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Effects of Memantine on Nitric Oxide Production and Hydroxyl Radical Metabolism during Cerebral Ischemia and Reperfusion in Mice
Abstract
Background:
The purpose of this study was to investigate the effects of memantine on brain ischemia. Because we can measure nitric oxide (NO) production and hydroxyl radical metabolism continuously, we investigated the effect of memantine on NO production and hydroxyl radical metabolism in cerebral ischemia and reperfusion.
Methods:
Memantine (25 µmol/kg) was administered intraperitoneally to 6 C57BL/6 mice 30 minutes before ischemia. Seven additional mice received no injection (controls). NO production and hydroxyl radical metabolism were continuously monitored using bilateral striatal microdialysis in vivo. Hydroxyl radical formation was monitored using the salicylate trapping method. Forebrain ischemia was produced in all mice by occluding the common carotid artery bilaterally for 10 minutes. Levels of the NO metabolites nitrite (NO2-) and nitrate (NO3-) were determined using the Griess reaction. Survival rates of hippocampal CA1 neurons were calculated and 8-hydroxydeoxyguanosine (8-OHdG)-immunopositive cells were counted to evaluate the oxidative stress in hippocampal CA1 neurons 72 hours after the start of reperfusion.
Results:
The regional cerebral blood flow was significantly higher in the memantine group than in the control group after reperfusion. Furthermore, the level of 2,3-dihydroxybenzoic acid was significantly lower in the memantine group than in the control group during ischemia and reperfusion. Levels of NO2- and NO3- did not differ significantly between the 2 groups. Although survival rates in the CA1 did not differ significantly, there were fewer 8-OHdG-immunopositive cells in animals that had received memantine than in control animals.
Conclusions:
These data suggest that memantine exerts partially neuroprotective effects against cerebral ischemic injury.
... The molecular structure of Memantine is shown in Figure 1(b). It is widely reported that Memantine significantly alleviates the declined cognitive function, abnormal behavior, and overall pathological changes in severe AD patients, accompanied by a decline in the total cost of health and social services [17]. Recently, it has been reported that Memantine has a significant beneficial effect on injured neurons by ameliorating inflammation and oxidative stress [17,18]. ...
... It is widely reported that Memantine significantly alleviates the declined cognitive function, abnormal behavior, and overall pathological changes in severe AD patients, accompanied by a decline in the total cost of health and social services [17]. Recently, it has been reported that Memantine has a significant beneficial effect on injured neurons by ameliorating inflammation and oxidative stress [17,18]. This evidence supports that Memantine is a promising neuroprotective agent. ...
... The cells were recovered in a 150 mm cell culture dish and split by trypsinization until 90% confluence was reached. For the experiment, 2 × 10 5 cells / well were cultured in a 6-well plate and treated with 50 μM oxaliplatin (Cat#HY-17371, MedChemExpress, USA) [4,5] in the presence or absence of Memantine (Cat#1189713-18-5, Chemgen bio-Tech Pioneer, USA) [17] at the concentrations of 5 or 10 µM for 24 hours. In this study, cells from passages 3 to 6 were used for experiments. ...
Oxaliplatin is an effective chemotherapeutic agent for the treatment of malignant tumors. However, severe oxaliplatin-induced neurotoxicity has been well documented. Memantine is a drug for the management of Alzheimer’s Disease (AD) due to its promising neuroprotective properties. We hypothesize that Memantine possesses a beneficial role against chemotherapy-induced neuronal damages. In this study, we established an oxaliplatin-induced neurotoxicity assay model in human SHSY-5Y neuronal cells and investigated the protective effect of Memantine. We showed that Memantine treatment ameliorated oxaliplatin-elevated intracellular production of reactive oxygen species (ROS), lipid product malondialdehyde (MDA), and NOX-2 expression. Memantine alleviated impairment of the mitochondrial membrane potential and ATP production by oxaliplatin. As a result, Memantine showed a protective role against oxaliplatin-induced cytotoxicity. Moreover, the terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) apoptosis assay revealed that Memantine protected oxaliplatin-induced apoptosis through mitigating the ratio of Bax/Bcl-2 and Caspase-3 cleavage. We concluded Memantine ameliorated the neurotoxicity of oxaliplatin in a mitochondrial-dependent pathway.
... Genetic knockdown of Fstl1 in the hippocampus has been associated with impairments in learning and memory, as well as abnormal neural oscillations and synaptic plasticity [89]. Another previous report suggested that FSTL1 may modulate the activation of glial cells by reducing the expression of proinflammatory cytokines such as TNF-α and IL-1β via blockage of the MAPK/p-ERK1/2 pathway in astrocytes and NF-κB pathway in microglia, thus playing an inhibitory role in early neuroinflammation [22,90,91]. Furthermore, FSTL1 has been shown to shift microglial phenotypes toward an anti-inflammatory, M2-like state, aiding in tissue repair through the production of anti-inflammatory cytokines [17,31]. ...
Background
Defects in the ataxin-2 (ATXN-2) protein and CAG trinucleotide repeat expansion in its coding gene, Atxn-2, cause the neurodegenerative disorder spinocerebellar ataxia type 2 (SCA2). While clinical studies suggest potential benefits of human-derived mesenchymal stem cells (hMSCs) for treating various ataxias, the exact mechanisms underlying their therapeutic effects and interaction with host tissue to stimulate neurotrophin expression remain unclear specifically in the context of SCA2.
Methods
Human bone marrow-derived MSCs (hMSCs) were injected into the cisterna magna of 26-week-old wild-type and SCA2 mice. Mice were assessed for impaired motor coordination using the accelerating rotarod, open field test, and composite phenotype scoring. At 50 weeks, the cerebellum vermis was harvested for protein assessment and immunohistochemical analysis.
Results
Significant loss of NeuN and calbindin was observed in 25-week-old SCA2 mice. However, after receiving multiple injections of hMSCs starting at 26 weeks of age, these mice exhibited a significant improvement in abnormal motor performance and a protective effect on Purkinje cells. This beneficial effect persisted until the mice reached 50 weeks of age, at which point they were sacrificed to study further mechanistic events triggered by the administration of hMSCs. Calbindin-positive cells in the Purkinje cell layer expressed bone-derived neurotrophic factor after hMSC administration, contributing to the protection of cerebellar neurons from cell death.
Conclusion
In conclusion, repeated administration of hMSCs shows promise in alleviating SCA2 symptoms by preserving Purkinje cells, improving neurotrophic support, and reducing inflammation, ultimately leading to the preservation of locomotor function in SCA2 mice.
... In contrast, amantadine-derived phenolic azo Schiff bases showed no antioxidant activity 124 . While the antioxidant properties of memantine are supported by some reports (in vitro inhibition of ROS production and lipid peroxidation; in vivo enhancement of the antioxidant barrier and reduction of protein and lipid oxidation) 35,[125][126][127][128][129][130] , data on glycation prevention remain inconclusive [130][131][132][133] . There are also no data on thromantadine, and the antiglycoxidative impact of rimantadine was not confirmed [134][135][136] . ...
An important drug used in the treatment of Parkinson’s disease is amantadine. We are the first to perform a comprehensive study based on various glycation and oxidation factors, determining the impact of amantadine on protein glycoxidation. Sugars (glucose, fructose, galactose) and aldehydes (glyoxal, methylglyoxal) were used as glycation agents, and chloramine T was used as an oxidant. Glycoxidation biomarkers in albumin treated with amantadine were generally not different from the control group (glycation/oxidation factors), indicating that the drug did not affect oxidation and glycation processes. Molecular docking analysis did not reveal strong binding sites of amantadine on the bovine serum albumin structure. Although amantadine poorly scavenged hydroxyl radical and hydrogen peroxide, it had significantly lower antioxidant and antiglycation effect than all protein oxidation and glycation inhibitors. In some cases, amantadine even demonstrated glycoxidant, proglycation, and prooxidant properties. In summary, amantadine exhibited weak antioxidant properties and a lack of antiglycation activity.
... The virtual screening was performed by using PyRx virtual screening tool between Gallic acid and above cerebrotoxic proteins which are involved in cerebral stroke they are like Bax (PDB ID: 5 W60), Tumor necrosis factor a (PDB ID : 5UUI), Caspases 3 (PDB ID : 2DKO), Interleukin (PDB ID : 1ALU), Nitric oxide synthase (PDB ID : 6CIC) compared to standard drugs such as Minocycline (Pub Chem Id : 54675783), Quercetin (Pub Chem Id:5280343) with Acid sensing ion channel (PDB ID : 3S3X); Memantine (Pub Chem Id: 4054) with Glutamate receptor (Pub Chem Id:5ZG2). Minocycline (Naderi et al., 2020), Quercetin (Wang et al., 2020), Memantine (Tanaka et al., 2018) reported significant neuroprotective effect on targeting reported cerebrotoxic proteins. Hence current study was using these compounds as standard. ...
... The virtual screening was performed by using PyRx virtual screening tool between Gallic acid and above cerebrotoxic proteins which are involved in cerebral stroke they are like Bax (PDB ID: 5 W60), Tumor necrosis factor a (PDB ID : 5UUI), Caspases 3 (PDB ID : 2DKO), Interleukin (PDB ID : 1ALU), Nitric oxide synthase (PDB ID : 6CIC) compared to standard drugs such as Minocycline (Pub Chem Id : 54675783), Quercetin (Pub Chem Id:5280343) with Acid sensing ion channel (PDB ID : 3S3X); Memantine (Pub Chem Id: 4054) with Glutamate receptor (Pub Chem Id:5ZG2). Minocycline (Naderi et al., 2020), Quercetin (Wang et al., 2020), Memantine (Tanaka et al., 2018) reported significant neuroprotective effect on targeting reported cerebrotoxic proteins. Hence current study was using these compounds as standard. ...
Current study was designed multiple occlusions and reperfusion of bilateral carotid arteries induced cerebral injury model and evaluated the protective effect of gallic acid on it. In silico study was involved to study gallic acid binding affinity on cerebrotonic proteins compared with standard drugs using Autodoc vina tool. Cerebral ischemia was induced by occlusion of bilateral common carotid arteries for 10 mins followed by 10 reperfusions (1 cycle), cycle was continued to 3 cycles (MO/RCA), then pathological changes were observed by estimation of brain antioxidants as superoxide dismutase, glutathione, catalase, oxidants like malonaldehyde, cerebral infarction area, histopathology, and study gallic acid treatment against cerebral injury. Gallic acid exhibited a strong binding affinity on targeted cerebrotoxic proteins. MO/RCA rat brain antioxidant levels were significantly decreased and increased MDA levels (p<0.0001), Infarction size compared to sham rats. Gallic acid treatment rat brain MDA levels significantly decreased (p<0.4476) and increased SOD (p<0.0001), CAT (p<0.0001), GSH (p<0.0001), cerebral infarction area when compared to MO/RCA group. Developed model showed significant cerebral ischemic injury in rats, injury was ameliorated by Gallic acid treatment and in silico approaches also inhibit the cerebrotoxic protein function by targeting on active sites.
... Large amounts of glutamate is released during the pathophysiology of distinct CNS disorders, leading to NMDAR stimulation [17]. Memantine, the NMDAR antagonist, has recently demonstrated a powerful brain-protective effect in the treatment of disparate CNS diseases such as cerebral ischemia [18,19], infantile rat hydrocephalus [20], traumatic brain injury [21,22] and subarachnoid hemorrhage [16,23]. ...
Memantine has demonstrated beneficial effects on several types of brain insults via therapeutic mechanisms mainly related to its activity as a receptor antagonist of N-methyl-D-aspartate. However, the influences of memantine on intracerebral hemorrhage (ICH) remain obscure. This research probed into the neurovascular protective mechanisms of memantine after ICH and its impacts on neuronal nitric oxide synthase (nNOS) ser1412 phosphorylation. ICH model was established by employing intrastriatal collagenase injection in rats. After modeling, rats were then allocated randomly into sham-operated (sham), vehicle-treated (ICH+V), and memantine-administrated (ICH+M) groups. Memantine (20 mg/kg/day) was intraperitoneally administered 30 min after ICH and thenceforth once daily. Rats were dedicated at 0.25, 6, 12, 24 h, 3 and 7 d post-ICH for measurement of corresponding indexes. Behavioral changes, brain edema, levels of nNOS ser1412 phosphorylation, peroxynitrite, matrix metalloproteinase (MMP)-9, NLRP3, IL-1β and numbers of dying neurons, as well as the cellular localization of gelatinolytic activity, were detected among the groups. Memantine improved the neurologic deficits and mitigated brain water content, levels of MMP-9, NLRP3, IL-1β and dying neurons. Additionally, treatment with memantine also reduced nNOS ser1412 phosphorylation and peroxynitrite formation compared with the ICH+V group at 24 h after ICH. In situ zymography simultaneously revealed that gelatinase activity was primarily colocalized with vessel walls and neurons. We concluded that memantine ameliorated blood-brain barrier disruption and neurologic dysfunction in an ICH rat model. The underlying mechanism might involve repression of nNOS ser1412 phosphorylation, as well as peroxynitrite-related MMP-9 and NLRP3 inflammasome activation.
... In another study in primary cultured cortical neurons, memantine (10 μM) significantly decreased the apoptosis and increased cell viability (Chen et al., 2016). Another source that can also help, are the results obtained in experiments that applied memantine before the artery occlusion and the results show that memantine decreased oxidative stress (Tanaka et al., 2018), the number of reactive microglia and astrocytes, endothelial cells damaged, matrix metallopeptidase 9 secretion, an enzyme involved in the degradation of extracellular matrix, and improved the BBB integrity (Chen et al., 2016). Furthermore, indirect evidence from the effects of memantine in another disease can also help. ...
Ischemic stroke is the most common type of cerebrovascular disease and is caused by an interruption of blood flow in the brain. In this disease, two different damage areas are identifying: the lesion core, in which cells quickly die; and the penumbra (surrounding the lesion core), in which cells are functionally weakened but may recover and restore their functions. The currently approved treatments for ischemic stroke are the recombinant tissue plasminogen activator and endovascular thrombectomy, but they have a short therapeutic window (4.5 and 6 hours after stroke onset, respectively) and a low percentage of stroke patients actually receive these treatments. Memantine is an approved drug for the treatment of Alzheimer's disease. Memantine is a noncompetitive, low affinity and use-dependent antagonist of N-methyl-D-aspartate glutamate receptor. Memantine has several advantages over developing a new drug to treat focal ischemic stroke, but the most important is that it has sufficient safe probes in preclinical models and humans, and if the preclinical studies provide more evidence about pharmacological actions in tissue protection and repair, this could help to increase the number of clinical trials. The present review summarizes the physiopathology of isquemic stroke and the pharmacological actions in neuroprotection and neuroplasticity of memantine in the post stroke stage of preclinical stroke models, to illustrate their potential to improve functional recovery in human patients.
... These data could be results of the antioxidative system response induced by propolis against free radical production in the case of I/R. Additionally, to assess the extent of oxidative DNA damage [26] we analyzed 8-OHdGimmunopositive cells in a rat model of renal I/R injury. The results showed that propolis significantly decreased the 8-OHdG formation in kidney cells. ...
... These data could be results of the antioxidative system response induced by propolis against free radical production in the case of I/R. Additionally, to assess the extent of oxidative DNA damage [26] we analyzed 8-OHdGimmunopositive cells in a rat model of renal I/R injury. The results showed that propolis significantly decreased the 8-OHdG formation in kidney cells. ...
Ischemia reperfusion (I/R) injury which causes kidney dysfunction is one of the most studied diseases directly linked to oxidative stress. In this regard, it is important to protect cells against damage by inducing antioxidant response. Herein, we aimed to evaluate the therapeutic roles and possible mechanisms of propolis and boric acid in kidney I/R injury based on relevant basic research and clinical studies. Sprague-Dawley rats were subjected to 50 min of ischemia followed by 3 h of reperfusion. Animals were randomly divided into a control group (the abdominal wall was just opened and closed), an I/R injury group, the propolis intervention group (200 mg/kg, intragastric administration, 1 h before ischemia), boric acid intervention group (14 mg/kg, intragastric administration 1 h before ischemia), and the propolis + boric acid intervention group (intragastric administration 1 h before ischemia). Kidney function, the antioxidant defensive system, and renal damage were assessed. In addition, the oxidative stress and inflammatory status were estimated in renal tissue. Furthermore, DNA damageand apoptosis were detected by immunohistochemistry. When compared with I/R group, propolis alone and especially propolis + boric acid groups significantly improved functional parameters. While the antioxidant response was increased, renal injury size and apoptosis were significantly decreased in both groups. Also, the MDA and TNF-α levels besides the 8-OHdG formation were downregulated. According to these outcomes, it can be said that especially propolis together with boric acid ameliorates kidney injury caused by I/R through acting as an antioxidant, anti-inflammatory, and antiapoptotic agent. In conclusion, propolis alone and its combination with boric acid could be developed as therapeutic agents against serious renal I/R injuries.
... We have already reported the effect of the NMDA-R antagonist memantine on hydroxyl radical metabolism during cerebral ischemia and reperfusion in mice. 22 In our previous study, we reported that hydroxyl radicals were significantly lower in the memantine-treated group than in the control group. Similarly, it was also noted that yokukansan reduced hydroxyl radical metabolites in this study. ...
Background: The purpose of this study was to investigate the effects of yokukansan on forebrain ischemia. Because we can measure nitric oxide production and hydroxyl radical metabolism continuously, we investigated the effect of yokukansan on nitric oxide production and hydroxyl radical metabolism in cerebral ischemia and reperfusion. Methods: Yokukansan (300 mg per kg per day) was mixed into feed and given to 8 mice for 10 days. Eight additional mice received normal feed (control). Nitric oxide production and hydroxyl radical metabolism were continuously monitored using the salicylate trapping method. Forebrain ischemia was produced in all mice by occluding the common carotid artery bilaterally for 10 minutes. Levels of the nitric oxide metabolites nitrite and nitrate were determined using the Griess reaction. Survival rates of hippocampal CA1 neurons were calculated and 8-hydroxydeoxyguanosine-immunopositive cells were counted to evaluate the oxidative stress in hippocampal CA1 neurons 72 hours after the start of reperfusion. Results: Arterial blood pressure and regional cerebral blood flow were not significantly different between the 2 groups. The level of nitrate was significantly higher in the yokukansan group than in the control group during ischemia and reperfusion. Levels of 2,3- and 2,5-dihydroxybenzoic acid were significantly lower in the yokukansan group than in the control group during ischemia and reperfusion. Although survival rates in the CA1 did not differ significantly, there were fewer 8-hydroxydeoxyguanosine-immunopositive cells in animals that had received yokukansan than in control animals. Conclusions: These data suggest that yokukansan exerts reducing hydroxyl radicals in cerebral ischemic injury.
Delayed encephalopathy after acute carbon monoxide poisoning (DEACMP) is a relatively rare inflammatory-associated neurometabolic complication. In this article, we present a case report of a 50-year-old male patient with a history of carbon monoxide poisoning. This acute poisoning, although successfully controlled during a stay in the intensive care unit of a local hospital, later led to persistent neurological symptoms. The patient was then treated in the inpatient unit of the rehabilitation clinic, where cognitive deterioration began to develop 20 days after admission. Subsequent examination using EEG and magnetic resonance imaging confirmed severe encephalopathy later complicated by SARS-CoV-2 infection with fatal consequences due to bronchopneumonia. Because currently there are no approved guidelines for the management of DEACMP, we briefly discuss the existing challenges for future studies, especially the application of rational immunosuppressive therapy already in the acute treatment phase of CO poisoning, which could prevent the development of a severe form of DEACMP.
It has been reported that apelin-13 possesses neuroprotective effects against cerebral ischemia/reperfusion injury (IRI). Disabilities in sense, movement and balance are the major stroke complications which, result in a high rate of mortality. Here, effects of intravenous (IV) injection of apelin-13 on the severity of neural death, infarct volume, neurological defects and its association with nitric oxide (NO) were investigated. A rat model of cerebral IRI was created by middle cerebral artery occlusion (MCAO) for 60 min and restoration of blood flow for 23 h. Animals were randomly assigned into six groups: sham, ischemia (MCAO), vehicle (MCAO + PBS) and three treatment groups (MCAO + apelin-13 in 10, 20, 40 μg/kg doses, IV). All injections were carried out via tail vein injection 5 min before reperfusion. Neural loss and infarct volume were evaluated by Nissl and 2,3,5-triphenyltetrazolium chloride (TTC) staining, respectively. Neurological defects were scored by standard modified criteria. Serum NO was measured by colorimetric method. Apelin-13 in doses of 20 and 40 μg/kg significantly reduced neural death, infarct volume and disturbance of sensory-motor balance compared to control and vehicle groups (p < 0.05). Serum NO levels reduced in MCAO groups compared to sham. Apelin-13 restored serum NO levels at 20 μg/kg dose (p < 0.05). Our data showed beneficial effect of IV injection of apelin-13 on sensory-motor balance defects by reducing neural death and restoration of serum NO levels. The present study shows the validity of apelin-13 in treatment of ischemic stroke in different administration methods.
We have previously designed and synthesized a series of novel memantine nitrates and some of them have shown neuroprotective effects; however, the detail mechanisms remain unknown. In this study, we demonstrated that MN-12, one of the memantine nitrates, concentration-dependently protected against glutamate-induced neurotoxicity in rat primary cultured cerebellar granule neurons (CGNs). Western blotting assays revealed that MN-12 might possess neuroprotective effects through the inhibition of ERK pathway and activation of PI3K/Akt pathway concurrently. Moreover, MN-12 concentration-dependently dilated pre-contracted rat middle cerebral artery through activation of NO-cGMP pathway ex vivo. In the 2-vessel occlusion (2VO) rat model, MN-12 alleviated the impairments of spatial memory and motor dysfunction possibly via neuroprotection and improvement of the cerebral blood flow. Furthermore, the results of preliminary pharmacokinetic studies showed that MN-12 might quickly distribute to the major organs including the brain, indicating that MN-12 could penetrate the blood-brain barrier. Taken together, MN-12 might provide multifunctional therapeutic benefits for dementia associated with Alzheimer’s disease, vascular dementia and ischemic stroke, via neuroprotection and vessel dilation to improve the cerebral blood flow.
Stroke is an important cause of mortality and disability worldwide. Immediately after stroke onset, the ischemic cascade initiates and deleteriously affects neural cells. Time to reperfusion therapy is a critical determinant of functional recovery in stroke patients. Although recent trials have shown the significant efficacy of endovascular thrombectomy, either alone or with intravenous tissue plasminogen activator, in improving the functional outcomes of stroke patients with large vessel occlusion, hours can pass before patients receive reperfusion therapy. Moreover, many patients do not meet the eligibility criteria to receive reperfusion treatments. Therefore, an adjunct and alternative agent that can protect ischemic neuronal tissue during the hyperacute phase until reperfusion therapy can be administered may prevent further brain damage and enhance functional recovery. Memantine is a US Food and Drug Administration approved drug for the treatment of Alzheimer’s disease. Memantine blocks overstimulated N-methyl- d -aspartate receptors and prevents neurotoxicity caused by massive glutamate release. Preclinical studies show that memantine decreases infarction volume and improves neurologic outcomes. However, few clinical studies have evaluated the safety and efficacy of memantine in stroke patients. This review article summarizes the current evidence for the role of memantine in the treatment of ischemic stroke and highlights areas for future research.
Excitotoxicity is a major contributor to cell death during the acute phase of ischemic stroke but aggressive pharmacological targeting of excitotoxicity has failed clinically. Here we investigated whether pre-treatment with low doses of memantine, within the range currently used and well tolerated for the treatment of Alzheimer’s disease, produce a protective effect in stroke. A co-culture preparation exposed to modelled ischemia showed cell death associated with rapid glutamate rises and cytotoxic Ca2+ influx. Cell death was significantly enhanced in the presence of high memantine concentrations. However, low memantine concentrations significantly protected neurons and glia via excitotoxic cascade interruption. Mice were systemically administered a range of memantine doses (0.02, 0.2, 2, 10 and 20 mg/kg/day) starting 24 hr prior to 60 min reversible focal cerebral ischemia and continuing for a 48 hr recovery period. Low dose (0.2 mg/kg/day) memantine treatment significantly reduced lesion volume (by 30-50%) and improved behavioural outcomes in stroke lesions that had been separated into either small/striatal or large/striato-cortical infarcts. However, higher doses of memantine (20 mg/kg/day) significantly increased injury. These results show that clinically established low doses of memantine should be considered for patients ‘at risk’ of stroke, while higher doses are contraindicated.
Hydroxylation of salicylate to2, 3- and2, 5–dihydroxy-benzoates (DHBs) is widely used as an index of hydroxyl radical (OH) formation in vivo and in vitro. Several recent studies indicate that peroxynitrite can lead to generation of DHBs from salicylate and it is uncertain as to whether or not OH' is involved. A similar problem may occur in the use of phenylalanine as an OH' detector. Hence formation of hydroxylation products from salicylate (or phenylalanine) may not in itself be a definitive index of OH' generation, especially in cases where such generation in physiological systems is decreased by inhibitors of nitric oxide syn-thase. Determination of salicylate (or phenylalanine) nitration products can allow distinction between peroxynitrite-dependent aromatic hydroxylation and that involving “real” OH.
Physiological levels of H2S exert neuroprotective effects, whereas high concentrations of H2S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous
H2S and NMDAR antagonism, we synthesized a novel H2S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H2S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular
sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine
after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons
more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons
were aggravated by sodium sulfide (Na2S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in
SH-SY5Y cells more markedly than did Na2S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral
infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest
that an H2S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy
for ischemic brain injury.
The effect of memantine administration on hippocampal neurons of the infantile rats with kaolin induced hydrocephalus was investigated. MATERIAL and
Hydrocephalus was induced by injecting kaolin into the cisterna magna of 3-weeks old Sprague-Dawley rat pups. One group received a single daily dose of 20mg/kg memantine i.p. following hydrocephalus induction for a period of two weeks. By the end of the two-week period, animals were radiologically evaluated by magnetic resonance imaging and then sacrificed to get their cerebrums removed. Both immunohistochemical analysis of nitric oxide synthase activity and quantification of spared neurons in CA1, CA2 and CA3 regions of hippocampus were performed.
In hydrocephalus-induced rats considerable neuronal loss associated with significantly increased nitric oxide synthase immunoreactivity were determined in all hippocampal regions. However, memantine treated rats showed significantly higher number of spared neuron counts and reduced nitric oxide synthase immunoreactivity in CA1 and CA2 regions compared with the non-treated rats.
The findings of the study show that hippocampal neurons may constitute important targets for injury secondary to hydrocephalic process in experimental infantile hydrocephalus. Early anti-excitotoxic treatment with memantine seems to have a neuroprotective effect especially in the CA1 and CA2 subunits of the hippocampus.
The purpose of this study was to clarify the kinetics of nitric oxide (NO) induced by either endothelial NO synthase (eNOS) or neuronal NO synthase (nNOS) after transient global forebrain ischemia. We investigated NO production and ischemic changes to hippocampal CA1 neurons in eNOS knockout (-/-) mice and nNOS (-/-) mice during cerebral ischemia and reperfusion. NO production was continuously monitored by in vivo microdialysis. Global forebrain ischemia was produced by occlusion of both common carotid arteries for 10 minutes. Levels of nitrite (NO(2)(-)) and nitrate (NO(3)(-)), as NO metabolites, in dialysate were determined using the Griess reaction. Two hours after the start of reperfusion, animals were perfused with 4% paraformaldehyde. Hippocampal CA1 neurons were divided into three phases (severely ischemic, moderately ischemic, surviving), and the ratio of surviving neurons to degenerated neurons was calculated as the survival rate. The relative cerebral blood flow (rCBF) was significantly higher in nNOS (-/-) mice than in control mice after reperfusion. Levels of NO(3)(-) were significantly lower in eNOS (-/-) mice and nNOS (-/-) mice than in control mice during ischemia and reperfusion. NO(3)(-) levels were significantly lower in nNOS (-/-) mice than in eNOS (-/-) mice after the start of reperfusion. Survival rate tended to be higher in nNOS (-/-) mice than in control mice, but not significantly. These in vivo data suggest that NO production in the striatum after reperfusion is closely related to activities of both nNOS and eNOS, and is mainly related to nNOS following reperfusion.
The potential of most N-methyl-D-aspartate antagonists as neuroprotectants is limited by side effects. We previously reported that memantine is an open-channel N-methyl-D-aspartate blocker with a faster off-rate than many uncompetitive N-methyl-D-aspartate antagonists such as dizocilpine maleate. This parameter correlated with memantine's known clinical tolerability in humans with Parkinson's disease. Memantine is the only N-methyl-D-aspartate antagonist that has been used clinically for excitotoxic disorders at neuroprotective doses. Therefore, we wanted to investigate further the basis of its clinical efficacy, safety, and tolerability. Here we show for the first time for any clinically-tolerated N-methyl-D-aspartate antagonist that memantine significantly reduces infarct size when administered up to 2 h after induction of hypoxia/ischemia in immature and adult rats. We found that at neuroprotective concentrations memantine results in few adverse side effects. Compared to dizocilpine maleate, memantine displayed virtually no effects on Morris water maze performance or on neuronal vacuolation. At concentrations similar to those in brain following clinical administration, memantine (6-10 microM) did not attenuate long-term potentiation in hippocampal slices and substantially spared the N-methyl-D-aspartate component of excitatory postsynaptic currents, while dizocilpine maleate (6-10 microM) or D-2-amino-5-phosphovalerate (50 microM) completely blocked these phenomena. We suggest that the favorable kinetics of memantine interaction with N-methyl-D-aspartate channels may be partly responsible for its high index of therapeutic safety, and make memantine a candidate drug for use in many N-methyl-D-aspartate receptor-mediated human CNS disorders.
We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO2−) and nitrate (NO3−) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO2− and NO3− were decreased by the intraperitoneal injection of NG-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, whereas NG-nitro-d-arginine methyl ester had no effect. l-Arginine by itself increased NO2− and NO3− levels and diminished the reduction of their levels caused by NG-nitro-l-arginine methyl ester. Direct infusion of l-glutamate, N-methyl-d-aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO2− and/or NO3− levels. The effects of N-methyl-d-aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l-glutamate and N-methyl-d-aspartate were inhibited by NG-nitro-l-arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N-methyl-d-aspartate receptor antagonist. These results suggest that NO2− and NO3− levels may be related to nitric oxide production in vivo.
Stroke is a major public health problem leading to high rates of death and disability in adults. Excessive stimulation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation are crucial for neuronal injury after stroke insult. However, directly inhibiting NMDARs or nNOS can cause severe side effects because they have key physiological functions in the CNS. Here we show that cerebral ischemia induces the interaction of nNOS with postsynaptic density protein-95 (PSD-95). Disrupting nNOS-PSD-95 interaction via overexpressing the N-terminal amino acid residues 1-133 of nNOS (nNOS-N(1-133)) prevented glutamate-induced excitotoxicity and cerebral ischemic damage. Given the mechanism of nNOS-PSD-95 interaction, we developed a series of compounds and discovered a small-molecular inhibitor of the nNOS-PSD-95 interaction, ZL006. This drug blocked the ischemia-induced nNOS-PSD-95 association selectively, had potent neuroprotective activity in vitro and ameliorated focal cerebral ischemic damage in mice and rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Moreover, it readily crossed the blood-brain barrier, did not inhibit NMDAR function, catalytic activity of nNOS or spatial memory, and had no effect on aggressive behaviors. Thus, this new drug may serve as a treatment for stroke, perhaps without major side effects.
In the vascular system, endothelium-derived relaxing factor (EDRF) is the name of the local hormone released from endothelial cells in response to vasodilators such as acetylcholine, bradykinin and histamine. It diffuses into underlying smooth muscle where it causes relaxation by activating guanylate cyclase, so producing a rise in cyclic GMP levels. It has been known for many years that in the central nervous system (CNS) the excitatory neurotransmitter glutamate can elicit large increases in cGMP levels, particularly in the cerebellum where the turnover rate of cGMP is low. Recent evidence indicates that cell-cell interactions are involved in this response. We report here that by acting on NMDA (N-methyl-D-aspartate) receptors on cerebellar cells, glutamate induces the release of a diffusible messenger with strikingly similar properties to EDRF. This messenger is released in a Ca2+-dependent manner and its activity accounts for the cGMP responses that take place following NMDA receptor activation. In the CNS, EDRF may link activation of postsynaptic NMDA receptors to functional modifications in neighbouring presynaptic terminals and glial cells.
In this study the effect of memantine, an antagonist at the N-methyl-D-aspartate receptor, on spatial learning deficit and on neuronal damage following transient cerebral ischemia was evaluated. Global ischemia was induced by four-vessel-occlusion (4VO) for 20 min in rats. Memantine was administered 20 min before induction of ischemia at a dose of 10 or 20 mg/kg. One week after surgery spatial learning was tested in the Morris water maze. Treatment with the higher dose of memantine reduced the increase in escape latency and in swim distance induced by 4VO. Neuronal damage in the CA1 sector of the hippocampus and in the striatum produced by 4VO was significantly attenuated by 20 mg/kg memantine. Treatment with the lower dose of memantine had no influence on the deficit in spatial learning and the neuronal damage resulting from ischemia. The present data demonstrate that treatment with a neuroprotective agent like memantine can reduce functional as well as morphological sequelae induced by ischemia.
Excessive accumulation of glutamate or other excitatory amino acids and the subsequent overactivity of NMDA receptors is currently thought to lead to neuronal injury in cerebral ischemia. Therefore, antagonists of the NMDA receptor may offer an approach for the treatment of ischemic brain injury. Dizocilpine (MK-801), an NMDA receptor-associated channel blocker, protects neurons in several rodent stroke models. However, this drug has numerous side effects and causes apoptosis of neonatal neurons. Recently, another NMDA receptor-associated channel blocker, memantine, has been shown to ameliorate NMDA-receptor mediated neurotoxicity in neuronal cell cultures and in focal cerebral ischemia models in adult rats without substantial side effects. Memantine has been used clinically in the treatment of Parkinson's disease and spasticity for a number of years. Here we tested the effects of memantine on focal stroke caused by photochemical thrombosis in neonatal rats and demonstrated a neuroprotective effect of memantine in this model. We also found excellent correlation between infarct size determined by magnetic resonance imaging (MRI) and histopathological analysis in the same animals. A single pre-ischemic dose of memantine (20 mg/kg) given 15 min prior to induction of stroke reduced the infarct size by 36.3% when compared to control animals treated with normal saline (P < 0.0001). At this dosage, memantine manifests few, if any, neurobehavioral side effects. Thus memantine appears to be both safe and effective in neonatal as well as adult animal models of stroke.
The direct measurement of hydroxyl radicals in vivo is extremely difficult. Therefore, the indirect determination of hydroxyl radicals using salicylate (2-hydroxybenzoate) is widely accepted. Reverse microdialysis with glutamate led to a dose-dependent production of hydroxyl free radicals indicated by the hydroxylation adduct of salicylate, namely 2,3-dihydroxybenzoic acid. The local stimulation of hydroxyl free radical formation seems to be suitable to investigate a radical-scavenging property of potential neuroprotective drugs. In vitro experiments using the Fenton reaction may be a helpful tool to assess whether or not a substance is able to act as a radical scavenger in a cell free environment, which is easy to handle and a simple screening method before in vivo experiments were performed. In the present study we present an in vivo approach using local application of glutamate into the striatum and an in vitro screening using the Fenton reaction to induce hydroxyl radical formation. The main goal is to reliable measure hydroxyl free radicals, which are the most reactive oxygen radicals in biology and medicine.
This study investigated whether memantine, a non-competitive NMDA receptor antagonist is neuroprotective after traumatic brain injury (TBI) induced in adult rats with a controlled cortical impact device. TBI led to significant neuronal death in the hippocampal CA2 and CA3 regions (by 50 and 59%, respectively), by 7 days after the injury. Treatment of rats with memantine (10 and 20 mg/Kg, i.p.) immediately after the injury significantly prevented the neuronal loss in both CA2 and CA3 regions. This is the first study showing the neuroprotective potential of memantine to prevent the TBI-induced neuronal damage.
The pathogenesis of stroke, trauma and chronic degenerative diseases, such as Alzheimer's disease (AD), has been linked to excitotoxic processes due to inappropriate stimulation of the N-methyl-D-aspartate receptor (NMDA-R). Attempts to use potent competitive NMDA-R antagonists as neuroprotectants have shown serious side-effects in patients. As an alternative approach, we were interested in the anti-excitotoxic properties of memantine, a well-tolerated low affinity uncompetitive NMDA-R antagonist presently used as an anti-dementia agent. We explored in a series of models of increasing complexity, whether this voltage-dependent channel blocker had neuroprotective properties at clinically relevant concentrations. As expected, memantine protected neurons in organotypic hippocampal slices or dissociated cultures from direct NMDA-induced excitotoxicity. However, low concentrations of memantine were also effective in neuronal (cortical neurons and cerebellar granule cells) stress models dependent on endogenous glutamate stimulation and mitochondrial stress, i.e. exposure to hypoxia, the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) or a nitric oxide (NO) donor. Furthermore, memantine reduced lethality and brain damage in vivo in a model of neonatal hypoxia-ischemia (HI). Finally, we investigated functional rescue (neuronal capacity to migrate along radial glia) by memantine in cerebellar microexplant cultures exposed to the indirect excitotoxin 3-nitropropionic acid (3-NP). Potent NMDA-R antagonists, such as (+)MK-801, are known to block neuronal migration in microexplant cultures. Interestingly, memantine significantly restored the number of neurons able to migrate out of the stressed microexplants. These findings suggest that inhibition of the NMDA-R by memantine is sufficient to block excitotoxicity, while still allowing some degree of signalling.
Hydroxylation of salicylate and phenylalanine as assays for hydroxyl radicals: a cautionary note visited for the third time
- B Halliwell
- H Kaur
Halliwell B, Kaur H. Hydroxylation of salicylate and
phenylalanine as assays for hydroxyl radicals: a cautionary
note visited for the third time. Free Radic Res 1997;27:239-244.