A research report is submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Sciences in Child Health Johannesburg, 2016 / Background: Leratong Hospital is a regional hospital in the West Rand of Johannesburg, South Africa. Statistics from maternity in 2008 showed high utilisation rates for delivery services at Leratong but a study on neonatal mortality was not yet done. It was therefore essential to measure and analyse the causes of new-born deaths so as to have policies to advance neonatal care. Objectives: To determine the neonatal mortality rate (NMR), the major neonatal causes of death and the occurrence of avoidable health factors. Methods: This was a prospective review of the clinical records of the 46 neonates who died within the 3 month period (15th April 2013 to the 15th July 2013). Data was obtained from neonatal admission and death registers. Information on the number oflive births was obtained from labour ward registers. Delegation books for nurses were checked to determine the number of nursing staff per shift as well as their allocation in different rooms. Neonate's age, birth weight, gender, race, place of origin, reason for admission and cause of death, were analysed. Health factors examined were access to high care services and to the neonatal ICU, number of staff on duty and the use of treatment guidelines. Questionnaires were used to collect information, and the consent to use clinical records was obtained from the mothers. Descriptive statistics were used to describe the frequencies and percentages of variables. Logistic regression of variables was applied to predict mortality. Results: The overall neonatal mortality rate at Leratong Hospital was lower than the rates found in South Africa and other studies in sub-Saharan Africa. Almost 37% of neonates died within 24 hours of admission. The three most common causes of death were: prematurity (39%), perinatal asphyxia (26%) and infection (20%). More than sixty per cent of deaths occurred in the admission room. Three-quarters of neonates who died (74%) were low birth weight neonates. A critical staff shortage (nurse: neonate rati02.: 1:10) was the most common modifiable factor (63% of deaths). Thirty seven per cent of neonates were denied access to ICU. The significant predictors of neonatal death were being born preterm (OR: 3.1, 95% CI 1.7-6.0), extremely low birth weight (OR: 27.5,95% CI 8.2-92.6), very low birth weight (OR: 5.0, 95% CI 2.1-12.3) and birth by caesarean section (OR: 3.2, 95% CI 1.6-6.2). Conclusions: The study found the neonatal mortality rate at Leratong Hospital in 2013 to be lower than rates recorded in South Africa. Our results showed that the most common causes of neonatal mortality were similar to those in other hospitals in sub-Saharan Africa and in South Africa. A high number of neonatal deaths were avoidable by providing high care services (including NCP AP and surfactant) and adequate number of nurses trained in newborn care in the admission room, improving access to neonatal ICU, early detection of perinatal asphyxia and improved neonatal resuscitation, and the supervision of medical doctors. / AC2016

Made available in DSpace on 2014-06-11T19:28:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-05-29Bitstream added on 2014-06-13T20:17:42Z : No. of bitstreams: 1 suarez_oax_me_botfm.pdf: 656572 bytes, checksum: abeb20dbfb7202f9f09aa053ef22fc6c (MD5) / As limitações dos métodos diagnósticos da asfixia perinatal levaram-nos a investigar se o monitoramento dos movimentos corporais do sono (MCS) e do ritmo de sono-vigília (S/V) pode ser utilizado como marcador diagnóstico deste processo patológico. Para tanto, os registros eletromiográfico e eletrocardiográfico de 8 ratos recém-nascidos, Wistar, com 6 a 48h de vida, foram obtidos durante 4 períodos experimentais: período controle - com ratos respirando ar atmosférico, por 30min; período de asfixia - com ratos submetidos a dois períodos de asfixia de 30min (T1 e T2), com seus corpos inteiramente envolvidos por filme de polivinil (PVC); e período de recuperação - com ratos respirando ar ambiente novamente, por 30min. A freqüência cardíaca diminuiu significantemente durante a asfixia, em relação à fase controle. Nós concluímos que o monitoramento dos MCS e do ritmo de sono-vigília se mostrou um parâmetro promissor no diagnóstico da asfixia perinatal e de suas complicações. Este fato, todavia, requer novas investigações para se estabelecer a real viabilidade do método para o uso rotineiro na prática clínica. / The limitations of current diagnostic methods of perinatal asphyxia induced to investigate if monitoring of body movements and of the sleep-wake rhythm can be used as diagnostic marker of this pathologic process. So, electromyogram and electrocardiogram records of 8 newborn Wistar rats, 6 to 48 hours postnatal, were obtained throughout 4 experimental periods: control period - with rats breathing room air for 30 min (ARpre); asphyxia periods - with rats submitted to two 30 min asphyxia periods (T1 and T2) with Its entire body covered with a polyvinyl sheet (PVC); and recovering period - with rats breathing room air for 30 min again. Asphyxia Heart rate was significantly decreased all over the asphyxia period compared with the control phase. In conclusion the monitoring of SBM, and of the sleep-wake rhythm seems to be of utmost value to the perinatal asphyxia diagnosis and its complications. This fact, however, requires new investigations to establish the real viability of the method for clinical practice routine.

Orientador: Katsumasa Hoshino / Banca: Lígia Maria Suppo de Souza Sugolo / Banca: Mônica Levy Andersen / Resumo: As limitações dos métodos diagnósticos da asfixia perinatal levaram-nos a investigar se o monitoramento dos movimentos corporais do sono (MCS) e do ritmo de sono-vigília (S/V) pode ser utilizado como marcador diagnóstico deste processo patológico. Para tanto, os registros eletromiográfico e eletrocardiográfico de 8 ratos recém-nascidos, Wistar, com 6 a 48h de vida, foram obtidos durante 4 períodos experimentais: período controle - com ratos respirando ar atmosférico, por 30min; período de asfixia - com ratos submetidos a dois períodos de asfixia de 30min (T1 e T2), com seus corpos inteiramente envolvidos por filme de polivinil (PVC); e período de recuperação - com ratos respirando ar ambiente novamente, por 30min. A freqüência cardíaca diminuiu significantemente durante a asfixia, em relação à fase controle. Nós concluímos que o monitoramento dos MCS e do ritmo de sono-vigília se mostrou um parâmetro promissor no diagnóstico da asfixia perinatal e de suas complicações. Este fato, todavia, requer novas investigações para se estabelecer a real viabilidade do método para o uso rotineiro na prática clínica. / Abstract: The limitations of current diagnostic methods of perinatal asphyxia induced to investigate if monitoring of body movements and of the sleep-wake rhythm can be used as diagnostic marker of this pathologic process. So, electromyogram and electrocardiogram records of 8 newborn Wistar rats, 6 to 48 hours postnatal, were obtained throughout 4 experimental periods: control period - with rats breathing room air for 30 min (ARpre); asphyxia periods - with rats submitted to two 30 min asphyxia periods (T1 and T2) with Its entire body covered with a polyvinyl sheet (PVC); and recovering period - with rats breathing room air for 30 min again. Asphyxia Heart rate was significantly decreased all over the asphyxia period compared with the control phase. In conclusion the monitoring of SBM, and of the sleep-wake rhythm seems to be of utmost value to the perinatal asphyxia diagnosis and its complications. This fact, however, requires new investigations to establish the real viability of the method for clinical practice routine. / Mestre

Background: South Africa aims to end all preventable deaths of children under the age of five as part of their commitment to the Sustainable Development Goals. More than half of these mortalities occur in the neonatal period with perinatal asphyxia as one of the leading causes. This study investigated and identified the characteristics of perinatal asphyxia and its contributing factors at a district hospital in Cape Town. Methods: A retrospective descriptive case series was performed and included all suspected cases of perinatal asphyxia referred from Mitchells Plain District Hospital (MPH)) to a specialised centre in the years 2016-2018. A data collection tool was used to extract information. Data was processed with SPSS to produce descriptive statistics and to investigate associations between variables using the Chi-square tests. Results: The study included 29 cases of suspected perinatal asphyxia. Ten (34.5%) had abnormal amplitude Electroencephalograms (aEEG's) indicative of Hypoxic Ischaemic Encephalopathy (HIE) and four (13.8%) demised before day seven of life. Non-operative deliveries (p=0.005), lack of a doctor at the time of delivery (p=0.004) and neonatal chest compressions (p=0.044) were associated with abnormal aEEG's. Babies with Thompson score of equal to or more than 12 (p=0.006), neonatal seizures (p=0.036) and delayed arrival at referral hospital (p=0.005) were associated with abnormal aEEG findings. Mortality was associated with Thompson score ≥12 (p=0.007) and the need for neonatal intubation at delivery (p=0.016). Conclusions: Significant reversable factors were identified in the peri-and postpartum periods. More capacitated staff would have the greatest impact on outcomes. The profile of HIE is exceedingly complex and challenges the resources and services of district level of care. Therefore, these factors should be targeted for future development and investment to improve outcomes from district hospitals.

L’encéphalopathie hypoxique-­‐ischémique cause des milliers de victimes à travers le monde chaque année. Les enfants survivants à un épisode hypoxique-­‐ischémique sont à risque de développer des problèmes neurologiques incapacitants comme une paralysie cérébrale, un retard mental, une épilepsie ou des troubles d’ordre comportemental. Les modèles animaux ont amélioré nos connaissances sur les mécanismes sous-­‐jacents aux dommages cérébraux, mais elles sont encore trop incomplètes pour être capables de prévenir les problèmes neurologiques. Ce projet vise à comprendre l’impact d’un épisode asphyxique périnatale associé à des convulsions ainsi que l’activation de l’adenosine monophosphate-­‐activated protein kinase (AMPK) sur les circuits GABAergiques inhibiteurs en développement chez la souris. Dans le but d’investiguer le sort des neurones inhibiteurs, appelés interneurones, suite à un épisode asphyxique périnatal associé à des convulsions avec des animaux transgéniques, nous avons pris avantage d’un nouveau modèle d’hypoxie permettant d’induire des convulsions chez la souris. Deux populations d’interneurones représentant ensemble environ 60% de tous les interneurones corticaux ont été étudiées, soit les cellules exprimant la parvalbumine (PV) et les cellules exprimant la somatostatine (SOM). L’étude stéréologique n’a montré aucune mort neuronale de ces deux populations d’interneurones dans l’hippocampe chez les souris hypoxique d’âge adulte. Par contre, le cortex des souris hypoxiques présentait des zones complètement ou fortement dépourvues de cellules PV alors que les cellules SOM n’étaient pas affectées. L’utilisation d’une lignée de souris transgénique exprimant une protéine verte fluorescente (GFP) dans les cellules PV nous a permis de comprendre que les trous PV sont le reflet de deux choses : 1) une diminution des cellules PV et 2) une immaturité des cellules PV restantes. Puisque les cellules PV sont spécifiquement affectées dans la première partie de notre étude, nous avons voulu étudier les mécanismes moléculaires sous-­‐jacents à cette vulnérabilité. L’AMPK est un senseur d’énergie qui orchestre le rétablissement des i niveaux d’énergie cellulaire dans le cas d’une déplétion énergétique en modulant des voies de signalisation impliquant la synthèse de protéines et l’excitabilité membranaire. Il est possible que l’activation d’AMPK suite à un épisode asphyxique périnatal associé à des convulsions soit néfaste à long-­‐terme pour le circuit GABAergique en développement et modifie l’établissement de l’innervation périsomatique d’une cellule PV sur les cellules pyramidales. Nous avons étudié cette hypothèse dans un modèle de culture organotypique en surexprimant la forme wild-­‐type (WT) de la sous-­‐unité α2 d’AMPK, ainsi qu’une forme mutée dominante négative (DN), dans des cellules PV individuelles. Nous avons montré que pendant la phase de formation synaptique (jours post-­‐natals équivalents EP 10-­‐18), la surexpression de la forme WT désorganise la stabilisation des synapses. De plus, l’abolition de l’activité d’AMPK semble augmenter le nombre de synapses périsomatiques faits par la cellule PV sur les cellules pyramidales pendant la phase de formation et semble avoir l’effet inverse pendant la phase de maturation (EP 16-­‐24). La neurotransmission GABAergique joue plusieurs rôles dans le cerveau, depuis la naissance jusqu’à l’âge adulte des interneurones, et une dysfonction des interneurones a été associée à plusieurs troubles neurologiques, comme la schizophrénie, l’autisme et l’épilepsie. La maturation des circuits GABAergiques se fait majoritairement pendant la période post-­‐natale et est hautement dépendante de l’activité neuronale et de l’expérience sensorielle. Nos résultats révèlent que le lourd fardeau en demande énergétique d’un épisode asphyxique périnatal peut causer une mort neuronale sélective des cellules PV et compromettre l’intégrité de leur maturation. Un des mécanismes sous-­‐ jacents possible à cette immaturité des cellules PV suite à l’épisode hypoxique est l’activation d’AMPK, en désorganisant leur profil d’innervation sur les cellules pyramidales. Nous pensons que ces changements dans le réseau GABAergique pourrait contribuer aux problèmes neurologiques associés à une insulte hypoxique. / Hypoxia-­‐ischemia encephalopathy causes thousands of victims each year around the world. Children surviving such hypoxia-­‐ischemia insults are at risk of developing disabling neurological problems such as cerebral palsy, epilepsy or cognitive problems. Animal models have improved our knowledge about the underlying mechanisms causing cerebral injury, but it is still too incomplete to be able to prevent neurological problems. This project aims to understand the impact of a perinatal asphyxic insult associated with seizures as well as the activation of adenosine monophosphate-­‐activated protein kinase (AMPK) on developing inhibitory GABAergic networks in mouse. With the objective to study the fate of inhibitory cells, called interneurons, following a perinatal asphyxia insult associated with seizures in transgenic animals, we took advantage of a new hypoxia model allowing us to induce seizures in mice. Cells expressing parvalbumin (PV) and cells expressing somatostatin (SOM) were studied as together they represent about 60% of all cortical interneurons. A stereological study showed no cell death within those two interneuron populations in the hippocampus of adult hypoxic mice. However, the cortex of hypoxic mice showed zones with complete or strongly lacking PV cells, whereas SOM cells were not affected. A transgenic mouse line allowed us to show that PV holes are the reflection of two things 1) a decrease in PV cells and 2) immaturity of surviving PV cells. Because PV cells a selectively affected in the first part of our study, we wanted to study the molecular mechanisms underlying this vulnerability. AMPK is a metabolic energy sensor that orchestrates the recovery of energy in cells undergoing energy depletion by modulating cellular pathways involved in protein synthesis and membrane excitability. It is possible that the activation of AMPK following a perinatal asphyxic insult associated with seizures is detrimental for developing GABAergic networks and modifies the establishment of perisomatic innervation of PV cells on excitatory pyramidal cells. We have studied this hypothesis in an organotypic system by overexpressing the wild-­‐type (WT) form and the dominant negative (DN) form of AMPK iv α 2 sub-­‐unit in individual PV cells. We have observed that during the synaptic formation phase (equivalent post-­‐natal day EP 10-­‐18), overexpressing WT AMPK disorganises synapse stabilisation. Moreover, abolishing AMPK activity with the transfection of AMPK DN seems to increase the number of perisomatic synapses made by PV cells onto pyramidal cells, and seems to have the inverse effect during the synaptic maturation phase (EP 18-­‐24). GABAergic neurotransmission plays many roles in the brain, from interneurons birth to adulthood, and a dysfunction in GABAergic neurotransmission has been associated with many neurological diseases such as schizophrenia, autism and epilepsy. GABAergic networks maturation mainly happens postnatally and is highly dependent on neural activity and sensory experience. Our results reveal that the heavy energetic burden caused by an asphyxic insult can cause selective PV cells death and interfere with their maturation. AMPK activation following an asphyxic insult could be one mechanism interfering with PV cells maturity by disorganising their pattern of innervation onto pyramidal cells. We think that these GABAergic networks alterations could contribute to the neurological problems associated to a hypoxic insult.

Les enfants qui ont subi une asphyxie périnatale modérée (MPA) risquent de développer des déficits cognitifs et comportementaux subtils et durables, notamment des troubles d'apprentissage et des problèmes émotionnels. Comprendre les mécanismes sous-jacents est une étape essentielle pour concevoir une thérapie ciblée. Déterminer comment le développement du cerveau est corrélé entre les humains et les rongeurs n'est pas simple, mais il existe également un alignement inter-espèces considérable en termes d'étapes clés du développement. Sur la base des changements biochimiques et neuroanatomiques au cours du développement précoce, le consensus général est qu'un cerveau de rongeur P8-10 correspond à peu près au cerveau d'un enfant à terme ; par conséquent, nous avons utilisé cette fenêtre temporelle comme référence pour développer un modèle préclinique de MPA chez la souris. Nous avons d'abord établi un protocole qui nous permet d'observer de manière fiable les crises induites par l'hypoxie chez les souris postnatales. Nous avons constaté que l'exposition de chiots P8-9 directement à 4 % d'O2 pendant 8 minutes induit de manière fiable des crises avec une latence d'environ 5 minutes chez 3 souches de souris (FVB, C57Bl/6, 129S6). Cet aspect est cliniquement pertinent car les convulsions sont la caractéristique néonatale la plus importante de l'encéphalopathie de stade 2 (modérée) telle que définie par l'échelle de Sarnat. Les souris MPA adultes présentent des séquelles à long terme sur des performances cognitives spécifiques, notamment des déficits de la mémoire de reconnaissance et de la flexibilité cognitive, mais aucune altération du comportement moteur et émotionnel. Le cortex préfrontal (PFC) régule la flexibilité cognitive et le comportement émotionnel. Les neurones qui libèrent la sérotonine (5-HT) projettent vers le PFC, et les composés modulant l'activité 5-HT influencent l'émotion et la cognition. On ne sait pas si les dérégulations de la 5-HT contribuent aux problèmes cognitifs induits par le MPA. Dans une première étude, nous avons trouvé que les niveaux d'expression de 5-HT, quantifiés par immunohistochimie, et de libération de 5-HT, quantifiés par microdialyse in vivo chez des souris éveillées, sont réduits dans le PFC de souris MPA adultes. Les souris MPA présentent également une régulation de la température corporelle altérée après l'injection de l'agoniste des récepteurs 5-HT1A, 8-OH-DPAT, suggérant la présence de déficits dans la fonction des auto-récepteurs 5-HT sur les neurones du raphé. Enfin, le traitement chronique de souris MPA adultes avec de la fluoxétine, un inhibiteur du transporteur de recapture de la 5-HT, ou l'agoniste des récepteurs 5-HT1A, la tandospirone, sauve la flexibilité cognitive et les troubles de la mémoire. Ensemble, ces données démontrent que le développement de la fonction du système 5-HT est vulnérable à une asphyxie périnatale modérée. L'hypofonctionnement de la 5-HT pourrait à son tour contribuer à une déficience cognitive à long terme à l'âge adulte, indiquant une cible potentielle pour les thérapies pharmacologiques. Les circuits GABAergiques comprennent une variété étonnante de différents types de cellules, qui sont probablement recrutées par différents événements comportementaux. Un sous-type important de cellules GABAergiques, les cellules positives à la parvalbumine (PV), génèrent des potentiels d'action à haute fréquence et synchronisent l'activité des neurones pyramidaux excitateurs. Les cellules PV sont particulièrement importantes pour la génération d'oscillations gamma, qui à leur tour régulent de nombreuses fonctions cognitives, notamment le traitement attentionnel axé sur les objectifs et la mémoire de travail. Des découvertes récentes indiquent que les cellules PV utilisent beaucoup plus d'énergie que les autres neurones corticaux, ce qui peut les rendre très vulnérables aux conditions de stress métabolique et oxydatif causées par le MPA. Nos données ont montré que l'expression de PV est altérée chez les souris MPA adultes. Nous avons en outre constaté que le niveau d'expression du récepteur de la neurotrophine p75NTR, qui limite la maturation des cellules PV au cours de la première semaine postnatale, est augmenté chez les souris MPA. La suppression génétique de p75NTR dans les neurones GABAergiques exprimant le facteur de transcription Nkx2.1, qui comprend les cellules PV, protège les souris de la perte de niveaux de PV et des effets cognitifs à long terme du MPA. Enfin, un traitement d'une semaine avec un inhibiteur de p75NTR commençant après le MPA sauve complètement les déficits d'activité cognitive et corticale chez les souris adultes. L'ensemble de ces données révèle une cible moléculaire potentielle pour le traitement des altérations cognitives causées par le MPA. / Children who experienced moderate perinatal asphyxia (MPA) are at risk of developing long lasting subtle cognitive and behavioral deficits, including learning disabilities and emotional problems. Understanding the underlying mechanisms is an essential step for designing targeted therapy. Determining how brain development correlates between humans and rodents is not straightforward, however there is also considerable cross-species alignment in terms of key developmental milestones. Based on biochemical and neuroanatomical changes during early development, the general consensus is that a P8-10 rodent brain corresponds roughly to the brain of a term infant; therefore, we used this time window as reference to develop a preclinical model of MPA in mouse. We first established a protocol that allows us to reliably observe hypoxia-induced seizures in postnatal mice. We found that exposing P8-9 pups directly to 4% O2 for 8 minutes reliably induces seizures with a latency of about 5’ in 3 mouse strains (FVB, C57Bl/6, 129S6). This aspect is clinically relevant as seizures are the most prominent neonatal hallmark of Stage 2 (Moderate) encephalopathy as defined by the Sarnat Scale. Adult MPA mice show long-term sequelae on specific cognitive performance, including deficits in recognition memory and cognitive flexibility, but no impairment in motor and emotional behavior. The prefrontal cortex (PFC) regulates cognitive flexibility and emotional behavior. Neurons that release serotonin (5-HT) project to the PFC, and compounds modulating 5-HT activity influence emotion and cognition. Whether 5-HT dysregulations contribute to MPA-induced cognitive problems is unknown. In a first study, we found that 5-HT expression levels, quantified by immunohistochemistry, and 5-HT release, quantified by in vivo microdialysis in awake mice, are reduced in PFC of adult MPA mice. MPA mice also show impaired body temperature regulation following injection of the 5-HT1A receptor agonist 8-OH-DPAT, suggesting the presence of deficits in 5-HT auto-receptor function on raphe neurons. Finally, chronic treatment of adult MPA mice with fluoxetine, an inhibitor of 5-HT reuptake transporter, or the 5-HT1A receptor agonist tandospirone rescues cognitive flexibility and memory impairments. All together, these data demonstrate that the development of 5-HT system function is vulnerable to moderate perinatal asphyxia. 5-HT hypofunction might in turn contribute to long-term cognitive impairment in adulthood, indicating a potential target for pharmacological therapies. GABAergic circuits comprise an astonishing variety of different cell types, which are likely recruited by different behavioral events. An important subtype of GABAergic cells, the fast-spiking, parvalbumin-positive (PV) cells, generate action potentials at high frequency and synchronize the activity of excitatory pyramidal neurons. PV cells are particularly important for the generation of gamma oscillations, which in turn regulate many cognitive functions including goal-directed attentional processing and working memory. Recent findings indicate that PV cells utilize much more energy than other cortical neurons, which may render them highly vulnerable to conditions of metabolic and oxidative stress caused by MPA. Our data showed that PV expression is impaired in adult MPA mice. We further found that the expression level of the neurotrophin receptor p75NTR, which limits PV cell maturation during the first postnatal week, is increased in MPA mice. Genetic deletion of p75NTR in GABAergic neurons expressing the transcription factor Nkx2.1, which include PV cells, protects mice from PV levels loss and the long-term cognitive effects of MPA. Finally, one week treatment with a p75NTR inhibitor starting after MPA completely rescues the cognitive and cortical activity deficits in adult mice. All together this data reveals a potential molecular target for the treatment of the cognitive alterations caused by MPA.