Ming-Yang Lee

Melatonin attenuates gray and white matter damage in a mouse model of transient focal cerebral ischemia

Abstract:  We have previously shown that melatonin reduces infarct volumes and enhances neurobehavioral and electrophysiological recoveries following transient middle cerebral artery (MCA) occlusion in rats. In the study, we examined whether melatonin would display neuroprotection against neuronal, axonal and oligodendrocyte pathology after 24 hr of reperfusion following 1 hr of MCA occlusion in mice. Melatonin (5 mg/kg) or vehicle was given intraperitoneally at the commencement of reperfusion. Neurological deficits were assessed 24 hr after ischemia. Gray matter damage was evaluated by quantitative histopathology. Axonal damage was determined with amyloid precursor protein and microtubule‐associated protein tau‐1 immunohistochemistry to identify postischemic disrupted axonal flow and oligodendrocyte pathology, respectively. Oxidative damage was assessed by 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) and 4‐hydroxynonenal (4‐HNE) immunohistochemistry. Relative to controls, melatonin‐treated animals not only had a significantly reduced volume of gray matter infarction by 42% (P < 0.001), but also exhibited a decreased score of axonal damage by 42% (P < 0.001) and a reduction in the volume of oligodendrocyte pathology by 58% (P < 0.005). Melatonin‐treated animals also had significantly reduced immunopositive reactions for 8‐OHdG and 4‐HNE by 53% (P < 0.001) and 49% (P < 0.001), respectively. In addition, melatonin improved sensory and motor neurobehavioral outcomes by 47 and 30%, respectively (P < 0.01). Thus, delayed (1 hr) treatment with melatonin reduced both gray and white matter damage and improved neurobehavioral outcomes following transient focal cerebral ischemia in mice. The finding of reduced oxidative damage observed with melatonin suggests that its major mechanisms of action are mediated through its antioxidant and radical scavenging activity.

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.

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 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.

Delayed Treatment with Nicotinamide Inhibits Brain Energy Depletion,Improves Cerebral Microperfusion, Reduces Brain Infarct Volume, but does not Alter Neurobehavioral Outcome Following Permanent Focal Cerebral Ischemia in Sprague Dawley Rats

Delayed treatment with nicotinamide (NAm) reduces infarction induced by middle cerebral artery occlusion (MCAO) in rats. This study explored some potential mechanisms by which delayed NAm treatment may confer protection in the brain of Sprague-Dawley rats following permanent MCAO (pMCAO). NAm (500 mg/kg) or vehicle was given 2 h after the onset of pMCAO. Cortical microperfusion, brain and rectal temperature were serially measured. Neurobehavioral examinations were performed at 24 h post-ischemia followed by sacrifice for histologic assessment. Some rats were also sacrificed at 4 h post-ischemia for analyses of ATP, ADP, AMP, and adenosine. Permanent MCAO induced spontaneous hyperthermia and a sharp decrease in cortical microperfusion, ATP concentration, and the sum of adenine nucleotides (p < 0.05). At 4 h post-ischemia, NAm improved ATP recovery, the sum of adenine nucleotides (p < 0.05) and attenuated the ischemia-induced systemic hyperthermia (p < 0.05) without affecting brain temperature or cortical microperfusion. At 24 h, NAm improved cortical microperfusion in the ischemic hemisphere and reduced total infarct volume (p < 0.05), but did not affect behavioral scores. The data suggest that NAm attenuated brain damage following pMCAo initially by improving cerebral bioenergetic metabolism during the sub-acute phase of ischemia, followed by a delayed improvement in microvascular perfusion.

Kinematics of cervical spine discectomy with and without bone grafting: Quantitative evaluation of late fusion in a sheep model

OBJECTIVE This study was conducted to evaluate the kinematic response of late fusion results for cervical spine discectomies with and without bone grafting. MATERIALS AND METHODS Fifteen Barbados Black Belly sheep underwent sham operations (Group A, n = 5), C2-C3 discectomies only (Group B, n = 5), and C2-C3 discectomies with autologous iliac bone grafting (Group C, n = 5). Ten months after surgery, the animals were killed. Fresh ligamentous spines (C1-C5) were subjected to the relevantly applied loads through a loading frame attached to the C1. Each vertebra (from C2 to C4) was attached with a set of three infrared light-emitting diodes to record the spatial location relating to each load application using a Selspot II system (Selcom Selective Electronics, Inc., Valdese, NC). The load-deformation data of the C2-C3 and C3-C4 motion segments were recorded and analyzed for the three groups. RESULTS At the C2-C3 motion segment, the results indicated that Group B displayed larger motion ranges of rotation and lateral bending loads than did the other two groups. Significantly larger motion ranges of rotation loads were found in Group B than in Group C (P<0.05, for both comparisons). In contrast, Group C had the smallest motion ranges of flexion, lateral bending, and rotation loads. At the C3-C4 motion segment, both groups that had undergone discectomies had a significantly larger motion range of flexion load compared with Group A (P<0.05, for both comparisons). A significant increase in the motion range of right axial rotation was found in Group B (P<0.05), but not in Group C, compared with Group A. Group B exhibited larger motion ranges responding to all six tested loads than did Group C. CONCLUSION The results indicate that anterior fusion after C2-C3 cervical discectomies, regardless of the presence or absence of bone grafting, decreases the motion range of flexion load at the C2-C3 motion segment, and contrary data were seen at the C3-C4 motion segment. For axial rotation loads, discectomies without bone grafting resulted in increased motion ranges of both C2-C3 and C3-C4 motion segments whereas discectomies with bone grafting did not. The data may have clinical relevance regarding the role of bone grafting in cases of cervical spine disease.

Anemic hypoxia in moderate intracerebral hemorrhage: the alterations of cerebral hemodynamics and brain metabolism.

To determine the influence of anemic hypoxia on cerebral hemodynamics and brain metabolism during pathological conditions of the brain, moderate-sized intracerebral hemorrhage (ICH) was created in canines with and without preoperatively inducing chronic anemia. The changes in cerebral perfusion pressure (CPP) and cerebral blood flow velocities (CBFv) were evaluated as well as the determinations for cerebral extraction fraction of oxygen (CEO2), arteriovenous oxygen content difference (AVDO2) and lactate (Lac) concentrations through the arterial and superior sagittal sinus (SSS) samples. Before ICH production, anemic animals (n = 8) showed a significant reduction in cerebral AVDO2 and arteriovenous Lac difference (AVDLac) but had higher CBFv as well as CEO2 than did nonanemic animals (n = 8). The CBFv began to decrease within 30 min after ICH in anemic but not in nonanemic animals, and the difference between the two groups was found to be significant at 2 h (P<0.05). Following ICH, anemic group also showed coupling reductions in CEO2 and AVDO2, indicating a decreased cerebral metabolic rate for oxygen (CMRO2) relative to the baseline data, compared with a constant CMRO2 in nonanemic group in which the CEO2, AVDO2, and CBFv remained relatively normal. Moreover, compared to the baseline data, a significant increase of the AVDLac was found in anemic but not in nonanemic group, although the former had lower Lac concentrations of the SSS than did the latter group throughout the whole observation period. We conclude that, in cases with chronically reduced Hct, cerebral hemodynamics and oxygenation remain in favorable conditions, thus decreasing Lac production of the brain. The findings suggest a lowered metabolic demand of the brain tissue due to reduced cerebral O2-carrying capacity. During the early phase of moderate ICH, the regulation capacity in cerebral hemodynamics and brain oxygenation tend to deteriorate in profound anemic hypoxia, which consequently leads to enhancing at least modest anaerobic glycolysis.

The Application of Transcranial Doppler Sonography in Patients with Chronic Subdural Haematoma

Summary The aim of this study was to evaluate the haemodynamic changes of the middle cerebral artery (MCA) and their clinical significance before and after surgical aspiration in patients with chronic subdural haematoma (CSDH). Nineteen patients with CSDH (17 unilateral and 2 bilateral) received transcranial Doppler sonography (TCD) examinations for cerebral blood flow velocity (CBFv) of the MCA prior to and 5 days after neurosurgical treatment. A total of 21 lesion and 10 non-lesion hemispheres were included. Cranial computerized tomography (CT) and clinical assessments were performed before and 3 months following surgery. The preoperative TCD study revealed that the lesion hemisphere had a modest decrease in CBFv in the MCA as compared to the non-lesion hemisphere. Postoperatively, the CBFv significantly improved in the lesion hemisphere, but not in the non-lesion hemisphere, compared to the preoperative data (P<0.005). The improvement in CBFv showed no significant correlation with brain shift and haematoma volume of the initial cranial CT. Additionally, two patients, who were proved to have a postoperative complication of subdural pneumocephalus, failed to attend follow-up examinations of TCD. Our results support TCD as an alternative follow-up examination for patients with CSDH, although it may not be sensitive enough as a preoperative screening tool. Postoperatively, improvements in the CBFv of the lesion hemisphere are characteristic. An unexplained difficulty of accessing cerebral basal arteries in follow-up TCD examinations should suggest pneumocephalus in the primary differential diagnosis.