Design and development / Respiratory distress syndrome (RDS) is a major cause of mortality and long-term morbidity annually affecting 14% preterm infants worldwide. Therapies have been developed to overcome this common disorder; however, limitations exist with these treatments that often lead to complications including bronchopulmonary dysplasia (BPD). One approach to address RDS is to implement a microfluidic oxygenator that serves as a respiratory support system for preterm neonates while the lungs fully develop, extra-uterine. This artificial lung assist device (LAD) is characterised by its non-invasiveness (given that it is connected via umbilical vessels), pumpless configuration, ambient air operation, portability and low priming volume. Furthermore, the LAD is formed by single oxygenator units (SOU) that are stacked in a parallel array which allows for usage on different body weights. The objective of this thesis is to design an electrochemical system to provide an in-situ enriched O2 environment able to supply 1.9 ml O2/min for use in the SOU while maintaining the simplicity of operation of the oxygenator. An inexpensive, electrically powered and compact device was envisioned allowing for a higher permeation flux to fully oxygenate the blood. Moreover, the system would be easy to manufacture, low maintenance and avoid the risk of gas contamination. In the initial work, different designs of electrolytic cells were developed and tested. The two- chamber design connected by a gel membrane showed an O2 production 10 times higher than with previous designs with 42 mg O2/L. Subsequently, different supporting electrolytes were tested. NaOH demonstrated a better performance and no degradation of the electrode in contrast to NaCl and Na2SO4. Stainless steel mesh (SSM) and graphite sheet electrodes were then tested; it was observed that stainless steel produced 3.4 times more dissolved oxygen (DO) than graphite with 28.3 mg O2/L. Experimentation with electrolysis of water showed that the DO in water reached stability 3 min after the electrolysis process was initiated measuring a change of DO of 29 mg/L at 3 A. Furthermore, an active oxygenation (AO) system was developed for in-vitro experiments via electrolysis of water and compared to a passive oxygenation (PO) system exposing blood to enriched O2 air and ambient air, respectively. It was demonstrated that AO provided 300% greater oxygenation to blood than PO. The electrolysis chamber designed for the microfluidic oxygenator allows the oxygenator to maintain its essential characteristics of simplicity and low cost while increasing the rate of oxygenation of blood. Preterm neonates suffering from RDS need an artificial lung that can partially support the oxygenation of their blood. Thus, combining the oxygenator with the O2 generation in-situ system enables a greater blood O2 uptake of 300% making possible the development of an efficient artificial lung. / Thesis / Master of Applied Science (MASc)

A clinical need exists for a blood oxygenator and pumping system for the support of neonates with respiratory deficiencies. Such systems now available for support of adults are not suitable for neonatal patients. In vitro evaluation of a new blood oxygenator and blood pumping system was performed. The data obtained suggested that this system may be applicable to neonates. In vivo studies with rabbits to further analyze the new system were done. Preliminary data from these studies indicate that the new blood oxygenator and blood pump system may be applicable to supporting neonates with respiratory deficiencies. Suggestions for future development of this system are presented. / Master of Science

Thesis (MMed) -- Stellenbosch University, 2010. / ENGLISH ABSTRACT: Introduction: Limited facilities exist at rural hospitals for the management of newborn infants with respiratory distress syndrome (RDS). Furthermore, the secondary and tertiary hospitals are under severe strain to accept all the referrals from rural hospitals. Many of these infants require intubation and ventilation with a resuscitation bag which must be sustained for hours until the transport team arrives. Not only is lung damage inflicted by the prolonged ventilation, but transferring the infant by helicopter and ambulance is expensive. CPAP (continuous positive airway pressure), a non-invasive form of ventilatory support, has been used successfully at regional (Level 2) and tertiary (Level 3) neonatal units, to manage infants with RDS. It is cost-effective for infants with mild to moderate grades of RDS to be managed at the rural hospital instead of being transferred to the regional secondary or tertiary hospital. CPAP was introduced to Ceres Hospital, a rural Level 1 hospital, in February 2008 for the management of infants with RDS. Aim: To determine the impact of CPAP on the management of infants with RDS in a rural level 1 hospital and whether it can reduce the number of referrals to regional hospitals. Study setting: Nursery at Ceres District Hospital, Cape Winelands District, Western Cape. Study design: Prospective cohort analytical study with an historic control group (HCG). Patients and Methods: The study group (SG) comprised all neonates with respiratory distress born between 27/02/2008 and 26/02/2010. The infants were initially resuscitated with a Neopuff® machine in labour-ward and CPAP was commenced for those with RDS. The survival and referral rates of the SG were compared to an historic control group (HCG) of infants born between 1/2/2006 to 31/01/2008 at Ceres Hospital. Results: During the 2 years of the study, 51 neonates received CPAP (34 <1800g, 17>1800g). Twenty (83%) of the SG infants between 1000g and 1800g and 23 (68%) of the infants between 500g and 1800g survived. Those <1800g that failed CPAP, had either a severe grade of RDS which required intubation and ventilation or were <1000g. Seventeen (33%) of the infants that received CPAP, were in the >1800g group. Thirteen (76%) of these infants were successfully treated with CPAP only. The four infants that failed CPAP suffered from congenital abnormalities and would not have benefited from CPAP. There was no statistically significant difference in the survival between the SG and HCG (80%) (p=0.5490) but the number of referrals decreased significantly from 21% in the HCG to 7% in the SG (p=0.0003). No complications related to CPAP treatment, such as pneumothorax, were noted. The nursing and medical staff quickly became proficient and confident in applying CPAP and were committed to the project. Conclusion: CPAP can be safely and successfully practised in infants with mild to moderate RDS in a rural Level 1 hospital. The survival rate stayed the same as the HCG, even though a higher risk infants were treated in the SG. The transfers were significantly reduced from 21% to 7%. This resulted in significant cost savings for the hospital. / AFRIKAANSE OPSOMMING: geen opsomming

Einleitung: Trotz jahrzehntelanger Forschung bleibt die lungenprotektive Beatmung bei Menschen mit Lungenkontusion und daraus resultierendem ARDS ein kontrovers diskutiertes Thema. Als bisheriger Goldstandard gilt das ARDSnet Protokoll. Es zeichnet sich durch ein niedriges Tidavolumen (6ml/kg), einen maximal zu erreichenden Plateaudruck von 30cmH2O und tabellarisch festgelegten Kombinationen von Positivem Endexspiratorischen Druck (PEEP) und Inspiratorischer Sauerstoffkonzentration (FiO2) aus. Die Generierung einer akzeptablen Oxygenierung bei minimaler Volu- und Barotraumabelastung ist das Ziel. Im Gegensatz dazu steht das OPEN LUNG Konzept (OLC), bei welchem zu Beginn der Therapie ein Rekrutierungsmanöver zum Öffnen aller Alveolen durchgeführt wird. Anschließend erfolgt die Beatmung mit titriertem, hohem PEEP Niveau, sodass die Lunge offen gehalten werden kann. Tidalvolumen und Sauerstoffpartialdruck werden so gering wie möglich eingestellt. Ziele der Arbeit: Ein Ziel dieser Arbeit war es, auf histologischer Basis zu evaluieren, ob es einen beatmungsassoziierten Unterschied der Lungenschädigung nach experimentell erfolgtem Thoraxtrauma am Modell Schwein gibt. Darüber hinaus sollten als zweites Ziel dieser Arbeit zwei histologische Analysemethoden in ihrer Anwendbarkeit überprüft und miteinander verglichen werden. Tiere, Material und Methoden: Als Versuchstiere gingen 17 weibliche, 10-12 Wochen alte Schweine der Deutschen Landrasse in den Versuch ein. Nach experimentell induziertem Thoraxtrauma (Fallrohr-assoziiert) wurden die Tiere in zwei Beatmungsgruppen randomisiert und über 24 Stunden nach ARDSnet oder OLC Protokoll beatmet. Nach erfolgter Euthanasie der Tiere am Ende des Versuches wurden Lungenproben aus insgesamt 9 Arealen der Lobi caudales (rechts dorsal, rechts medial, rechts ventral, rechts Kontusion, rechts Randkontusion, links dorsal, links medial, links ventral und links Contre Coup) gewonnen und fixiert. Als Analysemethoden standen der DAD Score (semiquantitative Analyse) und ein für diese Arbeit entwickelter Grid Score, der an Hand eines Punktegrids die Auswertung der Präparate erweitern und verfeinern sollte, zur Verfügung. Die statistische Auswertung des Datenpools erfolgte durch die Anwendung des Kolmororov-Smirnov-Tests, gefolgt von dem Mann-Whitney-U-Test (p<0,05). Für die Analyse der Interobserver- und Intraobserver-Übereinstimmung wurde die Bland-Altman-Methode genutzt. Ergebnisse: Es konnte ein klarer Unterschied im histologischen Schädigungsbild zwischen den Beatmungskonzepten OLC und ARDSnet in beiden Analysemethoden detektiert werden. Dieser beschränkte sich auf den Bereich „rechts dorsal“. In allen Kriterien des DAD Scores (intraalveoläres Ödem, Hämorrhagie und Inflammation) zeigte sich ein signifikant geringeres Schädigungsbild der Lunge im OLC verglichen mit dem ARDSnet Protokoll (Mittelwerte der Scoringpunkte für intraalveoläres Ödem: 1,2 im OLC und 4,2 im ARDSnet; Hämorrhagie: 1,2 im OLC und 3,3 im ARDSnet; Inflammation: 3,3 im OLC und 7,5 im ARDSnet). In der Grid Score Methode wurde diese Aussage bei den Kriterien „Luft“ (51,2% im OLC und 17,9% im ARDSnet), „Zellen“ (24,6% im OLC und 41,2% im ARDSnet), „Blut“ (1,9% im OLC und 12,5% im ARDSnet), „Ödem-Blut-Gemisch“ (2% im OLC und 9% im ARDSnet) und „Gewebe gesamt“ (48,8% im OLC und 82,1% im ARDSnet) bestätigt. Das Kriterium „Hyaline Membran“ wurde im OLC signifikant häufiger ermittelt (0,9% im OLC und 0,3% im ARDSnet). Im Kriterium „Ödem“ des Grid Scores konnte kein signifikanter Unterschied zwischen beiden Beatmungskonzepten evaluiert werden (19,6% im OLC und 19,3% im ARDSnet). Schlussfolgerung: Der histologisch sichtbare Lungenschaden nach Thoraxtrauma ist nach 24 Stunden OLC-Beatmung signifikant geringer als mit ARDSnet-Beatmung. Damit bietet das OLC die bessere Möglichkeit zur Regeneration. Da sekundäre Schäden bis hin zum Multiorganversagen auf ein Thoraxtrauma folgen können, sollte in weiteren Untersuchungen zusätzlich die Reaktion des gesamten Körpers verglichen werden. In Abhängigkeit des Schädigungsmusters ist dann eine entsprechende intensivmedizinische Behandlung zu wählen. Im Vergleich der Scoringmethoden kann zusammengefasst werden, dass beide Methoden durchaus die histologischen Unterschiede des Lungenschadens beider Beatmungskonzepte quantifizieren konnten, allerdings Vor- und Nachteile bergen. Deutlich wurde im Verlauf der Untersuchungen, dass in der Grid Score Methode einzelne Kriterien genauer definiert werden müssen. Generell verspricht die Grid Score Methode jedoch einen erheblichen Informationszuwachs im Vergleich zum DAD Score.:Abkürzungsverzeichnis 1 Einleitung 1 2 Literaturübersicht 2 2.1 Anatomie und Physiologie der Lunge 2 2.1.1 Makroskopischer Aufbau und Funktion 2 2.1.2 Histologie des Alveolarraumes 3 2.2 Thoraxtrauma und Lungenkontusion 5 2.2.1 Lungenkontusion 5 2.2.1.1 Definition und Charakter der Lungenkontusion 5 2.2.1.2 Geschichtlicher Hintergrund der Forschung zur Lungenkontusion 6 2.2.1.3 Modellmöglichkeiten zur Nachstellung einer Lungenkontusion in der Forschung 6 2.2.2 Thoraxtrauma und Lungenkontusion in der Tiermedizin- Vorkommen, Häufigkeit und Ursachen 7 2.3 Diffuse Alveolar Damage und Acute Respiratory Distress Syndrome- Definition und histologischer Hintergrund 9 2.3.1 Diffuse Alveolar Damage 9 2.3.2 Acute Respiratory Distress Syndrome 10 2.4 Therapiekonzepte einer Lungenkontusion 12 2.4.1 Das ARDS Netzwerk Protokoll 12 2.4.2 Das Open Lung Concept 13 2.4.3 Beatmungsassoziierte Schäden- VALI/VILI 14 2.4.4 Behandlung einer Lungenkontusion in der Tiermedizin 16 2.5 Histologische Analysen pulmonaler Schädigung 17 2.5.1 Der Diffuse Alveolar Damage Score 17 2.5.2 Der Grid Score zur Quantifizierung histologischer Veränderungen des Lungenparenchyms 19 3 Tiere, Material und Methoden 20 3.1 Versuchstiere 20 3.2 Versuchsablauf 21 3.2.1 Prämedikation und Instrumentierung 22 3.2.2 Ablauf aller Messmanöver eines Messzeitpunktes 25 3.2.3 Thoraxkontusion und Prähospitale Phase 27 3.2.4 Randomisierung und Einstellung der Beatmung 29 3.2.5 Euthanasie des Tieres und Probenentnahme 30 3.2.5.1 Sektion des Versuchstieres 30 3.2.5.2 Probenentnahme 31 3.2.5.3 Probenentnahme aus der Lunge zur histologischen Auswertung – Fokus dieser Arbeit 31 3.3 Aufbereitung des Gewebes für die histologische Auswertung 32 3.3.1 Fixierung und Einbettung 32 3.3.2 Hämatoxylin-Eosin-Färbung 33 3.3.3 Mikroskopie und Digitalisierung der histologischen Präparate 34 3.4 Auswertung der histologischen Schnitte 34 3.4.1 Diffuse Alveolar Damage Score nach SPIETH et al. 2007 und 2011 34 3.4.2 Grid Score 35 3.4.3 Statistische Auswertung 38 4 Ergebnisse 39 4.1 DAD Score Übersicht 39 4.2 Grid Score Übersicht 40 4.3 Parallele Betrachtung des DAD und GRID Scores im dorsalen Bereich des Lobus caudalis dexter (RD) 42 4.4 Ergänzende Betrachtung des dorsalen Bereiches des Lobus caudalis dexter (RD) durch die Grid Score Methode 44 4.5 Ergebnisse der erweiterten Grid Score Methode 46 4.6 Gesamtübersicht des dorsalen Bereiches des Lobus caudalis dexter (RD) 47 4.7 Probleme der Grid Score Methode und Neubewertung der Vorgehensweise 48 4.8 Evaluierung der Scoringmethoden mittels Inter- und Intraobserververgleichen 51   4.9 Ergebnisse der Gebiete „Kontusion- rechte Lunge“, „Randkontusion- rechte Lunge“ und „Contre-Coup Bereich der linken Lunge“ 54 4.10 Entwicklung eines ARDS nach Berlin Definition in den unterschiedlichen Beatmungsgruppen 55 5 Diskussion 57 5.1 Vergleich der Beatmungskonzepte auf histologischer Ebene 57 5.2 Vergleich der Methoden DAD Score und Grid Score 66 6 Zusammenfassung 69 7 Summary 71 8 Literaturverzeichnis 73 9 Anhang 81 10 Danksagung 87

We have demonstrated that simvastatin and sphingosine 1-phosphate (S1P) both attenuate increased vascular permeability in preclinical models of acute respiratory distress syndrome. However, the underlying mechanisms remain unclear. As Kruppel-like factor 2 (KLF2) serves as a critical regulator for cellular stress response in endothelial cells (EC), we hypothesized that simvastatin enhances endothelial barrier function via increasing expression of the barrier-promoting S1P receptor, S1PR1, via a KLF2-dependent mechanism. S1PR1 luciferase reporter promoter activity in human lung artery EC (HPAEC) was tested after simvastatin (5 mu M), and S1PR1 and KLF2 protein expression detected by immunoblotting. In vivo, transcription and expression of S1PR1 and KLF2 in mice lungs were detected by microarray profiling and immunoblotting after exposure to simvastatin (10 mg/kg). Endothelial barrier function was measured by trans-endothelial electrical resistance with the S1PR1 agonist FTY720-(S)-phosphonate. Both S1PR1 and KLF2 gene expression (mRNA, protein) were significantly increased by simvastatin in vitro and in vivo. S1PR1 promoter activity was significantly increased by simvastatin (P < 0.05), which was significantly attenuated by KLF2 silencing (siRNA). Simvastatin induced KLF2 recruitment to the S1PR1 promoter, and consequently, significantly augmented the effects of the S1PR1 agonist on EC barrier enhancement (P < 0.05), which was significantly attenuated by KLF2 silencing (P < 0.05). These results suggest that simvastatin upregulates S1PR1 transcription and expression via the transcription factor KLF2, and consequently augments the effects of S1PR1 agonists on preserving vascular barrier integrity. These results may lead to novel combinatorial therapeutic strategies for lung inflammatory syndromes.

A malária é uma doença causada pelo parasita do gênero Plasmodium e que foi responsável por cerca de 440.000 mortes em 2015. A síndrome do desconforto respiratório agudo (SDRA) é uma das principais complicações clínicas da malária. O modelo murino DBA/2 reproduz os sinais clínicos da SDRA observados em humanos, quando infectado com o Plasmodium berghei ANKA. Além disso, altos níveis da enzima heme oxigenase 1 (HO-1) foram observados em casos de malária cerebral e em SDRA em humanos. Os nossos dados indicam que os níveis da HO-1 estão aumentados em camundongos que desenvolvem SDRA associada à malária (SDRA-AM). Adicionalmente, a droga indutora de HO-1 (hemina) aumentou a sobrevivência e preveniu a SDRA-AM. Verificou-se também uma redução na permeabilidade pulmonar e nos níveis de VEGF, além de uma melhoria nos parâmetros respiratórios em animais tratados com hemina. Assim sendo, a indução da HO-1 antes do desenvolvimento da SDRA-AM é protetora e assim, a HO-1 pode ser um alvo de novos fármacos, como forma de prevenir o desenvolvimento da SDRA-AM em humanos. / Malaria is a serious disease, caused by the parasite of the genus Plasmodium, which was responsible to 440,000 deaths in 2015. Acute lung injury/ acute respiratory distress syndrome (ALI/ARDS) is one of the main clinical complications in severe malaria. The murine model DBA/2 reproduces the clinical signs of ALI/ARDS observed in humans, when infected with Plasmodium berghei ANKA. Additionally, high levels heme oxygenase 1 (HO-1) were reported in cases of cerebral malaria and in ALI/ARDS in humans. Our data have indicated that the HO-1 levels are increased in mice that develop malaria associated ALI/ARDS (MA-ALI/ARDS). Additionally, a HO-1 inducing drug (hemin) increased the survival rate and prevented mice from developing MA-ALI/ARDS in treated mice. Also, there was a decrease in the lung permeability and in lung VEGF levels, and an amelioration of respiratory parameters. Therefore, the induction of HO-1 before the development of MA-ALI/ARDS is protective, making this enzyme a possible target of new drugs to prevent the development of MA-ALI/ARDS in humans.

A malária é um problema de saúde global, que hoje acomete aproximadamente 207 milhões de pessoas, e que levou ao óbito cerca de 607.000 indíviduos apenas no ano de 2013. No Brasil, 99% dos casos concentram-se na Amazônia Legal onde infecções por Plasmodium vivax são as principais causas da doença e podem ser fatais. Infecções por Plasmodium spp. podem levar à um quadro respiratório grave, com complicações pulmonares denominadas lesão pulmonar aguda (LPA) e síndrome do desconforto respiratório agudo (SDRA). A LPA/SDRA é caracterizada pela lesão dos alvéolos e do parênquima pulmonar, com perda da função da barreira epitelial do alvéolo e do capilar pulmonar de células endoteliais e, consequentemente, presença de edema pulmonar de origem não-cardiogênica. A diminuição da capacidade de trocas gasosas, aumento da atividade leucocitária e de mediadores inflamatórios nos pulmões resultam em insuficiência respiratória. A dificuldade em se estudar a doença em humanos associado ao desconhecimento dos fatores envolvidos na síndrome faz com que essa disfunção pulmonar torne-se mal compreendida e leve cada vez mais pessoas a óbito. O objetivo central do presente trabalho foi reconhecer e caracterizar o perfil leucocitário pulmonar, os fatores inflamatórios e a morte celular que contribuem para o desenvolvimento da LPA/SDRA associada a malária. Neste trabalho foi utilizado como modelo experimental a associação entre camundongos da linhagem DBA/2 e o parasita murino Plasmodium berghei ANKA. Neste modelo 30-75% dos camundongos desenvolvem sintomas pulmonares agudos (LPA/SDRA) e os demais morrem tardiamente com hiperparasitemia (HP). Desenvolvemos a partir dos parâmetros respiratórios e da parasitemia um modelo preditivo para classificação dos animais em LPA/SDRA ou HP antes do momento da morte com alta sensibilidade e especificidade. Nossos resultados mostram que os macrófagos alveolares e os neutrófilos estão aumentados de maneira significativa nos animais classificados como LPA/SDRA e que a depleção dessas populações promove a sobrevida dos animais e o não desenvolvimento da síndrome. Verificamos também que as células TCD4+ e TCD8+ produzem grandes quantidades de IFN-? nos animais LPA/SDRA, contudo o sistema imunológico desses animais produz grandes quantidades de IL-10 como forma de regular a resposta inflamatória. Em nosso estudo mensuramos a apoptose no tecido pulmonar e verificamos que os animais com LPA/SDRA possuem um número maior de células morrendo em relação aos animais HP. Vimos também que a apoptose de neutrófilos e células dendríticas ocorrem de maneira significativa no lavado broncoalveolar dos animais LPA/SDRA. O estudo da expressão de genes pró e anti-apoptóticos mostrou que há o aumento da expressão de Casp-3, Casp-9, Bad, Bid, Bak, FADD, CAD e Ripk-1 nos animais LPA/SDRA, enquanto que Bcl-XL e Bcl2 estão mais expressos nos animais HP. / Malaria is a global health problem that now affects approximately 207 million people, and led to the deaths of about 607,000 individuals only in 2013. In Brazil, 99% of the cases are concentrated in the Amazon where infections by Plasmodium vivax are the major cause of morbidity and which can also be fatal. Infections with Plasmodium spp. can lead to a serious respiratory condition including pulmonary complications named as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). ALI /ARDS is characterized by damage to the alveoli and the lung parenchyma, loss of epithelial barrier function of the alveoli and pulmonary capillary endothelial cells and, consequently, pulmonary edema noncardiogenic origin. Decreased capacity for gas exchange, increased leukocyte activity and inflammatory mediators in the lungs resulting in respiratory failure. The difficulty in studying human disease associated with lack of knowledge of factors involved in the syndrome and makes the pulmonary dysfunction become misunderstood and take more people to death. The central objective of this study was to determine and characterize the lung leukocytes profile, inflammatory factors and cell death that contribute to the development of ALI / ARDS associated with malaria. In this work was used as an experimental model the association between DBA/2 mice strain and the Plasmodium berghei ANKA murine parasite. In this model, 30-75% of mice develop acute pulmonary symptoms (ALI/ARDS) and the others died too late with hyperparasitaemia (HP). Developed from the respiratory parameters and parasitaemia a predictive model for classification of animals in ALI/ARDS or HP before the moment of death with high sensitivity and specificity. Our results show that alveolar macrophages and neutrophils were increased significantly in animals classified as ALI/ARDS and the depletion of these populations promotes the survival of the animals and not development of the syndrome. We also observed that CD4+ and CD8+ T cells produce large amounts of IFN-? in animals ALI/ARDS, however the immune system of these animals produce large amounts of IL-10 in order to regulate the inflammatory response. In our study we measured apoptosis in lung tissue and found that animals with ALI/ARDS have a larger number of cells dying compared to HP animals. We also saw that apoptosis of neutrophils and dendritic cells occur significantly in BAL of animals ALI/ARDS. The study of the expression of pro and anti-apoptotic genes showed that there is increased expression of Casp-3, Casp-9, Bad, Bid, Bak, FADD, and Ripk CAD-1 in animals ALI/ARDS, as that Bcl XL and Bcl2 are more expressed in HP animals.

Introdução: Vários modelos experimentais para o estudo da síndrome do desconforto respiratório do tipo agudo (SDRA) foram desenvolvidos, sendo o modelo de lavagem pulmonar o mais utilizado. No entanto, a técnica originalmente descrita foi modificada por outros autores, tornando difícil a reprodutibilidade deste modelo experimental. Objetivos: Avaliar os efeitos do recrutamento pulmonar, do uso de volumecorrente fixo e do uso de diferentes volumes de lavagem na instalação do modelo experimental de SDRA, em relação ao número de lavagens necessárias para a obtenção do modelo experimental, a mortalidade e a estabilidade hemodinâmica durante o procedimento. Metodologia: Coelhos adultos da raça New-Zealand-White, foram divididos em 5 grupos de estudo, de acordo com a técnica utilizada para a lavagem pulmonar: 1- Volume-corrente (Vt) fixo de 10 ml/kg, volume de lavagem de 30 ml/kg, sem recrutamento pulmonar; 2- Pressão inspiratória (Pinsp) fixa, com um volume de lavagem de 30 ml/kg, sem recrutamento pulmonar; 3- Vt fixo, com um volume de lavagem de 25 ml/kg sem recrutamento pulmonar; 4- Pinsp fixa, com volume de lavagem de 25 ml/kg sem recrutamento pulmonar; 5- Vt fixo, com volume de lavagem de 30 ml/kg, com recrutamento pulmonar antes da primeira lavagem. Os animais foram submetidos a repetidas lavagens pulmonares com soro fisiológico aquecido em intervalos de 5 minutos, até se atingir o critério de definição de SDRA, estabelecido como uma relação PaO2/FiO2 <= 100. Resultados: Não foram encontradas diferenças entre os grupos em relação ao número de lavagens necessárias para a instalação do modelo experimental. O uso de recrutamento alveolar prévio e a utilização de pressão inspiratória fixa com volume de lavagem de 25 ml/kg foi associado a uma tendência à maior mortalidade. Embora não se tenha observado diferenças na estabilidade hemodinâmica entre os grupos de estudo, os animais ventilados com Pinsp fixa apresentaram uma pior ventilação alveolar com valores mais baixos de pH em relação aos animais ventilados com Vt fixo. Conclusões: A utilização de manobra de recrutamento alveolar, a uso de um volume-corrente fixo ou pressão inspiratória fixa entre as lavagens, e a utilização de diferentes volumes de lavagem (25 e 30 ml/kg) não modificaram o número de lavagens necessárias para a obtenção do modelo experimental de SDRA, assim como não modificaram a estabilidade hemodinâmica dos animais durante a realização do procedimento. Foi observada uma tendência à maior mortalidade com a realização da manobra de recrutamento alveolar e com o uso de pressão inspiratória fixa associado ao volume de lavagem de 25 ml/kg. / Background: Many experimental models were developed for the study of the acute respiratory distress syndrome (ARDS), and the lung lavage model is the more frequently used. The original technique was modified by many authors, resulting in difficulties for this experimental model reproducibility. Objectives: To evaluate the effects of the pulmonary recruitment, the use of fixed tidal-volume and different lavage volumes at the experimental ARDS model installation, regarding to the number of lung lavages necessary to obtain the experimental model, the mortality and the hemodynamic stability during the procedure. Methods: New-Zealand-White adult rabbits were divided into 5 study groups, according to the technique used: 1- Fixed tidal-volume (Vt) of 10 ml/kg, lavage volume of 30 ml/kg, no pulmonary recruitment; 2- Fixed inspiratory pressure (IP), lavage volume of 30 ml/kg, no pulmonary recruitment; 3- Fixed Vt, lavage volume of 25 ml/kg, no pulmonary recruitment; 4- Fixed IP, lavage volume of 25 ml/kg, no pulmonary recruitment; 5- Fixed Vt, lavage volume of 30 ml/kg, using pulmonary recruitment. The animals were submitted to repeated lung lavages with warm saline at 5 min interval until the ARDS definition(PaO2/FiO2 <= 100) be reached. Results: There was no differences among the study groups regarding the number of lung lavages necessary to obtain the experimental model. The use of alveolar recruitment before the first lavage and the use of fixed ventilatory pressure with 25 ml/kg lavage volume were associated with trend to a higher mortality rate. Although there were no differences regarding the hemodynamic stability among the study groups, animals ventilated with fixed inspiratory pressure had worse alveolar ventilation with higher levels of PaCO2 and lower pH. Conclusions: The use of alveolar recruitment maneuvers, the use of a fixed tidal-volume or inspiratory pressure between the lung lavages and the utilization of different lavage volumes did not change the number of lung lavages necessary to obtain the experimental model of ARDS or the hemodynamic stability of the animals during the procedure. It was observed a trend to an increased mortality rate with the recruitment maneuver and with the use of a fixed inspiratory pressure associated to the lavage volume of 25 ml/kg.

Introdução: Vários modelos experimentais para o estudo da síndrome do desconforto respiratório do tipo agudo (SDRA) foram desenvolvidos, sendo o modelo de lavagem pulmonar o mais utilizado. No entanto, a técnica originalmente descrita foi modificada por outros autores, tornando difícil a reprodutibilidade deste modelo experimental. Objetivos: Avaliar os efeitos do recrutamento pulmonar, do uso de volumecorrente fixo e do uso de diferentes volumes de lavagem na instalação do modelo experimental de SDRA, em relação ao número de lavagens necessárias para a obtenção do modelo experimental, a mortalidade e a estabilidade hemodinâmica durante o procedimento. Metodologia: Coelhos adultos da raça New-Zealand-White, foram divididos em 5 grupos de estudo, de acordo com a técnica utilizada para a lavagem pulmonar: 1- Volume-corrente (Vt) fixo de 10 ml/kg, volume de lavagem de 30 ml/kg, sem recrutamento pulmonar; 2- Pressão inspiratória (Pinsp) fixa, com um volume de lavagem de 30 ml/kg, sem recrutamento pulmonar; 3- Vt fixo, com um volume de lavagem de 25 ml/kg sem recrutamento pulmonar; 4- Pinsp fixa, com volume de lavagem de 25 ml/kg sem recrutamento pulmonar; 5- Vt fixo, com volume de lavagem de 30 ml/kg, com recrutamento pulmonar antes da primeira lavagem. Os animais foram submetidos a repetidas lavagens pulmonares com soro fisiológico aquecido em intervalos de 5 minutos, até se atingir o critério de definição de SDRA, estabelecido como uma relação PaO2/FiO2 <= 100. Resultados: Não foram encontradas diferenças entre os grupos em relação ao número de lavagens necessárias para a instalação do modelo experimental. O uso de recrutamento alveolar prévio e a utilização de pressão inspiratória fixa com volume de lavagem de 25 ml/kg foi associado a uma tendência à maior mortalidade. Embora não se tenha observado diferenças na estabilidade hemodinâmica entre os grupos de estudo, os animais ventilados com Pinsp fixa apresentaram uma pior ventilação alveolar com valores mais baixos de pH em relação aos animais ventilados com Vt fixo. Conclusões: A utilização de manobra de recrutamento alveolar, a uso de um volume-corrente fixo ou pressão inspiratória fixa entre as lavagens, e a utilização de diferentes volumes de lavagem (25 e 30 ml/kg) não modificaram o número de lavagens necessárias para a obtenção do modelo experimental de SDRA, assim como não modificaram a estabilidade hemodinâmica dos animais durante a realização do procedimento. Foi observada uma tendência à maior mortalidade com a realização da manobra de recrutamento alveolar e com o uso de pressão inspiratória fixa associado ao volume de lavagem de 25 ml/kg. / Background: Many experimental models were developed for the study of the acute respiratory distress syndrome (ARDS), and the lung lavage model is the more frequently used. The original technique was modified by many authors, resulting in difficulties for this experimental model reproducibility. Objectives: To evaluate the effects of the pulmonary recruitment, the use of fixed tidal-volume and different lavage volumes at the experimental ARDS model installation, regarding to the number of lung lavages necessary to obtain the experimental model, the mortality and the hemodynamic stability during the procedure. Methods: New-Zealand-White adult rabbits were divided into 5 study groups, according to the technique used: 1- Fixed tidal-volume (Vt) of 10 ml/kg, lavage volume of 30 ml/kg, no pulmonary recruitment; 2- Fixed inspiratory pressure (IP), lavage volume of 30 ml/kg, no pulmonary recruitment; 3- Fixed Vt, lavage volume of 25 ml/kg, no pulmonary recruitment; 4- Fixed IP, lavage volume of 25 ml/kg, no pulmonary recruitment; 5- Fixed Vt, lavage volume of 30 ml/kg, using pulmonary recruitment. The animals were submitted to repeated lung lavages with warm saline at 5 min interval until the ARDS definition(PaO2/FiO2 <= 100) be reached. Results: There was no differences among the study groups regarding the number of lung lavages necessary to obtain the experimental model. The use of alveolar recruitment before the first lavage and the use of fixed ventilatory pressure with 25 ml/kg lavage volume were associated with trend to a higher mortality rate. Although there were no differences regarding the hemodynamic stability among the study groups, animals ventilated with fixed inspiratory pressure had worse alveolar ventilation with higher levels of PaCO2 and lower pH. Conclusions: The use of alveolar recruitment maneuvers, the use of a fixed tidal-volume or inspiratory pressure between the lung lavages and the utilization of different lavage volumes did not change the number of lung lavages necessary to obtain the experimental model of ARDS or the hemodynamic stability of the animals during the procedure. It was observed a trend to an increased mortality rate with the recruitment maneuver and with the use of a fixed inspiratory pressure associated to the lavage volume of 25 ml/kg.

A malária é um problema de saúde global, que hoje acomete aproximadamente 207 milhões de pessoas, e que levou ao óbito cerca de 607.000 indíviduos apenas no ano de 2013. No Brasil, 99% dos casos concentram-se na Amazônia Legal onde infecções por Plasmodium vivax são as principais causas da doença e podem ser fatais. Infecções por Plasmodium spp. podem levar à um quadro respiratório grave, com complicações pulmonares denominadas lesão pulmonar aguda (LPA) e síndrome do desconforto respiratório agudo (SDRA). A LPA/SDRA é caracterizada pela lesão dos alvéolos e do parênquima pulmonar, com perda da função da barreira epitelial do alvéolo e do capilar pulmonar de células endoteliais e, consequentemente, presença de edema pulmonar de origem não-cardiogênica. A diminuição da capacidade de trocas gasosas, aumento da atividade leucocitária e de mediadores inflamatórios nos pulmões resultam em insuficiência respiratória. A dificuldade em se estudar a doença em humanos associado ao desconhecimento dos fatores envolvidos na síndrome faz com que essa disfunção pulmonar torne-se mal compreendida e leve cada vez mais pessoas a óbito. O objetivo central do presente trabalho foi reconhecer e caracterizar o perfil leucocitário pulmonar, os fatores inflamatórios e a morte celular que contribuem para o desenvolvimento da LPA/SDRA associada a malária. Neste trabalho foi utilizado como modelo experimental a associação entre camundongos da linhagem DBA/2 e o parasita murino Plasmodium berghei ANKA. Neste modelo 30-75% dos camundongos desenvolvem sintomas pulmonares agudos (LPA/SDRA) e os demais morrem tardiamente com hiperparasitemia (HP). Desenvolvemos a partir dos parâmetros respiratórios e da parasitemia um modelo preditivo para classificação dos animais em LPA/SDRA ou HP antes do momento da morte com alta sensibilidade e especificidade. Nossos resultados mostram que os macrófagos alveolares e os neutrófilos estão aumentados de maneira significativa nos animais classificados como LPA/SDRA e que a depleção dessas populações promove a sobrevida dos animais e o não desenvolvimento da síndrome. Verificamos também que as células TCD4+ e TCD8+ produzem grandes quantidades de IFN-? nos animais LPA/SDRA, contudo o sistema imunológico desses animais produz grandes quantidades de IL-10 como forma de regular a resposta inflamatória. Em nosso estudo mensuramos a apoptose no tecido pulmonar e verificamos que os animais com LPA/SDRA possuem um número maior de células morrendo em relação aos animais HP. Vimos também que a apoptose de neutrófilos e células dendríticas ocorrem de maneira significativa no lavado broncoalveolar dos animais LPA/SDRA. O estudo da expressão de genes pró e anti-apoptóticos mostrou que há o aumento da expressão de Casp-3, Casp-9, Bad, Bid, Bak, FADD, CAD e Ripk-1 nos animais LPA/SDRA, enquanto que Bcl-XL e Bcl2 estão mais expressos nos animais HP. / Malaria is a global health problem that now affects approximately 207 million people, and led to the deaths of about 607,000 individuals only in 2013. In Brazil, 99% of the cases are concentrated in the Amazon where infections by Plasmodium vivax are the major cause of morbidity and which can also be fatal. Infections with Plasmodium spp. can lead to a serious respiratory condition including pulmonary complications named as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). ALI /ARDS is characterized by damage to the alveoli and the lung parenchyma, loss of epithelial barrier function of the alveoli and pulmonary capillary endothelial cells and, consequently, pulmonary edema noncardiogenic origin. Decreased capacity for gas exchange, increased leukocyte activity and inflammatory mediators in the lungs resulting in respiratory failure. The difficulty in studying human disease associated with lack of knowledge of factors involved in the syndrome and makes the pulmonary dysfunction become misunderstood and take more people to death. The central objective of this study was to determine and characterize the lung leukocytes profile, inflammatory factors and cell death that contribute to the development of ALI / ARDS associated with malaria. In this work was used as an experimental model the association between DBA/2 mice strain and the Plasmodium berghei ANKA murine parasite. In this model, 30-75% of mice develop acute pulmonary symptoms (ALI/ARDS) and the others died too late with hyperparasitaemia (HP). Developed from the respiratory parameters and parasitaemia a predictive model for classification of animals in ALI/ARDS or HP before the moment of death with high sensitivity and specificity. Our results show that alveolar macrophages and neutrophils were increased significantly in animals classified as ALI/ARDS and the depletion of these populations promotes the survival of the animals and not development of the syndrome. We also observed that CD4+ and CD8+ T cells produce large amounts of IFN-? in animals ALI/ARDS, however the immune system of these animals produce large amounts of IL-10 in order to regulate the inflammatory response. In our study we measured apoptosis in lung tissue and found that animals with ALI/ARDS have a larger number of cells dying compared to HP animals. We also saw that apoptosis of neutrophils and dendritic cells occur significantly in BAL of animals ALI/ARDS. The study of the expression of pro and anti-apoptotic genes showed that there is increased expression of Casp-3, Casp-9, Bad, Bid, Bak, FADD, and Ripk CAD-1 in animals ALI/ARDS, as that Bcl XL and Bcl2 are more expressed in HP animals.