Neuroprotective effects of N-acetyl-cysteine and acetyl-L-carnitine after spinal cord injury in adult rats

Following the initial acute stage of spinal cord injury, a cascade of cellular and inflammatory responses will lead to progressive secondary damage of the nerve tissue surrounding the primary injury site. The degeneration is manifested by loss of neurons and glial cells, demyelination and cyst formation. Injury to the mammalian spinal cord results in nearly complete failure of the severed axons to regenerate. We have previously demonstrated that the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) can attenuate retrograde neuronal degeneration after peripheral nerve and ventral root injury. The present study evaluates the effects of NAC and ALC on neuronal survival, axonal sprouting and glial cell reactions after spinal cord injury in adult rats. Tibial motoneurons in the spinal cord were pre-labeled with fluorescent tracer Fast Blue one week before lumbar L5 hemisection. Continuous intrathecal infusion of NAC (2.4 mg/day) or ALC (0.9 mg/day) was initiated immediately after spinal injury using Alzet 2002 osmotic minipumps. Neuroprotective effects of treatment were assessed by counting surviving motoneurons and by using quantitative immunohistochemistry and Western blotting for neuronal and glial cell markers 4 weeks after hemisection. Spinal cord injury induced significant loss of tibial motoneurons in L4-L6 segments. Neuronal degeneration was associated with decreased immunostaining for microtubular-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker GFAP and microglial marker OX42 was increased. Treatment with NAC and ALC rescued approximately half of the motoneurons destined to die. In addition, antioxidants restored MAP2 and synaptophysin immunoreactivity. However, the perineuronal synaptophysin labeling was not recovered. Although both treatments promoted axonal sprouting, there was no effect on reactive astrocytes. In contrast, the microglial reaction was significantly attenuated. The results indicate a therapeutic potential for NAC and ALC in the early treatment of traumatic spinal cord injury.     

Neuroprotective effects of VEGF administration after focal cerebral ischemia/reperfusion: dose response and time window

Administration of vascular endothelial growth factor (VEGF) has been shown to increase cerebral blood flow and reduce neurological damage after experimental ischemic brain injury. The purpose of this study was to examine the optimal dose and time window for the neuroprotective effect of VEGF when administrated after focal ischemia/reperfusion injury in rabbits. Focal cerebral ischemia/reperfusion was induced by the middle cerebral artery occlusion (MCAO) method. In a dose response experiment, low (1.25 ng/μL), middle (2.5 ng/μL) and high (5.0 ng/μL) doses of VEGF were administered 2h after MCAO by the route of perifocal region. The VEGF at a dose of middle (2.5 ng/μL) displayed excellent effects on neuroprotective efficacy for focal cerebral ischemia/reperfusion injury. In another experiment, 2.5 ng/μL VEGF was administered at times varying from 2 to 8h after MCAO. Infarct volume, water content and neurological deficits were significantly reduced when VEGF was given at 2 and 3h after injury. The protective effect was less when the same dose was given at the later times. Thus, the present findings indicated that VEGF reduced ischemic neuronal danger with a therapeutic time window within the first 3h of transient MCAO and may be useful in the treatment of acute ischemic stroke in humans.     

Neuroprotective and growth-promoting effects of bone marrow stromal cells after cervical spinal cord injury in adult rats

Background aimsBone marrow stromal cells (BMSC) have been shown to provide neuroprotection after transplantation into the injured central nervous system. The present study investigated whether adult rat BMSC differentiated along a Schwann cell lineage could increase production of trophic factors and support neuronal survival and axonal regeneration after transplantation into the injured spinal cord.MethodsAfter cervical C4 hemi-section, 5-bromo-2-deoxyuridine (BrdU)/green fluorescent protein (GFP)-labeled BMSC were injected into the lateral funiculus at 1 mm rostral and caudal to the lesion site. Spinal cords were analyzed 2–13 weeks after transplantation.Results and ConclusionsTreatment of native BMSC with Schwann cell-differentiating factors significantly increased production of brain-derived neurotrophic factor in vitro. Transplanted undifferentiated and differentiated BMSC remained at the injection sites, and in the trauma zone were often associated with neurofilament-positive fibers and increased levels of vascular endothelial growth factor. BMSC promoted extensive in-growth of serotonin-positive raphaespinal axons and calcitonin gene-related peptide (CGRP)-positive dorsal root sensory axons into the trauma zone, and significantly attenuated astroglial and microglial cell reactions, but induced aberrant sprouting of CGRP-immunoreactive axons in Rexed's lamina III. Differentiated BMSC provided neuroprotection for axotomized rubrospinal neurons and increased the density of rubrospinal axons in the dorsolateral funiculus rostral to the injury site. The present results suggest that BMSC induced along the Schwann cell lineage increase expression of trophic factors and have neuroprotective and growth-promoting effects after spinal cord injury.     

Neuroprotective effects of Caffeic acid phenethyl ester on experimental traumatic brain injury in rats

The aim of this study was to evaluate the therapeutic efficacy of caffeic acid phenethyl ester (CAPE) with an experimental traumatic brain injury (TBI) model in rats. Twenty-four adult male Sprague–Dawley rats were randomly divided into three groups of 8 rats each: control, TBI, and TBI + CAPE treatment. In TBI and TBI + CAPE treatment groups, a cranial impact was delivered to the skull from a height of 7 cm at a point just in front of the coronal suture and over the right hemisphere. Rats were sacrificed at 4 h after the onset of injury. Brain tissues were removed for biochemical and histopathological investigation. To date, no biochemical and histopathological changes of neurodegeneration in the frontal cortex after TBI in rats by CAPE treatment have been reported. The TBI significantly increased tissue malondialdehyde (MDA) levels, and significantly decreased tissue superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, but not tissue catalase (CAT) activity, when compared with controls. The administration of a single dose of CAPE (10 μmol/kg) 15 min after the trauma has shown protective effect via decreasing significantly the elevated MDA levels and also significantly increasing the reduced antioxidant enzyme (SOD and GPx) activities, except CAT activity. In the TBI group, severe degenerative changes, shrunken cytoplasma and extensively dark picnotic nuclei in neurons, as well as vacuolization indicating tissue edema formation. The morphology of neurons in the CAPE treatment group was well protected. The number of neurons in the trauma alone group was significantly less than that of both the control and TBI +CAPE treatment groups. The caspase 3 immunopositivity was increased in degenerating neurons of the traumatic brain tissue. Treatment of CAPE markedly reduced the immunoreactivity of degenerating neurons. TBI caused severe degenerative changes, shrunken cytoplasma, severely dilated cisternae of endoplasmic reticulum, markedly swollen mitochondria with degenerated cristae and nuclear membrane breakdown with chromatin disorganization in neurons of the frontal cortex. In conclusion, the CAPE treatment might be beneficial in preventing trauma-induced oxidative brain tissue damage, thus showing potential for clinical implications. We believe that further preclinical research into the utility of CAPE may indicate its usefulness as a potential treatment on neurodegeneration after TBI in rats.     

Neuroprotective effect of neuroserpin in rat primary cortical cultures after oxygen and glucose deprivation and tPA

Besides its role as a thrombolytic agent, tissue plasminogen activator (tPA) triggers harmful effects in the brain parenchyma after stroke, such as inflammation, excitotoxicity and basal lamina degradation. Neuroserpin, a natural inhibitor of tPA, has shown neuroprotective effects in animal models of brain infarct. However, the molecular mechanisms of neuroserpin-mediated neuroprotection after brain ischemia remain to be well characterized. Then, our aim was to investigate such mechanisms in primary mixed cortical cell cultures after oxygen and glucose deprivation (OGD). Primary rat mixed cortical cultures containing both astrocytes and neurons were subjected to OGD for 150min and subsequently treated with either tPA (5μg/mL), neuroserpin (0.125, 0.25, 0.5 or 1μM), and tPA together with neuroserpin at the mentioned doses. Twenty-four hours after treatment, LDH release, caspase-3 activity, MCP-1, MIP-2, active MMP-9, GRO/KC and COX-2 were measured. Statistical differences were analyzed using Student's t-test or one-way ANOVA as appropriate. Treatment with tPA after OGD increased LDH release, active MMP-9, MCP-1 and MIP-2 (all p≤0.05), but not caspase-3, GRO/KC or COX-2 compared to control. Treatment with neuroserpin after OGD decreased LDH release and active MMP-9 (all p≤0.05). It had no effect on caspase-3 activity, or on MCP-1, MIP-2, GRO/KC or COX-2 expression compared to control. Administration of tPA together with neuroserpin decreased LDH release, active MMP-9 and MIP-2 (all p≤0.05) and showed no effect on MCP-1, GRO/KC or COX-2 compared to control. Our results suggest that neuroprotective activity of neuroserpin involves attenuation on tPA-mediated mechanisms of inflammation and BBB disruption after brain ischemia.     

Neuroprotective effect on brain injury by neurotoxins from the spider Phoneutria nigriventer

The role of calcium channels blockers in ischemic condition has been well documented. The PhTx3 neurotoxic fraction of the spider Phoneutria nigriventer venom is a broad-spectrum calcium channel blocker that inhibits glutamate release, calcium uptake and also glutamate uptake in synaptosomes. In the present study we describe the effect of PhTx3 (1.0 microg/mL), omega-conotoxin GVIA (1.0 micromol/L) and omega-conotoxin MVIIC (100 nmol/L) on neuroprotection of hippocampal slices and SN56 cells subjected to ischemia by oxygen deprivation and low glucose insult (ODLG). After the insult, cell viability in the slices and SN56 cells was assessed by confocal microscopy and epifluorescence, using live/dead kit containing calcein-AM and ethidium homodimer. Confocal images of CA1 region of the rat hippocampal slices subjected to ischemia insult and treated with omega-conotoxin GVIA, omega-conotoxin MVIIC and PhTx3 showed a percentage of dead cells of 68%, 54% and 18%, respectively. The SN56 cells subjected to ischemia were almost completely protected from damage by PhTx3 while with omega-conotoxin GVIA or omega-conotoxin MVIIC the cell protection was only partial. Thus, PhTx3 provided robust ischemic neuroprotection showing potential as a novel class of agents that targets multiple components and exerts neuroprotection in in vitro model of brain ischemia.     

Neuroprotective effects of furopyrazole derivative of benzylindazole analogs on C2 ceramide-induced apoptosis in cultured cortical neurons

Ceramide accumulation in neurons during various disorders is associated with acute and chronic neurodegeneration. Here we investigate the neuroprotective effects of furopyrazole derivative of benzylindazole analogs on C2 ceramide-induced cell death in primary cortical neurons. Among the 12 furopyrazole derivative of benzylindazole analogs tested, carbinol derivatives exhibited strongest neuroprotection against C2 ceramide-induced apoptosis. The results suggest that furopyrazole derivative of benzylindazole analogs can be developed as useful neuroprotectants against neurodegenerative diseases.     

Neuroprotective effects of grape seed extract on neuronal injury by inhibiting DNA damage in the gerbil hippocampus after transient forebrain ischemia

Grape seed extract (GSE) possess cardioprotective abilities by functioning as in vivo antioxidants and by virtue of their ability to directly scavenge ROS including hydroxyl and peroxyl radicals. In the present study, we investigated the neuroprotective effects of grape seed extract (GSE) in the gerbil hippocampus after 5 min transient forebrain ischemia. Neuronal cell density in GSE-treated ischemic animals was significantly increased as compared with vehicle-treated ischemic animals 4 days after ischemic insult. In the GSE-treated groups, about 60% of pyramidal cells of the sham-operated group were stained with cresyl violet 4 days after ischemic insult. In this study, we found that GSE had neuroprotective effects on neuronal injury by inhibiting DNA damage in the CA1 region after ischemia. In vehicle-treated groups, 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunoreactivity was significantly changed time-dependently, whereas the immunoreactivity in the GSE-treated group was similar to the sham-operated group. In addition, we confirmed that astrocytes and microglia did not show significant activation in the CA1 region 4 days after ischemia-reperfusion, because many CA1 pyramidal cells were not damaged. Therefore, these results suggest that GSE can protect ischemic neuronal damage by inhibiting DNA damage after transient forebrain ischemia.     

Contrasting effect of piracetam and proline on the release of 3H-D-aspartic acid from the cerebral cortical synaptosomes of rats

Piracetam (at concentrations of 10(-6) and 10(-5), but not 10(-4) and 5 X 10(-4) M) decreased K+-stimulated 3H-D-aspartate release. Proline enhanced K+-stimulated D-aspartate release, and its effect was antagonized by piracetam at a concentration that had no effect on K+-stimulated release. Quisqualic acid attenuated K+-stimulated D-aspartate release, with the effect antagonized by GDEE. GDEE also blocked the effect of piracetam, but not proline. The data are discussed in terms of the role of excitatory amino acid neurotransmission in the mechanisms of amnestic and antihypoxic piracetam action.     

CD38 exacerbates focal cytokine production, postischemic inflammation and brain injury after focal cerebral ischemia

Background

Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion.

Methodology/Principal Findings

We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8⁺ cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model.

Conclusion/Significance

CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke.     

Neuroprotective and neurotrophic effects of long term lithium treatment in mouse brain

Since the worldwide approval of lithium therapy in 1970, lithium has been used for its anti-manic, antidepressant, and anti-suicidal effects. The last decade has witnessed the following discoveries about its neuroprotective and neurotrophic properties, yet the therapeutic mechanisms at the cellular level remain not-fully defined. We have undertaken the present study to determine if chronic lithium treatment, at therapeutically relevant concentrations, exerts neurotrophic/neuroprotective effects in the mouse brain in vivo. For this purpose, 10 months aged mice were fed for 3 months on food pellets contained 1 g (L1 group) or 2 g (L2 group) lithium carbonate/kg, resulting in serum concentrations of 0.4 and 0.8 mM, respectively. The evaluation of lipid peroxidation level and the activities of catalase, superoxide-dismutase and glutathione-peroxidase showed that chronic Li administration, at therapeutic doses doesn’t induce oxidative stress in brain tissue. No changes in the expression levels of molecular chaperones, namely, the HSP70, and HSP90 heat shock proteins and the GRP94 glucose-regulated protein were detected. Moreover, this treatment has caused (1) an increase in the relative brain weight (2) a delay in the age induced cerebral glucose impairment (3) an enhancement of the neurogenesis in hippocampus and enthorinal cortex highlighted by silver impregnation. Under these experimental conditions, no modifications were observed in expression levels of GSK3 and of its downstream target β-catenin proteins. These results suggested that chronic Li administration, at therapeutic doses, has a neuroprotective/neurotrophic properties and its therapeutic mechanism doesn’t implicate GSK3 inactivation.     

Identification,enrichment, and isolation ofThiothrix spp. from environmental samples

We have developed a method to enrich, isolate, and identifyThiothrix spp. in environmental samples. This procedure employs low concentrations of organic compounds, the addition of reduced sulfur compounds (sulfide or thiosulfate), and preparation with spring water that containsThiothrix spp. The enrichment enhanced identification ofThiothrix spp. by promoting deposition of intracellular sulfur granules and inhibiting overgrowth by other bacteria. The relatively high calcium content of the spring water contributed to the culture procedure. With this technique,Thiothrix spp. were observed in two activated sludge systems, a municipal water storage tank, three springs, and four underground freshwater caves in the phreatic zone of the Floridan aquifer. Two differentThiothrix cultures have been isolated from a freshwater cave and a water storage tank by this procedure. It appears that media prepared with spring water known to supportThiothrix spp. can be designed to provide highly selective methods for isolation ofThiothrix spp. from a wide range of environments.Florida Agricultural Experimentation Station, Journal Series Number R-03446.     

Improvement of tactile discrimination performance and enlargement of cortical somatosensory maps after 5 Hz rTMS

Repetitive transcranial magnetic stimulation (rTMS) is increasingly used to investigate mechanisms of brain functions and plasticity, but also as a promising new therapeutic tool. The effects of rTMS depend on the intensity and frequency of stimulation and consist of changes of cortical excitability, which often persists several minutes after termination of rTMS. While these findings imply that cortical processing can be altered by applying current pulses from outside the brain, little is known about how rTMS persistently affects learning and perception. Here we demonstrate in humans, through a combination of psychophysical assessment of two-point discrimination thresholds and functional magnetic resonance imaging (fMRI), that brief periods of 5 Hz rTMS evoke lasting perceptual and cortical changes. rTMS was applied over the cortical representation of the right index finger of primary somatosensory cortex, resulting in a lowering of discrimination thresholds of the right index finger. fMRI revealed an enlargement of the right index finger representation in primary somatosensory cortex that was linearly correlated with the individual rTMS-induced perceptual improvement indicative of a close link between cortical and perceptual changes. The results demonstrate that repetitive, unattended stimulation from outside the brain, combined with a lack of behavioral information, are effective in driving persistent improvement of the perception of touch. The underlying properties and processes that allow cortical networks, after being modified through TMS pulses, to reach new organized stable states that mediate better performance remain to be clarified.     

Short-term effects of a disturbed light-dark cycle and environmental enrichment on aggression and stress-related parameters in male mice

In the laboratory setting, environmental factors have a major influence on the well-being of laboratory animals. The present study shows the importance of a semi-natural light-dark cycle. In this experiment one cohort of mice was kept with a continuous lighting for one week. After the first week the artificial light-dark cycle was 12:12 with lights on at 07:00 h. The second cohort of mice was kept with this 12:12 h light-dark cycle from the start. Half of each cohort received environmental enrichment. In order to analyse corticosterone levels, urine samples were collected. To measure agonistic behaviour, the behaviour of the mice was recorded on videotape immediately after cage cleaning. A significant difference in corticosterone levels between cohorts was found during disturbed lighting, but not after lighting conditions were reset to 12:12 h. In the first test week, mice subjected to disturbed lighting also showed a significantly shorter agonistic latency than control mice. This difference had disappeared when in the second test week all mice experienced 12:12 h lighting. No effects of enriched housing were found. This experiment has shown that disturbed lighting for socially-housed male mice caused physiological and behavioural changes indicative of stress, not only leading to much higher levels of corticosterone but also to shorter agonistic latency within the groups.     

Wnt signaling enhances neurogenesis and improves neurological function after focal ischemic injury

Stroke potently stimulates cell proliferation in the subventricular zone of the lateral ventricles with subsequent neuroblast migration to the injured striatum and cortex. However, most of the cells do not survive and mature. Extracellular Wnt proteins promote adult neurogenesis in the neurogenic niches. The aim of the study was to examine the efficacy of Wnt signaling on neurogenesis and functional outcome after focal ischemic injury. Lentivirus expressing Wnt3a-HA (LV-Wnt3a-HA) or GFP (LV-GFP) was injected into the striatum or subventricular zone of mice. Five days later, focal ischemic injury was induced by injection of the vasoconstrictor endothelin-1 into the striatum of the same hemisphere. Treatment with LV-Wnt3a-HA into the striatum significantly enhanced functional recovery after ischemic injury and increased the number of BrdU-positive cells that differentiated into mature neurons in the ischemic striatum by day 28. Treatment with LV-Wnt3a-HA into the subventricular zone significantly enhanced functional recovery from the second day after injury and increased the number of immature neurons in the striatum and subventricular zone. This was accompanied by reduced dissemination of the neuronal injury. Our data indicate that Wnt signaling appears to contribute to functional recovery after ischemic injury by increasing neurogenesis or neuronal survival in the striatum.     

Non-muscle myosin II regulates aortic stiffness through effects on specific focal adhesion proteins and the non-muscle cortical cytoskeleton

Non-muscle myosin II (NMII) plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in mammalian vascular function is not well understood. Here, we investigated the function of NMII in the biomechanical and signalling properties of mouse aorta. We found that blebbistatin, an inhibitor of NMII, decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate that in freshly isolated, contractile, aortic smooth muscle cells, the non-muscle myosin IIA (NMIIA) isoform is associated with contractile filaments in the core of the cell as well as those in the non-muscle cell cortex. However, the non-muscle myosin IIB (NMIIB) isoform is excluded from the cell cortex and colocalizes only with contractile filaments. Furthermore, both siRNA knockdown of NMIIA and NMIIB isoforms in the differentiated A7r5 smooth muscle cell line and blebbistatin-mediated inhibition of NM myosin II suppress agonist-activated increases in phosphorylation of the focal adhesion proteins FAK Y925 and paxillin Y118. Thus, we show in the present study, for the first time that NMII regulates aortic stiffness and stress and that this regulation is mediated through the tension-dependent phosphorylation of the focal adhesion proteins FAK and paxillin.     

Neuroprotective effects of allicin on spinal cord ischemia-reperfusion injury via improvement of mitochondrial function in rabbits

Allicin, the active substance of garlic, exerts a broad spectrum of pharmacological activities and is considered to have potential therapeutic applications. The present study was designed to investigate the beneficial effects of allicin against spinal cord ischemia-reperfusion (I/R) injury and its associated mechanisms. Male New Zealand white rabbits were pretreated with allicin (1, 10 and 50mg/kg) for two weeks, and exposed to infrarenal aortic occlusion-induced spinal cord I/R injury. We found that allicin significantly reduced the volume of the spinal cord infarctions, improved the histopathologic features and increased the number of motor neurons in a dose-dependent manner. This protection was associated with an improvement in neurological function, which was measured by the hind-limb motor function scores. Furthermore, allicin also significantly suppressed the accumulations of protein and lipid peroxidation products, and increased the activities of endogenous antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione S-transferase (GST). In addition, allicin treatment preserved the function of mitochondria respiratory chain complexes and inhibited the production of ROS and the release of mitochondrial cytochrome c in the spinal cord of this model. Collectively, these findings demonstrated that allicin exerts neuroprotection against spinal cord I/R injury in rabbits, which may be associated with the improvement of mitochondrial function.