Memantine Improves Recovery After Spreading Depolarization in Brain Slices and can be Considered for Future Clinical Trials.

BACKGROUND: Spreading depolarization (SD) has been identified as a key mediator of secondary lesion progression after acute brain injuries, and clinical studies are beginning to pharmacologically target SDs. Although initial work has focused on the N-Methyl-D-aspartate receptor antagonist ketamine, there is also interest in alternatives that may be better tolerated. We recently showed that ketamine can inhibit mechanisms linked to deleterious consequences of SD in brain slices. The present study tested the hypothesis that memantine improves recovery of brain slices after SD and explored the effects of memantine in a clinical case targeting SD. METHODS: For mechanistic studies, electrophysiological and optical recordings were made from hippocampal area CA1 in acutely prepared brain slices from mice. SDs were initiated by localized microinjection of K+ in conditions of either normal or reduced metabolic substrate availability. Memantine effects were assessed from intrinsic optical signals and extracellular potential recordings. For the clinical report, a subdural strip electrode was used for continuous electrocorticographic recording after the surgical evacuation of a chronic subdural hematoma. RESULTS: In brain slice studies, memantine (10-300 µM) did not prevent the initiation of SD, but impaired SD propagation rate and recovery from SD. Memantine reduced direct current (DC) shift duration and improved recovery of synaptic potentials after SD. In brain slices with reduced metabolic substrate availability, memantine reduced the evidence of structural disruption after the passage of SD. In our clinical case, memantine did not noticeably immediately suppress SD; however, it was associated with a significant reduction of SD duration and a reduction in the electrocorticographic (ECoG) suppression that occurs after SD. SD was completely suppressed, with improvement in neurological examination with the addition of a brief course of ketamine. CONCLUSIONS: These data extend recent work showing that N-Methyl-D-aspartate receptor antagonists can improve recovery from SD. These results suggest that memantine could be considered for future clinical trials targeting SD, and in some cases as an adjunct or alternative to ketamine.

Microtubule-dependent processes precede pathological calcium influx in excitotoxin-induced axon degeneration.

Axon degeneration and axonal loss is a feature of neurodegenerative disease and injury and occurs via programmed pathways that are distinct from cell death pathways. While the pathways of axonal loss following axon severing are well described, less is known about axonal loss following other neurodegenerative insults. Here we use primary mouse cortical neuron cultures grown in compartmentalized chambers to investigate the role of calcium in the degeneration of axons that occurs following a somal insult by the excitotoxin kainic acid. Calcium influx has been implicated in both excitotoxicity and axon degeneration mechanisms, however the link between a somal insult and axonal calcium increase is unclear. Live imaging of axons demonstrated that pharmacologically preventing intracellular calcium increases through the endoplasmic reticulum or mitochondria significantly (p < 0.05) reduced axon degeneration. Live calcium-imaging with the Ca2+ indicator Fluo-4 demonstrated that kainic acid exposure to the soma resulted in a rapid, and transient, increase in calcium in the axon, which occured even at low kainic acid concentrations that do not cause axon degeneration within 24 h. However, this calcium transient was followed by a gradual increase in axonal calcium, which was associated with axonal loss. Furthermore, treatment with a range of doses of the microtubule stabilizing drug taxol, which protects against axon fragmentation in this model, prevented this gradual calcium increase, suggesting that the intra-axonal calcium changes are downstream of microtubule associated events. Biochemical analysis of taxol treated neurons demonstrated a shift in microtubule post-translational modifications, with a significant (p < 0.05) increase in acetylated tubulin and a significant (p < 0.05) decrease in tyrosinated tubulin, suggestive of a more stable microtubule pool. Together our results suggest that axonal degeneration following excitotoxicity is dependent on an increase in axonal calcium, which is downstream of a microtubule-dependent event.

Retinal control of lens-induced astigmatism in chicks.

PURPOSE: Astigmatism is a refractive error due to meridional differences in refractive powers of lens or cornea. The resulting failure to focus image points in a single plane causes blurred vision at all distances. In this study, using an animal model of lens-induced astigmatism, we tested the hypothesis that induced astigmatism is due to processing of astigmatic retinal image information by the brain, which causes distorted growth in the anterior segment via centrifugal neural projections. METHODS: To induce astigmatism, +4.00DS/-8.00DC crossed-cylinder-lens goggles were affixed over the right eyes of 7-day-old chicks (P7), with the -8.00DC axis oriented vertically (at 90°) or horizontally (180°) (n = 12 each); the left eyes were without goggles (non-goggled). For all experiments, refractive errors of both eyes were measured by streak retinoscopy, before and after 1 week of lens wear. To test whether neuronal pathways between retina and brain are required, axonal conduction within the eye was blocked by intravitreal injections of tetrodotoxin (TTX; 7 µL of 10-4M) in phosphate-buffered saline (PBS), or of PBS alone (7 µL); fellow open eyes received PBS alone. Pupillary light reflex (PLR) and optokinetic response (OKR) were measured, to assess the efficacy and duration of TTX action. To test whether retinal circuitry is required, groups of chicks (n = 12 each) were treated at P7 by intravitreal injection of 20 µL of mixed excitotoxins (2 µmol N-methyl-D-aspartate, 0.2 µmol quisqualic acid, 0.2 µmol kainic acid; in water) into goggled or non-goggled eyes, to compromise retinal circuitry needed for emmetropization. RESULTS: Crossed-cylinder goggles reliably induced refractive astigmatism. Maximum astigmatic error was induced when the cylindrical axis was oriented at 90° (vertically). TTX effectively blocked nerve conduction within the eye for 48 h after injection. Goggled eyes developed astigmatism after treatment with TTX or PBS, but not after excitotoxins. CONCLUSION: Our hypothesis was rejected. In this model, the compensatory astigmatism induced by crossed-cylinder lenses is intrinsic to the eye, and mediated by visual processing in the retina.

Excitotoxins, Mitochondrial and Redox Disturbances in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). There is increasing evidence that MS is not only characterized by immune mediated inflammatory reactions, but also by neurodegenerative processes. There is cumulating evidence that neurodegenerative processes, for example mitochondrial dysfunction, oxidative stress, and glutamate (Glu) excitotoxicity, seem to play an important role in the pathogenesis of MS. The alteration of mitochondrial homeostasis leads to the formation of excitotoxins and redox disturbances. Mitochondrial dysfunction (energy disposal failure, apoptosis, etc.), redox disturbances (oxidative stress and enhanced reactive oxygen and nitrogen species production), and excitotoxicity (Glu mediated toxicity) may play an important role in the progression of the disease, causing axonal and neuronal damage. This review focuses on the mechanisms of mitochondrial dysfunction (including mitochondrial DNA (mtDNA) defects and mitochondrial structural/functional changes), oxidative stress (including reactive oxygen and nitric species), and excitotoxicity that are involved in MS and also discusses the potential targets and tools for therapeutic approaches in the future.

Riluzole and novel naphthalenyl substituted aminothiazole derivatives prevent acute neural excitotoxic injury in a rat model of temporal lobe epilepsy.

Epileptogenic seizures, or status epilepticus (SE), leads to excitotoxic injury in hippocampal and limbic neurons in the kainic acid (KA) animal model of temporal lobe epilepsy (TLE). Here, we have further characterized neural activity regulated methylaminoisobutryic acid (MeAIB)/glutamine transport activity in mature rat hippocampal neurons in vitro that is inhibited by riluzole (IC50 = 1 µM), an anti-convulsant benzothiazole agent. We screened a library of riluzole derivatives and identified SKA-41 followed by a second screen and synthesized several novel chlorinated aminothiazoles (SKA-377, SKA-378, SKA-379) that are also potent MeAIB transport inhibitors in vitro, and brain penetrant following systemic administration. When administered before KA, SKA-378 did not prevent seizures but still protected the hippocampus and several other limbic areas against SE-induced neurodegeneration at 3d. When SKA-377 - 379, (30 mg/kg) were administered after KA-induced SE, acute neural injury in the CA3, CA1 and CA4/hilus was also largely attenuated. Riluzole (10 mg/kg) blocks acute neural injury. Kinetic analysis of SKA-378 and riluzoles' blockade of Ca2+-regulated MeAIB transport in neurons in vitro indicates that inhibition occurs via a non-competitive, indirect mechanism. Sodium channel NaV1.6 antagonism blocks neural activity regulated MeAIB/Gln transport in vitro (IC50 = 60 nM) and SKA-378 is the most potent inhibitor of NaV1.6 (IC50 = 28 µM) compared to NaV1.2 (IC50 = 118 µM) in heterologous cells. However, pharmacokinetic analysis suggests that sodium channel blockade may not be the predominant mechanism of neuroprotection here. Riluzole and our novel aminothiazoles are agents that attenuate acute neural hippocampal injury following KA-induced SE and may help to understand mechanisms involved in the progression of epileptic disease.

Symptomatic and asymptomatic domoic acid exposure in zebrafish (Danio rerio) revealed distinct non-overlapping gene expression patterns in the brain.

Domoic acid (DA) is a naturally produced neurotoxin synthesized by marine diatoms in the genus Pseudo-nitzschia. DA accumulates in filter-feeders such as shellfish, and can cause severe neurotoxicity when contaminated seafood is ingested, resulting in Amnesic Shellfish Poisoning (ASP) in humans. Overt clinical signs of neurotoxicity include seizures and disorientation. ASP is a significant public health concern, and though seafood regulations have effectively minimized the human risk of severe acute DA poisoning, the effects of exposure at asymptomatic levels are poorly understood. The objective of this study was to determine the effects of exposure to symptomatic and asymptomatic doses of DA on gene expression patterns in the zebrafish brain. We exposed adult zebrafish to either a symptomatic (1.1 ± 0.2 µg DA/g fish) or an asymptomatic (0.31 ± 0.03 µg DA/g fish) dose of DA by intracelomic injection and sampled at 24, 48 and 168 h post-injection. Transcriptional profiling was done using Agilent and Affymetrix microarrays. Our analysis revealed distinct, non-overlapping changes in gene expression between the two doses. We found that the majority of transcriptional changes were observed at 24 h post-injection with both doses. Interestingly, asymptomatic exposure produced more persistent transcriptional effects - in response to symptomatic dose exposure, we observed only one differentially expressed gene one week after exposure, compared to 26 in the asymptomatic dose at the same time (FDR <0.05). GO term analysis revealed that symptomatic DA exposure affected genes associated with peptidyl proline modification and retinoic acid metabolism. Asymptomatic exposure caused differential expression of genes that were associated with GO terms including circadian rhythms and visual system, and also the neuroactive ligand-receptor signaling KEGG pathway. Overall, these results suggest that transcriptional responses are specific to the DA dose and that asymptomatic exposure can cause long-term changes. Further studies are needed to characterize the potential downstream neurobehavioral impacts of DA exposure.

Cycad ß-N-methylamino-L-alanine (BMAA), methylazoxymethanol, genotoxicity, and neurodegeneration.

Cycad-associated neurodegenerative disease is more strongly correlated with the gymnosperm's major neurotoxin cycasin (methylazoxymethanol glucoside) than with the minor neurotoxin ß-N-methylamino-L-alanine (L-BMAA).

Identifying the primary site of pathogenesis in amyotrophic lateral sclerosis - vulnerability of lower motor neurons to proximal excitotoxicity.

There is a desperate need for targeted therapeutic interventions that slow the progression of amyotrophic lateral sclerosis (ALS). ALS is a disorder with heterogeneous onset, which then leads to common final pathways involving multiple neuronal compartments that span both the central and peripheral nervous system. It is believed that excitotoxic mechanisms might play an important role in motor neuron death in ALS. However, little is known about the mechanisms by which excitotoxicity might lead to the neuromuscular junction degeneration that characterizes ALS, or about the site at which this excitotoxic cascade is initiated. Using a novel compartmentalised model of site-specific excitotoxin exposure in lower motor neurons in vitro, we found that spinal motor neurons are vulnerable to somatodendritic, but not axonal, excitotoxin exposure. Thus, we developed a model of somatodendritic excitotoxicity in vivo using osmotic mini pumps in Thy-1-YFP mice. We demonstrated that in vivo cell body excitotoxin exposure leads to significant motor neuron death and neuromuscular junction (NMJ) retraction. Using confocal real-time live imaging of the gastrocnemius muscle, we found that NMJ remodelling preceded excitotoxin-induced NMJ degeneration. These findings suggest that excitotoxicity in the spinal cord of individuals with ALS might result in a die-forward mechanism of motor neuron death from the cell body outward, leading to initial distal plasticity, followed by subsequent pathology and degeneration.

Age-Dependent Resistance to Excitotoxicity in Htt CAG140 Mice and the Effect of Strain Background.

Mouse strain background can influence vulnerability to excitotoxic neuronal cell death and potentially modulate phenotypes in transgenic mouse models of human disease. Evidence supports a contribution of excitotoxicity to the selective death of medium spiny neurons in Huntington's disease (HD). Here, we assess whether strain differences in excitotoxic vulnerability influence striatal cell death in a knock-in mouse model of HD. Previous studies that evaluated resistance to excitotoxic lesions in several mouse models of HD had variable outcomes. In the present study, we directly compare one model on two different background strains to test the contribution of strain to excitotoxicity-mediated neurodegeneration. Mice of the FVB/N strain, which are highly vulnerable to excitotoxicity, become extremely resistant to quinolinic acid-induced striatal neurodegeneration with age, when carrying a huntingtin (Htt) allele expressing a HD transgene (CAG140). The resistance is much greater than the age-dependent resistance that has been previously reported in YAC128 mice. By 12 months of age, both heterozygous and homozygous FVB.CAG140 mice displayed virtually complete resistance to quinolinic acid-induced striatal neurodegeneration. A similar resistance develops in CAG140 mice on a C57BL/6N background although the effect size is smaller because C57BL/6N mice are already resistant due to genetic background. In a direct comparison with the YAC128 mice, FVB.CAG140 mice have greater resistance. FVB.CAG140 mice are also resistant to neurodegeneration following kainic acid-induced status epilepticus suggesting the existence of a common cellular mechanism that provides protection against multiple types of excitotoxic insult. These findings establish FVB.CAG140 mice as a useful model to investigate the cellular and molecular mechanisms that confer neuroprotection against excitotoxicity.