A S100B é uma proteína ligante de cálcio produzida e secretada por astrócitos e tem sido relacionada à interação neurônio-glia. Nosso primeiro objetivo foi investigar um possível efeito autócrino da S100B na captação de glutamato. A utilização do anticorpo anti-S100B reduziu a captação de glutamato após 30 minutos de incubação, sem afetar a integridade e viabilidade celular. Além disso, baixas concentrações de S100B (menos de 0,1 ng/mL) estimularam a captação de glutamato avaliada imediatamente após a troca de meio. Este dado reforça a importância dos astrócitos na transmissão glutamatérgica, particularmente, a função da S100B na neuroproteção contra dano excitotóxico. De fato, a S100B extracelular protege os neurônios quanto ao dano excitotóxico, enquanto concentrações tóxicas de glutamato têm demonstrado reduzir a secreção de S100B em astrócitos e fatias cerebrais, por mecanismos desconhecidos. Nosso segundo objetivo foi investigar os mecanismos possivelmente envolvidos neste efeito em cultura primária de astrócitos hipocampais utilizando agonistas glutamatérgicos e inibidores da captação de glutamato. Nossos resultados sugerem que a secreção de S100B está inversamente ligada à captação de glutamato. Além disso, a secreção da S100B parece ser estimulada por serotonina e estudos clínicos têm sugerido que a elevação da S100B está positivamente relacionada à resposta terapêutica dos antidepressivos, particularmente, os inibidores da recaptação da serotonina. Desta forma, nosso terceiro objetivo foi medir a secreção da S100B em culturas de astrócitos expostas à fluoxetina. Nós observamos um significativo aumento da secreção da S100B pela fluoxetina, efeito aparentemente dependente de PKA. Este dado reforça a importância da fluoxetina, independente da serotonina e receptores serotoninérgicos, para a atividade antidepressiva, bem como a função putativa da S100B nas doenças depressivas. Em paralelo, a padronização de uma técnica de ELISA para GFAP (a principal proteína marcadora de astrócitos), nós observamos que a fosforilação in vitro da GFAP purificada ou de amostras biológicas com PKA, indicam que a fosforilação aumenta o reconhecimento pelo anticorpo policlonal anti GFAP da DAKO. Estes resultados favorecem o entendimento de rápidas alterações na imunoreatividade da GFAP. / S100B is a calcium-binding protein expressed and secreted by astrocytes, which has been implicated in glial-neuronal communication. Our first aim was to investigate a possible autocrine role of S100B in glutamate uptake activity. Antibody anti-S100B addition decreased glutamate uptake measured 30 min after medium replacement, without affect cell integrity or viability. Moreover, low levels of S100B (less than 0.1 ng/mL) stimulated glutamate uptake measured immediately after medium replacement. This finding reinforces the importance of astrocytes in the glutamatergic transmission, particularly the role of S100B neuroprotection against excitotoxic damage. In fact, extracellular S100B protects hippocampal neurons from excitotoxic damage, whilst toxic levels of glutamate to neurons have been shown to reduce S100B secretion in astrocytes and brain slices, by unknown mechanisms. Our second aim was to investigate which mechanisms are possibly involved in this effect in primary cultures of hippocampal astrocytes using glutamate agonists and glutamate uptake inhibitors. Our findings suggest that S100B secretion is inversely coupled to glutamate uptake. Moreover, S100B secretion appears to be stimulated by serotonin and clinical studies have suggested that serum elevation of S100B is positively correlated with therapeutic antidepressant response, particularly selective serotonin reuptake inhibitors. Our third aim was to measure S100B secretion is astrocyte cultures exposed to fluoxetine. We observed a significant increment of S100B release by fluoxetine, apparently dependent on PKA. These data reinforce the importance of fluoxetine, independent of serotonin and serotonin receptors, for antidepressant activity, as well as the putative role of S100B in depressive disorders. In parallel, standardizing an ELISA for GFAP (the main protein marker for mature astrocytes) we found that in vitro phosphorylation of purified GFAP or biological samples with PKA indicate that GFAP phosphorylation improves the recognition by the polyclonal antibody anti-GFAP from DAKO. These results provide support to the understanding of fast changes in the GFAP-immunoreactivity.

A S100B é uma proteína ligante de cálcio produzida e secretada por astrócitos e tem sido relacionada à interação neurônio-glia. Nosso primeiro objetivo foi investigar um possível efeito autócrino da S100B na captação de glutamato. A utilização do anticorpo anti-S100B reduziu a captação de glutamato após 30 minutos de incubação, sem afetar a integridade e viabilidade celular. Além disso, baixas concentrações de S100B (menos de 0,1 ng/mL) estimularam a captação de glutamato avaliada imediatamente após a troca de meio. Este dado reforça a importância dos astrócitos na transmissão glutamatérgica, particularmente, a função da S100B na neuroproteção contra dano excitotóxico. De fato, a S100B extracelular protege os neurônios quanto ao dano excitotóxico, enquanto concentrações tóxicas de glutamato têm demonstrado reduzir a secreção de S100B em astrócitos e fatias cerebrais, por mecanismos desconhecidos. Nosso segundo objetivo foi investigar os mecanismos possivelmente envolvidos neste efeito em cultura primária de astrócitos hipocampais utilizando agonistas glutamatérgicos e inibidores da captação de glutamato. Nossos resultados sugerem que a secreção de S100B está inversamente ligada à captação de glutamato. Além disso, a secreção da S100B parece ser estimulada por serotonina e estudos clínicos têm sugerido que a elevação da S100B está positivamente relacionada à resposta terapêutica dos antidepressivos, particularmente, os inibidores da recaptação da serotonina. Desta forma, nosso terceiro objetivo foi medir a secreção da S100B em culturas de astrócitos expostas à fluoxetina. Nós observamos um significativo aumento da secreção da S100B pela fluoxetina, efeito aparentemente dependente de PKA. Este dado reforça a importância da fluoxetina, independente da serotonina e receptores serotoninérgicos, para a atividade antidepressiva, bem como a função putativa da S100B nas doenças depressivas. Em paralelo, a padronização de uma técnica de ELISA para GFAP (a principal proteína marcadora de astrócitos), nós observamos que a fosforilação in vitro da GFAP purificada ou de amostras biológicas com PKA, indicam que a fosforilação aumenta o reconhecimento pelo anticorpo policlonal anti GFAP da DAKO. Estes resultados favorecem o entendimento de rápidas alterações na imunoreatividade da GFAP. / S100B is a calcium-binding protein expressed and secreted by astrocytes, which has been implicated in glial-neuronal communication. Our first aim was to investigate a possible autocrine role of S100B in glutamate uptake activity. Antibody anti-S100B addition decreased glutamate uptake measured 30 min after medium replacement, without affect cell integrity or viability. Moreover, low levels of S100B (less than 0.1 ng/mL) stimulated glutamate uptake measured immediately after medium replacement. This finding reinforces the importance of astrocytes in the glutamatergic transmission, particularly the role of S100B neuroprotection against excitotoxic damage. In fact, extracellular S100B protects hippocampal neurons from excitotoxic damage, whilst toxic levels of glutamate to neurons have been shown to reduce S100B secretion in astrocytes and brain slices, by unknown mechanisms. Our second aim was to investigate which mechanisms are possibly involved in this effect in primary cultures of hippocampal astrocytes using glutamate agonists and glutamate uptake inhibitors. Our findings suggest that S100B secretion is inversely coupled to glutamate uptake. Moreover, S100B secretion appears to be stimulated by serotonin and clinical studies have suggested that serum elevation of S100B is positively correlated with therapeutic antidepressant response, particularly selective serotonin reuptake inhibitors. Our third aim was to measure S100B secretion is astrocyte cultures exposed to fluoxetine. We observed a significant increment of S100B release by fluoxetine, apparently dependent on PKA. These data reinforce the importance of fluoxetine, independent of serotonin and serotonin receptors, for antidepressant activity, as well as the putative role of S100B in depressive disorders. In parallel, standardizing an ELISA for GFAP (the main protein marker for mature astrocytes) we found that in vitro phosphorylation of purified GFAP or biological samples with PKA indicate that GFAP phosphorylation improves the recognition by the polyclonal antibody anti-GFAP from DAKO. These results provide support to the understanding of fast changes in the GFAP-immunoreactivity.

A S100B é uma proteína ligante de cálcio produzida e secretada por astrócitos e tem sido relacionada à interação neurônio-glia. Nosso primeiro objetivo foi investigar um possível efeito autócrino da S100B na captação de glutamato. A utilização do anticorpo anti-S100B reduziu a captação de glutamato após 30 minutos de incubação, sem afetar a integridade e viabilidade celular. Além disso, baixas concentrações de S100B (menos de 0,1 ng/mL) estimularam a captação de glutamato avaliada imediatamente após a troca de meio. Este dado reforça a importância dos astrócitos na transmissão glutamatérgica, particularmente, a função da S100B na neuroproteção contra dano excitotóxico. De fato, a S100B extracelular protege os neurônios quanto ao dano excitotóxico, enquanto concentrações tóxicas de glutamato têm demonstrado reduzir a secreção de S100B em astrócitos e fatias cerebrais, por mecanismos desconhecidos. Nosso segundo objetivo foi investigar os mecanismos possivelmente envolvidos neste efeito em cultura primária de astrócitos hipocampais utilizando agonistas glutamatérgicos e inibidores da captação de glutamato. Nossos resultados sugerem que a secreção de S100B está inversamente ligada à captação de glutamato. Além disso, a secreção da S100B parece ser estimulada por serotonina e estudos clínicos têm sugerido que a elevação da S100B está positivamente relacionada à resposta terapêutica dos antidepressivos, particularmente, os inibidores da recaptação da serotonina. Desta forma, nosso terceiro objetivo foi medir a secreção da S100B em culturas de astrócitos expostas à fluoxetina. Nós observamos um significativo aumento da secreção da S100B pela fluoxetina, efeito aparentemente dependente de PKA. Este dado reforça a importância da fluoxetina, independente da serotonina e receptores serotoninérgicos, para a atividade antidepressiva, bem como a função putativa da S100B nas doenças depressivas. Em paralelo, a padronização de uma técnica de ELISA para GFAP (a principal proteína marcadora de astrócitos), nós observamos que a fosforilação in vitro da GFAP purificada ou de amostras biológicas com PKA, indicam que a fosforilação aumenta o reconhecimento pelo anticorpo policlonal anti GFAP da DAKO. Estes resultados favorecem o entendimento de rápidas alterações na imunoreatividade da GFAP. / S100B is a calcium-binding protein expressed and secreted by astrocytes, which has been implicated in glial-neuronal communication. Our first aim was to investigate a possible autocrine role of S100B in glutamate uptake activity. Antibody anti-S100B addition decreased glutamate uptake measured 30 min after medium replacement, without affect cell integrity or viability. Moreover, low levels of S100B (less than 0.1 ng/mL) stimulated glutamate uptake measured immediately after medium replacement. This finding reinforces the importance of astrocytes in the glutamatergic transmission, particularly the role of S100B neuroprotection against excitotoxic damage. In fact, extracellular S100B protects hippocampal neurons from excitotoxic damage, whilst toxic levels of glutamate to neurons have been shown to reduce S100B secretion in astrocytes and brain slices, by unknown mechanisms. Our second aim was to investigate which mechanisms are possibly involved in this effect in primary cultures of hippocampal astrocytes using glutamate agonists and glutamate uptake inhibitors. Our findings suggest that S100B secretion is inversely coupled to glutamate uptake. Moreover, S100B secretion appears to be stimulated by serotonin and clinical studies have suggested that serum elevation of S100B is positively correlated with therapeutic antidepressant response, particularly selective serotonin reuptake inhibitors. Our third aim was to measure S100B secretion is astrocyte cultures exposed to fluoxetine. We observed a significant increment of S100B release by fluoxetine, apparently dependent on PKA. These data reinforce the importance of fluoxetine, independent of serotonin and serotonin receptors, for antidepressant activity, as well as the putative role of S100B in depressive disorders. In parallel, standardizing an ELISA for GFAP (the main protein marker for mature astrocytes) we found that in vitro phosphorylation of purified GFAP or biological samples with PKA indicate that GFAP phosphorylation improves the recognition by the polyclonal antibody anti-GFAP from DAKO. These results provide support to the understanding of fast changes in the GFAP-immunoreactivity.

abstract: Alzheimer's Disease (AD) is the sixth leading cause of death in the United States and the most common form of dementia. Its cause remains unknown, but it is known to involve two hallmark pathologies: Amyloid Beta plaques and neurofibrillary tangles (NFTs). Several proteins have been implicated in the formation of neurofibrillary tangles, including Tau and S100B. S100B is a dimeric protein that is typically found bound to Ca(II) or Zn(II). These experiments relate to the involvement of S100B in Alzheimer's Disease-related processes and the results suggest that future research of S100B is warranted. Zn(II)-S100B was found to increase the rate at which tau assembled into paired helical filaments, as well as affect the rate at which tubulin polymerized into microtubules and the morphology of SH-SY5Y neuroblastoma cells after 72 hours of incubation. Zn(II)-S100B also increased the firing rate of hippocampal neurons after 36 hours of incubation. Together, these results suggest several possibilities: Zn(II)-S100B may be a key part of the formation of paired helical filaments (PHFs) that subsequently form NFTs. Zn(II)-S100B may also be competing with tau to bind tubulin, which could lead to an instability of microtubules and subsequent cell death. This finding aligns with the neurodegeneration that is commonly seen in AD and which could be a result of this microtubule instability. Ultimately, these results suggest that S100B is likely involved in several AD-related processes, and if the goal is to find an efficient and effective therapeutic target for AD, the relationship between S100B, particularly Zn(II)-S100B, and tau needs to be further studied. / Dissertation/Thesis / M.S. Applied Biological Sciences 2013

Identification et caractérisation comme cible de la S100B d'une protéine impliquée dans la distribution subcellulaire des mitochondries <br />Les mitochondries appartiennent à un réseau dynamique et doivent être positionnées de manière stratégique dans la cellule. 15 % des mitochondries sont en contact étroit avec le réticulum endoplasmique (RE), et il a été proposé que la calciprotéine S100B régule les interactions entre le RE et les mitochondries. Cependant, une localisation perimitochondriale de la S100B n'a jamais été montrée, ni sa cible mitochondriale identifiée. Après avoir montré dans les cellules progénitrices oligodendrogliales (CPO) que la S100B interagit avec les mitochondries, nous avons identifié par une approche de Far Western une protéine comme cible de la S100B. Aucune étude préalable ne porte sur la fonction de cette protéine, qui par ailleurs appartient à la famille AAA (Atpases Associées à des Activités variées). Nous l'avons nommée MSBP pour « Mitochondria S100B Binding Protein ». MSBP est transmembranaire, localisée aux sites de contacts entre les membranes internes et externes des mitochondries, le domaine N-terminal exposé côté cytosolique, et la boîte ATPase du côté C-terminal protégée à l'intérieur de la mitochondrie. Comparée aux autres membres de la famille S100, la S100B interagit de façon spécifique avec la protéine MSBP, dont elle inhibe de manière dépendante du calcium l'oligomérisation. Dans les CPO, il avait été montré que l'invalidation de la S100B induit un ralentissement de leur différenciation. Nous montrons ici que la diminution de l'expression de sa cible MSBP par une approche siRNA a un effet similaire. <br />La fonction cellulaire de MSBP a été analysée plus en détail dans la lignée cellulaire gliale U373, l'invalidation de l'expression de la protéine MSBP par siRNA et l'inhibition de son activité ATPase par une mutation ponctuelle ont des effets opposés sur la distribution des mitochondries : la protéine MSBP est nécessaire à l'ancrage des mitochondries à un pôle du noyau, tandis que son activité ATPase permet la libération des mitochondries de ce centre organisateur périnucléaire. L'invalidation de l'expression de la protéine MSBP, outre la dispersion des mitochondries vers la périphérie, s'accompagne d'une diminution des interactions entre le RE et les mitochondries et d'une diminution des transferts de phosphatidylserine (PS) qui nécessitent une interaction entre ces organelles.<br /><br />Expression des protéines MSBP dans les gliomes <br />Les gènes codant pour les protéines MSBP1 et MSBP2 sont localisés à l'extrémité distale du chromosome 1 au locus 1p36, qui se caractérise par une perte d'hétérozygotie dans les oligodendrogliomes. Nous montrons que l'expression de la protéine MSBP2 pourrait être utilisée comme un marqueur pour différencier les glioblastomes des oligodendrogliomes : alors que la protéines MSBP2 est détectée dans les glioblastomes, elle ne l'est pas dans les oligodendrogliomes. D'autre part, dans la lignée cellulaire HS683 dérivée d'un oligodendrogliome, l'absence d'expression de la protéine MSBP2 résulte d'une délétion homozygote qui tronque la partie 5' du gène codant pour MSBP2. L'étendue de cette délétion homozygote recouvre une région très restreinte, et ne concerne que deux gènes, dont le gène codant MSBP2. Bien plus, dans la lignée U373, l'inhibition de l'expression des gènes MSBP par siRNA favorise la prolifération, et la croissance indépendante de l'ancrage en Soft agar, tandis que leur surexpression se caractérise par un effet opposé. Ces résultats suggèrent que l'invalidation du gène MSBP2 pourrait participer à la transformation cellulaire et favoriser ainsi le développement des oligodendrogliomes.

Fragestellung: Gegenstand dieser Untersuchung ist der diagnostische Nutzen der Neuro-Biomarker S100B-Protein und Neuronenspezifische Enolase (NSE) bei leichten Schädel-Hirn-Verletzungen im Kindesalter. Es wird untersucht, ob anhand der posttraumatischen S100B- und NSE-Serum-Konzentrationen Kinder mit einer Schädelprellung und einem leichten Schädel-Hirn-Trauma (SHT) differenziert werden können. Material und Methode: In einer prospektiven, klinischen Studie werden die posttraumatischen S100B- und NSE-Serum-Konzentrationen von Kindern im Alter zwischen 6 Monaten und 15 Jahren mit einer Schädelprellung oder einem leichten SHT untersucht. Kinder mit extrakraniellen Begleitverletzungen und Begleiterkrankungen sind ausgeschlossen. Die Blutentnahme erfolgt innerhalb von 6 Stunden nach dem Trauma. Es werden 2 diagnostische Gruppen definiert: Gruppe 1: asymptomatische Schädelprellungen (Glasgow-Coma-Scale [GCS] 15 Punkte), Gruppe 2: leichte SHT (GCS 13-15 Punkte) mit klinischen Zeichen einer Gehirnerschütterung (kurze Bewusstlosigkeit, Amnesie, Übelkeit, Erbrechen, Somnolenz, Kopfschmerzen, Schwindel, Sehstörungen, Kreislaufreaktion). Die S100B- und NSE- Konzentrationen werden zwischen beiden Diagnosegruppen verglichen. Die Korrelation zwischen S100B und NSE sowie zwischen den Markern und dem Alter der Kinder, dem Zeitraum zwischen Trauma und Blutentnahme, dem GCS-Wert und dem Vorhandensein von Kopfplatzwunden wird analysiert. Ergebnisse: 148 Kinder sind in die Studie eingeschlossen (53 Kinder mit einer Schädelprellung und 95 mit einem leichten SHT). Nach Adjustierung der gemessenen Marker-Konzentrationen auf Unterschiede im Alter und Zeitraum zwischen Trauma und Blutentnahme unterscheiden sich die S100B- und NSE-Konzentrationen nicht signifikant zwischen Kindern mit einer Schädelprellung und einem leichten SHT. Zwischen den S100B- und NSE-Konzentrationen besteht eine signifikant positive Korrelation. Beide Marker korrelieren signifikant negativ mit dem Alter und dem Entnahmezeitraum. Der GCS-Wert und das Vorhandensein von Kopfplatzwunden zeigen keinen Effekt auf die Marker-Konzentrationen. Schlussfolgerung: Die posttraumatischen S100B- und NSE-Serum-Konzentrationen zeigen keinen diagnostischen Nutzen bei der Differenzierung zwischen Kindern mit einer Schädelprellung und Kindern mit einem leichten SHT. S100B und NSE sind altersabhängige Marker.

Le pronostic de l’arrêt cardiaque (AC) reste dramatique. Diagnostiquer sa cause rapidement et prédire précocement son pronostic ("pronostication") de manière fiable permettrait de mieux guider les traitements initiaux, en évitant de traiter futilement les patients avec faible probabilité d’évolution favorable ou à l’inverse de permettre d’intensifier le traitement de patients avec forte probabilité d’évolution favorable. Les biomarqueurs, dont l’utilité diagnostique et pronostique reste débattue, semblent actuellement insuffisamment sensibles et précis, surtout dans les 1ères heures après la reprise de l’activité circulatoire spontanée (RACS). Dans l’algorithme pronostique, seule la Neuron Specific Enolase (NSE) est validée après le 3ème jour post-AC et en 2ème intention. Notre première étude a montré que la valeur diagnostique des biomarqueurs "spécifiques" des lésions cérébrales en post-AC (protéine S100B : S100 et surtout NSE) était insuffisante, à l’admission en réanimation, pour étayer précisément le diagnostic de cause neurologique d’AC. Si la coronarographie précoce est l’outil diagnostique de référence de l’AC de probable cause cardiaque, les biomarqueurs ne peuvent remplacer le scanner cérébral pour diagnostiquer une cause neurologique d’AC. La deuxième étude a évalué, au 1er jour post-AC, S100 et NSE avec 2 témoins d’œdème cérébral proposés comme outils pronostiques : le diamètre de l’enveloppe du nerf optique (DENO) par échographie et le rapport de dédifférenciation substance grise / substance blanche (DSG/B) par scanner cérébral. Même si une relation directe ne peut être affirmée formellement entre ces paramètres, l’élargissement du DENO à J1 post-AC était corrélé aux lésions cérébrales, surtout l’œdème cérébral et les lésions neuronales suspectés sur l’élévation de la NSE (à l’admission et à J1) et la baisse de DSG/B. Si NSE, DSG/B et DENO à J1 étaient liés, S100, plus spécifique de la glie, n’était pas corrélée au DENO ni au DSG/B. NSE et S100 à l’admission, à J1 et J2 post-RACS et DENO à J1 étaient associées à la mortalité hospitalière. La troisième étude évaluait la valeur pronostique des biomarqueurs à la phase précoce de l’AC (NSE et S100 étant prélevées en médiane 220 min après la RASC). S100, réalisée en aveugle des cliniciens, était le biomarqueur le plus précis à l’admission en réanimation pour prédire correctement le pronostic défavorable à la sortie de l’hôpital et à 3 mois après AC, par rapport au lactate, pH et créatininémie, et surtout à la NSE. Les variations de S100 dans le temps permettaient d’affiner cette prédiction. S100 à l’admission était un facteur indépendant du pronostic défavorable à la sortie de l’hôpital, avec la durée sans massage cardiaque, le rythme initial non-choquable, le lactate initial et la présence de convulsion clinique. Selon les recommandations, la pronostication nécessite théoriquement d’être différée et multimodale, les biomarqueurs seuls n’étant pas recommandés, surtout précocement. Les biomarqueurs ne peuvent constituer une alternative, en comparaison à l’imagerie, pour l’aide diagnostique de la cause d’AC. A l’inverse, certains biomarqueurs comme la S100 après admission pourraient facilement et spécifiquement discriminer les AC ayant une certitude de pronostic défavorable. Associée à d’autres outils prédictifs clinico-radiologiques, la S100 pourrait être incorporée dans des algorithmes permettant de guider les thérapeutiques initiales. Une pronostication correcte précoce pourrait éviter des traitements invasifs inutiles, ou au contraire optimiser certaines thérapeutiques agressives. Le choix de méthodes recommandées et automatisées de contrôle ciblé de la température, très efficaces mais invasives et onéreuses, ou l’indication d’utiliser -ou pas- une assistance cardio-circulatoire extra-corporelle pourrait bénéficier d’une telle stratégie précoce de sélection des patients. / Outcome of cardiac arrest (CA) remains dramatic. To quickly diagnose the cause of CA and establish a reliable outcome prediction (prognostication) as early as possible could help to guide initial treatments. It could avoid futile treatments in patients with low chance of survival or of good neurological recovery, or conversely allow treatment optimization in patients expected to have a high likelihood of good neurological outcome. Usefulness of biomarkers to guide clinicians in finding the CA diagnosis and helping prognostication is debated. Biomarkers are considered as not sensitive and accurate enough, especially within the first hours after return of spontaneous circulation (ROSC). Their use is only recommended in prognostication for Neuron Specific Enolase (NSE) as a second line tool and after the third day from CA. Our first study confirmed that biomarkers “specific” of brain injury (S100B protein: S100 and moreover NSE) cannot sufficiently discriminate the neurological cause of CA on ICU admission. If early coronary angiogram is the standard for diagnosing a probable cardiac cause of CA, biomarkers cannot replace brain computed-tomography (CT) in CA from a neurological cause. The second study evaluated, during the 1st day after ROSC, the link between biomarkers (S100 and NSE) and 2 surrogates of brain oedema recently proposed as outcome predictors: echography of the optic nerve sheath diameter (ONSD), and grey to white matter attenuation ratio (GWR) on brain CT-scan. Even though we cannot conclude on a definitive relationship between these parameters, ONSD enlargement at day 1 was associated with specific brain damage after CA, such as brain oedema and mostly axonal injuries, as reflected by increases in NSE (on admission and at day 1) and low GWR measurements. Whereas NSE, GWR and ONSD at day 1 were correlated, S100, which is more specific of glial injuries, did not reach significance. NSE and S100 on admission, at days 1 and 2 after ROSC, as well as ONSD at day 1, were associated with survival at hospital discharge. The third study evaluated the prognostic value of several biomarkers in the early phase after CA (NSE and S100 being sampled at median 220 min after ROSC). S100, blinded to physicians, was the biomarker with the best accuracy after ICU admission to correctly predict unfavourable outcome at hospital discharge and at 3 months after CA, compared with all other biomarkers such as lactate, pH, creatinine, and especially NSE. S100 variations during the first day after admission refined prognostication. Initial S100 was an early independent predictive factor associated with unfavourable outcome at hospital discharge, with the no-flow duration, initial lactate value, initial non-shockable rhythm, and the presence of clinical seizure. According to guidelines, prognostication theoretically needs to be delayed and multimodal, biomarkers alone not being recommended especially in the early phase after CA. Biomarkers cannot seem to be an alternative option compared to imaging to precisely diagnose the CA cause. By contrast, some biomarkers, such as S100 after admission, could easily and specifically discriminate CA patients with certainty of unfavourable outcome. Associated with other predictive tools (clinical or using imaging), biomarkers could interestingly be incorporated in early decisional algorithms to optimally guide initial therapies. This correct patient classification could help to avoid unuseful treatments versus to maximize aggressive therapies. The choice of recommended servo-controlled targeted temperature management devices, very efficient but invasive and expensive, or the indication -or not- of a cardio-circulatory assist device implementation should be guided in the early stage after ROSC using this simple strategy of patient selection.

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Submitted by FLAVIA DUTRA DE TOLEDO (flavia.dutra@unesp.br) on 2018-10-16T12:49:00Z No. of bitstreams: 1 TOLEDO_FLAVIA_DUTRA_TESE.pdf: 1343341 bytes, checksum: 04c961b0d4c2c7913f24f0ef86d634f4 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-10-17T14:19:04Z (GMT) No. of bitstreams: 1 toledo_fd_dr_bot.pdf: 1343341 bytes, checksum: 04c961b0d4c2c7913f24f0ef86d634f4 (MD5) / Made available in DSpace on 2018-10-17T14:19:04Z (GMT). No. of bitstreams: 1 toledo_fd_dr_bot.pdf: 1343341 bytes, checksum: 04c961b0d4c2c7913f24f0ef86d634f4 (MD5) Previous issue date: 2018-08-24 / Introdução: A última atualização demográfica do Instituto Brasileiro de Geografia e Estatística realizada em 2017 mostrou que a população acima de 60 anos representava 14,6% dos 207,1 milhões de habitantes do Brasil, o que corresponde a aproximadamente 30 milhões de idosos, com expectativa de vida de 75,8 anos. Entre 2007 e 2011, o número acumulado de internações por fraturas de fêmur em pacientes acima de 60 anos, nos hospitais do Sistema Único de Saúde, foi de 175.781 O delirium pós-operatório é uma complicação comum nos pacientes idosos, e está associado a hospitalizações prolongadas, maiores taxas de institucionalização após a alta, deterioração cognitiva prolongada, diminuição da capacidade funcional, além de ser fator independente de mortalidade em 6-12 meses. Estudo disponível na literatura mostra que 14 a 24% dos pacientes idosos com fratura de quadril apresentam delirium já na admissão hospitalar, sendo que a prevalência durante a internação chega a 56%. Os mecanismos fisiopatológicos do desenvolvimento do delirium pós-operatório (DPO) e do déficit cognitivo pós-operatório têm sido estudados em nível molecular, porém ainda com pouco ganho. Maiores esforços de pesquisa são colocados na identificação de biomarcadores diagnósticos e prognósticos que estejam relacionados com mecanismos moleculares que levam ao DPO. Objetivos: Primariamente, relacionar os níveis séricos de dois biomarcadores de neuroinflamação (S100B e enolase neurônio-específica) com a ocorrência de delirium pré e pós-operatório em pacientes idosos submetidos a cirurgias de correção de fratura de quadril (fêmur proximal). Como objetivo secundário, foi proposto avaliar a influência dos fatores sexo, hipotensão perioperatória, classificação de risco anestésico ASA, uso de fármacos anestésicos para sedação (midazolam e fentanil), além de duração e tipo de cirurgia realizada (artroplastia de quadril ou osteossíntese de fêmur) sobre os níveis dos biomarcadores. Métodos: Foi realizado estudo clínico observacional prospectivo com pacientes acima de 60 anos internados no HCFMB para correção de fratura de quadril (terço proximal do fêmur) no período de maio de 2017 a abril de 2018; todos os pacientes assinaram o Termo de Consentimento Livre e Esclarecido. Os pacientes foram avaliados em dois momentos: dentro das 24 horas que antecederam a cirurgia (M1) e, novamente, 24 horas após o final da cirurgia (M2). Em cada momento, foi realizado o diagnóstico de presença ou ausência de delirium por meio da escala CAM (Confusion Assessment Method), instrumento validado em Português, e foram coletadas amostras de sangue para dosagem de S100B e Enolase plasmáticos. As dosagens dos biomarcadores foram realizadas por método ELISA. Resultados: Dos 42 pacientes analisados, 33 eram do sexo feminino e 9 do sexo masculino (78,57% e 21,4%, respectivamente), com idade média de 71,97 ± 8,68 anos. Delirium ocorreu em 11,9% dos pacientes em M1 e 16,7% em M2. Não houve variação dos níveis médios de S100B entre pacientes com e sem delirium, nem dentro do mesmo momento, quanto de um momento para outro (p=0,12). Os valores médios de NSE foram estatisticamente diferentes quando comparados os pacientes sem delirium pré-operatório e aqueles com delirium pós-operatório (p=0,002); entretanto, pacientes que não tiveram delirium em qualquer momento também apresentaram variação significante do marcador entre M1 e M2 (p=0,004). Ainda, pacientes com delirium pós-operatório tiveram níveis mais altos de NSE dosados em M2 (p=0,02). Mulheres apresentaram maiores níveis de S100B (p=0,03), enquanto as médias de NSE foram maiores entre homens (p=0,04). Não houve influência da classificação ASA tanto nos níveis de S100B quanto de NSE (p=0,67 e p=0,12, respectivamente). Níveis mais altos de S100B foram verificados em pacientes que apresentaram algum episódio de hipotensão perioperatória (p=0,035), porém não houve relação entre este último evento e a ocorrência de delirium pós-operatório (p=0,65). Quanto ao uso de sedação intraoperatória, doses maiores de midazolam foram administradas aos pacientes que não desenvolveram DPO (p=0,001), enquanto as doses de fentanil não diferiram entre pacientes com e sem DPO (p=0,21). Não houve relação entre o tipo de procedimento cirúrgico executado e duração média da cirurgia (p=0,89 e p=0,15, respectivamente) com a ocorrência de DPO. Conclusões: Não foi encontrada associação entres os níveis do marcador S100B e a presença de delirium em qualquer momento; entretanto, níveis pré-operatórios aumentados de NSE foram relacionados à ocorrência de delirium pós-operatório. Pacientes com episódios de hipotensão perioperatória apresentaram níveis mais altos de S100B na avaliação pós-operatória. São necessárias investigações futuras quanto ao nível de expressão destas proteínas por outros tecidos e se existem outras vias de sinalização celular ainda desconhecidas, para que assim possamos entender melhor o comportamento destes biomarcadores e a sua relação com a patogênese do delirium. / Background: According to the latest census from IBGE (Instituto Brasileiro de Geografia e Estatística), population over 60 years-old accounted for 14,3% of a total 204,9 million habitants in Brazil. From 2007 to 2011, 175.781 elder patients were admitted after a hip fracture in brazilian public hospitals. Post-operative delirium (POD) is an usual complication in the elderly and is associated to longer hospital stay, cognitive impairment, functional decline and increased 6-12 months mortality rate. Delirium rates between the elder population can be as high as 14 to 24% already at admission, being the prevalence as high as 56% during hospital stay. Pathophysiologic mechanisms of delirium have been studied at molecular level, but research efforts are still needed in order to develop sensitive and specific early markers for this condition, so that diagnosis and effective treatment would be readily given. Objectives: This research project aims to correlate two neuroinflamation biomarkers serum levels (S100B protein and Neuron Specific Enolase - NSE) and the incidence of pre and postoperative delirium in elderly patients undergoing hip fracture repair. Also, we intent to evaluate how gender, ASA Physical Status classification and perioperative hypotension may contribute to changes in the levels of these biomarkers. Methods: An observational prospective study was proposed envolving patients aged 60 or more who were admitted at Clinics Hospital of Botucatu Medical School (Botucatu, São Paulo, Brazil) for hip fracture repair, from May, 2017 to April, 2018. All patients signed the Informed Consent. The CAM (Confusion Assessment Method) short scale was used to assess the presence of delirium first at admission and again, 24 hours after surgery. Blood samples were also collected at two moments, by the patient’s admission to the operating room and then 24 hours after surgery, to assess NSE and S100B plasmatic levels. S100B and NSE levels were measured on the Synergy HT Biotec analyzer using ELISA technique (Elabscience ® kits). Results: Of 42 patients included, 33 were female and 9 male (78.57% vs 21.4%), with mean age of 71.97 ± SD 8.68 years). Delirium occurred in 11.9% of patients at M1 and 16.7% at M2. S100B mean levels did not significantly change between patients with and without delirium, neither at the same moment (p=0.95 and p=0.96, respectively) nor from M1 to M2 (p=0,12). NSE levels in delirious and non-delirious patients at M1 were elevated at M2 (p=0.04); however, higher NSE levels at M1 were significantly related to emergence of delirium at M2 (p=0.002). Moreover, despite of the fact that NSE levels increased at M2 in patients without delirium (p=0.004), delirious patients at M2 had significantly higher NSE levels (p=0.02). S100B levels were higher in female patients (p=0.03), whereas NSE was higher in male ones (p=0.04). Perioperative hypotension was linked to higher S100B levels at M2 (p=0.035), but not to postoperative delirium 9p=0,65). ASA classification had no influence on biomarkers levels. Regarding the use of intraoperative sedation, higher amounts of midazolam were administered to patients who did not further develop POD (p=0.001); fentanyl doses did not differ between patients with or without POD (p=0.21). Surgical technique (hip replacement or osteosynthesis) and surgery duration had no influence on POD occurrence (p=0.89 and p=0.15, respectively). Conclusions: No association was found between S100B levels and delirium, but increased preoperative NSE levels were related to emergence of postoperative delirium. Patients with records showing episodes of perioperative hypotension had higher postoperative levels of S100B. Further investigation of yet unidentified tissue expression and signaling pathways of these proteins are needed in order for them to be used as predictors of delirium in clinical practice.

Calorie restriction (CR) is one of the few treatments that has been observed to significantly extend life in a wide variety of species. While its life-extending properties are still being investigated in primates, there is general agreement that it reduces oxidative stress and inflammation. Interestingly, there is some evidence that it may ameliorate or delay the onset of a number of neurodegenerative diseases, including age-related white matter degeneration. The processes underlying its neuroprotective effects in non-human primates are unknown, but oxidative stress and inflammation are potential contributors to age-related white matter pathologies that characterize aging in the monkey brain and correlate with cognitive decline. To determine if CR reduces damage due to oxidative stress and inflammation in the monkey brain, brains from four calorie restricted monkeys and four matched controls brains were processed for immunohistochemical analysis using an antibody against the pro-inflammatory protein S100b. S100b is a widely expressed calcium-binding cytoplasmic protein associated with neurological insults like ischemia, atrophy, and neurofibrillary tangles and plaques in Alzheimer's disease. It is primarily expressed in astrocytes, but is also expressed to a lesser extent in microglia, oligodendrocytes, and some neuronal populations. Stereology was used to estimate density of S100b labeling in the cingulum, corpus callosum and visual cortex. No significant difference between calorie restricted animals and controls was found. More specific markers of oxidative stress and inflammation may be more effective in revealing any significant differences between CR and control brains. Potential alternatives include antibodies against 4-hydroxynonenal, a lipid peroxidation product, and encephalitogenic peptides of myelin basic protein, which are only exposed to the extracellular environment when myelin is damaged.