradation) was significantly increased in and around the lesion site as early as 4 hr following injury with continued elevation at 48 hr compared to sham controls. Likewise, calpain expression was significantly increased in both the lesion site and penumbra at 4 and 48 hr after injury. Using double immunofluorescent labeling for calpain and cell-specific markers, this increase in calpain expression was found to be due in part to activated glial/inflammatory cells such as astrocytes, microglia, and infiltrating macrophages in these areas. Thus, since calpain degrades many myelin and axonal structural proteins, the increased activity and expression of this enzyme may be responsible for destruction of myelinated axons adjacent to the lesion site following SCI.