Effect of prothymosin α on neuroplasticity following cerebral ischemia­reperfusion injury.

Prothymosin α (ProT), a highly acidic nuclear protein with multiple cellular functions, has shown potential neuroprotective properties attributed to its anti­necrotic and anti­apoptotic activities. The present study aimed to investigate the beneficial effect of ProT on neuroplasticity after ischemia­reperfusion injury and elucidate its underlying mechanism of action. Primary cortical neurons were either treated with ProT or overexpressing ProT by gene transfection and exposed to oxygen­glucose deprivation for 2 h in vitro. Immunofluorescence staining for ProT and MAP­2 was performed to quantify ProT protein expression and assess neuronal arborization. Mice treated with vehicle or ProT (100 µg/kg) and ProT overexpression in transgenic mice received middle cerebral artery occlusion for 50 min to evaluate the effect of ProT on neuroplasticity­associated protein following ischemia­reperfusion injury. The results demonstrated that in cultured neurons ProT significantly increased neurite lengths and the number of branches, accompanied by an upregulation mRNA level of brain­derived neurotrophic factor. Furthermore, ProT administration improved the protein expressions of synaptosomal­associated protein, 25 kDa and postsynaptic density protein 95 after ischemic­reperfusion injury in vivo. These findings suggested that ProT can potentially induce neuroplasticity effects following ischemia­reperfusion injury.

Therapeutic window for YC­1 following glutamate­induced neuronal damage and transient focal cerebral ischemia.

3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), has been demonstrated to inhibit platelet aggregation, vascular contraction and hypoxia­inducible factor 1 activity in vitro and in vivo. The present study investigated the neuroprotective efficacy of YC­1 in cultured neurons exposed to glutamate­induced excitotoxicity and in an animal model of stroke. In a cortical neuronal culture model, YC­1 demonstrated neurotoxicity at a concentration >100 µM, and YC­1 (10­30 µM) achieved potent cytoprotection against glutamate­induced neuronal damage. Additionally, YC­1 (30 µM) effectively attenuated the increase in intracellular Ca2+ levels. Delayed treatment of YC­1 (30 µM) also protected against glutamate­induced neuronal damage and cell swelling in cultured neurons, though only at 4 h post­treatment. In addition, immediate treatment of YC­1 (30 µM) following the exposure of cortical neurons to glutamate (300 µM) produced a marked reduction in intracellular pH. Delayed treatment of YC­1 (25 mg/kg) protected against ischemic brain damage in vivo, though only when administered at 3 h post­insult. Thus, YC­1 exhibited neuroprotection against glutamate-induced neuronal damage and in mice subjected to transient focal cerebral ischemia. This neuroprotection may be mediated via its ability to limit the glutamate­induced excitotoxicity. However, the neuroprotective therapeutic window of YC­1 is only at 3 h in vivo and 4 h in vitro, which may, at least in part, be attributed to its ability to reduce the intracellular pH in the early phase of ischemic stroke. Although YC­1 provided the potential for clinical therapy, the treatment time point must be carefully evaluated following ischemia.

Melatonin improves neuroplasticity by upregulating the growth-associated protein-43 (GAP-43) and NMDAR postsynaptic density-95 (PSD-95) proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to transient focal cerebral ischemia even during a long-term recovery period.

Recent evidence shows that the NMDAR postsynaptic density-95 (PSD-95), growth-associated protein-43 (GAP-43), and matrix metalloproteinase-9 (MMP-9) protein enhance neuroplasticity at the subacute stage of stroke. Here, we evaluated whether melatonin would modulate the PSD-95, GAP-43, and MMP-9 proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to experimental stroke. Adult male Sprague-Dawley rats were treated with melatonin (5 mg/kg) or vehicle at reperfusion onset after transient occlusion of the right middle cerebral artery (tMCAO) for 90 min. Animals were euthanized for Western immunoblot analyses for the PSD-95 and GAP-43 proteins and gelatin zymography for the MMP-9 activity at 7 days postinsult. Another set of animals was sacrificed for histologic and Golgi-Cox-impregnated sections at 28 days postinsult. In cultured neurons exposed to glutamate excitotoxicity, melatonin significantly upregulated the GAP-43 and PSD-95 expressions and improved dendritic aborizations (P<0.05, respectively). Relative to controls, melatonin-treated stroke animals caused a significant improvement in GAP-43 and PSD-95 expressions as well as the MMP-9 activity in the ischemic brain (P<0.05). Consequently, melatonin also significantly promoted the dendritic spine density and reduced infarction in the ischemic brain, and improved neurobehaviors as well at 28 days postinsult (P<0.05, respectively). Together, melatonin upregulates GAP-43, PSD-95, and MMP-9 proteins, which likely accounts for its actions to improve neuroplasticity in cultured neurons exposed to glutamate excitotoxicity and to enhance long-term neuroprotection, neuroplasticity, and brain remodeling in stroke rats.

Nicotinamide inhibits nuclear factor-kappa B translocation after transient focal cerebral ischemia.

OBJECTIVE: We explored the putative anti-inflammatory effects of nicotinamide against experimental stroke. DESIGN: Prospective laboratory study. SETTING: Research laboratory in a university teaching hospital. SUBJECTS: Adult male Sprague-Dawley rats (250-300 g). INTERVENTIONS: The antioxidant, radical scavenging, and anti-inflammatory actions of nicotinamide were evaluated using a panel of acellular assays and lipopolysaccharide-stimulated RAW 264.7 and BV2 cells. Animals were subjected to transient middle cerebral artery occlusion for 90 mins. Nicotinamide (500 mg/kg) or vehicle was given intravenously at reperfusion onset. MEASUREMENTS AND MAIN RESULTS: Nicotinamide effectively inhibited nuclear factor-κB translocation and binding activity as well as the production of tumor necrosis factor-α, nitrite/nitrate, and interleukin-6 in the lipopolysaccharide-stimulated RAW 264.7 and BV2 cells (p < .05, respectively) but exhibited weak antioxidant and radical-scavenging actions. Relative to controls, nicotinamide-treated animals had significant reductions in neutrophil and macrophage/activated microglial infiltration in the ischemic brain by 53% and 77% (p < .05, respectively). Additionally, nicotinamide significantly attenuated phosphorylation of nuclear factor-κB's inhibitory protein, nuclear factor-κB translocation and binding activity, and the synthesis of inducible nitric oxide in the ischemic brain (p < .05, respectively). Consequently, nicotinamide effectively reduced brain infarction and improved neurobehavioral outcome by 43% and 50% (p < .05, respectively). CONCLUSIONS: Nicotinamide effectively attenuated postischemic nuclear factor-kappa]B activation and exhibited robust anti-inflammatory actions against ischemic stroke.

Cinnamophilin offers prolonged neuroprotection against gray and white matter damage and improves functional and electrophysiological outcomes after transient focal cerebral ischemia.

OBJECTIVE: We have previously shown that cinnamophilin ([8R, 8'S]-4, 4'-dihydroxy-3, 3'-dimethoxy-7-oxo-8, 8'-neolignan) exhibited potent antioxidant, radical-scavenging, and anti-inflammatory actions and reduced acute ischemic brain damage, even when it was given up to 6 hrs postinsult. Here, we characterized the long-lasting neuroprotection of cinnamophilin against gray and white matter damage and its beneficial effects on electrophysiological and functional outcomes in a model of stroke. DESIGN: Prospective laboratory animal study. SETTING: Research laboratory in a university teaching hospital. SUBJECTS: Adult male Sprague-Dawley rats (240-290 g). INTERVENTIONS: Under controlled conditions of normoxia, normocarbia, and normothermia, spontaneously breathing, halothane-anesthetized (1.0-1.5%) rats were subjected to transient middle cerebral artery occlusion for 90 mins. Cinnamophilin (80 mg/kg) or vehicle was given intravenously at reperfusion onset. MEASUREMENTS AND MAIN RESULTS: Physiological parameters, including arterial blood gases and cortical blood perfusion, somatosensory-evoked potentials, and neurobehavioral outcomes, were serially examined. Animals were euthanized at 7 days or 21 days postinsult. Gray matter and white matter (axonal and myelin) damage were then evaluated by quantitative histopathology and immunohistochemistry against phosphorylated component-H neurofilaments and myelin basic protein, respectively. After the follow-up period of 7 and 21 days, our results showed that cinnamophilin significantly decreased gray matter damage by 31.6% and 34.9% (p < .05, respectively) without notable adverse effects. Additionally, cinnamophilin effectively reduced axonal and myelin damage by 46.3-68.6% (p < .05) and 25.2-28.1% (p < .05), respectively. Furthermore, cinnamophilin not only improved the ipsilateral field potentials (p < .05, respectively), but also reduced the severity of contralateral electrophysiological diaschisis (p < .05). Consequently, cinnamophilin improved sensorimotor outcomes up to 21 days postinsult (p < .05, respectively). CONCLUSIONS: Administration with cinnamophilin provides long-lasting neuroprotection against gray and white matter damage and improves functional and electrophysiological outcomes after ischemic stroke. The results suggest a need for further studies to characterize the potential of cinnamophilin in the field of ischemic stroke.

Melatonin protects against transient focal cerebral ischemia in both reproductively active and estrogen-deficient female rats: the impact of circulating estrogen on its hormetic dose-response.

Melatonin (5-15 mg/kg) protects male animals against ischemic stroke. We explored the potential interactions and synergistic neuroprotection of melatonin and estrogen using a panel of lipid peroxidation and radical-scavenging assays, primary neuronal cultures subjected to oxygen-glucose deprivation (OGD), and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Neuroprotective efficacy of melatonin was also evaluated in both reproductively active and ovariectomized female rats subjected to transient focal cerebral ischemia. Relative to melatonin or estradiol (E2) alone, a combination of the two agents exhibited robust, synergistic antioxidant and radical-scavenging actions (P<0.05, respectively). Additionally, the two agents, when combined at large doses, showed synergistic inhibition in the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in the LPS-stimulated RAW 264.7 cells (P<0.05, respectively). Alternatively, co-treatment with melatonin and E2 independently, but not combined, showed a U-shaped dose-responsive (hormetic) cytoprotection for neuronal cultures subjected to OGD. When combined at a dosage either positively or negatively skewed from each optimal dosage, however, co-treatment caused synergistic neuroprotection. Relative to vehicle-injected controls, melatonin given intravenously at 1-5 mg/kg, but not 0.1 or 15 mg/kg, significantly reduced brain infarction and improved neurobehavioral outcomes (P<0.05, respectively) in reproductively active female rats. In ovariectomized stroke rats, melatonin was only effective at a large dosage (15-50 mg/kg). These results demonstrate complex interactions and synergistic antioxidant, radical-scavenging, and anti-inflammatory actions between estradiol and melatonin, and highlight the potential need to rectify the melatonin's hormetic dose-response by the level of circulating estradiol in the treatment of female stroke patients.