Yu-Chang Hung

Cerebral blood flow velocity and vasomotor reactivity before and after shunting surgery in patients with normal pressure hydrocephalus.

Summary The purpose of this study was to evaluate pre- and post-shunting haemodynamic changes and their correlation with the clinical results in normal pressure hydrocephalus (NPH). Accordingly, eleven demented patients with clinical signs suggestive of NPH received examinations of cerebral blood flow velocity (BFV) and vasomotor reactivity (VMR) by transcranial Doppler sonography with carbogen testing before and after shunt treatment. Computerized tomography (CT), clinical assessment and neuropsychological grading were performed prior to and at 3 months following surgery. A control group consisting of 10 patients was included to establish baseline data. The pre-operative CBF studies in the anterior cerebral artery (ACA) and the middle cerebral artery (MCA) revealed the NPH patients did not have significant decreases of BFVs, but had significant decreases of carbogen VMR (P<0.05). After shunting, there were no significant changes of the BFVs as compared with the pre-shunting data. The post-shunting VMR of the ACA was significantly higher than the pre-shunting one (p<0.05), but there was no variation in that of the MCA. Both the values of post-shunting VMR in ACA and the post-shunting increase in VMR in MCA of the 7 shunt-responsive patients who improved mentally and in other symptoms were significantly higher than those of patients without improvement (p<0.05). In addition, the five patients with gait improvement showed significantly higher values of post-shunting VMR of ACA and the post-shunting increase of VMR for both ACA and MCA when compared with those patients without gait improvement (p<0.05, respectively). Our study supports the view that patients with NPH had various degrees of impaired VMR in both the ACA and the MCA, but showed insignificant reduction in BFVs, indicating a compensatory mechanism of CBF over time to accommodate the subnormal state of cerebral perfusion pressure. Shunt placement would improve the VMR in responsive patients. Postoperatively, an increase of VMR tends to accompany improvement of the functional state: that in the MCA alone is associated with symptomatic improvement in mental function and that increase in VMR in both the ACA and the MCA with improvement in gait, respectively.

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.

Two primary brain tumors, meningioma and glioblastoma multiforme, in opposite hemispheres of the same patient

We report a case with double primary intracranial tumors of different cell types without phacomatosis. The patient was hospitalized due to progressive memory impairment, headaches, dysarthria and right hemiparesis. Initial computed tomographic (CT) examinations revealed a large hyperdense tumor over the right frontal lobe, suggestive of an extra-axial meningioma. Additionally, there was unusual brain edema in the contralateral hemisphere that subsequently proved to originate from an intrinsic tumor. Staged craniotomies were used to treat the patient. Pathological examinations confirmed the two tumors to be a meningioma and a glioblastoma multiforme, respectively. The patient made an uneventful recovery after treatment. Although meningioma and glioma represent two common primary intracranial tumors, the simultaneous development of the two tumors is rare. A randomly occurring event most likely accounted for this linkage in the patient. We suggest that extraordinary brain edema far remote from the primary brain lesion warrants special attention for identifying other potentially undetected lesions.

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.

Optimal Percoll concentration facilitates flow cytometric analysis for annexin V/propidium iodine‐stained ischemic brain tissues

We sought to determine the optimal Percoll concentration for ischemic rat brain prepared for flow cytometric (FC) measurements. Animals were subjected to the right middle cerebral artery (MCA) occlusion, and were euthanized at 3, 12, 24, and 72 h after reperfusion onset. The brains were processed by different concentrations (unisolated, 20, 25, 30, or 40%) of Percoll and stained with annexin V/propidium iodine (PI). Ischemic brain damage was evaluated by FC analysis and image analysis for histologic sections. The relative susceptibility of different phenotypes of cells to necrotic and apoptotic damage were evaluated by the FC analyses for the immunohistochemistry, PI, and the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling (TUNEL)‐processed brain tissues. Our results showed that FC analysis effectively detected the extent and maturation of apoptotic/necrotic brain damage, and the results were consistent with those determined from histologic brain sections. Neuron was more vulnerable to apoptosis than glia, whereas both cellular phenotypes were compatible in susceptibility for necrotic cell death. Percoll at a low concentration (20%) could effectively remove tissue debris without affecting membranous integrity of the injured neurons. Conversely, high percentages of Percoll (30–40%) substantially increased membranous damage for the injured cells. These results supported the application of FC to determine the extent and progression in time, as well as relative phenotypes of apoptotic/necrotic cell deaths following ischemic damage. We highlighted the use of Percoll at low percentages to facilitate the removal of tissue debris and to improve membrane integrity preservation for the injured neurons.

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.

Melatonin inhibits matrix metalloproteinase-9 (MMP-9) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells and a mouse model of meningitis.

Abstract:  We explored anti‐inflammatory potential of melatonin against the lipopolysaccharide (LPS)‐induced inflammation in vivo and in vitro. RAW 264.7 and BV2 cells were stimulated by LPS, followed by the treatment with melatonin or vehicle at various time intervals. In a mouse model of meningitis induced by LPS, melatonin (5 mg/kg) or vehicle was intravenously injected at 30 min postinsult. The activity of matrix metalloproteinase‐2 (MMP‐2) and metalloproteinase‐9 (MMP‐9) was determined by gelatin zymography. Nuclear factor‐kappa B (NFκB) translocation and binding activity were determined by immunocytochemistry and electrophoretic mobility shift assay (EMSA). Our results showed that either pretreatment or cotreatment with melatonin at 50–500 μm effectively inhibited the LPS‐induced proMMP‐9 activation in the RAW 264.7 and BV2 cells, respectively (P < 0.05). This melatonin‐induced proMMP‐9 inhibition remained effective when treatment was delayed up to 2 and 6 hr postinsult for RAW 264.7 and BV2 cells, respectively (P < 0.05 for both groups). Additionally, melatonin significantly attenuated the rises of circulatory and cerebral MMP‐9 activity, respectively (P < 0.05) and reduced the loss of body weight (P < 0.05) in mice with meningitis. Moreover, melatonin (50 μm) effectively inhibited nuclear factor‐kappa B (NFκB) translocation and binding activity in the LPS‐treated RAW 264.7 and BV2 cells, respectively (P < 0.05). These results demonstrate direct inhibitory actions of melatonin against postinflammatory NFκB translocation and MMP‐9 activation and highlight its ability to inhibit systemic and cerebral MMP‐9 activation following brain inflammation.