RESUMO
Novel digital endpoints gathered via wearables, small devices, or algorithms hold great promise for clinical trials. However, implementation has been slow because of a lack of guidelines regarding the validation process of these new measurements. In this paper, we propose a pragmatic approach toward selection and fit-for-purpose validation of digital endpoints. Measurements should be value-based, meaning the measurements should directly measure or be associated with meaningful outcomes for patients. Devices should be assessed regarding technological validity. Most importantly, a rigorous clinical validation process should appraise the tolerability, difference between patients and controls, repeatability, detection of clinical events, and correlation with traditional endpoints. When technically and clinically fit-for-purpose, case building in interventional clinical trials starts to generate evidence regarding the response to new or existing health-care interventions. This process may lead to the digital endpoint replacing traditional endpoints, such as clinical rating scales or questionnaires in clinical trials. We recommend initiating more data-sharing collaborations to prevent unnecessary duplication of research and integration of value-based measurements in clinical care to enhance acceptance by health-care professionals. Finally, we invite researchers and regulators to adopt this approach to ensure a timely implementation of digital measurements and value-based thinking in clinical trial design and health care. SIGNIFICANCE STATEMENT: Novel digital endpoints are often cited as promising for the clinical trial of the future. However, clear validation guidelines are lacking in the literature. This paper contains pragmatic criteria for the selection, technical validation, and clinical validation of novel digital endpoints and provides recommendations for future work and collaboration.
Assuntos
Ensaios Clínicos como Assunto/métodos , Biomarcadores/análise , Biomarcadores/metabolismo , Determinação de Ponto Final/métodos , Humanos , Reprodutibilidade dos TestesRESUMO
The phosphodiesterases (PDEs) are a superfamily of enzymes that regulate spatio-temporal signaling by the intracellular second messengers cAMP and cGMP. PDE2A is expressed at high levels in the mammalian brain. To advance our understanding of the role of this enzyme in regulation of neuronal signaling, we here describe the distribution of PDE2A in the rat brain. PDE2A mRNA was prominently expressed in glutamatergic pyramidal cells in cortex, and in pyramidal and dentate granule cells in the hippocampus. Protein concentrated in the axons and nerve terminals of these neurons; staining was markedly weaker in the cell bodies and proximal dendrites. In addition, in both hippocampus and cortex, small populations of non-pyramidal cells, presumed to be interneurons, were strongly immunoreactive. PDE2A mRNA was expressed in medium spiny neurons in neostriatum. Little immunoreactivity was observed in cell bodies, whereas dense immunoreactivity was found in the axon tracts of these neurons and their terminal regions in globus pallidus and substantia nigra pars reticulata. Immunostaining was dense in the medial habenula, but weak in other diencephalic regions. In midbrain and hindbrain, immunostaining was restricted to discrete regions of the neuropil or clusters of cell bodies. These results suggest that PDE2A may modulate cortical, hippocampal and striatal networks at several levels. Preferential distribution of PDE2A into axons and terminals of the principal neurons suggests roles in regulation of axonal excitability or transmitter release. The enzyme is also in forebrain interneurons, and in mid- and hindbrain neurons that may modulate forebrain networks and circuits.
Assuntos
Encéfalo/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Animais , Elementos Antissenso (Genética) , Autorradiografia , Vasos Sanguíneos/enzimologia , Encéfalo/anatomia & histologia , Mapeamento Encefálico , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/enzimologia , Dendritos/enzimologia , Imunofluorescência , Hipocampo/anatomia & histologia , Hipocampo/enzimologia , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Hibridização In Situ , Neostriado/anatomia & histologia , Neostriado/enzimologia , Neurônios/enzimologia , Células Piramidais/enzimologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Medula Espinal/enzimologiaRESUMO
Medically unexplained physical symptoms (MUPS) are physical symptoms for which no relevant organic pathology can be found. Patients with MUPS commonly present to the emergency department (ED) but are rarely considered in emergency medicine teaching or literature. Management of these patients is frequently more challenging than where there is an obvious organic pathology. This review provides the emergency physician with background knowledge regarding the classification and aetiology of MUPS. It then provides strategies for more effective management, such as exploring the contribution of psychosocial factors with patients, explaining negative test results, and providing reassurance and avoiding creating iatrogenic anxiety. Early recognition of the fact that symptoms may not result from organic disease and an appreciation of the role of psychosocial factors may improve outcomes by reducing unnecessary investigation and admission, and avoiding reinforcement that encourages further similar presentations and unhelpful coping mechanisms.
Assuntos
Serviços Médicos de Emergência , Transtornos Somatoformes , Atitude Frente a Saúde , Serviços Médicos de Emergência/estatística & dados numéricos , Humanos , Relações Médico-Paciente , Medicina Psicossomática/normas , Transtornos Somatoformes/diagnóstico , Transtornos Somatoformes/psicologia , Transtornos Somatoformes/terapia , Terminologia como AssuntoRESUMO
PDE10A is a recently identified phosphodiesterase that is highly expressed by the GABAergic medium spiny projection neurons of the mammalian striatum. Inhibition of PDE10A results in striatal activation and behavioral suppression, suggesting that PDE10A inhibitors represent a novel class of antipsychotic agents. In the present studies we further elucidate the localization of this enzyme in striatum of rat and cynomolgus monkey. We find by confocal microscopy that PDE10A-like immunoreactivity is excluded from each class of striatal interneuron. Thus, the enzyme is restricted to the medium spiny neurons. Subcellular fractionation indicates that PDE10A is primarily membrane bound. The protein is present in the synaptosomal fraction but is separated from the postsynaptic density upon solubilization with 0.4% Triton X-100. Immuno-electron microscopy of striatum confirms that PDE10A is most often associated with membranes in dendrites and spines. Immuno-gold particles are observed on the edge of the postsynaptic density but not within this structure. Our studies indicate that PDE10A is associated with post-synaptic membranes of the medium spiny neurons, suggesting that the specialized compartmentation of PDE10A enables the regulation of intracellular signaling from glutamatergic and dopaminergic inputs to these neurons.
Assuntos
Corpo Estriado/citologia , Neurônios/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Frações Subcelulares/enzimologia , Animais , Western Blotting/métodos , Calbindina 2 , Colina O-Acetiltransferase/metabolismo , Corpo Estriado/enzimologia , Masculino , Microscopia Imunoeletrônica/métodos , Neurônios/ultraestrutura , Óxido Nítrico Sintase Tipo I/metabolismo , Parvalbuminas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/metabolismo , Frações Subcelulares/ultraestrutura , Sinaptossomos/enzimologia , Sinaptossomos/ultraestruturaRESUMO
This paper forms the second part of the debate on prehospital thrombolysis (PHT). It is argued that large scale studies have failed to show a benefit for PHT, even when the time saved over conventional treatment was considerably greater than would be the case in the UK urban setting. In practice, a relatively small proportion of the total population receiving thrombolysis would receive PHT. Other strategies to reduce time to thrombolysis can benefit all patients and are likely to be more cost effective and safer.
Assuntos
Serviços Médicos de Emergência , Infarto do Miocárdio/tratamento farmacológico , Terapia Trombolítica , Serviços Urbanos de Saúde , Inglaterra , Humanos , TempoRESUMO
Global brain ischemia causes cell death in the CA1 region of the hippocampus 3-5 days after reperfusion. The biological pathway leading to such delayed neuronal damage has not been established. By using differential display analysis, we examined expression levels of poly(A) RNAs isolated from hippocampal extracts prepared from rats exposed to global ischemia and found an up-regulated transcript, clone 17a. Northern blot analysis of clone 17a showed an approximately 35-fold increase in the ischemic brain at 24 h after four-vessel occlusion. Rapid amplification of cDNA ends of clone 17a revealed a family of genes (160-540 base pairs) that had the characteristics of rodent B(2) sequences. In situ hybridization demonstrated that the elevated expression of this gene was localized predominantly in the CA1 pyramidal neurons. The level of expression in the CA1 region decreased dramatically between 24 and 72 h after ischemia. The elevated expression of clone 17a was not observed in four-vessel occlusion rats treated with the compound LY231617, an antioxidant known to exert neuroprotection in rats subjected to global ischemia. Since delayed neuronal death has the characteristics of apoptosis, we speculate that clone 17a may be involved in apoptosis. We examined the expression level of clone 17a in in vitro models of apoptosis using cerebellar granule neurons that were subjected to potassium removal, glutamate toxicity, or 6-hydroxydopamine treatment and found that clone 17a transcripts were induced in cerebellar granule neurons by glutamate or 6-hydroxydopamine stimulation but not potassium withdrawal.
Assuntos
Hipocampo/metabolismo , Ataque Isquêmico Transitório/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Hipocampo/patologia , Ataque Isquêmico Transitório/patologia , Dados de Sequência Molecular , Oxidopamina/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
Stimulation of metabotropic glutamate receptors in vitro has been shown to accelerate the breakdown of amyloid precursor protein (APP) to form increased production of non-amyloidogenic secreted APP (sAPP). The mechanism whereby this occurs is not entirely clear but it is presumed to be linked to generation of diacylglycerol and activation of protein kinase C because other neurotransmitter receptors such as m1 and m3 muscarinic receptors, known to be coupled to this second messenger cascade, likewise increase sAPP production. Although it is presumed that a reciprocal relationship exists between the formation of amyloid beta protein (Abeta) and the production of sAPP, recent evidence suggests alternative processing can occur. Given the fact that much of the observations on APP metabolism have been made in vitro we sought to investigate the effect of metabotropic receptor activation on Abeta in vivo in a species known to contain the same amino acid sequence of Abeta as found in humans. Intrahippocampal injection of the mGluR agonist 1S,3R-ACPD in guinea pigs produced neurodegeneration of CA1 hippocampal pyramidal neurons at 12 h postinjection. Immunocytochemistry of sections from ACPD injected animals using selective antibodies to Abeta revealed the presence of punctate intraneuronal granules in pyramidal neurons of the hippocampus. These structures appeared to be localized within the nucleus and were particularly prominent in neurons within the region of neurodegeneration. Immunoreactivity was not observed in vehicle injected controls nor in sections from ACPD injected animals stained with preadsorbed antiserum. Abeta immunodetection was correlated with the onset of neurodegeneration since animals evaluated at 1 h and 4 h postinjection lacked both Abeta immunoreactivity as well as neurodegeneration. Evaluation of animals injected with NMDA revealed neurodegeneration but no Abeta immunoreactivity suggesting Abeta formation did not appear to be due to non-selective excitotoxicity. Staining of sections with antibodies directed to various regions of APP demonstrated increased C-terminal APP immunoreactivity in pyramidal neurons in the vicinity of degeneration. These data support recent in vitro studies illustrating that Abeta can be found intracellularly within neurons.
Assuntos
Amiloide/metabolismo , Hipocampo/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Cobaias , Hipocampo/patologia , Imuno-Histoquímica , Masculino , Microinjeções , Neurônios/metabolismo , Neurônios/patologiaRESUMO
BACKGROUND AND PURPOSE: Nuclear factor-kappaB (NF-kappaB) is an oxidative stress responsive transcription factor that is transiently activated in most forebrain neurons in response to transient global ischemia. However, in hippocampal CA1 neurons destined to die, NF-kappaB remains persistently activated. The present study was performed to determine whether an antioxidant (LY231617) that afforded neuroprotection in previous studies had any effect on NF-kappaB activation in hippocampal CA1 neurons after global ischemia. METHODS: Rats were subjected to 30 minutes of forebrain ischemia by 4-vessel occlusion (4-VO) and killed at 24 and 72 hours after ischemia. LY231617 was administered orally at a dose of 50 mg/kg 30 minutes before 4-VO and again 4 hours after 4-VO. Neuronal damage was evaluated in sections stained with cresyl violet. Other sections were immunostained with antibodies to NF-kappaB p50 to assess nuclear localization. An electrophoretic mobility shift assay was performed on nuclear extracts from sham- and LY231617-treated rats at 24 and 72 hours after ischemia. RESULTS: The administration of LY231617 had a significant protective effect on hippocampal CA1 neurons at 72 hours after ischemia (control group, 16 +/- 7 neurons/mm; treated group, 294 +/- 35 neurons/mm, P<.02) and prevented nuclear translocation of activated NF-kappaB as normally seen at 72 hours after ischemia in untreated controls. In contrast, the untreated controls showed activated NF-kappaB at 72 hours after ischemia. At 24 hours after ischemia, both the control group and the LY231617 group showed intense nuclear localization of NF-kappaB. CONCLUSIONS: Activation of NF-kappaB in vitro has been reported to promote proapoptotic as well as antiapoptotic mechanisms, depending on the cell type being investigated. In the present in vivo study, the role of the transient activation of NF-kappaB observed at 24 hours may be responsible for the induction of protective factors in neurons that survive the ischemic insult, whereas the persistent activation of NF-kappaB in hippocampal neurons could be responsible for the induction of proteins that result in CA1 neuronal death.
Assuntos
Antioxidantes/farmacologia , Hidroxitolueno Butilado/análogos & derivados , NF-kappa B/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Hidroxitolueno Butilado/farmacologia , Proteínas de Ligação a DNA/metabolismo , Hipocampo/irrigação sanguínea , Proteínas Nucleares/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Ativação Transcricional/efeitos dos fármacosRESUMO
Peripheral benzodiazepine receptors (PBRs) are expressed in a variety of tissues but are normally found at low levels in the brain. Following various types of nerve injury, a reactive gliosis results that exhibits a high expression of this receptor. To further characterize the expression of PBRs following neuronal injury, we evaluated PBR expression in the facial nucleus following facial nerve axotomy (FNA). Injury to a peripheral nerve results in a complex series of metabolic and morphological changes around the injured neuron. Transections of the facial nerve results in a rapid activation of both astrocytes and microglia around axotomized motor neurons. FNA resulted in an increase in the staining for both astrocytes (glial fibrillary acidic protein) and activated microglia (OX42). There was also a reduction in synaptic contacts with the motor nucleus as evidenced by reduced staining for the synaptic marker, synaptophysin. In sections labeled with [3H]-PK11195, the subsequent autoradiograms displayed marked increases in the labeling for PBRs. This increase was observed at 5, 7 and 10 days after nerve transection. The increase was primarily in the level of expression (Bmax), with no change in the affinity of the ligand (Kd). The increase in PBR expression after FNA supports the hypothesis that PBRs can be used as a sensitive marker for CNS injury.
Assuntos
Encéfalo/metabolismo , Nervo Facial/metabolismo , Nervo Facial/fisiologia , Neurônios Motores/fisiologia , Receptores de GABA-A/biossíntese , Animais , Autorradiografia , Axotomia , Encéfalo/fisiopatologia , Química Encefálica , Isoquinolinas/metabolismo , Ligantes , Masculino , Modelos Neurológicos , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/fisiologiaRESUMO
The oxidative stress responsive transcription factor nuclear factor-kappa B (NF-kappa B) consists of a p50 (50 kDa) and p65/RelA (65 kDa) component and can be activated in vitro by TNF alpha, IL1 beta, hydrogen peroxide and oxygen radicals. All of the above factors are also known to be elevated at certain times after transient global ischemia. The present study was performed to determine if NF-kappa B was activated in vivo by transient global forebrain ischemia. Adult male rats were subjected to 30 min of 4-vessel occlusion (4-VO) and sacrificed at selected post-ischemic time points. Levels of NF-kappa B p50 and p65 subunits were determined by immunocytochemistry, Western blot and electrophoretic mobility-shift analysis. The enhancer complex was also confirmed by immuno-gel-shift analysis. Specific labeling of DNA strand breaks and DNA fragmentation was examined in situ by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Western blot analysis of hippocampus showed induction of p50 and p65. A time course of NF-kappa B induction in hippocampus showed a p50-specific band at 6 h that increased in intensity over 12, 48 h and then decreased by 96 h post-ischemia. Immunocytochemistry revealed at 24 h post-ischemia that p65 and p50 immunoreactivity was present in neuronal nuclei of hippocampal CA1 neurons as well as all other hippocampal regions and several other forebrain regions which were not vulnerable to transient forebrain ischemia. At 72 h post-ischemia, nuclear NF-kappa B immunoreactivity had disappeared in all brain areas except in hippocampal CA1 neurons which were degenerating. No evidence for DNA fragmentation as revealed by TUNEL staining could be observed at 24 h. However, at 72 h, hippocampal CA1 neurons were heavily labeled. The results of this study demonstrate that global forebrain ischemia causes a transient activation of NF-kappa B in many forebrain regions. NF-kappa B remains persistently activated in the vulnerable hippocampal CA1 sector. Because of the persistent activation of NF-kappa B in these neurons, the possibility exists that NF-kappa B has a role in programmed cell death in hippocampal CA1 neurons.
Assuntos
Isquemia Encefálica/metabolismo , Fragmentação do DNA , NF-kappa B/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Eletroforese/métodos , Hipocampo/citologia , Imuno-Histoquímica , Masculino , Neurônios/fisiologia , Ratos , Ratos WistarRESUMO
BACKGROUND AND PURPOSE: After global ischemia, brain levels of hydrogen peroxide, oxygen radicals, and the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are increased. Oxygen radicals, TNF-alpha, and IL-1 beta are known to activate nuclear factor-kappa B (NF-kappa B) in vitro. The present study was performed to determine whether NF-kappa B was activated in vivo by global ischemia in hippocampal CA1 neurons. METHODS: Adult male rats were subjected to 30 minutes of four-vessel occlusion and killed 72 hours later. Levels of NF-kappa B p50 and p65 subunits in hippocampus were determined by immunocytochemistry, Western blot, and gel-shift analysis. Specific labeling of DNA strand breaks was demonstrated by means of an Apoptag apoptosis detection kit. RESULTS: Labeling of DNA strand breaks was present at 72 hours. Chromatin compaction and segregation, a characteristic of apoptosis, was observed in sections stained with hematoxylin and eosin. NF-kappa B p50 and p65 immunoreactivity localized only to nuclei of CA1 neurons at 72 hours after reperfusion. Induction of the activated p50 and p65 subunits was confirmed by Western blot and electromobility shift analysis. The results demonstrate that NF-kappa B is activated selectively in hippocampal CA1 neurons at 72 hours after four-vessel occlusion, which is at the approximate time of CA1 neuronal cell death. CONCLUSIONS: Transient forebrain ischemia resulted in a marked activation of nuclear NF-kappa B in the highly vulnerable CA1 sector. Intense nuclear localization of NF-kappa B was associated only with dying neurons; regions of the hippocampus that were not vulnerable to four-vessel occlusion did not exhibit nuclear NF-kappa B localization. The elevation of NF-kappa B in degenerating CA1 neurons may be associated mechanistically with apoptotic or necrotic cell death.
Assuntos
Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , NF-kappa B/metabolismo , Neurônios/fisiologia , Prosencéfalo/fisiopatologia , Animais , Apoptose , Isquemia Encefálica/genética , Dano ao DNA , Hipocampo/patologia , Hipocampo/fisiopatologia , Masculino , Prosencéfalo/patologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Hippocampal CA1 neurons are highly susceptible to short periods of transient global ischemia. We have previously reported in a rat model of transient forebrain global ischemia that activation and nuclear localization of NF-kB occurs in the CA1 neurons at 24 and 72 h post reperfusion. Events following NF-kB activation would ultimately determine whether damaged cells will undergo programmed cell death. We have selected bcl-x gene expression for study because there is increasing evidence that proteins encoded by the bcl-2 gene family (bcl-2, bcl-x, bax etc) play a role in the regulation of programmed cell death. We have observed that the bcl-x gene promoter contains a putative consensus sequence for NF-kB/CS4 responsive activation. We also can show that other members of the bcl-2 multigene family contain the NF-kB/CS4 sequence in their five prime regulatory regions. In this study, we show that NF-kB p50 and NF-kB p65 act in synergy to transactivate the bcl-x promoter in co-transfected 293 cells. We also report that following ischemia and NF-kB activation, bcl-x messenger RNA levels increase in the CA1 hippocampal region. As a result of this transcriptional increase, surprisingly, it is bcl-xs, the apoptotic form of bcl-x, that is elevated. These results suggest that activation of NF-kB can lead to increased expression of bcl-x as manifested by the increase in the short form of bcl-x.
Assuntos
Hipocampo/metabolismo , Ataque Isquêmico Transitório/metabolismo , Prosencéfalo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/fisiologia , Sequência Consenso , Regulação da Expressão Gênica/genética , Genes Reporter , Hipocampo/irrigação sanguínea , Hipocampo/citologia , NF-kappa B/análise , NF-kappa B/genética , Oxirredução , Plasmídeos , Regiões Promotoras Genéticas/genética , Prosencéfalo/irrigação sanguínea , Prosencéfalo/citologia , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Proteína bcl-XRESUMO
The Ca(2+)-sensitive 85 kDa cytosolic PLA2 (cPLA2) is a receptor-regulated enzyme that may initiate the cascade of events leading to the production of free fatty acids and lysophospholipids for subsequent conversion to eicosanoids and PAF. At least two early events are necessary for full activation of cPLA2: (1) increased concentration of cytosolic free Ca2+ promoting association of cPLA2 with its membrane phospholipid substrate and (2) phosphorylation by stimulated proline-directed kinases converting cPLA2 into an enzyme of enhanced catalytic efficiency. Moreover, pro-inflammatory cytokines, such as IL-1 and TNF may induce de novo synthesis of cPLA2 thus further potentiating the mobilization of arachidonic acid and subsequent production of eicosanoids and PAF. Increased levels of fatty acids and PLA2-derived products, including eicosanoids and PAF are amongst the hallmarks of cerebral ischemia and reperfusion, and thought to mediate pathophysiological alterations and cellular processes which may lead to cell injury and death. There is substantial evidence to indicate that cPLA2 is present in the brain and appears most abundant in astrocytes. Therefore, cPLA2 may be an important component in the cascade of events leading to acute and delayed destructive cellular processes in the brain and accordingly represents an attractive target for the development of novel therapies to prevent brain damage triggered by ischemic and inflammatory insults.
Assuntos
Encéfalo/metabolismo , Metabolismo dos Lipídeos , Fosfolipases A/metabolismo , Animais , Citoplasma/metabolismo , Humanos , Fosfolipases A2RESUMO
BACKGROUND AND PURPOSE: Phospholipid breakdown has been reported to be an early event in the brain after global cerebral ischemia. Our earlier observations showing the localization of cytosolic phospholipase A2 (cPLA2) to astrocytes in aged human brains and the intense glial activation observed after global forebrain ischemia prompted us to investigate the cellular localization of cPLA2 in the rat brain subjected to global ischemia. METHODS: Immunohistochemistry was performed in sections through the dorsal hippocampus in rats subjected to 30 minutes of four- vessel occlusion. PLA2 was localized with the use of a highly selective antiserum. Double immunofluorescent localization was performed to colocalize cPLA2 with various glial cell types. cPLA2 levels were also measured by enzymatic assay and Western blot analysis. RESULTS: A marked induction of cPLA2 was observed in activated microglia and astrocytes in the CA1 hippocampal region at 72 hours after ischemia. Only a subset of astrocytes and microglia were immunoreactive for cPLA2. Twenty-four hours after ischemia, numerous cPLA2 immunoreactive astrocytes were observed. Western blot analysis of hippocampal homogenates at 72 hours after ischemia showed induction of a 100-kD band that comigrated with purified human cPLA2, and a threefold induction in cPLA2 activity was demonstrated by enzymatic assay. CONCLUSIONS: These results indicate that both reactive astrocytes and microglia contain elevated levels of cPLA2. Induction of cPLA2 was confined to areas of neurodegeneration and likely precedes its onset. The results suggest that reactive glia may play a role in the pathophysiology of delayed neuronal death after transient global forebrain ischemia.
Assuntos
Isquemia Encefálica/enzimologia , Neuroglia/enzimologia , Fosfolipases A/análise , Prosencéfalo/irrigação sanguínea , Animais , Astrócitos/enzimologia , Western Blotting , Isquemia Encefálica/genética , Morte Celular , Citosol/enzimologia , Doxorrubicina/análogos & derivados , Regulação Enzimológica da Expressão Gênica , Hipocampo/enzimologia , Humanos , Masculino , Microglia/enzimologia , Degeneração Neural , Fosfolipases A/genética , Fosfolipases A2 , Prosencéfalo/enzimologia , Ratos , Ratos WistarRESUMO
Phospholipase A2 (PLA2) is the key enzyme that initiates the arachidonic acid cascade, which leads to the generation of multiple eicosanoid products. Many of these products are believed to play an important role in the inflammatory process. Activation of PLA2 is observed under pathological conditions where inflammation is present. Cytosolic PLA2 (cPLA2) is activated by very low levels of calcium and is thought to control receptor-mediated eicosanoid production and to participate in intracellular signal transduction processes. In view of the presence of numerous inflammatory mediators and acute phase proteins in the Alzheimer's disease (AD) brain, localization of cPLA2 in AD brain was evaluated and compared to that observed in nonneurologically diseased controls. In this study, a monoclonal antibody raised against cPLA2 was used to immunostain tissue sections of human cerebral cortex. Five AD cases and six neurologically normal cases were evaluated in the occipital cortex and the cerebellum. Two of the AD cases were also examined in other cortical regions. Granular-like staining with anti-cPLA2 was found to be associated with astrocytes in the cortex of both control and AD cases. Colocalization with GFAP confirmed that cPLA2 immunoreactivity is associated almost exclusively with protoplasmic astrocytes. Staining was abolished when sections were labeled with antibody that had been preadsorbed with purified cPLA2. In AD brain, cPLA2 immunoreactive astrocytes were greater in number and more intensely stained than those in control cases. cPLA2 immunoreactivity was virtually absent in the cerebelium of AD and control cases, despite the presence in this region of diffuse amyloid in two AD cases and amyloid angiopathy in a third case. In the cortex, cPLA2 immunoreactive astrocytes were detected in regions that contained numerous A beta deposits. The finding of elevated levels of cPLA2 immunoreactivity in AD brain supports the hypothesis that there is an active inflammatory process occurring in AD.
Assuntos
Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Citosol/enzimologia , Isoenzimas/análise , Proteínas do Tecido Nervoso/análise , Fosfolipases A/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Ácido Araquidônico/metabolismo , Astrócitos/enzimologia , Biomarcadores , Encéfalo/patologia , Cerebelo/enzimologia , Cerebelo/patologia , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Ativação Enzimática , Humanos , Técnicas Imunoenzimáticas , Inflamação , Fosfolipases A2 , Transdução de SinaisRESUMO
The mechanisms underlying the response of the brain to ischemia are not fully understood. Biochemical and morphological changes following neocortical infarction can be investigated in rats using a model of focal cerebral ischemia induced by unilateral occlusion of the middle cerebral artery (MCA). Evaluation of ischemic damage often employs conventional histologic stains. Immunocytochemistry can be used as a valuable tool in this model to define changes in specific proteins of interest. In this study, an antiserum raised against insulin-like growth factor II (IGF-II) receptor was used to evaluate changes of IGF-II receptor immunoreactivity in the cerebral cortex of rats 4 and 7 days following permanent MCA occlusion. IGF-II receptor immunoreactivity was found to be associated with neocortical pyramidal neurons within the core of the ischemic infarct itself. The staining intensity was markedly elevated above that observed in nonischemic neurons. Immunopositive neurons exhibited a punctate staining pattern. These neurons appeared to correspond to argentophilic neurons, as defined by modified Bielschowsky silver staining. Evaluation of other neuronal markers revealed the absence of immunoreactivity for neuron-specific enolase and for tyrosine hydroxylase within the ischemic area. These observations show an increase in a specific growth factor receptor within neurons in the ischemic core of a focal infarct several days following permanent focal infarction, a time when neurons are presumed to be dead. The significance and the potential role of IGF-II receptor in lesion-induced plasticity are discussed.
Assuntos
Isquemia Encefálica/metabolismo , Infarto Cerebral/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Neurônios/metabolismo , Receptores de Somatomedina/metabolismo , Animais , Isquemia Encefálica/patologia , Infarto Cerebral/patologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Endogâmicos SHRRESUMO
In mammalian brain the expression of peripheral benzodiazepine receptors (PBRs) can be markedly induced following different types of neuronal injury. PBRs are believed to be expressed on non-neuronal cells in the brain, yet the specific cell type that expresses these receptors following CNS insult has not been defined. In the present study, we investigated the effects of transient global forebrain ischemia on PBRs by autoradiographic localization of 3H-PK11195 binding. The distribution of PBRs was compared to glial fibrillary acidic protein (GFAP) as a marker for astrocytes and OX42 as a marker for microglia. Five to 6 d following four-vessel occlusion (4-VO), an increase in PBRs was seen in the CA1 region of all 15 brains examined. In brains from rats subjected to 4-VO, microglia were selectively activated in stratum pyramidale of the CA1 layer. In contrast, astrocytes appeared to be activated in multiple hippocampal cell layers including stratum radiatum and stratum oriens. Activated astrocytes were also found in regions that did not exhibit increased 3H-PK11195 binding. In some brains, selected regions of secondary lesion, specifically necrotic thalamic nuclei and the isocortex were found to be strongly immunoreactive for OX42 but lacked GFAP immunoreactive cells. In adjacent sections, these same regions displayed high densities of 3H-PK1195 binding. These observations lend further support to the application of 3H-PK11195 binding as a marker of neuronal injury in the brain. Furthermore, the data strongly suggest that activated microglia rather than astrocytes express PBRs following ischemic insults.
Assuntos
Ataque Isquêmico Transitório/metabolismo , Microglia/metabolismo , Prosencéfalo/irrigação sanguínea , Animais , Anticorpos Monoclonais , Astrócitos/metabolismo , Autorradiografia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Ataque Isquêmico Transitório/patologia , Isoquinolinas/metabolismo , Masculino , Prosencéfalo/patologia , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismo , Distribuição TecidualRESUMO
Calcium-sensitive cytosolic phospholipase A2 (cPLA2) is responsible for receptor-mediated liberation of arachidonic acid, and thus plays an important role in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. In this study we have investigated the cellular distribution of cPLA2 in brain using a monoclonal antibody raised against cPLA2 to immunostain tissue sections of human cerebral cortex. We have localized cPLA2 in astrocytes of the gray matter. Colocalization with glial fibrillary acidic protein (GFAP) confirmed that cPLA2 is associated predominantly with protoplasmic astrocytes. Astrocytes of the white matter, on the other hand, were not immunoreactive. In experiments using different human astrocytoma cell lines we found that cPLA2 can be immunochemically localized in UC-11 MG cells, but cannot be detected in U-373 MG cells. This finding is consistent with the observation that cPLA2 mRNA as well as cPLA2 enzymatic activity can be readily measured in UC-11 MG astrocytoma cells, yet cannot be detected in U-373 MG cells. Our data suggest that the astrocyte is a primary source of cPLA2 in the brain and provide further evidence for the importance of this cell type in inflammatory processes in the brain.
Assuntos
Astrócitos/enzimologia , Encéfalo/enzimologia , Cálcio/fisiologia , Citosol/enzimologia , Fosfolipases A/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Astrocitoma/enzimologia , Encéfalo/citologia , Neoplasias Encefálicas/enzimologia , Feminino , Proteína Glial Fibrilar Ácida/imunologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Fosfolipases A2 , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The cerebral deposition of amyloid beta protein (A beta P) is an early pathogenetic event in Alzheimer's disease (AD). Recent studies suggest both neurotoxic and neurotrophic effects of A beta P in vitro. Because progressive A beta P deposition and surrounding neuritic dystrophy occur spontaneously in primates, we evaluated the in vivo effects of synthetic A beta P in monkey cortex. Experimental and control (reverse or substituted) peptides were stereotactically injected into multiple neocortical sites of adult rhesus monkeys in a vehicle of either artificial cerebrospinal fluid or acetonitrile. After 2 weeks, all injection sites were identified and characterized. A beta P antibodies specifically detected the injected A beta P1-40 peptide. Serial sections stained with silver and antineurofilament protein demonstrated comparable degrees of degenerating neurons, dystrophic neurites, and axonal spheroids associated with both experimental and control peptide injections. Alz 50 staining was sparse or absent in all sites. Similar results were obtained in an animal killed 3 months after injection. We conclude that specific cellular changes closely resembling the pathology of Alzheimer's disease were not detected in these acute experiments, and that control and experimental A beta P peptides produced indistinguishable effects.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Córtex Cerebral/patologia , Neurônios/patologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/análise , Animais , Córtex Cerebral/efeitos dos fármacos , Modelos Animais de Doenças , Macaca mulatta , Masculino , Microinjeções , Dados de Sequência Molecular , Neurônios/efeitos dos fármacosRESUMO
The distribution of beta-amyloid precursor protein (APP) was examined immunocytochemically in rats subjected to focal cerebral ischemia by permanent occlusion of the middle cerebral artery. At 4 and 7 days post-occlusion, APP immunoreactivity was preferentially localized within axonal swellings, dystrophic neurites and neuronal perikarya all along the periphery of the infarct. Immunolabeling was observed with antibodies generated against N-terminal, midregion, and C-terminal domains of APP. No immunoreactivity was observed with antisera directed against beta-amyloid protein (beta A4) itself. This pathological accumulation of APP is consistent with alterations of APP recently described in other models of neurodegeneration and implies a role for this protein in the response to CNS injury.