Tsung-Ying Chen

Acute administration of Ginkgo biloba extract (EGb 761) affords neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rats.

We examined the neuroprotective action of a standardized extract of Ginkgo biloba leaves (EGb 761) in permanent and transient middle cerebral artery (MCA) occlusion models in Sprague‐Dawley rats. Forty‐four animals were given either EGb 761 (50–200 mg/kg) or vehicle intraperitoneally, 1 hr before permanent MCA occlusion, to evaluate the dose‐response effects. An additional 58 animals received EGb 761 (200 mg/kg) or vehicle, 0.5– 4 hr after permanent MCA occlusion, for establishing the therapeutic window. Delayed treatment was also employed in 110 animals treated with either EGb 761 (100–200 mg/kg) or vehicle at 2–3 hr following transient focal cerebral ischemia induced by MCA occlusion for 2 hr. Neurobehavioral scores were determined 22–24 hr after permanent MCA occlusion and either 3 or 7 days after transient MCA occlusion, and brain infarction volumes were measured upon sacrifice. Local cortical blood flow (LCBF) was serially measured in a subset of animals receiving EGb 761 (100–200 mg/kg) or vehicle, 0.5 hr and 2 hr after permanent and transient MCA occlusion, respectively. Relative to vehicle‐treated controls, rats pretreated with EGb761 (100 and 200 mg/kg) had significantly reduced infarct volumes, by 36% and 49%, respectively, and improved sensory behavior (P < 0.05). Delayed treatment with EGb 761 also significantly reduced brain infarction, by 20–29% and 31%, when given up to 2 and 3 hr following transient and permanent MCA occlusion, respectively, whereas improved neurobehavioral scores were noted up to 2 hr after the onset of MCA occlusion (P < 0.05). LCBF was significantly improved in the ipsilateral cortex following the EGb 761 treatment, whereas a higher dose showed a more sustained effect. In conclusion, EGb 761 protected against transient and permanent focal cerebral ischemia and was effective after a prolonged reperfusion period even when therapy is delayed up to 2 hr. This neuroprotection may be at least partially attributed to the beneficial effects of selectively improved LCBF in the area at risk of infarction.

Intravenous administration of melatonin reduces the intracerebral cellular inflammatory response following transient focal cerebral ischemia in rats

Abstract:  We have previously shown that exogenous melatonin improves the preservation of the blood–brain barrier (BBB) and neurovascular unit following cerebral ischemia–reperfusion. Recent evidence indicates that postischemic microglial activation exaggerates the damage to the BBB. Herein, we explored whether melatonin mitigates the cellular inflammatory response after transient focal cerebral ischemia for 90 min in rats. Melatonin (5 mg/kg) or vehicle was given intravenously at reperfusion onset. Immunohistochemistry and flow cytometric analysis were used to evaluate the cellular inflammatory response at 48 hr after reperfusion. Relative to controls, melatonin‐treated animals did not have significantly changed systemic cellular inflammatory responses in the bloodstream (P > 0.05). Melatonin, however, significantly decreased the cellular inflammatory response by 41% (P < 0.001) in the ischemic hemisphere. Specifically, melatonin effectively decreased the extent of neutrophil emigration (Ly6G‐positive/CD45‐positive) and macrophage/activated microglial infiltration (CD11b‐positive/CD45‐positive) by 51% (P < 0.01) and 66% (P < 0.01), respectively, but did not significantly alter the population composition of T lymphocyte (CD3‐positive/CD45‐positive; P > 0.05). This melatonin‐mediated decrease in the cellular inflammatory response was accompanied by both reduced brain infarction and improved neurobehavioral outcome by 43% (P < 0.001) and 50% (P < 0.001), respectively. Thus, intravenous administration of melatonin upon reperfusion effectively decreased the emigration of circulatory neutrophils and macrophages/monocytes into the injured brain and inhibited focal microglial activation following cerebral ischemia–reperfusion. The finding demonstrates melatonins inhibitory ability against the cellular inflammatory response after cerebral ischemia–reperfusion, and further supports its pleuripotent neuroprotective actions suited either as a monotherapy or an add‐on to the thrombolytic therapy for ischemic stroke patients.

MELATONIN DECREASES NEUROVASCULAR OXIDATIVE/NITROSATIVE DAMAGE AND PROTECTS AGAINST EARLY INCREASES IN THE BLOOD–BRAIN BARRIER PERMEABILITY AFTER TRANSIENT FOCAL CEREBRAL ISCHEMIA IN MICE

Abstract:  We have recently shown that melatonin decreases the late (24 hr) increase in blood–brain barrier (BBB) permeability and the risk of tissue plasminogen activator‐induced hemorrhagic transformation following ischemic stroke in mice. In the study, we further explored whether melatonin would reduce postischemic neurovascular oxidative/nitrosative damage and, therefore, improve preservation of the early increase in the BBB permeability at 4 hr after transient focal cerebral ischemia for 60 min in mice. Melatonin (5 mg/kg) or vehicle was given intraperitoneally at the beginning of reperfusion. Hydroethidine (HEt) in situ detection and immunohistochemistry for nitrotyrosine were used to evaluate postischemic accumulation in reactive oxygen and nitrogen species, respectively, in the ischemic neurovascular unit. BBB permeability was evaluated by spectrophotometric and microscopic quantitation of Evans Blue leakage. Relative to controls, melatonin‐treated animals not only had a significantly reduced superoxide accumulation in neurovascular units in boundary zones of infarction, by reducing 35% and 54% cytosolic oxidized HEt in intensity and cell‐expressing percentage, respectively (P < 0.001), but also exhibited a reduction in nitrotyrosine by 52% (P < 0.01). Additionally, melatonin‐treated animals had significantly reduced early postischemic disruption in the BBB permeability by 53% (P < 0.001). Thus, melatonin reduced postischemic oxidative/nitrosative damage to the ischemic neurovascular units and improved the preservation of BBB permeability at an early phase following transient focal cerebral ischemia in mice. The findings further highlight the ability of melatonin in anatomical and functional preservation for the ischemic neurovascular units and its relevant potential in the treatment of ischemic stroke.

Melatonin attenuates the postischemic increase in blood-brain barrier permeability and decreases hemorrhagic transformation of tissue-plasminogen activator therapy following ischemic stroke in mice

Abstract:  Melatonin protects against transient middle cerebral artery (MCA) occlusion and may be suited as an add‐on therapy of tissue plasminogen activator (t‐PA) thrombolysis. Herein, we examined whether melatonin would reduce postischemic increase in the blood–brain barrier (BBB) permeability and, therefore, attenuate the risk of hemorrhagic transformation after t‐PA therapy in experimental stroke. Twelve mice were subjected to transient occlusion of the MCA for 1 hr, followed by 24 hr of reperfusion. Melatonin (5 mg/kg, i.p.) or vehicle was given at the beginning of reperfusion. BBB permeability was evaluated by quantitation of Evans Blue leakage. An additional 32 mice underwent photothrombotic occlusion of the distal MCA, and were administered vehicle or t‐PA (10 mg/kg, i.v.), alone or in combination with melatonin (5 mg/kg, i.p.), at 6 hr postinsult. The animals were then killed after 24 hr for the determination of infarct and hemorrhage volumes. Relative to controls, melatonin‐treated animals had significantly reduced BBB permeability (by 52%; P < 0.001). Additionally, we found that at 6 hr after photo‐irradiation, either t‐PA or melatonin, or a combined administration of t‐PA plus melatonin, did not significantly affect brain infarction (P > 0.05), compared with controls. Mice treated with t‐PA alone, however, had significantly increased hemorrhagic formation (P < 0.05), and the event was effectively reversed by co‐treatment with melatonin (P < 0.05). Thus, melatonin improved postischemic preservation of the BBB permeability and a decreased risk of adverse hemorrhagic transformation after t‐PA therapy for ischemic stroke. The findings further highlight melatonins potential role in the field of thrombolytic treatment for ischemic stroke patients.

Delayed treatment with melatonin enhances electrophysiological recovery following transient focal cerebral ischemia in rats

Abstract:  Melatonin has been reported to reduce infarct volumes induced by transient middle cerebral artery (MCA) occlusion. We examined whether melatonin could improve electrophysiological and neurobehavioral recoveries in rats after 72 hr of reperfusion following 1.5 hr of MCA occlusion. Melatonin (5 mg/kg) or vehicle was given intravenously at the commencement of reperfusion. Neurobehavioral outcome was serially examined, and somatosensory evoked potentials (SSEP) were recorded prior to ischemia and at 72 hr after the onset of reperfusion. Brain infarction was assessed upon killing. Before ischemia‐reperfusion, stable SSEP waveforms were consistently recorded after individual fore‐ or hindpaw stimulation. The amplitude between the first positive (P1) and the first negative (N1) peaks and the P1 latency did not differ significantly between controls and melatonin‐treated animals. At 72 hr of reperfusion, controls had severely depressant SSEPs recorded from ischemic fore‐ and hindpaw cortical fields, and the amplitudes decreased to 36 and 35% of baselines, respectively (P < 0.001). These animals also had transcallosal electrophysiological diaschisis in the SSEPs recorded at the contralateral hindpaw cortical field (P < 0.01). Relative to controls, melatonin‐treated animals not only had significantly improved amplitudes of the SSEPs recorded from both ischemic fore‐ and hindpaw cortical fields, by 33 and 37% of baselines, respectively (P < 0.001), but also exhibited diminished transcallosal electrophysiological diaschisis following ischemia‐reperfusion. In addition, melatonin improved sensory and motor neurobehavioral outcomes by 40 and 28%, respectively (P < 0.001), and reduced cortical and striatal infarct sizes by 32 and 40%, respectively (P < 0.05). Thus, delayed intravenous administration with melatonin both enhances electrophysiological and neurobehavioral recoveries and reduces cortical and striatal infarct sizes after cerebral ischemia and reperfusion injury.

Melatonin decreases matrix metalloproteinase‐9 activation and expression and attenuates reperfusion‐induced hemorrhage following transient focal cerebral ischemia in rats

Abstract:  We have previously shown that melatonin reduces postischemic rises in the blood–brain barrier (BBB) permeability and improves neurovascular dysfunction and hemorrhagic transformation following ischemic stroke. It is known that activation of the matrix metalloproteinases (MMPs) plays a crucial role in the pathogenesis of brain edema and hemorrhagic transformation after ischemic stroke. We, herein, investigated whether melatonin would ameliorate MMP‐2 and MMP‐9 activation and expression in a rat model of transient focal cerebral ischemia. Adult male Sprague–Dawley rats were subjected to a 90‐min middle cerebral artery (MCA) occlusion using an intraluminal filament. Melatonin (5 mg/kg) or vehicle was intravenously injected upon reperfusion. Brain infarction and hemorrhage within infarcts were measured, and neurological deficits were scored. The activity and expression of MMP‐2 and MMP‐9 were determined by zymography, in situ zymography and Western immunoblot analysis. Cerebral ischemia–reperfusion induced increased pro‐MMP‐9 and MMP‐9 activity and expression 24 hr after reperfusion onset. Relative to controls, melatonin‐treated animals, however, had significantly reduced levels in the MMP‐9 activity and expression (P < 0.01), in addition to reduced brain infarct volume and hemorrhagic transformation as well as improved sensorimotor neurobehavioral outcomes. No significant change in MMP‐2 activity was observed throughout the course experiments. Our results indicate that the melatonin‐mediated reductions in ischemic brain damage and reperfusion‐induced hemorrhage are partly attributed to its ability to reduce postischemic MMP‐9 activation and increased expression, and further support the fact that melatonin is a suitable as an add‐on to thrombolytic therapy for ischemic stroke patients.

Melatonin inhibits postischemic matrix metalloproteinase-9 (MMP-9) activation via dual modulation of plasminogen/plasmin system and endogenous MMP inhibitor in mice subjected to transient focal cerebral ischemia

Abstract:  We have shown that melatonin attenuated matrix metalloproteinase‐9 (MMP‐9) activation and decreased the risk of hemorrhagic transformation following cerebral ischemia‐reperfusion. Herein, we investigate the possible involvement of the plasminogen/plasmin system and endogenous MMPs inhibitor underlying the melatonin‐mediated MMP‐9 inhibition. Mice were subjected to 1‐hr ischemia and 48‐hr reperfusion of the right middle cerebral artery. Melatonin (5 mg/kg) or vehicle was intravenously injected upon reperfusion. Brain infarction and hemorrhagic transformation were measured. Extracellular matrix damage was determined by Western immunoblot analysis for laminin protein. The activity and expression of MMP‐2 and MMP‐9 were determined by gelatin zymography, in situ zymography, and Western immunoblot analysis. In addition, the activities of tissue and urokinase plasminogen activators (tPA and uPA) were evaluated by plasminogen‐dependent casein zymography. Endogenous plasminogen activator inhibitor (PAI) and tissue inhibitors of MMP (TIMP‐1) were investigated using enzyme‐linked immunosorbent assay (ELISA) and Western immunoblot analysis, respectively. Cerebral ischemia‐reperfusion induced increased MMP‐9 activity and expression at 12–48 hr after reperfusion onset. Relative to controls, melatonin‐treated animals had significantly decreased MMP‐9 activity and expression (P < 0.05), in addition to reduced brain infarction and hemorrhagic transformation as well as improved laminin protein preservation. This melatonin‐mediated MMP‐9 inhibition was accompanied by reduced uPA activity (P < 0.05), as well as increased TIMP‐1 expression and PAI activity (P < 0.05, respectively). These results demonstrate the melatonin’s pluripotent mechanisms for attenuating postischemic MMP‐9 activation and neurovascular damage, and further support it as an add‐on to thrombolytic therapy for ischemic stroke patients.

Melatonin improves presynaptic protein, SNAP-25, expression and dendritic spine density and enhances functional and electrophysiological recovery following transient focal cerebral ischemia in rats

Abstract:  Synapto‐dendritic dysfunction and rearrangement takes place over time at the peri‐infarct brain after stroke, and the event plays an important role in post‐stroke functional recovery. Here, we evaluated whether melatonin would modulate the synapto‐dendritic plasticity after 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. Local cerebral blood perfusion, somatosensory electrophysiological recordings and neurobehavioral tests were serially measured. Animals were sacrificed at 7 days after tMCAO. The brain was processed for Nissl‐stained histology, Golgi–Cox‐impregnated sections, or Western blotting for presynaptic proteins, synaptosomal‐associated protein of 25 kDa (SNAP‐25) and synaptophysin (a calcium‐binding protein found on presynaptic vesicle membranes). Relative to controls, melatonin‐treated animals had significantly reduced infarction volumes (P < 0.05) and improved neurobehavioral outcomes, as accessed by sensorimotor and rota‐rod motor performance tests (P < 0.05, respectively). Melatonin also significantly improved the SNAP‐25, but not synaptophysin, protein expression in the ischemic brain (P < 0.05). Moreover, melatonin significantly improved the dendritic spine density and the somatosensory electrophysiological field potentials both in the ischemic brain and the contralateral homotopic intact brain (P < 0.05, respectively). Together, melatonin not only effectively attenuated the loss of presynaptic protein, SANP‐25, and dendritic spine density in the ischemic territory, but also improved the reductions in the dendritic spine density in the contralateral intact brain. This synapto‐dendritic plasticity may partly account for the melatonin‐mediated improvements in functional and electrophysiological circuitry after 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.

Abstract:  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.

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.