Estudo cinético de um episódio agudo de translocação bacteriana e de suas repercussões imunológicas e microcirculatórias em ratos

Introdução: Muitos pacientes ainda morrem devido a infecções. Crescentes relatos da literatura atual têm atribuído ao intestino o papel de agravamento de doenças graves, e/ou a sua participação na gênese da sepse por mecanismo de translocação bacteriana (TB). Objetivo: Avaliar de forma cinética a TB e suas repercussões microcirculatórias e imunológicas. Método: Ratos Wistar-EPM (n=162) foram aleatoriamente distribuídos em grupo Sham (n=72) e grupo TB (n=90), e avaliados nos períodos de 2h, 6h, 24h, 72h, 7 e 14 dias, em relação a índice de translocação bacteriana; microscopia intravital; perfusão tecidual; e componentes celulares e humorais da linfa mesentérica por linfograma, citometria de fluxo e CBA-Flex. Resultado: A TB ocorreu somente no grupo TB e foi expressiva nas primeiras 24 horas tornando-se negativa somente com 7 dias. A conseqüência de um episódio de TB repercutiu na celularidade e citocinas pró e antiinflamatórias da linfa mesentérica associado a lesões da microcirculação e hipoperfusão tecidual de forma local e sistêmica. A citometria de fluxo mostrou que a linfa mesentérica eferente pós TB difere significativamente do grupo Sham quanto a número e subpopulação de linfócitos. Conclusão: Um episódio agudo de TB determinou uma recuperação máxima bacteriana com 6 horas e sua completa depuração entre 3 e 7 dias, além de provocar alterações da microcirculação intestinal e sistêmica associadas à ativação do GALT, principalmente no período de permanência das bactérias translocadas no hospedeiro, sendo a via linfática mesenterial uma importante rota na intercomunicação imunológica entre o ambiente intestinal e sistêmico.

Applicazioni cliniche e sperimentali dell’ecografia toracica in pneumologia: la diagnostica precoce delle patologie pleuropolmonari

Lung ultrasound use is increasing in respiratory medicine thanks to its development in the latest years. Actually it allows to study diseases of the chest wall (traumas, infections, neoplasms), diaphragm (paralysis, ipokinesis), pleura (effusions, pneumothorax, thickenings, neoplasms) and lung parenchyma (consolidations, interstitial syndromes, peripheral lesions). One of the most useful application of chest ultrasound is the evaluation of effusions. However, no standardized approach for ultrasound-guided thoracenthesis is available. Our study showed that our usual ultrasonographic landmark (“V-point”) could be a standard site to perform thoracenthesis: in 45 thoracenthesis no pneumothorax occurred, drainage was always successful at first attempt. Values of maximum thickness at V-point and drained fluid volume showed a significative correlation. Proteins concentration of ultrasound patterns of effusions (anechoic, ipoechoic, moving echoic spots, dense moving spots, hyperechoic) were compared to those of the macroscopic features of fluids showing connection between light-yellow fluid and echoic moving spots pattern and between ipoechoic/dense moving spots and cloudy-yellow/serum-haematic fluids. These observations suggest that ultrasound could predict chemical-physical features of effusions. Lung ultrasound provides useful information about many disease of the lung, but actually there is not useful in obstructive bronchial diseases. Analysing diaphragmatic kinetics using M-mode through transhepatic scan we described a similarity between diaphragm excursion during an expiratory forced maneuver and the volume/time curve of spirometry. This allowed us to identify the M-mode Index of Obstruction (MIO), an ultrasound-analogue of FEV1/VC. We observed MIO values of normal subjects (9) and obstructed patients (9) comparing the two groups. FEV1/VC and MIO showed a significant correlation suggesting that MIO may be affected by airways obstruction; MIO values were significatively different between normal and obstructed so that it could identify an obstructive syndrome. The data show that it is possible to suspect the presence of obstructive syndrome of the airways using ultrasonography of the diaphragm.

Risultati funzionali a lungo termine dopo sutura o plicatura del diaframma

Obbiettivo. Analizzare la funzionalità polmonare e diaframmatica dopo interventi di plicatura del diaframma con rete di rinforzo peri-costale eseguiti per relaxatio e riparazione di ernia transdiaframmatica cronica mediante riduzione e sutura diretta. Metodi. Dal 1996 al 2010, 10 pazienti con relaxatio unilaterale del diaframma e 6 pazienti con ernia transdiaframmatica cronica misconosciuta sono stati sottoposti a chirurgia elettiva. Gli accertamenti preoperatori e al follow-up di 12 mesi includevano prove di funzionalità respiratoria, misura della pressione massimale inspiratoria alla bocca in clino e ortostatismo, emogasanlisi, TC del torace e dispnea score. Risultati. I pazienti dei due gruppi non differivano in termini di funzionalità respiratoria preoperatoria nè di complicanze postoperatorie; al follow-up a 12 mesi il gruppo Eventrazione mostrava un significativo aumento del FEV1% (+18,2 – p<0.001), FVC% (+12,8 – p<0.001), DLCO% (+6,84 – p=0,04) e pO2 (+9,8 mmHg – p<0.001). Al contrario nrl gruppo Ernia solo il miglioramento della pO2 era significativo (+8.3 – p=0.04). Sebbene la massima pressione inspiratoria (PImax) fosse aumentata in entrambi i gruppi al follow-up, i pazienti operati per ernia mostravano un miglioramento limitato con persistente caduta significativa della PImax dall’ortostatismo al clinostatismo (p<0.001). Il Transitional dyspnoea score è stato concordante con tali miglioramenti pur senza differenze significative tra i due gruppi. La TC del torace ha evidenziato una sopraelevazione dell’emidiaframma suturato, senza recidiva di ernia, mentre i pazienti sottoposti a plicatura hanno mantenuto l’ipercorrezione. Conclusioni. L’utilizzo di un rinforzo protesico è sicuro e sembra assicurare risultati funzionali migliori a distanza in termini di flussi respiratori e di movimento paradosso del diaframma (valutato mediante PImax). Lacerazioni estese del diaframma coinvolgenti le branche principali di suddivisione del nervo frenico si associano verosimilmente a una relaxatio che può quindi ridurre il guadagno funzionale a lungo termine se non adeguatamente trattata mediante l’utilizzo di un rinforzo protesico.

Modalita Nava come tecnica di monitoraggio e di ventilazione artificiale in terapia intensiva.

NAVA®, dall’inglese Neurally Adjusted Ventilatory Assist, è una tecnica di monitoraggio e di ventilazione e rappresenta una sofisticata innovazione tecnologica in quanto consente un’assistenza ventilatoria costantemente in armonia con le esigenze del paziente, grazie alla rilevazione diretta dell’attività elettrica del diaframma. Da un punto di vista pratico-operativo, NAVA è un modulo integrativo che si inserisce nelle apparecchiature di ventilazione già esistenti integrandone al meglio le funzionalità. A tale modulo è collegato un sondino nasogastrico nella cui parte distale vi sono elettrodi bipolari. Il sondino viene inserito nell’esofago vicino al diaframma e permette l’acquisizione del segnale Edi (Diaphragmatic Electrical Activity), relativo all’attività elettrica del diaframma. L’attività di quest ultimo è strettamente correlata alla dinamica respiratoria del paziente, in quanto il diaframma, contraendosi, determina il flusso d’aria all’interno delle vie aree. Il segnale Edi acquisito viene usato per interfacciarsi con il ventilatore e grazie speciali algoritmi, il segnale guida il ventilatore permettendo un’assistenza ventilatoria proporzionale e sincrona agli sforzi respiratori del paziente. NAVA è tra le nuove apparecchiature sanitarie ed elettromedicali che la Banca Popolare dell’Emilia Romagna ha recentemente donato al Centro Grandi Ustionati dell’Ospedale Bufalini di Cesena per un valore complessivo di oltre 120.000 euro. NAVA e gli altri strumenti donati sono apparecchiature di ultima generazione destinate a migliorare le possibilità di sopravvivenza dei pazienti più critici, con ustioni e ferite alla cute molto gravi, che necessitano di un’assistenza intensiva. In questo elaborato, nel Capitolo 1, viene presentata la Fisiologia dell’apparato respiratorio e a seguire, nel Capitolo 2, viene descritta la Ventilazione meccanica convenzionale, ancora oggi molto utilizzata. Successivamente, nel Capitolo 3 è illustrata la nuova modalità NAVA. Proseguendo, nel Capitolo 4 si apre un confronto tra le principali differenze tra la NAVA e le precedenti modalità di ventilazione. L’elaborato si conclude con la speranza che NAVA, un’innovazione senza precedenti, non sia limitata ad un investimento potenzialmente utile nel presente della terapia intensiva, ma che la ricerca ad essa correlata possa, in un imminente futuro, aprire la strada a nuove tecnologie ancora più efficienti nella salvaguardia dei pazienti in terapia intensiva.

Aspetti Ultrasonografici in Corso di Sviluppo Placentare e Adattamento Cardiovascolare nella Cavalla Gravida: ecografia bidimensionale, ecocardiografia ed ecocontrastografia (CEUS)

In corso di gravidanza normale avvengono modificazioni emodinamiche centrali e periferiche volte a garantire le crescenti richieste nutritive dell'unità feto-placentare. L’ecografia con mezzo di contrasto (CEUS-Contrast Enhanced Ultrasonography) a base di microbolle offre una nuova opportunità di monitorare e quantificare la perfusione utero-placentare in condizioni normali e patologiche. L’ecocardiografia è stata ampiamente usata in medicina umana per valutare l’adattamento morfo-funzionale cardiaco materno durante la gravidanza. Gli scopi di questo lavoro prospettico sono stati di applicare, per la prima volta nella specie equina, un mezzo di contrasto di II generazione (Sonovue®), al fine quantificare la perfusione utero-placentare in corso di gravidanza normale, valutandone gli effetti sul benessere materno-fetale e di descrivere le modificazioni nei parametri ecocardiografici morfometrici e funzionali cardiaci, in particolare relativi alla funzione del ventricolo sinistro nel corso di una gravidanza fisiologica. Due fattrici sane di razza Trottatore sono state monitorate ecograficamente in maniera seriale durante l’intero corso della gravidanza, tramite esame bidimensionale, ecocontrastografia dell'unità utero-placentare, flussimetria Doppler delle arterie uterine, ecocardiografia materna in modalità bidimensionale, M-mode, Doppler e Tissue Doppler Imaging. I neonati sono stati clinicamente monitorati e gli invogli fetali esaminati. Il pattern di microperfusione utero-placentare è valutabile quali-quantitativamente tramite la CEUS e dimostra un’aumento del flusso a livello di microvascolarizzazione uterina con l'avanzare della gravidanza; non è stata rilevata la presenza di microbolle a livello di strutture fetali nè effetti dannosi sul benessere materno-fetale. In questo studio sono state osservate delle modificazioni cardiache materne in corso di gravidanza fisiologica, relative all'aumento della FC, del CO ed in particolare all'aumento delle dimensioni dell'atrio sinistro ed a modificazioni nelle onde di velocità di flusso e tissutali di riempimento del ventricolo sinistro.

Vergleich der retinalen und dermalen mikrovaskulären endothelialen Funktion bei Gesunden, Insulinresistenten und Typ-2-Diabetikern

Die vorliegende klinische Studie hatte zum Ziel, die mikrovaskuläre Endothelfunktion retinaler und dermaler Gefäße von Insulinresistenten und Typ 2- Diabetikern ohne Zeichen einer diabetischen Retinopathie mit einer gesunden insulinsensitiven nicht-diabetischen Kontrollgruppe hinsichtlich früher morphologischer und funktioneller Veränderungen zu vergleichen.rnrnMethode:rnEs wurden 54 Patienten ohne Nachweis einer diabetischen Retinopathie eingeschlossen und in 3 Gruppen entsprechend ihren metabolischen Ergebnissen eingeteilt: 1.) Gruppe K (Kontrollgruppe) setzte sich aus gesunden, nicht-diabetischen, insulin-sensitiven rn(HOMA ≤ 2) Probanden mit einem BMI ≤ 28 kg/m2 zusammen; 2.) Gruppe IR bestand aus den nicht-diabetischen, insulin-resistenten (HOMAs > 2), übergewichtigen Patienten mit einem BMI > 28 kg/m2 und 3.) Gruppe DM war definiert als Patienten mit einem manifesten Typ 2-Diabetes mellitus.rnrnDie mikrovaskuläre Funktion der Retina wurde mittels eines Laserdoppler-Verfahrens (Heidelberg Retina Flowmeter) untersucht und hierbei der retinale Blutfluss und das Verhältnis der Gefäßwand zum Lumen (WLR, wall-to-lumen-ratio) basal und nach Flickerlicht-Stimulation (10 Hz, Photo Stimulator 750) gemessen. Letzterer gilt als Marker für vaskuläre Schädigung. rnZusätzlich wurde die dermale Mikrozirkulation (Blutfluss, O2-Sättigung) als weiterer Faktor der mikrovaskulären Endothelfunktion in den 3 Studiengruppen untersucht und miteinander verglichen.rnErgebnisse:rnEs zeigte sich kein signifikanter Unterschied des retinalen Blutflusses zwischen den 3 Gruppen weder basal noch nach Flickerlicht-Stimulation. Es zeigte sich keine Korrelation zwischen der mikrovaskulären Funktion der Haut und der Retina. rnDie arterielle WLR zeigte nur geringe Unterscheide zwischen den 3 Gruppen.rnrnMit zunehmendem Grad der Insulinresistenz wurde jedoch eine Reduktion des basalen als auch des flickerlicht-stimulierten retinalen Blutflusses deutlich, dabei zeigte sich unerwarteter Weise eine Abnahme der WLR.rnrnDer (prä-ischämische) muskuläre Blutfluss war in der IR-Gruppe signifikant geringer als in der K-Gruppe. Auch war die postischämische dermale O2-Sättigung in der DM und IR-Gruppe signifikant niedriger im Vergleich zur K-Gruppe. Jedoch war die postischämische hyperämische dermale Reaktion in der IR und DM-Gruppe nur geringgradig weniger als in der K-Gruppe. rnrnSchlussfolgerung:rnEine Korrelation zwischen der Entwicklung der Insulinresistenz und retinaler sowie dermaler mikrovaskulärer endothelialer Funktion wurde bei der Studie deutlich. Mithilfe des neuen Verfahrens der Laser Scanner Flowmeter zur Messung der retinalen Endothelfunktion lassen sich sehr frühe morphologische Veränderungen des mikrovaskulären Blutflusses erfassen. rnDie fehlende Korrelation zwischen retinaler und dermaler mikrovaskulärer Funktion als auch die geringen Unterschiede der WLR sollte Gegenstand weiterer Studien seinrn

3D-ultrastructure, functions and stress responses of rhogocytes from the freshwater gastropods Biomphalaria glabrata and Lymnaea stagnalis

Rhogocytes, also termed ‘pore cells’, exist free in the hemolymph or embedded in the connective tissue of different body parts of molluscs, notably gastropods. These unique cells can be round, elongated or irregularly shaped, and up to 30 μm in diameter. Their hallmark is the so-called slit apparatus: i.e. pocket-like invaginations of the plasma membrane creating extracellular lacunae, bridged by cytoplasmic bars. These bars form distinctive slits of ca. 20 nm width. A slit diaphragm composed of proteins establishes a molecular sieve with holes of 20 x 20 nm. Different functions have been assigned to this special molluscan cell type, notably biosynthesis of the hemolymph respiratory protein hemocyanin. It has further been proposed, but not proven, that in the case of red-blooded snail species rhogocytes might synthesize the hemoglobin. However, the secretion pathway of these hemolymph proteins, and the functional role of the enigmatic slit apparatus remained unclear. Additionally proposed functions of rhogocytes, such as heavy metal detoxification or hemolymph protein degradation, are also not well studied. This work provides more detailed electron microscopical, histological and immunobiochemical information on the structure and function of rhogocytes of the freshwater snails Biomphalaria glabrata and Lymnaea stagnalis. By in situ hybridization on mantle tissues, it proves that B. glabrata rhogocytes synthesize hemoglobin and L. stagnalis rhogocytes synthesize hemocyanin. Hemocyanin is present, in endoplasmic reticulum lacunae and in vesicles, as individual molecules or pseudo-crystalline arrays. The first 3D reconstructions of rhogocytes are provided by means of electron tomography and show unprecedented details of the slit apparatus. A highly dense material in the cytoplasmic bars close to the diaphragmatic slits was shown, by immunogold labeling, to contain actin. By immunofluorescence microscopy, the protein nephrin was localized at the periphery of rhogocytes. The presence of both proteins in the slit apparatus supports the previous hypothesis, hitherto solely based on similarities of the ultrastructure, that the molluscan rhogocytes are phylogenetically related to mammalian podocytes and insect nephrocytes. A possible secretion pathway of respiratory proteins that includes a transfer mechanism of vesicles through the diaphragmatic slits is proposed and discussed. We also studied, by electron microscopy, the reaction of rhogocytes in situ to two forms of animal stress: deprivation of food and cadmium contamination of the tank water. Significant cellular reactions to both stressors were observed and documented. Notably, the slit apparatus surface and the number of electron-dense cytoplasmic vesicles increased in response to cadmium stress. Food deprivation led to an increase in hemocyanin production. These observations are also discussed in the framework of using such animals as potential environmental biomarkers.

Hemodynamic parameters change earlier than tissue oxygen tension in hemorrhage

BACKGROUND: Untreated hypovolemia results in impaired outcome. This study tests our hypothesis whether general hemodynamic parameters detect acute blood loss earlier than monitoring parameters of regional tissue beds. MATERIALS AND METHODS: Eight pigs (23-25 kg) were anesthetized and mechanically ventilated. A pulmonary artery catheter and an arterial catheter were inserted. Tissue oxygen tension was measured with Clark-type electrodes in the jejunal and colonic wall, in the liver, and subcutaneously. Jejunal microcirculation was assessed by laser Doppler flowmetry (LDF). Intravascular volume was optimized using difference in pulse pressure (dPP) to keep dPP below 13%. Sixty minutes after preparation, baseline measurements were taken. At first, 5% of total blood volume was withdrawn, followed by another 5% increment, and then in 10% increments until death. RESULTS: After withdrawal of 5% of estimated blood volume, dPP increased from 6.1% +/- 3.0% to 20.8% +/- 2.7% (P < 0.01). Mean arterial pressure (MAP), mean pulmonary artery pressure (PAP) and pulmonary artery occlusion pressure (PAOP) decreased with a blood loss of 10% (P < 0.01). Cardiac output (CO) changed after a blood loss of 20% (P < 0.05). Tissue oxygen tension in central organs, and blood flow in the jejunal muscularis decreased (P < 0.05) after a blood loss of 20%. Tissue oxygen tension in the skin, and jejunal mucosa blood flow decreased (P < 0.05) after a blood loss of 40% and 50%, respectively. CONCLUSIONS: In this hemorrhagic pig model systemic hemodynamic parameters were more sensitive to detect acute hypovolemia than tissue oxygen tension measurements or jejunal LDF measurements. Acute blood loss was detected first by dPP.

VEGF-A promotes intussusceptive angiogenesis in the developing chicken chorioallantoic membrane

To assess the impact of vascular endothelial growth factor (VEGF) on intussusceptive angiogenesis.

Respiratory muscle activity related to flow and lung volume in preterm infants compared with term infants

Infants with chronic lung disease (CLD) have a capacity to maintain functional lung volume despite alterations to their lung mechanics. We hypothesize that they achieve this by altering breathing patterns and dynamic elevation of lung volume, leading to differences in the relationship between respiratory muscle activity, flow and lung volume. Lung function and transcutaneous electromyography of the respiratory muscles (rEMG) were measured in 20 infants with CLD and in 39 healthy age-matched controls during quiet sleep. We compared coefficient of variations (CVs) of rEMG and the temporal relationship of rEMG variables, to flow and lung volume [functional residual capacity (FRC)] between these groups. The time between the start of inspiratory muscle activity and the resulting flow (tria)--in relation to respiratory cycle time--was significantly longer in infants with CLD. Although FRC had similar associations with tria and postinspiratory activity (corrected for respiratory cycle time), the CV of the diaphragmatic rEMG was lower in CLD infants (22.6 versus 31.0%, p = 0.030). The temporal relationship of rEMG to flow and FRC and the loss of adaptive variability provide additional information on coping mechanisms in infants with CLD. This technique could be used for noninvasive bedside monitoring of CLD.

Techniques for assessing cardiac output and fetal cardiac function

Fetal echocardiography was initially used to diagnose structural heart disease, but recent interest has focused on functional assessment. Effects of extracardiac conditions on the cardiac function such as volume overload (in the recipient in twin-twin transfusion syndrome), a hyperdynamic circulation (arterio-venous malformation), cardiac compression (diaphragmatic hernia, lung tumours) and increased placental resistance (intrauterine growth restriction and placental insufficiency) can be studied by ultrasound and may guide decisions for intervention or delivery. A variety of functional tests can be used, but there is no single clinical standard. For some specific conditions, however, certain tests have shown diagnostic value.

A transgenic model for conditional induction and rescue of portal hypertension reveals a role of VEGF-mediated regulation of sinusoidal fenestrations

Portal hypertension (PH) is a common complication and a leading cause of death in patients with chronic liver diseases. PH is underlined by structural and functional derangement of liver sinusoid vessels and its fenestrated endothelium. Because in most clinical settings PH is accompanied by parenchymal injury, it has been difficult to determine the precise role of microvascular perturbations in causing PH. Reasoning that Vascular Endothelial Growth Factor (VEGF) is required to maintain functional integrity of the hepatic microcirculation, we developed a transgenic mouse system for a liver-specific-, reversible VEGF inhibition. The system is based on conditional induction and de-induction of a VEGF decoy receptor that sequesters VEGF and preclude signaling. VEGF blockade results in sinusoidal endothelial cells (SECs) fenestrations closure and in accumulation and transformation of the normally quiescent hepatic stellate cells, i.e. provoking the two processes underlying sinusoidal capillarization. Importantly, sinusoidal capillarization was sufficient to cause PH and its typical sequela, ascites, splenomegaly and venous collateralization without inflicting parenchymal damage or fibrosis. Remarkably, these dramatic phenotypes were fully reversed within few days from lifting-off VEGF blockade and resultant re-opening of SECs' fenestrations. This study not only uncovered an indispensible role for VEGF in maintaining structure and function of mature SECs, but also highlights the vasculo-centric nature of PH pathogenesis. Unprecedented ability to rescue PH and its secondary manifestations via manipulating a single vascular factor may also be harnessed for examining the potential utility of de-capillarization treatment modalities.

Neurally adjusted ventilatory assist in patients with critical illness-associated polyneuromyopathy

Diaphragmatic electrical activity (EA(di)), reflecting respiratory drive, and its feedback control might be impaired in critical illness-associated polyneuromyopathy (CIPM). We aimed to evaluate whether titration and prolonged application of neurally adjusted ventilatory assist (NAVA), which delivers pressure (P (aw)) in proportion to EA(di), is feasible in CIPM patients.

Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.

Microvascular response to shock wave application in striated skin muscle

This study aims to quantify by intravital microscopy the microhemodynamic response after extracorporeal shock wave application (ESWA) to the physiologic microcirculation of the mouse dorsal skinfold chamber.