Опыт ингаляционного применения илопроста при хирургическом лечении хронической посттромбэмболической легочной гипертензии

The paper describes a case of inhaled iloprost use in a female patient with severe respiratory failure after pulmonary artery thromboendarterectomy, who was on venovenous extracorporeal membrane oxygenation. To reduce pulmonary hypertension and to prevent reperfusion syndrome, the patient received inhaled iloprost in a dose of 5 ^g every 3 hours during surgery and in the first two days after surgery. On day 14 after surgery, extracorporeal membrane oxygenation was stopped as there were satisfactory respiratory and hemodynamic parameters. Four days later, the patient was weaned from mechanical ventilation. The length of stay in the intensive care unit was 24 days. The patient was discharged from hospital in a satisfactory condition. Thus, the perioperative use of iloprost could reduce pulmonary artery pressure by twice; however, reperfusion syndrome could not prevent significant respiratory failure. The data available in the literature on the use of ilo-prost in patients after pulmonary artery thromboendarterectomy are single and their results are ambiguous. There is a need for large-scale multicenter studies in this group of patients. Key words: chronic postthromboembolic pulmonary hypertension, pulmonary artery thromboendarterectomy, iloprost, extracorporeal membrane oxygenation.В статье описан клинический случай ингаляционного применения илопроста у больной с тяжелой дыхательной недостаточностью после тромбэндартерэктомии из легочной артерии, находящейся на вено-венозной экстракорпоральной мембранной оксигенации. С целью снижения легочной гипертензии и профилактики реперфузионных повреждений больной во время операции и первые двое суток после операции проводилась ингаляция илопроста в дозе 5 мкг каждые 3 часа. На 14-е сутки после операции на фоне удовлетворительных показателей дыхания и гемодинамики экстракорпоральная мембранная оксигенация прекращена. Спустя 4 суток больная была отлучена от аппарата искусственной вентиляции легких. Срок пребывания в палате реанимации составил 24 дня. Больная была выписана из стационара в удовлетворительном состоянии. Таким образом, периоперационное использование илопроста позволило снизить давление в легочной артерии в 2 раза, однако реперфузионные повреждения не позволили избежать выраженной дыхательной недостаточности. Данные литературы по применению илопроста у пациентов после тромбэндар-терэктомии из легочной артерии единичны, а их результаты неоднозначны. Необходимы большие многоцентровые исследования данной группы пациентов. Ключевые слова: хроническая посттромбэмболическая легочная гипертен-зия, тромбэндартерэктомия из легочной артерии, илопрост, экстракорпоральная мембранная оксигенация

Validation of an LC-MS/MS Method to Quantify the New TRPC6 Inhibitor SH045 (Larixyl N-methylcarbamate) and Its Application in an Exploratory Pharmacokinetic Study in Mice

TRPC6 (transient receptor potential cation channels; canonical subfamily C, member 6) is widespread localized in mammalian tissues like kidney and lung and associated with progressive proteinuria and pathophysiological pulmonary alterations, e.g., reperfusion edema or lung fibrosis. However, the understanding of TRPC6 channelopathies is still at the beginning stages. Recently, by chemical diversification of (+)-larixol originating from Larix decidua resin traditionally used for inhalation, its methylcarbamate congener, named SH045, was obtained and identified in functional assays as a highly potent, subtype-selective inhibitor of TRPC6. To pave the way for use of SH045 in animal disease models, this study aimed at developing a capable bioanalytical method and to provide exploratory pharmacokinetic data for this promising derivative. According to international guidelines, a robust and selective LC-MS/MS method based on MRM detection in positive ion mode was established and validated for quantification of SH045 in mice plasma, whereby linearity and accuracy were demonstrated for the range of 2–1600 ng/mL. Applying this method, the plasma concentration time course of SH045 following single intraperitoneal administration (20 mg/kg body weight) revealed a short half-life of 1.3 h. However, the pharmacological profile of SH045 is promising, as five hours after administration, plasma levels still remained sufficiently higher than published low nanomolar IC50 values. Summarizing, the LC-MS/MS method and exploratory pharmacokinetic data provide essential prerequisites for experimental pharmacological TRPC6 modulation and translational treatment of TRPC6 channelopathies

Postoperative Intensive Care Management of Aortic Repair

Vascular surgery patients have multiple comorbidities and are at high risk for perioperative complications. Aortic repair surgery has greatly evolved in recent years, with an increasing predominance of endovascular techniques (EVAR). The incidence of cardiac complications is significantly reduced with endovascular repair, but high-risk patients require postoperative ST-segment monitoring. Open aortic repair may portend a prohibitive risk of respiratory complications that could be a contraindication for surgery. This risk is greatly reduced in the case of an endovascular approach, and general anesthesia should be avoided whenever possible in the case of endovascular repair. Preoperative renal function and postoperative kidney injury are powerful determinants of short- and long-term outcome, so that preoperative risk stratification and secondary prevention are critical tasks. Intraoperative renal protection with selective renal and distal aortic perfusion is essential during open repair. EVAR has lower rates of postoperative renal failure compared to open repair, with approximately half the risk for acute kidney injury (AKI) and one-third of the risk of hemodialysis requirement. Spinal cord ischemia used to be the most distinctive and feared complication of aortic repair. The risk has significantly decreased since the beginning of aortic surgery, with advances in surgical technique and spinal protection protocols, and is lower with endovascular repair. Endovascular repair avoids extensive aortic dissection and aortic cross-clamping and is generally associated with reduced blood loss and less coagulopathy. The intensive care physician must be aware that aortic repair surgery has an impact on every organ system, and the importance of early recognition of organ failure cannot be overemphasized

The study and manipulation of piglet gonocytes

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model

An evaluation of the therapeutic potential of human amniotic epithelial cells during ex-vivo donor lung perfusion

Ph. D. Thesis.Introduction: Ex Vivo Lung Perfusion (EVLP) provides a normothermic isolated environment for the evaluation and reconditioning of donor lungs deemed unsuitable for immediate transplantation and offers a unique opportunity to administer advanced therapeutics, such as cell-based therapies. Human Amniotic Epithelial Cells (hAECs) have been shown to have immunomodulatory properties that could reduce injury in donor lungs. Our aim was to assess the anti-inflammatory actions of hAECs when administered during EVLP to lungs declined for transplant due to poor organ function. Methods: hAECs were isolated from term placenta through enzymatic digestion. In in vitro studies, THP-1 derived macrophage phagocytosis and activation was determined after treatment with hAECs for 6 hours. Neutrophils were migrated through an IL-1b activated Human Microvascular Endothelial Cells (HMEC)-1 monolayer, after treatment of hAECs. In ex vivo perfusion studies; human lungs declined for transplant were split, with 150 x 106 hAECs or the HTR-8/SVneo cell line administered to each single lung and perfused concurrently for up to 4 hours (n=3). Serial samples of perfusate and tissue biopsies were collected for ELISA, qPCR and immunofluorescence (IF). Results: hAECs were isolated with 94 ± 4% purity, with an average isolation yielding 134.2 x 106 with viability >90%. hAECs reduced neutrophil transendothelial migration (p=0.0128). hAEC treatment of macrophages led to an increase in phagocytosis observed in vitro and a decrease in CXCL8 (p=0.0465) and TNFa (p=0.0158) expression. hAEC-treated lungs had significantly reduced TNFa expression in the tissue (p=0.0415). IF staining demonstrated reduced expression of CXCL8 and 3- nitrotyrosine in the hAEC-treated lungs compared to the HTR cell treated lungs. Conclusion: In vitro assays demonstrated the potential of hAECs to minimise proinflammatory macrophage activation and neutrophil migration. hAEC-treated lungs led to a reduction in pro-inflammatory cytokine production and oxidative stress. hAECs may offer a therapeutic approach to reduce inflammation in donor lungs during EVLP.NIHR Blood and Transplant Research Unit in Organ Donation and Transplantation (BTRU-ODT

Investigation of the mechanisms of acute lung injury, using an isolated perfused mouse lung

Acute lung injury (ALI) is a severe inflammatory lung disease with high mortality. Previous studies revealed several important concepts in ALI, including cellular interaction between lung-marginated leukocytes and pulmonary endothelium, and decompartmentalisation of soluble mediators. However, there are inherent limitations within both in vivo and in vitro models to identify the detailed mechanism underlying these concepts. In this PhD project, we attempted to address these unanswered questions, using an in situ isolated perfused mouse lung (IPL). Specifically, we aimed to 1) develop, characterise, and optimise the mouse IPL model; 2) investigate soluble and cellular aspects of two models of ALI that are particularly amenable to study using the IPL, namely ventilator-induced lung injury (VILI) and ischaemia-reperfusion injury. From a physiological viewpoint, VILI consists of 2 primary components, high-stretch and atelectasis. Modelling atelectasis-related injury in vivo is difficult due to negative pleural pressure. We took advantage of the zero pleural pressure of open-chest IPL system to develop an atelectasis-related VILI model. Comparison of this ‘atelectrauma’ and a high-stretch ‘volutrauma’ model demonstrated that both cause lung oedema and pulmonary inflammation, but the inflammatory impact was different between them. Volutrauma, but not atelectrauma, facilitated systemic cytokine release, in which lung-marginated monocytes seem to play an important role. This finding in the VILI model drove us to further investigate the role of these monocytes in an ischaemia-reperfusion model, which is clinically highly relevant and simulates a lung transplantation setting. Our results suggested that lung-marginated monocytes may also contribute to develop ischaemia-reperfusion injury, potentially involving TNF upregulation. Through this PhD project, we have successfully developed a technically very challenging mouse IPL model. We utilised the unique features of the IPL to develop experimental models that we believe will be strong tools to fill the gap between in vivo physiological significance and in vitro mechanistic understanding