Masamitsu Shimazawa

Toll-like receptor 4 (TLR4), but not TLR3 or TLR9, knock-out mice have neuroprotective effects against focal cerebral ischemia

Toll-like receptors (TLRs) are signaling receptors in the innate immune system that is a specific immunologic response to systemic bacterial infection. We investigated whether cerebral ischemia induced by the middle cerebral artery occlusion (MCAO) for 2 h differed in mice that lack a functional TLR3, TLR4, or TLR9 signaling pathway. TLR4, but not TLR3 or TLR9, knock-out (KO) mice had significantly smaller infarct area and volume at 24 h after ischemia-reperfusion (I/R) compared with wild-type mice. In addition, TLR4 KO mice improved in neurological deficits after I/R compared with wild-type mice. Moreover, we investigated the expression of TLR4 in the ischemic brain with immunohistochemistry. The number of TLR4-positive cells gradually increased from 1 h after MCAO to 22 h after I/R. We also examined the localization of TLR4 in the ischemic area. TLR4 was localized with CD11b-positive microglial cells in the ischemic striatum and the number of CD11b-positive microglial cells was smaller in TLR4 KO mice than in wild-type mice. In addition, we investigated the translocation of NF-κB among TLR3, 4, and 9 KO mice after I/R injury using western blotting. NF-κBs p65 subunit was decreased in TLR4 KO mice compared to wild-type mice, but not TLR3 or 9 KO mice. These data suggest that TLR4 knockout, but not TLR3 or TLR9 knockout, may play a neuroprotective role in ischemic brain injury induced by MCAO in mice.

The Experimental and Clinical Pharmacology of Propofol, an Anesthetic Agent with Neuroprotective Properties

Propofol (2,6‐diisopropylphenol) is a versatile, short‐acting, intravenous (i.v.) sedative‐hypnotic agent initially marketed as an anesthetic, and now also widely used for the sedation of patients in the intensive care unit (ICU). At the room temperature propofol is an oil and is insoluble in water. It has a remarkable safety profile. Its most common side effects are dose‐dependent hypotension and cardiorespiratory depression. Propofol is a global central nervous system (CNS) depressant. It activates γ‐aminobutyric acid (GABAA) receptors directly, inhibits the N‐methyl‐d‐aspartate (NMDA) receptor and modulates calcium influx through slow calcium‐ion channels. Furthermore, at doses that do not produce sedation, propofol has an anxiolytic effect. It has also immunomodulatory activity, and may, therefore, diminish the systemic inflammatory response believed to be responsible for organ dysfunction. Propofol has been reported to have neuroprotective effects. It reduces cerebral blood flow and intracranial pressure (ICP), is a potent antioxidant, and has antiinflammatory properties. Laboratory investigations revealed that it might also protect brain from ischemic injury. Propofol formulations contain either disodium edetate (EDTA) or sodium metabisulfite, which have antibacterial and antifungal properties. EDTA is also a chelator of divalent ions such as calcium, magnesium, and zinc. Recently, EDTA has been reported to exert a neuroprotective effect itself by chelating surplus intracerebral zinc in an ischemia model. This article reviews the neuroprotective effects of propofol and its mechanism of action.

Minocycline inhibits oxidative stress and decreases in vitro and in vivo ischemic neuronal damage

The neuroprotective effects of minocycline-which is broadly protective in neurologic-disease models featuring cell death and is being evaluated in clinical trials-were investigated both in vitro and in vivo. For the in vivo study, focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice. Minocycline at 90 mg/kg intraperitoneally administered 60 min before or 30 min after (but not 4 h after) the occlusion reduced infarction, brain swelling, and neurologic deficits at 24 h after the occlusion. For the in vitro studies, we used cortical-neuron cultures from rat fetuses in which neurotoxicity was induced by 24-h exposure to 500 microM glutamate. Furthermore, the effects of minocycline on oxidative stress [such as lipid peroxidation in mouse forebrain homogenates and free radical-scavenging activity against diphenyl-p-picrylhydrazyl (DPPH)] were evaluated to clarify the underlying mechanism. Minocycline significantly inhibited glutamate-induced cell death at 2 microM and lipid peroxidation and free radical scavenging at 0.2 and 2 microM, respectively. These findings indicate that minocycline has neuroprotective effects in vivo against permanent focal cerebral ischemia and in vitro against glutamate-induced cell death and that an inhibition of oxidative stress by minocycline may be partly responsible for these effects.

Neuroprotection by Brazilian Green Propolis against In vitro and In vivo Ischemic Neuronal Damage

We examined whether Brazilian green propolis, a widely used folk medicine, has a neuroprotective function in vitro and/or in vivo. In vitro, propolis significantly inhibited neurotoxicity induced in neuronally differentiated PC12 cell cultures by either 24 h hydrogen peroxide (H2O2) exposure or 48 h serum deprivation. Regarding the possible underlying mechanism, propolis protected against oxidative stress (lipid peroxidation) in mouse forebrain homogenates and scavenged free radicals [induced by diphenyl-p-picrylhydrazyl (DPPH). In mice in vivo, propolis [30 or 100 mg/kg; intraperitoneally administered four times (at 2 days, 1 day and 60 min before, and at 4 h after induction of focal cerebral ischemia by permanent middle cerebral artery occlusion)] reduced brain infarction at 24 h after the occlusion. Thus, a propolis-induced inhibition of oxidative stress may be partly responsible for its neuroprotective function against in vitro cell death and in vivo focal cerebral ischemia.

Fasudil, a Rho kinase (ROCK) inhibitor, protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons

BACKGROUND AND PURPOSE Recently, fasudil, a Rho kinase (ROCK) inhibitor, was reported to prevent cerebral ischemia in vivo by increasing cerebral blood flow and inhibiting inflammatory responses. However, it is uncertain whether a ROCK inhibitor can directly protect neurons against ischemic damage. Our purpose was to evaluate both the involvement of ROCK activity in ischemic neuronal damage and any direct neuroprotective effect of fasudil against cerebral infarction. METHODS In vivo, focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice, and the resulting infarction was evaluated 24 h later. ROCK expression and activity were assessed using Western blotting and immunohistochemistry. In vitro, the effects of fasudil and hydroxyfasudil (a main metabolite of fasudil) were examined on oxygen-glucose deprivation (OGD)-induced PC12 cell death and on glutamate-induced neurotoxicity in primary cerebral neuronal culture. RESULTS ROCK expression and activity increased in the striatum, especially in axons, in the early phase of ischemia. Fasudil reduced this ROCK activity and protected against cerebral infarction in vivo. Hydroxyfasudil inhibited OGD-induced PC12 cell death, and fasudil and hydroxyfasudil each attenuated glutamate-induced neurotoxicity in vitro. CONCLUSIONS These findings indicate that ROCK plays a pivotal role in the mechanism underlying ischemic neuronal damage and that a direct effect of fasudil on neurons may be partly responsible for its protective effects against such damage.

Damage of photoreceptor-derived cells in culture induced by light emitting diode-derived blue light

Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm(2)). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light.

Docosahexaenoic acid (DHA) has neuroprotective effects against oxidative stress in retinal ganglion cells.

We examined the radical-scavenging activity of docosahexaenoic acid (DHA) and its effects on the neuronal cell death induced by oxidative or hypoxic stress in cultured retinal ganglion cells (RGC-5, a rat ganglion cell-line transformed using E1A virus). The radical-scavenging activity [hydrogen peroxide (H(2)O(2)), superoxide anion (O(2)*(-)), and hydroxyl radical (*OH)] of DHA in RGC-5 cells was measured using the ROS-sensitive probes CM-H(2)DCFDA and APF. DHA concentration-dependently scavenged the intracellular radical productions induced by H(2)O(2) radical, O(2)*(-), and *OH (minimum effective DHA concentrations 0.1, 10 and 100 microM, respectively). Cell damage was induced by H(2)O(2), oxygen-glucose deprivation (OGD), or tunicamycin (an endoplasmic reticulum-stress inducer), and cell viability was assessed by Hoechst 33342 nuclear staining or by the tetrazolium salt (WST-8) cell-viability assay. H(2)O(2) (0.3 mM for 24 h), 4-h OGD exposure followed by 18-h reoxygenation, or tunicamycin at 2 microg/ml for 24 h induced apoptotic cell death accompanied by nuclear condensation and/or fragmentation, and each maneuver decreased cell viability. Treatment with DHA at 0.1 and 1 microM significantly inhibited the decrease in cell viability induced by H(2)O(2). Treatment with DHA at 0.1, 1, or 10 microM significantly inhibited the decrease in cell viability induced by OGD/reoxygenation exposure. However, DHA (0.1 to 10 microM) had no effect on the decrease in cell viability induced by tunicamycin. These results indicate that DHA may be protective against oxidative or hypoxic stress-induced cell damage in retinal ganglion cells.

Involvement of CHOP, an ER-stress apoptotic mediator, in both human sporadic ALS and ALS model mice.

Endoplasmic reticulum (ER) stress-induced neuronal death may play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, whether CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), an ER-stress apoptotic mediator, is involved in the pathogenesis of ALS is controversial. Here we demonstrate the expression levels and localization of CHOP in spinal cords of both sporadic ALS patients and ALS transgenic mice by immunohistochemistry. In the spinal cords of sporadic ALS patients, CHOP was markedly up-regulated but typically expressed at low levels in those of the control. Likewise, CHOP expression increased at 14 (symptomatic stage) and 18 to 20 weeks (end stage) in ALS transgenic mice spinal cords. Furthermore, localizations of CHOP were merged in motor neurons and glial cells, such as oligodendrocytes, astrocytes, and microglia. These results indicate that the up-regulation of CHOP in motor neurons and glial cells may play pivotal roles in the pathogenesis of ALS.

Bilberry and its main constituents have neuroprotective effects against retinal neuronal damage in vitro and in vivo.

Our aim was to determine whether a Vaccinium myrtillus (bilberry) anthocyanoside (VMA) and/or its main anthocyanidin constituents (cyanidin, delphinidin, and malvidin) can protect retinal ganglion cells (RGCs) against retinal damage in vitro and in vivo. In RGC cultures (RGC-5, a rat ganglion cell-line transformed using E1A virus) in vitro, cell damage and radical activation were induced by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Cell viability was measured using a water-soluble tetrazolium salt assay. Intracellular radical activation within RGC-5 cells was evaluated using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H(2)DCFDA). Lipid peroxidation was assessed using the supernatant fraction of mouse forebrain homogenates. In mice in vivo, we evaluated the effects of VMA on N-methyl-D-aspartic acid (NMDA)-induced retinal damage using hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) stainings. VMA and all three anthocyanidins (i) significantly inhibited SIN-1-induced neurotoxicity and radical activation in RGC-5, (ii) concentration-dependently inhibited lipid peroxidation in mouse forebrain homogenates. Intravitreously injected VMA significantly inhibited the NMDA-induced morphological retinal damage and increase in TUNEL-positive cells in the ganglion cell layer. Thus, VMA and its anthocyanidins have neuroprotective effects (exerted at least in part via an anti-oxidation mechanism) in these in vitro and in vivo models of retinal diseases.

Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye

The purpose of this study was to evaluate the potential of submicron-sized liposomes (ssLips) as a novel system for delivering ocular drugs to the eyes posterior segment. Fluorescence emission of coumarin-6 formulated into ssLip was obvious in that segment in mice after eyedrop administration of the liposomal suspension. Such fluorescence was not observed after administration of either multilamellar vesicles or dimethyl sulfoxide (DMSO) solution containing the same amount of coumarin-6. The highest fluorescence of ssLip occurred 30 min after eyedrop administration, and all fluorescence disappeared after 180 min. The ssLip based on l-alpha-distearoyl phosphatidylcholine (DSPC ssLip) showed higher fluorescence emission in the retina than that based on egg phosphatidylcholine (EPC ssLip). These results confirmed that the magnitude of fluorescence in the retina was closely related to both liposome rigidity and particle size. Images of the entire eye showed that ssLip was delivered via the non-corneal pathway after administration. The liposomes tested in ocular cells showed little cytotoxicity. These results suggest that ssLip can be used to deliver drugs to the posterior segment of the eye.