Núria de Vera

Changes in polyamine levels in rat brain after systemic kainic acid administration: relationship to convulsant activity and brain damage

Abstract: We have examined the effects of systemic kainic acid (KA) administration (9 mg/kg, i.p.) on rat behavior, brain damage, and polyamine levels and the action of the specific ornithine decarboxylase inhibitor α‐difluoromethylornithine (DFMO) on these effects. KA elicited convulsant activity in 63% of the animals. In the acute convulsant phase (1–3 h after KA), a rapid decline (−39% at 3 h) of spermidine content in frontal cortex was found. After the acute convulsant phase, levels of hippocampal spermidine and spermine were reduced (−70 and −66%, respectively, at 8 h). A dramatic increase of putrescine content (681, 1,382, and 336% at 8h, 24h, and 9 days, respectively, after KA) was found, associated with histological signs of cortical brain damage (ischemia and necrosis). There was a close relationship between the concentration of putrescine and signs of delayed toxicity (body weight losses) 24 h and 9 days after KA. DFMO partially antagonized the convulsant activity and reduced the increased putrescine levels to ∼50% of values in KA‐treated animals at 24 h but did not change the pattern of histological damage. The role of polyamines in the early and late phases of KA‐induced neurotoxicity is discussed.

Seizures and neuronal damage induced in the rat by activation of group I metabotropic glutamate receptors with their selective agonist 3,5-dihydroxyphenylglycine.

While it is well documented that the overactivation of ionotropic glutamate receptors leads to seizures and excitotoxic injury, little is known about the role of metabotropic glutamate receptors (mGluRs) in epileptogenesis and neuronal injury. Intracerebroventricular (i.c.v.) infusion of the group I mGluR specific agonist (R,S)‐3,5‐dihydroxyphenylglycine (3,5‐DHPG) (1.5 μmol) to conscious rats produced severe and delayed seizures (onset at 4 hr) in 70% of the animals. The i.c.v. infusion of the group I mGluR non‐selective agonist 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) (2 μmol) produced a similar rate of severe seizures, but with an early onset (0.6 hr). The analysis of motor activity showed that 3,5‐DHPG elicited higher central stimulatory action than did 1S,3R‐ACPD. Histopathological analysis of the hippocampus showed that 3,5‐DHPG produced severe neuronal damage mainly in the CA1 pyramidal neurons and, to a lesser extent, in the CA3. Although 1S,3R‐ACPD infusion also induced a slight injury of the CA1 and CA3 pyramidal neurons, damage was greater in the CA4 and dentate gyrus cells. In conclusion, the in vivo activation of group I mGluRs with the selective agonist 3,5‐DHPG produces hyperexcitatory effects that lead to seizures and neuronal damage, these effects being more severe than those observed after infusion of the non‐selective agonist 1S,3R‐ACPD. J. Neurosci. Res. 51:339–348, 1998. © 1998 Wiley‐Liss, Inc.

Modified C-reactive protein is expressed by stroke neovessels and is a potent activator of angiogenesis In Vitro

Native C‐reactive protein (nCRP) is a pentameric oligo‐protein and an acute phase reactant whose serum expression is increased in patients with inflammatory disease. We have identified by immunohistochemistry, significant expression of a tissue‐binding insoluble modified version or monomeric form of CRP (mCRP) associated with angiogenic microvessels in peri‐infarcted regions of patients studied with acute ischaemic stroke. mCRP, but not nCRP was expressed in the cytoplasm and nucleus of damaged neurons. mCRP co‐localized with CD105, a marker of angiogenesis in regions of revascularisation. In vitro investigations demonstrated that mCRP was preferentially expressed in human brain microvessel endothelial cells following oxygen‐glucose deprivation and mCRP (but not column purified nCRP) associated with the endothelial cell surface, and was angiogenic to vascular endothelial cells, stimulating migration and tube formation in matrigel more strongly than fibroblast growth factor‐2. The mechanism of signal transduction was not through the CD16 receptor. Western blotting showed that mCRP stimulated phosphorylation of the key down‐stream mitogenic signalling protein ERK1/2. Pharmacological inhibition of ERK1/2 phosphorylation blocked the angiogenic effects of mCRP. We propose that mCRP may contribute to the neovascularization process and because of its abundant presence, be important in modulating angiogenesis in both acute stroke and later during neuro‐recovery.

In Vitro and In Vivo Activation of Astrocytes by Amyloid-β is Potentiated by Pro-Oxidant Agents

Alzheimers disease (AD) is a devastating age-related neurodegenerative disease. Age is the main risk factor for sporadic AD, which is the most prevalent type. Amyloid-beta peptide (Abeta) neurotoxicity is the proposed first step in a cascade of deleterious events leading to AD pathology and dementia. Glial cells play an important role in these changes. Astrocytes provide vital support to neurons and modulate functional synapses. Therefore, the toxic effects of Abeta on astrocytes might promote neurodegenerative changes that lead to AD. Aging reduces astrocyte antioxidant defenses and induces oxidative stress. We studied the effects of Abeta(42) on cultures of human astrocytes in the presence or absence of the following pro-oxidant agents: buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, and FeSO(4), which liberates redox active iron. Pro-oxidant conditions potentiated Abeta toxicity, as shown by the generation of free radicals, inflammatory changes, and apoptosis. Similar treatments were assessed in rats in vivo. A combination of Abeta(40) and Abeta(42) or Abeta(42) alone was infused intracerebroventricularly for 4 weeks. Other animal groups were also infused with BSO and FeSO(4). A long-term analysis that ended 4 months later showed greater cognitive impairment in the Morris water maze task, which was induced by Abeta plus pro-oxidant agent treatments. Pro-oxidant agents also potentiated brain tissue pathology. This was demonstrated in histological studies that showed highly increased astrocyte reactivity in AD-vulnerable areas, Abeta deposits, and oxidative damage of AD-sensitive hippocampal neurons. To increase our understanding of AD, experimental models should be used that mimic age-related brain changes, in which age-related oxidative stress potentiates the effects of Abeta.

Polyamines in the basal ganglia of human brain. Influence of aging and degenerative movement disorders.

The distribution of polyamines in the human basal ganglia was examined, using dansyl-derivatives and high pressure liquid chromatography with fluorimetric detection. A heterogeneous distribution of putrescine, spermidine (SD) and spermine (SM) was observed in control brains. A consistent negative correlation between SD and SM content and age was found in different brain areas. These results suggest an involvement of polyamines in age-related changes occurring in white-matter. When the influence of degenerative movement disorders -Parkinsons disease, Huntingtons disease (HD) and progressive supranuclear palsy- was analyzed, significant changes were observed only in HD, where a decrease in the concentration of SM was found in the putamen. These results suggest that in advanced stages of neurodegenerative processes, polyamines maintain their regulation. Only in the presence of severe atrophy, SM concentration is reduced.

A complementary diffusion tensor imaging (DTI)-histological study in a model of Huntington's disease

In vivo diffusion tensor imaging (DTI) was performed on the quinolinic acid (QUIN) rat model of Huntingtons disease, together with behavioral assessment of motor deficits and histopathological characterization. DTI and histology revealed the presence of a cortical lesion in 53% of the QUIN animals (QUIN(+ctx)). Histologically, QUIN(+ctx) were distinguished from QUIN(-ctx) animals by increased astroglial reaction within a subregion of the caudate putamen and loss of white matter in the external capsula. Although both techniques are complementary, the quantitative character of DTI makes it possible to pick up subtle differences in tissue microstructure that are not identified with histology. DTI demonstrated differential changes of fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) in the internal and external capsula, and within a subregion of the caudate putamen. It was suggested that FA increased due to a selective loss of the subcortical connections targeted by degenerative processes at the early stage of the disease, which might turn the striatum into a seemingly more organized structure. When tissue degeneration becomes more severe, FA decreased while AD, RD and MD increased.

Identification of pro-angiogenic markers in blood vessels from stroked-affected brain tissue using laser-capture microdissection

BackgroundAngiogenesis correlates with patient survival following acute ischaemic stroke, and survival of neurons is greatest in tissue undergoing angiogenesis. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion, enhanced neuronal survival and improved recovery.ResultsHere, we have isolated active (CD105/Flt-1 positive) and inactive (CD105/Flt-1 minus (n=5) micro-vessel rich-regions from stroke-affected and contralateral tissue of patients using laser-capture micro-dissection. Areas were compared for pro- and anti-angiogenic gene expression using targeted TaqMan microfluidity cards containing 46 genes and real-time PCR. Further analysis of key gene de-regulation was performed by immunohistochemistry to define localization and expression patterns of identified markers and de novo synthesis by human brain microvessel endothelial cells (HBMEC) was examined following oxygen-glucose deprivation (OGD). Our data revealed that seven pro-angiogenic genes were notably up-regulated in CD105 positive microvessel rich regions. These were, beta-catenin, neural cell adhesion molecule (NRCAM), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hepatocyte growth factor-alpha (HGF-alpha), monocyte chemottractant protein-1 (MCP-1) and and Tie-2 as well as c-kit. Immunohistochemistry demonstrated strong staining of MMP-2, HGF-alpha, MCP-1 and Tie-2 in stroke-associated regions of active remodeling in association with CD105 positive staining. In vitro, OGD stimulated production of Tie-2, MCP-1 and MMP-2 in HBMEC, demonstrated a de novo response to hypoxia.ConclusionIn this work we have identified concurrent activation of key angiogenic molecules associated with endothelial cell migration, differentiation and tube-formation, vessel stabilization and stem cell homing mechanisms in areas of revascularization. Therapeutic stimulation of these processes in all areas of damaged tissue might improve morbidity and mortality from stroke.

Spermine induces cell death in cultured human embryonic cerebral cortical neurons through N-methyl-D-aspartate receptor activation

The polyamines putrescine, spermidine, and spermine play important roles in cell proliferation, differentiation, and modulation of ion channel receptors. However, the function of increased concentrations of these compounds in brain injury and disease is unclear, in that they have been proposed as being both neuroprotective and neurotoxic. The effects of spermine and putrescine were studied in human primary cerebral cortical cultures containing both neurons and glia. No toxic effects were induced at 8 days in vitro (DIV) by either of the two polyamines at concentrations ranging from 0.3 μM to 2 mM. However, when the oxidative metabolism of spermine that generates toxic byproducts was induced by the presence of fetal calf serum, spermine caused cellular death with an LC50 of approximately 50 μM. At 14 DIV, the coapplication of spermine 2 mM and glutamate 5 mM induced neuron cell death, but the effect of applying both components separately was null. Both spermine and glutamate were toxic to older neurons (26–42 DIV cultures), and here the coapplication of glutamate was found always to intensify the effect of spermine. Spermine showed greater toxicity than glutamate in neurons. Another effect observed is that glutamate, but not spermine, induced astrocyte swelling. Spermine toxicity was inhibited by both MK801 and ifenprodil, indicating a mechanism involving N‐methyl‐D‐aspartate (NMDA) receptor activation. Moreover, a strong spermine modulation of the NMDA receptor was demonstrated by the inhibition of glutamate toxicity by ifenprodil. Putrescine induced minor effects also as a neurotoxic agent. In conclusion, neuronal death by spermine can be induced by its toxic byproducts as well as through NMDA receptor action. The present results confirm the potentially harmful role of the polyamines in excitotoxicity‐related human disorders.

Silent information regulator 1 protects the liver against ischemia-reperfusion injury: Implications in steatotic liver ischemic preconditioning

Ischemia–reperfusion (IR) injury is an important problem in liver surgery especially when steatosis is present. Ischemic preconditioning (PC) is the only surgical strategy that has been applied in patients with steatotic livers undergoing warm ischemia. Silent information regulator 1 (SIRT1) is a histone deacetylase that regulates various cellular processes. This study evaluates the SIRT1 implication in PC in fatty livers. Homozygous (Ob) Zucker rats were subjected to IR and IR + PC. An additional group treated with sirtinol or EX527 (SIRT1 inhibitors) before PC was also realized. Liver injury and oxidative stress were evaluated. SIRT1 protein levels and activity, as well as other parameters involved in PC protective mechanisms (adenosine monophosphate protein kinase, eNOS, HSP70, MAP kinases, apoptosis), were also measured. We demonstrated that the protective effect of PC was due in part to SIRT1 induction, as SIRT1 inhibition resulted in increased liver injury and abolished the beneficial mechanisms of PC. In this study, we report for the first time that SIRT1 is involved in the protective mechanisms induced by hepatic PC in steatotic livers.

RGD-based cell ligands for cell-targeted drug delivery act as potent trophic factors.

UNLABELLED Integrin-binding, Arg-Gly-Asp (RGD)-containing peptides are the most widely used agents to deliver drugs, nanoparticles, and imaging agents. Although in nature, several protein-mediated signal transduction events depend on RGD motifs, the potential of RGD-empowered materials in triggering undesired cell-signaling cascades has been neglected. Using an RGD-functionalized protein nanoparticle, we show here that the RGD motif acts as a powerful trophic factor, supporting extracellular signal-regulated kinase 1/2 (ERK1/2)-linked cell proliferation and partial differentiation of PC12 cells, a neuronlike model. FROM THE CLINICAL EDITOR This work focuses on RGD peptides, which are among the most commonly used tags for targeted drug delivery. They also promote protoneurite formation and expression of neuronal markers (MAP2) in model PC12 cells, which is an unexpected but relevant event in the functionalization of drugs and their nanocarriers.