In-Red 2022 - VIII Congreso Nacional de Innovación Educativa y Docencia en Red, Jul 6, 2022
The educational model has developed new transformations, including the introduction of new pedago... more The educational model has developed new transformations, including the introduction of new pedagogical methodologies such as the Flipped Classroom (FC). FC is a tool that provides greater autonomy for the student, as well as greater assimilation of knowledge. The main objective of the study is the application of FC in practice, for greater acquisition of basic skills in the application of knowledge, communication and interpretation of results in order to make judgements. The participants were students of nutrition and dietetics, and the practices were carried out in two ways; in one group the practices were taught with the traditional method methodology (TM) and in another group the application of FC. At the end of the practices, a questionnaire was carried out to evaluate the acquisition of knowledge and the degree of satisfaction. The results obtained showed a significant increase in the maximum number of correctly answered questions (FC) and a higher degree of satisfaction of the students who did the practicals using FC. As a conclusion, we could indicate that the application of FC in health sciences shows better results in the acquisition of knowledge than didactic lecture methodologies.
THE BENEFITS OF postmenopausal estrogen replacement therapy for protecting the brain against the ... more THE BENEFITS OF postmenopausal estrogen replacement therapy for protecting the brain against the neuronal death associated with neurological diseases and disorders remain controversial. Global ischemia, arising during cardiac arrest or surgery in humans or induced experimentally in animals, elicits selective, delayed neuronal death; pyramidal neurons of the hippocampal CA1 are particularly vulnerable (1, 2). The ischemia-induced loss of CA1 neurons is associated with cognitive deficits. 17�-Estradiol (E2), the primary estrogen produced and secreted by the ovaries, and other estrogens afford neuroprotection in experimental models of global and focal ischemia (3–9). Recent work from this laboratory demonstrates that E2 at levels considered physiological for postmenopausal women prevents the activation of apoptotic signaling cascades and ameliorates global ischemia-induced neuronal loss in male gerbils (7). However, the cellular targets that mediate the protective actions of E2 on hipp...
ex] ischemia I neuronal death I glutamate receptors I a-amino-3-hydroxy-5methyl-4-isoxazole-propi... more ex] ischemia I neuronal death I glutamate receptors I a-amino-3-hydroxy-5methyl-4-isoxazole-propionic acid receptors I ischemic tolerance Ml GlI Ischemic tolerance is a well-known phenomenon in which brief im ischemic episodes (ischemic preconditioning) afford robust pe protection to hippocampal CA1 pyramidal neurons against ral
The importance of postmenopausal estrogen replacement therapy in affording protection against the... more The importance of postmenopausal estrogen replacement therapy in affording protection against the selective and delayed neuronal death associated with cardiac arrest or cardiac surgery in women remains controversial. Here we report that exogenous estrogen at levels that are physiological for hormone replacement in postmenopausal women affords protection against global ischemia-induced neuronal death and prevents activation of apoptotic signaling cascades in the hippocampal CA1 of male gerbils. Global ischemia induced a marked increase in activated caspase-3 in CA1, evident at 6 hr after ischemia. Global ischemia induced a marked upregulation of the proapoptotic neurotrophin receptor p75NTRin CA1, evident at 48 hr. p75NTRexpression was induced primarily in terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling-positive cells, indicating expression in neurons undergoing apoptosis. Global ischemia also induced a marked downregulation of mRNA encoding the AMPA receptor Glu...
A subset of genes implicated in genetic and acquired neurological disorders encode proteins essen... more A subset of genes implicated in genetic and acquired neurological disorders encode proteins essential to neural patterning and neurogenesis. The gene silencing transcription factor neuronal repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF) plays a critical role in elaboration of the neuronal phenotype. In neural progenitor and non-neural cells, REST acts by repression of a subset of neural genes important to synaptic plasticity and synaptic remodeling, including the AMPA receptor (AMPAR) subunit GluR2. Here we show that global ischemia triggers REST mRNA and protein expression. REST suppresses GluR2 promoter activity and gene expression in neurons destined to die. Because the GluR2 subunit governs AMPAR Ca2+permeability, these changes are expected to have profound effects on neuronal survival. In keeping with this concept, acute knockdown of the REST gene by antisense administration prevents GluR2 suppression and rescues post-ischemi...
Regulation of neuronal NMDA receptors (NMDARs) by group I metabotropic glutamate receptors (mGluR... more Regulation of neuronal NMDA receptors (NMDARs) by group I metabotropic glutamate receptors (mGluRs) is known to play a critical role in synaptic transmission. The molecular mechanisms underlying mGluR1-mediated potentiation of NMDARs are as yet unclear. The present study shows that in Xenopus oocytes expressing recombinant receptors, activation of mGluR1 potentiates NMDA channel activity by recruitment of new channels to the plasma membrane via regulated exocytosis. Activation of mGluR1␣ induced (1) an increase in channel number times channel open probability, with no change in mean open time, unitary conductance, or reversal potential; (2) an increase in charge transfer in the presence of NMDA and the open channel blocker MK-801, indicating an increased number of functional NMDARs in the cell membrane; and (3) increased NR1 surface expression, as indicated by cell surface Western blots and immunofluorescence. Botulinum neurotoxin A or expression of a dominant negative mutant of synaptosomal associated protein of 25 kDa molelcular mass (SNAP-25) greatly reduced mGluR1␣-mediated potentiation, indicating that receptor trafficking occurs via a SNAP-25-mediated form of soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor-dependent exocytosis. Because group I mGluRs are localized to the perisynaptic region in juxtaposition to synaptic NMDARs at glutamatergic synapses in the hippocampus, mGluR-mediated insertion of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.
Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippoca... more Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cellspecific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbuminpositive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/Ϫ) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cellspecific patterns of global ischemia-induced neuronal death.
Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in ... more Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.
We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves t... more We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-β-estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-β-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERβ agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERβ (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 17, 2004
Apoptosis is an evolutionarily conserved process critical to tissue development and tissue homeos... more Apoptosis is an evolutionarily conserved process critical to tissue development and tissue homeostasis in eukaryotic organisms and, when dysregulated, causes inappropriate cell death. Global ischemia is a neuronal insult that induces delayed cell death with many features of apoptosis. Ischemic preconditioning affords robust protection of CA1 neurons against a subsequent severe ischemic challenge. The molecular mechanisms underlying ischemic tolerance are unclear. Here we show that ischemia induces pronounced caspase-3 activity in naive neurons that die and in preconditioned neurons that survive. Preconditioning intervenes downstream of proteolytic processing and activation of caspase-3 (a protease implicated in the execution of apoptosis) and upstream of the caspase-3 target caspase-activated DNase (CAD, a deoxyribonuclease that catalyzes DNA fragmentation) to arrest neuronal death. We further show that global ischemia promotes expression of the pro-survival inhibitor-of-apoptosis (...
In isolated goat middle cerebral artery segments, 5-hydroxytryptamine (5-HT, 10-8-3 x 10-5 M) cau... more In isolated goat middle cerebral artery segments, 5-hydroxytryptamine (5-HT, 10-8-3 x 10-5 M) caused concentration-dependent contractions, with ECs0 = 2.1 (1.9-2.5) x 10-7 M and Em~x = 60 + 2% of 50 mM KCl-induced contraction. 2. Mechanical removal of endotbelium significantly increased the Em,x (91 + 8%) and did not change the ECs0 value of 5-HT-elicited contractions. 3. Incubation of unrubbed arteries with the irreversible inhibitor of EDRF, gossypol (10-5 M), significantly increased the contractile response to 5-HT (E,~ = 77 + 4%). 4. Incubation of unrubbed arteries with the competitive inhibitor of the NO synthesis, N°-nitro-Larginine (L-NOARG) (10-5 M), significantly enhanced the arterial response to 5-HT (Em~ = 71 + 5%). The effects of L-NOARG were reversed by L-arginine (10-4 M) but not by D-arginine (10-4 M). 5. Pretreatment with the inhibitor of cyclooxygenase, indomethacin (10-5 M), significantly increased the response of unrubbed arteries to 5-HT, with an Em~of 69 + 3%. 6. These results suggest that endothelium modulates the constrictor effect of 5-HT in goat cerebral arteries by producing both EDRF, probably NO, and prostacyclin.
Maturation Phenomenon in Cerebral Ischemia V, 2004
Animals subjected to sublethal transient global ischemia (preconditioning) exhibit neuroprotectio... more Animals subjected to sublethal transient global ischemia (preconditioning) exhibit neuroprotection against subsequent global ischemia-induced neuronal death in the hippocampal CAI (ischemic tolerance). The molecular mechanisms underlying ischemic tolerance are, however, unclear
Ischemia is the condition or state in which a tissue such as brain is subjected to hypoxia or low... more Ischemia is the condition or state in which a tissue such as brain is subjected to hypoxia or low oxygen because of an obstruction of the arterial blood supply or inadequate blood flow. 1 Brain ischemia can be broadly divided into two main classifications, global ischemia and focal ischemia. Global ischemia is the condition or state in which blood flow to the entire brain is transiently blocked, resulting in delayed, selective neuronal death. Focal ischemia , or cerebral infarction, is the condition or state in which a specific area of brain tissue undergoes injury as a consequence of a temporary or permanent obstruction of local blood supply. Focal ischemia results in death of both neurons and non-neuronal cells in contiguous areas of brain, usually representing a single vascular territory. In this chapter, we present the current understanding of the molecular and cellular mechanisms of neuronal death associated with brain ischemia. Table 42.1 defines the abbreviations used in the text.
Transient global ischemia induces a delayed rise in intracellular Zn2+, which may be mediated via... more Transient global ischemia induces a delayed rise in intracellular Zn2+, which may be mediated via glutamate receptor 2 (GluR2)-lacking AMPA receptors (AMPARs), and selective, delayed death of hippocampal CA1 neurons. The molecular mechanisms underlying Zn2+toxicityin vivoare not well delineated. Here we show the striking finding that intraventricular injection of the high-affinity Zn2+chelator calcium EDTA (CaEDTA) at 30 min before ischemia (early CaEDTA) or at 48-60 hr (late CaEDTA), but not 3-6 hr, after ischemia, afforded robust protection of CA1 neurons in ∼50% (late CaEDTA) to 75% (early CaEDTA) of animals. We also show that Zn2+acts via temporally distinct mechanisms to promote neuronal death. Early CaEDTA attenuated ischemia-induced GluR2 mRNA and protein downregulation (and, by inference, formation of Zn2+-permeable AMPARs), the delayed rise in Zn2+, and neuronal death. These findings suggest that Zn2+acts at step(s) upstream from GluR2 gene downregulation and implicate Zn2+...
Proceedings of the National Academy of Sciences, 2002
Animals subjected to sublethal transient global ischemia (ischemic preconditioning) exhibit neuro... more Animals subjected to sublethal transient global ischemia (ischemic preconditioning) exhibit neuroprotection against subsequent global ischemia-induced neuronal death in the hippocampal CA1 (ischemic tolerance). The molecular mechanisms underlying ischemic tolerance are unclear. Here we report that ischemic preconditioning induced a small, transient down-regulation of GluR2 mRNA expression and greatly attenuated subsequent ischemia-induced GluR2 mRNA and protein down-regulation and neuronal death. Ischemic preconditioning and GluR2 antisense knockdown acted synergistically to increase cell death. Sublethal antisense knockdown did not protect against subsequent ischemic insults or antisense knockdown. These findings indicate that ischemic preconditioning acts at step(s) upstream from suppression of GluR2 gene expression to afford neuroprotection and implicate transcriptional regulation of GluR2 expression in the adaptive mechanisms associated with ischemic tolerance.
The role of endothelium-related factors in the pathogenesis of cerebral vasospasm after subarachn... more The role of endothelium-related factors in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) has gained interest since the discovery of endothelin-1 (ET-1). We have examined, before and after SAH, the responsiveness of the cerebrovascular bed of the goat to ET-1, the sources of Ca2+ in ET-1-induced responses, and the ability of the Ca2+ entry blocker nicardipine to counteract them. Before SAH, injection of ET-1 into the cerebral circulation increased cerebrovascular resistance, thereby producing dose-dependent reductions in cerebral blood flow (CBF), which were prevented by nicardipine. In isolated middle cerebral arteries, ET-1 induced concentration-dependent contractions, which were equally inhibited in Ca(2+)-free medium (without or with ethylene glycol tetraacetic acid) and by the Ca2+ entry blocker nicardipine. On the third day after SAH, CBF was reduced by 28% and cerebrovascular resistance increased by 39%. At the same time, both ET-1-induced reductions in CBF and the constricting effects of ET-1 in vitro were enhanced. The ability of nicardipine to increase CBF and to inhibit the effects of ET-1 was impaired as a result of reduced dependence of cerebral arteries on extracellular Ca2+. On the seventh day after SAH, CBF and cerebrovascular resistance returned to control values, and effects of ET-1 became normal. It is suggested that the hyperreactivity to ET-1 of the cerebrovascular bed induced by SAH could have a role in the development of vasospasm, which could reduce the vascular effects of Ca2+ entry blockers after SAH.
Endothelial dysfunction is claimed to play a role in the pathogenesis of delayed cerebral vasospa... more Endothelial dysfunction is claimed to play a role in the pathogenesis of delayed cerebral vasospasm after subarachnoid hemorrhage (SAH). We have examined the effect of experimental SAH on the modulatory action of endothelial and nonendothelial nitric oxide (NO) in the contractile response of goat middle cerebral artery to 5-hydroxytryptamine (5-HT). We compared the 5-HT-induced contractile responses of cerebral arteries from control goats and from goats with SAH that had been experimentally induced 3 days earlier by delivery of autologous arterial blood into the subarachnoid space. Contractile responses were examined by recording the isometric tension in isolated cerebral arteries. To assess the influence of endothelium, this cell layer was mechanically removed in some of the arteria, segments (rubbed arteries) from both control goats and goats with SAH. In arteries from control goats, contractile responses to 5-HT were significantly higher in rubbed arteries than in arteries with intact endothelium; 5-HT-induced contractions were significantly enhanced by a competitive inhibitor of NO synthesis, NG-nitro-l-arginine, in arteries both with and without endothelium. In arteries from goats with SAH, 5-HT contracted cerebral arteries without showing significant differences between segments with endothelium and those that had been rubbed; in both cases, 5-HT-induced contractions were significantly higher than those obtained in arteries from control goats. NG-Nitro-l-arginine significantly enhanced the contractile response to 5-HT of cerebral arteries from goats with SAH. These results suggest that cerebral arteries after SAH exhibit hyperreactivity to 5-HT via a mechanism that involves the absence of the modulatory role of endothelial NO, that SAH does not modify the modulatory role of nonendothelial NO, and that impairment of the modulatory action of endothelial NO on vascular responses to 5-HT could contribute to the pathogenesis of cerebral vasospasm after SAH.
The effects of MK-801 on the cerebral arteries and the possible involvement of the endothelium in... more The effects of MK-801 on the cerebral arteries and the possible involvement of the endothelium in such a response were examined using two experimental approaches: in vivo, by recording cerebral blood flow (CBF) in the unanesthetized goat, and in vitro, by recording isometric tension in goat and human cerebral arteries. Injection of increasing doses (3, 10, 30, and 100 micrograms) of MK-801 directly into the cerebroarterial supply elicited decreases in CBF and increases in cerebral vascular resistance (CVR; for the highest dose tested CBF decreased by 16 +/- 10% and CVR increased by 18 +/- 10%, p < 0.05). Administration of MK-801 as a single intravenous bolus (0.2 mg kg-1) reproduced that vasoconstrictor response (CBF decreased by 17 +/- 9% and CVR increased by 46 +/- 33%, p < 0.05), and it was followed by a phase of sustained tachycardia (26 +/- 15% increase in resting heart rate, p < 0.01) and hypertension (34 +/- 17% increase in resting mean arterial blood pressure, p < 0.05). In the in vitro experiments, addition of cumulative concentrations (10(-6) to 3 x 10(-4) M) of MK-801 elicited concentration-related contractions of goat and human cerebral arteries at both resting and active tone (10(-5) M prostaglandin F2 alpha).(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Cerebral Blood Flow & Metabolism, 2000
Lipid peroxidation and the cytotoxic by-product 4-hydroxynonenal (4-HNE) have been implicated in ... more Lipid peroxidation and the cytotoxic by-product 4-hydroxynonenal (4-HNE) have been implicated in neuronal perikaryal damage. This study sought to determine whether 4-HNE was involved in white matter damage in vivo and in vitro. Immunohistochemical studies detected an increase in cellular and axonal 4-HNE within the ischemic region in the rat after a 24-hour period of permanent middle cerebral artery occlusion. Exogenous 4-HNE (3.2 nmol) was stereotaxically injected into the subcortical white matter of rats that were killed 24 hours later. Damaged axons detected by accumulation of β-amyloid precursor protein (β-APP) were observed transversing medially and laterally away from the injection site after intracerebral injection of 4-HNE. In contrast, in the vehicle-treated animals, axonal damage was restricted to an area immediately surrounding the injection site. Exogenous 4-HNE produced oligodendrocyte cell death in culture in a time-dependent and a concentration-dependent manner. After...
In-Red 2022 - VIII Congreso Nacional de Innovación Educativa y Docencia en Red, Jul 6, 2022
The educational model has developed new transformations, including the introduction of new pedago... more The educational model has developed new transformations, including the introduction of new pedagogical methodologies such as the Flipped Classroom (FC). FC is a tool that provides greater autonomy for the student, as well as greater assimilation of knowledge. The main objective of the study is the application of FC in practice, for greater acquisition of basic skills in the application of knowledge, communication and interpretation of results in order to make judgements. The participants were students of nutrition and dietetics, and the practices were carried out in two ways; in one group the practices were taught with the traditional method methodology (TM) and in another group the application of FC. At the end of the practices, a questionnaire was carried out to evaluate the acquisition of knowledge and the degree of satisfaction. The results obtained showed a significant increase in the maximum number of correctly answered questions (FC) and a higher degree of satisfaction of the students who did the practicals using FC. As a conclusion, we could indicate that the application of FC in health sciences shows better results in the acquisition of knowledge than didactic lecture methodologies.
THE BENEFITS OF postmenopausal estrogen replacement therapy for protecting the brain against the ... more THE BENEFITS OF postmenopausal estrogen replacement therapy for protecting the brain against the neuronal death associated with neurological diseases and disorders remain controversial. Global ischemia, arising during cardiac arrest or surgery in humans or induced experimentally in animals, elicits selective, delayed neuronal death; pyramidal neurons of the hippocampal CA1 are particularly vulnerable (1, 2). The ischemia-induced loss of CA1 neurons is associated with cognitive deficits. 17�-Estradiol (E2), the primary estrogen produced and secreted by the ovaries, and other estrogens afford neuroprotection in experimental models of global and focal ischemia (3–9). Recent work from this laboratory demonstrates that E2 at levels considered physiological for postmenopausal women prevents the activation of apoptotic signaling cascades and ameliorates global ischemia-induced neuronal loss in male gerbils (7). However, the cellular targets that mediate the protective actions of E2 on hipp...
ex] ischemia I neuronal death I glutamate receptors I a-amino-3-hydroxy-5methyl-4-isoxazole-propi... more ex] ischemia I neuronal death I glutamate receptors I a-amino-3-hydroxy-5methyl-4-isoxazole-propionic acid receptors I ischemic tolerance Ml GlI Ischemic tolerance is a well-known phenomenon in which brief im ischemic episodes (ischemic preconditioning) afford robust pe protection to hippocampal CA1 pyramidal neurons against ral
The importance of postmenopausal estrogen replacement therapy in affording protection against the... more The importance of postmenopausal estrogen replacement therapy in affording protection against the selective and delayed neuronal death associated with cardiac arrest or cardiac surgery in women remains controversial. Here we report that exogenous estrogen at levels that are physiological for hormone replacement in postmenopausal women affords protection against global ischemia-induced neuronal death and prevents activation of apoptotic signaling cascades in the hippocampal CA1 of male gerbils. Global ischemia induced a marked increase in activated caspase-3 in CA1, evident at 6 hr after ischemia. Global ischemia induced a marked upregulation of the proapoptotic neurotrophin receptor p75NTRin CA1, evident at 48 hr. p75NTRexpression was induced primarily in terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling-positive cells, indicating expression in neurons undergoing apoptosis. Global ischemia also induced a marked downregulation of mRNA encoding the AMPA receptor Glu...
A subset of genes implicated in genetic and acquired neurological disorders encode proteins essen... more A subset of genes implicated in genetic and acquired neurological disorders encode proteins essential to neural patterning and neurogenesis. The gene silencing transcription factor neuronal repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF) plays a critical role in elaboration of the neuronal phenotype. In neural progenitor and non-neural cells, REST acts by repression of a subset of neural genes important to synaptic plasticity and synaptic remodeling, including the AMPA receptor (AMPAR) subunit GluR2. Here we show that global ischemia triggers REST mRNA and protein expression. REST suppresses GluR2 promoter activity and gene expression in neurons destined to die. Because the GluR2 subunit governs AMPAR Ca2+permeability, these changes are expected to have profound effects on neuronal survival. In keeping with this concept, acute knockdown of the REST gene by antisense administration prevents GluR2 suppression and rescues post-ischemi...
Regulation of neuronal NMDA receptors (NMDARs) by group I metabotropic glutamate receptors (mGluR... more Regulation of neuronal NMDA receptors (NMDARs) by group I metabotropic glutamate receptors (mGluRs) is known to play a critical role in synaptic transmission. The molecular mechanisms underlying mGluR1-mediated potentiation of NMDARs are as yet unclear. The present study shows that in Xenopus oocytes expressing recombinant receptors, activation of mGluR1 potentiates NMDA channel activity by recruitment of new channels to the plasma membrane via regulated exocytosis. Activation of mGluR1␣ induced (1) an increase in channel number times channel open probability, with no change in mean open time, unitary conductance, or reversal potential; (2) an increase in charge transfer in the presence of NMDA and the open channel blocker MK-801, indicating an increased number of functional NMDARs in the cell membrane; and (3) increased NR1 surface expression, as indicated by cell surface Western blots and immunofluorescence. Botulinum neurotoxin A or expression of a dominant negative mutant of synaptosomal associated protein of 25 kDa molelcular mass (SNAP-25) greatly reduced mGluR1␣-mediated potentiation, indicating that receptor trafficking occurs via a SNAP-25-mediated form of soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor-dependent exocytosis. Because group I mGluRs are localized to the perisynaptic region in juxtaposition to synaptic NMDARs at glutamatergic synapses in the hippocampus, mGluR-mediated insertion of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.
Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippoca... more Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cellspecific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbuminpositive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/Ϫ) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cellspecific patterns of global ischemia-induced neuronal death.
Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in ... more Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.
We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves t... more We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-β-estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-β-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERβ agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERβ (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 17, 2004
Apoptosis is an evolutionarily conserved process critical to tissue development and tissue homeos... more Apoptosis is an evolutionarily conserved process critical to tissue development and tissue homeostasis in eukaryotic organisms and, when dysregulated, causes inappropriate cell death. Global ischemia is a neuronal insult that induces delayed cell death with many features of apoptosis. Ischemic preconditioning affords robust protection of CA1 neurons against a subsequent severe ischemic challenge. The molecular mechanisms underlying ischemic tolerance are unclear. Here we show that ischemia induces pronounced caspase-3 activity in naive neurons that die and in preconditioned neurons that survive. Preconditioning intervenes downstream of proteolytic processing and activation of caspase-3 (a protease implicated in the execution of apoptosis) and upstream of the caspase-3 target caspase-activated DNase (CAD, a deoxyribonuclease that catalyzes DNA fragmentation) to arrest neuronal death. We further show that global ischemia promotes expression of the pro-survival inhibitor-of-apoptosis (...
In isolated goat middle cerebral artery segments, 5-hydroxytryptamine (5-HT, 10-8-3 x 10-5 M) cau... more In isolated goat middle cerebral artery segments, 5-hydroxytryptamine (5-HT, 10-8-3 x 10-5 M) caused concentration-dependent contractions, with ECs0 = 2.1 (1.9-2.5) x 10-7 M and Em~x = 60 + 2% of 50 mM KCl-induced contraction. 2. Mechanical removal of endotbelium significantly increased the Em,x (91 + 8%) and did not change the ECs0 value of 5-HT-elicited contractions. 3. Incubation of unrubbed arteries with the irreversible inhibitor of EDRF, gossypol (10-5 M), significantly increased the contractile response to 5-HT (E,~ = 77 + 4%). 4. Incubation of unrubbed arteries with the competitive inhibitor of the NO synthesis, N°-nitro-Larginine (L-NOARG) (10-5 M), significantly enhanced the arterial response to 5-HT (Em~ = 71 + 5%). The effects of L-NOARG were reversed by L-arginine (10-4 M) but not by D-arginine (10-4 M). 5. Pretreatment with the inhibitor of cyclooxygenase, indomethacin (10-5 M), significantly increased the response of unrubbed arteries to 5-HT, with an Em~of 69 + 3%. 6. These results suggest that endothelium modulates the constrictor effect of 5-HT in goat cerebral arteries by producing both EDRF, probably NO, and prostacyclin.
Maturation Phenomenon in Cerebral Ischemia V, 2004
Animals subjected to sublethal transient global ischemia (preconditioning) exhibit neuroprotectio... more Animals subjected to sublethal transient global ischemia (preconditioning) exhibit neuroprotection against subsequent global ischemia-induced neuronal death in the hippocampal CAI (ischemic tolerance). The molecular mechanisms underlying ischemic tolerance are, however, unclear
Ischemia is the condition or state in which a tissue such as brain is subjected to hypoxia or low... more Ischemia is the condition or state in which a tissue such as brain is subjected to hypoxia or low oxygen because of an obstruction of the arterial blood supply or inadequate blood flow. 1 Brain ischemia can be broadly divided into two main classifications, global ischemia and focal ischemia. Global ischemia is the condition or state in which blood flow to the entire brain is transiently blocked, resulting in delayed, selective neuronal death. Focal ischemia , or cerebral infarction, is the condition or state in which a specific area of brain tissue undergoes injury as a consequence of a temporary or permanent obstruction of local blood supply. Focal ischemia results in death of both neurons and non-neuronal cells in contiguous areas of brain, usually representing a single vascular territory. In this chapter, we present the current understanding of the molecular and cellular mechanisms of neuronal death associated with brain ischemia. Table 42.1 defines the abbreviations used in the text.
Transient global ischemia induces a delayed rise in intracellular Zn2+, which may be mediated via... more Transient global ischemia induces a delayed rise in intracellular Zn2+, which may be mediated via glutamate receptor 2 (GluR2)-lacking AMPA receptors (AMPARs), and selective, delayed death of hippocampal CA1 neurons. The molecular mechanisms underlying Zn2+toxicityin vivoare not well delineated. Here we show the striking finding that intraventricular injection of the high-affinity Zn2+chelator calcium EDTA (CaEDTA) at 30 min before ischemia (early CaEDTA) or at 48-60 hr (late CaEDTA), but not 3-6 hr, after ischemia, afforded robust protection of CA1 neurons in ∼50% (late CaEDTA) to 75% (early CaEDTA) of animals. We also show that Zn2+acts via temporally distinct mechanisms to promote neuronal death. Early CaEDTA attenuated ischemia-induced GluR2 mRNA and protein downregulation (and, by inference, formation of Zn2+-permeable AMPARs), the delayed rise in Zn2+, and neuronal death. These findings suggest that Zn2+acts at step(s) upstream from GluR2 gene downregulation and implicate Zn2+...
Proceedings of the National Academy of Sciences, 2002
Animals subjected to sublethal transient global ischemia (ischemic preconditioning) exhibit neuro... more Animals subjected to sublethal transient global ischemia (ischemic preconditioning) exhibit neuroprotection against subsequent global ischemia-induced neuronal death in the hippocampal CA1 (ischemic tolerance). The molecular mechanisms underlying ischemic tolerance are unclear. Here we report that ischemic preconditioning induced a small, transient down-regulation of GluR2 mRNA expression and greatly attenuated subsequent ischemia-induced GluR2 mRNA and protein down-regulation and neuronal death. Ischemic preconditioning and GluR2 antisense knockdown acted synergistically to increase cell death. Sublethal antisense knockdown did not protect against subsequent ischemic insults or antisense knockdown. These findings indicate that ischemic preconditioning acts at step(s) upstream from suppression of GluR2 gene expression to afford neuroprotection and implicate transcriptional regulation of GluR2 expression in the adaptive mechanisms associated with ischemic tolerance.
The role of endothelium-related factors in the pathogenesis of cerebral vasospasm after subarachn... more The role of endothelium-related factors in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) has gained interest since the discovery of endothelin-1 (ET-1). We have examined, before and after SAH, the responsiveness of the cerebrovascular bed of the goat to ET-1, the sources of Ca2+ in ET-1-induced responses, and the ability of the Ca2+ entry blocker nicardipine to counteract them. Before SAH, injection of ET-1 into the cerebral circulation increased cerebrovascular resistance, thereby producing dose-dependent reductions in cerebral blood flow (CBF), which were prevented by nicardipine. In isolated middle cerebral arteries, ET-1 induced concentration-dependent contractions, which were equally inhibited in Ca(2+)-free medium (without or with ethylene glycol tetraacetic acid) and by the Ca2+ entry blocker nicardipine. On the third day after SAH, CBF was reduced by 28% and cerebrovascular resistance increased by 39%. At the same time, both ET-1-induced reductions in CBF and the constricting effects of ET-1 in vitro were enhanced. The ability of nicardipine to increase CBF and to inhibit the effects of ET-1 was impaired as a result of reduced dependence of cerebral arteries on extracellular Ca2+. On the seventh day after SAH, CBF and cerebrovascular resistance returned to control values, and effects of ET-1 became normal. It is suggested that the hyperreactivity to ET-1 of the cerebrovascular bed induced by SAH could have a role in the development of vasospasm, which could reduce the vascular effects of Ca2+ entry blockers after SAH.
Endothelial dysfunction is claimed to play a role in the pathogenesis of delayed cerebral vasospa... more Endothelial dysfunction is claimed to play a role in the pathogenesis of delayed cerebral vasospasm after subarachnoid hemorrhage (SAH). We have examined the effect of experimental SAH on the modulatory action of endothelial and nonendothelial nitric oxide (NO) in the contractile response of goat middle cerebral artery to 5-hydroxytryptamine (5-HT). We compared the 5-HT-induced contractile responses of cerebral arteries from control goats and from goats with SAH that had been experimentally induced 3 days earlier by delivery of autologous arterial blood into the subarachnoid space. Contractile responses were examined by recording the isometric tension in isolated cerebral arteries. To assess the influence of endothelium, this cell layer was mechanically removed in some of the arteria, segments (rubbed arteries) from both control goats and goats with SAH. In arteries from control goats, contractile responses to 5-HT were significantly higher in rubbed arteries than in arteries with intact endothelium; 5-HT-induced contractions were significantly enhanced by a competitive inhibitor of NO synthesis, NG-nitro-l-arginine, in arteries both with and without endothelium. In arteries from goats with SAH, 5-HT contracted cerebral arteries without showing significant differences between segments with endothelium and those that had been rubbed; in both cases, 5-HT-induced contractions were significantly higher than those obtained in arteries from control goats. NG-Nitro-l-arginine significantly enhanced the contractile response to 5-HT of cerebral arteries from goats with SAH. These results suggest that cerebral arteries after SAH exhibit hyperreactivity to 5-HT via a mechanism that involves the absence of the modulatory role of endothelial NO, that SAH does not modify the modulatory role of nonendothelial NO, and that impairment of the modulatory action of endothelial NO on vascular responses to 5-HT could contribute to the pathogenesis of cerebral vasospasm after SAH.
The effects of MK-801 on the cerebral arteries and the possible involvement of the endothelium in... more The effects of MK-801 on the cerebral arteries and the possible involvement of the endothelium in such a response were examined using two experimental approaches: in vivo, by recording cerebral blood flow (CBF) in the unanesthetized goat, and in vitro, by recording isometric tension in goat and human cerebral arteries. Injection of increasing doses (3, 10, 30, and 100 micrograms) of MK-801 directly into the cerebroarterial supply elicited decreases in CBF and increases in cerebral vascular resistance (CVR; for the highest dose tested CBF decreased by 16 +/- 10% and CVR increased by 18 +/- 10%, p < 0.05). Administration of MK-801 as a single intravenous bolus (0.2 mg kg-1) reproduced that vasoconstrictor response (CBF decreased by 17 +/- 9% and CVR increased by 46 +/- 33%, p < 0.05), and it was followed by a phase of sustained tachycardia (26 +/- 15% increase in resting heart rate, p < 0.01) and hypertension (34 +/- 17% increase in resting mean arterial blood pressure, p < 0.05). In the in vitro experiments, addition of cumulative concentrations (10(-6) to 3 x 10(-4) M) of MK-801 elicited concentration-related contractions of goat and human cerebral arteries at both resting and active tone (10(-5) M prostaglandin F2 alpha).(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Cerebral Blood Flow & Metabolism, 2000
Lipid peroxidation and the cytotoxic by-product 4-hydroxynonenal (4-HNE) have been implicated in ... more Lipid peroxidation and the cytotoxic by-product 4-hydroxynonenal (4-HNE) have been implicated in neuronal perikaryal damage. This study sought to determine whether 4-HNE was involved in white matter damage in vivo and in vitro. Immunohistochemical studies detected an increase in cellular and axonal 4-HNE within the ischemic region in the rat after a 24-hour period of permanent middle cerebral artery occlusion. Exogenous 4-HNE (3.2 nmol) was stereotaxically injected into the subcortical white matter of rats that were killed 24 hours later. Damaged axons detected by accumulation of β-amyloid precursor protein (β-APP) were observed transversing medially and laterally away from the injection site after intracerebral injection of 4-HNE. In contrast, in the vehicle-treated animals, axonal damage was restricted to an area immediately surrounding the injection site. Exogenous 4-HNE produced oligodendrocyte cell death in culture in a time-dependent and a concentration-dependent manner. After...
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Papers by Teresa Jover