A nutrient combination designed to enhance synapse formation and function improves outcome in experimental spinal cord injury.

Spinal cord injury leads to major neurological impairment for which there is currently no effective treatment. Recent clinical trials have demonstrated the efficacy of Fortasyn® Connect in Alzheimer's disease. Fortasyn® Connect is a specific multi-nutrient combination containing DHA, EPA, choline, uridine monophosphate, phospholipids, and various vitamins. We examined the effect of Fortasyn® Connect in a rat compression model of spinal cord injury. For 4 or 9 weeks following the injury, rats were fed either a control diet or a diet enriched with low, medium, or high doses of Fortasyn® Connect. The medium-dose Fortasyn® Connect-enriched diet showed significant efficacy in locomotor recovery after 9 weeks of supplementation, along with protection of spinal cord tissue (increased neuronal and oligodendrocyte survival, decreased microglial activation, and preserved axonal integrity). Rats fed the high-dose Fortasyn® Connect-enriched diet for 4 weeks showed a much greater enhancement of locomotor recovery, with a faster onset, than rats fed the medium dose. Bladder function recovered quicker in these rats than in rats fed the control diet. Their spinal cord tissues showed a smaller lesion, reduced neuronal and oligodendrocyte loss, decreased neuroinflammatory response, reduced astrocytosis and levels of inhibitory chondroitin sulphate proteoglycans, and better preservation of serotonergic axons than those of rats fed the control diet. These results suggest that this multi-nutrient preparation has a marked therapeutic potential in spinal cord injury, and raise the possibility that this original approach could be used to support spinal cord injured patients.

Distribution of pH changes in mouse neonatal hypoxic-ischaemic insult.

We assessed the distribution in brain pH after neonatal hypoxic-ischaemic insult and its correlation with local injury. Postnatal day 7 mice were injected with neutral red and underwent left carotid occlusion and exposure to 8% oxygen. Images captured from the cut surface of snap-frozen brain were used to calculate the pH from the blue-green absorbance ratios. Carotid occlusion alone had no effect, but combined with hypoxia caused rapid, biphasic pH decline, with the first plateau at 15-30 min, and the second at 60-90 min. The ipsilateral dorsal cortex, hippocampus, striatum and thalamus were most affected. Contralateral pH initially showed only 30% of the ipsilateral decline, becoming more acidotic with increasing duration. Systemic blood analysis revealed, compared with hypoxia alone, that combined insult caused a 63% decrease in blood glucose (1.3 ± 0.2 mM), a 2-fold increase in circulating lactate (17.7 ± 2.9 mM), a reduction in CO(2) to 1.9 ± 0.1 kPa and a drop in pH (7.26 ± 0.06). Re-oxygenation resulted in the normalisation of systemic changes, as well as a global alkaline rebound in brain pH at 4-6 h. A topographic comparison of brain injury showed only a partial correlation with pH changes, with the severest injury occurring in the ipsilateral hippocampus and sparing acidic parts of the contralateral cortex.

TNF gene cluster deletion abolishes lipopolysaccharide-mediated sensitization of the neonatal brain to hypoxic ischemic insult.

In the current study, we explored the role of TNF cluster cytokines on the lipopolysaccharide (LPS)-mediated, synergistic increase in brain injury after hypoxic ischemic insult in postnatal day 7 mice. Pretreatment with moderate doses of LPS (0.3 µg/g) resulted in particularly pronounced synergistic injury within 12 h. Systemic application of LPS alone resulted in a strong upregulation of inflammation-associated cytokines TNFα, LTß, interleukin (IL) 1ß, IL6, chemokines, such as CXCL1, and adhesion molecules E-Selectin, P-Selectin and intercellular adhesion molecule-1 (ICAM1), as well as a trend toward increased LTα levels in day 7 mouse forebrain. In addition, it was also associated with strong activation of brain blood vessel endothelia and local microglial cells. Here, deletion of the entire TNF gene cluster, removing TNFα, LTß and LTα completely abolished endotoxin-mediated increase in the volume of cerebral infarct. Interestingly, the same deletion also prevented endothelial and microglial activation following application of LPS alone, suggesting the involvement of these cell types in bringing about the LPS-mediated sensitization to neonatal brain injury.

Activation and deactivation of periventricular white matter phagocytes during postnatal mouse development.

Brain microglia are related to peripheral macrophages but undergo a highly specific process of regional maturation and differentiation inside the brain. Here, we examined this deactivation and morphological differentiation in cerebral cortex and periventricular subcortical white matter, the main "fountain of microglia" site, during postnatal mouse development, 0-28 days after birth (P0-P28). Only macrophages in subcortical white matter but not cortical microglia exhibited strong expression of typical activation markers alpha5, alpha6, alphaM, alphaX, and beta2 integrin subunits and B7.2 at any postnatal time point studied. White matter phagocyte activation was maximal at P0, decreased linearly over P3 and P7 and disappeared at P10. P7 white matter phagocytes also expressed high levels of IGF1 and MCSF, but not TNFalpha mRNA; this expression disappeared at P14. This process of deactivation followed the presence of ingested phagocytic material but correlated only moderately with ramification, and not with the extent of TUNEL+ death in neighboring cells, their ingestion or microglial proliferation. Intravenous fluosphere labeling revealed postnatal recruitment and transformation of circulating leukocytes into meningeal and perivascular macrophages as well as into ramified cortical microglia, but bypassing the white matter areas. In conclusion, this study describes strong and selective activation of postnatally resident phagocytes in the P0-P7 subcortical white matter, roughly equivalent to mid 3rd trimester human fetal development. This presence of highly active and IGF1- and MCSF-expressing phagocytes in the neighborhood of vulnerable white matter could play an important role in the genesis of or protection against axonal damage in the fetus and premature neonate.

Implantation of c-mycER TAM immortalized human mesencephalic-derived clonal cell lines ameliorates behavior dysfunction in a rat model of Parkinson's disease.

Human neural stem cells offer the hope that a cell therapy treatment for Parkinson's disease (PD) could be made widely available. In this study, we describe two clonal human neural cell lines, derived from two different 10-week-old fetal mesencephalic tissues and immortalized with the c-mycER(TAM) transgene. Under the growth control of 4-hydroxytamoxifen, both cell lines display stable long-term growth in culture with a normal karyotype. In vitro, these nestin-positive cells are able to differentiate into tyrosine hydroxylase (TH)-positive neurons and are multipotential. Implantation of the undifferentiated cells into the 6-OHDA substantia nigral lesioned rat model displayed sustained improvements in a number of behavioral tests compared with noncell-implanted, vehicle-injected controls over the course of 6 months. Histological analysis of the brains showed survival of the implanted cells but no evidence of differentiation into TH-positive neurons. An average increase of approximately 26% in host TH immunoreactivity in the lesioned dorsal striatum was observed in the cell-treated groups compared to controls, with no difference in loss of TH cell bodies in the lesioned substantia nigra. Further analysis of the cell lines identified a number of expressed trophic factors, providing a plausible explanation for the effects observed in vivo. The exact mechanisms by which the implanted human neural cell lines provide behavioral improvements in the PD model are not completely understood; however, these findings provide evidence that cell therapy can be a potent treatment for PD acting through a mechanism independent of dopaminergic neuronal cell replacement.

Short-term supplementation with plant extracts rich in flavonoids protect nigrostriatal dopaminergic neurons in a rat model of Parkinson's disease.

OBJECTIVE: Antioxidants from plants were known to reduce the oxidative stress by scavenging free radicals, chelating metal ions and reducing inflammation. As increased oxidative stress was implicated in the nigrostriatal dopaminergic neuronal loss in Parkinson's disease (PD), we have assessed whether the plant extracts protects the nigrostriatal dopaminergic neurons in the animal model of PD. METHODS: Male adult Sprague-Dawley rats were treated orally between 10 am-11 am each day with the extracts from tangerine peel, grape seeds, cocoa and red clover for four days. One hour after the final dosing, the left medial forebrain bundle was lesioned by infusing the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA; 12 microg) under anaesthesia. Seven days post-lesion, the number of dopaminergic cells in the substantia nigra pars compacta and the levels of dopamine and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striata were quantified and compared with the vehicle-treated groups. RESULTS: Compared to the unlesioned side, 6-OHDA lesions significantly reduced the number of dopaminergic cells and the levels of dopamine and its metabolites DOPAC and HVA in the vehicle-treated animals. Pretreatment of animals with extracts of tangerine peel (rich in polymethoxylated flavones; 35 mg/kg/day), cocoa-2 (rich in procyanidins; 100 mg/kg/day) and red clover (rich in isoflavones; 200 mg/kg/day) significantly attenuated the 6-OHDA-induced dopaminergic loss. However, no significant protection was seen in animals supplemented with red and white grape seeds (rich in catechins; 100 mg/kg/day), and cocoa-1 (rich in catechins; 100 mg/kg/day). CONCLUSIONS: Pre-treatment of plant extracts rich in polymethoxylated flavones, procyanidins and isoflavones but not catechins protected the nigrostriatal dopaminergic neurons in the rat model of PD.

Subtype selective antagonism of substantia nigra pars compacta Group I metabotropic glutamate receptors protects the nigrostriatal system against 6-hydroxydopamine toxicity in vivo.

Evidence suggests that increased glutamatergic input to the substantia nigra pars compacta as a result of hyperactivity of subthalalmic nucleus output pathways may contribute to the progressive degeneration of nigral dopaminergic neurones in Parkinson's disease (PD), a debilitating neurodegenerative disorder which affects approximately 1% of people aged over 65. Substantial electrophysiological evidence suggests that the excitation of nigral dopaminergic neurones is regulated by the activation of Group I metabotropic glutamate receptors (mGluR), comprising mGluR1 and mGluR5 subtypes. As activation of these receptors by endogenous glutamate may promote multiple cascades leading to excitotoxic neuronal death, it may be hypothesised that functional antagonism of Group I mGluR should be neuroprotective and could form the basis of a novel neuroprotective treatment for PD. To investigate this hypothesis, the neuroprotective potential of the selective competitive mGlu1 antagonist (+)-2-methyl-4-carboxyphenylglycine ((S)-(+)-alpha-amino-4-carboxy-2-methlybenzeneacetic acid; LY367385) and the selective allosteric mGlu5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) was tested in a rodent 6-hydroxydopamine (6-OHDA) model of PD in vivo. Both acute and subchronic intranigral administration of either LY367385 or MPEP resulted in significant neuroprotection of nigral tyrosine hydroxylase immunoreactive cell bodies, which correlated closely with prevention of striatal monoamine depletion following 6-OHDA lesioning. This neuroprotective action of LY367385 and MPEP displayed a clear concentration-dependent effect, suggesting a receptor-mediated mechanism of action. LY367385 produced robust neuroprotection at all concentrations tested (40, 200 and 1000 nmol in 4 microL), whilst MPEP displayed a bell-shaped neuroprotective profile with significant neuroprotection at low concentrations (2 and 10 nmol in 4 microL) but not at higher concentrations (50 nmol). Importantly, subchronic intranigral administration of MPEP and LY367385 appeared to slow the degeneration of remaining nigral dopaminergic neurones and prevented further striatal dopamine depletion in animals with established 6-OHDA induced nigrostriatal lesions, suggesting that these compounds may significantly influence disease progression in this model.

Selective activation of group III metabotropic glutamate receptors by L-(+)-2-amino-4-phosphonobutryic acid protects the nigrostriatal system against 6-hydroxydopamine toxicity in vivo.

Evidence from several studies suggests that the progressive degeneration of dopaminergic (DA) neurones of the substantia nigra pars compacta (SNc) in Parkinson's disease (PD) may in part be due to excessive release of glutamate from subthalamic projections onto nigral DA neurones. Previous in vitro studies have demonstrated that selective activation of Group III metabotropic glutamate receptors (mGluR) negatively modulates excitatory transmission in the SNc and is neuroprotective against glutamate-mediated toxicity. Consistent with this, we have reported preliminary data indicating that the selective group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) can also protect the nigrostriatal system against 6-hydroxydopamine (6-OHDA) toxicity in vivo. We have now extended these preliminary studies in this model and report here that both acute and subchronic intranigral injections of l-AP4 provide significant protection of the nigrostriatal system against 6-OHDA toxicity. This neuroprotection displays a bell-shaped profile with a clear concentration-dependent relationship. In contrast, when administered to animals 7 days post-6-OHDA lesioning, l-AP4 significantly protects the functionality but not the integrity of the nigrostriatal system. We further demonstrate that neuroprotection by l-AP4 in vivo is reversed by coadministration of the selective Group III mGluR antagonist (RS)-alpha-methylserine-O-phosphate, confirming a receptor-mediated mechanism of action. These data provide further compelling evidence that selective activation of Group III mGluR is neuroprotective in an in vivo experimental model of PD, a finding that may have important implications for the future treatment of this disease.

Assessment of the polyphenolic composition of the organic extracts of Mauritian black teas: a potential contributor to their antioxidant functions.

There is increasing interest in the emerging view that tea improves the antioxidant status in vivo and thereby helps to lower risk of certain types of cancer, coronary heart disease and stroke and its component biofactors could provide prophylactic potential for these diseases. The polyphenolic composition and the antioxidant properties of organic extracts (acetone/methanol) of Mauritian commercial black teas were evaluated. HPLC data of the individual compounds revealed remarkably high levels (+)-Catechin ((+)-C), (-)-epicatechin ((-)-EC), (-)-epicatechin 3-gallate ((-)-ECG), (-)-epigallocatechin ((-)-EGC), (-)-epigallocatechin 3-gallate ((-)-EGCG) and gallic acid. Analysis of hydrolysed extracts indicated that quercetin was the dominant flavonol aglycone with traces of myricetin and kaempferol. Based on the Ferric Reducing Antioxidant Power (FRAP) and the Trolox Equivalent Antioxidant Capacity (TEAC) assays Extra tea from Bois Chéri exhibited the highest antioxidant potential. Linear regression analyses showed that the antioxidant capacities of the organic extracts are strongly influenced by total phenols (TEAC: r=0.95 and FRAP: r=0.96) and to a lesser extent by total proanthocyanidin and total flavonoid contents. Catechins and gallic acid seem to add up to the overall antioxidant capacity of black tea extracts. The fresh tea leaves had high levels of total phenols, total flavonoids, total proanthocyanidin and exhibited greater antioxidant potential when compared with black teas. Organic extracts of endemic teas represent useful source of phenolic antioxidants supplements for prophylactic use.

Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson's disease.

Although the cause of dopaminergic cell death in Parkinson's disease (PD) remains unknown, oxidative stress has been strongly implicated. Because of their ability to combat oxidative stress, diet derived phenolic compounds continue to be considered as potential agents for long-term use in PD. This study was aimed at investigating whether the natural phenolic compounds curcumin, naringenin, quercetin, fisetin can be neuroprotective in the 6-OHDA model of PD. Unilateral infusion of 6-OHDA into the medial forebrain bundle produced a significant loss of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) as well as a decreased of dopamine (DA) content in the striata in the vehicle-treated animals. Rats pretreated with curcumin or naringenin showed a clear protection of the number of TH-positive cells in the SN and DA levels in the striata. However, neither pretreatment with quercetin nor fisetin had any effects on TH-positive cells or DA levels. The ability of curcumin and naringenin to exhibit neuroprotection in the 6-OHDA model of PD may be related to their antioxidant capabilities and their capability to penetrate into the brain.

Bioactivity of a peptide derived from acetylcholinesterase: involvement of an ivermectin-sensitive site on the alpha 7 nicotinic receptor.

A peptide fragment of 14 amino acids, derived from the C-terminus of acetylcholinesterase (AChE), might underlie the now well-established noncholinergic effects of the enzyme. This peptide is bioactive in a variety of systems including acute (brain slices) and chronic (organotypic culture) preparations of hippocampus, a pivotal area in Alzheimer's disease (AD); invariably, the action of the peptide is mediated specifically via an as yet unknown receptor. In this study, the allosteric alpha 7 agent, ivermectin (IVM), had a modest inhibitory effect, whilst that of the peptide was significantly more marked. However, ivermectin rendered ineffective the toxicity of high doses of the peptide, that is, when the two were co-applied, only the smaller effects of ivermectin were seen. Ivermectin, therefore, is presumably acting at a site that is identical to, or at least strongly interactive with, the normal binding site for AChE-peptide. This observation could have important implications for eventual therapeutic targeting of the action of AChE-peptide, in neurodegeneration.

Effects of orally administered levodopa on mesencephalic dopaminergic neurons undergoing a degenerative process.

Although the issue of in vivo levodopa toxicity appears to be settled by now in the light of recent findings, a crucial aspect was not accounted for the experiments designed to tackle that question. Levodopa could in fact be non-toxic on surviving dopamine neurons, but that could not be the case when the drug is administered at the same time those neurons are undergoing degeneration, which is what happens in the clinical setting. Dopaminergic neurons could in that situation be more vulnerable to levodopa's potential toxic action. Our aim was to determine if oral administration of levodopa is toxic for mesencephalic dopaminergic neurons that are actively involved in a degenerative process. We induced delayed retrograde degeneration of the nigrostriatal system in rats by injecting 6-hydroxydopamine (6-OHDA) intrastriatally. Treatment was started the day after the injection. Dopaminergic markers were histologically studied at the striatal and nigral levels, to determine degree of damage of the nigrostriatal dopaminergic system in levodopa- and vehicle-treated rats. No significant differences between levodopa or vehicle-treated rats were found in: (i) striatal immunoautoradiographic labeling for tyrosine hydroxylase (TH) and the membrane dopamine transporter (DAT); (ii) cell counts of TH-immunoreactive (TH-ir) neurons remaining in the substantia nigra and ventral tegmental area (VTA); (iii) surface area of remaining TH-immunoreactive neurons in the substantia nigra. The present experiments demonstrate that levodopa does not enhance delayed retrograde degeneration of dopaminergic neurons induced by intrastriatal administration of 6-OHDA.